MASARYK UNIVERSITY
FACULTY OF SCIENCE
Department of Experimental Biology
Section of Animal Physiology and Immunology
Modulation of professional phagocyte activity
HABILITATION THESIS
Field: Animal Physiology
BRNO 2019 RNDr. MILAN ČÍŽ, Ph.D.
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I would like to thank to all the people who contributed to the formation of this thesis. First
of all to my mentor Antonín Lojek, all colleagues from Institute of Biophysics, CAS (Brno);
Department of Experimental Biology, Faculty of Science, Masaryk University (Brno);
Institute of Experimental Pharmacology & Toxicology, SAS (Bratislava) and Institute of
Organic Chemistry with Centre for Phytochemistry, BAS (Plovdiv) for their intellectual
contribution and formation of friendly environment for my personal and professional
development. Further I would like to thank to all my former and current students for their
fundamental contribution to the experimental part of the thesis. Finally I would like to thank
to my wife Hana and the whole family for their support.
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TABLE OF CONTENTS
Abstract.................................................................................................................................. 6
1 Introduction.................................................................................................................. 7
1.1 Neutrophils.............................................................................................................. 7
1.2 Monocytes and macrophages.................................................................................. 9
2 Effects of serotonin on professional phagocytes........................................................ 11
3 Effects of Polyphenols and polysaccharides of plant origin on professional
phagocytes ........................................................................................................................... 14
3.1 Antioxidant properties of plant polyphenols ........................................................ 14
3.2 Immunomodulatory properties of plant polyphenols............................................ 16
3.3 Immunomodulatory properties of plant polysaccharides...................................... 18
4 Summary .................................................................................................................... 20
5 Future perspectives..................................................................................................... 21
5.1 Effects of histone post-translational modifications on the functional variability of
neutrophils ....................................................................................................................... 21
5.2 Plant polyphenol effects on professional phagocytes ........................................... 22
5.3 Plant and mushroom polysaccharide effects on professional phagocytes ............ 23
6 References.................................................................................................................. 24
7 List of supplements .................................................................................................... 30
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ABSTRACT
Neutrophils and monocytes/macrophages, so called professional phagocytes, as integral
constituents of innate immunity serve as an essential first-line of defence against microbial
pathogens and foreign substances at sites of inflammation. Upon activation, these cells
among other functional manifestations produce reactive oxygen and nitrogen species. While
respiratory burst is important for the elimination of invading pathogens, the overproduction
of reactive oxygen species or the impairment of endogenous antioxidant defences may lead
to a damage of membrane lipids, DNA, proteins, and lipoproteins which results in various
autoimmune and inflammatory diseases. Thus, a modulation of professional phagocyte
activity is very important task in physiology and medicine. We have been studying the
possibilities of professional phagocyte modulation by both endogenous (serotonin) and
exogenous (polyphenols and polysaccharides of plant origin as food constituents) mediators.
Our studies bring the evidence that serotonin released from platelets is a very important
modulator of professional phagocyte activity. Its inhibitory activity is manifested by the
decrease in the generation of reactive oxygen species due to the inhibition of
myeloperoxidase activity and by direct scavenging of reactive oxygen species. The effects
of serotonin on professional phagocytes are also partially mediated by 5-HTR2 receptor
expressed on monocytes/macrophages but not on neutrophils. We also introduced and
optimized several methods to investigate antioxidant properties of plant extracts and
individual plant polyphenols. We confirmed that many medicinal plants, vegetables and
berries are rich sources of polyphenol compounds and free radical scavengers. Furthermore,
we demonstrated that various plant polyphenols and polysaccharides exerted evident
immunomodulatory activity. Thus, these compounds represent effective naturally occurring
substances with potent pharmacological effects on respiratory burst of professional
phagocytes useful for treatment of compromised immune system and control of
inflammation.
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1 INTRODUCTION
Innate immunity serves as an essential first-line of defence against microbial pathogens and
foreign substances. Neutrophils are the typical effector cells of the innate immune response
because they are the first leukocytes to be recruited to an inflammatory site. In response to a
variety of stimuli, neutrophils adhere to and migrate through the endothelium to reach the
inflamed tissue where they engulf invading microorganisms and destroy them through
multiple oxidative and non-oxidative mechanisms (Futosi et al. 2013, Kolaczkowska and
Kubes 2013, Sadik et al. 2011). Neutrophils together with monocytes and macrophages are
called professional phagocytes because of their primary function – engulfing and destroying
potentially injurious particles.
1.1 NEUTROPHILS
Neutrophils play a vital role in innate immune reactions. They are recruited to sites of
infection and constitute the first line of defence (Selders et al. 2017, Timar et al. 2013). Based
on the short life-span and predefined set of functions, neutrophils were most often considered
as one population. Thus, functional and mechanistic studies on neutrophils generally deal
with them as a homogenous population of terminally differentiated cells (Chakravarti et al.
2009). However, a growing number of studies are showing the phenotype and functional
heterogeneity of neutrophils existing simultaneously both under healthy and acute immune
response conditions. Rather than being an end-stage uniform cell population, neutrophils can
show a great level of plasticity and develop distinct phenotypes or subpopulations under a
wide range of physiological (e.g. age) and pathological (e.g. inflammation, infection, cancer)
conditions. This makes neutrophils not only passive by-standers but showing their
importance in orchestrating function of other immune cells and over all inflammatory
response (Beyrau et al. 2012, Kamp et al. 2012, Sagiv et al. 2015, Welin et al. 2013).
Neutrophils are the most abundant white blood cell type in the blood stream of humans,
comprising about 50–70% of all leukocytes (Kruger et al. 2015). In response to infection or
injury they migrate to the site of inflammation to neutralize the invading microorganisms or
to eliminate tissue injury (Serhan et al. 2007). Neutrophil recruitment from the blood stream
is triggered by inflammatory stimuli such as chemokines, cytokines, or lipid mediators. The
inflammatory microenvironment triggers a process leading to the transmigration of
neutrophils through the vessel wall, known as extravasation (Mocsai et al. 2015). After the
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phagocytosis of invading microorganisms, neutrophils die by programmed cell death and are
ingested by tissue macrophages (Maderna and Godson 2003).
Fig. 1: Cascade of reactive oxygen species generation. eNOS - endothelial nitric oxide
synthase, GSH – reduced glutathione, GSSG - oxidized glutathione, MPO myeloperoxidase,
Prx - peroxiredoxin, SOD - superoxide dismutase, Trx - thioredoxin
(Vasicek et al. 2017)
Reactive oxygen species produced by phagocytes (Fig. 1) after their activation are one of the
most important microbicidal mechanisms associated with protection against invading
microorganisms and also seem to have important physiological roles in priming the immune
system as second messengers (Filippin et al. 2008, Halliwell 2007, Oktyabrsky and
Smirnova 2007). The process of reactive oxygen species production by neutrophils is known
as the respiratory burst and follows an activation of the enzyme NADPH oxidase (Fig. 2).
NADPH oxidase is a multicomponent enzyme system composed of heterodimeric
membrane-associated flavocytochrome b588 protein, which is composed of gp91phox and
p22phox subunits, three cytosolic proteins (p47phox, p67phox, and p40phox) and small
GTPase (Rac1 or Rac2) (Babior 1999). NADPH oxidase produces superoxide anion radical
which is rapidly reduced, either spontaneously or enzymatically, to hydrogen peroxide.
Hydrogen peroxide is the principal substrate for another important enzyme myeloperoxidase
which is stored in the azurophil granules and released by activated neutrophils to produce
other reactive oxygen species such as hypochlorous acid with potent cytotoxic properties
(Kolarova et al. 2013, Rudolph et al. 2012).
While respiratory burst is important for the elimination of invading microorganisms, the
overproduction of reactive oxygen species or the impairment of endogenous antioxidant
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defences may result in damaging effects on the host’s own cells and tissues (Freitas et al.
2009). The inappropriate production of reactive oxygen species by neutrophils is associated
with oxidative damage to membrane lipids, DNA, proteins, and lipoproteins which results
in various autoimmune and inflammatory diseases (Perecko et al. 2013, Sadik et al. 2011).
Fig. 2: Signalling pathways leading to the assembly and activation of NADPH oxidase in
neutrophils. AC - adenylate cyclase, DAG - diacyl-glycerol, fMLP - N-Formylmethionineleucyl-phenylalanine,
IP3 - inositol trisphosphate, MAPK - mitogen-activated protein
kinases, OZP - opsonized zymosan particles, PIP2 – phosphatidylinositol 4,5bisphosphate,
PKC - protein kinase C, PLC - phospholipase C, PMA - 4-phorbol-12myristate-13-acetate
(Vasicek et al. 2017)
1.2 MONOCYTES AND MACROPHAGES
Monocytes are generated from hematopoietic stem cells in bone marrow, released into
circulation and further differentiated into tissue macrophages and dendritic cells (Murray
and Wynn 2011, Shi and Pamer 2011). Cells of the monocyte/macrophage lineage are
important for host antimicrobial defence (Serbina et al. 2008) and are implicated in many
inflammatory diseases (Geissmann et al. 2008). Monocytes and macrophages together with
neutrophils belong to professional phagocytes which express various types of receptors on
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their surface to recognize signals that are not found in healthy cells and tissues (Gundra et
al. 2014, Matzinger and Kamala 2011). Upon activation, mononuclear phagocytes produce
reactive oxygen and nitrogen species by NADPH oxidase and inducible NO synthase,
respectively (Ambrozova et al. 2011). Nevertheless, the complex mechanisms of respiratory
burst and other functional manifestations of mononuclear phagocytes are different from
those in neutrophils. Neutrophils exhibit more rapid rates of phagocytosis and a more intense
respiratory burst response than macrophages. The endocytic and phagosome maturation
pathways in macrophages are substituted in neutrophils by the rapid delivery of preformed
granules to phagosomes (Nordenfelt and Tapper 2011).
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2 EFFECTS OF SEROTONIN ON PROFESSIONAL PHAGOCYTES
Serotonin (5-hydroxytryptamine), known mainly as one of the neurotransmitters of the
central nervous system, is also present in a variety of peripheral tissues, including several
constituents of the immune system. As the major secretory product of activated platelets, it
has been widely reported as a regulator of various constituents of the immune system and
immune functions. This modulation is complex and the data available are rather
controversial (Mohammad-Zadeh 2008, Olivier 2015, Wu et al. 2019, Xia et al. 2019).
We found that human platelets significantly decreased the amount of reactive oxygen species
produced by stimulated neutrophils and that the reduction was partially mediated by
serotonin released from platelets during their activation. We further investigated whether
platelet-induced inhibition of neutrophil respiratory burst could occur independently of
serotonin release and whether it could be pharmacologically modulated. We observed that
the inhibition of reactive oxygen species production was not accompanied with release of
serotonin by non-stimulated platelets and was more profound after addition of chloroquine
(a serotonin liberating drug) to platelet-neutrophil samples. Chloroquine itself did not affect
neutrophil reactive oxygen species production, but it caused a release of serotonin from
platelets to concentrations sufficient for inhibition of neutrophil reactive oxygen species
production. Our results indicated that non-stimulated platelets decreased neutrophil
production of reactive oxygen species by a serotonin-independent mechanism, while their
inhibitory effect could be enhanced pharmacologically through chloroquine-induced release
of serotonin. Furthermore, the effectiveness of chloroquine was found to be altered in the
presence of platelets. This indicated that under in vivo conditions, the effect of chloroquine
on one type of cells could be modified in the presence of cells of another type (Jancinova et
al. 2003).
Our next experiments clearly demonstrated that serotonin in a dose dependent manner was
also a potent inhibitor of reactive oxygen species production by rat whole blood phagocytes.
Since the effect of serotonin on phagocytes is complex, our experiments were focused on
elucidating the possible individual mechanisms of serotonin activity. Our results suggested
that the modulatory effect of serotonin on an respiratory burst of rat whole blood phagocytes
might occur through the activation of the 5-HTR2 serotonin receptor subtype since DOI
hydrochloride, a selective 5-HTR2 receptor agonist, had an inhibitory effect on the reactive
oxygen species production similar to that of serotonin. However, the antagonist of 5-HTR2
receptor did not block the effect of serotonin. Therefore, we could speculate that other
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mechanisms are involved in the serotonin dependent modulation of rat whole blood
phagocyte activity. Another probable explanation could be the direct scavenging activity of
serotonin against free radicals produced during respiratory burst of phagocytes (Okenkova
et al. 2007). It was previously already shown that serotonin could act as a true scavenger of
reactive oxygen species generated during the respiratory burst of stimulated phagocytes
(Schuff-Werner et al. 1995, Vial et al. 1995).
The aim of the following study was to clarify the mechanisms of serotonin effects on various
parameters of respiratory burst of human phagocytes using various luminol-enhanced
chemiluminescence methods. It was proved again that serotonin inhibited the
chemiluminescence response of human phagocytes in a dose dependent manner. Serotonin
was also found to exert a dose dependent inhibition of myeloperoxidase activity. The
hypothesis that the inhibitory activity of serotonin might be also receptor mediated was
evaluated using various serotonin receptor agonists and antagonists. Only DOI
hydrochloride, a selective agonist of 5-HTR2 receptor, exerted similar effects on phagocytes
as serotonin. It could be concluded that serotonin inhibitory activity on human phagocytes
could be explained by the decrease in the generation of reactive oxygen species (due to the
inhibition of myeloperoxidase activity) and direct scavenging of reactive oxygen species
already produced. The effect of serotonin on phagocytes might be also partially mediated by
5-HTR2 receptor (Ciz et al. 2007).
The goal of next experiments was to elucidate whether serotonin reduced the respiratory
burst of professional phagocytes via 5-HTR receptors or due to its antioxidative properties.
In this case, the effects of serotonin on total human leukocytes were compared with its effects
on isolated neutrophils. The ability of cells to produce reactive oxygen species was tested
using luminol-enhanced chemiluminescence analysis. In accordance with our hypothesis, it
was observed that serotonin and its 5-HTR2 agonist DOI hydrochloride were able to inhibit
the production of reactive oxygen species in activated rat and human total leukocytes, but
not in isolated human neutrophils. The interpretation of these results could suggest the
involvement of 5-HTR2 receptors expressed not on the neutrophils but on the surface of
other leukocytes including cells of monocyte/macrophage lineage. Moreover, some of the
observed inhibitory effects of serotonin on the respiratory burst of phagocytes were partially
caused by its reactive oxygen species scavenging activity (Pracharova et al. 2010).
In recent experiments, we showed that not only serotonin but also its structurally related
metabolites such as N-acetyl-serotonin and melatonin were able to reduce oxidative stress
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and inhibit production of inflammatory cytokines by murine RAW264.7 macrophages. In a
comparison of the tested compounds, serotonin and N-acetylserotonin were more effective
reactive oxygen species scavengers than melatonin. Other effects of tested compounds were
found to be very similar (Vasicek et al. 2019).
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3 EFFECTS OF POLYPHENOLS AND POLYSACCHARIDES OF
PLANT ORIGIN ON PROFESSIONAL PHAGOCYTES
A significant attention in medicine is paid to approaches designed either to enhance innate
immune mechanisms non-specifically with the aim to increase the defence against microbial
infections or to inhibit immune mechanisms involved in chronic or acute inflammation.
Thus, the effects of natural substances which can modulate the metabolic activity of
professional phagocytes and other immune cells without exerting any unfavourable side
effects have recently been investigated. Suitable candidates for this purpose might be
compounds contained in root, leaf and/or fruit extracts from herbs, fruits and vegetables.
These include especially polyphenols and polysaccharides (Lojek et al. 2014).
3.1 ANTIOXIDANT PROPERTIES OF PLANT POLYPHENOLS
We introduced into our laboratory and optimized several methods such as total peroxyl
radical-trapping parameter (TRAP), oxygen radical absorbance capacity (ORAC) and
hydroxyl radical averting capacity (HORAC) methods to investigate the antioxidant
properties of plant extracts and individual plant polyphenols. Although we found a good
correlation among all the methods for assessing antioxidant capacity and a good correlation
with polyphenol content, using more than one antioxidant assay was strongly recommended
since the combination of individual methods described the antioxidant properties of the
sample in more detail. Nevertheless, ORAC assay was found to be the most sensitive method
to measure chain-breaking antioxidant activity (hydrogen atom transfer pathway) in plant
extracts while HORAC assay measured mainly metal-chelating antioxidant capacity
(preventive antioxidants). As a supplement, the Folin–Ciocalteu assay (single electron
transfer pathway) should be a part of antioxidant profile measurement (Ciz et al. 2010).
Another parameter which significantly influenced the outcomes of studies investigating the
antioxidant properties of plant extracts (extractability of polyphenol compounds and
antioxidant activity of samples) was the extraction system used. When the ORAC parameter
and total polyphenol content of twenty five Bulgarian medicinal plants were examined, the
higher results were obtained for 80 % acetone extractions when compared to water extraction
(Kratchanova et al. 2010). Another approach was to use solid-phase extraction to obtain
anthocyanin-rich extracts from various berry species and their crude extracts. During solidphase
extraction the majority of impurities (sugars and acids) present in the crude extracts
were separated (Denev et al. 2010). It was concluded that the solvent used affected
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significantly the polyphenol content and the antioxidant activity of the extracts and therefore
it was recommended to use more than one extraction system for better assessment of the
antioxidant activity of natural products.
Using the optimized methods for plant polyphenol extraction and antioxidant capacity
evaluation we confirmed that many medicinal plants (Kratchanova et al. 2010), vegetables
(Ciz et al. 2010) and berries (Denev et al. 2010) tested were rich sources of polyphenol
compounds and free radical scavengers. Generally, there was a significant linear correlation
between the concentration of total polyphenols and the antioxidant capacity in investigated
samples.
In another study the antioxidant properties of four wine polyphenols (flavonoids catechin,
epicatechin, and quercetin, and hydroxystilbene resveratrol) were studied. All three
flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical
and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly
lower. All polyphenols also decreased production of reactive oxygen species by RAW264.7
macrophages. We concluded that the higher number of hydroxyl substituents is an important
structural feature of flavonoids in respect to their scavenging activity against reactive oxygen
species and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) was
important for inhibition of reactive oxygen species and nitric oxide production by RAW
264.7 macrophages. (Ciz et al. 2008) Resveratrol (3,5,4’-trihydroxystilbene) was also shown
to possess antioxidant activities in vitro in another study. It dose-dependently inhibited the
generation of peroxyl and hydroxyl radicals, peroxides, and lipid peroxidation products in
various cell free systems (Nosal et al. 2014).
Small fruits are a rich source of bioactive substances, including polyphenols, and are
therefore suitable raw materials for production of functional foods. In next study, we tested
the antioxidative properties of six fruits: rosehip (Rosa canina), black chokeberry (Aronia
melanocarpa), hawthorn (Crataegus monogyna), blackcurrant (Ribes nigrum), blueberry
(Vaccinium myrtillus) and rowanberry (Sorbus aucuparia) using various methods. The
antioxidant activity data indicated that all the fruits tested, and particularly rosehip and
hawthorn, were very suitable raw materials for the production of foods with strong
antioxidant properties (Denev et al. 2014b).
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3.2 IMMUNOMODULATORY PROPERTIES OF PLANT
POLYPHENOLS
Regardless of their direct scavenging activity, we also studied the immunomodulatory
activity of various plant constituents in various biological models. We observed that
pinosylvin, a stilbenoid that is synthetized in plants during fungal infections, was able to
decrease concentration of reactive oxygen and nitrogen species, along with its capacity to
enhance the efficacy of methotrexate in arthritis treatment in neutrophils of rats with
adjuvant arthritis and in RAW 264.7 macrophages (Jancinova et al. 2010). Another naturally
occurring phenolic derivative of resveratrol, pterostilbene, possessed comparable
antioxidant properties as resveratrol in human neutrophils and in cell free system. Reducing
the respiratory burst of human neutrophils during their activation in vitro with pterostilbene
does not include protein kinase C phosphorylation pathway. Pterostilbene showed dose
dependent activation/inhibition of caspase-3 enzyme activity (Perecko et al. 2010). Since
little was known about pterostilbene effects on neutrophils during inflammation in vivo, the
effect of pterostilbene on neutrophil activity was investigated in experimental arthritis model
in following study. Lewis rats were injected by a single intradermal injection of heat-killed
Mycobacterium butyricum in Freund’s adjuvant to develop arthritis. In the pterostilbene
treated arthritic rats, the treatment significantly lowered the number of neutrophils in blood
without significant downregulation of neutrophil respiratory burst. Pterostilbene only
nonsignificantly increased the antioxidant capacity in arthritic animals. These results
indicated that pterostilbene affected reactive oxygen species in vitro and in the animal model
of inflammation by different mechanisms. It can be concluded that the inhibitory effects of
stilbene derivatives and their bioavailability were structure dependent (Perecko et al. 2013).
We further demonstrated that resveratrol in a concentration-dependent manner inhibited the
respiratory burst of human whole blood professional phagocytes stimulated with various
activators. Results from isolated human neutrophils revealed that resveratrol was active
extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen
species. It was documented that resveratrol significantly decreased phosphorylation of
protein kinase C. Inhibition of nitric oxide production and inducible NO synthase protein
expression in RAW 264.7 cells indicated possible interference of resveratrol with reactive
nitrogen radical generation in professional phagocytes (Nosal et al. 2014).
Another study provided a comprehensive data on the antioxidant, antimicrobial and
neutrophil-modulating activities of extracts from six medicinal plants - blackberry (Rubus
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fruticosus) leaves, black chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus
monogyna) leaves, lady’s mantle (Alchemilla glabra) aerial parts, meadowsweet
(Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. Obtained results
indicated that all herbs studied were rich sources of chain-breaking, chelating and lipid
peroxidation inhibiting antioxidants, rendering high antioxidant activity measured by several
methods. Besides that the herb extracts could prevent oxidative stress in the gastrointestinal
tract trough modulation of neutrophils and an antimicrobial effect against a broad spectrum
of human pathogens (Denev et al. 2014a).
Small fruits are a rich source of several classes of polyphenol compounds and differences in
their polyphenol profile led to different biological activities. In next study, we tested the
effects of rosehip (Rosa canina), black chokeberry (Aronia melanocarpa), hawthorn
(Crataegus monogyna), blackcurrant (Ribes nigrum), blueberry (Vaccinium myrtillus) and
rowanberry (Sorbus aucuparia) on the production of reactive oxygen species by professional
phagocytes and their antimicrobial properties against eleven human pathogens. All extracts,
especially those from anthocyanin-rich fruits, inhibited reactive oxygen species production
in activated phagocytes, indicating that extracts interfered with the signalling cascade of
phagocyte activation upstream to the protein kinase C activation. Some of the extracts
revealed strong antimicrobial properties against selected human pathogens (Denev et al.
2014b). The recent study reported data on antioxidant, antimicrobial and neutrophilmodulating
activities of various polyphenolic preparations from black chokeberry (Aronia
melanocarpa) fruits: crude extract, purified extract standardized to 20% and 40%
anthocyanins, and proanthocyanidins; as well as of pure compounds (chlorogenic acid,
cyanidin-3-O-galactoside, epicatechin, rutin and quercetin) present in black chokeberries.
The highest antioxidant activity in cell free systems was observed for minor phenolic
components such as quercetin and epicatechin. According to the amount of individual
phenolics in the fruits, proanthocyanidins were the major contributor to the total antioxidant
activity of fresh black chokeberries. Studied polyphenols and preparations had no effect on
the spontaneous production of reactive oxygen species in human neutrophils. Greater effects
on the production of reactive oxygen species were observed in activated neutrophils,
especially for quercetin and rutin. The antimicrobial activity test against ten pathogens
showed that black chokeberry proanthocyanidins are the most potent antimicrobial agents in
the fruit. The study confirmed our hypothesis that black chokeberry polyphenols differed in
their antioxidant, antimicrobial and neutrophil modulating activities (Denev et al. 2019).
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3.3 IMMUNOMODULATORY PROPERTIES OF PLANT
POLYSACCHARIDES
We observed that leek (Allium ampeloprasum) polysaccharides modulated professional
phagocytes via different mechanisms. Leek pectic polysaccharides significantly decreased
the production of reactive oxygen species by human neutrophils. On the other hand,
polysaccharides isolated from alcohol insoluble substances of leek exhibited potent
macrophage-activating properties in a concentration-dependent manner resulting in an
increased generation of nitric oxide in RAW 264.7 cells via the induction of inducible NO
synthase. Moreover, polysaccharides extracted from alcohol insoluble substances with water
showed the ability to fix serum complement, especially through the alternative pathway. The
polysaccharide with the highest complement-fixing activity was characterized by the highest
content of uronic acids and the highest molecular weight. Nevertheless, the molecular weight
was not the only parameter that determined the macrophage immunomodulatory activity of
leek polysaccharides (Nikolova et al. 2013).
Other polysaccharide complexes containing mainly pectic polysaccharides were isolated
from the aerial parts of common purslane (Portulaca oleracea), and from the flowers of
common lavender (Lavandula angustifolia) and silver linden (Tilia tomentosa) by boiling
water extraction and ethanol precipitation. These samples also exerted stimulating activity
on phagocytes of the monocyte/macrophage lineage (CD14+ and CD64+ cells) and induced
production of reactive oxygen species. Despite that these extracts stimulated also T-cell
populations (CD4+/CD25+ and CD8+/CD25+ cells) and induced IL-6 production in human
leukocytes (Georgiev et al. 2017). The polysaccharide extracts with immunomodulatory
activity from lavender flowers were further chemically characterised as two pectic and one
polyphenolic fractions. One of the pectic polysaccharide fractions (52.4 kDa) contained
mainly low-acetylated and high-methoxylated homogalacturonans, and smaller
rhamnogalacturonan I backbone fragments rich in 1,3,5-branched arabinan and
arabinogalactan II side chains. The other pectic polysaccharide fraction (21.8 kDa) contains
predominantly similarly esterified homogalacturonans, followed by rhamnogalacturonan I
with arabinogalactan II structures and rhamnogalacturonan II. The prevalence of catechin
and epicatechin in polyphenolic polysaccharide fraction indicated that they form weak
interactions with pectins. In this set of experiments we confirmed that lavender pectins
activated innate and adaptive immune response through the complement system, neutrophils,
and macrophages. On the other hand, the observed inhibitory effects of pectins against the
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activated reactive oxygen species production and the suppression of nitric oxide generation
suggested anti-inflammatory activity of the studied pectins (Georgiev et al. 2017b).
Similarly, silver linden pectins with immunomodulation properties were analysed in more
details. Linden pectins induced reactive oxygen species and nitric oxide generation in nonstimulated
whole blood phagocytes and macrophages, but suppressed activated reactive
oxygen species generation, inducible NO synthase expression and nitric oxide production
(Georgiev et al. 2017c).
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4 SUMMARY
 Our studies bring the evidence that serotonin released from platelets is a very
important mediator of professional phagocyte activity. Serotonin inhibitory activity
on professional phagocytes is manifested by the decrease in the generation of reactive
oxygen species (due to the inhibition of myeloperoxidase activity) and direct
scavenging of reactive oxygen species already produced. The effects of serotonin on
professional phagocytes are also partially mediated by 5-HTR2 receptor expressed
on monocytes/macrophages but not on neutrophils. Thus, serotonin plays a principal
role in modulation of inflammation, regulation of function of immune cells and
protection of cells against the oxidative stress.
 We introduced and optimized several methods such as total peroxyl radical-trapping
parameter (TRAP), oxygen radical absorbance capacity (ORAC) and hydroxyl
radical averting capacity (HORAC) methods to investigate the antioxidant properties
of plant extracts and individual plant polyphenols. Using the optimized methods for
plant polyphenol extraction and antioxidant capacity evaluation we confirmed that
many medicinal plants, vegetables and berries tested were rich sources of polyphenol
compounds and free radical scavengers.
 We observed the immunomodulatory activity of various plant polyphenols in various
biological models. These compounds, namely stilbenes and proanthocyanidins
represented effective naturally occurring substances with potent pharmacological
effects on respiratory burst of human neutrophils and nitric oxide production by
macrophages.
 We demonstrated that various herbal materials contain immunomodulating pectic
polysaccharides that could be useful for treatment of compromised immune system
and control of inflammation. It was shown for the first time that acetylated and highly
glucuronidated rhamnogalacturonans I from Tilia tomentosa blossoms may exhibit
immunomodulating properties via neutrophil and monocyte/macrophage activation.
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5 FUTURE PERSPECTIVES
5.1 EFFECTS OF HISTONE POST-TRANSLATIONAL
MODIFICATIONS ON THE FUNCTIONAL VARIABILITY OF
NEUTROPHILS
Post-translational modifications can substantially and rapidly alter the phenotype of
neutrophils and influence their function. This suggests that regulation of the phenotype and
function of neutrophils represents a common mechanism for modulating innate or adaptive
immunity (Galli et al. 2011). Histone proteins are modified in response to various external
signals, however, their mechanisms are still not completely elucidated. One such
modification is citrullination, which is catalysed by peptidyl arginine deiminases (PADs), a
unique family of enzymes that catalyses the hydrolysis of peptidyl-arginine to form peptidylcitrulline
on histones and other biologically relevant proteins. Overexpression and/or
increased PAD activity is observed in several diseases, including rheumatoid arthritis
(Bicker and Thompson 2013, Neeli et al. 2008).
The role of neutrophil-derived reactive oxygen species in induction of neutrophil
extracellular traps (NETs) has been studied and discussed. Generally there are both reactive
oxygen species-dependent and reactive oxygen species-independent pathways leading to
NET formation. It has been recently reported, that inhibitors of NADPH oxidase and
myeloperoxidase disrupt NET formation (Zhou et al. 2018, Tao et al. 2018). Furthermore, it
has been shown that PAD4 is physically associated with p47phox and p67phox cytosolic
subunits of the neutrophil NADPH oxidase. After activation, PAD4 induced NADPH
oxidase subunit citrullination and their dissociation from PAD4, altered NADPH oxidase
assembly and production of reactive oxygen species (Hu et al. 2019).
We hypothesise that various activators enhance expression of PAD4, modulates its activity
and production of citrullinated histone H3 protein and cytosolic NADPH oxidase subunits
in neutrophils. Histone H3 citrullination leads to chromatin decondensation. This is
manifested by neutrophil functional variability. Altered reactive oxygen species production
also modulates the rate of chromatin decondensation and neutrophil extracellular trap
release. A comprehensive survey of the receptors and signalling pathways that regulate
PAD4 activation will be important for our understanding of innate immunity. Identification
of signalling intermediates in PAD4 activation may also lead to the generation of
pharmaceuticals to target histone H3 or NADPH oxidase subunit citrullination-related
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pathogenesis. The expected result will be an obtaining of new and original knowledge which
will serve as a source information for further research in biology and medicine with possible
application in clinical practice.
Fig. 3: Proposal hypothesis scheme
5.2 PLANT POLYPHENOL EFFECTS ON PROFESSIONAL
PHAGOCYTES
Neutrophil lifespan constitutes a sensitive balance between their function and their potential
to inflict tissue damage. In the absence of inflammatory stimuli, neutrophils continuously
undergo apoptosis. On the other hand, NETs are formed in response to a variety of proinflammatory
stimuli, microorganisms and pathogens. We hypothesize that plant
polyphenols as strong antioxidants and reactive oxygen species scavengers will inhibit
neutrophil NETosis and promote their apoptosis. As such, anthocyanins, hydroxycinnamic
acids and proanthocyanidins from plants may be a novel class of neutrophil apoptosisinducing
anti-inflammatory compounds for the treatment of human inflammatory diseases.
On the other hand, treatment with plant polyphenols may be considered as therapeutic
strategy for neutrophil-mediated diseases, in which NETosis play a role. The main aim of
the future studies will be to describe the antioxidative and anti-inflammatory effects of plant
polyphenols with respect to their role in modulation of neutrophil apoptosis and NETosis
and to clarify underlying molecular and signalling mechanisms.
23
5.3 PLANT AND MUSHROOM POLYSACCHARIDE EFFECTS ON
PROFESSIONAL PHAGOCYTES
The search for attractive sources of polysaccharides, the discovery of their new biological
effects, and the elucidation of intimate biochemical mechanisms of their action are major
scientific problems in the field of carbohydrate chemistry and glycobiology. The general
goal of the future project proposal will be to investigate the structure of purified herbal and
mushroom polysaccharides and the biochemical mechanisms of immunomodulation of
phagocytes by them. Neutrophils and macrophages play a significant role not only in our
own immune defence, but also in the development of an immunomodulatory response.
Generally, it is hypothesized that immunomodulatory effects of polysaccharides on
neutrophils and macrophages may have protective effects in inflammatory, infectious,
allergic, tumour, autoimmune and gastrointestinal diseases.
24
6 REFERENCES
Ambrozova, G., M. Pekarova and A. Lojek (2011). "The effect of lipid peroxidation products
on reactive oxygen species formation and nitric oxide production in lipopolysaccharidestimulated
RAW 264.7 macrophages." Toxicol In Vitro 25(1): 145-152.
Babior, B. M. (1999). "NADPH oxidase: an update." Blood 93(5): 1464-1476.
Beyrau, M., J. V. Bodkin and S. Nourshargh (2012). "Neutrophil heterogeneity in health and
disease: a revitalized avenue in inflammation and immunity." Open Biol 2(11): 120134.
Bicker, K. L. and P. R. Thompson (2013). "The protein arginine deiminases: Structure,
function, inhibition, and disease." Biopolymers 99(2): 155-163.
Chakravarti, A., D. Rusu, N. Flamand, P. Borgeat and P. E. Poubelle (2009).
"Reprogramming of a subpopulation of human blood neutrophils by prolonged exposure to
cytokines." Lab Invest 89(10): 1084-1099.
Ciz, M., D. Komrskova, L. Pracharova, K. Okenkova, H. Cizova, A. Moravcova, V.
Jancinova, M. Petrikova, A. Lojek and R. Nosal (2007). "Serotonin modulates the oxidative
burst of human phagocytes via various mechanisms." Platelets 18(8): 583-590.
Ciz, M., M. Pavelkova, L. Gallova, J. Kralova, L. Kubala and A. Lojek (2008). "The
influence of wine polyphenols on reactive oxygen and nitrogen species production by murine
macrophages RAW 264.7." Physiol Res 57(3): 393-402.
Ciz, M., H. Cizova, P. Denev, M. Kratchanova, A. Slavov and A. Lojek (2010). "Different
methods for control and comparison of the antioxidant properties of vegetables." Food
Control 21(4): 518-523.
Denev, P., M. Ciz, G. Ambrozova, A. Lojek, I. Yanakieva and M. Kratchanova (2010).
"Solid-phase extraction of berries' anthocyanins and evaluation of their antioxidative
properties." Food Chem 123(4): 1055-1061.
Denev, P., M. Kratchanova, M. Ciz, A. Lojek, O. Vasicek, D. Blazheva, P. Nedelcheva, L.
Vojtek and P. Hyrsl (2014a). "Antioxidant, antimicrobial and neutrophil-modulating
activities of herb extracts." Acta Biochim Pol 61(2): 359-367.
Denev, P., M. Kratchanova, M. Ciz, A. Lojek, O. Vasicek, P. Nedelcheva, D. Blazheva, R.
Toshkova, E. Gardeva, L. Yossifova, P. Hyrsl and L. Vojtek (2014b). "Biological activities
25
of selected polyphenol-rich fruits related to immunity and gastrointestinal health." Food
Chem 157: 37-44.
Denev, P., M. Ciz, M. Kratchanova and D. Blazheva (2019). "Black chokeberry (Aronia
melanocarpa) polyphenols reveal different antioxidant, antimicrobial and neutrophilmodulating
activities." Food Chem 284: 108-117.
Filippin, L. I., R. Vercelino, N. P. Marroni and R. M. Xavier (2008). "Redox signalling and
the inflammatory response in rheumatoid arthritis." Clin Exp Immunol 152(3): 415-422.
Freitas, M., J. L. Lima and E. Fernandes (2009). "Optical probes for detection and
quantification of neutrophils' oxidative burst. A review." Anal Chim Acta 649(1): 8-23.
Futosi, K., S. Fodor and A. Mocsai (2013). "Neutrophil cell surface receptors and their
intracellular signal transduction pathways." Int Immunopharmacol 17(3): 638-650.
Galli, S. J., N. Borregaard and T. A. Wynn (2011). "Phenotypic and functional plasticity of
cells of innate immunity: macrophages, mast cells and neutrophils." Nat Immunol 12(11):
1035-1044.
Geissmann, F., C. Auffray, R. Palframan, C. Wirrig, A. Ciocca, L. Campisi, E. NarniMancinelli
and G. Lauvau (2008). "Blood monocytes: distinct subsets, how they relate to
dendritic cells, and their possible roles in the regulation of T-cell responses." Immunol Cell
Biol 86(5): 398-408.
Georgiev, Y. N., M. H. Ognyanov, H. Kiyohara, T. G. Batsalova, B. M. Dzhambazov, M.
Ciz, P. N. Denev, H. Yamada, B. S. Paulsen, O. Vasicek, A. Lojek, H. Barsett, D. Antonova
and M. G. Kratchanova (2017a). "Acidic polysaccharide complexes from purslane, silver
linden and lavender stimulate Peyer's patch immune cells through innate and adaptive
mechanisms." Int J Biol Macromol 105(Pt 1): 730-740.
Georgiev, Y. N., B. S. Paulsen, H. Kiyohara, M. Ciz, M. H. Ognyanov, O. Vasicek, F. Rise,
P. N. Denev, H. Yamada, A. Lojek, V. Kussovski, H. Barsett, A. I. Krastanov, I. Z.
Yanakieva and M. G. Kratchanova (2017b). "The common lavender (Lavandula angustifolia
Mill.) pectic polysaccharides modulate phagocytic leukocytes and intestinal Peyer's patch
cells." Carbohydr Polym 174: 948-959.
Georgiev, Y. N., B. S. Paulsen, H. Kiyohara, M. Ciz, M. H. Ognyanov, O. Vasicek, F. Rise,
P. N. Denev, A. Lojek, T. G. Batsalova, B. M. Dzhambazov, H. Yamada, R. Lund, H.
26
Barsett, A. I. Krastanov, I. Z. Yanakieva and M. G. Kratchanova (2017c). "Tilia tomentosa
pectins exhibit dual mode of action on phagocytes as beta-glucuronic acid monomers are
abundant in their rhamnogalacturonans I." Carbohydr Polym 175: 178-191.
Gundra, U. M., N. M. Girgis, D. Ruckerl, S. Jenkins, L. N. Ward, Z. D. Kurtz, K. E. Wiens,
M. S. Tang, U. Basu-Roy, A. Mansukhani, J. E. Allen and P. Loke (2014). "Alternatively
activated macrophages derived from monocytes and tissue macrophages are phenotypically
and functionally distinct." Blood 123(20): e110-122.
Halliwell, B. (2007). "Biochemistry of oxidative stress." Biochem Soc Trans 35(Pt 5): 1147-
1150.
Hu, Q., H. Shi, T. Zeng, H. Liu, Y. Su, X. Cheng, J. Ye, Y. Yin, M. Liu, H. Zheng, X. Wu,
H. Chi, Z. Zhou, J. Jia, Y. Sun, J. Teng and C. Yang (2019). "Increased neutrophil
extracellular traps activate NLRP3 and inflammatory macrophages in adult-onset Still's
disease." Arthritis Res Ther 21(1): 9.
Jancinova, V., R. Nosal, A. Lojek, M. Ciz, G. Ambrozova, D. Mihalova, K. Bauerova, J.
Harmatha and T. Perecko (2010). "Formation of reactive oxygen and nitrogen species in the
presence of pinosylvin - an analogue of resveratrol." Neuroendocrinol Lett 31: 79-83.
Kamp, V. M., J. Pillay, J. W. Lammers, P. Pickkers, L. H. Ulfman and L. Koenderman
(2012). "Human suppressive neutrophils CD16bright/CD62Ldim exhibit decreased
adhesion." J Leukoc Biol 92(5): 1011-1020.
Kolaczkowska, E. and P. Kubes (2013). "Neutrophil recruitment and function in health and
inflammation." Nat Rev Immunol 13(3): 159-175.
Kolarova, H., A. Klinke, S. Kremserova, M. Adam, M. Pekarova, S. Baldus, J. P. Eiserich
and L. Kubala (2013). "Myeloperoxidase induces the priming of platelets." Free Radic Biol
Med 61: 357-369.
Kratchanova, M., P. Denev, M. Ciz, A. Lojek and A. Mihailov (2010). "Evaluation of
antioxidant activity of medicinal plants containing polyphenol compounds. Comparison of
two extraction systems." Acta Biochim Pol 57(2): 229-234.
Kruger, P., M. Saffarzadeh, A. N. Weber, N. Rieber, M. Radsak, H. von Bernuth, C.
Benarafa, D. Roos, J. Skokowa and D. Hartl (2015). "Neutrophils: Between host defence,
immune modulation, and tissue injury." PLoS Pathog 11(3): e1004651.
27
Lojek, A., P. Denev, M. Ciz, O. Vasicek and M. Kratchanova (2014). "The effects of
biologically active substances in medicinal plants on the metabolic activity of neutrophils."
Phytochem Rev 13(2): 499-510.
Maderna, P. and C. Godson (2003). "Phagocytosis of apoptotic cells and the resolution of
inflammation." Biochim Biophys Acta 1639(3): 141-151.
Matzinger, P. and T. Kamala (2011). "Tissue-based class control: the other side of
tolerance." Nat Rev Immunol 11(3): 221-230.
Mocsai, A., B. Walzog and C. A. Lowell (2015). "Intracellular signalling during neutrophil
recruitment." Cardiovasc Res 107(3): 373-385.
Mohammad-Zadeh, L. F., L. Moses and S. M. Gwaltney-Brant (2008). "Serotonin: a
review." J Vet Pharmacol Ther 31(3): 187-199.
Murray, P. J., and T. A. Wynn (2011). “Protective and pathogenic functions of macrophage
subsets.” Nat Rev Immunol 11: 723-737.
Neeli, I., S. N. Khan and M. Radic (2008). "Histone deimination as a response to
inflammatory stimuli in neutrophils." J Immunol 180(3): 1895-1902.
Nikolova, M., G. Ambrozova, M. Kratchanova, P. Denev, V. Kussovski, M. Ciz and A.
Lojek (2013). "Effects of Pectic Polysaccharides Isolated from Leek on the Production of
Reactive Oxygen and Nitrogen Species by Phagocytes." J Med Food 16(8): 711-718.
Nordenfelt, P. and H. Tapper (2011). "Phagosome dynamics during phagocytosis by
neutrophils." J Leukoc Biol 90(2): 271-284.
Nosal, R., K. Drabikova, V. Jancinova, T. Perecko, G. Ambrozova, M. Ciz, A. Lojek, M.
Pekarova, J. Smidrkal and J. Harmatha (2014). "On the Molecular Pharmacology of
Resveratrol on Oxidative Burst Inhibition in Professional Phagocytes." Oxid Med Cell
Longev: 706269
Okenkova, K., A. Lojek, L. Kubala and M. Ciz (2007). “Modulation of rat blood phagocyte
activity by serotonin.” Chem Listy 101: 245-246.
Oktyabrsky, O. N. and G. V. Smirnova (2007). "Redox regulation of cellular functions."
Biochemistry (Mosc) 72(2): 132-145.
Olivier, B. (2015). "Serotonin: a never-ending story." Eur J Pharmacol 753: 2-18.
28
Perecko, T., K. Drabikova, L. Rackova, M. Ciz, M. Podborska, A. Lojek, J. Harmatha, J.
Smidrkal, R. Nosal and V. Jancinova (2010). "Molecular targets of the natural antioxidant
pterostilbene: effect on protein kinase C, caspase-3 and apoptosis in human neutrophils in
vitro." Neuroendocrinol Lett 31: 84-90.
Perecko, T., K. Drabikova, A. Lojek, M. Ciz, S. Ponist, K. Bauerova, R. Nosal, J. Harmatha
and V. Jancinova (2013). "The Effects of Pterostilbene on Neutrophil Activity in
Experimental Model of Arthritis." Biomed Res Int: 106041.
Rudolph, V., T. Keller, A. Schulz, F. Ojeda, T. K. Rudolph, S. Tzikas, C. Bickel, T.
Meinertz, T. Munzel, S. Blankenberg and S. Baldus (2012). "Diagnostic and prognostic
performance of myeloperoxidase plasma levels compared with sensitive troponins in
patients admitted with acute onset chest pain." Circ Cardiovasc Genet 5(5): 561-568.
Sadik, C. D., N. D. Kim and A. D. Luster (2011). "Neutrophils cascading their way to
inflammation." Trends Immunol 32(10): 452-460.
Sagiv, J. Y., J. Michaeli, S. Assi, I. Mishalian, H. Kisos, L. Levy, P. Damti, D. Lumbroso,
L. Polyansky, R. V. Sionov, A. Ariel, A. H. Hovav, E. Henke, Z. G. Fridlender and Z. Granot
(2015). "Phenotypic diversity and plasticity in circulating neutrophil subpopulations in
cancer." Cell Rep 10(4): 562-573.
Schuff-Werner, P., W. Splettstosser, F. Schmidt and G. Huether (1995). "Serotonin acts as
a radical scavenger and is oxidized to a dimer during the respiratory burst of human
mononuclear and polymorphonuclear phagocytes." Eur J Clin Invest 25(7): 477-484.
Selders, G. S., A. E. Fetz, M. Z. Radic and G. L. Bowlin (2017). "An overview of the role
of neutrophils in innate immunity, inflammation and host-biomaterial integration." Regen
Biomater 4(1): 55-68.
Serhan, C. N., S. D. Brain, C. D. Buckley, D. W. Gilroy, C. Haslett, L. A. O'Neill, M. Perretti,
A. G. Rossi and J. L. Wallace (2007). "Resolution of inflammation: state of the art,
definitions and terms." FASEB J 21(2): 325-332.
Shi, C., and E. G. Pamer (2011). “Monocyte recruitment during infection and inflammation.”
Nat Rev Immunol 11: 762-774.
Tao, L., M. Xu, X. Dai, T. Ni, D. Li, F. Jin, H. Wang, L. Tao, B. Pan, J. R. Woodgett, Y.
Qian and Y. Liu (2018). "Polypharmacological Profiles Underlying the Antitumor Property
29
of Salvia miltiorrhiza Root (Danshen) Interfering with NOX-Dependent Neutrophil
Extracellular Traps." Oxid Med Cell Longev 2018: 4908328.
Timar, C. I., A. M. Lorincz and E. Ligeti (2013). "Changing world of neutrophils." Pflugers
Arch 465(11): 1521-1533.
Vasicek, O., T. Perecko, V. Jancinova, S. Pazourekova, R. Nosal, R., and M. Ciz (2017).
“Chemiluminescence: A Sensitive Method for Detecting the Effects of Histamine Receptor
Agonists/Antagonists on Neutrophil Oxidative Burst.”, in: Tiligada, E., Ennis, M. (Eds.),
Histamine Receptors as Drug Targets. Humana Press Inc, Totowa, pp. 183-208.
Vasicek, O., A. Lojek and M. Ciz (2019). “Serotonin and its metabolites reduce oxidative
stress in murine RAW264.7 macrophages and prevent inflammation.” Submitted to J Physiol
Biochem.
Vial, T., R. Tedone, C. Patriarca and J. Descotes (1995). "Effect of serotonin on the
chemiluminescence response of rat peripheral blood leucocytes." Int J Immunopharmacol
17(10): 813-819.
Welin, A., F. Amirbeagi, K. Christenson, L. Bjorkman, H. Bjornsdottir, H. Forsman, C.
Dahlgren, A. Karlsson and J. Bylund (2013). "The human neutrophil subsets defined by the
presence or absence of OLFM4 both transmigrate into tissue in vivo and give rise to distinct
NETs in vitro." PLoS One 8(7): e69575.
Wu, H., T. H. Denna, J. N. Storkersen and V. A. Gerriets (2019). "Beyond a
neurotransmitter: The role of serotonin in inflammation and immunity." Pharmacol Res 140:
100-114.
Xia, Y., S. Chen, S. Zeng, Y. Zhao, C. Zhu, B. Deng, G. Zhu, Y. Yin, W. Wang, R. Hardeland
and W. Ren (2019). "Melatonin in macrophage biology: Current understanding and future
perspectives." J Pineal Res 66(2): e12547.
Zhou, Y., L. L. An, R. Chaerkady, N. Mittereder, L. Clarke, T. S. Cohen, B. Chen, S. Hess,
G. P. Sims and T. Mustelin (2018). "Evidence for a direct link between PAD4-mediated
citrullination and the oxidative burst in human neutrophils." Sci Rep 8(1): 15228.
30
7 LIST OF SUPPLEMENTS
1. Jancinova V, Drabikova K, Nosal R, Petrikova M, Ciz M, Lojek A, Danihelova E
(2003) Inhibition of FMLP-stimulated neutrophil chemiluminescence by blood
platelets increased in the presence of the serotonin-liberating drug chloroquine.
Thrombosis Research 109: 293-298
2. Okenkova K, Lojek A, Kubala L, Ciz M (2007) Modulation of rat blood phagocyte
activity by serotonin. Chemické Listy 101: 245-246
3. Ciz M, Komrskova D, Pracharova L, Okenkova K, Cizova H, Moravcova A, Jancinova
V, Petrikova M, Lojek A, Nosal R (2007) Serotonin modulates the oxidative burst of
human phagocytes via various mechanisms. Platelets 18: 583-590
4. Pracharova L, Okenkova K, Lojek A, Ciz M (2010) Serotonin and its 5-HT2 receptor
agonist DOI hydrochloride inhibit the oxidative burst in total leukocytes but not in
isolated neutrophils. Life Sciences 86: 518-523
5. Vasicek O, Lojek A, Ciz M (2019) Serotonin and its metabolites reduce oxidative
stress in murine RAW264.7 macrophages and prevent inflammation. Submitted to
Journal of Physiol Biochem.
6. Ciz M, Pavelkova M, Gallova L, Kralova J, Kubala L, Lojek A (2008) The influence
of wine polyphenols on reactive oxygen and nitrogen species production by murine
macrophages RAW 264.7. Physiological Research 57: 393-402
7. Kratchanova M, Denev P, Ciz M, Lojek A, Mihailov A (2010) Evaluation of
antioxidant activity of medicinal plants containing polyphenol compounds.
Comparison of two extraction systems. Acta Biochimica Polonica 57: 229-234
8. Ciz M, Cizova H, Denev P, Kratchanova M, Slavov A, Lojek A (2010) Different
methods for control and comparison of the antioxidant properties of vegetables. Food
Control 21: 518-523
9. Denev P, Ciz M, Ambrozova G, Lojek A, Yanakieva I, Kratchanova M (2010) Solidphase
extraction of berries' anthocyanins and evaluation of their antioxidative
properties. Food Chemistry 123: 1055-1061
10. Jancinova V, Nosal R, Lojek A, Ciz M, Ambrozova G, Mihalova D, Bauerova K,
Harmatha J, Perecko T (2010) Formation of reactive oxygen and nitrogen species in
31
the presence of pinosylvin - an analogue of resveratrol. Neuroendocrinology Letters
31: 79-83
11. Perecko T, Drabikova K, Rackova L, Ciz M, Podborska M, Lojek A, Harmatha J,
Smidrkal J, Nosal R, Jancinova V (2010) Molecular targets of the natural antioxidant
pterostilbene: effect on protein kinase C, caspase-3 and apoptosis in human neutrophils
in vitro. Neuroendocrinology Letters 31: 84-90
12. Perecko T, Drabikova K, Lojek A, Ciz M, Ponist S, Bauerova K, Nosal R, Harmatha
J, Jancinova V (2013) The Effects of Pterostilbene on Neutrophil Activity in
Experimental Model of Arthritis. Biomed Research International, Artn 106041
13. Denev P, Kratchanova M, Ciz M, Lojek A, Vasicek O, Blazheva D, Nedelcheva P,
Vojtek L, Hyrsl P (2014) Antioxidant, antimicrobial and neutrophil-modulating
activities of herb extracts. Acta Biochimica Polonica 61: 359-367
14. Denev P, Kratchanova M, Ciz M, Lojek A, Vasicek O, Nedelcheva P, Blazheva D,
Toshkova R, Gardeva E, Yossifova L, Hyrsl P, Vojtek L (2014) Biological activities
of selected polyphenol-rich fruits related to immunity and gastrointestinal health. Food
Chemistry 157: 37-44
15. Nosal R, Drabikova K, Jancinova V, Perecko T, Ambrozova G, Ciz M, Lojek A,
Pekarova M, Smidrkal J, Harmatha J (2014) On the Molecular Pharmacology of
Resveratrol on Oxidative Burst Inhibition in Professional Phagocytes. Oxidative
Medicine and Cellular Longevity, Artn 706269
16. Denev P, Ciz M, Kratchanova M, Blazheva D (2019) Black chokeberry (Aronia
melanocarpa) polyphenols reveal different antioxidant, antimicrobial and neutrophilmodulating
activities. Food Chemistry 284: 108-117
17. Nikolova M, Ambrozova G, Kratchanova M, Denev P, Kussovski V, Ciz M, Lojek A
(2013) Effects of Pectic Polysaccharides Isolated from Leek on the Production of
Reactive Oxygen and Nitrogen Species by Phagocytes. Journal of Medicinal Food 16:
711-718
18. Georgiev YN, Ognyanov MH, Kiyohara H, Batsalova TG, Dzhambazov BM, Ciz M,
Denev PN, Yamada H, Paulsen BS, Vasicek O, Lojek A, Barsett H, Antonova D,
Kratchanova MG (2017) Acidic polysaccharide complexes from purslane, silver
32
linden and lavender stimulate Peyer's patch immune cells through innate and adaptive
mechanisms. International Journal of Biological Macromolecules 105: 730-740
19. Georgiev YN, Paulsen BS, Kiyohara H, Ciz M, Ognyanov MH, Vasicek O, Rise F,
Denev PN, Yamada H, Lojek A, Kussovski V, Barsett H, Krastanov AI, Yanakieva
IZ, Kratchanova MG (2017) The common lavender (Lavandula angustifolia Mill.)
pectic polysaccharides modulate phagocytic leukocytes and intestinal Peyer's patch
cells. Carbohydrate Polymers 174: 948-959
20. Georgiev YN, Paulsen BS, Kiyohara H, Ciz M, Ognyanov MH, Vasicek O, Rise F,
Denev PN, Lojek A, Batsalova TG, Dzhambazov BM, Yamada H, Lund R, Barsett H,
Krastanov AI, Yanakieva IZ, Kratchanova MG (2017) Tilia tomentosa pectins exhibit
dual mode of action on phagocytes as beta-glucuronic acid monomers are abundant in
their rhamnogalacturonans I. Carbohydrate Polymers 175: 178-191
Regular Article
Inhibition of FMLP-stimulated neutrophil chemiluminescence by blood
platelets increased in the presence of the serotonin-liberating
drug chloroquine
Viera Jancˇinova´a,*, Katarı´na Dra´bikova´a
, Radomı´r Nosa´l’a
, Margita Petrı´kova´a
,
Milan Cˇ ı´zˇb
, Antonı´n Lojekb
, Edita Danihelova´c
a
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Du´bravska´ cesta 9, 841 04 Bratislava, Slovak Republic
b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
c
Institute of Haematology and Transfusiology, Bratislava, Slovak Republic
Received 8 November 2002; received in revised form 14 March 2003; accepted 17 March 2003
Abstract
Introduction: Previously, we reported that human blood platelets significantly decreased the concentration of reactive oxygen species
(chemiluminescence) produced by Ca2 +
-ionophore-stimulated neutrophils and that the reduction was partially mediated by serotonin
liberated from platelets during their activation. The aim of the present study was to investigate whether platelet inhibition can occur
independently of serotonin liberation and whether it can be pharmacologically enhanced. Materials and methods: Chemiluminescence was
measured after stimulation of human neutrophils with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in the presence of
luminophore luminol. Concentration of platelet serotonin was estimated fluorometrically. Results: Platelets, added to neutrophils in the
physiological cell ratio 50:1, decreased neutrophil chemiluminescence by 47%. The inhibition was not accompanied with liberation of
platelet serotonin and rose after addition of chloroquine to platelet–neutrophil samples. In the absence of platelets, this drug did not affect
neutrophil chemiluminescence. Chloroquine actively liberated serotonin; amine concentrations found in platelet supernatants were sufficient
to inhibit neutrophil chemiluminescence. Conclusions: The presented results indicate that unstimulated platelets decreased neutrophil
chemiluminescence by a serotonin-independent mechanism, yet their inhibitory effect could be enhanced pharmacologically through
chloroquine-induced serotonin liberation.
D 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Blood platelets; Neutrophils; Chloroquine; Chemiluminescence; Serotonin; FMLP
1. Introduction
New activities of blood platelets are still appearing
despite the fact that they have been in the focus of interest
for more than 160 years. Of these, inhibition of neutrophil
functions seems to be noteworthy since it could indicate
platelet involvement in tissue protection against toxic effects
of neutrophil products. They include particularly reactive
oxygen species and proteolytic enzymes, which if released
inappropriately, may induce serious tissue damage, as found
in respiratory distress syndrome [1], ischaemia/reperfusion
injury [2], renal failure [3], rheumatoid arthritis [4], gastrointestinal
inflammation [5], or in cerebral infarction [6].
Blood platelets could represent a unique protective
mechanism, active selectively at sites exposed to toxic
effects of neutrophil products. As found in patients suffering
from myocardial ischaemia/reperfusion injury, binding of
platelets to neutrophils decreased production of superoxide
anion [7] or conversely, lower platelet reactivity was associated
with more severe inflammatory characteristics [8].
Under in vitro conditions, blood platelets diminished formation
of reactive oxygen species and/or chemiluminescence
of neutrophils [9,10], reduced elastase secretion and
0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0049-3848(03)00239-1
Abbreviations: A23187, Ca2+
-ionophore A23187; ADP, adenosine 5Vdiphosphate;
ATP, adenosine 5V-triphosphate; EDTA, ethylene diamine
tetraacetic acid; FMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine;
PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate;
PRP, platelet-rich plasma.
* Corresponding author. Tel.: +421-2-594-10-672; fax: +421-2-547-75-
928.
E-mail address: exfavega@savba.sk (V. Jancˇinova´).
Thrombosis Research 109 (2003) 293–298
myeloperoxidase activity, and impaired intracellular killing
[11,12]. As we reported previously, chemiluminescence (i.e.
concentration of reactive oxygen species) produced by
Ca2 +
-ionophore A23187 (A23187)-stimulated neutrophils
decreased significantly in the presence of blood platelets,
and the reduction was partially mediated by serotonin
liberated from platelets during their activation [13,14]. The
aim of the present study was to investigate whether platelet
inhibition can occur independently of serotonin liberation
and whether it can be pharmacologically enhanced.
2. Materials and methods
2.1. Materials
N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP),
dextran (average mol. wt 464,000), luminol (5-amino-2,3dihydro-1,4-phthalazinedione),
hypoxanthine, xanthine oxidase,
and hydrogen peroxide were from Sigma-Aldrich
Chemie (Deisenhofen, Germany); Lymphoprep (density
1.077 g/ml) from Nycomed Pharma (Oslo, Norway); serotonin
(5-hydroxytryptamine creatinine sulphate) from Koch
Light (Colbrook Bucks, UK); and chloroquine phosphate
from ACO (Molndal, Sweden). All other chemicals of
analytical grade were from available commercial sources;
deionised water was used as a solvent.
2.2. Isolation of blood platelets
Isolation was performed as described previously [15].
Briefly, fresh blood was obtained from healthy male donors
(20–50 years) who had not received any medication for at
least 7 days. Blood samples were anticoagulated with 3.8%
trisodium citrate (blood: citrate ratio = 9:1) and centrifuged
at 260 Â g for 15 min. Platelet-rich plasma (PRP) was
removed, mixed with a solution containing 4.5% citric acid
and 6.6% glucose (50 Al/1 ml PRP), and centrifuged at
1070 Â g for 10 min. Platelets were resuspended in an equal
volume of Tyrode’s solution (136.9 mmol/l NaCl, 2.7 mmol/
l KCl, 11.9 mmol/l NaHCO3, 0.4 mmol/l NaH2PO4Á2H2O,
1 mmol/l MgCl2Á6H2O, 5.6 mmol/l glucose) containing
5.4 mmol/l ethylene diamine tetraacetic acid (EDTA),
pH = 6.5. After 10-min stabilisation, the suspension was
centrifuged at 1070 Â g for 6 min and platelets were
resuspended in the same buffer without EDTA (pH = 7.4)
to obtain 2 Â 105
(chemiluminescence assay) or 5 Â 105
(serotonin determination) platelets per 1 Al.
2.3. Isolation of neutrophils
Neutrophils were isolated according to the method
described by Dra´bikova´ et al. [16]. After removal of PRP
(see Isolation of blood platelets), the blood volume was
reconstituted with 0.9% NaCl. Then, a 3% dextran solution
(1% final concentration) was added and blood was allowed
to sediment (1 Â g) for 25 min at 22 jC. The neutrophil/
dextran mixture was centrifuged at 500 Â g for 10 min and
the pellet was resuspended in phosphate-buffered saline
(PBS: 137 mmol/l NaCl, 2.7 mmol/l KCl, 8.1 mmol/l
Na2HPO4, 1.5 mmol/l KH2PO4, pH = 7.4). Neutrophil suspension
(3 ml) was layered on lymphoprep (3 ml) and
centrifuged for 30 min at 500 Â g. Contaminating red blood
cells were removed by hypotonic lysis (3 ml of ice-cold
deionised water followed after 45 s by 3 ml of 1.8% NaCl
and 4 ml of PBS). After centrifugation (500 Â g, 10 min),
neutrophils in PBS were stained with Tu¨rk solution, counted
under light microscope, and adjusted to 104
neutrophils per
1 Al. Final suspension contained more than 96% of viable
cells, as evaluated by trypan blue exclusion, and was used
maximally for 2 h—as long as control chemiluminescence
kept constant.
2.4. Chemiluminescence assay
Chemiluminescence of neutrophils was measured in a
lumiaggregometer, model 500 (Chrono-log., USA). The
reaction mixture consisted of 200 Al of neutrophils
(2 Â 106
), 175 Al PBS, 50 Al 18 mmol/l CaCl2, 25 Al
1 mmol/l MgCl2, and 500 Al platelets (108
), or Tyrode’s
solution. After 1 min of incubation at 37 jC and stirring
at 1000 rpm, 20 Al of chloroquine (1, 10 and 100 Amol/l
final concentration), and 2 min later, 20 Al of luminol
(5 Amol/l final concentration) was added. Samples were
incubated for further 3 min and stimulated with 10 Al of
FMLP (final concentration 0.1 Amol/l). Time-dependent
alterations in intensity of the chemiluminescence signal
were recorded in the form of curves, the presented results
(mV) referring to peak values.
A slightly modified protocol was used when the effect of
serotonin was studied. To prevent its degradation, serotonin
(20 Al) was added 1 min before FMLP.
Chemiluminescence of cell-free systems was measured in
a luminometer Immunotech LM-01T (Immunotech, Czech
Republic) at 37 jC for 60 min. Samples for the analysis of
hydrogen peroxide chemiluminescence contained 30 Al of
serotonin or chloroquine (0.1–100 Amol/l final concentration),
30 Al of luminol (final concentration 1 mmol/l), 60 Al
of 0.006% H2O2, and 180 Al of deionised water. When
chemiluminescence of hydroxyl radical was measured, 30 Al
of 10 mmol/l FeSO4 was added to this reaction mixture.
Formation of superoxide anion was initiated by the reaction
of 210 Al 7.35 mmol/l hypoxanthine with 30 Al of xanthine
oxidase (0.084 U/ml), and its chemiluminescence was
measured in the presence of 30 Al of luminol and of 30 Al
of serotonin (or chloroquine).
2.5. Serotonin determination
A slight modification of the method described by Nosa´l’
et al. [17] was used for the determination of platelet
serotonin. Isolated platelets (1100 Al, 5.5 Â 108
) were treatV.
Jancˇinova´ et al. / Thrombosis Research 109 (2003) 293–298294
ed with 50 Al of chloroquine (1, 10 and 100 Amol/l final
concentration). After 5 min of incubation at 37 jC, 50 Al of
FMLP (0.1 Amol/l final concentration) was added. Serotonin
liberation was stopped after 5 min by cooling the samples to
4 jC and by immediate centrifugation at 14000 Â g and
4 jC for 2 min. For determination, 400 Al of supernatant,
2600 Al of 0.02 N HCl, 200 Al of 10% w/w ZnSO4, and 100
Al of 1N NaOH were mixed and centrifuged for 10 min at
1670 Â g. Native fluorescence of serotonin was measured in
a Perkin-Elmer Fluorescence Spectrometer, Model 203, at
300 and 332 nm excitation and emission wavelength,
respectively. Concentration of extracellular serotonin was
calculated to platelet number (108
) and sample volume
(1 ml) used in the chemiluminescence assay.
When the effect of neutrophils on extracellular serotonin
concentration was measured, the reaction mixture consisted
of 1000 Al of platelets (4 Â 108
), 430 Al PBS, 100 Al 18
mmol/l CaCl2, 50 Al 1 mmol/l MgCl2, and 400 Al neutrophils
(8 Â 106
). After 6 min of incubation at 37 jC, samples
were stimulated for 5 min with 20 Al FMLP (final concentration
of 0.1 Amol/l), and 800 Al of aliquots of supernatants
was used for serotonin determination.
2.6. Data analysis
Commercially available computer statistic programmes
were used for calculations of means and standard errors of
the mean (S.E.M.). Statistical significance of differences
between means was established by Student’s t-test and
p V .05 was taken as statistically significant.
3. Results
Fig. 1 shows platelet-dependent inhibition of FMLPstimulated
neutrophil chemiluminescence and its amplification
by chloroquine. Incubation of neutrophils with blood
platelets in the physiological cell ratio 1:50 declined their
chemiluminescence from 26.86 F 3.37 to 14.33 F 2.06 mV.
Application of 10 or 100 Amol/l chloroquine simultaneously
with platelets induced further reduction of the chemiluminescence
signal to 11.13 F 2.73 or 7.40 F 1.14 mV, respec-
tively.
Co-incubation of platelets with neutrophils and/or with
FMLP was not accompanied with liberation of serotonin—
a potential platelet-derived chemiluminescence inhibitor.
Even in the presence of neutrophils, extracellular serotonin
concentration decreased from 0.0079 F 0.0026 to 0.0008 F
0.0005 Amol/l (without FMLP) or from 0.0069 F 0.0028 to
0.0041 F 0.0011 Amol/l (with FMLP). Significant serotonin
discharge resulted however from treatment of platelets
with chloroquine (10 and 100 Amol/l), in the absence as
well as in the presence of FMLP (Table 1). Maximum extraplatelet
(extracellular) serotonin concentrations exceeded
0.1 Amol/l.
The ability of chloroquine to inhibit chemiluminescence
depended on the presence of platelets. When platelets were
omitted, chloroquine (1–100 Amol/l) did not reduce chemiluminescence
of FMLP-stimulated neutrophils and neither
did it that of cell-free systems, with the exception of 28%
inhibition of hydroxyl radical chemiluminescence observed
in the presence of 100 Amol/l concentration (Fig. 2).
On the other hand, serotonin was found to be a potent
inhibitor. This amine in the concentration of 0.1 Amol/l (i.e.
in the concentration estimated in supernatants of chloroFig.
1. Inhibition of FMLP-stimulated neutrophil chemiluminescence by
blood platelets. Increase of the inhibition by chloroquine. Mean F S.E.M.,
n = 7, *p < .05, **p < .01 (vs. chemiluminescence of neutrophils + platelets
in the absence of chloroquine).
Table 1
Liberation of platelet serotonin by 1, 10, and 100 Amol/l chloroquine; effect
of FMLP
Extracellular serotonin (Amol/l)
No stimulus FMLP
Platelets 0.0075 F 0.0040 0.0152 F 0.0022
Platelets + chloroquine 1 0.0067 F 0.0031 0.0212 F 0.0036
Platelets + chloroquine 10 0.0252 F 0.0064** 0.0511 F 0.0109*
Platelets + chloroquine 100 0.1075 F 0.0246** 0.1450 F 0.0124**
Extracellular serotonin concentration was corrected for 108
platelets, and
sample volume of 1 ml as used in the chemiluminescence assay.
Mean F S.E.M., n = 10–12.
* p < .05.
** p < .01 (vs. platelets).
Fig. 2. Effect of chloroquine on chemiluminescence of superoxide anion
(O2
SÀ
), hydroxyl radical (OHS
), hydrogen peroxide (H2O2), and of FMLPstimulated
neutrophils. Mean F S.E.M., n = 6.
V. Jancˇinova´ et al. / Thrombosis Research 109 (2003) 293–298 295
quine-treated platelets) decreased neutrophil chemiluminescence
by 28% (Fig. 3). Significant reduction by 77%, 87%,
and 86% was observed in the presence of 1, 10, and 100
Amol/l serotonin, respectively. Chemiluminescence of individual
oxygen metabolites was inhibited in the following
sequence (percentage of inhibition caused by 10 and 100
Amol/l of serotonin is given in parenthesis): superoxide
anion (69% and 97%)>hydroxyl radical (34% and
82%)>hydrogen peroxide (0% and 63%). In comparison
to chemiluminescence originated from neutrophils, 10 times
higher serotonin concentrations were needed to inhibit
chemiluminescence of cell-free systems.
4. Discussion
4.1. Platelet inhibition of neutrophil chemiluminescence by
a serotonin-independent mechanism
The presented results indicated that blood platelets
reduced neutrophil chemiluminescence by a serotonin-independent
mechanism. In contrast to platelets activated by
A23187 [14], the concentration of serotonin liberated in the
presence of FMLP (0.015 Amol/l) was not sufficient to
explain the 47% inhibition of chemiluminescence (Figs. 1
and 3, Table 1). Moreover, platelets did not inhibit chemiluminescence
of cell-free systems (our preliminary results
are not published as of yet), thus involvement of their
scavenging effect (mediated by serotonin or other plateletderived
substances) seems to be less likely.
As we found previously, platelet inhibition of FMLPstimulated
chemiluminescence disappeared in the presence
of horseradish peroxidase [18], i.e. at increased extracellular
concentration of peroxidase. It indicates that decreased
chemiluminescence signal could result from inhibition of
myeloperoxidase liberation by platelets. This enzyme is
essentially involved in luminol excitation [19,20] and its
importance was proved by the absence of chemiluminescence
in patients with normal radical formation and myeloperoxidase
deficiency [21]. Interference of platelets with
liberation of myeloperoxidase could be mediated by several
mechanisms—action of platelet derived inhibitory protein
[12], platelet–neutrophil contact mechanism [11], or platelet
induced polymerisation of actin in neutrophils [22,23] could
all be taken into account.
Interference of platelets with superoxide anion formation
in FMLP-stimulated neutrophils seems to be less likely
since it was found to require completely stimulated platelets,
releasing adenosine 5V-triphosphate (ATP) and adenosine 5Vdiphosphate
(ADP) from their dense granules, as well as a
long-time (30 min) platelet–neutrophil co-incubation
[9,24,25]; these requirements were not fulfilled in our
experiments. Moreover, platelet inhibition was reversed by
increased extracellular peroxidase.
4.2. Amplification of platelet effect by chloroquine; involvement
of serotonin
In accordance with previous findings [26–28], chloroquine
was not found to considerably inhibit chemiluminescence
of FMLP-stimulated neutrophils or of cell-free systems
(Fig. 2). The inhibitory effect of this drug appeared in the
presence of blood platelets and was dose-dependent; chemiluminescence
reduced by platelets declined further by 11%,
26%, and 48% after addition of chloroquine in the concentration
of 1, 10, and 100 Amol/l, respectively (Fig. 1). The
additional inhibition of neutrophil chemiluminescence may
have been mediated by substances released from platelets in
the presence of chloroquine. Particular involvement of the
antioxidant and scavenger serotonin [29–31] seems to be
highly acceptable because this amine was found to be
displaced from platelet-dense granules after accumulation
of basic drugs including chloroquine [17,32,33]. Moreover,
serotonin in the concentration of 0.1 Amol/l, estimated in
supernatants of chloroquine-treated platelets, decreased
chemiluminescence of neutrophils by 28% (Table 1, Fig. 3).
Serotonin can inhibit neutrophil chemiluminescence by
its scavenging effect—i.e. through elimination of existing
radicals [34]. The different affinity of serotonin to particular
oxygen metabolites, indicated by our measurements (Fig. 3),
could explain why chemiluminescence stimulated with
FMLP was inhibited by serotonin more effectively than that
stimulated with opsonised zymosan [30] or with phorbol 12myristate
13-acetate (PMA) [29]. Namely, FMLP-induced
chemiluminescence was found to be formed by superoxide
anion and hydroxyl radical, whereas hydrogen peroxide is
prevalent in neutrophils stimulated with opsonised zymosan
or with PMA [35]. The interference of serotonin with the
formation of reactive oxygen metabolites [36] may represent
another mechanism potentially participating in the inhibitory
effect of serotonin. This was indicated by the fact that
inhibition of neutrophil chemiluminescence by serotonin
started at 100 times lower concentrations than the scavenging
effect of this amine (Fig. 3).
Reduction of radical concentration, mediated by platelet
serotonin, may participate in the anti-inflammatory activity
Fig. 3. Chemiluminescence of superoxide anion (O2
S À
), hydroxyl radical
(OHS
), hydrogen peroxide (H2O2), and of FMLP-stimulated neutrophils in
the presence of serotonin. Mean F S.E.M., n = 6.
V. Jancˇinova´ et al. / Thrombosis Research 109 (2003) 293–298296
of chloroquine. This may be operative in patients with
rheumatoid arthritis with local chloroquine concentration
of 10–100 Amol/l [37]. However, during antimalarial therapy,
chloroquine concentrations are substantially lower [38],
and thus this chloroquine effect would not impair bactericidal
activity of neutrophils.
4.3. Pharmacology of platelet–neutrophil interactions
Inflammation and atherogenesis were found to be multicellular
processes, both accompanied with centralisation and
activation of blood platelets and neutrophils at the same site
[39]. Existing mutual platelet–neutrophil interactions, both
stimulatory and inhibitory [40], can weaken the effects of
drugs and complicate therapy of these pathological states.
On the other hand, pharmacological intervention aimed at
inhibition of potentially harmful amplificatory pathways
and/or at the enhancement of protective inhibitory mechanisms
appears to be more effective, rational, and safe than
the current strategy.
Our results are indicative of several facts. Firstly, platelet-mediated
reduction of radical concentration (the natural
protective mechanism?) could be amplified by serotonin
liberating drug(s). Secondly, besides inhibition of platelet
functions [17], protection of tissues against aggressive
neutrophil products could explain chloroquine benefit in
thromboembolic prophylaxis [41]. Finally, effectiveness of
chloroquine was found to be altered in the presence of
platelets. Lower concentrations and shorter incubation with
neutrophils were sufficient to inhibit FMLP chemiluminescence,
compared to the chloroquine effect in the absence of
platelets [27,28]. This indicated that under in vivo conditions,
the effect of a drug on one type of cells could be
modified in the presence of cells of another type. Accurate
prediction of drug effect in the whole organism does therefore
call for the use of multicellular test systems.
Acknowledgements
This work was supported in part by grants No.2/1012/21
(Scientific Grant Agency VEGA) and No.049/198 (Slovak–
Czech Intergovernmental Research–Technical Coopera-
tion).
References
[1] Geerts L, Jorens PG, Willems J, De Ley M, Slegers H. Natural inhibitors
of neutrophil function in acute respiratory distress syndrome.
Crit Care Med 2001;29:1920–4.
[2] Jordan JE, Zhao Z-Q, Vinten-Johansen J. The role of neutrophils in
myocardial ischemia–reperfusion injury. Cardiovasc Res 1999;43:
860–78.
[3] Heinzelmann M, Mercer-Jones MA, Passmore JC. Neutrophils and
renal failure. Am J Kidney Dis 1999;34:384–99.
[4] Mur E, Zabernigg A, Hilbe W, Eisterer W, Halder W, Thaler J. Oxidative
burst of neutrophils in patients with rheumatoid arthritis: influence
of various cytokines and medication. Clin Exp Rheumatol
1997;15:233–7.
[5] Kountouras J, Chatzopoulos D, Zavos C. Reactive oxygen metabolites
and upper gastrointestinal diseases. Hepato-Gastroenterol 2001;
48:743–51.
[6] Akopov SE, Simonian NA, Grigorian GS. Dynamics of polymorphonuclear
leukocyte accumulation in acute cerebral infarction and their
correlation with brain tissue damage. Stroke 1996;27:1739–43.
[7] Serrano CV, Ramires JAF, Venturinelli M, Arie S, D’Amico E, Zweier
JL, et al. Coronary angioplasty results in leukocyte and platelet activation
with adhesion molecule expression. Evidence of inflammatory
responses in coronary angioplasty. J Am Coll Cardiol 1997;29:
1276–83.
[8] Jaremo P, Hansson G, Nilsson O. Elevated inflammatory parameters
are associated with lower platelet density in acute myocardial infarctions
with ST-elevation. Thromb Res 2000;100:471–8.
[9] Reinisch CM, Dunzendorfer S, Pechlaner C, Ricevuti G, Wiedermann
CJ. The inhibition of oxygen radical release from human neutrophils
by resting platelets is reversed by administration of acetylsalicylic
acid or clopidogrel. Free Radic Res 2001;34:461–6.
[10] Lo¨sche W, Temmler U, Redlich H, Vickers J, Krause S, Spangenberg
P. Inhibition of leukocyte chemiluminescence by platelets: role of
platelet-bound fibrinogen. Platelets 2001;12:15–9.
[11] Lo¨sche W, Dressel M, Krause S, Redlich H, Spangenberg P, Heptinstall
S. Contact-induced modulation of neutrophil elastase secretion
and phagocytic activity by platelets. Blood Coagul Fibrinolysis 1996;
7:210–3.
[12] Del Principe D, Menichelli A, Di Giulio S, De Matteis W, Giordani
M, Pentassuglio AM, et al. Stimulated platelets release factor(s) affecting
the in vitro response of human polymorphonuclear cells. J Leukoc
Biol 1990;48:7–14.
[13] Dra´bikova´ K, Jancˇinova´ V, Nosa´l’ R, Danihelova´ E. Human blood
platelets, PMN leukocytes and their interactions in vitro. Responses to
selective and non-selective stimuli. Gen Physiol Biophys 2000;19:
393–404.
[14] Jancˇinova´ V, Nosa´l’ R, Dra´bikova´ K, Danihelova´ E. Cooperation
of chloroquine and blood platelets in inhibition of polymorphonuclear
leukocyte chemiluminescence. Biochem Pharmacol 2001;62:
1629–36.
[15] Nosa´l’ R, Jancˇinova´ V. Cationic amphiphilic drugs and platelet phospholipase
A2 (cPLA2). Thromb Res 2002;105:339–45.
[16] Dra´bikova´ K, Nosa´l’ R, Jancˇinova´ V, Cˇ izˇ M, Lojek A. Reactive
oxygen metabolite production is inhibited by histamine and H1-antagonist
dithiaden in human PMN leukocytes. Free Radic Res 2002;
36:975–80.
[17] Nosa´l’ R, Jancˇinova´ V, Danihelova´ E. Chloroquine: a multipotent
inhibitor of human platelets in vitro. Thromb Res 2000;98:411–21.
[18] Jancˇinova´ V, Dra´bikova´ K, Nosa´l’ R, Danihelova´ E. Platelet-dependent
modulation of polymorphonuclear leukocyte chemiluminescence.
Platelets 2000;11:278–85.
[19] Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil
phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood
1998;92:3007–17.
[20] Dahlgren C, Karlsson A. Respiratory burst in human neutrophils.
J Immunol Methods 1999;232:3–14.
[21] DeChatelet LR, Long GD, Shirley PS, Bass DA, Thomas MJ, Henderson
FW, et al. Mechanism of the luminol-dependent chemiluminescence
of human neutrophils. J Immunol 1982;129:1589–93.
[22] Bengtsson T, Zalavary S, Stendahl O, Grenegard M. Release of oxygen
metabolites from chemoattractant-stimulated neutrophils is inhibited
by resting platelets: role of extracellular adenosine and actin
polymerization. Blood 1996;87:4411–23.
[23] Herbertsson H, Bengtsson T. Role of platelets and the arachidonic
acid pathway in the regulation of neutrophil oxidase activity. Scand J
Clin Lab Invest 2001;61:641–50.
[24] Aziz KA, Zuzel M. Regulation of polymorphonuclear leukocyte function
by platelets. Saudi Med J 2001;22:526–30.
V. Jancˇinova´ et al. / Thrombosis Research 109 (2003) 293–298 297
[25] Carulli G, Barsotti G, Cupisti A, Minnucci S, Gianfaldoni ML, Agostini
B, et al. Platelet-neutrophil interactions in uremic patients: effects
on neutrophil superoxide anion production and chemiluminescence.
Nephron 1995;69:248–52.
[26] Labro MT, Babin-Chevaye C. Effects of amodiaquine, chloroquine,
and mefloquine on human polymorphonuclear neutrophil function in
vitro. Antimicrob. Agents Chemother 1988;32:1124–30.
[27] Styrt B, Klempner MS. Inhibition of neutrophil oxidative metabolism
by lysosomotropic weak bases. Blood 1986;67:334–42.
[28] Neal TM, Vissers MCM, Winterbourn CC. Inhibition by nonsteroidal
anti-inflammatory drugs of superoxide production and granule enzyme
release by polymorphonuclear leukocytes stimulated with immune
complexes of formyl-methionyl-leucyl-phenylalanine. Biochem Pharmacol
1987;36:2511–7.
[29] Schuff-Werner P, Splettsto¨sser W. Antioxidative properties of serotonin
and the bactericidal function of polymorphonuclear phagocytes.
Adv Exp Med Biol 1999;467:321–5.
[30] Vial T, Tedone R, Patriarca C, Descotes J. Effect of serotonin on the
chemiluminescence response of rat peripheral blood leucocytes. Int J
Immunopharmacol 1995;17:813–9.
[31] Huether G, Fettko¨tter I, Keilhoff G, Wolf G. Serotonin acts as a
radical scavenger and is oxidized to a dimer during the respiratory
burst of activated microglia. J Neurochem 1997;69:2096–101.
[32] Costa JL, Fay DD, Kirk KL. Quinacrine and other basic amines in
human platelets: subcellular compartmentation and effect on serotonin.
Res Commun Chem Pathol Pharmacol 1984;43:25–42.
[33] Nosa´l’ R, Ericsson O¨ , Sjo¨qvist F, Dˇ urisˇova´ M. Distribution of chloroquine
in human blood fractions. Methods Find Exp Clin Pharmacol
1988;10:581–7.
[34] Huether G, Schuff-Werner P. Platelet serotonin acts as a locally releasable
antioxidant. In: Filippini GA, Costa CVL, Bertazzo A, editors.
Recent advances in tryptophan research. Tryptophan and
serotonin pathways. New York: Plenum; 1996. p. 299–306.
[35] Takahashi R, Edashige K, Sato EF, Inoue M, Matsuno T, Utsumi K.
Luminol chemiluminescence and active oxygen generation by activated
neutrophils. Arch Biochem Biophys 1991;285:325–30.
[36] Salman-Tabcheh S, Gue´rin MC, Torreilles J. Potential role of the
peroxidase-dependent metabolism of serotonin in lowering the polymorphonuclear
leukocyte bactericidal function. Free Radic Res
1996;24:61–8.
[37] French JK, Hurst NP, O’Donnell ML, Betts WH. Uptake of chloroquine
and hydroxychloroquine by human blood leucocytes in vitro:
relation to cellular concentrations during antirheumatic therapy. Ann
Rheum Dis 1987;46:42–5.
[38] Salmon D, Verdier F, Malhotra K, Pussard E, Clavier F, Le Bras J,
et al. Absence of effect of chloroquine in vivo on neutrophil oxidative
metabolism in human subjects. J Antimicrob Chemother 1990;
25:367–70.
[39] Sullivan GW, Sarembock IJ, Linden J. The role of inflammation in
vascular diseases. J Leukoc Biol 2000;67:591–602.
[40] Siminiak T, Flores NA, Sheridan DJ. Neutrophil interactions with
endothelium and platelets: possible role in the development of cardiovascular
injury. Eur Heart J 1995;16:160–70.
[41] Wallace DJ. The use of chloroquine and hydroxychloroquine for noninfectious
conditions other than rheumatoid arthritis or lupus: a critical
review. Lupus 1996;5(Suppl 1):S59–64.
V. Jancˇinova´ et al. / Thrombosis Research 109 (2003) 293–298298
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DownloadedBy:[Ciz,Milan]At:06:5527November2007
Platelets, December 2007; 18(8): 583–590
Serotonin modulates the oxidative burst of human phagocytes via various
mechanisms
MILAN Cˇ I´Zˇ 1
, DANIELA KOMRSKOVA´ 1
, LUCIE PRACHARˇ OVA´ 1
,
KATERˇ INA OKE´ NKOVA´ 1
, HANA Cˇ I´Zˇ OVA´ 1
, ANETA MORAVCOVA´ 1
,
VIERA JANCˇ INOVA´ 2
, MARGITA PETRI´KOVA´ 2
, ANTONI´N LOJEK1
, &
RADOMI´R NOSA´ Lˇ 2
1
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic and 2
Institute of
Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia
(Received 13 March 2007; revised 24 April 2007; accepted 24 May 2007)
Abstract
Serotonin, the major secretory product of activated platelets, has been widely reported as regulating various constituents of
the immune system and immune functions. This modulation is complex and the data available are rather controversial. The
aim of the present study was to clarify the mechanisms of serotonin action on human phagocytes. The effect of serotonin in
a concentration range of 10À7
M–10À3
M on various parameters of oxidative burst of phagocytes was studied using various
luminol-enhanced chemiluminescence methods. Serotonin inhibited the chemiluminescence response of the cells in a dose
dependent manner. The effect of serotonin on the activity of myeloperoxidase was studied in further experiments. In this
case, serotonin again exerted a dose dependent inhibition of the myeloperoxidase activity. The hypothesis that the
inhibitory activity of serotonin might be also receptor mediated was evaluated using various serotonin receptor agonists and
antagonists. None of the agonists studied exerted any direct antioxidative properties. Only (Æ)-DOI hydrochloride, a
selective 5-HTR2 agonist, exerted similar effects on phagocytic cells as serotonin. It can be concluded that serotonin could
affect the oxidative burst of phagocytes. Responsibility for its inhibitory effects lies with both the decrease in the generation
of reactive oxygen species (due to the inhibition of myeloperoxidase activity) and with direct scavenging of reactive oxygen
species. The effect of serotonin on phagocytes is also partially mediated by 5-HTR2 receptor.
Keywords: Antioxidant, chemiluminescence, phagocytes, serotonin
Introduction
Oxidative stress and oxidation-induced tissue
damage represent a serious problem for medicine.
The oxidative injury can potentially occur due to
either an excess production of reactive oxygen and
nitrogen species or a decrease in antioxidant
defences. It is suggested that an excessive production
of phagocyte-derived reactive oxygen and nitrogen
metabolites plays a role in a number of various
diseases [1] connected to oxidative stress. Our
previous results showed that the mobilization of
neutrophils taking place in an inflammatory response
is a very important component in the pathophysiology
of ischemia/reperfusion [2–5].
A wide cooperation between platelets and professional
phagocytes exists in the sites of inflammation
and endothelial cell damage. Cathepsin G [6] and
platelet activating factor [7] are among the major
neutrophil-derived activators of platelets. On the
other hand, vasodilatation produced by activated
platelets could be profoundly impaired by activated
leukocytes, probably via superoxide anion generated
by activated neutrophils [8]. Platelets affect neutrophil
activation by releasing serotonin, histamine,
thromboxane A2, platelet-derived growth factor,
lipoxygenase products, proteases, and adenosine
[9–11].
Serotonin (5-hydroxytryptamine, 5-HT) is formed
by the hydroxylation and decarboxylation of tryptophan.
The greatest concentration of serotonin (90%)
is found in the cells of the gastrointestinal tract.
Most of the remainder of the body’s serotonin is
found in platelets and the central nervous system.
Vasoconstriction is a classic response to the administration
of serotonin. The function of serotonin is
exerted upon its interaction with specific receptors
[12]. Most of these receptors are coupled to
G-proteins that affect the activities of either adenylate
cyclase or phospholipase C.
Correspondence: Milan Cˇ ı´zˇ, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, 612 65 Brno, Czech Republic.
Tel: þ420 541 517 207. Fax: þ420 541 211 293. E-mail: milanciz@ibp.cz
ISSN 0953–7104 print/ISSN 1369–1635 online ß 2007 Informa UK Ltd.
DOI: 10.1080/09537100701471865
DownloadedBy:[Ciz,Milan]At:06:5527November2007
Specific uptake of serotonin by macrophages may
regulate various constituents of the immune system
and immune functions [13–14]. Serotonin, the major
secretory product of activated platelets, was widely
reported to modulate the function of neutrophils.
This modulation is complex and the data available is
rather controversial [15–17]. Serotonin could act as a
true scavenger of the reactive oxygen species that are
generated during the respiratory burst of stimulated
phagocytes [16], and caused the aggregation and
degranulation of neutrophils [18], as well as inhibited
the migration of mononuclear leucocytes [19]. It was
previously shown that serotonin modulated
interferon-gamma-induced phagocytosis in bone
marrow macrophages through a 5-HT receptormediated
mechanism [20]. Taking all these factors
into consideration, the accumulating evidence suggests
that serotonin is a very important mediator of
mutual interactions between platelets and professional
phagocytes.
The major aim of the present study was to provide
new information on the effects of serotonin on
phagocytic cells. The effect of serotonin in a
concentration range of 10À7
M–10À3
M on various
parameters of oxidative burst of phagocytes was
studied using various luminol-enhanced chemiluminescence
(CL) methods.
Materials and methods
The effect of serotonin (serotonin creatinine sulfate
salt monohydrate, Sigma, St-Louis, USA) in a
concentration range of 10À3
M–10À7
M on various
parameters of oxidative burst of phagocytes was
studied using various luminol-enhanced CL
methods. Reagents and chemicals were purchased
from either Sigma (USA) or local distributors.
Whole blood chemiluminescence
Heparinized (50 IU/ml) blood samples were
obtained from healthy male volunteers with informed
consent. Sampling procedure was in accordance with
the ethical standards of the responsible committee of
the Institute of Biophysics on human experimentation
and with the Helsinki Declaration of 1975, as
revised in 1983. The number of leukocytes in the
blood and their relative differentiation counts were
determined using Coulter counter STKS (Coulter
Corporation, Mianui, USA). Luminol-enhanced
chemiluminescence of human phagocytes in the
whole blood was measured using an LM-01 microplate
luminometer (Immunotech, Prague, Czech
Republic). The principle of the method is based on
luminol interaction with the phagocyte-derived
oxidizing species, which results in large measurable
amounts of light at a peak wavelength of 425 nm.
Measurement details have been described previously
[21]. Briefly, the reaction mixture consisted of 10 ml
whole blood, 1 mM luminol (stock solution of
10 mM luminol in 0.2 M borate buffer) and one of
the activators: 62.5 mg/mL opsonized zymosan particles
(OZP), 9.55 mM calcium ionophore A23187
(Ca-I), 0.81 mM phorbol-12-myristate-13-acetate
(PMA) or 1.14 mM N-formyl-Met-Leu-Phe (fMLP).
The total reaction volume of 200 ml was adjusted with
Hanks balanced salt solution. The assays were run in
duplicates. The CL emission expressed as relative
light units (RLU) was recorded continuously for
60 min at 37
C.
Activity of myeloperoxidase
Since luminol-enhanced CL of phagocytes is widely
considered to be dependent on a reaction of the
myeloperoxidase (MPO) system, the effect of serotonin
on the activity of MPO was studied in further
experiments. Lysed HL-60 cells were used as a source
of MPO and the activity of the enzyme was evaluated
using bromide-dependent CL reaction [22].
TRAP (total peroxyl radical-trapping antioxidant
parameter) analysis
The luminol-enhanced chemiluminescence was used
to follow up peroxyl radical reaction. The chemiluminescence
signal is driven by the production of
luminol derived radicals from thermal decomposition
of 2,2-azo-bis-2-amidinopropane hydrochlorid
(ABAP, Polysciences, Inc., Warrington, USA). This
method was described previously in Cˇ ı´zˇova´ et al.
[21]. The TRAP (total peroxyl radical-trapping
antioxidant parameter) value is determined from
the duration of the period of time during which the
serum sample resists lipid peroxidation due to the
present antioxidants. A known quantity (8.0 nM) of
trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2carboxylic
acid, Sigma-Aldrich Chemie GmbH,
Steinheim, USA), a water-soluble analogue of
tocopherol was used as a reference inhibitor instead
of serum.
Antioxidant properties of serotonin in various
chemical systems
Reactive oxygen species (ROS) production was
measured by luminol-enhanced chemiluminescence
in a microtitre plate computer-driven luminometer
LMT-01 (Immunotech) and analysed for light
emission expressed in RLU. The inhibition of the
CL signal was assessed as a comparison of integral
CL signals of samples and control. Samples were not
stirred while being measured. The CL assays
were performed in a total volume of 300 ml for
30 minutes at 37
C [23]. The hypoxanthine/xanthine
oxidase system was used to generate Á OÀ
2 .
The reaction mixture contained hypoxanthine at a
final concentration of 5 mM, xanthine oxidase at
584 M. Cˇ ı´zˇ et al.
DownloadedBy:[Ciz,Milan]At:06:5527November2007
a final concentration of 7.3 mU/ml and luminol
at a final concentration of 0.8 mM. The reaction
was initiated by adding xanthine oxidase. The
hydrogen peroxide/ferrous sulphate system (H2O2/
FeSO4) was used for the production of OH. The
reaction mixture contained H2O2 at a final concentration
of 2 mM, FeSO4 at final concentrations of 0.1
and 0.001 mM and luminol at a final concentration
of 0.8 mM. Finally, the hydrogen peroxide system
(H2O2 itself) was employed. The reaction mixture
contained H2O2 at a final concentration of 2 mM and
luminol at a final concentration of 0.8 mM.
Statistical evaluation
All data are expressed as the mean Æ standard error
of the mean (SEM), n ¼ 8. The data were analysed
by one-way analysis of variance (ANOVA) followed
by a Students t-test with a level of significance of
p50.05.
Results
The effect of serotonin in a concentration range of
10À7
M–10À3
M on various parameters of oxidative
burst of phagocytes was studied using various
luminol-enhanced chemiluminescence methods.
Serotonin inhibited the CL response of opsonized
zymosan particle – OZP (Figure 1A), phorbol
myristate acetate – PMA (Figure 1B), formylMethionyl-Leucyl-Phenylalanine
– fMLP (Figure 1C)
and calcium ionophore – Ca-I (Figure 1D) activated
phagocytes in human whole blood in a dose
dependent manner. The effect of serotonin on the
oxidative burst of human whole blood phagocytes
expressed as the peak (maximum) of the CL
response of phagocytes is shown in Table I.
Since luminol-enhanced CL of phagocytes is
widely considered to be dependent on a reaction of
the myeloperoxidase system, the effect of serotonin
on the activity of MPO was studied in further
experiments. Serotonin exerted a dose dependent
inhibition of the MPO activity: 17% inhibition
(10À6
M serotonin), 27% inhibition (10À5
M serotonin),
95% inhibition (10À4
M serotonin), and 99%
inhibition (10À3
M serotonin). The lowest concentration
of serotonin (10À7
M) did not inhibit the
activity of MPO at all (Figure 2).
Antioxidant properties of serotonin were studied
in various chemical systems producing individual
ROS: the thermal decomposition of ABAP
(produced peroxyl radical) hypoxanthine/xanthine
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Time (min)
CL(RLU.103
)
(A)
0
5
10
15
20
25
30
35
40
Time (min)
CL(RLU.103)
(B)
0
10
20
30
40
50
60
70
0 10 20 30
Time (min)
CL(RLU.103
)
(C)
0
10
20
30
40
50
60
70
0 10 20 30
Time (min)
CL(RLU.103
)
(D)
Figure 1. The effect of serotonin in concentrations of 10À7
(circle), 10À6
(cross), 10À5
(triangle), 10À4
(square), and 10À3
(diamond) on
the oxidative burst of human whole blood phagocytes activated by OZP (A), PMA (B), fMLP (C), and Ca-I (D). The smooth line
represents control without serotonin.
Serotonin and oxidative burst of human phagocytes 585
DownloadedBy:[Ciz,Milan]At:06:5527November2007
oxidase system (produced superoxide anion), hydrogen
peroxide/ferrous sulphate system (produced
hydroxyl radical) and hydrogen peroxide itself.
Only the two highest concentrations of serotonin
showed a peroxyl radical-scavenging effect evaluated
as the TRAP parameter (Figure 3). The TRAP
values were 410 mmol/l and 4959 mmol/l at concentrations
of 10À4
M and 10À3
M, respectively. As for
the other ROS, serotonin (in a dose-dependent
manner) exerted the strongest antioxidant potential
against superoxide anion radical (Figure 4A) and
hydroxyl radical (Figure 4C) and the weakest
antioxidant potential against hydrogen peroxide
(Figure 4B). The percentage of inhibition was as
follows for individual serotonin concentrations:
10À6
M – 22%, 0%, and 0% (CL driven by superoxide
anion, hydroxyl radical and hydrogen peroxide,
respectively); 10À5
M – 69%, 34%, and 0%;
10À4
M – 97%, 82%, and 63%; 10À3
M – 100%,
98%, and 96%. Serotonin at the concentration of
10À7
M did not inhibit CL in any of the chemical
systems mentioned above.
The hypothesis that the inhibitory activity
of serotonin might also be receptor
mediated was evaluated using various
serotonin receptor agonists ((Æ)-8-Hydroxy-2-(di-npropylamino)tetralin
hydrobromide; (Æ)-DOI
hydrochloride; 1-(3-Chlorophenyl)biguanide hydrochloride;
Cisapride; 5-Carboxamidotryptamine
maleate salt; O-Methylserotonin hydrochloride) and
antagonists. None of the agonists studied exerted any
direct antioxidative properties. Only (Æ)-DOI hydrochloride,
a selective 5-HTR2 serotonin receptor
agonist, exerted similar effects on phagocytic cells
as serotonin (Figure 5, Table I).
Discussion
Our previous study investigated the effect of
activated blood platelets and chloroquine on
concentrations of reactive oxygen species produced
by polymorphonuclear leukocytes stimulated with
calcium ionophore A23187. A significant reduction
of chemiluminescence was observed in the presence
of platelets (added to polymorphonuclear leukocytes
in the physiological cell ratio 50 : 1) and of
chloroquine (10 and 100 micromol/L). Although
chloroquine decreased effectively both the extra as
well as the intracellular part of the chemiluminescence
signal, the activity of platelets occurred largely
outside polymorphonuclear leukocytes. Serotonin
liberated from platelets by calcium ionophore
appeared to be involved in the inhibition of
chemiluminescence [17]. Moreover, platelet activity
on neutrophils might also depend on the extent of
platelet activation, as non-activated platelets (in the
presence of fMLP) were found to potentiate
neutrophil-generated chemiluminescence, while
platelets activated with calcium ionophore displayed
the opposite effect. The interference of platelets with
the formation and liberation of superoxide anion was
indicated by platelet-modified isoluminol chemiluminescence.
Superoxide dismutase with catalase and
sodium azide were used, respectively, to differentiate
the intracellular and the extracellular part of the
chemiluminescence signal. Platelets were found to be
capable of modifying both components of chemiluminescence,
i.e. oxygen metabolites produced on the
plasma membrane as well as on membranes of
intracellular granules [24].
It was clearly demonstrated in our recent experiments
that serotonin is a potent inhibitor of the
oxidative burst of human phagocytes. Since the effect
of serotonin on phagocytes is complex, our experiments
were focused to elucidate possible individual
mechanisms of serotonin activity. It was previously
shown that serotonin could act as a true scavenger of
reactive oxygen species generated during the respiratory
burst of stimulated phagocytes [16], caused
aggregation and degranulation of neutrophils [18],
and inhibited the migration of mononuclear
leucocytes [19].
We have observed that the effect of serotonin
could be at least partially caused by the inhibition of
Serotonin (mol.l−1
)
CL(mV.106
)
*
** **
10−7 10−6 10−5 10−4 10−3
1.0
0.8
0.6
0.4
0.2
0.0
C
Figure 2. The effect of serotonin in a concentration range
of 10À7
–10À3
on the activity of myeloperoxidase evaluated as
a bromide-dependent chemiluminescence. ANOVA: p50.01,
*indicates p50.05 from the control and **indicates p50.01
from the control evaluated by t-test.
0
1
10
100
1000
10000
100000
Serotonin (mol.l−1
)
TRAP(µmol.l−1
)
10−7 10−6 10−5 10−4 10−3
Figure 3. Total peroxyl radical-scavenging antioxidative
parameter of serotonin in a concentration range of 10À7
–10À3
.
ANOVA, p50.01.
586 M. Cˇ ı´zˇ et al.
DownloadedBy:[Ciz,Milan]At:06:5527November2007
0
1
2
3
Serotonin [mol.l−1]
CL[RLU.s.106]
**
**
**
(A)
C
0
1
2
3
CL[RLU.s.106]
**
**
(B)
0
2
4
6
8
CL[RLU.s.106]
**
*
**
(C)
10−7 10−6 10−5 10−4 10−3
Serotonin [mol.l−1]
C 10−7 10−6 10−5 10−4 10−3
Serotonin [mol.l−1]
C 10−7 10−6 10−5 10−4 10−3
Figure 4. Antioxidative properties of serotonin (10À7
–10À3
) in various chemical systems producing individual reactive oxygen species:
hypoxanthine/xanthine oxidase system (produced superoxide anion (A)), hydrogen peroxide/ferrous sulphate system (produced hydroxyl
radical (C)) and hydrogen peroxide itself (B).
Serotonin and oxidative burst of human phagocytes 587
DownloadedBy:[Ciz,Milan]At:06:5527November2007
myeloperoxidase activity. The oxidation of serotonin
by the myeloperoxidase intermediates compounds
I and II was investigated previously by Dunford
and Hsuanyu [25]. Rapid scan spectra demonstrated
that both compound I and compound II
oxidized serotonin via one-electron processes.
A direct competition of serotonin with chloride for
myeloperoxidase compound I oxidation was
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40 50 60
Time (min)
CL(RLU.103)
(A)
0
5
10
15
20
25
30
35
40
0 10 20 30 40 50 60
Time (min)
CL(RLU*103)
(B)
0
10
20
30
40
50
60
70
0 10 20 30
Time (min)
0 10 20 30
Time (min)
CL(RLU.103)
(C)
0
10
20
30
40
50
60
70
CL(RLU.103)
(D)
Figure 5. The effect of (Æ)-DOI hydrochloride in concentrations of 10À7
(circle), 10À6
(cross), 10À5
(triangle), 10À4
(square), and 10À3
(diamond) on the oxidative burst of human whole blood phagocytes activated by OZP (A), PMA (B), fMLP (C), and Ca-I (D). The
smooth line represents control without serotonin.
Table I. The effect of serotonin and (Æ)-DOI hydrochloride in concentrations of 10À7
to 10À3
M on the oxidative burst of human whole
blood phagocytes activated by OZP, PMA, fMLP, and Ca-I. The data represent the peak (maximum) of the CL response of phagocytes
expressed as meansÆSEM. The asterisks indicate the statistically significant differences from the control at p50.05.
Opsonized Zymosan
Particles (OZP)
Phorbol-12-Myristate-
13-Acetate (PMA)
N-formyl-MetLeu-Phe
(fMLP)
Calcium ionophore
A23187 (Ca-I)
Serotonin
Control 78.92 Æ 23.03 37.74 Æ 5.61 89.54 Æ 14.78 70.17 Æ 9.90
10À7
M 54.61 Æ 8.08 36.41 Æ 3.83 77.74 Æ 7.56 56.45 Æ 10.82
10À6
M 43.49 Æ 4.91 26.43 Æ 3.85 76.84 Æ 11.89 47.57 Æ 7.48
10À5
M 17.96 Æ 2.91 11.64 Æ 2.19* 26.60 Æ 4.05* 14.84 Æ 4.68*
10À4
M 4.20 Æ 0.80* 7.00 Æ 1.58* 13.83 Æ 4.40* 7.70 Æ 3.96*
10À3
M 0.72 Æ 0.29* 0.52 Æ 0.18* 5.95 Æ 2.18* 4.09 Æ 2.35*
(Æ)-DOI hydrochloride
Control 78.00 Æ 31.12 28.97 Æ 9.10 70.72 Æ 19.61 53.73 Æ 12.88
10À7
M 79.49 Æ 37.17 27.29 Æ 10.96 58.81 Æ 14.57 38.51 Æ 14.02
10À6
M 77.37 Æ 23.46 33.40 Æ 8.72 56.90 Æ 13.37 30.34 Æ 10.35
10À5
M 36.27 Æ 3.91 34.01 Æ 4.15 21.55 Æ 2.17 6.46 Æ 1.99*
10À4
M 0.02 Æ 0.00* 0.62 Æ 0.10* 0.02 Æ 0.00* 0.02 Æ 0.00*
10À3
M 0.02 Æ 0.00* 0.01 Æ 0.00* 0.04 Æ 0.01* 0.03 Æ 0.01*
588 M. Cˇ ı´zˇ et al.
DownloadedBy:[Ciz,Milan]At:06:5527November2007
observed. These findings accord with the results of
Salman-Tabcheh et al. [26]. They found that
serotonin significantly and dose-dependently
suppressed the luminol-enhanced chemiluminescence
signal generated by polymorphonuclear
leukocytes activated with phorbol myristate acetate,
but did not modify either lucigenin-enhanced CL or
the reduction of superoxide dismutase-inhibitable
cytochrome c. Their results suggested that serotonin
metabolism was mediated by hydrogen peroxide and
myeloperoxidase which was released by activated
polymorphonuclear leukocytes.
In another set of our experiments, we showed that
the inhibition of chemiluminescence signal of
phagocytes might be caused by the direct scavenging
of reactive oxygen species already produced. We
have found that serotonin directly scavenged peroxyl
radical, superoxide anion radical, hydroxyl radical
and to a lesser extent hydrogen peroxide as well.
Schuff-Werner et al. [27] and Huether et al. [28]
observed that serotonin was oxidized by the reactive
oxygen species released by stimulated phagocytes.
This oxidation was prevented in the presence of
other antioxidants. The major serotonin oxidation
product was isolated by gel chromatography and
identified by mass-spectrometry as a serotonin
dimer, probably 5,5’-dihydroxy-4,4’-bitryptamine.
They concluded that serotonin released from
activated thrombocytes at the sites of inflammation
and endothelial cell damage acts as a true scavenger
of reactive oxygen species generated during the
respiratory burst of stimulated phagocytes. SchuffWerner
and Splettstoesser found that the modulation
of the bactericidal function of neutrophils by
serotonin was complex and depended upon the
amount of serotonin: at concentrations normally
present at sites of tissue injury and consecutive
thrombus formation (10À6
to 10À5
M), bacterial
killing increased by about 50%. In contrast, at higher
concentrations (10À3
to 10À2
M) an adverse effect
could be observed: the elimination of opsonized
S. aureus was reduced by 30 to 90% [29]. It is
obvious from our results that serotonin rather in
concentrations which may be locally reached at the
immediated sites of serotonin release by activated
platelets, e.g. at sites of inflammation or endothelial
damage (10À3
–10À4
M) than in concentrations
found in whole blood (10À6
M and lower) [27]
could affect the phagocyte derived reactive oxygen
species. Thus, serotonin would not interfere with
phagocytes in vivo under physiological conditions,
but it could control the overproduction of phagocytederived
reactive oxygen species in limited areas
under non-physiological conditions.
The hypothesis that the inhibitory activity
of serotonin might also be receptor mediated
was evaluated using various serotonin receptor
agonists ((Æ)-8-Hydroxy-2-(di-n-propylamino)tetralin
hydrobromide; (Æ)-DOI hydrochloride;
1-(3-Chlorophenyl)biguanide hydrochloride;
Cisapride; 5-Carboxamidotryptamine maleate salt;
O-Methylserotonin hydrochloride) and antagonists.
Only (Æ)-DOI hydrochloride, a selective 5-HTR2
agonist, exerted similar effects on phagocytic cells as
serotonin. None of the agonists studied exerted any
direct antioxidative properties.
Conclusion
It can be concluded that serotonin could affect the
oxidative burst of phagocytes. Both decreasing the
generation of ROS including inhibition of myeloperoxidase
activity and direct scavenging of ROS were
responsible for its inhibitory effects. The hypothesis
that the inhibitory activity of serotonin might also be
receptor mediated was evaluated using various
serotonin receptor agonists and antagonists. Only
(Æ)-DOI hydrochloride, a selective 5-HTR2 agonist,
exerted similar effects on phagocytic cells as serotonin.
Taking all these facts into consideration, the
evidence to date suggests that serotonin is a very
important mediator of mutual interactions between
platelets and phagocytes.
Acknowledgements
This study was executed within a research plan
AVOZ50040507 and was supported by grants No.
524/04/0897 of the Grant Agency of the Czech
Republic, No. 2/7019/27 of the VEGA grant agency
and No. SK-CZ-06606 of the Slovak-Czech
Intergovernmental cooperation.
References
1. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser
J. Free radicals and antioxidants in normal physiological
functions and human disease. Int J Biochem Cell Biol
2007;39:44–84.
2. Lojek A, Cˇ ı´zˇ M, Slavı´kova´ H, Dusˇkova´ M, Vondra´cˇek J,
Kubala L, Ra´cz I, Hamar J. Leukocyte mobilization, chemiluminescence
response, and antioxidative capacity of the blood in
intestinal ischemia and reperfusion. Free Radic Res
1997;27:359–367.
3. Slavı´kova´ H, Lojek A, Hamar J, Dusˇkova´ M, Kubala L,
Vondra´cˇek J, Cˇ ı´zˇ M. Total antioxidant capacity of serum
increased in early but not late period after intestinal ischemia in
rats. Free Radical Biol Med 1998;25:9–18.
4. Kubala L, Cˇ ı´zˇ M, Vondra´cˇek J, Cˇ ı´zˇova´ H, Cˇ erny´ J, Neˇmec P,
Studenı´k P, Dusˇkova´ M, Lojek A. Peri- and post-operative
course of cytokines and the metabolic activity of neutrophils in
human liver transplantation. Cytokine 2001;16:97–101.
5. Kubala L, Cˇ ı´zˇ M, Vondra´cˇek J, Cˇ erny´ J, Neˇmec P, Studenı´k P,
Cˇ ı´zˇova´ H, Lojek A. Perioperative and postoperative course of
cytokines and the metabolic activity of neutrophils in human
cardiac operations and heart transplantation. J Thorac Cardiov
Surg 2002;124:1122–1129.
6. Evangelista V, Piccardoni P, White JG, de Gaetano G, Cerletti
C. Cathepsin G-dependent platelet stimulation by activated
polymorphonuclear leukocytes and its inhibition by antiproteinases:
Role of P-selectin-mediated cell-cell adhesion. Blood
1993;81:2947–2957.
Serotonin and oxidative burst of human phagocytes 589
DownloadedBy:[Ciz,Milan]At:06:5527November2007
7. Zhou W, Javors MA, Olson MS. Platelet-activating factor as
an intercellular signal in neutrophil- dependent platelet
activation. J Immunol 1992;149:1763–1769.
8. Kaul S, Waack BJ, Padgett RC, Brooks RM, Heistad DD.
Interaction of human platelets and leukocytes in modulation
of vascular tone. Am J Physiol 1994;266:H1706–1714.
9. Jean T, Bodinier MC. Mediators involved in inflammation:
Effects of Daflon 500 mg on their release. Angiology
1994;45:554–559.
10. Sirois MG, de Lima WT, de Brum Fernandes AJ, Johnson RJ,
Plante GE, Sirois P. Effect of PAF on rat lung vascular
permeability: Role of platelets and polymorphonuclear leucocytes.
Br J Pharmacol 1994;111:1111–1116.
11. Siminiak T, Flores NA, Sheridan DJ. Neutrophil interactions
with endothelium and platelets: Possible role in the development
of cardiovascular injury. Eur Heart J 1995;16:160–170.
12. Essman EJ. Serotonin receptors on pulmonary alveolar
macrophages. Ric Clin Lab 1985;15:19–24.
13. Frank MG, Johnson DR, Hendricks SE, Frank JL. Monocyte
5-HT1A receptors mediate pindobind suppression of natural
killer cell activity: Modulation by catalase. Int
Immunopharmacol 2001;1:247–253.
14. Jackson JC, Walker RF, Brooks WH, Roszman TL. Specific
uptake of serotonin by murine macrophages. Life Sci
1988;42:1641–1650.
15. Tokmakov AA, Kykhova MP, Koshkina OV, Vasil’ev V. The
effect of serotonin on the functional activity of monocytes.
Tsitologiia 1999;33:81–87.
16. Schuff-Werner P, Splettstoesser W. Antioxidative properties
of serotonin and the bactericidal function of polymorphonuclear
phagocytes. Adv Exp Med Biol 1999;467:321–325.
17. Jancˇinova´ V, Nosa´lˇ R, Dra´bikova´ K, Danihelova´ E.
Cooperation of chloroquine and blood platelets in inhibition
of polymorphonuclear leukocyte chemiluminescence.
Biochem Pharmacol 2001;62:1629–1636.
18. Renesto P, Chignard M. Progressive inactivation of cathepsin
G and elastase released from activated neutrophils in vitro:
Lack of participation of the neutrophil alpha 1-proteinase
inhibitor. J Lab Clin Med 1994;123:906–913.
19. Bondesson L, Nordlind K, Liden S, Sundstrom E. Inhibiting
effects of serotonin and serotonin antagonists on the migration
of mononuclear leucocytes. Immunopharmacol
Immunotoxicol 1993;15:243–250.
20. Sternberg EM, Wedner HJ, Leung MK, Parker CW. Effect of
serotonin (5-HT) and other monoamines on murine macrophages:
modulation of interferon-gamma induced phagocytosis.
J Immunol 1987;138:4360–4365.
21. Cˇ ı´zˇova´ H, Lojek A, Kubala L, Cˇ ı´zˇ M. The effect of intestinal
ischemia duration on changes in plasma antioxidant defence
status in rats. Physiol Res 2004;53:523–531.
22. Haqqani AS, Dandhu JK, Birnboim HC. A myeloperoxidasespecific
assay based upon bromide-dependent chemiluminescence
of luminol. Anal Biochem 1999;273:126–132.
23. Komrskova´ D, Lojek A, Hrba´cˇ J, Cˇ ı´zˇ M. A comparison of
chemical systems for luminometric determination of antioxidant
capacity towards individual reactive oxygen species.
Luminescence 2006;21:239–244.
24. Jancˇinova´ V, Dra´bikova´ K, Nosa´lˇ R, Danihelova´ E. Plateletdependent
modulation of polymorphonuclear leukocyte
chemiluminescence. Platelets 2000;11:278–285.
25. Dunford HB, Hsuanyu Y. Kinetics of oxidation of serotonin
by myeloperoxidase compounds I and II. Biochem. Cell Biol
1991;77:449–457.
26. Salman-Tabcheh S, Guerin MC, Torreilles J. Potential role of
the peroxidase-dependent metabolism of serotonin in lowering
the polymorphonuclear leukocyte bactericidal function.
Free Radic Res 1996;24:61–68.
27. Schuff-Werner P, Splettstosser W, Schmidt F, Huether G.
Serotonin acts as a radical scavenger and is oxidized to a
dimer during the respiratory burst of human mononuclear
and polymorphonuclear phagocytes. Eur J Clin Invest
1995;25:477–484.
28. Huether G, Fettkotter I, Keilhoff G, Wolf G. Serotonin acts
as a radical scavenger and is oxidized to a dimer during the
respiratory burst of activated microglia. J Neurochem
1997;69:2096–2101.
29. Schuff-Werner P, Splettstoesser W. Antioxidative properties
of serotonin and the bactericidal function of polymorphonuclear
phagocytes. Adv Exp Med Biol 1999;467:321–325.
590 M. Cˇ ı´zˇ et al.
Serotonin and its 5-HT2 receptor agonist DOI hydrochloride inhibit the oxidative
burst in total leukocytes but not in isolated neutrophils
Lucie Prachařová, Kateřina Okénková, Antonín Lojek, Milan Číž ⁎
Department of Free Radical Pathophysiology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic
a b s t r a c ta r t i c l e i n f o
Article history:
Received 3 December 2009
Accepted 3 February 2010
Keywords:
Chemiluminescence
Leukocyte
Neutrophil
Oxidative burst
Serotonin
Serotonin receptor
Aims: Serotonin (5-HT) is capable of reducing the oxidative burst of professional phagocytes. In this study,
we investigated whether 5-HT mediates this modulation via 5-HT receptors (5-HTR) or whether this is due
instead to 5-HT antioxidative properties.
Main methods: The leukocytes or polymorphonuclear leukocytes (PMNL) were isolated from human blood,
and their ability to produce reactive oxygen species (ROS) after 5-HT or its agonist treatment was tested by
luminol-enhanced chemiluminescence (CL) analysis.
Key findings: It was found that 5-HTR2 agonist DOI hydrochloride does not have any antioxidative properties,
despite its ability to inhibit the CL response of activated human total leukocytes. On the other hand, DOI
hydrochloride was unable to inhibit the CL response of activated human PMNL. It seems that the reduction of
the oxidative burst of professional phagocytes was evoked by the activation of 5-HTR not on the neutrophil
surface but on the surface of different leukocytes, which produced anti-inflammatory cytokines with NADPH
oxidase activity modulating properties.
Significance: Platelets and activated PMNL are in tight contact at sites of inflammation. 5-HT released from
platelets might have a protective function against PMNL-derived oxidative stress and oxidative damages.
© 2010 Elsevier Inc. All rights reserved.
Introduction
Professional phagocytes are essential components of the innate nonspecific
immune response both in humans and animals. In blood, they
comprise monocytes and the most frequent neutrophils. Neutrophils
are accumulated in areas of infection, where they contribute to the
killing of invading microorganisms by the generation of reactive oxygen
species (ROS) through NADPH oxidase activity (Brechard et al. 2005).
The excessive production of phagocyte-derivated ROS plays a role in a
number of different diseases (Roberts and Sindhu 2009).
Serotonin (5-hydroxytryptamine, 5-HT) is synthesized by the
hydroxylation and decarboxylation of the amino acid tryptophan in
enterochromaffin cells of the gastrointestinal tract and in the cells of
the central nervous system. The highest concentration of 5-HT in the
body (about 95%) is found in the cells of the gastrointestinal tract, of
which 90% are within enterochromaffin cells and 10% within enteric
neurons. The rest of 5-HT is found in the brain. All the serotonin in the
blood and platelets is derived from the gastrointestinal tract (Lesurtel
et al. 2008).
A wide variety of the physiological effects of 5-HT on cells are
mediated by a polymorphic group of specific serotoninergic membrane
receptors (5-HTR) classified into 7 families. In humans, these
families have been subdivided into 15 distinct receptor subtypes.
With the exception of a ligand-gated cation channel (5-HTR3), most
5-HTR are coupled to G-proteins and linked either to adenylate
cyclase (5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HT4, 5-HT6,
and 5-HT7) or to phospholipase C/protein kinase C (5-HTR2A, 5-HTR2B
and 5-HTR2C) (Nichols and Nichols 2008; Hoyer et al. 2002).
Wide cooperation exists between platelets and professional phagocytes
in sites of inflammation and endothelial cell damage. Activation
of platelets leads to their aggregation and to a rapid release of 5-HT in
micromolar concentrations in proximity to blood cells (Mossner and
Lesch 1998). It has been widely reported that 5-HT modulates the
function of professional phagocytes. However, this modulation is complex,
and the data available are rather controversial (Jancinova et al.
2001; Schuff-Werner and Splettstoesser 1999; Tokmakov et al.
1991). 5-HT could act as a true scavenger of ROS generated during a
respiratory burst of stimulated leukocytes (Schuff-Werner and Splettstoesser
1999), could be caused by the aggregation and degranulation of
neutrophils (Renesto and Chignard 1994), or by inhibitingthe migration
of mononuclear leukocytes (Bondesson et al. 1993). It was shown in our
previous study that 5-HT inhibits the chemiluminiscence response of
neutrophils in human whole blood (Ciz et al. 2007). That inhibition was
partially due to the direct quenching activity of 5-HT against ROS, and to
the inhibition of myeloperoxidase activity.
Taking all these facts into consideration, accumulating evidence
suggests that 5-HT is a very important mediator of mutual interactions
between platelets and professional phagocytes. However, additional
Life Sciences 86 (2010) 518–523
⁎ Corresponding author. Tel.: +420 541 517 104; fax: +420 541 211 293.
E-mail address: milanciz@ibp.cz (M. Číž).
0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2010.02.003
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
work is needed to clarify the mechanisms of its action on neutrophils. In
this study, the hypothesis that the inhibitory activity of 5-HT on the
respiratory burst of professional phagocytes might also be receptor
mediated was evaluated using various 5-HTR agonists in rat and human
total leukocytes and isolated polymorphonuclear leukocytes (PMNL).
Materials and methods
Reagents
5-HT (serotonin creatinine sulfate salt monohydrate), 5-HT agonists:
(±)-8-Hydroxy-2-(dipropylamino) tetralin (5-HT1 receptor
agonist), (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) hydrochloride
(5-HT2 receptor agonist), 1-(3-Chlorophenyl) biguanide
hydrochloride (5-HT3 receptor agonist), Cisapride (5-HT4 receptor
agonist), 5-Carboxamidotryptamine maleate salt (5-HT1, 5, 7 receptor
agonist), and oxidative burst activators were obtained from Sigma
(USA) and luminol from Molecular Probes (USA). A generator of
peroxyl radicals 2,2-azo-bis-2-amidinopropane hydrochloride (ABAP)
and reference inhibitor 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic
acid (trolox) from Sigma (USA) were used. Dextran-T500
(Pharmacia, Sweden) and telebrix N 300 (Léčiva, Czech Republic)
were used for total leukocyte and PMNL isolation. Anaesthetic for
rats was mixed from 10 ml of Narkamon (ketamini hydrochloridum)
and 0.5 ml of Rometar (xylazinum hydrochloridum) from Spofa a.s.
(Czech Republic). All other chemicals were purchased from local
distributors.
Total peroxyl radical-trapping antioxidant parameter analysis (TRAP)
The TRAP analysis is based on the measurement of the luminolenhanced
chemiluminescence (CL) (Cloez-Tayarani et al. 2003) in a
chemical system where peroxyl radicals are produced at a constant
rate by a thermal decomposition of ABAP. The TRAP value is determined
from the duration of the time period during which the sample
diminished the CL signal. Trolox, a water-soluble analogue of αtocopherol,
was used as a reference inhibitor (Pavelkova and Kubala
2004).
Isolation of rat total leukocytes
This study was performed in accordance with the National Institute
of Health's guidelines for the care and use of laboratory animals.
Male Wistar rats were kept under controlled light and temperature
conditions; water and food were given ad libitum. The animals were
subjected to the experimental procedure at the age of 3 months (body
weight 250–300 g). Rats were intraperitoneally anaesthetized with
ketamine/xylazine (20/2 mg per 100 g body weight).
Heparinized blood (50 IU/ml) from the anaesthetized rats was
obtained via a heart puncture. Erythrocytes were removed after 1 h
sedimentation in Dextran separation solution. The leukocyte-rich
plasma (buffy coat) was washed twice in HBSS, and total leukocytes
were adjusted to 1×106
cells×ml−1
(Pavelkova and Kubala 2004).
Total leukocyte/PMNL isolation from human whole blood
The heparinized blood (50 IU/ml) from healthy volunteers (n=5)
was obtained by antecubital venipuncture. Erythrocytes were removed
after 1 h sedimentation in Dextran separation solution. For
isolation of total leukocytes, the leukocyte-rich plasma (buffy coat)
was centrifugated (300 g, 20 °C, and 5 min). The pellet was exposed to
the hypotonic lysis of contaminating erythrocytes; the rest of the total
leukocyte was washed in HBSS, and total leukocytes were adjusted to
1×106
cells×ml−1
. For isolation of PMNL, Ficoll-Hypaque density
gradient centrifugation was used (400 g, 20 °C, and 40 min). The
pellet composed of erythrocytes and neutrophils (80–93%), eosinophils
(1.3–4%), and basophils (b2.7%) was exposed to hypotonic shock
to take out the contaminating erythrocytes. The obtained PMNLs were
washed in HBSS, and PMNLs were adjusted to 1×105
cells×ml−1
(Drabikova et al. 2002).
Total leukocyte/PMNL chemiluminescence
The CL of rat total leukocytes and human total leukocytes and
PMNL were measured using the microtitre plate luminometer Orion II
(Berthold Detection systems GmbH, Germany). The principle of the
method was previously described (Ciz et al. 2007). Each reaction
mixture consisted of 1×105
total leukocytes or 1×104
PMNL, 1 mM
luminol (stock solution prepared in borate buffer), one of the
activators (opsonized zymosan particles — OZP in a concentration of
62.5 μg×ml−1
, phorbol-12-myristate-13-acetate — PMA in a concentration
of 0.81 μg×ml−1
, calcium ionophore — CaI in a concentration
of 9.55 μg×ml−1
or N-formyl-methionyl-leucyl-phenylalanin — fMLP
in a concentration of 1.14 μg×ml−1
) and 5-HT or its agonist in various
concentrations between 1×10−7
and 1×10−4
M. Preparation of the
stock solutions of luminol and oxidative burst activators was
previously described (Pavelkova and Kubala 2004); 5-HT and its
Table 1
The antioxidative properties of 5-HTR agonists expressed as the TRAP mean values±
S.E.M. For comparison, the TRAP value of the reference inhibitor trolox is shown.
5-HT agonists TRAP (nmol×ml−1
)
(±)-8-Hydroxy-2-(dipropylamino) tetralin 7.3±7.3
Cisapride 11.0±6.5
1-(3-Chlorophenyl) biguanide hydrochloride 16.8±10.4
(±)-DOI hydrochloride 20.3±18.3
5-Carboxamidotryptamine maleate salt 13.0±10.5
Trolox 800.0±0.0
Table 2
The effect of 5-HT and its 5-HTR agonists on the oxidative burst of rat total leukocytes
activated by CaI (A), fMLP (B), PMA (C) and OZP (D). The results are expressed as a
percentage of the control±S.E.M. with level of significance analyzed by ANOVA,
pb0.01**, pb0.05*.
10−6
M 10− 5
M 10−4
M
A)
5-HT 46±15 26±8 9±4*
(±)-DOI hydrochloride 83±37 39±21 5±5**
(±)-8-Hydroxy-2-(dipropylamino) tetralin 66±20 33±9 7±5
Cisapride 45±8 48±12 7±3
1-(3-Chlorophenyl) biguanide hydrochloride 74±23 73±38 5±2
5-Carboxamidotryptamine maleate salt 91±38 85±41 10±7
B)
5-HT 99±11 52±11 3±1**
(±)-DOI hydrochloride 93±15 30±10* 0±1**
(±)-8-Hydroxy-2-(dipropylamino) tetralin 131±45 128±36 6±1**
Cisapride 110±12 149±33 81±8
1-(3-Chlorophenyl) biguanide hydrochloride 103±13 99±18 11±3**
5-Carboxamidotryptamine maleate salt 109±13 107±14 25±6*
C)
5-HT 103±15 76±18 30±7*
(±)-DOI hydrochloride 107±12 86±12 0±1**
(±)-8-Hydroxy-2-(dipropylamino) tetralin 85±21 81±15 49±29
Cisapride 97±13 98±9 79±5
1-(3-Chlorophenyl) biguanide hydrochloride 100±12 108±10 82±13
5-Carboxamidotryptamine maleate salt 109±13 108±10 74±4
D)
5-HT 88±14 44±9 2±0**
(±)-DOI hydrochloride 84±17 19±4** 0±0**
(±)-8-Hydroxy-2-(dipropylamino) tetralin 95±10 73±12 3±1**
Cisapride 81±9 88±11 70±12
1-(3-Chlorophenyl) biguanide hydrochloride 93±15 77±16 5±1**
5-Carboxamidotryptamine maleate salt 82±8 92±18 45±10
519L. Prachařová et al. / Life Sciences 86 (2010) 518–523
agonists were diluted in HBSS to a concentration of 10 mM. HBSS was
used to adjust the total reaction volume to 250 μl.
ATP test of cell viability
Cell viability was determined luminometrically by using the ATP
Cell Viability test (BioThema, Sweden). Total leukocytes isolated from
human blood were incubated in 96 well plates at 1×105
cells/well for
30 min with 5-HT or DOI hydrochloride (10−4
M) at 37 °C. After
incubation, somatic cell ATP releasing reagent was added. After
15 min shaking, the ATP reagent was added, and the luminescence
was immediately measured. The intensity of CL was used for the
evaluation of the toxic effects of 5-HT or DOI hydrochloride on total
leukocytes (Ambrozova et al. 2009).
Statistical analysis
All samples were measured in duplicate. The results are expressed
as the mean+standard error of the mean (S.E.M.), n=5. The level of
significance was analyzed by one-way analysis of variance (ANOVA),
followed by a Student t-test, pb0.01**, or pb0.05*.
Results
Antioxidative properties of 5-HT and its receptor agonists
The antioxidative properties of 5-HT and its agonists in the
concentration of 10−4
M were tested by TRAP analysis. Our results
confirmed the fact that 5-HT is a very strong antioxidant (data not
shown). On the other hand, the selected 5-HT agonists (±)-8-Hydroxy-
2-(dipropylamino) tetralin, DOI hydrochloride, 1-(3-Chlorophenyl)
biguanide hydrochloride, Cisapride, and 5-Carboxamidotryptamine
maleate salt, did not exert any antioxidative properties expressed as
TRAP values (nmol×ml−1
) and shown in Table 1.
Effect of 5-HT and its receptor agonists on rat total leukocytes
The effect of 5-HT and its agonists in a concentration range of
10− 6
M–10− 4
M on the oxidative burst of rat total leukocytes was
tested. The results showed that the most effective inhibitor of rat
total leukocyte CL reaction was DOI hydrochloride (Table 2). This
compound exerted the inhibition activity at a concentration of 10−4
M
for all types of phagocyte activators and also at a concentration of
10− 5
M when fMLP and OZP were used as phagocyte activators.
Based on these results, DOI hydrochloride was chosen for the
experiments that followed.
The effect of 5-HT and DOI hydrochloride on cell viability
Cell viability after 5-HT and DOI hydrochloride treatment was tested
by ATP test. After 5-HT (10−4
M) treatment, the viability of human total
leukocytes was 97±0.7% of control (human total leukocytes without
treatment); and, after DOI hydrochloride (10− 4
M) treatment, it was
96±1.7% of control. This means that both 5-HT and DOI hydrochloride
at a concentration of 10−4
M did not have any toxic effect on the
studied cells.
Fig. 1. The effect of 5-HT (10−7
–10−4
M) on the oxidative burst of human total leukocytes activated by CaI (A), fMLP (B), PMA (C) and OZP (D). The results are expressed as a
percentage of the control, with the level of significance analyzed by ANOVA, pb0.01**, pb0.05*.
520 L. Prachařová et al. / Life Sciences 86 (2010) 518–523
Effect of 5-HT and DOI hydrochloride on human total leukocytes
The absolute values of the integrals obtained from the measurements
of CL responses of human total leukocytes activated with
CaI were 102±12, fMLP 125±20, PMA 168±39, and with OZP were
477±152 RLU×min×106
. Then, the oxidative burst of human total
leukocytes was measured after incubation (10min at room temperature)
with either 5-HT or DOI hydrochloride. The data showed that
5-HT significantly decreased the CL response of human total leukocytes
in a dose-dependent manner for all types of phagocyte activators
(CaI, fMLP, PMA, OZP). The CL response was significantly decreased at
the concentrations of 10−5
M–10−4
M for all types of activators.
Furthermore, CaI and OZP activated CL responses were significantly
inhibited at the concentration of 10−6
M and CaI activated CL response
even at the concentration of 10−7
M (Fig. 1).
The effect of DOI hydrochloride was similar. We observed
decreased CL response in a dose-dependent manner. A significant
decrease was detected at a concentration of 10−4
M for all types of
activation and for CaI and fMLP also at a concentration of 10−5
M
(Fig. 2).
Effect of 5-HT and DOI hydrochloride on human PMNL
The absolute value of the integrals obtained from the measurement
of CL response of human PMNL activated with OZP was 449±120
RLU×min×106
. The luminol-enhanced CL of isolated human PMNL was
not significantly decreased in the presence of either 5-HT or DOI
hydrochloride (Fig. 3). The 5-HT exerted a very low inhibition of
PMNL chemiluminescence response: 4% inhibition (10−6
M 5-HT),
8% inhibition (10−5
M 5-HT), and 31% inhibition (10−4
M 5-HT). The
effect of DOI hydrochloride was similar: 5% inhibition (10−5
M DOI
hydrochloride) and 28% inhibition (10−4
M DOI hydrochloride).
Discussion
It is known from previous studies that 5-HT, liberated from
activated platelets, is involved in the reduction of the oxidative burst
in activated human professional phagocytes, measured as a luminolenhanced
CL (Jancinova et al. 2001; Ciz et al. 2007). This effect of 5-HT
could, at least partially, be caused by its scavenging feature, which has
been discussed in several studies (Schuff-Werner et al. 1995; Huether
et al. 1997). The aim of this study was to elucidate the effect of 5-HT
on the oxidative burst of professional phagocytes and to establish
whether 5-HTR is also involved in this process. It is known that 5-HTR
is positively coupled through the Gq-protein to phospholipase C and
phospholipase A2, and their activation leads to increased accumulation
of inositol phosphates and intracellular Ca2+
(Van Oekelen et al.
2003). These events could lead to NADPH oxidase activity modification
and subsequent inhibition of ROS production which was observed
as the reduction of luminol-enhanced CL response in our
previous experiments.
First, the antioxidative properties of selected 5-HT agonists with a
different specificity to the 5-HT receptor families were tested by TRAP
analysis. None of these studied agonists exerted any direct antioxidative
properties. Thus, their potential effect on the oxidative burst of
Fig. 2. The effect of DOI hydrochloride (10−7
–10−4
M) on the oxidative burst of human total leukocytes activated by CaI (A), fMLP (B), PMA (C) and OZP (D). The results are
expressed as a percentage of the control, with level of significance analyzed by ANOVA, pb0.01**.
521L. Prachařová et al. / Life Sciences 86 (2010) 518–523
activated rat and human total leukocytes cannot be caused by their
scavenging of produced ROS.
DOI hydrochloride was the most effective 5-HTR agonist which
was capable of causing a modification of the oxidative burst of rat total
leukocytes in a dose-dependent manner, respectively the reduction of
CL response in activated rat total leukocytes, similarly to 5-HT. We
suppose that DOI hydrochloride as well as 5-HT influenced the
oxidative burst of activated rat total leukocytes via 5-HTR2.
The following experiments were focused on human immune cells
and the effect of 5-HT and its 5-HTR2 agonists, DOI hydrochloride.
First, the influence of 5-HT and DOI hydrochloride on the modification
of the oxidative burst, respectively the reduction of CL response in
human activated total leukocytes, was tested. Our results showed that
5-HT and DOI hydrochloride were both potent inhibitors of the CL
reaction of activated human total leukocytes. These results again
indicate the involvement of 5-HTR2.
Next, the influence of DOI hydrochloride and 5-HT on the CL
response of isolated human PMNL was tested. DOI hydrochloride,
similarly to 5-HT, in the concentration range of 10−7
–10−4
M, was
unable to significantly decrease the CL response of activated PMNL.
The fact that this observation was not caused by overactivation of
isolated PMNL was evidenced by the equal absolute values of the
integrals obtained from CL measurement of activated human total
leukocytes and PMNL. According to these results, we suppose that
involvement of 5-HTR2 during the oxidative burst is not directly
through the receptors on the neutrophil surface as we speculated
above. But, it seems that 5-HT influenced the oxidative burst indirectly,
via activation of 5-HTR2 on the other leukocytes.
Distribution of 5-HTR on immune cells was described in several
studies. While an expression of 5-HTR on the neutrophil surface has
not yet been described, it is known that 5-HTR2 are expressed on
the peripheral leukocytes (lymphocytes and monocytes) surface
(Mossner and Lesch 1998; Idzko et al. 2004; Durk et al. 2005). Both
lymphocytes and monocytes have been described as strong producers
of pro-inflammatory and anti-inflammatory cytokines, the production
of which could be modulated by 5-HT as has been described by several
authors (Idzko et al. 2004; Aune et al. 1994; Arzt et al. 1991; Kubera
et al. 2000; Menard et al. 2007). For example, 5-HT and its agonists
cause a decreased production of pro-inflammatory cytokines such as
interleukin 2 (IL-2) and interferon-γ (INF-γ) by memory T cells (Aune
et al. 1994), interferon-γ and tumor necrosis factor (TNF) by human
blood leukocytes (Arzt et al. 1991; Kubera et al. 2000), IL-12 and
tumor necrosis factor (TNF) by dendritic cells (Idzko et al. 2004)
and macrophages. On the other hand, 5-HT can increase antiinflammatory
cytokine IL-10 production by macrophages (Menard
et al. 2007), or IL-6 by human vascular smooth muscle cells (Ito et al.
2000). A number of studies exist that explain the mechanisms of 5-HT
modulation of cytokine production as a result of 5-HTR2 activation
(Cloez-Tayarani et al. 2003; Kubera et al. 2000; Kubera et al. 2005).
According to these, we suppose that 5-HT or DOI hydrochloride can
modify macrophage cytokine production towards anti-inflammatory
cytokines and that these cytokines exert their anti-inflammatory
properties by down-regulation of the oxidative burst, as was
described previously (Pilette et al. 2002; Dang et al. 2006).
It seems that 5-HT accumulated at inflammatory sites acts as a
protector against oxidative stress-induced tissue damages. In a physiological
concentration that is about 10−7
M and lower, 5-HT does not
have any influence on the oxidative burst of professional phagocytes;
but, in a pathophysiological concentration that is about 10−5
M
(Mossner and Lesch 1998), 5-HT exerts a reduction of the oxidative
burst and, in this way, averts the formation of oxidative tissue damages.
Conclusion
In accordance with our hypothesis, it was observed that 5-HT and
its 5-HTR2 agonist DOI hydrochloride were able to inhibit the luminolenhanced
CL response in activated rat and human total leukocytes,
but not in isolated human PMNL. The interpretation of these results
could suggest the indirect involvement of 5-HTR2 expressed not on
the neutrophil surface but on the surface of other leukocytes. These
leukocytes might, after 5-HT or DOI hydrochloride stimulation, be
able to produce anti-inflammatory cytokines that cause a downregulation
of the oxidative burst of PMNL. However, more investigations
are needed to understand the connection between the activation
of 5-HTR2 and the reduction of the oxidative burst in human
professional phagocytes. Moreover, some of the observed inhibitory
effects of 5-HT on the oxidative burst of PMNL might be partially
caused by other 5-HT receptors, namely 5-HTR3 and 5-HTR4.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was conducted under the research plans
AVOZ50040507 and AVOZ50040702 and was financially supported
by grant 524/07/1511 (Grant Agency of the Czech Republic). The
support and sponsorship by COST Action CM0603 on “Free Radicals in
Chemical Biology (CHEMBIORADICAL)” is kindly acknowledged.
Fig. 3. The effect of 5-HT (A) and DOI (B) hydrochloride (10−6
–10−4
M) on the
oxidative burst of human PMNL activated by OZP. The results are expressed as a
percentage of the control.
522 L. Prachařová et al. / Life Sciences 86 (2010) 518–523
References
Ambrozova G, PekarovaM, Lojek A. Effect of polyunsaturated fatty acids on the reactive
oxygen and nitrogen species production by raw 264.7 macrophages. European
Journal of Nutrition, (in press). doi:10.1007/s00394-009-0057-3.
Arzt E, Costas M, Finkielman S, Nahmod VE. Serotonin inhibition of tumor necrosis factoralpha
synthesis by human monocytes. Life Sciences 48 (26), 2557–2562, 1991.
Aune TM, Golden HW, McGrath KM. Inhibitors of serotonin synthesis and antagonists of
serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated
immunity in vivo. The Journal of Immunology 153 (2), 489–498, 1994.
Bondesson L, Nordlind K, Liden S, Sundstrom E. Inhibiting effects of serotonin and
serotonin antagonists on the migration of mononuclear leucocytes. Immunopharmacology
Immunotoxicology 15 (2–3), 243–250, 1993.
Brechard S, Bueb J, Tschirhart EJ. Interleukin-8 primes oxidative burst in neutrophil-like
HL-60 through changes in cytosolic calcium. Cell Calcium 37 (6), 531–540, 2005.
Ciz M, Komrskova D, Pracharova L, Okenkova K, Cizova H, Moravcova A, Jancinova V,
Petrikova M, Lojek A, Nosal R. Serotonin modulates the oxidative burst of human
phagocytes via various mechanisms. Platelets 18 (8), 583–590, 2007.
Cloez-Tayarani I, Petit-Bertron AF, Venters HD, Cavaillon JM. Differential effect of
serotonin on cytokine production in lipopolysaccharide-stimulated human
peripheral blood mononuclear cells: involvement of 5-hydroxytryptamine2A
receptors. International Immunology 15 (2), 233–240, 2003.
Dang PM, Elbim C, Marie JC, Chiandotto M, Gougerot-Pocidalo MA, El-Benna J. Antiinflammatory
effect of interleukin-10 on human neutrophil respiratory burst
involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a
decrease in ERK1/2 activity. The FASEB Journal 20 (9), 1504–1506, 2006.
Drabikova K, Nosal R, Jancinova V, Ciz M, Lojek A. Reactive oxygen metabolite
production is inhibited by histamine and H1-antagonist dithiaden in human PMN
leukocytes. Free Radical Research 36 (9), 975–980, 2002.
Durk T, Panther E, Muller T, Sorichter S, Ferrari D, Pizzirani C, Di Virgilio F, Myrtek D,
Norgauer J, Idzko M. 5-Hydroxytryptamine modulates cytokine and chemokine
production in LPS-primed human monocytes via stimulation of different 5-HTR
subtypes. International Immunology 17 (5), 599–606, 2005.
Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of
5-HT receptors. Pharmacology Biochemistry and Behavior 71 (4), 533–554, 2002.
Huether G, Fettkotter I, Keilhoff G, Wolf G. Serotonin acts as a radical scavenger and is
oxidized to a dimer during the respiratory burst of activated microglia. Journal of
Neurochemistry 69 (5), 2096–2101, 1997.
Idzko M, Panther E, Stratz C, Muller T, Bayer H, Zissel G, Durk T, Sorichter S, Di Virgilio F,
Geissler M, Fiebich B, Herouy Y, Elsner P, Norgauer J, Ferrari D. The serotoninergic
receptors of human dendritic cells: identification and coupling to cytokine release.
The Journal of Immunology 172 (10), 6011–6019, 2004.
Ito T, Ikeda U, Shimpo M, Yamamoto K, Shimada K. Serotonin increases interleukin-6
synthesis in human vascular smooth muscle cells. Circulation 102 (20), 2522–2527,
2000.
Jancinova V, Nosal R, Drabikova K, Danihelovaa E. Cooperation of chloroquine and blood
platelets in inhibition of polymorphonuclear leukocyte chemiluminescence.
Biochemical Pharmacology 62 (12), 1629–1636, 2001.
Kubera M, Kenis G, Bosmans E, Scharpe S, Maes M. Effects of serotonin and serotonergic
agonists and antagonists on the production of interferon-gamma and interleukin-
10. Neuropsychopharmacology 23 (1), 89–98, 2000.
Kubera M, Maes M, Kenis G, Kim YK, Lason W. Effects of serotonin and serotonergic
agonists and antagonists on the production of tumor necrosis factor alpha and
interleukin-6. Psychiatry Research 134 (3), 251–258, 2005.
Lesurtel M, Soll C, Graf R, Clavien PA. Role of serotonin in the hepato-gastrointestinal tract:
an old molecule for new perspectives. Cellular and Molecular Life Sciences 65 (6),
940–952, 2008.
Menard G, Turmel V, Bissonnette EY. Serotonin modulates the cytokine network in
the lung: involvement of prostaglandin E2. Clinical and Experimental Immunology
150 (2), 340–348, 2007.
Mossner R, Lesch KP. Role of serotonin in the immune system and in neuroimmune
interactions. Brain Behavior and Immunity 12 (4), 249–271, 1998.
Nichols DE, Nichols CD. Serotonin receptors. Chemical Reviews 108 (5), 1614–1641,
2008.
Pavelkova M, Kubala L. Luminol-, isoluminol- and lucigenin-enhanced chemiluminescence
of rat blood phagocytes stimulated with different activators. Luminescence
19 (1), 37–42, 2004.
Pilette C, Ouadrhiri Y, Van Snick J, Renauld JC, Staquet P, Vaerman JP, Sibille Y. IL-9 inhibits
oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human
monocytes through TGF-beta. The Journal of Immunology 168 (8), 4103–4111, 2002.
Renesto P, Chignard M. Progressive inactivation of cathepsin G and elastase released
from activated neutrophils in vitro: lack of participation of the neutrophil alpha 1proteinase
inhibitor. Journal of Laboratory and Clinical Medicine 123 (6), 906–913,
1994.
Roberts CK, Sindhu KK. Oxidative stress and metabolic syndrome. Life Sciences 84
(21–22), 705–712, 2009.
Schuff-Werner P, Splettstoesser W. Antioxidative properties of serotonin and the
bactericidal function of polymorphonuclear phagocytes. Advances in Experimental
Medicine and Biology 467, 321–325, 1999.
Schuff-Werner P, Splettstosser W, Schmidt F, Huether G. Serotonin acts as a radical
scavenger and is oxidized to a dimer during the respiratory burst of human mononuclear
and polymorphonuclear phagocytes. European Journal of Clinical Investigation
25 (7), 477–484, 1995.
Tokmakov AA, Kykhova MP, Koshkina OV, Vasil'ev V. The effect of serotonin on the
functional activity of monocytes. Tsitologiia 33 (1), 81–87, 1991.
Van Oekelen D, Luyten WH, Leysen JE. 5-HT2A and 5-HT2C receptors and their atypical
regulation properties. Life Sciences 72 (22), 2429–2449, 2003.
523L. Prachařová et al. / Life Sciences 86 (2010) 518–523
Title page
Serotonin and its metabolites reduce oxidative stress in murine RAW264.7 macrophages and
prevent inflammation
Ondřej Vašíček1
, Antonín Lojek1
, Milan Číž1,2*
1
Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, 612 65,
Brno, Czech Republic
2
Department of Animal Physiology and Immunology, Institute of Experimental Biology,
Masaryk University, Kotlarska 2, 611 37, Brno, Czech Republic
* Corresponding author. Tel.: +420 541 517 104. E-mail address: milanciz@ibp.cz
A short title:
Serotonin and its metabolites prevent inflammation
Keywords:
Serotonin; N-acetylserotonin; melatonin; RAW264.7 macrophages; reactive oxygen species;
nitric oxide; cytokines
Abstract
In this study we focused on comparing the effects of serotonin and its metabolites on the
functions of RAW264.7 cells (emphasis on oxidative burst and production of nitric oxide and
cytokines), thereby expanding the scope of existing knowledge with advent of novel findings
in this field. Changes in production of reactive oxygen species (ROS) by RAW264.7 cells
after treatment with serotonin, N-acetylserotonin and melatonin were determined using the
chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied
compounds on the CL response, the antioxidant properties of all respective compounds were
measured using TRAP and amperometrical method. Nitric Oxide (NO) production was
measured by Griess reagent and inducible NO synthase (iNOS) expression by Western blot.
Cytokine production was assessed using the Mouse Cytokine Panel A Array kit and ELISA.
We showed that all tested compounds were able to reduce oxidative stress, as well as inhibit
production of inflammatory cytokines by macrophages. Of the tested compounds, serotonin
and N-acetylserotonin were markedly better antioxidants than melatonin. In comparison, other
effects of tested compounds were very similar. It can be concluded that antioxidant capacity
of tested compounds is a major advantage in the early stages of inflammation. Since plasma
concentrations of N-acetylserotonin and melatonin are lower than serotonin, it can be deduced
that, serotonin plays a key role in modulation of inflammation and the regulatory functions of
immune cells; while also protecting cells against oxidative stress.
Key points:
Serotonin and related compounds reduce oxidative stress in macrophages.
Serotonin modulates inflammation mainly via its antioxidative properties.
Serotonin and N-acetylserotonin are better antioxidants than melatonin.
Introduction
Serotonin, otherwise known as 5-hydroxytryptamine (5-HT), is one of the most extensively
studied neurotransmitters of the central nervous system (CNS). However, serotonin is also
present in a variety of peripheral tissues, including several constituents of the immune system
[50,47,71,31,49,55]. Serotonin, an indolic monoamine compound metabolized from the
essential amino acid tryptophan, also functions within the CNS where it is synthesized and
stored in presynaptic neurons (serotoninergic neurons, pineal gland and catecholaminergic
neurons) [15]. Serotonin in the enteric nervous system (ENS), is synthesized mainly by
enterochromaffin (EC) cells lining the lumen of the gastrointestinal tract, subsequently
released into the plasma. Directly from the plasma, it is further readily taken up by platelets
during their life cycle - thus representing a major storage site for serotonin outside the CNS
[59,46,20]. Serotonin is inactivated via combination of oxidative deamination (monoamine
oxidase; MAO), conjugation with sulfuric and glucuronic acids, N-acetylation or 5-Omethylation
processes. Cleavage of the amine group (and adjacent hydrogen atom) by MAO
forms the primary metabolic pathway for serotonin in the body. Inactive 5hydroxyindoleacetic
acid (5-HIAA), a major catabolite of MAO metabolism, is then excreted
mainly into the urine [45,25]. On the other hand, two highly specific enzymes, serotonin-Nacetyltransferase
and hydroxyindol-O-methyltransferase, known to catalyze serotonin, are
also involved in synthesis of N-acetylserotonin and melatonin, primarily in the pineal gland
[36,47]. Melatonin plays an important role in regulation of various physiologic functions,
such as circadian rhythms, reproduction, mood, and immune functions. Moreover,
exogenously administred melatonin has been demonstrated to express various clinical effects
such as sleep modulating, analgesic, anxiolytic, antiinflammatory, and antioxidative activity
[43]. It was also shown that circadian secretion of melatonin synchronizes immune cells via
melatonin receptors [48]. Several studies have shown that both N-acetylserotonin and
melatonin are also produced at extrapineal sites, such as by immunocompetent cells, whereby
local concentrations can reach considerably high levels [9,44,60].
In addition to the neuromodulatory effects, all three tested compounds are able to interact with
immune cells, thereby exerting their direct influence on the immune system [50,42,8,10,58].
Evidence suggests that serotonin is capable of regulating immune function through several
serotonin receptor subtypes [52,4,53]. Other studies reveal the role of serotonin in
upregulating the activity of phagocytes [21], modulating the differentiation of human
monocytes [35] and the proliferation of T-cells [3,79,37]. Furthermore, it was concurred that
serotonin is involved in modulation of cytokine and chemokine production [19,13,27],
migration and recruitment of immune cells [51,18], and modulation of oxidative burst
mechanisms [61,12]. Melatonin, as well as serotonin, is recognized for having many effects
on immune cells. Carrillo-Vico et al. [10], Calvo et al. [8], and most recently summarized
these effects in detailed reviews [24,77,78]. In addition to the expression of melatonin
receptors, immune cell are capable to synthetise melatonin and thus induce changes in various
immune cell proportions, their viability, trafficking, metabolism and cytokine production in
the tumor microenvironment [48]. Huang et al. (2019) found that melatonin treatment
significantly decreases the expression of TNF-, IL-6 and IFN- and increases the production
of IL-10 and TGF- in dendritic cells. Melatonin also significantly inhibits the production of
TNF- in CD8 T cells [29]. Meanwhile, further information about the effects of Nacetylserotonin
as well as melatonin or serotonin on immune cells largely remains incomplete.
Several studies define the capacity of N-acetylserotonin to inhibit NO production and TNF-α
synthesis or attenuate the production of superoxide anion radical [58,38]. Oxenkrug [56] also
summarized the antioxidant effects, suggesting potential mechanisms of action characteristic
of N-acetylserotonin.
Consideration given to these facts and so, this study focused on comparing effects of
serotonin and its metabolites on the functions of RAW264.7 cells (especially oxidative burst,
production of NO and cytokine production). Therefore, we maintain strong beliefs that our
findings significantly enhance current knowledge in the field.
Methods
Serotonin, N-acetylserotonin and melatonin
Serotonin hydrochloride, N-acetylserotonin and melatonin were purchased from SigmaAldrich,
USA. All compounds were tested according to clinically relevant concentrations of
serotonin in different fluid compartments within the body: 10-8
M, physiological
concentration in plasma; 10-6
M, in sites of inflammation (primarily contained in dense
granules of activated platelets); and 10-3
M, pharmacological concentration of serotonin (the
approximate concentration of serotonin inside dense granules of platelets). Melatonin was
solubilized in ethanol (final concentration <0,2%). Furthermore, in the concentrations used,
all compounds studied were deemed non-toxic to RAW 264.7 cells, as proven by results of
the ATP tests (data not shown).
Cell culture
RAW 264.7 cells (American Type Culture Collection, USA), a mouse macrophage cell line,
were grown in Dulbecco’s Modified Eagle Medium (DMEM; PAN, Germany), supplemented
with 10 % of fetal bovine serum (PAN, Germany) and 1 % combination
penicillin/streptomycin. Cells were maintained at 37 °C in an atmosphere comprised of 5 %
CO2 and 95 % air.
Chemiluminescence (CL) assay of macrophages
The CL of macrophages was measured using an LM-01 microplate luminometer
(Immunotech, Prague, Czech Republic). Protocol was designed based on the method
described previously by Vasicek et al. [72]. Briefly: each reaction mixture consisted of 1x105
macrophages, one of the tested compounds (serotonin, N-acetylserotonin or melatonin) in
various concentrations (10-3
M, 10-6
M or 10-8
M, respectively) and lipopolysaccharide (LPS)
from E.coli (Sigma-Aldrich, USA) in the concentration of 100 ng/ml. 24 h incubation was
followed by addition of 1 mM luminol (Sigma-Aldrich, USA, 09253). Hank's Balanced Salt
Solution (HBSS) was then used to adjust for the required total reaction volume of 225 µl. The
reaction was initiated by adding 25 µl of one of the activators: opsonized zymosan particles
(OZP; Sigma-Aldrich, USA, L8274) in a concentration of 62.5 μM, or phorbol 12-myristate
13-acetate (PMA; Sigma-Aldrich, USA, P8139) in a concentration of 0.81 μM) and the
samples were immediately analyzed. Data acquisition was based on integral values of CL
over 90 minutes, whereby they were converted to a percentage of the control as final results.
Total peroxyl radical-trapping antioxidant parameter analysis (TRAP)
TRAP analysis is based on the measurement of luminol-enhanced CL in a chemical system
where peroxyl radicals are produced. Published by Denev in 2014 [16], brief outline of the
basic method includes the following steps: the reaction mixture contained 160 μl of Phosphate
Buffered Saline (PBS; pH 7.4), 16.8 μl of 10 mM luminol, and 6.7 μl of a tested compound.
Samples were subsequently incubated at 37 °C in a temperature-controlled carrousel of the
luminometer for 10 minutes. 16.8 μl of 400 mM 2,2'-dimethyl-2,2'-azodipropanimidamiddihydrochlorid
(ABAP, Sigma-Aldrich 44,091-4) were added to commence the generation of
peroxyl radicals. 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox; SigmaAldrich,
USA), was used as a reference inhibitor at a concentration of 400 μM. TRAP value
was determined by measurement of the time taken for the CL signal to diminish completely
by addition of the tested compounds.
Electrochemical detection of nitric oxide scavenging
Evaluation of the scavenging properties and capabilites against nitric oxide (NO) of studied
compounds was performed in a chemical system using an amperometrical method as
described previously by Pekarova [57]. The microsensor (AmiNO 700, Innovative
Instruments, USA) was connected to the ISO-NO MARK II potentiostat (WPI, USA).
Measurement was performed using distilled water saturated with pure NO gas (according to
the WPI manual). Temperature of the system was kept at 37 °C. Direct injection of 15 μl of
the NO-saturated water into the glass vial containing 5 ml of either tested compounds diluted
in PBS; or just PBS (control), caused rapid increase in the detected signal, which was
followed by a subsequent gradual decrease of the NO-induced signal until it reached the
background level. Index of scavenging (ratio of the signal peak height at time of NO injection,
to; time taken for re-entry and the signal to return to baseline) was used for evaluation of
radical scavenging properties of the tested compounds.
Measurement of nitrite concentration by Griess reaction
Changes in NO production were measured indirectly, by virtue of the accumulation of nitrites
(the end-product of NO metabolism) in a medium using Griess assay, as described previously
in various publications [41,2]. Cells were incubated in 6-well plates at concentration of
1 × 106
cells/well; with tested compounds and LPS (100ng/ml) for 24 h at 37°C in 5% CO2.
Positive control cells were treated only by LPS. At the end of the incubation period, culture
media was collected from the respective wells and centrifuged (16000g/4 °C/5 min). 150 μl of
centrifuged supernatant was mixed with an equal volume of Griess reagent (Sigma-Aldrich,
USA, G4410) and the mixture was incubated at room temperature (RT) in the dark for
30 min. Absorbance was measured at 546 nm; using sodium nitrite as the standard for the
calibration curve.
Western blot analysis of iNOS protein expression
After the treatment procedure, RAW264.7 cells were gently washed with cold PBS and lysed
using lysis buffer (1% sodium dodecyl sulfate (SDS); 100 mM Tris, pH 7.4; 10% glycerol)
containing phosphatase and protease inhibitor cocktail tablets (Roche, Germany). Protein
concentrations were determined using BCA™ protein assay (Thermo Scientific, USA, 23223)
and bovine serum albumin (Amresco, 0332, USA) was used as a standard. The same amount
of protein (40µg) from each lysate supplemented with Laemli buffer (200mM Tris-HCl, pH
6,8; 3% SDS; 30% glycerol; 0,03 bromphenol blue; 3% ß-mercaptoethanol; 200mM DTT)
was subjected to SDS-polyacrylamide gel electrophoresis, using 10 % running gel [22].
Electrophoresis was performed at 60 V for 15 minutes and then at 120V for 1.5 hour.
Separated proteins were transferred to PVDF membranes (2 hours at 330 mA) that were
blocked in 5% non-fat milk in Tris-buffered saline, 0.1% Tween 20 (TBS-T). Then, the
membranes were incubated with monoclonal antibodies against anti-iNOS/NOS Type II
mouse monoclonal antibody (BD transduction laboratories, USA, 610431; 1:5000), phosphoprotein
kinase C (PKC)α/βII and phosphor-PKCδ (Cell signaling technology, USA, 9375,
9374,; 1:2000) and ECL™ Anti-mouse IgG horseradish peroxidase linked whole antibody
(Biosciences, USA, 7076; 1:2000) were used. The equal loading of proteins was confirmed by
determination of β-actin (Santa Cruz Biotechnology, sc-47778, USA, 1:5000). The
immunoreactive bands were detected using an ECL™ detection reagent kit (Pierce, USA) and
exposed to radiographic film (AGFA, Belgium). Relative protein levels were quantified by
scanning densitometry, using the Image J™ program, and the individual band density value
was expressed in arbitrary units.
Cytokine assay
Mouse cytokine array panel A array kit (R&D Systems, Minneapolis, USA) was used for
determination of the relative levels of cytokines and chemokines, proceed according to the
manufacturer´s instructions. Mouse IL-6 and TNF-α DuoSet ELISA kits (R&D Systems,
USA) were used for more accurate measurement of selected cytokines, processed according to
the manufacturer´s instructions.
Statistical analysis
Samples were measured in duplicates. Results are reported as mean ± standard error of the
mean (SEM). Results were normalized with reference to the control cells in each run to
account for variability due to individual cell passages. Data were statistically analyzed using a
one-way analysis of variance (ANOVA), which was followed by Dunnett´s multiple
comparison test was performed using GraphPad Prism version 6.01 for Windows, GraphPad
Software, La Jolla California USA, www.graphpad.com. P < 0.05 was taken to indicate
significant differences between data mean values.
Results
The effect of serotonin, N-acetylserotonin and melatonin on ROS production by macrophages
First, we were able to detect the effect of tested compounds on ROS production by RAW
264.7 cells. When PMA was used as the activator, the tested compounds in concentrations 10-
6
M and 10-8
M did not significantly change the CL signal, however, concentrations of 10-3
M
dramatically reduced it. Serotonin and N-acetylserotonin reduced the CL signal down to 5 %
of control and; melatonin to 52 % of control (Fig. 1, upper panel). Similarly, when OZP was
used as the activator, no significant changes in the CL signal were detected for concentrations
amounting to 10-8
M and 10-6
M, but the concentration 10-3
M again, reduced the CL signal.
Serotonin and N-acetylserotonin reduced the CL signal significantly to 18 % and 20 % of the
control, respectively. Less dramatically, melatonin decreased the CL signal to just 90 % of
control (Fig. 1, bottom OZP). When LPS was used in combination with PMA and OZP,
newly obtained CL signals (Fig. 1, black bars) matched previous results (Fig. 1, white bars).
Tested compounds had no significant effects on inhibition of the phosphorylation of
PKCα/βII, PKCδ (data not shown).
Serotonin is known for its highly effective antioxidant capacity, therefore, we determined
antioxidant capacity of all tested compounds using TRAP method, which largely shares many
methodological similarities to CL measurement. Results from TRAP reveal that serotonin
(3399±29 mmol/ml) and N-acetylserotonin (2977±306 mmol/ml) are excellent antioxidants.
Melatonin, in comparison with the parallel compounds is a markedly weaker antioxidant
(760±263 mmol/ml) (Fig. 2).
The effect of serotonin, N-acetylserotonin and melatonin on NO production by macrophages
Here, we focused on NO production, an important signaling molecule of macrophages. The
LPS-activated macrophages significantly increase NO production and level of nitrite in
medium. Serotonin, N-acetylserotonin and melatonin decreased the nitrite formation in a
concentration-dependent manner (Fig. 3). Concentrations of 10-6
M serotonin significantly
reduced nitrite formation to 28 % of control; and just 1% of the control at 10-3
M. Nacetylserotonin
had a similar reduction potential profile to serotonin; in that, the concentration
of 10-6
M reduced nitrite formation to 30 % of control and to just 3 % of control, at 10-3
M.
Melatonin also had a similar effects to serotonin and N-acetylserotonin: 10-6
M, reduced
nitrites formation to 27 % of control. In the case of melatonin concentration of 10-3
M
reduced the signal to 14 % of control. Lower concentrations of tested compounds did not
affect the formation of nitrites in medium (Fig. 3).
Concentration dependent production of nitrites was confirmed by the expression of iNOS
protein. To demonstrate presence of iNOS protein expression, corresponding Western blots
were chosen (Fig. 4) after treatment with serotonin, N-acetylserotonin or melatonin. Looking
at the ratio of optical densities of iNOS and β-actin, it is evident that, all tested compounds in
concentration 10-3
M and 10-6
M, significantly decrease the expression of iNOS to
approximately 50 % of control. Compounds tested at 10-8
M did not significantly affect the
iNOS expression (Fig. 5).
No significant changes in NO scavenging properties of tested compounds were observed
using the amperometric method (data not shown).
The effect of serotonin, N-acetylserotonin and melatonin cytokine production by macrophages
The last part of this study was focused on the effects of tested compounds on production of
cytokines by RAW264.7 cells. Overall changes in cytokine production were evaluated using
Mouse Cytokine Array Kit. Non-stimulated macrophages produced IP-10 (CXCL10/CRG-2),
G-CSF, IL-1ra, KC, JE, sICAM-1, MIP-1α, MIP-1β, MIP-2, RANTES and; to a lesser extent
also TNF-α. When RAW264.7 cells were stimulated by LPS, there nascent production of IL-
6, M-CSF, TIMP-1, TNF-α and to a lower extent IFN-γ was observed. If RAW264.7 cells
were treated with 10-3
M serotonin, reduction in IL-6, M-CSF, IFN-γ, TIMP-1 production was
also noted. When LPS-stimulated RAW264.7 cells were treated with N-acetylserotonin (10-3
M), the results mimicked to some degree, the effect of serotonin. In addition, a reduction in
sICAM-1 was visible. When LPS-stimulated RAW264.7 cells were treated with melatonin
(10-3
M), TIMP-1 and IFN- γ were reduced completely, but IL-6 and M-CSF changed only
moderately (Fig. 6).
According to these results, we focused on IL-6 and TNF-α cytokines for further evaluation.
For accurate measurement of changes in production of these cytokines, ELISA was used. All
tested compounds had notably similar inhibitory effects on production of IL-6. The highest
concentration used of tested compounds, significantly decreased IL-6 from 1100 pg/ml to 77
± 14 pg/ml for serotonin; 100 ± 10 pg/ml for N-acetylserotonin and; 178 ± 4 pg/ml for
melatonin. Tested compounds in concentrations 10-6
M, decreased IL-6 production to 760 ±
68 pg/ml for serotonin; 830 ± 31 pg/ml for N-acetylserotonin and 772 ± 39 pg/ml for
melatonin. No significant changes in IL-6 production were observed when testing 10-8
M (Fig.
7). All compounds in concentration 10-3
M, significantly decreased TNF-α from 7385 ± 228
pg/ml to 3502 ± 187 pg/ml for serotonin; 5304 ± 187 pg/ml for N-acetylserotonin and 4007 ±
163 pg/ml for melatonin. All compounds in concentration 10-6
M, decreased the TNF-α
production, however, only N-acetylserotonin and melatonin were remarkable. No significant
changes in TNF-α production were observed using 10-8
M samples (Fig. 7).
Discussion
Serotonin and its metabolites (N-acetylserotonin and melatonin) play an important role in
coordination of the immune system, known for their involvement in regulation of phagocyte
responses [8,11,50]. In this study, we compared the effects of serotonin, N-acetylserotonin
and melatonin on important biological responses of RAW 264.7 mouse macrophage cell line.
Our results show that all tested compounds were able to significantly reduce ROS formation;
only at the upper limit of the range of the respective concentrations tested. To distinguish
whether tested compounds affect the NADPH oxidase pathway, different types of activators
were used. OZP (a typical receptor binding stimulus) triggers the signaling cascade during
phagocytosis via complement and/or Fc receptors whereas; PMA (a typical receptorbypassing
stimulus) directly activates PKC. LPS was used as a typical stimulant of
macrophages via TLR4 receptors. We found that reduction of CL signal was not caused by
direct inhibition of phosphorylation of PKC isoforms, but rather, their ROS-scavenging
activity. Our results clearly point to the considerably higher ROS-scavenging activity of
serotonin and N-acetylserotonin, compared to melatonin. Moreover, Schuff-Werner et al.
(1995), showed that serotonin acts locally, as a releasable antioxidant [67]. Further works
reveal a crucial role of serotonin in phagocytosis and its intimate relationship with respiratory
burst in human polymorphonuclear phagocytes [66,67,30]. Williams et al. (1998) [74], on the
contrary, showed that melatonin did not affect production of ROS and did not have
antioxidant properties, although; they used lower concentrations. Serotonin (through serotonin
transporter), N-acetylserotonin and melatonin (mainly due to their amphiphilicity) can easily
cross cell membranes and may influence for example, intracellular redox processes [76] or
serotonylation [64,73].
Another important role of macrophages is undoubtedly NO production. NO plays a pivotal
role in the immune system; fundamentally involved in the pathogenesis and control of
infectious diseases, association with tumor formation, autoimmune processes or chronic
degenerative diseases [5,7,14,6]. Moreover, NO can react with ROS, most notably superoxide
anion, to form peroxynitrite anion, which can potentially affect luminol-enhanced CL, too
[62]. Our results showed that all tested compounds were able to inhibit NO production in a
concentration-dependent manner. We observed different effects on NO production, only at the
highest tested concentrations of respective compounds. Variability, we assume, may be
caused by their different scavenging mechanisms [54]. Shimpo et al. (1997) [65], observed
similar effect of serotonin on vascular smooth muscle cells. They supposed that the inhibitory
effect of serotonin was mediated via the 5-HT2 receptor subfamily. Klemm et al. (1995) [38],
showed that, N-acetylserotonin was able to inhibit nitrite formation in murine macrophages
stimulated by a combination of LPS and IFNγ. Several authors have further established the
effect of melatonin on NO production in various experimental models. Tamura et al. (2009)
[70], demonstrated that melatonin inhibited NO production, induced by LPS in cultured rat
endothelial cells and aorta rings. Other authors elucidate the inhibitory effect of melatonin on
NO production in murine macrophages [1,17,23,81]. However, none have compared the
effects of serotonin and its metabolites on NO production. Recently, Hyeon et al. (2017) [32]
indicated that agomelatine, a structural analog of melatonin, suppressed an LPS-induced
generation of iNOS-derived NO via the repression of NF-κB, STAT1; STAT3 activation and
SOCS1 induction. Similarly, Jeon et al. [34], showed that N-arachidonoyl serotonin (NA-
5HT) at non-cytotoxic concentrations suppressed LPS-induced formation of nitric oxide, as
well as expression of inducible NO synthase in RAW264.7 murine cells. NA-5HT efficiently
reversed LPS-induced phosphorylative activation of nuclear factor-κB pathway, probably
through suppression of the mitogen-activated protein kinases (MAPKs) or
phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways.
According to the correspondence by the innate immune cells, LPS activates the signaling
pathway such as NF-κB via the stimulation of TLR4 which consequently releases not only
NO but proinflammatory cytokines including IL-1β, TNF-α, and IL-6 [63] . Based on the
results and measurements of cytokine profiling, it is obvious that serotonin, N-acetylserotonin
and melatonin possess robust anti-inflammatory properties. Interestingly, tested compounds
were able to inhibit early response to cytokines such as TNF-α and IL-6. Conversely, the
same compounds were able to partially inhibit production of IL-10, a potent cytokine with
reputable anti-inflammatory properties, subsequently repressing expression of inflammatory
cytokines, like for example, TNF-α, IL-6 and IL-1 [75]. Kubera et al.(2000) [39], showed in
blood samples, that the effect of serotonin on IL-10 production was dependent on the age of
volunteers. Several authors found that serotonin, via serotonin receptors (mainly 5-HT2A
subtypes) inhibited TNF-α and IL-6; while IL-1β production as a consequence, was increased
[40,13,80]. N-acetylserotonin and melatonin were also mentioned in connection with their
anti-inflammatory effects [8,11,74,58]. Recently, Jeon et al. (2016) [34], reported that another
serotonin metabolite, NA-5HT at non-cytotoxic concentrations, suppressed LPS-induced
formation of TNF- and selected interleukins in RAW264.7 cells.
Latest studies have also addressed the effects of serotonin on phagocytes, despite their rather
controversial results [28]. Earlier in 1980s, Sternberg et al. [68,69], showed that serotonin had
dose dependent inhibitory or stimulatory effects on murine macrophages. Generally, it seems
that, serotonin had suppressive effects at high concentrations and stimulatory effects at lower
(physiological) concentrations. As described by Jackson et al. (1988) [33], via expression of
serotonergic components like SERT (serotonin transporter) and other serotonin receptors on
immune cells, serotonin is able to exert a plethora of complex modulatory effects on
monocytes and macrophages. Since, expression of several subtypes of serotonin receptor by
monocytes and macrophages have additionally been reported: 5-HT2 [68], 5-HT1A [26]; 5HT1E,
5-HT2A, 5-HT3, 5-HT4, and 5-HT7 [19].
In summary, we showed that all tested compounds were able to reduce oxidative stress and
inhibit production of inflammatory cytokines by macrophages. In a comparison of the tested
compounds, serotonin and N-acetylserotonin were more effective antioxidants than melatonin.
Other effects of tested compounds were found to be very similar. It seems that their
antioxidant capacity is a major advantage in the early stages of inflammation. Plasma
concentrations of N-acetylserotonin or melatonin are relatively lower than serotonin. It can
therefore be deemed, that serotonin plays a principal role in modulation of inflammation,
regulation of function of immune cells and protection of cells against the detriment of
oxidative stress.
Statement of Contribution
All authors contributed equally to the design and implementation of the research and to the
analysis of the results. M.C. supervised the project, O.V. performed the majority of
experiments and designed the figures. All authors discussed the results and contributed to the
final manuscript.
Acknowledgments
The authors would like to thank Sumeet Gulati for checking the English of the manuscript.
Declaration of Interest
The study was supported by the MEYS of the Czech Republic (LD14030).
References
1. Aparicio-Soto M, Alarcon-de-la-Lastra C, Cardeno A, Sanchez-Fidalgo S, SanchezHidalgo
M (2014) Melatonin modulates microsomal PGE synthase 1 and NF-E2related
factor-2-regulated antioxidant enzyme expression in LPS-induced murine
peritoneal macrophages. British journal of pharmacology 171:134-144.
doi:10.1111/bph.12428
2. Arias-Negrete S, Jimenez-Romero LA, Solis-Martinez MO, Ramirez-Emiliano J, Avila EE,
Cuellar-Mata P (2004) Indirect determination of nitric oxide production by reduction
of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli
MC1061. Anal Biochem 328:14-21. doi:10.1016/j.ab.2004.01.026
3. Aune TM, McGrath KM, Sarr T, Bombara MP, Kelley KA (1993) Expression of 5HT1a
receptors on activated human T cells. Regulation of cyclic AMP levels and T cell
proliferation by 5-hydroxytryptamine. J Immunol 151:1175-1183
4. Barnes NM, Sharp T (1999) A review of central 5-HT receptors and their function.
Neuropharmacology 38:1083-1152
5. Bogdan C (2001) Nitric oxide and the immune response. Nature immunology 2:907-916.
doi:10.1038/ni1001-907
6. Bogdan C (2015) Nitric oxide synthase in innate and adaptive immunity: an update. Trends
in immunology 36:161-178. doi:10.1016/j.it.2015.01.003
7. Bogdan C, Rollinghoff M, Diefenbach A (2000) The role of nitric oxide in innate
immunity. Immunological reviews 173:17-26
8. Calvo JR, Gonzalez-Yanes C, Maldonado MD (2013) The role of melatonin in the cells of
the innate immunity: a review. Journal of pineal research 55:103-120.
doi:10.1111/jpi.12075
9. Carrillo-Vico A, Calvo JR, Abreu P, Lardone PJ, Garcia-Maurino S, Reiter RJ, Guerrero
JM (2004) Evidence of melatonin synthesis by human lymphocytes and its
physiological significance: possible role as intracrine, autocrine, and/or paracrine
substance. FASEB journal : official publication of the Federation of American
Societies for Experimental Biology 18:537-539. doi:10.1096/fj.03-0694fje
10. Carrillo-Vico A, Guerrero JM, Lardone PJ, Reiter RJ (2005) A review of the multiple
actions of melatonin on the immune system. Endocrine 27:189-200.
doi:10.1385/ENDO:27:2:189
11. Carrillo-Vico A, Lardone PJ, Alvarez-Sanchez N, Rodriguez-Rodriguez A, Guerrero JM
(2013) Melatonin: buffering the immune system. International journal of molecular
sciences 14:8638-8683. doi:10.3390/ijms14048638
12. Ciz M, Komrskova D, Pracharova L, Okenkova K, Cizova H, Moravcova A, Jancinova V,
Petrikova M, Lojek A, Nosal R (2007) Serotonin modulates the oxidative burst of
human phagocytes via various mechanisms. Platelets 18:583-590.
doi:10.1080/09537100701471865
13. Cloez-Tayarani I, Petit-Bertron AF, Venters HD, Cavaillon JM (2003) Differential effect
of serotonin on cytokine production in lipopolysaccharide-stimulated human
peripheral blood mononuclear cells: involvement of 5-hydroxytryptamine2A
receptors. International immunology 15:233-240
14. Coleman JW (2001) Nitric oxide in immunity and inflammation. International
immunopharmacology 1:1397-1406
15. Dahlstroem A, Fuxe K (1964) Evidence for the Existence of Monoamine-Containing
Neurons in the Central Nervous System. I. Demonstration of Monoamines in the Cell
Bodies of Brain Stem Neurons. Acta physiologica Scandinavica
Supplementum:SUPPL 232:231-255
16. Denev P, Kratchanova M, Ciz M, Lojek A, Vasicek O, Blazheva D, Nedelcheva P, Vojtek
L, Hyrsl P (2014) Antioxidant, antimicrobial and neutrophil-modulating activities of
herb extracts. Acta biochimica Polonica 61:359-367
17. Deng WG, Tang ST, Tseng HP, Wu KK (2006) Melatonin suppresses macrophage
cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52
acetylation and binding. Blood 108:518-524. doi:10.1182/blood-2005-09-3691
18. Duerschmied D, Suidan GL, Demers M, Herr N, Carbo C, Brill A, Cifuni SM, Mauler M,
Cicko S, Bader M, Idzko M, Bode C, Wagner DD (2013) Platelet serotonin promotes
the recruitment of neutrophils to sites of acute inflammation in mice. Blood 121:1008-
1015. doi:10.1182/blood-2012-06-437392
19. Durk T, Panther E, Muller T, Sorichter S, Ferrari D, Pizzirani C, Di Virgilio F, Myrtek D,
Norgauer J, Idzko M (2005) 5-Hydroxytryptamine modulates cytokine and chemokine
production in LPS-primed human monocytes via stimulation of different 5-HTR
subtypes. International immunology 17:599-606. doi:10.1093/intimm/dxh242
20. Endo Y, Shibazaki M, Nakamura M, Takada H (1997) Contrasting effects of
lipopolysaccharides (endotoxins) from oral black-pigmented bacteria and
Enterobacteriaceae on platelets, a major source of serotonin, and on histamine-forming
enzyme in mice. The Journal of infectious diseases 175:1404-1412
21. Freire-Garabal M, Nunez MJ, Balboa J, Lopez-Delgado P, Gallego R, Garcia-Caballero T,
Fernandez-Roel MD, Brenlla J, Rey-Mendez M (2003) Serotonin upregulates the
activity of phagocytosis through 5-HT1A receptors. British journal of pharmacology
139:457-463. doi:10.1038/sj.bjp.0705188
22. Georgiev YN, Paulsen BS, Kiyohara H, Ciz M, Ognyanov MH, Vasicek O, Rise F, Denev
PN, Lojek A, Batsalova TG, Dzhambazov BM, Yamada H, Lund R, Barsett H,
Krastanov AI, Yanakieva IZ, Kratchanova MG (2017) Tilia tomentosa pectins exhibit
dual mode of action on phagocytes as beta-glucuronic acid monomers are abundant in
their rhamnogalacturonans I. Carbohydrate polymers 175:178-191.
doi:10.1016/j.carbpol.2017.07.073
23. Gilad E, Wong HR, Zingarelli B, Virag L, O'Connor M, Salzman AL, Szabo C (1998)
Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in
murine macrophages: role of inhibition of NFkappaB activation. FASEB journal :
official publication of the Federation of American Societies for Experimental Biology
12:685-693
24. Hardeland R (2018) Melatonin and inflammation-Story of a double-edged blade. Journal
of pineal research 65:e12525. doi:10.1111/jpi.12525
25. Helander A, Beck O, Borg S (1992) Determination of urinary 5-hydroxytryptophol by
high-performance liquid chromatography with electrochemical detection. Journal of
chromatography 579:340-345
26. Hellstrand K, Hermodsson S (1993) Serotonergic 5-HT1A receptors regulate a cell
contact-mediated interaction between natural killer cells and monocytes. Scandinavian
journal of immunology 37:7-18
27. Hellstrand K, Kylefjord H, Asea A, Hermodsson S (1992) Regulation of the natural killer
cell response to interferon-alpha by biogenic amines. Journal of interferon research
12:199-206
28. Herr N, Bode C, Duerschmied D (2017) The Effects of Serotonin in Immune Cells.
Frontiers in cardiovascular medicine 4:48. doi:10.3389/fcvm.2017.00048
29. Huang SH, Liao CL, Chen SJ, Shi LG, Lin L, Chen YW, Cheng CP, Sytwu HK, Shang
ST, Lin GJ (2019) Melatonin possesses an anti-influenza potential through its immune
modulatory effect. J Funct Foods 58:189-198. doi:10.1016/j.jff.2019.04.062
30. Huether G, Schuff-Werner P (1996) Platelet serotonin acts as a locally releasable
antioxidant. Advances in experimental medicine and biology 398:299-306
31. Hung AS, Tsui TY, Lam JC, Wai MS, Chan WM, Yew DT (2011) Serotonin and its
receptors in the human CNS with new findings - a mini review. Current medicinal
chemistry 18:5281-5288
32. Hyeon JY, Choi EY, Choe SH, Park HR, Choi JI, Choi IS, Kim SJ (2017) Agomelatine, a
MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic
properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of
proinflammatory mediators in murine macrophages. Archives of oral biology 82:11-
18. doi:10.1016/j.archoralbio.2017.05.015
33. Jackson JC, Walker RF, Brooks WH, Roszman TL (1988) Specific uptake of serotonin by
murine macrophages. Life sciences 42:1641-1650
34. Jeon HL, Yoo JM, Lee BD, Lee SJ, Sohn EJ, Kim MR (2016) Anti-Inflammatory and
Antioxidant Actions of N-Arachidonoyl Serotonin in RAW264.7 Cells. Pharmacology
97:195-206. doi:10.1159/000443739
35. Katoh N, Soga F, Nara T, Tamagawa-Mineoka R, Nin M, Kotani H, Masuda K,
Kishimoto S (2006) Effect of serotonin on the differentiation of human monocytes
into dendritic cells. Clinical and experimental immunology 146:354-361.
doi:10.1111/j.1365-2249.2006.03197.x
36. Kema IP, de Vries EG, Muskiet FA (2000) Clinical chemistry of serotonin and
metabolites. Journal of chromatography B, Biomedical sciences and applications
747:33-48
37. Khan NA, Poisson JP (1999) 5-HT3 receptor-channels coupled with Na+ influx in human
T cells: role in T cell activation. Journal of neuroimmunology 99:53-60
38. Klemm P, Hecker M, Stockhausen H, Wu CC, Thiemermann C (1995) Inhibition by Nacetyl-5-hydroxytryptamine
of nitric oxide synthase expression in cultured cells and in
the anaesthetized rat. British journal of pharmacology 115:1175-1181
39. Kubera M, Kenis G, Bosmans E, Scharpe S, Maes M (2000) Effects of serotonin and
serotonergic agonists and antagonists on the production of interferon-gamma and
interleukin-10. Neuropsychopharmacology : official publication of the American
College of Neuropsychopharmacology 23:89-98. doi:10.1016/S0893-133X(99)00150-
5
40. Kubera M, Maes M, Kenis G, Kim YK, Lason W (2005) Effects of serotonin and
serotonergic agonists and antagonists on the production of tumor necrosis factor alpha
and interleukin-6. Psychiatry research 134:251-258.
doi:10.1016/j.psychres.2004.01.014
41. Lojek A, Ciz M, Pekarova M, Ambrozova G, Vasicek O, Moravcova J, Kubala L,
Drabikova K, Jancinova V, Perecko T, Pecivova J, Macickova T, Nosal R (2011)
Modulation of metabolic activity of phagocytes by antihistamines. Interdisciplinary
toxicology 4:15-19. doi:10.2478/v10102-011-0004-z
42. Markus RP, Ferreira ZS, Fernandes PA, Cecon E (2007) The immune-pineal axis: a
shuttle between endocrine and paracrine melatonin sources. Neuroimmunomodulation
14:126-133. doi:10.1159/000110635
43. Marra A, McGrane TJ, Henson CP, Pandharipande PP (2019) Melatonin in Critical Care.
Crit Care Clin 35:329-340. doi:10.1016/j.ccc.2018.11.008
44. Martins E, Jr., Ferreira AC, Skorupa AL, Afeche SC, Cipolla-Neto J, Costa Rosa LF
(2004) Tryptophan consumption and indoleamines production by peritoneal cavity
macrophages. Journal of leukocyte biology 75:1116-1121. doi:10.1189/jlb.1203614
45. McIsaac W, Page IH (1959) The metabolism of serotonin (5-hydroxytryptamine). The
Journal of biological chemistry 234:858-864
46. McNicol A, Israels SJ (1999) Platelet dense granules: structure, function and implications
for haemostasis. Thrombosis research 95:1-18
47. Mohammad-Zadeh LF, Moses L, Gwaltney-Brant SM (2008) Serotonin: a review. Journal
of veterinary pharmacology and therapeutics 31:187-199. doi:10.1111/j.1365-
2885.2008.00944.x
48. Moradkhani F, Moloudizargari M, Fallah M, Asghari N, Heidari Khoei H, Asghari MH
(2019) Immunoregulatory role of melatonin in cancer. J Cell Physiol.
doi:10.1002/jcp.29036
49. Morrissette DA, Stahl SM (2014) Modulating the serotonin system in the treatment of
major depressive disorder. CNS spectrums 19 Suppl 1:57-67; quiz 54-57, 68.
doi:10.1017/S1092852914000613
50. Mossner R, Lesch KP (1998) Role of serotonin in the immune system and in
neuroimmune interactions. Brain, behavior, and immunity 12:249-271.
doi:10.1006/brbi.1998.0532
51. Muller T, Durk T, Blumenthal B, Grimm M, Cicko S, Panther E, Sorichter S, Herouy Y,
Di Virgilio F, Ferrari D, Norgauer J, Idzko M (2009) 5-hydroxytryptamine modulates
migration, cytokine and chemokine release and T-cell priming capacity of dendritic
cells in vitro and in vivo. PloS one 4:e6453. doi:10.1371/journal.pone.0006453
52. Nichols DE, Nichols CD (2008) Serotonin receptors. Chemical reviews 108:1614-1641.
doi:10.1021/cr078224o
53. Noda M, Higashida H, Aoki S, Wada K (2004) Multiple signal transduction pathways
mediated by 5-HT receptors. Molecular neurobiology 29:31-39.
doi:10.1385/MN:29:1:31
54. Noda Y, Mori A, Liburdy R, Packer L (1999) Melatonin and its precursors scavenge nitric
oxide. Journal of pineal research 27:159-163
55. Olivier B (2015) Serotonin: A never-ending story. European journal of pharmacology
753:2-18. doi:10.1016/j.ejphar.2014.10.031
56. Oxenkrug G (2005) Antioxidant effects of N-acetylserotonin: possible mechanisms and
clinical implications. Annals of the New York Academy of Sciences 1053:334-347.
doi:10.1196/annals.1344.029
57. Pekarova M, Lojek A, Martiskova H, Vasicek O, Bino L, Klinke A, Lau D, Kuchta R,
Kadlec J, Vrba R, Kubala L (2011) New role for L-arginine in regulation of inducible
nitric-oxide-synthase-derived superoxide anion production in raw 264.7 macrophages.
TheScientificWorldJournal 11:2443-2457. doi:10.1100/2011/321979
58. Perianayagam MC, Oxenkrug GF, Jaber BL (2005) Immune-modulating effects of
melatonin, N-acetylserotonin, and N-acetyldopamine. Annals of the New York
Academy of Sciences 1053:386-393. doi:10.1196/annals.1344.033
59. Pletscher A (1968) Metabolism, transfer and storage of 5-hydroxytryptamine in blood
platelets. British journal of pharmacology and chemotherapy 32:1-16
60. Pontes GN, Cardoso EC, Carneiro-Sampaio MM, Markus RP (2006) Injury switches
melatonin production source from endocrine (pineal) to paracrine (phagocytes) melatonin
in human colostrum and colostrum phagocytes. Journal of pineal research
41:136-141. doi:10.1111/j.1600-079X.2006.00345.x
61. Pracharova L, Okenkova K, Lojek A, Ciz M (2010) Serotonin and its 5-HT(2) receptor
agonist DOI hydrochloride inhibit the oxidative burst in total leukocytes but not in
isolated neutrophils. Life sciences 86:518-523. doi:10.1016/j.lfs.2010.02.003
62. Radi R, Cosgrove TP, Beckman JS, Freeman BA (1993) Peroxynitrite-induced luminol
chemiluminescence. The Biochemical journal 290 ( Pt 1):51-57
63. Rossol M, Heine H, Meusch U, Quandt D, Klein C, Sweet MJ, Hauschildt S (2011) LPSinduced
cytokine production in human monocytes and macrophages. Critical reviews
in immunology 31:379-446
64. Shajib MS, Khan WI (2015) The role of serotonin and its receptors in activation of
immune responses and inflammation. Acta Physiol (Oxf) 213:561-574.
doi:10.1111/apha.12430
65. Shimpo M, Ikeda U, Maeda Y, Kurosaki K, Okada K, Saito T, Shimada K (1997)
Serotonin inhibits nitric oxide synthesis in rat vascular smooth muscle cells stimulated
with interleukin-1. European journal of pharmacology 338:97-104
66. Schuff-Werner P, Splettstoesser W (1999) Antioxidative properties of serotonin and the
bactericidal function of polymorphonuclear phagocytes. Advances in experimental
medicine and biology 467:321-325
67. Schuff-Werner P, Splettstosser W, Schmidt F, Huether G (1995) Serotonin acts as a
radical scavenger and is oxidized to a dimer during the respiratory burst of human
mononuclear and polymorphonuclear phagocytes. European journal of clinical
investigation 25:477-484
68. Sternberg EM, Trial J, Parker CW (1986) Effect of serotonin on murine macrophages:
suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic
receptor antagonists. J Immunol 137:276-282
69. Sternberg EM, Wedner HJ, Leung MK, Parker CW (1987) Effect of serotonin (5-HT) and
other monoamines on murine macrophages: modulation of interferon-gamma induced
phagocytosis. J Immunol 138:4360-4365
70. Tamura EK, Cecon E, Monteiro AW, Silva CL, Markus RP (2009) Melatonin inhibits
LPS-induced NO production in rat endothelial cells. Journal of pineal research 46:268-
274. doi:10.1111/j.1600-079X.2008.00657.x
71. Tops M, Russo S, Boksem MA, Tucker DM (2009) Serotonin: modulator of a drive to
withdraw. Brain and cognition 71:427-436. doi:10.1016/j.bandc.2009.03.009
72. Vasicek O, Lojek A, Jancinova V, Nosal R, Ciz M (2014) Role of histamine receptors in
the effects of histamine on the production of reactive oxygen species by whole blood
phagocytes. Life sciences 100:67-72
73. Walther DJ, Peter JU, Winter S, Holtje M, Paulmann N, Grohmann M, Vowinckel J,
Alamo-Bethencourt V, Wilhelm CS, Ahnert-Hilger G, Bader M (2003) Serotonylation
of small GTPases is a signal transduction pathway that triggers platelet alpha-granule
release. Cell 115:851-862
74. Williams JG, Bernstein S, Prager M (1998) Effect of melatonin on activated macrophage
TNF, IL-6, and reactive oxygen intermediates. Shock 9:406-411
75. Williams LM, Ricchetti G, Sarma U, Smallie T, Foxwell BM (2004) Interleukin-10
suppression of myeloid cell activation--a continuing puzzle. Immunology 113:281-
292. doi:10.1111/j.1365-2567.2004.01988.x
76. Wolfler A, Abuja PM, Schauenstein K, Liebmann PM (1999) N-acetylserotonin is a better
extra- and intracellular antioxidant than melatonin. FEBS Lett 449:206-210.
doi:S0014-5793(99)00435-4 [pii]
77. Wu H, Denna TH, Storkersen JN, Gerriets VA (2019) Beyond a neurotransmitter: The
role of serotonin in inflammation and immunity. Pharmacol Res 140:100-114.
doi:10.1016/j.phrs.2018.06.015
78. Xia Y, Chen S, Zeng S, Zhao Y, Zhu C, Deng B, Zhu G, Yin Y, Wang W, Hardeland R,
Ren W (2019) Melatonin in macrophage biology: Current understanding and future
perspectives. Journal of pineal research 66:e12547. doi:10.1111/jpi.12547
79. Yin J, Albert RH, Tretiakova AP, Jameson BA (2006) 5-HT(1B) receptors play a
prominent role in the proliferation of T-lymphocytes. Journal of neuroimmunology
181:68-81. doi:10.1016/j.jneuroim.2006.08.004
80. Yu B, Becnel J, Zerfaoui M, Rohatgi R, Boulares AH, Nichols CD (2008) Serotonin 5hydroxytryptamine(2A)
receptor activation suppresses tumor necrosis factor-alphainduced
inflammation with extraordinary potency. The Journal of pharmacology and
experimental therapeutics 327:316-323. doi:10.1124/jpet.108.143461
81. Zhang S, Li W, Gao Q, Wei T (2004) Effect of melatonin on the generation of nitric oxide
in murine macrophages. European journal of pharmacology 501:25-30.
doi:10.1016/j.ejphar.2004.08.015
Figures
Figure 1: The effects of serotonin, N-acetylserotonin and melatonin on PMA- and OZPactivated
ROS production in murine macrophages of RAW 264.7 cell line. The macrophages
were pre-incubated for 24h with tested compounds and addition of 100 ng/ml LPS (black
bars) or control medium (white bars) in dark, 37°C and 5% CO2. After 24 h incubation,
luminol was added. HBSS was then used to adjust the total reaction volume to 225 µl. The
reaction was initiated by adding 25 µl of one of the activators (OZP or PMA). The results
from five independent experiments were integrated, converted to a percentage of the control
and expressed as the mean ± SEM. Dashed line indicates 100 % of control. Data were
statistically analyzed using ANOVA, which was followed by Dunnett’s multiple comparison
test (** p < 0,01).
Figure 2: The antioxidant properties of serotonin, N-acetylserotonin and melatonin expressed
as TRAP mean values ± SEM. Dashed lines indicate the level of negative control. Data were
statistically analyzed using ANOVA, which was followed by Dunnett’s multiple comparison
test (** p < 0,01 and * p < 0,05).
Figure 3: The effects of serotonin, N-acetylserotonin and melatonin on nitrite production in
murine macrophages of RAW264.7 cell line, stimulated by 100 ng/ml LPS. The results from
five independent experiments were integrated, converted to a percent of the control and
expressed as the mean ± SEM. Dashed lines indicate 100 % of control. Data were statistically
analyzed using ANOVA, which was followed by Dunnett’s multiple comparison test (** p <
0,01).
Figure 4: The effects of serotonin, N-acetylserotonin and melatonin on protein expression of
iNOS in murine macrophages of RAW264.7 cell line. Figure showing a representive gel from
western blot analysis.
Figure 5: The effects of serotonin, N-acetylserotonin and melatonin on protein expression of
iNOS of murine macrophages RAW264.7 cell line stimulated by 100 ng/ml LPS. The results
from five independent experiments are expressed as the ratio of optical densities of iNOS and
β-actin mean ± SEM. Data were statistically analyzed using ANOVA, which was followed by
Dunnett’s multiple comparison test (** p < 0,01 and * p < 0.05).
Figure 6: The effects of serotonin, N-acetylserotonin and melatonin on cytokine production
of murine macrophages RAW264.7 cell line stimulated by 100 ng/ml LPS. The results from
one experiment (in duplicate) are expressed as the ratio of optical densities of reference spots
and cytokine spots mean ± SD calculate from duplicate.
Figure 7: The effect of serotonin, N-acetylserotonin and melatonin on IL-6 and TNF-α
production in murine macrophages of RAW264.7 cell line, stimulated by 100 ng/ml LPS. The
results from five independent experiments are expressed as the mean ± SEM. Data were
statistically analyzed using ANOVA, which was followed by Dunnett’s multiple comparison
test (** p < 0,01 and * p < 0.05).
Fig 1 Click here to access/download;Line Figure;Figure_01.tif
Fig 2 Click here to access/download;Line Figure;Figure_02.tif
Fig 3 Click here to access/download;Line Figure;Figure_03.tif
Fig 4 Click here to access/download;Line Figure;Figure_04.tif
Fig 5 Click here to access/download;Line Figure;Figure_05.tif
Fig 6 Click here to access/download;Line Figure;Figure_06.tif
Fig 7 Click here to access/download;Line Figure;Figure_07.tif
PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)
© 2008 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic
Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres
Physiol. Res. 57: 393-402, 2008
The Influence of Wine Polyphenols on Reactive Oxygen and Nitrogen
Species Production by Murine Macrophages RAW 264.7
M. ČÍŽ, M. PAVELKOVÁ, L. GALLOVÁ, J. KRÁLOVÁ, L. KUBALA, A. LOJEK
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
Received August 14, 2008
Accepted February 27, 2008
On-line April 25, 2007
Summary
The aim was to study the antioxidant properties of four wine
polyphenols (flavonoids catechin, epicatechin, and quercetin, and
hydroxystilbene resveratrol). All three flavonoids exerted
significant and dose-dependent scavenging effects against
peroxyl radical and nitric oxide in chemical systems. The
scavenging effect of resveratrol was significantly lower. All
polyphenols decreased production of reactive oxygen species
(ROS) by RAW264.7 macrophages. Only quercetin quenched ROS
produced by lipopolysaccharide-stimulated RAW264.7
macrophages incubated for 24 h with polyphenols. Quercetin and
resveratrol decreased the release of nitric oxide by these cells in
a dose-dependent manner which corresponded to a decrease in
iNOS expression in the case of quercetin. In conclusion, the
higher number of hydroxyl substituents is an important structural
feature of flavonoids in respect to their scavenging activity
against ROS and nitric oxide, while C-2,3 double bond (present in
quercetin and resveratrol) might be important for inhibition of
ROS and nitric oxide production by RAW 264.7 macrophages.
Key words
Antioxidants • Polyphenols • Macrophages • Oxidative stress •
Nitric oxide
Corresponding author
Milan Číž, Institute of Biophysics, Academy of Sciences of the
Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.
Fax: +420-541211293. E-mail: milanciz@ibp.cz
Introduction
The overproduction of reactive oxygen and
nitrogen species (ROS and RNS, respectively) by
phagocytes causes oxidative damage to membrane lipids,
DNA, proteins and lipoproteins. These reactions have
functional consequences, which may be deleterious to
cells and tissues. Thus, the inhibition of ROS and RNS
production is a popular target for the attenuation of many
inflammatory diseases (Shen et al. 2002).
Dietary polyphenols with antioxidative effects
from fruit and vegetables play an important role in a
prevention of the oxidative stress (Mojžišová and Kuchta
2001, Osawa 1999). Another important source of
polyphenolic antioxidants is wine, particularly red wine.
It has been demonstrated that polyphenols from wine
have not only antioxidative but also anti-inflammatory
effects (Oak et al. 2005) and that they can prevent
cardiovascular diseases (Babal et al. 2006). It is
furthermore suggested that they prevent free radicalmediated
lipid peroxidation of low density lipoproteins
(LDL), which is associated with cell aging and chronic
disesases such as atherosclerosis (Dell'Agli et al. 2004,
Cook and Samman 1996, Oak et al. 2005, Rajdl et al.
2006). It is postulated that the antioxidant and free radical
scavenging properties of phenolic compounds, present in
red wine, may partly explain the ”French paradox”, i.e.
the fact that French people have low incidence of
coronary heart disease, despite having a diet high in fat
and being heavy smokers (Aruoma 1994). The main
polyphenolic compounds in red wine belong to two major
classes: flavonoids and stilbenes. Of the flavonoids, (+)catechin,
(–)-epicatechin and quercetin and of the
stilbenes, trans-resveratrol, are the most abundant
polyphenols in wine. Exact mechanisms by which
flavonoids protect against oxidative stress-mediated
diseases (such as atherosclerosis) are still a matter of
debate (Benito et al. 2004).
The aim of this experiment was to study the
antioxidant properties of four wine polyphenols (catechin
394 Číž et al. Vol. 57
– CAT, epicatechin – EPI, quercetin – QUE, resveratrol –
RES) against peroxyl radical and nitric oxide, their
immediate and long-term effects on the production of
reactive oxygen and nitrogen species by RAW 264.7
macrophages, and their influence on the expression of
inducible nitric oxide synthase. Special attention was paid
to differentiate between scavenging and inhibiting
activity of the studied polyphenols with respect to their
chemical structure. As far as we know, there are no
similar investigations on the same selection of
polyphenols, especially concerning the effects of
polyphenols on various parameters linked to the
generation of nitric oxide. The advantage of the study is
that it took into consideration many factors: kind and
dose of polyphenols, time of their action, and different
reactive metabolites.
Methods
Materials
Murine RAW 264.7 macrophage cell line was
obtained from the American Type Culture Collection
(ATCC, Manassas, Virginia, USA). Cells were
maintained in a Dulbecco`s Eagle medium (DMEM)
(Sigma-Aldrich, St. Louis, Missouri, USA) supplemented
with a 10 % fetal bovine serum, gentamycin (0.045 mg/l),
glucose (3.5 g/l) and NaHCO3 (1.5 g/l). The stock
solution of lipopolysaccharide (LPS) from Escherichia
coli serotype 0111:B4 (Sigma-Aldrich, St. Louis,
Missouri, USA) was made up at 10-3
g/l in Hanks'
balanced salt solution without phenol red (HBSS, pH
7.4). The final concentration of LPS in a reaction mixture
was 10-7
g/l. Polyphenols (CAT, EPI, QUE and RES)
were purchased from Sigma-Aldrich (St. Louis, Missouri,
USA). Stock solutions (2x10-2
mol/l) were always
prepared fresh, in 99.8 % ethanol. Then, concentrations
of 2x10-4
, 5x10-4
, 1x10-3
, 2x10-3
mol/l were prepared in
RPMI 1640 and added to the reaction mixture to obtain
final concentrations of 10-5
, 2.5x10-5
, 5x10-5
and 10-4
mol/l, respectively.
ABAP [2,2-azo-bis(2-amidinopropane) hydrochloride]
was purchased from Polyscience (Warrington,
Pennsylvania, USA) and Trolox (6-hydroxy-2,5,7,8,tetramethyl-chroman-2-carboxylic
acid) from SigmaAldrich
(St. Louis, Missouri, USA). The stock solution of
10-2
mol/l luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)
(Molecular Probes, Eugene, Oregon, USA) was
prepared in 0.2 mol/l sodium borate buffer, pH 9.0
(1.24 g of H3BO3 and 7.63 g of Na2B4O7 ⋅10H2O in one
liter of redistilled water). All other reagents were
purchased from Sigma-Aldrich (St. Louis, Missouri,
USA). PMA (phorbol 12-myristate 13-acetate) was
dissolved in dimethylsulphoxide to obtain 3x10-3
mol/l
stock solution.
Total radical-trapping antioxidant parameter (TRAP)
The luminol-enhanced chemiluminescence (CL)
assay for TRAP was measured using a Luminometer
1251 (BioOrbit, Turku, Finland). The method is based on
the measurement of peroxyl radicals produced at a
constant rate by a thermal decomposition of ABAP. The
TRAP value is determined from the time period during
which the CL signal is diminished by antioxidants
(Slavíková et al. 1998). Trolox, a water-soluble analogue
of α-tocopherol, was used as a reference inhibitor for the
calculation of the TRAP value.
The reaction mixture contained 475 µl of sodium
phosphate buffered saline (PBS 10-1
mol/l, pH 7.4), 50 µl
of 10-2
mol/l luminol in 10-1
mol/l borate buffer
(pH 10.0), and 20 µl of examined compound. The
cuvettes were incubated at 37 °C in the temperaturecontrolled
carousel of the luminometer for 10 min. Then,
50 µl of 4x10-1
mol/l ABAP (prepared in 10-1
mol/l PBS)
was added. TRAP value for the sample was obtained
from the equation: TRAP = n [Trolox]τsample /ƒ τTrolox,
where n is the stoichiometric factor of Trolox (2.0), τ is
the time period of diminished chemiluminescence, and ƒ
is the dilution factor of the sample.
Scavenging of nitric oxide
Tested compounds were diluted to reach final
concentrations of 10-5
– 10-4
mol/l in a final volume of
10 ml of PBS. 1 μl of saturated nitric oxide solution was
added and the concentration of nitric oxide was measured
amperometrically using the ISO-NO Mark II isolated
nitric oxide meter and ISO-NOP sensor (both from WPI,
Sarasota, FL, USA). The method is based on a diffusion
of NO through a selective membrane covering the sensor
and its oxidation at the working electrode, resulting in an
electrical current. The scavenging effect of tested
compounds was evaluated based on a decrease in NO
concentration in reaction mixture as compared to the
control. The data are expressed as a redox current in pA.
Experimental design
When the immediate effect of polyphenols was
studied, 2 x 105
of RAW 264.7 cells in 100 µl of DMEM
were allowed to adhere for 60 min to the edge of each of
2008 Immunomodulatory Effects of Wine Polyphenols 395
the 96-well plate and then all chemicals were added
immediately before CL was measured.
When the long-term effect was studied, 96-well
cultivation plates (2x105
cells per well) and 6-well
cultivation plates (2x106
cells per well) were used for CL
measurement and Western-blot analysis (and Griess
reaction), respectively. Adhered cells (1 h) were
preincubated with one of the polyphenols at an indicated
concentration for 1 h prior to the 24 h of incubation with
LPS (10-4
g/l). Adherence as well as incubation steps
proceeded at 37 °C with 5 % CO2. After 24 h the 96-wells
were gently washed with HBSS and CL was measured
(as described below). The supernatants of the 6-well
plates were removed and used for the determination of
nitrites by Griess reaction as described below. Cells were
used for a Western-blot analysis as described below.
Chemiluminescence assay
The luminol-enhanced CL of RAW 264.7
macrophages was measured using microtitre plate
Luminometer LM-01T (Immunotech, Prague, Czech
Republic) as described previously (Lojek et al. 1997).
The principle of the method is based on a luminol
interaction with the phagocyte-derived ROS, which
results in large measurable amounts of light. Each well
(in 96-well culture plates) contained 2x105
RAW 264.7
cells, luminol (at a final concentration of 10-3
mol/l) and
PMA (at a final concentration of 8x10-7
mol/l), which was
selected on the basis of previous results (Lojek et al.
1997) and one of the polyphenols at final concentrations
of 0, 10-5
, 2.5x10-5
, 5x10-5
and 10-4
mol/l. The total
reaction volume of 250 μl was adjusted with HBSS. The
assays were run in duplicates. Light emission expressed
as relative light units (RLU) was recorded continuously at
37 °C for 60 min. Intensity of the CL reaction is
expressed as the integral of the obtained kinetic curves
which correspond to the total amount of light produced
during the time of measurements.
Determination of nitric oxide
The production of nitric oxide (NO) was
estimated indirectly as the accumulation of nitrites
(NO2
),
the metabolic end-product of NO metabolism, in
the medium using the Griess reagent as described
previously (Migliorini et al. 1991). Sodium nitrite was
used as a standard. 150 µl of culture supernatant was
added to 150 µl of Griess reagent (Sigma-Aldrich, St.
Louis, Missouri, USA), then incubated for 15 min in a
dark at room temperature and absorbance was measured
at 532 nm on a SLT Rainbow spectrophotometer (Tecan,
Crailsheim, Germany).
Detection of inducible nitric oxide synthase (iNOS) by
Western blot
Cells were washed with cold PBS, scraped and
then lysed in the lysis buffer (1 % sodium dodecyl
sulphate – SDS, 10-1
mol/l Tris pH 7.4, 10 % glycerol,
10-3
mol/l sodium ortho-vanadate, 10-3
mol/l phenylmethanesulfonyl
fluoride). Protein concentrations were
measured with the DC protein assay (Bio-Rad, Hercules,
California, USA) using bovine serum albumin as a
standard. Equal amounts of proteins (20 µg) were applied
on SDS-polyacrylamide gel electrophoresis. Proteins
were electrically transferred from the gel to a
nitrocelulose membrane and immunoblotted with rabbit
antiserum against the murine iNOS (Transduction Lab,
Lexington, Kentucky, USA). Horseradish peroxidaseconjugated
with anti-rabbit IgG antibody was used as a
secondary antibody. The blots were visualized using
ECL+ kit (Amersham, Arlington Heights, Illinois, USA)
and exposed to CP-B X-ray films (Agfa, Brno, Czech
Republic).
Viability
Viability was set using ATP kit SL (BioThema
AB, Hanince, Sweden) on microtitre plate Luminometer
LM-01T (Immunotech, Prague, Czech Republic).
Statistical analysis
All experiments were done in duplicates and
repeated six times. All data are reported as means ±
S.E.M. The data were analyzed using the non-parametric
Wilcoxon test or one-way analysis of variance (ANOVA)
using Statistica for windows 5.0 (Statsoft, USA). P≤0.01
value was considered to be significant.
Results
Immediate effect of polyphenols
The total antioxidant capacity of four
polyphenols studied (CAT, EPI, QUE and RES)
measured as the ability to scavenge chemicaly generated
peroxyl radical is shown in Figure 1. All polyphenols
showed a significant antioxidant capacity, which
increased in a dose-dependent manner. Flavonoids (CAT,
EPI, QUE) were observed to show an approximately
fourfold higher antioxidant capacity than hydroxystilbene
RES.
396 Číž et al. Vol. 57
Fig. 1. Total antioxidant capacity of
polyphenols. TRAP is expressed as µmol of
the peroxyl radical trapped per one liter of
polyphenolic solution. The changes of the
parameter were found to be significant at
the level of p = 0.01 using ANOVA test.
Statistically significant contrasts between
the values are marked by different capital
letters.
Fig. 2. The ability of polyphenols to
scavenge NO determined ampero-metrically
in a chemical system. The concentration of
NO is expressed as a redox current in
picoampers (pA).
Similar results were obtained when the ability of
tested compounds to scavenge nitric oxide was evaluated.
Flavonoids (CAT, EPI, and especially QUE) were
observed to markedly scavenge NO. However, resveratrol
scavenged NO only mildly in the highest tested
concentration (Fig. 2).
The ability of polyphenols to reduce the
oxidative stress caused by biologically generated radicals
was studied using murine macrophages RAW 264.7. All
polyphenols dose-dependently inhibited the chemiluminescence
produced by the PMA-stimulated RAW 264.7
(Fig. 3). The effect of RES was the highest among all
compounds tested, because even the lowest concentration
markedly inhibited chemiluminescence. Thus, RES seems
to be a more potent inhibitor of the oxidative burst than
flavonoids.
Long-term effect of polyphenols
LPS (10-4
g/l) increased the ROS production by
190 % (51.2 x 104
vs. 17.6 x 104
RLU in untreated
controls), as measured by PMA-activated CL. CAT, EPI
and RES did not have any inhibitory effect on ROS
production (data not shown). Conversely, QUE
significantly decreased the ROS production of LPSstimulated
RAW 264.7 in a dose-dependent manner
(Fig. 4A).
LPS evoked a 30-fold induction of nitrite
production as opposed to the untreated control (34.4x10-6
vs. 1.1x10-6
mol/l NO2
-
/2x106
cells, respectively). This
induction was inhibited by QUE (2.5x10-5
-10-4
mol/l)
treatment in a dose-dependent manner (Fig. 4B). RES at
concentrations of 5x10-5
and 10-4
mol/l cut the NO
production by 7 and 26 %, respectively. However, no
significant inhibition by CAT and EPI was found (data
not shown).
The cytotoxicity of polyphenols in RAW 264.7
cells was examined by luminometrical detection of ATP
concentration. None of the polyphenols affected the
2008 Immunomodulatory Effects of Wine Polyphenols 397
Fig. 3. Activity of polyphenols to scavenge
reactive metabolites produced by RAW
264.7 cells (measured by chemiluminiscence).
The changes of the parameter
were found to be significant at the level of
p≤0.01 using Wilcoxon test. Statistically
significant difference between the values
are marked by different capital letters.
Fig. 4. The effect of quercetin (QUE) on
chemiluminescence activity and nitrite
production by RAW 264.7 cells. Cells were
pretreated with the indicated concentrations
of QUE for 1 h before being incubated with
LPS (10-4
g/l) for 24 h. Control cells were
incubated with vehicle alone. The changes
of the parameter were found to be
significant at the level of p≤0.01 using
Wilcoxon test. Statistically significant
contrasts between the values are marked
by different capital letters. (A) Inhibition of
reactive metabolite production by QUE in
LPS-stimulated RAW 264.7 macrophages.
Before measurement, supernatant was
removed and cells were washed twice with
PBS. Phorbol-myristate acetate was used to
activate the oxidative burst of RAW 264.7
measured by chemiluminiscence. (B)
Inhibition of nitrite production by QUE in
LPS-stimulated RAW 264.7 cells. The
cultured supernatants were subsequently
isolated and analyzed for nitrite production.
Fig. 5. Inhibition of iNOS protein expression
by QUE. Cultures were set up as described
in the legend to Fig. 4. Equal loading of
proteins was verified by β-actin
immunoblotting. One of three representative
experiments is shown.
viability of RAW 264.7 cells (data not shown).
In view of the involvement of iNOS in the
inflammatory process, we monitored iNOS protein
expression by Western blot in RAW 264.7 exposed to
polyphenols. As shown in Figure 5, expression of the
iNOS protein was not detectable in unstimulated cells,
but markedly increased 24 h after LPS (10-4
g/l)
treatment. Treatment with QUE showed a concentrationdependent
inhibition of iNOS protein expression in LPSstimulated
RAW 264.7 cells. CAT, EPI and RES did not
influence iNOS expression in LPS-activated RAW 264.7
cells (data not shown).
Discussion
In our paper, four wine polyphenols (CAT, EPI,
398 Číž et al. Vol. 57
QUE and RES) were studied from the viewpoint of their
antioxidant capacity, their ability to scavenge biologically
produced ROS and RNS and their influence on nitric
oxide production and iNOS expression. Concentrations of
polyphenols used in our experiments ranged between
10-4
– 10-5
M which corresponded to the red wine content
of individual compounds: 191 mg/l (6 x 10-4
M) CAT,
82 mg/l (3x10-4
M) EPI, 8-16 mg/l (2.5-5.0 x 10-5
M)
QUE, and 1-8 mg/l (0.5-3.5 x 10-5
M) RES (Pendurthi
and Rao 2002). The knowledge of absorption,
biodistribution and metabolism of polyphenols is partial
and incomplete. Some polyphenols are bioactive
compounds that are absorbed from the gut in their native
or modified form. They are subsequently metabolized to
products detected in the plasma and then excreted. At
concentrations found in vivo in human plasma (low
nanomolar range), the effect of polyphenols is negligible
in comparison with endogenous protection mechanisms
against oxidative stress (Huisman et al. 2004).
In agreement with other authors (Cuendet et al.
2000, Nakao et al. 1998, Scott et al. 1993) we observed a
significant antioxidant capacity against peroxyl radical of
all studied polyphenols. Some authors have even found
that QUE and CAT showed a greater efficacy to scavenge
peroxyl radical on a mole to mole basis than the
antioxidant nutrients vitamin C, vitamin E, and betacarotene
(Rice-Evans 1995). In our study, flavonoids
(CAT, EPI, QUE) showed an approximately four-fold
higher ability to scavenge peroxyl radical than
hydroxystilbene RES. The antioxidant potential of
polyphenols depends on the number and arrangement of
the hydroxyl groups and the extent of structure
conjugation (Robak et al. 1988). They can donate
hydrogen atom from their hydroxyl groups and stabilize
the phenoxy radical formed by delocalization of the
unpaired electron within the aromatic structure. It is wellknown
that aromatic compounds containing hydroxyl
groups, especially those having an O-dihydroxy group on
ring B, appear to be important scavengers as reported for
flavonoids (Fauconneau et al. 1997). RES, a stilbene, is
also known to have a strong antiradical activity, which is
due to the presence of a conjugated double bond, which
makes the electrons more delocalized (Khanduja and
Bhardwaj 2003). The higher TRAP of flavonoids found
in our experiments may be caused by the fact that
flavonoids contain multiple hydroxyl groups (five OH
groups) in comparison to RES (three OH groups), which
are able to donate hydrogen atoms to peroxyl radicals and
so have a greater potential to act as a scavenger of
peroxyl radicals. This is also supported by the findings of
López et al. (2003), who reported QUE to have higher
antioxidant capacity against peroxyl radical than RES. On
the other hand, Yilmaz and Toledo (2004) found the RES
to be a more potent scavenger of chemically generated
peroxide radicals than CAT and EPI.
Since cellular systems generate a variety of
radicals including superoxide, hydroxyl and peroxyl
radicals, nitric oxide and peroxynitrite, other experiments
were performed to demonstrate the ability of flavonoids
and RES to scavenge biologically generated radicals.
All tested compounds dose-dependently
scavenged the free radicals that were produced by the
PMA-stimulated RAW 264.7 cells. RES, interestingly,
seems to be a more potent scavenger, although its TRAP
was the lowest. The efficiency of individual polyphenols
to scavenge peroxyl radical and inhibit the oxidative burst
in macrophages was not always the same because of a
different specificity of a polyphenol to scavenge peroxyl
radical particularly and other free radicals involved in the
process of an oxidative burst of macrophages. The TRAP
test provides information on the reactivity of phenolic
compounds with only one radical in a buffered solution.
On the other hand, in the PMA-stimulated macrophage
CL assay, a combination of many ROS is present.
Moreover, the mechanisms of flavonoid actions may
differ from that of stilbenes. In addition to their
antioxidative properties, some polyphenols act as metal
chelating agents and inhibit the superoxide-derived
Fenton reaction, which is an important source of the most
reactive hydroxyl radicals. Various authors consider
chelation of metal ions as the main mechanism of
polyphenolic action (Iwahashi 2000, Morel et al. 1994),
while others consider ROS scavenging to dominate in the
antioxidant effects (Fremont et al. 1999). Belguendouz et
al. (1997) reported that RES protects LDL against
peroxidative degradation mainly by chelating copper
whereas flavonoids are better scavengers of free radicals.
The ability to inhibit the Fenton reaction could explain
the effectiveness of RES against biologically generated
radicals in contrast to the system, where radicals are
chemically produced in a copper-free buffered solution.
All phenomena previously described are
responsible for a short-term response mediated by wine
polyphenols. Recent studies have pointed out that
polyphenolic compounds from several sources may also
have long-term effects, as they are able to modulate gene
expression in different transformed cell lines and in
macrophages (Dell'Agli et al. 2004). We used a bacterial
2008 Immunomodulatory Effects of Wine Polyphenols 399
LPS for the induction of inflammation processes in RAW
264.7. LPS, as an outer membrane component of bacteria,
triggers the generation of reactive oxygen intermediates
as well as the secretion of a variety of inflammatory
mediators, such as nitric oxide. In our experiment, a
190 % increase in ROS production was observed when
RAW 264.7 cells were incubated with LPS (24 h) in
comparison with an untreated control. The incubation of
cells with QUE diminished their LPS-activated
production of ROS in a concentration-dependent manner
(Fig. 4A). We found that QUE, in a concentration of 10-4
mol/l, reduced the ROS release even to the level of nonLPS-treated
cells. Conversely, CAT, EPI and RES did
not decrease the amount of ROS produced by activated
macrophages. This indicates that QUE is the most
effective modulator of oxidative stress in the long-term.
The large amount of NO produced in response to
bacterial lipopolysaccharide plays an important role in
endotoxemia and inflammatory conditions (Bellot et al.
1996). Therefore, drugs that inhibit NO generation by
inhibiting iNOS expression or its enzyme activity may be
beneficial in treating diseases caused by an
overproduction of NO (Stoclet et al. 1998). To
investigate the effect of polyphenols on NO production,
we measured the accumulation of nitrite, the stable
metabolite of NO, in culture media. We found a huge NO
release by LPS-stimulated cells as opposed to untreated
control. The results are in full agreement with the data of
others (Shen et al. 2002, Wadsworth and Koop 1999,
Wadsworth et al. 2001).
When LPS-activated cells were incubated with
QUE or RES, significant and dose-dependent decrease of
the NO production was observed after 24 h. It seems
likely that QUE and RES inhibit NO production by
several mechanisms. Suppression of NO release may be
attributed to direct NO scavenging activity, which was
previously suggested to be due to their ability to scavenge
an exogenous NO donor sodium nitropruside (SNP) in
vitro (Chan et al. 2000). Direct NO scavenging activity of
studied flavonoids was also proved in our experiments.
Another possible mechanism is a modulation of iNOS
protein expression. To investigate, whether QUE and
RES are able to decrease iNOS protein expression,
Western blot analysis of RAW 264.7 exposed to LPS and
one of these polyphenols was monitored. As shown in
Figure 5, expression of the iNOS protein was not
detectable in unstimulated cells, but markedly increased
24 h after LPS treatment. Treatment with QUE showed a
concentration-dependent inhibition of iNOS protein
expression in LPS-stimulated RAW 264.7 cells. This
finding is in agreement with results of Jung and Sung
(2004) who found an inhibitory effect of 5 µmol/l QUE
on the expression of iNOS and COX-2 enzymes in
lipopolysaccharide-activated RAW 264.7 cells. The
results of Kim et al. (1999) indicated that the inhibitory
activity of studied flavonoids was not due to direct
inhibition of iNOS enzyme activity as measured by
[3
H]citrulline formation from [3
H]arginine. The
regulation of iNOS expression is complex, but appears to
occur primarily at the level of transcription. Activation of
mitogen-activated protein kinases (MAPKs) and the
redox-sensitive transcription factors, nuclear factor-κB
(NF-κB) and activator protein 1 (AP-1) are key events in
the signal transduction pathways mediating iNOS
induction in macrophages exposed to LPS (Sherman et
al. 1993, Xie et al. 1994). Wadsworth et al. (2001) have
found that QUE inhibited p38 MAPK activity and caused
the inhibition of iNOS mRNA expression which resulted
in decreasing iNOS protein level and NO release in LPSstimulated
RAW 264.7 macrophages. It is not clear
whether QUE acts more by inhibiting iNOS expression or
by direct scavenging of NO. Chan et al. (2000) speculate
that QUE and RES may act more by scavenging of NO
radicals than by inhibition of iNOS gene expression. The
rationale for this deduction is that these compounds were
not very effective in reducing iNOS mRNA, while they
readily scavenged NO produced by SNP. In our study,
inhibition of iNOS protein expression was in parallel with
the comparable inhibition of NO production, thus
agreeing with the results obtained by Chen et al. (2001),
Chan et al. (2000) and Wadsworth et al. (2001).
Therefore we propose that the suppression of NO by
QUE was mainly mediated by inhibition of iNOS protein
expression. As stated earlier, NO plays an important role
in the pathogenesis of various inflammatory diseases.
Therefore, the inhibitory effect of QUE on iNOS gene
expression suggests that this is one of the mechanisms
responsible for the anti-inflammatory action of QUE.
Although RES at higher concentrations
decreased NO production, it did not influence iNOS
expression in LPS-activated RAW 264.7 cells. Our
results agree with those of Cho et al. (2002), who found
that a higher concentration of RES than needed for the
inhibition of NO production was required for the iNOS
expression and NF-κB translocation. In contrast,
resveratrol in a concentration of 30 μmol/l was found to
reduce the amount of cytosolic iNOS protein and steady
state mRNA levels (Tsai et al. 1999), probably due to
400 Číž et al. Vol. 57
inhibition of phosphorylation as well as degradation of
IκBα (inhibitory protein bound to NF-κB in its
unstimulated form in the cytosol), and a reduced nuclear
content of NF-κB subunits. Thus, resveratrol (and other
polyphenolic compounds) may inhibit the enhanced
expression of iNOS in inflammation through downregulation
of NF-κB binding activity. Bi et al. (2005)
reported that despite the inhibition of LPS-induced
degradation of IκBα, resveratrol may inhibit iNOS
expression also via suppression of p38 MAPK in
microglial cells. The speculation that RES may act more
by scavenging of NO radicals and suppressing the
generation of RNS than by the inhibition of iNOS gene
expression (Chan et al. 2000, Manna et al. 2000) was not
proved in our experiments since RES exhibited only
minor NO-scavenging activity in the highest used
concentration. Despite that, resveratrol was previously
referred to inhibit neutrophil generation of oxidants like
superoxide anion and hypochlorous acid (Cavallaro et al.
2003).
In conclusion, this study provides the evidence
for in vitro antioxidative effects of wine polyphenols
(CAT, EPI, QUE and RES). It seems that the higher
number of hydroxyl substituents is an important
structural feature of flavonoids (QUE, CAT, EPI) when
compared to hydroxystilbenes (RES) in respect to their
direct scavenging activity against ROS and RNS while
C-2,3 double bond (present in QUE and RES) might be
important for inhibition of ROS and NO production. Only
QUE significantly decreased the ROS and NO production
in LPS-stimulated RAW 264.7 cells in a dose-dependent
manner due to its unique chemical structure.
Conflict of Interest
There is no conflict of interest.
Acknowledgements
This study was executed within a research plan
AVOZ50040507 and was supported by grants of the
Grant Agency of the Czech Republic No. 524/04/0897
and the Grant Agency of the Academy of Sciences of the
Czech Republic No. B6004204 and 1QS500040507.
References
ARUOMA OI: Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol 32: 671-683, 1994.
BABÁL P, KRISTOVÁ V, ČERNÁ A, JANEGA P, PECHÁŇOVÁ O, DANIHEL L, ANDRIANTSITOHAINA R:
Red wine polyphenols prevent endothelial damage induced by CCl4 administration. Physiol Res 55: 245-251,
2006.
BELGUENDOUZ L, FREMONT L, LINARD A: Resveratrol inhibits metal ion-dependent and independent
peroxidation of porcine low-density lipoproteins. Biochem Pharmacol 53: 1347-1355, 1997.
BELLOT JL, PALMERO M, GARCIA-CABANES C, ESPI R, HARITON C, ORTS A: Additive effect of nitric oxide
and prostaglandin-E2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit. Inflamm Res 45: 203-208,
1996.
BENITO S, BUXADERAS S, MITJAVILA MT: Flavonoid metabolites and susceptibility of rat lipoproteins to
oxidation. Am J Physiol 287: H2819-H2824, 2004.
BI XL, YANG JY, DONG YX, WANG JM, CUI YH, IKESHIMA T, ZHAO YQ, WU CF: Resveratrol inhibits nitric
oxide and TNF-alpha production by lipopolysaccharide-activated microglia. Int Immunopharmacol 5: 185-193,
2005.
CAVALLARO A, AINIS T, BOTTARI C, FIMIANI V: Effect of resveratrol on some activities of isolated and in whole
blood human neutrophils. Physiol Res 52: 555-562, 2003.
CHAN MM, MATTIACCI JA, HWANG HS, SHAH A, FONG D: Synergy between ethanol and grape polyphenols,
quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol
60: 1539-1548, 2000.
CHEN YC, SHEN SC, LEE WR, HOU WC, YANG LL, LEE TJ: Inhibition of nitric oxide synthase inhibitors and
lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and
quercetin pentaacetate in RAW 264.7 macrophages. J Cell Biochem 82: 537-548, 2001.
CHO DI, KOO NY, CHUNG WJ, KIM TS, RYU SY, IM SY, KIM KM: Effects of resveratrol-related hydroxystilbenes
on the nitric oxide production in macrophage cells: structural requirements and mechanism of action. Life Sci
71: 2071-2082, 2002.
2008 Immunomodulatory Effects of Wine Polyphenols 401
COOK NC, SAMMAN S: Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. J Nutr
Biochem 7: 66-76, 1996.
CUENDET M, POTTERAT O, SALVI A, TESTA B, HOSTETTMANN K: A stilbene and dihydrochalcones with
radical scavenging activities from Loiseleuria procumbens. Phytochemistry 54: 871-874, 2000.
DELL'AGLI M, BUSCIALA A, BOSISIO E: Vascular effects of wine polyphenols. Cardiovasc Res 63: 593-602, 2004.
FAUCONNEAU B, WAFFO-TEGUO P, HUGUET F, BARRIER L, DECENDIT A, MERILLON JM: Comparative
study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures
using in vitro tests. Life Sci 61: 2103-2110, 1997.
FREMONT L, BELGUENDOUZ L, DELPAL S: Antioxidant activity of resveratrol and alcohol-free wine polyphenols
related to LDL oxidation and polyunsaturated fatty acids. Life Sci 64: 2511-2521, 1999.
HUISMAN A, VAN DE WIEL A, RABELINK TJ, VAN FAASSEN EE: Wine polyphenols and ethanol do not
significantly scavenge superoxide nor affect endothelial nitric oxide production. J Nutr Biochem 15: 426-432,
2004.
IWAHASHI H: Some polyphenols inhibit the formation of pentyl radical and octanoic acid radical in the reaction
mixture of linoleic acid hydroperoxide with ferrous ions. Biochem J 346: 265-273, 2000.
JUNG WJ, SUNG MK: Effects of major dietary antioxidants on inflammatory markers of RAW 264.7 macrophages.
Biofactors 21: 113-117, 2004.
KHANDUJA K, BHARDWAJ A: Stable free radical scavenging and antiperoxidative properties of resveratrol
compared in vitro with some other bioflavonoids. Indian J Biochem Biophys 40: 416-422, 2003.
KIM HK, CHEON BS, KIM YH, KIM SY, KIM HP: Effects of naturally occurring flavonoids on nitric oxide
production in the macrophage cell line RAW 264.7 and their structure–activity relationships. Biochem
Pharmacol 58: 759-765, 1999.
LOJEK A, ČÍŽ M, MARNILA P, DUŠKOVÁ M, LILIUS E-M: Measurement of whole blood phagocyte
chemiluminescence in the Wistar rat. J Biolumin Chemilumin 12: 225-231, 1997.
LOPEZ M, MARTINEZ F, DEL VALLE C, FERRIT M, LUQUE R: Study of phenolic compounds as natural
antioxidants by a fluorescence method. Talanta 60: 609-616, 2003.
MANNA SK, MUKHOPADHYAY A, AGGARWAL BB: Resveratrol suppresses TNF-induced activation of nuclear
transcription factors NF-kappa B, activator protein-1, and apoptosis: potential role of reactive oxygen
intermediates and lipid peroxidation. J Immunol 164: 6509-6519, 2000.
MIGLIORINI P, CORRADIN G, CORRADIN SB: Macrophage NO2
production
as a sensitive and rapid assay for the
quantitation of murine IFN-gamma. J Immunol Methods 139: 107-114, 1991.
MOJŽIŠOVÁ G, KUCHTA M: Dietary flavonoids and risk of coronary heart disease. Physiol Res 50: 529-535, 2001.
MOREL I, LESCOAT G, CILLARD P, CILLARD J: Role of flavonoids and iron chelation in antioxidant action.
Methods Enzymol 234: 437-443, 1994.
NAKAO M, TAKIO S, ONO K: Alkyl peroxyl radical-scavenging activity of catechins. Phytochemistry 49: 2379-2382,
1998.
OAK MH, EL BEDOUI J, SCHINI-KERTH VB: Antiangiogenic properties of natural polyphenols from red wine and
green tea. J Nutr Biochem 16: 1-8, 2005.
OSAWA T: Protective role of dietary polyphenols in oxidative stress. Mech Ageing Dev 111: 133-139, 1999.
RAJDL D, RACEK J, TREFIL L, SIALA K: Effect of white wine consumption on oxidative stress markers and
homocysteine levels. Physiol Res 56: 203-212, 2007.
PENDURTHI UR, RAO LVM: Effect of wine phenolics and stilbene analogues on tissue factor expression in
endothelial cells. Thromb Res 106: 205-211, 2002.
RICE-EVANS C: Plant polyphenols: free radical scavengers or chain-breaking antioxidants? Biochem Soc Symp 61:
103-116, 1995.
ROBAK J, SHRIDI F, WOLBIS M, KROLIKOWSKA M: Screening of the influence of flavonoids on lipoxygenase
and cyclooxygenase activity, as well as on nonenzymic lipid oxidation. Pol J Pharmacol Pharm 40: 451-458,
1988.
SCOTT BC, BUTLER J, HALLIWELL B, ARUOMA OI: Evaluation of the antioxidant actions of ferulic acid and
catechins. Free Radic Res Commun 19: 241-253, 1993.
402 Číž et al. Vol. 57
SHEN SC, LEE WR, LIN HY, HUANG HC, KO CH, YANG LL, CHEN YC: In vitro and in vivo inhibitory activities
of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production.
Eur J Pharmacol 446: 187-194, 2002.
SHERMAN MP, AEBERHARD EE, WONG VZ, GRISCAVAGE JM, IGNARRO LJ: Pyrrolidine dithiocarbamate
inhibits induction of nitric oxide synthase activity in rat alveolar macrophages. Biochem Biophys Res Commun
191: 1301-1308, 1993.
SLAVÍKOVÁ H, LOJEK A, HAMAR J, DUŠKOVÁ M, KUBALA L, VONDRÁČEK J, ČÍŽ M: Total antioxidant
capacity of serum increased in early but not late period after intestinal ischemia in rats. Free Radic Biol Med
25: 9-18, 1998.
STOCLET JC, MULLER B, ANDRIANTSITOHAINA R, KLESCHYOV A: Overproduction of nitric oxide in
pathophysiology of blood vessels. Biochem-Moscow 63: 826-832, 1998.
TSAI S-T, LIN-SHIAU S-Y, LIN J-K: Suppression of nitric oxide synthase and the down-regulation of the activation of
NFκB in macrophages by resveratrol. Br J Pharmacol 126: 673 – 680, 1999.
WADSWORTH TL, KOOP DR: Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory
cytokine expression in RAW 264.7 macrophages. Biochem Pharmacol 57: 941-949, 1999.
WADSWORTH TL, MCDONALD TL, KOOP DR: Effects of Ginkgo biloba extract (EGb 761) and quercetin on
lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factor-alpha. Biochem
Pharmacol 62: 963-974, 2001.
XIE QW, KASHIWABARA Y, NATHAN C: Role of transcription factor NF-kappa B/Rel in induction of nitric oxide
synthase. J Biol Chem 269: 4705-4708, 1994.
YILMAZ Y, TOLEDO RT: Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin,
and gallic acid. J Agric Food Chem 52: 255-260, 2004.
Regular paper
Evaluation of antioxidant activity of medicinal plants containing
polyphenol compounds. Comparison of two extraction systems
Maria Kratchanova1
, Petko Denev1, Milan Ciz2, Antonin Lojek2 and Atanas Mihailov3
1Institute of Organic Chemistry with Centre of Phytochemistry – BAS, Laboratory of Biologicaly Active Substances, Plovdiv, Bulgaria; 2Institute of
Biophysics of the AS CR, Brno, Czech Republic; 3Phytotherapyst, Sofia, Bulgaria
This study investigates the influence of extraction system
on the extractability of polyphenol compounds and
antioxidant activity of various medicinal plants. Oxygen
radical absorbance capacity (ORAC) and total polyphenol
content of 25 Bulgarian medicinal plants subjected to
water or 80 % acetone extractions were investigated and
compared. The type of extragent significantly influenced
the efficiency of the polyphenol extraction and the antioxidant
activity. In all cases ORAC results and total polyphenol
content were higher for acetone extraction than
for water extraction. The acetone extract of peppermint
had the highest ORAC value — 2917 µmol Trolox equivalent
(TE)/g dry weight (DW) and polyphenol content —
20216 mg/100 g DW. For water extraction thyme exhibited
the highest ORAC antioxidant activity — 1434 µmol
TE/g DW. There was a significant linear correlation between
the concentration of total polyphenols and ORAC
in the investigated medicinal plants. It can be concluded
that the solvent used affects significantly the polyphenol
content and the antioxidant activity of the extract and
therefore it is recommended to use more than one extraction
system for better assessment of the antioxidant
activity of natural products. Several of the investigated
herbs contain substantial amounts of free radical scavengers
and can serve as a potential source of natural antioxidants
for medicinal and commercial uses.
Keywords: medicinal plants, ORAC, polyphenols
Received: 12 March, 2010; revised: 26 May, 2010; accepted: 08 June,
2010; available on-line: 09 June, 2010
INTRODUCTION
A growing amount of evidence indicates a role of
reactive oxygen species (ROS) such as peroxyl radicals
(ROO•), hydroxyl radical (НО•), superoxide anion (О2
•–)
and singlet oxygen (1О2) in the pathophysiology of aging
and different degenerative diseases such as cancer, cardiovascular
diseases, Alzheimer’s disease and Parkinson’s
disease (Davies, 2000; Fenkel & Holbrook, 2000). Living
cells possess a protective system of antioxidants which
prevents excessive formation and enables the inactivation
of ROS. The antioxidants protect from the potentially
damaging oxidative stress, which is a result of an
imbalance between the formation of ROS and the body
antioxidant defense. Antioxidants have also been used in
food industry to prevent deterioration, nutritional losses
and off-flavoring in various foods, especially those containing
polyunsaturated fatty acids. Recently, interest
has increased considerably in finding naturally occurring
antioxidants for use in foods because of their potential
in health promotion and disease prevention, and their
high safety and consumer acceptability (Gorinstein et al.,
2003).
In search of novel sources of antioxidants in the last
years, medicinal plants have been extensively studied for
their antioxidant activity. From ancient times, herbs have
been used in many areas, including nutrition, medicine,
flavoring, beverages, cosmetics, etc. The ingestion of
fresh fruit, vegetables and tea rich in natural antioxidants
has been associated with prevention of cancer and cardiovascular
diseases (Willcox et al., 2004). The higher intake
of plant foods correlates with lower risk of mortality
from these diseases (Johnson, 2001). Approximately
60 % of the commercially available anti-tumoral and antiinfective
agents are of natural origin (Cragg et al., 1997).
Polyphenols are the most significant compounds for
the antioxidant properties of plant raw materials. The
antioxidant activity of polyphenols is mainly due to their
redox properties, which allow them to act as reducing
agents, hydrogen donors, singlet oxygen quenchers, metal
chelators and reductants of ferryl hemoglobin (RiceEvans
et al., 1995; 1997; Prior et al., 2005; Lopez et al.,
2007; Ciz et al., 2008; Gebicka & Banasiak, 2009).
Investigation of natural products is a research field
with great potential and is especially important in countries
possessing great biodiversity, like Bulgaria. About
600 plant species from the Bulgarian flora are recognized
as medicinal and are traditionally used in ethnopharmacology
and phytotherapy (Dimkov, 1979; Petkov, 1982).
There are many reports in the literature about the antioxidant
properties of medicinal plants (Zheng & Wang,
2001; Djeridane et al., 2006; Katalinic et al., 2006; Wojdylo
et al., 2007), but there are only few papers reporting
data about the antioxidant properties of Bulgarian herbs
using methods such as DPPH and ABTS (Ivanova et al.,
2005; Kiselova et al., 2006). The current study employs
the oxygen radical absorbance capacity (ORAC) method,
which has been found to be the most relevant one for
biologic samples (Wang et al., 2004; Huang et al., 2005;
Prior et al., 2005). Different extraction systems were
used to extract antioxidant components from the plant
material and often it is difficult to compare the results
for the antioxidant properties even for the same plant
material. Water (Zheng & Wang, 2001; Ivanova et al.,
2005; Katalinic et al., 2006; Kiselova et al., 2006), metha-

e-mail: lbas@plov.omega.bg
Abbreviations: AAPH, 2,2-azobis-(2-amidino-propane)dihydrochloride;
ABTS, 2,2’azinobis(3-ethylbenzothiazoline-6-sulfonic acid; AUC,
area under the curve; SD, standard deviation; DW, dry weight; FL,
fluorescein; ORAC, oxygen radical absorbance capacity; TE, Trolox
equivalents.
Vol. 57, No. 2/2010
229–234
on-line at: www.actabp.pl
230											 2010M. Kratchanova and others
nol (Shan et al., 2005; Wojdylo et al., 2007) and ethanol
(Djeridane et al., 2006) have been widely used. In very
few cases only, more than one extragent or sequential
multi-solvent extractions were preferred (Su et al., 2007;
Wojcikowski et al., 2007). It has been recognized that
the extraction solvent may significantly alter the antioxidant
activity estimation (Zhou & Yu, 2004). In the present
work, two extragents were used for the extraction
of plant antioxidants — water and 80 % acetone. Plant
extracts made with water are nutritionally more relevant,
moreover, herbs are traditionally ingested as hot-water
infusions. On the other hand, acetone is preferred for
more exhaustive extraction of polyphenol compounds
and it was of particular interest to compare the polyphenol
content and ORAC antioxidant activity in water infusions
and acetone extracts.
The objective of the current study was to investigate
the influence of the extraction agent on the extractability
of polyphenol components and the antioxidant activity
of 25 Bulgarian medicinal plants. These two parameters
were evaluated in water and 80 % acetone extracts of
plants. Results from this study will lead to a better characterization
of the antioxidant properties of the mediciTable
1. Medicinal plants commonly used in traditional medicine
Botanical name Family Common name
Part of plant
used
Medical use
Achillea millefolium Asteraceae Yarrow Flowers Antiseptic, anti-inflammatory, stomach ulcer, gastrointestinal
disorders, liver diseases
Arctium lappa Asteraceae Greater bur-
dock
Roots Diuretic, kidney stones, rheumatism, gastritis,
stomach ulcer, gout
Betula pendula Betulaceae Birch Leaves Diuretic, kidney disorders, bladder disorders
Calendula officinalis Asteraceae Marigold Flowers Anti-inflammatory, pain-relieving, local treatment
of wounds, duodenum and stomach ulcer, gastrointestinal
disorders
Cichohrium intybus Asteraceae Chicory Aerial parts Digestive, chologogue, liver diseases
Clinopodium vulgare Labiatae Wild basil Leaves Immunostimulant, cardio-tonic, verruca.
Crataegus monogyna Rosaceae Hawthorn Flowers,
leaves
Promotes capillary formation and heart microcirculation,
cardiovascular diseases, ischemia
Glycyrrhiza glabra Fabaceae Liquorice Roots Adaptogen, anticancer
Humulus lupulus Cannabaceae Hop Flowers Sedative, digestive, menstrual disorders
Hypericum perforatum Hypericaceae St. John’s wort Aerial parts Anti-inflammatory, astringent, antibacterial, diuretic,
ulcer, colitis, gastritis
Laurus nobilis Lauraceae Laurel leaves Leaves Immunostimulant, antidiabetic, stomatitis, sinusitis
Matricaria chamomilla Asteraceae Chamomile Flowers Anti-inflammatory, antiseptic, sedative, throat and
mouth inflammations, gastrointestinal disorders,
influenza, pharyngitis, laryngitis
Melissa officinalis Labiatae Common balm Leaves Sedative, gastrointestinal disorders
Mentha piperita Labiatae Peppermint Leaves Spasmolytic, antiseptic, gastric disorders, indigestion,
neuralgia, myalgia, antivomiting
Mentha spicata Labiatae Spearmint Leaves Hormone regulating, spasmolytic, antiseptic, gastric
disorders, indigestion, neuralgia, myalgia
Ocimum basilicum Labiatae Basil Leaves Antiseptic, spasmolytic, expectorant gastrointestinal
diseases, antitussive
Rubus idaeus Rosaceae Raspberry Leaves Anti-inflammatory, antiseptic, antidiarrheic, gastrointestinal
disorders
Salvia officinalis Labiatae Sage Leaves Anti-inflammatory, antiseptic, inflammations of
throat and mouth
Sideritis scardica Labiatae Mountain tea Aerial parts Expectorant, antitussive, bronchitis, cough
Taraxacum officinale Asteraceae Dandelion Aerial parts Diuretic, cholagogue, appetizer
Thymus vulgaris Labiatae Thyme Aerial parts Expectorant, spasmolytic, antibacterial, antitusive,
asthma, emphysema, whooping-cough, diseases
of respiratory tract
Tilia cordata Tiliaceae Lime Flowers Anti-inflammatory, expectorant
Tribulus terrestris Zygophyllaceae Caltrop Aerial parts Hormone regulating, sperm
promoting, prevents ovary cysts
Trigonella foenum-graecum Fabaceae Fenugreek Seeds Anticancer, metabolic syndrome and diabetes
Urtica dioica Urticaceae Nettle Leaves Hormone regulating, prostate cancer prevention,
podagra, diabetes, allergies, anaemia
Vol. 57 						 231Antioxidant activity of medicinal plantsVol. 57 						 231
nal plants investigated and will reveal which of them are
the best sources of dietary antioxidants.
MATERIALS AND METHODS
Chemicals. Fluorescein disodium salt, 2,2-azobis-(2amidino-propane)dihydrochloride
(AAPH), 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox),
and gallic acid were obtained from Sigma-Aldrich (Steinheim,
Germany). Folin-Ciocalteu’s phenol reagent was
purchased from Merck (Darmstadt, Germany). All other
solvents used were of analytical grade and purchased
from local distributors.
Plants. All medicinal plants used were either obtained
from local pharmacies (Plovdiv, Bulgaria) or collected
from nature in 2008. The choice of the plants investigated
was based on their use in the traditional medicine.
In total, 25 medicinal plants were investigated (Table 1).
Plants were dried, packed in paper bags and stored at
ambient temperature prior to the analysis.
Extraction. All plant materials were subjected to extractions
with acetone and with water. For the acetone
extraction, 10 g of the plant material was powdered in a
laboratory mill, then 0.5 g of the powder was transferred
into extraction tubes and mixed with 20 ml of the extragent
(80 % acetone in 0.2 % formic acid). Extraction was
conducted on an orbital shaker at room temp. for one
hour. After that, the samples were centrifuged (6 000 × g)
and supernatants were removed. The solid residue was
subjected to the second extraction under the same conditions.
Both supernatants were combined and analyzed
for antioxidant activity and total polyphenol content.
Water infusions were prepared in compliance with
the traditional preparation which is close to home conditions.
For that purpose 5 g of the herb powder was
added to 200 ml water (90 ºC). Aerial parts of the plants
were incubated for 15 min, whereas roots were incubated
for 45 min. The slurry was centrifuged (6 000 × g) and
supernatants were used for further analysis.
ORAC assay. ORAC was measured according to
the method of Ou et al. (2001) with some modifications
(Ciz et al., 2010). The method measures the antioxidant
scavenging activity against peroxyl radical generated by
thermal decomposition of AAPH at 37 °C. Fluorescein
(FL) was used as the fluorescent probe. The loss
of fluorescence of FL was an indication of the extent
of damage from its reaction with the peroxyl radical.
The protective effect of an antioxidant was measured
by assessing the area under the fluorescence decay
curve (AUC) relative to that of a blank in which no
antioxidant has present. Solutions of AAPH, fluorescein
and Trolox were prepared in a phosphate buffer
(75 mmol/l, pH 7.4). Samples were diluted in the phosphate
buffer as well. Reaction mixture (total volume
200 μl) contained FL — (170  μl, final concentration
5.36 × 10–8 mol/l), AAPH — (20 μl, final concentration
51.51 mmol/l), and sample — 10 μl. The FL solution
and sample were incubated at 37 °C for 20 min
directly in a microplate reader, and AAPH (dissolved in
buffer at 37 °C) was added. The mixture was incubated
for 30 s before the initial fluorescence was measured.
After that, the fluorescence readings were taken at the
end of every cycle (1 min) after shaking. For the blank,
10 μl of phosphate buffer was used instead of the extract.
The antioxidant activity was expressed in micromole
Trolox equivalents per gram of dry weight (DW).
Trolox solutions (6.25, 12.5, 25, 50 and 100 μmol/l)
were used for defining the standard curve.
Total polyphenol compounds analysis. Total
polyphenols were determined according to the method
of Singleton and Rossi (1965) with Folin-Ciocalteu’s
reagent. Gallic acid was employed as calibration standard
and results were expressed as gallic acid equivalents
(GAE) per 100 g DW.
RESULTS AND DISCUSSION
It is of particular interest to investigate the antioxidant
properties of medicinal plants, especially those traditionally
used in folk medicine. More than one extraction system
is recommendable for detailed assessment of the antioxidant
properties of medicinal plants. It was found in
a recent study by Su et al. (2007) that the ORAC values
of acetone extracts were higher than those for methanolic
extracts for several herbs. Therefore, aiming at the
maximum extractability of the polyphenol compounds,
we chose to extract raw materials with acetone. On the
other hand, the traditional ingestion of medicinal plants
and their clinical usage usually requires their extraction
with water. Table 2 shows the ORAC antioxidant activity
of the investigated medicinal plants extracted by acetone
and water (ORACac and ORACw, respectively).
Table 2. Antioxidant activity of 25 medicinal plants.
Comparison between 80 % acetone (ac) and water (w) extraction.
Results are presented as mean ± S.D.
Medicinal plant
ORACac
µmol TE/g
ORACw
µmol TE/g
Ratio
ORACw/ORACac
%
Peppermint 2917 ± 52 1409 ± 62 48.3
Hawthorn 2163 ± 89 364 ± 28 16.8
Thyme 1637 ± 59 1434 ± 54 87.6
Wild basil 1437 ± 60 844 ± 41 58.7
Birch 1185 ± 73 142 ± 18 12.0
Raspberry 1156 ± 80 608 ± 35 52.6
St. John’s wort 1141 ± 93 629 ± 41 55.1
Common balm 1121 ± 60 996 ± 26 88.8
Lime 1020 ± 88 97 ± 11 9.5
Sage 966 ± 69 609 ± 54 63.0
Yarrow 842 ± 80 394 ± 30 46.8
Laurel leaves 837 ± 81 170 ± 12 20.3
Caltrop 819 ± 56 272 ± 16 33.2
Camomile 814 ± 72 469 ± 25 57.6
Mountain tea 778 ± 77 294 ± 21 37.8
Hop 749 ± 62 260 ± 19 34.7
Spearmint 748 ± 57 598 ± 31 79.9
Liquorice 670 ± 48 213 ± 12 31.8
Marigold 407 ± 57 247 ± 17 60.7
Basil 402 ± 40 271 ± 18 67.4
Chicory 398 ± 22 132 ± 14 33.2
Dandelion 381 ± 16 193 ± 16 50.7
Greater burdock 365 ± 31 323 ± 20 88.5
Fenugreek 327 ± 28 320 ± 12 97.9
Nettle 162 ± 11 141 ± 10 87.0
232											 2010M. Kratchanova and others
Since polyphenols significantly contribute to the overall
antioxidant activity, it was reasonable to determine
their total amount in the selected medicinal plants.
The total polyphenol content in the medicinal plants
is shown in Table 3. It is evident that in all cases the
ORAC values and total polyphenol content
obtained with acetone extraction were higher
than the respective results for water extraction.
The observed differences could be explained
by the different polarity of the polyphenol
compounds present in the investigated
medicinal herbs. This observation complies
with the findings of Wojcikowski et al. (2007)
who used sequential three-solvent extraction
for herb polyphenols. The antioxidant activity
of the samples varied significantly for both
acetone and water extracts in our study. The
greatest ORACac value was found in peppermint,
while the highest ORACw value was
found in thyme, followed by peppermint.
Since the ORAC method is preferred for the
measurement of the antioxidant activity of
foods and biological samples, it is surprising
that in the literature there are ORAC data just
for several of the medicinal plants investigated
in the current study. Ninfali et al. (2005)
performed a comprehensive evaluation of
different foods and spices using the ORAC
method. On the basis of fresh weight, they
reported ORAC values for thyme (274.26
µmol TE/g), sage (320.04 µmol TE/g) and
common balm (59.97 µmol TE/g). As the reported
data are based on fresh weight, it is
difficult to compare them with our results.
Zheng and Wang (2001) determined ORAC
of five herbs from the current study, but
again the results were expressed on the basis
of fresh weight. Moreover, they used Rphycoerythrin
as a fluorescent agent, which
could significantly alter the ORAC results in
their study (Ou et al., 2001). In another recent
study, Wojcikowski et al. (2007) investigated
the ORAC antioxidant activity of 55 medicinal
plants after sequential three-solvent extraction.
Since this presumes very exhaustive
extraction, it can explain the higher ORAC
values obtained by them for several herbs
from our study: liquorice — 1029 µmol TE/g
(670 µmol TE/g in our study), basil — 524.7
µmol TE/g compared with 402 µmol TE/g, and nettle
— 430.4 µmol TE/g against 162 µmol TE/g. Despite
the more exhaustive sequential extraction, three herbs
in our work showed ORAC values several times higher
Table 3. Polyphenol content of 25 medicinal plants.
Comparison between 80 % acetone (ac) and water (w) extraction. Results are
presented as mean ± S.D.
Medicinal plant
Polyphenolsac
mg/100g
Polyphenolsw
mg/100g
Ratio PFw/ PFac
%
Peppermint 20216 ± 359 9356 ± 204 46.3
Hawthorn 7104 ± 111 1903 ± 181 26.8
Thyme 11409 ± 171 8583 ± 241 75.2
Wild basil 9468 ± 128 4645 ± 201 49.1
Birch 5542 ± 201 1197 ± 124 21.6
Raspberry 7759 ± 216 4932 ± 164 63.6
St. John’s wort 11283 ± 74 6428 ± 152 57.0
Common balm 11885 ±109 8240 ± 207 69.3
Lime 9296 ± 427 787 ± 43 8.5
Sage 5295 ± 148 3845 ± 65 72.6
Yarrow 5728 ± 232 1968 ± 84 34.4
Laurel leaves 7081 ± 299 1766 ± 52 24.9
Caltrop 5681 ± 200 2790 ± 101 49.1
Camomile 4665 ± 137 1790 ± 45 38.4
Mountain tea 3984 ± 201 2044 ± 21 51.3
Hop 5728 ± 262 1697 ± 25 29.6
Spearmint 4522 ± 102 3713 ± 46 82.1
Liquorice 3452 ± 98 1548 ± 72 44.8
Marigold 2141 ± 115 1537 ± 33 71.8
Basil 2391 ± 38 1816 ± 52 76.0
Chicory 1821 ± 63 786 ± 46 43.2
Dandelion 2206 ± 58 1577 ± 51 71.5
Greater burdock 2742 ± 112 2531 ± 68 92.3
Fenugreek 1692 ± 105 1445 ± 41 85.4
Nettle 958 ± 43 776 ± 43 81.0
Figure 1. Correlation between total phenolic content and ORAC antioxidant activity in acetone (A) and water (B) extracts obtained
from 25 medicinal plants.
Vol. 57 						 233Antioxidant activity of medicinal plants
than the ones in the Wojcikowski’s study. For example,
our results for yarrow — 842 µmol TE/g, sage — 966
µmol TE/g and dandelion — 381 µmol TE/g were 3.2fold,
2.7-fold and 3.9-fold higher, respectively, than the
ORAC values for the same herbs in the above-mentioned
paper. The differences in the antioxidant activity
between the same materials can be attributed to some
environmental factors such as climate, location and temperature
which can significantly affect the accumulation
of the antioxidant components in plant material. From
the investigated 55 herbs in the same study, the root
of black cohosh (Cimisifuga racemosa) showed the highest
ORAC value of 1264.9 µmol TE/g. In our study, four
medicinal plants (peppermint, hawthorn, thyme and wild
basil) revealed higher antioxidant activity, and another
four (birch, raspberry, St. John’s wort and common
balm) showed comparable ORAC values to that result.
Our study reports the ORAC values of antioxidant capacity
of several plants for the first time — those for
wild basil (Clinopodium vulgare) leaves, birch (Betula pendula)
leaves, caltrop (Tribulus terrestris) aerial parts, mountain
tea (Sideritis scardica) aerial parts, hop (Humulus lupulus)
flowers, marigold (Calendula officinalis) flowers and greater
burdock (Arctium lappa) roots.
Several studies have investigated the relationship between
the antioxidant activity and the content of polyphenol
compounds in herbs. Some authors have reported
good linear correlation between these two parameters
(Zheng & Wang 2001; Shan et al., 2005; Djeridane et al.,
2006; Katalinic et al., 2006), whereas others have not observed
such correlation (Kahkonen et al., 1999). Figure
1 depicts the correlation between the total polyphenols
and the ORAC values of the medicinal plants investigated
in our study. The correlation coefficient between
ORAC and total polyphenol content was R = 0.875 for
acetone extracts and R = 0.950 for water extracts. These
correlations suggest that the ORAC antioxidant activity
could be attributed to the polyphenol compounds.
However, there are several discrepancies in the correlation.
Such an example is hawthorn whose high ORAC
value does not match its low polyphenol content. Several
explanations could be used to account for that. First,
it has been reported that polyphenol compounds differ
significantly in their antioxidant properties which are determined
by several structural features of the polyphenol
molecule (Ou et al., 2002). Second, the investigated
medicinal plants probably contain other substances with
antioxidant effect apart from the polyphenols. Moreover,
the amount of polyphenols does not represent the potential
synergism or antagonism between the individual
compounds in the samples, which depends on their
structure and mutual interactions.
A recent study by Prior et al. (2007) demonstrated that
the consumption of certain foods was associated with
increased plasma ORAC in the postprandial state, while
the consumption of an energy source of macronutrients
containing no antioxidants was associated with a decline
in the plasma antioxidant capacity. The authors estimated
that according to the energy intake of the diet, 5 000–
15 000 μmol TE are necessary to supply daily human
antioxidant needs. The ORAC values reported in the
current study are several times higher than the ORAC
values of many fruits and vegetables (Ou et al., 2002; Wu
et al., 2004; Ciz et al., 2010). This means that the studied
medicinal herbs exhibited a higher antioxidant activity
and contained more polyphenols than the common vegetables
and fruit. In the search for natural antioxidants,
herbs turned out to be a suitable source of dietary antioxidants.
The differences between the ORAC values and
polyphenol content obtained after acetone and water extractions
indicate that the traditional way of ingestion of
herbs does not fully utilize the available antioxidants in
the plant material. These antioxidant compounds could
be isolated and then used as antioxidant functional foods
(Grajek et al., 2005).
CONCLUSION
It can be concluded that the extracting solvent affects
significantly the polyphenol compound content and the
antioxidant activity measured and therefore it is recommended
to use more than one extraction system for
better assessment of the antioxidant activity of natural
products. Several of the Bulgarian medicinal plants tested
are rich sources of polyphenol compounds and free
radical scavengers. Some medicinal plants thus can be
considered as promising sources of natural antioxidants
for medicinal and commercial uses.
Acknowledgements
This study was supported by grants IF-02-66/2007,
OC08058 MEYS CR and COST B35 Action.
REFERENCES
Ciz M, Pavelkova M, Gallova L, Kralova J, Kubala L, Lojek A (2008)
The influence of wine polyphenols on reactive oxygen and nitrogen
species production by murine macrophages RAW 264.7. Physiol Res
57: 393–402.
Ciz M, Cizova H, Denev P, Kratchanova M, Slavov A, Lojek A (2010)
Different methods for control and comparison of the antioxidant
properties of vegetables. Food Control 21: 518–523.
Cragg GM, Newman DJ, Weiss RB (1997) Coral reefs, forests, and
thermal vents: the worldwide exploration of nature for novel antitumor
agents. Semin Oncol 24: 156–163.
Davies KJ (2000) Oxidative stress, antioxidant defenses and damage
removal, repair and replacement systems. IUBMB Life 50: 279–289.
Dimkov P (1979) Bulgarska Narodna Medicina, p 677. BAN, Sofia.
Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, Vidal
N (2006) Antioxidant activity of some Algerian medicinal plants extracts
containing phenolic compounds. Food Chem 97: 654–660.
Fenkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology
of aging. Nature 408: 240–247.
Gebicka L, Banasiak E (2009) Flavonoids as reductants of ferryl hemoglobin.
Acta Biochim Pol 56: 509–513.
Gorinstein S, Yamamoto K, Katrich E, Leontowicz H, Lojek A, Leontowicz
M, Ciz M, Goshev I, Shalev U, Trakhtenberg S (2003)
Antioxidative properties of Jaffa sweeties and grapefruit and their
influence on lipid metabolism and plasma antioxidative potential in
rats. Biosci Biotechnol Biochem 67: 907–910.
Grajek W, Olejnik A, Sip A (2005) Probiotics, prebiotics and antioxidants
as functional foods. Acta Biochim Pol 52: 665–671.
Huang D, Ou B, Prior RL (2005) The chemistry behind antioxidant
capacity assays. J Agric Food Chem 53: 1841–1856.
Ivanova D, Gerova D, Chervenkov T, Yankova T (2005) Polyphenols
and antioxidant capacity of Bulgarian medicinal plants. J Ethnopharm
69: 145–150.
Johnson IT (2001) Antioxidants and antitumour properties. In Antioxidants
in food, pp 100–123. Woodhead Publishing Ltd, Cambridge.
Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala
TS (1999) Antioxidant activity of plant extracts containing phenolic
compounds. J Agric Food Chem 47: 3954–3962.
Katalinic V, Milos M, Kulisic T, Jukic M (2006) Screening of 70 medicinal
plants for antioxidant capacity and total phenols. Food Chem
94: 550–557.
Kiselova Y, Ivanova D, Chervenkov T, Gerova D, Galunska B, Yankova
T (2006) Correlation between the in vitro antioxidant activity and
polyphenol content of aqueous extracts from Bulgarian herbs. Phytotherapy
Res 20: 961–965.
Lopez D, Pavelkova M, Gallova L, Simonetti P, Gardana C, Lojek A,
Loaiza R, Mltjavila MT (2007) Dealcoholized red and white wines
decrease oxidative stress associated with inflammation in rats. Br J
Nutr 98: 611–619.
234											 2010M. Kratchanova and others
Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M (2005) Antioxidant
capacity of vegetables, spices and dressings relevant to nutrition.
Br J Nutr 93: 257–266.
Ou B, Hampsch-Woodill M, Prior RL (2001) Development and validation
of an improved oxygen radical absorbance capacity assay
using fluorescein as the fluorescent probe. J Agric Food Chem 49:
4619–4626.
Ou B, Huang D, Hampsch-Woodill M, Flanagan JA, Deemer EK
(2002) Analysis of antioxidant activities of common vegetables
employing oxygen radical absorbance capacity (ORAC) and ferric
reducing antioxidant power (FRAP) assays: a comparative study. J
Agric Food Chem 50: 3122–3128.
Petkov V (1982) Modern phytotherapy, p 516. Meditzina, Sofia.
Prior RL, Wu X, Schaich K (2005) Standardized methods for the determination
of antioxidant capacity and phenolics in foods and dietary
supplements. J Agric Food Chem 53: 4290–4302.
Prior RL, Hoang H, Gu L, Wu X, Jacob R, Sotoudeh G, Kader A,
Cook R (2007) Plasma antioxidant capacity changes following a
meal as a measure of the ability of a food to alter in vivo antioxidant
status. J Am Coll Nutr 26: 170–181.
Rice-Evans C, Miller N, Bolwell P, Bramley P, Pridham J (1995) The
relative antioxidant activities of plant derived polyphenolic flavonoids.
Free Radic Res 22: 375–383.
Rice-Evans C, Miller N, Paganga G (1997) Antioxidant properties of
phenolic compounds. Trends Plant Sci 2: 152–159.
Shan B, Yizhong Z, Sun M, Corke H (2005) Antioxidant capacity of
26 spice extracts and characterization of their phenolic constituents.
J Agric Food Chem 53: 7749–7759.
Singleton V, Rossi J (1965) Colorimetry of total phenolics with phosphomolibdic-phosphotungstic
acid reagents. Am J Enol Vitic 16:
144–158.
Su L, Yin JJ, Charles D, Zhou K, Moore J, Yu L (2007) Total phenolic
contents, chelating capacities, and radical-scavenging properties
of black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf.
Food Chem 100: 990–997.
Wang CC, Chu CY, Chu KO, Choy KW, Khaw KS, Rogers MS,
Pang CP (2004) Trolox-equivalent antioxidant capacity assay versus
oxygen radical absorbance capacity assay in plasma. Clin Chem 50:
952–954.
Willcox JK, Ash SL, Catignani GL (2004) Antioxidants and prevention
of chronic disease. Crit Rev Food Sci Nutr 44: 275–295.
Wojcikowski K, Stevenson L, Leach D, Wohlmuth H, Gobe G (2007)
Antioxidant capacity of 55 medicinal herbs traditionally used to
treat the urinary system: a comparison using a sequential three-solvent
extraction process. J Alt Compl Med 13: 103–110.
Wojdylo A, Oszmiansky J, Czemerys R (2007) Antioxidant activity and
phenolic compounds in 32 selected herbs. Food Chem 105: 940–949.
Wu X, Gu L, Holden J, Haytowitz D, Gebhardt S, Beecher G, Prior R
(2004) Development of a database for total antioxidant capacity in
foods: a preliminary study. J Food Comp Anal 17: 407–422.
Zheng W, Wang Y (2001) Antioxidant activity and phenolic compounds
in selected herbs. Food Chem 49: 5165–5170.
Zhou K, Yu L (2004) Effects of extraction solvent on wheat antioxidant
activity estimation. Lebens Wissen Techn 37: 717–721.
Different methods for control and comparison of the antioxidant properties
of vegetables
Milan Cˇízˇ a,*, Hana Cˇízˇová a
, Petko Denev b
, Maria Kratchanova b
, Anton Slavov b
, Antonín Lojek a
a
Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic
b
Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 95 Vasil Aprilov Blvd,
4002 Plovdiv, Bulgaria
a r t i c l e i n f o
Article history:
Received 12 March 2009
Received in revised form 22 July 2009
Accepted 31 July 2009
Keywords:
Antioxidant activity
Polyphenols
Vegetables
a b s t r a c t
The present study investigates the antioxidant properties of selected vegetables, using the total peroxyl
radical-trapping parameter (TRAP), oxygen radical absorbance capacity (ORAC) and hydroxyl radical
averting capacity (HORAC) methods. ORAC, TRAP and HORAC values well correlated with polyphenol content.
A good correlation was found also between the methods for measuring antioxidant capacity. Nevertheless,
ORAC has been found to be the most sensitive method to measure chain-breaking antioxidant
activity. Although we have found a good correlation between TRAP, ORAC and HORAC, using more than
one antioxidant assay is recommended for more detailed understanding the principles of antioxidant
properties of samples.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Reactive oxygen species (ROS) contribute to cellular aging, possibly
through the destabilisation of membranes in cells. During
evolution, the organisms have developed an antioxidant defence
system to cope with oxidative stress. Antioxidants can be divided
into two groups – preventive antioxidants and chain-breaking
antioxidants. The first group comprises metal chelators such as
metallothionein, neuromelanin, transferin and other proteins involved
in transition metal transport and storage and antioxidant
enzymes such as catalase, superoxide dismutase, glutathione
reductase etc. Free-radical scavengers pertain to the second group.
They scavenge free radicals and stop the propagation of free radical
chain reactions. The most significant chain-breaking antioxidants
are vitamins C and E, carotenoids and polyphenols (Davies, 2000;
Finkel & Holbrook, 2000; Prior, 2003). Polyphenols are generally divided
into hydrolyzable tannins and phenylpropanoids, such as lignins,
flavonoids, and condensed tannins. The largest and best
studied group of polyphenols are flavonoids. Flavonoids are a class
of secondary plant metabolites that are thought to exert beneficial
health effects through their antioxidant and chelating properties
being the major contributor to the antioxidant capacity of vegetables
(Heim, Tagliaferro, & Bobilya, 2002; Williams, Spencer, & RiceEvans,
2004). In addition to antioxidant function, flavonoids may
also modulate cell signalling pathways and could have marked effects
on cell function by altering protein and lipid phosphorylation
and modulating gene expression (Lotito & Frei, 2006).
It is of a great interest to consumers and nutritionists to quantify
the antioxidant properties of various foods because antioxidants
are clearly important to human life. Moreover, the
attention should be also paid to the processing methods in order
to preserve the desirable antioxidant properties of foods (Gorinstein
et al., 2009). Antioxidant function can be defined as the ability
of a compound to reduce pro-oxidant agents (Prior & Cao,
1999). There are many different methods for determining antioxidant
function which rely on different generators of free radicals,
acting by different mechanisms (Huang, Ou, & Prior, 2005; Prior,
Wu, & Schaich, 2005). In the literature, antioxidant properties are
denoted as antioxidant capacity (Cao, Alessio, & Cutler, 1993; Pellegrini,
Re, Yang, & Rice-Evans, 1999), antioxidant power (Benzie &
Strain, 1999) and antioxidant potential (Ghiselli, Serafini, Maiani,
Azzini, & FerroLuzzi, 1995). From the current point of view, the
mix of methods should be used for assessing antioxidant activities
in vitro to cover all the aspects of antioxidant efficacy (Aruoma,
2003; Schlesier, Harwat, Böhm, & Bitsch, 2002).
The present study employs three different methods for assessing
antioxidant properties – oxygen radical absorbance capacity
(ORAC), total radical-trapping antioxidative parameter (TRAP)
and hydroxyl (HO) radical averting capacity (HORAC). The first
two methods assess the peroxyl radical chain-breaking ability of
antioxidants by hydrogen atom transfer pathway. The third method
– HORAC measures their metal-chelating radical prevention
activity (Prior et al., 2005). Despite the methods employed in this
study there are many other approaches to evaluate antioxidant
0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2009.07.017
* Corresponding author. Tel.: +420 541 517 104; fax: +420 541 211 293.
E-mail address: milanciz@ibp.cz (M. Cˇízˇ).
Food Control 21 (2010) 518–523
Contents lists available at ScienceDirect
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journal homepage: www.elsevier.com/locate/foodcont
properties of fruits and vegetables such as 2,20
-azinobis-(3-ethylbenzothiazoline-6-sulfonate)
(ABTS) radical assay, diphenylpicrylhydrazyl
(DPPH) radical assay, b-carotene bleaching
assay, ferric reducing antioxidant power (FRAP) assay, cupric ion
reducing antioxidant capacity (CUPRAC) assay etc. (Ao, Li, Elzaawely,
Xuan, & Tawata, 2008; Baydar, Ozkan, & Yasar, 2007; Deba,
Xuan, Yasuda, & Tawata, 2008; Gorinstein et al., 2009).
The aim of the study was to compare the antioxidant function of
selected vegetables of Bulgarian origin and commonly consumed
by the Bulgarian population as an important constituent of their
traditional food. Three different antioxidant assays were chosen
for the measurement of antioxidant function in vegetable samples
and compared. The total polyphenol content of selected vegetables
was measured as well, so as to evaluate its contribution to their total
antioxidant function. The objective of the study was to supply
both new methodological background for the food quality control
in food industry and new information on the antioxidant function
of selected vegetables for nutritionists and general public.
2. Materials and methods
2.1. Chemicals and instruments
2,20
-azobis (2-amidino-propane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox),
fluorescein disodium salt, cobalt (II) fluoride tetrahydrate and gallic
acid were obtained from Sigma–Aldrich (Steinheim, Germany).
Folin–Ciocalteu’s phenol reagent was purchased from Merck
(Darmstadt, Germany). Picollinic acid was purchased from Fluka
(Deisenhofen, Germany). All other solvents used were of analytical
grade and obtained from local producers.
ORAC and HORAC analyses were carried out on a FLUOstar Galaxy
plate reader (BMG Labtechnology, Offenburg, Germany), excitation
wavelength = 485 nm and emission wavelength = 520 nm.
TRAP assay was conducted on Luminometer Orion II (Berthold Dection
System GmbH, Pforzheim, Germany).
2.2. Food sample preparation
Vegetables were purchased from the local supermarkets at the
time of peak production, after that immediately frozen and kept
at À18 °C. Altogether 22 vegetables of Bulgarian origin were analyzed:
celery leaves (Apium graveolens var. dulce), parsley leaves
(Petroselinum crispum), chilli pepper (Capsicum frutescens), capsicum
(Capsicum annuum ‘Gorogled’), lovage (Levisticum officinale),
goathorn pepper (C. annuum), dill (Anethum graveolens), red pepper
(C. annuum), broccoli (Brassica oleracea var. italica), eggplant (Solanum
melongena), green bean (Phaseolus vulgaris), green onion (Allium
fistulosum), radish (Raphanus sativus), gumbo (Abelmoschus
esculentus), red beet (Beta vulgaris), green pepper (C. annuum), potato
(Solanum tuberosum), celery root (A. graveolens var. rapaceum),
tomato (Lycopersicon esculentum), carrot (Daucus carota), cucumber
(Cucumis sativus), and vegetable marrow (Cucurbita pepo).
The edible parts of the vegetables (2 g) were unfrozen and
homogenized in a laboratory blender before the analyses. Samples
were transferred into extraction tubes and mixed with 20 ml of the
extragent (80% acetone in 0.2% formic acid). Extraction was conducted
on an orbital shaker at room temperature for 1 h. After that
the sample was centrifuged at 6240 g and supernatant was removed.
The solid residue was subjected to a second extraction under
the same conditions. Both supernatants were combined and
further used for total polyphenol and antioxidant function determinations
after a dilution with a phosphate buffer.
2.3. TRAP assay
The luminol-enhanced chemiluminescence (CL) was used to follow
up the peroxyl radical reaction and the principle was described
previously in Cˇízˇová, Lojek, Kubala, and Cˇízˇ. (2004) and Uotila,
Kirkkola, Rorarius, Tuimala, and Metsä-Ketelä. (1994). The CL signal
is driven by the production of luminol derived radicals from
thermal decomposition of AAPH. The TRAP value is determined
from the duration of the time period (Tsample) during which the
sample quenched the CL signal due to the present antioxidants. A
known quantity (8.0 nM) of trolox, a water-soluble analogue of
tocopherol was used as a reference inhibitor (Ttrolox) instead of
the sample. The calculation of the TRAP value is represented by
the equation:
TRAP ¼ 2:0½TroloxŠTsample=f Á TTrolox;
where 2.0 is the stoichiometric factor of trolox (the number of peroxyl
radicals trapped per one molecule of trolox) and f is the dilution
of the sample.
2.4. ORAC assay
The ORAC assay measures the antioxidant scavenging function
against peroxyl radical induced by AAPH at 37 °C. Fluorescein is
used as a fluorescent probe. The loss of fluorescence of fluorescein
is an indication of the extent of damage from its reaction with the
peroxyl radical (Gomes, Fernandes, & Lima, 2005; Huang, Ou,
Hampsch-Woodill, Flanagan, & Deemer, 2002; Ou, HampschWoodill,
& Prior, 2001). Working solution of fluorescein (70 nM)
was prepared by dissolving fluorescein disodium salt in phosphate
buffer (75 mM, pH = 7.4).
The total reaction mixture volume was 200 ll and all solutions
were prepared in a phosphate buffer (75 mM, pH = 7.4). One-hundred
and seventy micro-litres of fluorescein solution (60 nM final
concentration) and 10 ll of the sample were placed in the well of
the microplate and incubated at 37 °C directly in the FLUOstar
plate reader for 10 min. After the incubation 20 ll of AAPH
(51.5 mM final concentration) was added rapidly using a multichannel
pipette to start the reaction. The fluorescence was recorded
every minute and the microplate was automatically
shaken prior to each reading. A blank using phosphate buffer instead
of the antioxidant and calibration solutions of Trolox (12.5,
20, 50, and 100 lM) as antioxidant were also carried out in each
assay. The final ORAC values were calculated using a regression
equation between the Trolox concentration and the net area under
the curve (AUC). The net AUC corresponding to the sample was calculated
by subtracting the AUC corresponding to the blank. ORAC
values were expressed as lmol Trolox equivalents per gram of
fresh weight (FW) of the samples.
2.5. HORAC assay
The HORAC assay developed by Ou et al. (2002) measures the
metal-chelating activity of antioxidants in the conditions of Fenton-like
reactions employing a Co(II) complex and hence the protecting
ability against formation of hydroxyl radical.
Hydrogen peroxide solution of 0.55 M was prepared in distilled
water. 4.6 mM Co(II) was prepared as follows: 15.7 mg of
CoF2Á4H2O and 20 mg of picolinic acid were dissolved in 20 ml of
distilled water.
Fluorescein – 170 ll (60 nM, final concentration) and 10 ll of
sample were incubated in 37 °C for 10 min. directly in the FLUOstar
plate reader. After incubation 10 ll H2O2 (27.5 mM, final concentration)
and 10 ll of Co(II) (230 lM final concentration) solutions
were added subsequently. The initial fluorescence was measured
M. Cˇízˇ et al. / Food Control 21 (2010) 518–523 519
after which the readings were taken every minute after shaking.
For the blank sample, phosphate buffer solution was used. 100,
200, 600, 800 and 1000 lM gallic acid solutions (in phosphate buffer
75 mM, pH = 7.4) were used for building the standard curve.
The AUC were calculated as they were for the ORAC assay. The final
HORAC values were calculated using a regression equation between
the gallic acid concentration and the net area under the
curve. One HORAC unit is assigned to the net protection area provided
by 1 lM gallic acid and the activity of the sample is expressed
as lmol gallic acid equivalents (GAE) per gram of fresh
weight of the samples.
2.6. Total polyphenol content analysis
The total polyphenol content was determined by the Folin–Ciocalteu
method. Gallic acid was employed as calibration standard
and results were expressed as gallic acid equivalents. Food extracts
(1 ml) or gallic acid standard solutions were mixed with 10 ml
deionized water and 1.0 ml of Folin–Ciocalteu phenol reagents.
After 5 min, 2.0 ml of 20% sodium carbonate was added to the mixture.
After 1 h in darkness the absorbance at 750 nm was measured.
The concentration of total polyphenols was expressed as
GAE per 100 g of fresh weight (Stoilova, Krastanov, Stoyanova, Denev,
& Gargova, 2007).
3. Statistical analysis
All experiments were repeated six times. The results are expressed
as the mean ± standard deviation (SD). One-way analysis
of variance (ANOVA) and Student’s t-test were used to evaluate
the differences of the mean between groups, and Newman–Keuls
test was used to analyse the contrasts. Pearson correlation coefficient
(r) was used to express correlations and linear regressions
were calculated. A p value of <0.05 was taken to be significant.
4. Results
The antioxidant capacity expressed as the TRAP, ORAC and
HORAC values of selected vegetables as well as the amount of total
polyphenols of these vegetables in rank order are shown in Table 1.
Using the ORAC assay we obtained a hierarchy of antioxidant
capacity ranging from 113.0 to 1.2 lmol TE/g of fresh weight.
Interestingly, the highest antioxidant function was found in the
samples of green parts of vegetables belonging to the Apiaceae family
with the exception of dill. On the other hand, the antioxidant
function of the root vegetables of the Apiaceae family was significantly
lower. The most widely used vegetables in Bulgarian cuisine
(peppers, tomato, and cucumber) belonged to the weakest ones
from the point of view of their antioxidant function.
The TRAP values ranged from 68.1 to 0.0 lmol TE/g of fresh
weight with hierarchy similar to ORAC on the first five positions
and the last three positions. The range of HORAC values was from
82.9 to 0.0 GAE/g fresh weight.
The total polyphenol content in the vegetable extracts analyzed
was in the range of 605.6–20.0 mg GAE/100 g fresh weight. The
highest content of total polyphenols was in accordance with the results
of antioxidant function determination found in celery leaves
and parsley leaves, followed by various types of peppers (Capsicum
sp., excepting green pepper), lovage and dill. The lowest total polyphenol
content was recorded in tomato, carrot, cucumber and
vegetable marrow corresponding to their low antioxidant function.
There was a direct relationship between the total polyphenol
content and antioxidant function in the extracts of various vegetables.
ORAC, TRAP and HORAC values were significantly linearly correlated
to the total polyphenol content: r = 0.95, r = 0.97 and
r = 0.94, respectively (see Fig. 1).
In addition, there was a good correlation between the methods
for measuring peroxyl radical-trapping antioxidant function of
vegetables – TRAP vs. ORAC, r = 0.96. A good correlation was also
found between methods for measuring the peroxyl radical-trapping
capacity and hydroxyl radical averting capacity – TRAP vs.
HORAC, r = 0.94 and ORAC vs. HORAC, r = 0.94 (see Fig. 2).
5. Discussion
Polyphenols constitute one of the most numerous and ubiquitous
groups of plant metabolites, and are an integral part of the human
diet. Vegetables are known to possess a variety of antioxidant
effects and properties. Flavonols (such as quercetin, myricetin,
Table 1
The rank order of antioxidant activity expressed as ORAC (lmol TE/g FW), TRAP (lmol TE/g FW) and HORAC (lmol GAE/g FW) of vegetable extracts and the rank order of total
polyphenol content (mg GAE/100 g FW) in vegetable extracts. There are no significant differences among values marked with the same superscript letters in individual columns.
Vegetables ORAC TRAP HORAC Total polyphenols
Value Rank Value Rank Value Rank Value Rank
Celery leaves 113.5a
± 6.1 1 68.1a
± 4.8 1 55.0b
± 2.9 2 605.6a
± 9.4 1
Parsley leaves 108.6b
± 13.1 2 67.2a
± 8.9 2 82.9a
± 1.2 1 599.7b
± 0.4 2
Lovage 57.3c
± 5.0 3 28.4b
± 1.3 3 19.2d
± 2.1 4 267.0e
± 2.1 5
Chilli pepper 36.1d
± 6.5 4 23.7cd
± 3.1 5 17.7de
± 0.4 5 298.6c
± 1.8 3
Goathorn pepper 30.6e
± 1.4 5 24.8c
± 1.2 4 13.7f
± 1.8 7 260.5f
± 5.0 6
Radish 23.6f
± 1.7 6 11.4h
± 2.2 11 15.9e
± 2.0 6 89.9j
± 1.7 13
Capsicum 19.9fg
± 1.4 7 20.9de
± 1.2 6 21.3c
± 5.0 3 286.7d
± 5.1 4
Eggplant 16.2gh
± 2.0 8 13.4gh
± 1.5 10 5.5h
± 0.7 15 102.9i
± 4.7 9
Broccoli 16.1gh
± 1.2 9 3.4ijk
± 0.1 17 7.2h
± 1.6 10 102.1i
± 5.9 10
Celery root 15.3ghi
± 1.2 10 1.0k
± 0.4 19 7.5h
± 0.6 9 43.8m
± 2.3 18
Green onion 14.7ghi
± 1.5 11 3.5ijk
± 0.5 16 5.7h
± 0.6 13 92.7j
± 0.9 12
Gumbo 14.6ghi
± 0.8 12 7.4i
± 0.8 12 5.7h
± 0.5 14 89.8j
± 4.0 14
Green bean 14.5ghi
± 1.2 13 16.9fg
± 0.5 8 5.7h
± 0.5 12 101.5i
± 3.3 11
Red beet 12.6hi
± 1.6 14 17.9ef
± 0.3 7 6.1h
± 1.0 11 81.5k
± 1.6 15
Dill 10.5hij
± 1.1 15 13.7h
± 1.5 9 11.3g
± 0.8 8 150.4g
± 0.9 7
Potato 10.3hij
± 1.3 16 3.8ijk
± 0.1 15 2.9i
± 0.2 17 65.5l
± 1.1 17
Red pepper 9.3ij
± 0.9 17 5.9ij
± 0.1 13 5.3h
± 0.3 16 115.7h
± 1.5 8
Green pepper 5.6jk
± 0.3 18 2.8jk
± 1.9 18 0.9i
± 0.3 20 80.7k
± 3.6 16
Tomato 5.4jk
± 0.3 19 4.4ijk
± 0.5 14 1.9i
± 0.3 18 41.3m
± 2.2 19
Carrot 4.8jk
± 1.1 20 n.d. 20 n.d. 21 35.2n
± 2.0 20
Vegetable marrow 2.9k
± 0.3 21 n.d. 21 1.8i
± 0.1 19 20.0p
± 1.7 22
Cucumber 1.2k
± 0.2 22 n.d. 22 n.d. 22 24.2o
± 2.4 21
520 M. Cˇízˇ et al. / Food Control 21 (2010) 518–523
kaempherol) and flavones (e.g. apigenin, luteolin) in plant materials
are closely associated with their antioxidant function mainly
due to their redox properties exerted by various possible mechanisms:
free-radical scavenging activity, transition-metal-chelating
activity, and/or singlet-oxygen-quenching capacity (Lotito & Frei,
2006; Shan, Cai, Sun, & Corke, 2005). There are many papers
attempting to rank the antioxidant properties of different plant
materials using different methods (Pellegrini et al., 2003; Proteggente
et al., 2002) including ORAC (Ou, Hampsch-Woodill, et al.,
2002; Velioglu, Mazza, Gao, & Oomah, 1998; Wang, Cao, & Prior,
1996; Wu et al., 2004). The antioxidant capacity assays measure
the combined effect of many antioxidants present in the sample,
which are able to scavenge free radicals generated in the assays.
Interactions between antioxidants are also reflected in the assay
value. Both ORAC and TRAP are indicators of the free-radical scavenging
ability of antioxidants against peroxyl radical, using the
same mechanism of hydrogen atom transfer. On the other hand,
HORAC measures the metal-chelating properties of antioxidants
thereby expressing a radical prevention ability of the sample.
According to Ou, Hampsch-Woodill, et al. (2002) and Ou, Huang,
Hampsch-Woodill, Flanagan, and Deemer (2002) pure flavonoids
can differ significantly in their ORAC and HORAC values. Nevertheless
in contrast to Ou, Hampsch-Woodill, et al. (2002) and Ou,
Huang, et al. (2002), who did not find any general agreement
among the antioxidant methods used, our data correlated well
(see Fig. 2). The data obtained using the ORAC and TRAP assays
(Fig. 2A) had the best correlation coefficient (r = 0.96). Also the
TRAP and HORAC values (r = 0.94, Fig. 2B) and ORAC and HORAC
values (r = 0.94, Fig. 2C) correlated strongly. A slight variation
among the results obtained by the ORAC, HORAC and TRAP assays
might relate to the different conditions of measurement and the
sensitivity of the assays. Therefore several methods should be used
in parallel to elucidate the complex field of antioxidants and oxidation
(Ou, Huang, et al., 2002).
In such complex samples as vegetable extracts where many
individual polyphenols occur, some discrepancies between polyphenol
content and antioxidant properties could be expected. Nevertheless
in the work reported here, the data unequivocally show
that ORAC, TRAP and HORAC values (see Table 1) are strictly
dependent on polyphenol content (r = 0.95, r = 0.97 and r = 0.94,
respectively, see Fig. 1). These data are in accordance with that of
other authors, who have shown that a high total polyphenol content
increases antioxidant function and that there is a linear correlation
between polyphenol content and antioxidant function
(Gorinstein, Martin-Belloso, et al., 2003; Gorinstein, Yamamoto,
et al., 2003).
A
0
20
40
60
80
100
120
0 100 200 300 400 500 600 700
Polyphenols (mg GAE/100g FW)
ORAC(µmolTE/gFW)
B
0
20
40
60
80
100
120
0 100 200 300 400 500 600 700
Polyphenols (mg GAE/100g FW)
TRAP(µmolTE/gFW)
C
0
20
40
60
80
100
120
0 100 200 300 400 500 600 700
Polyphenols (mg GAE/100g FW)
HORAC(µmolGAE/gFW)
Fig. 1. Correlations among total polyphenol content expressed as mg GAE/100 g FW
of vegetable extracts and various measurements of antioxidant activity: (A) ORAC
expressed as lmol TE/g FW of vegetable extracts (y = 0.1754x À 3.6334; r = 0.950),
(B) TRAP expressed as lmol TE/g FW of vegetable extracts (y = 0.1113x À 2.6021;
r = 0.969) and (C) HORAC expressed as lmol GAE/g FW of vegetable extracts
(y = 0,1101x À 4.4667; r = 0.935).
A
0
20
40
60
80
100
120
0 20 40 60 80 100 120
ORAC (µmol TE/g FW)
TRAP(µmolTE/gFW)
B
0
20
40
60
80
100
120
0 20 40 60 80 100 120
TRAP (µmol TE/g FW)
HORAC(µmolGAE/gFW)
C
0
20
40
60
80
100
120
0 20 40 60 80 100 120
ORAC (µmol TE/g FW)
HORAC(µmolGAE/gFW)
Fig. 2. Correlations among various types of measurements of antioxidant activity of
vegetable extracts: (A) TRAP vs. ORAC (y = 0.5991x + 0.5859; r = 0.962), (B) HORAC
vs. TRAP (y = 0.9596x À 1.4438; r = 0.936), and (C) HORAC vs. ORAC
(y = 0.6012x À 1.5316; r = 0.942).
M. Cˇízˇ et al. / Food Control 21 (2010) 518–523 521
In our experiments, it was impossible to detect the antioxidant
capacity using TRAP method in samples where the quantity of
polyphenols was equal to or less than 35 mg per 100 g of fresh
weight. The ORAC assay is more sensitive and reflects antioxidant
properties even of such a low quantity of polyphenols. The advantage
of the ORAC assay is that it combines both the inhibition time
and inhibition degree of the radical generation, as it takes the oxidation
reaction to completion and uses the area under the curve to
quantify the antioxidant capacity (Prior et al., 2003).
The antioxidant properties depend on several structural features
of the molecule of polyphenols in its base structure and are primarily
attributed to the high reactivity of hydroxyl substituents. The
B-ring hydroxyl configuration is the most significant determinant
of scavenging of ROS. 30
,40
-di OH (catechol) structure in the
B-ring strongly enhances lipid peroxidation inhibition. A free 3-hydroxyl
group and 30
,40
-catechol (dihydroxy) structure, a C2@C3 double
bond, and a 4-oxo group on the C ring endow the flavonoid with
potent antioxidant function. The superiority of quercetin in inhibiting
both metal and nonmetal-induced oxidative damage is partially
ascribed to its free 3-OH substituent which is thought to increase
the stability of the flavonoid radical (Davies, 2000). Fenton-induced
oxidation is strongly inhibited by flavonoids with catechol, 4-oxo,
and 50
-OH arrangements (Cao, Sofic, & Prior, 1997; Cheng & Breen,
2000; Wang et al., 2006) which is superior to isoforms that lack
these features.
Because fruit and vegetables are the major antioxidant sources
in our daily diet, an estimation of daily antioxidant capacity intake
from these foods is beneficial (Davies, 2000). Although the poor
bioavailability of flavonoids is widely discussed (Prior et al.,
2005) the availability of databases of the flavonoid content is
important to identify the major contributors to the antioxidant
activities of the flavonoid-rich dietary components (Proteggente
et al., 2002). As we have shown here, aromatic vegetable such as
parsley, dill and lovage with high polyphenol content and high
ORAC, HORAC and TRAP values could be excellent sources of antioxidant
and should be a part of everyday diet.
Although we have found a good correlation among all the methods
used here for assessing antioxidant capacity, using more than
one antioxidant assay is strongly recommended – a single method
will provide basic information about antioxidant properties, but a
combination of methods describes the antioxidant properties of
the sample in more detail.
Although these individual assays can be used for screening the
antioxidant properties of the food sample with similar results, we
recommend using the ORAC assay combined with HORAC for evaluating
overall antioxidant capacity. The ORAC has been found to be
the most sensitive method to measure chain-breaking antioxidant
function (hydrogen atom transfer pathway) while the HORAC measures
metal-chelating antioxidant capacity (preventive antioxidant).
As a supplement, the Folin–Ciocalteu assay (single electron
transfer pathway) should be a part of antioxidant profile
measurement.
Acknowledgements
This study was supported by Grant Nos. AV0Z50040507 and
AV0Z50040702 of the Academy of Sciences of the Czech Republic,
Grant No. 1QS500040507 GA AS CR, and Grant No. TK-X-1607/06
NSFB of the Bulgarian Ministry of Education and Science.
References
Ao, C. W., Li, A. P., Elzaawely, A. A., Xuan, T. D., & Tawata, S. (2008). Evaluation of
antioxidant and antibacterial activities of Ficus microcarpa L. fil extract. Food
Control, 19(10), 940–948.
Aruoma, O. I. (2003). Methodological consideration for characterizing potential
antioxidant action of bioactive components in plant foods. Mutation
Research(523–524), 9–20.
Baydar, N. G., Ozkan, G., & Yasar, S. (2007). Evaluation of the antiradical and
antioxidant potential of grape extracts. Food Control, 18(9), 1131–1136.
Benzie, I. F. F., & Strain, J. J. (1999). Ferric reducing antioxidant power assay: Direct
measure of total antioxidant function of biological fluids and modified version
for simultaneous measurement of total antioxidant power and ascorbic acid
concentration. Methods in Enzymology, 299, 15–27.
Cao, G. H., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical absorbancy capacity
assay for antioxidants. Free Radical Biology and Medicine, 14(3), 303–311.
Cao, G. H., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of
flavonoids: Structure-activity relationships. Free Radical Biology and Medicine,
22(5), 749–760.
Cheng, I. F., & Breen, K. (2000). On the ability of four flavonoids, baicilein, luteolin,
naringenin, and quercetin to suppress the fenton reaction of the iron-ATP
complex. Biometals, 13(1), 77–83.
Cˇízˇová, H., Lojek, A., Kubala, L., & Cˇízˇ, M. (2004). The effect of intestinal ischemia
duration on changes in plasma antioxidant defense status in rats. Physiological
Research, 53(5), 523–531.
Davies, K. J. A. (2000). Oxidative stress, antioxidant defenses, and damage removal,
repair, and replacement systems. IUBM Life, 50(4–5), 279–289.
Deba, F., Xuan, T. D., Yasuda, M., & Tawata, S. (2008). Chemical composition and
antioxidant, antibacterial and antifungal activities of the essential oils from
Bidens pilosa Linn. var. Radiata. Food Control, 19(4), 346–352.
Finkel, T., & Holbrook, N. J. (2000). Oxidants, oxidative stress and the biology of
ageing. Nature, 408(6809), 239–247.
Ghiselli, A., Serafini, M., Maiani, G., Azzini, E., & FerroLuzzi, A. (1995). A
fluorescence-based method for measuring total plasma antioxidant capability.
Free Radical Biology and Medicine, 18(1), 29–36.
Gomes, A., Fernandes, E., & Lima, J. L. F. C. (2005). Fluorescence probes used for
detection of reactive oxygen species. Journal of Biochemical and Biophysical
Methods, 65(2–3), 45–80.
Gorinstein, S., Jastrzebski, Z., Leontowicz, H., Leontowicz, M., Namiesnik, J., Najman,
K., et al. (2009). Comparative control of the bioactivity of some frequently
consumed vegetables subjected to different processing conditions. Food Control,
20(4), 407–413.
Gorinstein, S., Martin-Belloso, O., Katrich, E., Lojek, A., Cˇízˇ, M., Gligelmo-Miguel, N.,
et al. (2003). of the contents of the main biochemical compounds and the
antioxidant activity of some Spanish olive oils as determined by four different
radical scavenging tests. Journal of Nutritional Biochemistry, 14(3), 154–159.
Gorinstein, S., Yamamoto, K., Katrich, E., Leontowicz, H., Lojek, A., Leontowicz, M.,
et al. (2003). Antioxidative properties of Jaffa sweeties and grapefruit and their
influence on lipid metabolism and plasma antioxidative potential in rats.
Bioscience Biotechnology and Biochemistry, 67(4), 907–910.
Heim, K. E., Tagliaferro, A. R., & Bobilya, D. J. (2002). Flavonoid antioxidants:
Chemistry, metabolism and structure-activity relationship. Journal of Nutritional
Biochemistry, 13(10), 572–584.
Huang, D. J., Ou, B. X., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002).
Development and validation of oxygen radical absorbance capacity assay for
lipophilic antioxidants using randomly methylated beta-cyclodextrin as the
solubility enhancer. Journal of Agricultural and Food Chemistry, 50(7),
1815–1821.
Huang, D. J., Ou, B. X., & Prior, R. L. (2005). The chemistry behind antioxidant
capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841–1856.
Lotito, S. B., & Frei, B. (2006). Consumption of flavonoid-rich foods and increased
plasma antioxidant capacity in humans: Cause, consequence, or
epiphenomenon? Free Radical Biology and Medicine, 41(12), 1727–1746.
Ou, B., Hampsch-Woodill, M., Flanagan, J., Deemer, E. K., Prior, R. L., & Huang, D. J.
(2002). Novel fluorometric assay for hydroxyl radical prevention capacity using
fluorescein as the probe. Journal of Agricultural and Food Chemistry, 50(10),
2772–2777.
Ou, B. X., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of
an improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescence probe. Journal of Agricultural and Food Chemistry, 49(10),
4619–4626.
Ou, B. X., Huang, D. J., Hampsch-Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002).
Analysis of antioxidant activities of common vegetables employing oxygen
radical absorbance capacity (ORAC) and ferric reducing antioxidant power
(FRAP) assays: A comparative study. Journal of Agricultural and Food Chemistry,
50(11), 3122–3128.
Pellegrini, N., Re, R., Yang, M., & Rice-Evans, C. (1999). Screening of dietary
carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying
the 2,20
-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid) radical cation
decolorization assay. Methods in Enzymology, 299, 379–389.
Pellegrini, N., Serafini, M., Colombi, B., Del Rio, D., Salvatore, S., Bianchi, M., et al.
(2003). Total antioxidant capacity of plant foods, beverages and oils consumed
in Italy assessed by tree different in vitro assays. Journal of Nutrition, 133(9),
2812–2819.
Prior, R. L. (2003). Fruits and vegetables in the prevention of cellular oxidative
damage. American Journal of Clinical Nutrition, 78(3), 570S–578S.
Prior, R. L., & Cao, G. (1999). Invivo total antioxidant capacity: Comparison of
different analytical methods. Free Radical Biology and Medicine, 27(11/12),
1173–1181.
Prior, R. L., Hoang, H., Gu, L. W., Wu, X. L., Bacchiocca, M., Howard, L., et al. (2003).
Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical
522 M. Cˇízˇ et al. / Food Control 21 (2010) 518–523
absorbance capacity (ORACFL)) of plasma and other biological and food samples.
Journal of Agricultural and Food Chemistry, 51(11), 3273–3279.
Prior, R. L., Wu, X. L., & Schaich, K. (2005). Standardized methods for the
determination of antioxidant capacity and phenolics in foods and dietary
supplements. Journal of Agricultural and Food Chemistry, 53(10), 4290–4302.
Proteggente, A. R., Pannala, A. S., Paganga, G., Van Buren, L., Wagner, E., Wiseman, S.,
et al. (2002). The antioxidant activity of regularly consumed fruit and
vegetables reflects their phenolic and vitamin C composition. Free Radical
Research, 36(2), 217–233.
Schlesier, K., Harwat, M., Böhm, V., & Bitsch, R. (2002). Assessment of antioxidant
activity by using different in vitro methods. Free Radical Research, 36(2),
177–187.
Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice
extracts and characterization of their phenolic constituents. Journal of
Agricultural and Food Chemistry, 53(20), 7749–7759.
Stoilova, I., Krastanov, A., Stoyanova, A., Denev, P., & Gargova, S. (2007). Antioxidant
activity of a ginger extract (Zingiber officinale). Food Chemistry, 102(3), 764–770.
Uotila, J. T., Kirkkola, A. L., Rorarius, M., Tuimala, R. J., & Metsä-Ketelä, T. (1994). The
total peroxyl radical-trapping ability of plasma and cerebrospinal-fluid in
normal and preeclamptic parturients. Free Radical Biology and Medicine, 16(5),
581–590.
Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and
total phenolics in selected fruits, vegetables, and grain products. Journal of
Agricultural and Food Chemistry, 46(10), 4113–4117.
Wang, H., Cao, G. H., & Prior, R. L. (1996). Total antioxidant capacity of fruits. Journal
of Agricultural and Food Chemistry, 44(3), 701–705.
Wang, L. S., Tu, Y. C., Lian, T. W., Hung, J. T., Yen, J. H., & Wu, M. J. (2006). Distinctive
antioxidant and antiinflammatory effects of flavonols. Journal of Agricultural and
Food Chemistry, 54(26), 9798–9804.
Williams, R. J., Spencer, J. P. E., & Rice-Evans, C. (2004). Flavonoids: Antioxidants or
signalling molecules? Free Radical Biology and Medicine, 36(7), 838–849.
Wu, X. L., Gu, L. W., Holden, J., Haytowitz, D. B., Gebhardt, S. E., Beecher, G., et al.
(2004). Development of a database for total antioxidant capacity in foods: A
preliminary study. Journal of Food Composition and Analysis, 17(3–4), 407–422.
M. Cˇízˇ et al. / Food Control 21 (2010) 518–523 523
Solid-phase extraction of berries’ anthocyanins and evaluation of their
antioxidative properties
Petko Denev a
, Milan Ciz b
, Gabriela Ambrozova b
, Antonin Lojek b
, Irina Yanakieva a
, Maria Kratchanova a,*
a
Institute of Organic Chemistry with Centre of Phytochemistry – BAS, Laboratory of Biologically Active Substances, 139 Ruski Blvd., Plovdiv 4000, Bulgaria
b
Institute of Biophysics of the AS CR, v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 12 January 2010
Received in revised form 26 March 2010
Accepted 11 May 2010
Keywords:
Anthocyanins
SPE
Berry extracts
ORAC
HORAC
TRAP
Lipid peroxidation
NO-scavenging
a b s t r a c t
Solid-phase extraction (SPE) was used to obtain anthocyanin-rich extracts from five berry species: chokeberry,
elderberry, black currant, blackberry and blueberry. During SPE more than 94.4% of the sugars and
more than 88.5% of the acids present in the crude extracts were separated. The SPE resulted in 90–95.6%
anthocyanins recovery. The antioxidative properties of the anthocyanin-rich extracts were tested by
measuring their oxygen radical absorption capacity (ORAC), hydroxyl radical averting capacity (HORAC),
total peroxyl radical trapping antioxidant parameter (TRAP), scavenging of nitric oxide and inhibition of
lipid peroxidation. Elderberry extract revealed the highest ORAC value of 5783 lmol TE/g. Chokeberry
extract was the most potent inhibitor of lipid peroxidation and had the highest TRAP value of 4051 lmol
TE/g. Blueberry extract had the highest HORAC result – 1293 lmol GAE/g and was the most powerful
scavenger of NO. The high antioxidant activity according to all antioxidant assays revealed opportunities
to apply these preparations as antioxidants.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Anthocyanins comprise of a large group of water-soluble pigments
which pertain to the flavonoid class. They give red, blue,
purple and black colours to many fruits, vegetables and flowers.
There is an increasing interest in anthocyanins not only as natural
food colourants (Bridle & Timberlake, 1997) but also as pharmaceutical
products because of various therapeutic effects. Anthocyanins
reveal potent P-vitamin activity, reduce capillary fragility and
increase their permeability (Wagner, 1985). They take part in collagen
reticulation and inhibit the enzyme degradation of collagen
during inflammation (Ronziere, Herbage, Garrone, & Frey, 1981).
Anthocyanins are applied in prevention and treatment of glaucoma
and other eyesight disorders (Ghosh & Konishi, 2007). In model
systems, anthocyanin-rich extracts reveal cardio-protective effect
(Bell & Gochenaur, 2006). Inhibition of cancer-cells growth by such
extracts has also been reported (Zhao, Giusti, Malik, Moyer, & Magnuson,
2004). These biological effects are in part due to their antioxidant
capacity (Kahkonen & Heinonen, 2003). Anthocyanins are
potent radical scavengers and are very effective in the inhibition
of low density lipoprotein oxidation, which is a key step for
atherosclerosis development (Satue-Gracia, Heinonen, & Frankel,
1997). Among all common fruits and vegetables in the diet, berries,
especially those with dark blue or red colours, have the highest
content of anthocyanins and antioxidant capacities (Wu et al.,
2004) which makes them a suitable raw material for anthocyanin
extraction especially in a large scale.
The use of anthocyanins for pharmaceutical purposes requires
their separation and purification. Their polarity makes them soluble
in several types of polar solvents, such as methanol, ethanol,
acetone, and water. Solvent extraction of anthocyanins is the initial
step prior to quantification, purification, separation, and characterisation
and generally involves the use of an acidified extragent.
Common extraction procedures are non-selective and yield pigment
solutions with large amounts of by-products, such as sugars,
sugar alcohols, pectin, organic acids, amino acids and proteins.
Some of these impurities may accelerate anthocyanin degradation
or cause problems in further processing steps, such as freeze drying
or spray drying. Furthermore, the low content of anthocyanins
in raw materials hinders the commercial large scale production of
these pigments. Purification by solid-phase extraction (SPE) is a
relatively simple method allowing the elimination of non-phenolic
impurities. Currently SPE gains wider application as a rapid and
economical process and because different cartridges with a great
variety of sorbents can be used. In addition, it can also be automated
reducing the processing time. Different SPE conditions have
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.05.061
* Corresponding author. Tel./fax: +359 32 642759.
E-mail address: lbas@plov.omega.bg (M. Kratchanova).
Food Chemistry 123 (2010) 1055–1061
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
already been used successfully for purification of anthocyanins
(Kraemer-Schafhalter, Fuchs, & Pfannhauser, 1998; Nicoue, Savard,
& Belkacemi, 2007). The absorbent Amberlite XAD7 has proved to
be very suitable for chokeberry anthocyanin purification. The SPE
process includes application of the berry extracts into the SPE column,
elution of the impurities and finally, elution of the anthocyanin
fractions.
The aim of the current work was to further extend the application
of SPE for the purification of anthocyanins from different berries
and to investigate the antioxidant properties of the
anthocyanin-rich extracts. The antioxidant activity of the extracts
obtained was evaluated by determining their oxygen radical
absorption capacity (ORAC), hydroxyl radical averting capacity
(HORAC), total peroxyl radical trapping antioxidant parameter
(TRAP), scavenging of nitric oxide (NO) and inhibition of lipid peroxidation.
The methods chosen embrace different aspects of the
antioxidant action and give a comprehensive picture about the
antioxidant potential of the extracts produced.
2. Materials and methods
2.1. Chemicals
Fluorescein disodium salt, 2,2-azobis-(2-amidino-propane)dihydrochloride
(AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (Trolox), gallic acid, thiobarbituric acid and
Amberlite XAD7 were obtained from Sigma–Aldrich (Steinheim,
Germany). Folin–Ciocalteu’s phenol reagent was purchased from
Merck (Darmstadt, Germany). All other solvents used were of analytical
grade and purchased from local distributors.
2.2. Raw materials
Chokeberry (Aronia melanocarpa), elderberry (Sambucus nigra),
black currants (Ribes nigrum), blackberry (Rubus fruticosus) and
blueberry (Vaccinium myrtillus) were supplied at the time of peak
production from the region of Rodopi Mountains, Bulgaria. Chokeberry
and black currant were cultivated whereas elderberry, blackberry
and blueberry were wild-grown. Fruits were immediately
frozen and kept at À18 °C until analysed.
2.3. Extraction
For the extraction of anthocyanins, different extraction systems
have been used (Nicoue et al., 2007). Due to their polarity, anthocyanins
are usually extracted by methanol, ethanol, acetone and
water. Although methanol and acetone are quite effective extragents,
their application in foods is limited due to their toxicity. Ethanol
is more suitable for food application but at the same time is
more difficult to eliminate in the purification process. Using the
absorbent Amberlite XAD7 Kraemer-Schafhalter et al. (1998) observed
that the application of ethanolic chokeberry extract to SPE
resulted in incomplete anthocyanin retention and moreover colour
pigments evolved in two separated peaks, the first one overlapping
with that of the sugars. In our study we used the same filling material
which excluded the possibility to use ethanol as extragent.
Generally, the presence of acid is required for improved anthocyanin
extractability. The use of acid stabilizes anthocyanins in the
flavylium cation form, which is red at low pH. However, a solvent,
acidified with hydrochloric acid, may hydrolyse acylated anthocyanins.
To avoid, or at least, minimise the breakdown of acylated
anthocyanins, organic acids, such as acetic, citric, or tartaric acids,
which are easier to eliminate during anthocyanin concentration,
have been preferred (Perez-Magarino, Ortega-Heras, & Cano-Mozo,
2008). Bearing in mind that these extracts are intended for food
and pharmaceutical uses we chose 1% citric acid solution in water
as an extraction agent. For the extraction 50 g of the fruits were
weighed accurately, defrosted and homogenised in a laboratory
blender. Samples were transferred into extraction tubes and mixed
with 150 ml of 1% citric acid solution in water. Extraction was conducted
on an orbital shaker at 60 °C for 1 h. In a preliminary study
we found out that these conditions provide maximum extractability
of anthocyanins and these remain stable after 1 h extraction
(unpublished results). After that, samples were centrifuged
(20 min, 6200g) and the obtained extracts were used for SPE.
2.4. HPLC
High performance liquid chromatography (HPLC) of the sugars
and citric acid was performed on Waters 484 system, connected
to a refractometric Waters R401 detector and Aminex HPX – 87H
column (300 Â 7.8 mm, BioRad), eluent 0.004 mol/l H2SO4, flow
0.5 ml/min, temperature 23 °C. The standard compounds were purchased
from Sigma–Aldrich (Steinheim, Germany).
2.5. Total phenolic compound analysis
Total phenolics were determined according to the method of
Singleton and Rossi (1965) with Folin–Ciocalteu’s reagent. Gallic
acid was employed as calibration standard and results were expressed
as gallic acid equivalents (GAE) per gram dry weight.
2.6. Anthocyanins determination
Anthocyanins were determined by the pH-differential method
(Lee, 2005). Anthocyanin pigments change colour with pH. The
absorption of the sample was measured at pH 1.0 and pH 4.5.
The difference in absorbance is proportional to the anthocyanin
content. The total anthocyanin content was expressed as cyanidin-3-glucoside
equivalents and calculated via the following
formula:
Anthocyanin content ðmg=lÞ ¼
A Â MW Â DF Â 1000
e  L
where A = (A510 nm pH 1.0 À A700 nm pH 1.0) À (A510 nm pH
4.5 À A700 nm pH 4.5), MW = cyanidin-3-glucoside molecular
weight (449.2); DF = dilution factor; e = cyanidin-3-glucoside molar
absorbtivity (26,900); L = cell pathlength (usually 1 cm).
2.7. Solid-phase extraction
The SPE material (Amberlite XAD7) was soaked with bidistillated
water and then loaded into the column. After that, the column
was rinsed with bidistillated water and the crude extract was applied.
The impurities were eluted with bidistillated water. Ten millilitres
of fractions of the eluate were collected until no soluble dry
solids were detected. After that, anthocyanins were eluted with
96% ethanol. To represent the elution profile of anthocyanins,
10 ml fractions were collected and analysed. The dense part of
the anthocyanin-containing ethanol was concentrated via rotary
evaporation at 50 °C. After that, concentrated extracts were freeze
dried to obtain a powdered dry product. SPE was performed on
SUPELCO VISIPER system, connected to a vacuum pump (KNF
lab) with 50 ml glass cartridges. Dry extracts obtained after SPE
were kept at À18 °C until analysed. Before the analyses, extracts
were dissolved in phosphate buffered saline pH 7.4 (PBS) in concentration
2 mg sample/1 ml PBS. The SPE procedure was repeated
three times for all berry extracts.
1056 P. Denev et al. / Food Chemistry 123 (2010) 1055–1061
2.8. ORAC assay
ORAC was measured according to the method of Ou, HampschWoodill,
and Prior (2001) with some modifications. The method
measures the antioxidant scavenging activity against peroxyl
radical induced by 2,20
-azobis-(2-amidino-propane)dihydrochloride
(AAPH) at 37 °C. Fluorescein (FL) was used as the fluorescent
probe. The loss of fluorescence of FL was an indication of the extent
of damage from its reaction with the peroxyl radical. The protective
effect of an antioxidant was measured by assessing the area
under the fluorescence decay curve (AUC) as compared to that of
blank in which no antioxidant is present. Solutions of AAPH, fluorescein
and Trolox were prepared in a phosphate buffer
(75 mmol/l, pH 7.4). Samples were diluted in phosphate buffer as
well. Reaction mixture (total volume 200 ll) contained FL –
(170 ll, final concentration 5.36 Â 10À8
mol/l), AAPH – (20 ll, final
concentration 51.5 mmol/l), and sample – 10 ll. FL solution and
sample were incubated at 37 °C for 20 min, and AAPH (dissolved
in 37 °C buffer) was added. The mixture was incubated for 30 s before
the initial fluorescence was measured. After that, the fluorescence
readings were taken at the end of every cycle after shaking.
For the blank, 10 ll of phosphate buffer was used instead of a sample.
Antioxidant activity was expressed in Trolox equivalents. Trolox
solutions (6.25; 12.5; 25; 50 and 100 lmol/l) were used for
defining the standard curve. One ORAC unit is assigned to the net
protection area, provided by a Trolox solution with concentration
of 1 lmol/l. The final ORAC values were calculated using a regression
equation between the Trolox concentration and the net area
under the curve. Results were expressed as micromole Trolox
equivalents per gram of dry extract.
2.9. HORAC assay
HORAC measures the metal-chelating activity of antioxidants
under the conditions of Fenton-like reactions employing a Co(II)
complex and hence the protecting ability against formation of hydroxyl
radical (Ou et al., 2002). Hydrogen peroxide solution of
0.55 mol/l was prepared in distilled water. Co(II) (4.6 mmol/l)
was prepared as follows: 15.7 mg of CoF2Á4H2O and 20 mg of picolinic
acid were dissolved in 20 ml of distilled water. Fluorescein –
170 ll (60 nmol/l, final concentration) and 10 ll of sample were
incubated at 37 °C for 20 min directly in the FLUOstar plate reader.
After incubation, 10 ll H2O2 (27.5 mmol/l, final concentration) and
10 ll of Co(II) (230 lmol/l, final concentration) solutions were
added. The initial fluorescence was measured, after which the
readings were taken every minute after shaking. For the blank
sample, a phosphate buffer solution was used. Gallic acid solutions
of 100, 200, 400, 500 and 600 lmol/l (in phosphate buffer
75 mmol/l, pH 7.4) were used for building the standard curve.
The final HORAC values were calculated using a regression equation
between gallic acid concentration and the net area under the
curve. One HORAC unit was assigned to the net protection area
provided by 1 lmol/l gallic acid and the activity of the sample is
expressed as lmol gallic acid equivalents (GAE) per gram of dry extract.
ORAC and HORAC analyses were carried out using a FLUOstar
OPTIMA plate reader (BMG LABTECH, Offenburg, Germany), excitation
wavelength of 485 nm and emission wavelength of 520 nm
were used.
2.10. TRAP assay
The luminol-enhanced chemiluminescence (CL) was used to
monitor the peroxyl radical reaction as described previously
(Cˇízˇová, Lojek, Kubala, & Cˇízˇ, 2004). The CL signal is driven by the
production of luminol derived radicals from thermal decomposition
of AAPH. The TRAP value was determined from the duration
of the time period (Tsample) during which the sample quenched
the CL signal due to the present antioxidants. A known quantity
(8.0 nmol) of trolox, a water-soluble analogue of tocopherol was
used as a reference inhibitor (Ttrolox). The calculation of the TRAP
value is represented by the equation:
TRAP ¼ 2:0½TroloxŠTsample=f TTrolox;
where 2.0 is the stoichiometric factor of trolox (the number of peroxyl
radicals trapped per one molecule of trolox) and f is the dilution
of the sample. The activity of the sample is expressed as
Trolox equivalents per gramme dry extract. TRAP assay was conducted
on Luminometer Orion II (Berthold Dection System GmbH,
Pforzheim, Germany).
2.11. NO-scavenging activity
The potential ability of extracts to scavenge NO in chemical systems
was tested by the electrochemical measurement of NO. This
chemical system consisted of 1 ml of PBS (control sample) or
1 ml of PBS with one of tested extracts (permanent mixing), and
the temperature was kept at 37 °C. NO was measured using three
electrode systems as described by Hrbac et al. (2007). A porphyrinic
microsensor working electrode, counter electrode and a reference
electrode were connected to the ISO-NO MARK II
potentiostat (WPI, USA). The injection of the 2 ll NO-saturated
water into the measurement glass vial (final concentration of
NO = 2.38 lmol/l) caused the rapid increase with a subsequent
gradual decrease of the NO induced signal until it reached the
background current. In our experiments, the electrochemical signal
was followed up for 1000 s to obtain kinetic curves. The scavenging
properties of the tested extract were represented as a very rapid
decrease of the NO induced signal. The scavenging properties of
tested extracts were evaluated as the time needed for reaching
again the background current.
2.12. Lipid peroxidation inhibition
0.9 ml of 0.5 mmol/l a-linolenic acid (Sigma–Aldrich, Steinheim,
Germany) was mixed with 0.1 ml sample. Then, a system
generating hydroxyl radical (0.1 ml Co(II) and 0.1 ml hydrogen peroxide
– for detailed description see section HORAC) was added for
the induction of lipid peroxidation and the mixture was incubated
for 2 h in 37 °C. After the end of incubation, the concentration of
thiobarbituric acid-reactive substances (TBARS) was measured as
the index of lipid peroxidation as described previously (Slavíková
et al., 1998) with some modifications. Briefly, 0.5 ml of the analysed
mixture was added to 1 ml of the mixture consisting of
0.6% thiobarbituric acid, 0.25 N HCl and 15% trichloracetic acid at
a ratio of 1:1:1 (v/v). Then the samples were incubated in a water
bath (100 °C) for 45 min. After cooling to ambient temperature,
1.5 ml of n-butanol were added and the mixture was shaken vigorously.
The samples were centrifuged (5 min, 1500g), and the absorbance
of the upper layer was measured at 532 nm. 1,1,3,3Tetraethoxypropane
(Sigma–Aldrich, Steinheim, Germany) at a final
concentration of 0.1 lmol/l was used as a standard. Lipid peroxidation
was expressed in nmol of TBARS per 1 ml of the
mixture a-linolenic acid/analysed sample.
2.13. Statistical analysis
All data are expressed as the mean ± standard deviation (SD).
One-way analysis of variance (ANOVA) and Newman–Keuls test
were used to evaluate the differences of the mean between groups.
A P-value of 0.05 was considered significant. Pearson correlation
P. Denev et al. / Food Chemistry 123 (2010) 1055–1061 1057
coefficient (R) was used to show correlations and linear regressions
were calculated.
3. Results and discussion
3.1. Extraction of berries and solid-phase extraction of the crude
extracts
Among most fruits of common use, the chosen berries are distinctive
with their anthocyanin content (Wu et al., 2006). This
determined our choice in chokeberry, elderberry, black currant,
blackberry and blueberry as anthocyanin sources. The characteristics
of berry extracts are shown in Table 1. The obtained extracts
differed significantly in their sugar composition and anthocyanins
content. The amount of anthocyanins varied from 0.45 g/l for
blackberry to 1.69 g/l for elderberry crude extract. It is evident
from the data in Table 1 that anthocyanins comprised only a small
part of the soluble dry solids of the extracts – about 0.98% in blackberry,
1.26% in chokeberry, 1.67% in black currant, 1.77% in blueberry
and 3.52% in elderberry crude extracts. All extracts
contained fructose, glucose and sucrose (except blackberry).
Chokeberry and blueberry extracts contained sorbitol as well. Citric
acid and other organic acids were also present in the crude extracts.
The majority of citric acid in the crude extracts comes from
the extraction agent, since citric acid is used to improve anthocyanins
extractability. After extraction the obtained crude extracts
Table 1
Characteristics of berry crude extracts obtained after extraction of 50 g fruits with 150 ml 1% solution of citric acid in water.
Fruit Volume
(ml)
Dry solids (%) Polyphenols
(g/l)
Anthocyanins
(g/l)
Sugars (g/l) Citric
acid (g/l)
Total
acidity (g/l)
Ratio anthocyanins/
dry solids (%)
Sucrose Fructose Glucose Sorbitol
Chokeberry 175 5.7 2.60 0.72 0.16 9.58 6.29 20.1 10.5 13.2 1.26
Elderberry 178 4.8 2.66 1.69 0.28 7.12 8.31 – 13.2 14.2 3.52
Black currant 168 4.9 2.02 0.82 0.95 10.7 12.7 – 19.0 19.3 1.67
Blackberry 170 4.6 1.57 0.45 – 14.0 14.3 – 11.9 13.3 0.98
Blueberry 164 3.5 1.37 0.62 0.04 6.64 7.90 1.94 10.5 12.4 1.77
Fig. 1. SPE of berry extracts. Column volume – 50 ml, SPE material Amberlite XAD7. (A) Chokeberry extract: volume 30 ml, dry solids – 57 g/l, sugars – 36.1 g/l, citric acid –
10.5 g/l. (B) Elderberry extract: volume 15 ml, dry solids – 48.3 g/l, sugars – 15.7 g/l, citric acid – 13.2 g/1. (C) Black currant extract: volume 30 ml, dry solids – 49.6 g/l, sugars
24.3 g /l, citric acid – 19.0 g/l. (D) Blackberry extract: volume 50 ml, dry solids – 45.9 g/l, sugars – 28.3 g /l, citric acid – 11.9 g/l. (E) Blueberry extract: volume 30 ml, dry solids
– 35.6 g/l, sugars – 16.5 g /l, citric acid – 10.5 g/l .
1058 P. Denev et al. / Food Chemistry 123 (2010) 1055–1061
were subjected to SPE. The choice of SPE material is crucial for the
good separation of anthocyanins. Various SPE materials have been
applied for that purpose (Kraemer-Schafhalter et al., 1998; Nicoue
et al., 2007). Kraemer-Schafhalter et al. (1998) performed a comparison
of 16 different SPE materials for purification of chokeberry
anthocyanins. These included reversed phase silica gels and macroreticular
non-ionic acrylic polymer absorbents. The absorbent
Amberlite XAD7 was found to be very suitable for chokeberry
anthocyanin purification. The applicability of Amberlite XAD 7
for SPE of different berry extracts was extended in our study. The
volume of extract applied to the SPE column depended on the loading
capacity of the SPE material and the amount of anthocyanins
present in the sample. It was found that for the column used and
packing material, quantities of anthocyanins higher than 25 mg
led to column overloading. This amount determined the volume
of each extract to be applied for SPE. Elution profiles of the soluble
dry solids, sugars and anthocyanins during SPE are shown in Fig 1.
The quantity of sugars is given as the sum of glucose, fructose, sucrose
and sorbitol in the sample. Depending on the volume of the
applied extract, the elution profile was different. In all cases, there
was good separation of sugar and acids from anthocyanins despite
the different anthocyanin profile of the investigated raw materials.
Anthocyanins differ in their polarity and often a gradient elution is
performed in order to separate the anthocyanins. This leads to several
anthocyanin fractions of a different profile. In the current
work, the separation of the anthocyanins in the column was excellent
in all extracts studied. Under the SPE conditions used, the elution
was faster, anthocyanin fractions were more concentrated, no
gradient elution was required and only a single eluent was used. By
measuring the presence of soluble dry solids in the rinsing waters,
the purification process could be controlled in an easy manner.
This makes the SPE process simple to perform which is of a great
importance especially in cases of enlarged scale. The anthocyanin
fractions were concentrated by evaporation of the alcohol and then
subjected to freeze drying to obtain powdered products. The characteristics
of the powdered extracts obtained are shown on Table 2.
The content of anthocyanins in the dry extracts varied from 9.6%
for blackberry to 24.6% for elderberry extracts. Very good purification
from sugars was achieved. The amount of removed sugars varied
from 94.4% of elderberry extract to 98.1% of total amount of
sugars of blackcurrant and blueberry extracts. The same observation
was made for the removal of citric acid – over 88.5%. The
residual citric acid in the dry extracts is not undesirable. Anthocyanins
are more stable in the presence of acids, moreover citric acid
acts as a metal ion chelator and may show a protective effect on
anthocyanins during storage and processing (Timberlake & Bridle,
1980, chap. 5). Very good recovery of anthocyanins 90–95.6%
was achieved during the SPE process. This shows that the chosen
SPE material is suitable for all anthocyanins regardless of the aglycone
type.
3.2. Antioxidant activity
Many methods for determination of antioxidant activity have
been developed and reviewed. Various methods rely on the generation
of different radicals acting by different mechanisms (Huang,
Ou, & Prior, 2005) and usually it is recommendable to evaluate the
antioxidant properties of the investigated materials using several
assays (Ciz et al., 2010). In our study, the antioxidant properties
of the purified extracts were analysed via five different methods
– ORAC, TRAP, HORAC, NO-scavenging and lipid peroxidation inhibition.
The chosen methods embrace different aspects of the antioxidant
action and give a comprehensive view on the antioxidant
potential of the investigated extracts. The first two methods assess
the radical scavenging activity of the sample against peroxyl radicals,
whereas HORAC method measures the metal-chelating activity
of antioxidants under the conditions of Fenton-like reactions
and hence the protecting ability against formation of hydroxyl radical.
Since lipids are very susceptible to lipid peroxidation, we also
tested the ability of the samples to prevent the peroxidation of
polyunsaturated fatty acids induced by hydroxyl radical. In addition
to reactive oxygen species, reactive nitrogen species (produced
mainly by macrophages) are one of the important
microbicidal tools in the process of inflammation during the fight
against pathogenic microorganisms, bacteria and tumour cells. Nitric
oxide, a member of reactive nitrogen species, is an important
molecule involved in the regulation of many physiological and
microbicidal processes. However, the overproduction of NO occurs
in several chronic inflammatory diseases, such as bronchitis, osteoarthritis
and rheumatoid arthritis (Pekarova et al., 2009). For this
reason the elimination of NO from the inflammatory site is expected
to exhibit a very beneficial therapeutic effect. The scavenging
properties of our samples were tested using electrochemical
analysis, which is considered to be a reliable method for verifying
NO-scavenging.
All five berry extracts exhibited strong radical scavenging activities
measured by ORAC and TRAP methods. ORAC values were
quite high from 3949 to 5783 lmol TE/g (Table 3). Elderberry
and blueberry extracts showed the highest levels of peroxyl radical
scavenging via the ORAC method, while chokeberry extract was
the most potent antioxidant measured by the TRAP method. There
are several papers reporting antioxidant properties of anthocyanin-rich
extracts. Espin, Soler-Rivas, Wichers, and Garcia-Viguera
(2000) reported that a chokeberry extract, measured by the DPPHÅ
method, showed higher radical scavenging activity compared to
that of BHA-butylated hydroxyanisole and BHT-butylated
hydroxytoluene. Using gel filtration as a method for anthocyanin
Table 2
Characteristics of anthocyanin-rich berry extracts after SPE.
Fruit Yield of dry extracta
(g) Polyphenols (mg/g) Anthocyaninsa
(mg/g) Anthocyanin recoverya
(%) Sugars (mg/g) Citric acid (mg/g)
Chokeberry 0.92 ± 0.12 352 115 ± 8.2 91.2 ± 1.02 119 229
Elderberry 1.17 ± 0.09 283 246 ± 12.3 95.6 ± 2.10 124 289
Black currant 0.81 ± 0.10 239 158 ± 6.4 93.1 ± 1.82 87.2 421
Blackberry 0.72 ± 0.13 245 95.8 ± 11.2 90.2 ± 4.06 206 310
Blueberry 0.66 ± 0.04 267 139 ± 10.8 90.0 ± 0.80 80.2 303
a
Data are presented as means from three independent SPE ± standard deviation.
Table 3
ORAC, TRAP and HORAC antioxidant activity of anthocyanin-rich berry extracts. Data
are presented as means from six independent measurements ± standard deviation.
There are no significant differences among values marked with the same superscript
letters in individual columns.
Extract ORAC (lmol TE/g) TRAP (lmol TE/g) HORAC
(lmol GAE/g)
Chokeberry 5165 ± 223B
4051 ± 82D
1265 ± 50B
Elderberry 5783 ± 202C
3230 ± 62C
1264 ± 74B
Black currant 3949 ± 121A
2132 ± 28A
874 ± 50A
Blackberry 4042 ± 88A
2771 ± 42B
834 ± 50A
Blueberry 5646 ± 102C
2860 ± 76B
1293 ± 55B
P. Denev et al. / Food Chemistry 123 (2010) 1055–1061 1059
purification Elisia, Hu, Popovich, and Kitts (2007) obtained an
anthocyanin-enriched blackberry extract which showed a slightly
higher ORAC value – 4885 lmol TE/g than the blackberry extract
reported in our study. In one of the first attempts to quantify dietary
antioxidant needs of the body Prior et al. (2007) demonstrated
that consumption of certain berries and fruits such as blueberries,
mixed grape and kiwifruit was associated with increased ORAC
plasma antioxidant capacity in the postprandial state and consumption
of an energy source of macronutrients containing no
antioxidants was associated with a decline in plasma antioxidant
capacity. They estimated that according to the energy intake of
the diet, 5000–15,000 lmol TE/g are necessary to cover human
daily antioxidant needs. However, further long-term clinical studies
are needed to clarify the connection between the increment of
plasma antioxidant capacity and the potential decreased risk of
chronic degenerative disease. The ORAC values of the anthocyanin-rich
extracts studied are proof that these extracts present a
good source of dietary antioxidants. In the literature scarce data
are available about the HORAC values of foods (Ou et al., 2002).
To our knowledge, such data for anthocyanin-rich extracts is lacking.
Hydroxyl radicals (HOÅ
) are highly reactive and can be generated
via the Fenton reaction. Antioxidants, which are able to act
as metal chelators, may be able to prevent the formation of hydroxyl
radical, thus acting as a preventive antioxidant. Since dry extracts
contain some citric acid, which can act as metal chelator it
was reasonable to check whether citric acid contributes to the
HORAC antioxidant activity of the extracts. In model system, pure
citric acid did not show HORAC antioxidant activity even in concentration
50 times higher that the concentration of citric acid in
the dried anthocyanin extracts. This means that in the conditions
of HORAC assay citric acid does not show metal-chelating properties.
The elderberry and blueberry extracts obtained in our study
revealed four and eight times higher HORAC values, respectively,
than the data for elderberry and blueberry fruits (Ou et al.,
2002). All the investigated extracts showed several times higher
HORAC activity than apple, peach, sour cherry, grapes, cranberry,
raspberry, strawberry (Ou et al., 2002) and many vegetables (Ciz
et al., 2010). Similar results were obtained in the experiments
further carried where all investigated extracts showed high potential
to prevent lipid peroxidation and the following rank order of
potency was obtained: chokeberry > elderberry > black currant >
blueberry > blackberry (Fig 2). Very high scavenging activity
against NO was exhibited by all investigated extracts (Fig. 3). These
results are original and of particular interest because, according to
our knowledge, the direct measurement of the NO-scavenging
activity of anthocyanin-enriched extracts has not been performed
as yet and some authors only speculated that the NO-scavenging
activity of vegetable extracts could be caused by a high content
of total phenolic compounds and anthocyanins (Bor, Chen, & Yen,
2006).
Generally, antioxidant activity correlated better with the polyphenol
content than to the anthocyanin content. The correlation
between polyphenols and antioxidant activity was as follows:
TRAP (r = 0.95); lipid peroxidation inhibition (r = 0.86); HORAC
(r = 0.68); ORAC and NO-scavenging activity (r = 0.51). The highest
correlation between antioxidant activity and anthocyanin content
was found for ORAC (r = 0.52) and HORAC (r = 0.38). Taking in mind
that these were partially purified extracts of natural colourants, the
existence of several radical scavenger compounds in these extracts
was possible. It is well known that berries contain a large amount
of phenolic compounds that act as antioxidants besides anthocyanins
(Prior et al., 1998). Other possible reason for this observation
could be the different antioxidant potential of different anthocyanins
which is determined by their structural characteristics (Zheng
& Wang, 2003).
4. Conclusions
Solid-phase extraction (SPE) using the absorbent Amberlite
XAD7 is a fast, simple and reliable method for purification of
anthocyanins from complex extracts, containing impurities such
as sugars, acids, proteins, pectin, etc. The amount of the anthocyanins
in the dried extracts after SPE was 9.6–24.6%. During SPE,
more than 94% of the sugars present in the extracts were eluted.
A good anthocyanin recovery was observed in the purification process
as well. The obtained anthocyanin-rich extracts showed high
antioxidant efficacy in all five assays of antioxidant activity determination:
ORAC, TRAP, HORAC, scavenging of nitric oxide and inhibition
of lipid peroxidation. This reveals the possibilities for
application of these preparations as effective antioxidants.
Acknowledgements
This study was supported by Grants IF-02-66/2007, OC08058
MEYS and COST B35 Action.
Fig. 2. Inhibition of induced lipid peroxidation by analysed extracts. Data are
presented as means from at least three independent measurements ± standard
deviation. There are no significant differences among values marked with the same
superscript letters.
Fig. 3. Scavenging of nitric oxide by analysed samples. The representative curve
shows the scavenging effect of chokeberry extract in comparison with control
amperometrical signal. Inserted table shows values obtained for all analysed
samples. Data are presented as means from at least three independent measurements
± standard deviation. There are no significant differences among values
marked with the same superscript letters.
1060 P. Denev et al. / Food Chemistry 123 (2010) 1055–1061
References
Bell, D. R., & Gochenaur, K. (2006). Direct vasoactive and vasoprotective properties
of anthocyanin-rich extracts. Journal of Applied Physiology, 100, 1164–1170.
Bor, J. Y., Chen, H. Y., & Yen, G. C. (2006). Evaluation of antioxidant activity and
inhibitory effect on nitric oxide production of some common vegetables. Journal
of Agricultural and Food Chemistry, 54(5), 1680–1686.
Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours –
Selected aspects. Food Chemistry, 58, 103–109.
Ciz, M., Cizova, H., Denev, P., Kratchanova, M., Slavov, A., & Lojek, A. (2010). Different
methods for control and comparison of the antioxidant properties of vegetables.
Food Control, 21, 518–523.
Cˇízˇová, H., Lojek, A., Kubala, L., & Cˇízˇ, M. (2004). The effect of intestinal ischemia
duration on changes in plasma antioxidant defense status in rats. Physiological
Research, 53(5), 523–531.
Elisia, I., Hu, C., Popovich, D. G., & Kitts, D. D. (2007). Antioxidant assessment of an
anthocyanin-enriched blackberry extract. Food Chemistry, 101, 1052–1058.
Espin, J. C., Soler-Rivas, C., Wichers, H. J., & Garcia-Viguera, C. (2000). Anthocyaninbased
natural colorants: A new source of antiradical activity for foodstuff.
Journal of Agricultural and Food Chemistry, 48, 1588–1592.
Ghosh, D., & Konishi, T. (2007). Anthocyanins and anthocyanin-rich extracts: Role in
diabetes and eye function. Asia Pacific Journal of Clinical Nutrition, 16(2),
200–208.
Hrbac, J., Gregor, C., Machova, M., Kralova, J., Bystron, T., Ciz, M., et al. (2007). Nitric
oxide sensor based on carbon fiber covered with nickel porphyrin layer
deposited using optimized electropolymerization procedure.
Bioelectrochemistry, 71, 46–53.
Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity
assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856.
Kahkonen, M., & Heinonen, M. (2003). Antioxidant activity of anthocyanins and
their aglycons. Journal of Agricultural and Food Chemistry, 51, 628–633.
Kraemer-Schafhalter, A., Fuchs, H., & Pfannhauser, W. (1998). Solid-phase extraction
(SPE) a comparison of 16 materials for the purification of anthocyanins from
Aronia melanocarpa var Nero. Journal of the Science of Food and Agriculture, 78,
435–440.
Lee, J. (2005). Determination of total monomeric anthocyanin pigment content of
fruits juices, beverages, natural colorants and wines by the pH differential
method: Collaborative study. Journal of AOAC International, 88(5), 1269–1278.
Nicoue, E., Savard, S., & Belkacemi, K. (2007). Anthocyanins in wild blueberries of
Quebec: Extraction and identification. Journal of Agricultural and Food Chemistry,
55, 5626–5635.
Ou, B., Hampsch-Woodill, M., Flanagan, J., Deemer, E. K., Prior, R. L., & Huang, D. J.
(2002). Novel fluorometric assay for hydroxyl radical prevention capacity using
fluorescein as the probe. Journal of Agricultural and Food Chemistry, 50(10),
2772–2777.
Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescence probe. Journal of Agricultural and Food Chemistry, 49(10),
4619–4626.
Pekarova, M., Kralova, J., Kubala, L., Ciz, M., Lojek, A., Gregor, C., et al. (2009).
Continuous electrochemical monitoring of nitric oxide production in murine
macrophage cell line RAW 264.7. Analytical and Bioanalytical Chemistry, 394(5),
1497–1504.
Perez-Magarino, S., Ortega-Heras, M., & Cano-Mozo, E. (2008). Optimization of a
solid-phase extraction method using copolymer sorbents for isolation of
phenolic compounds in red wines and quantification by HPLC. Journal of
Agricultural and Food Chemistry, 56, 11560–11570.
Prior, R. L., Cao, G., Martin, A., Sofic, E., McEwen, J., O’Brien, C., et al. (1998).
Antioxidant capacity as influenced by total phenolic and anthocyanin content,
maturity, and variety of Vaccinium species. Journal of Agricultural and Food
Chemistry, 46(7), 2686–2693.
Prior, R. L., Hoang, H., Gu, L., Wu, X., Jacob, R., Sotoudeh, G., et al. (2007). Plasma
antioxidant capacity changes following a meal as a measure of the ability of a
food to alter in vivo antioxidant status. Journal of the American College of
Nutrition, 26(2), 170–181.
Ronziere, M. C., Herbage, D., Garrone, R., & Frey, J. (1981). Influence of some
flavonoids on reticulation of collagen fibrils in vitro. Biochemical Pharmacology,
30, 1771–1776.
Satue-Gracia, M. T., Heinonen, M., & Frankel, E. N. (1997). Anthocyanins as
antioxidants on human low-density lipoprotein and lecithinliposome systems.
Journal of Agricultural and Food Chemistry, 45, 3362–3367.
Singleton, V., & Rossi, J. (1965). Colorimetry of total phenolic with
phosphomolibdiphosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Slavíková, H., Lojek, A., Hamar, J., Dušková, M., Kubala, L., Vondrácˇek, J., et al. (1998).
Total antioxidant capacity of serum increased in early but not in late period
after intestinal ischemia in rats. Free Radical Biology and Medicine, 25, 9–18.
Timberlake, C. F., & Bridle, P. (1980). In J. Walford (Ed.), Developments in food colours
(1st ed.). London: Applied Science Publishers.
Wagner, H. (1985). New plant phenolics of pharmaceutical interest. Annual
proceedings of phytochemical society of Europe (Vol. 25, pp. 409–425). Oxford:
Clarendon Press.
Wu, X., Beecher, G., Holden, J., Haytowitz, D., Gebhardt, S. E., & Prior, R. L. (2004).
Lipophilic and hydrophilic antioxidant capacities of common foods in the
United States. Journal of Agricultural and Food Chemistry, 52, 4026–4037.
Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L.
(2006). Concentrations of anthocyanins in common foods in the United States
and estimation of normal consumption. Journal of Agricultural and Food
Chemistry, 54, 4069–4075.
Zhao, C., Giusti, M., Malik, M., Moyer, M., & Magnuson, B. (2004). Effects of
commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic
colonic cell growth. Journal of Agricultural and Food Chemistry, 52, 6122–6128.
Zheng, W., & Wang, S. Y. (2003). Oxygen radical absorbing capacity of phenolics in
blueberries, cranberries, chokeberries, and lingonberries. Journal of Agricultural
and Food Chemistry, 51(2), 502–509.
P. Denev et al. / Food Chemistry 123 (2010) 1055–1061 1061
To cite this article: Neuroendocrinol Lett 2010; 31(Suppl.2):79–83
ORIGINALARTICLE
Neuroendocrinology Letters  Volume 31  Suppl. 2  2010
Formation of reactive oxygen and nitrogen
species in the presence of pinosylvin
– an analogue of resveratrol
Viera Jančinová 1, Rado Nosáľ 1, Antonín Lojek 2, Milan Číž 2, Gabriela Ambrožová 2,
Danica Mihalová 1, Katarína Bauerová 1, Juraj Harmatha 3, Tomáš Perečko 1
1 	Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava,
Slovak Republic
2 	Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
3 	Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Praha,
Czech Republic
Correspondence to: PharmDr. Viera Jančinová, PhD.
Institute of Experimental Pharmacology and Toxicology,
Slovak Academy of Sciences
Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic
tel: +421-2-59410672; fax: +421-2-54775928; e-mail: viera.jancinova@savba.sk
Submitted: 2010-09-17	 Accepted: 2010-11-22	 Published online: 2010-00-00
Key words: pinosylvin; reactive oxygen and nitrogen species; adjuvant arthritis;
methotrexate; neutrophils; macrophages
Neuroendocrinol Lett 2010; 31(Suppl.2):79–83  PMID: 21187828  NEL31S210A13  © 2010 Neuroendocrinology Letters • www.nel.edu
Abstract OBJECTIVE: Formation of reactive oxygen species in neutrophils of rats with
adjuvant arthritis and generation of nitric oxide in RAW 264.7 macrophages were
analysed in the presence of pinosylvin.
METHODS AND RESULTS: The method of chemiluminescence was used for the
detection of reactive oxygen species in blood of rats with adjuvant arthritis. Pinosylvin
(50 mg/kg, daily, p.o.) and methotrexate (0.4 mg/kg, twice a week, p.o.)
were applied separately or in a combination over a period of 28 days from the day
of immunisation. Adjuvant arthritis was accompanied by a significantly increased
number of neutrophils, by elevated concentration of oxidants in blood and by
excessive responsiveness of neutrophils to stimulation with PMA. In rats treated
with methotrexate, all these changes were significantly reduced and the inhibition
became more pronounced when methotrexate was applied in the combination
with pinosylvin; the monotherapy with pinosylvin did not induce any detectable
changes in the parameters tested. Under in vitro conditions, pinosylvin inhibited
formation of nitric oxide (NO) in macrophages, as demostrated by the decreased
concentration of nitrite – the end-product of NO metabolism (assessed by Griess’
method), by the reduced expression of inducible NO synthase (detected by
Western blot), and by the failure of pinosylvin to scavenge nitric oxide (measured
amperometrically in cell-free system).
CONCLUSION: The observed ability of pinosylvin to decrease concentration
of reactive oxygen and nitrogen species, along with its capacity to enhance the
efficacy of methotrexate in arthritis treatment may shed more light into the pharmacological
potential of this prospective natural substance.
80 Copyright © 2010  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
Viera Jančinová, Rado Nosáľ, Antonín Lojek, Milan Číž, Gabriela Ambrožová, Danica Mihalová, Katarína Bauerová, Juraj Harmatha, Tomáš Perečko
Abbreviations:
NO 	 - nitric oxide
iNOS 	 - inducible NO synthase
NFκB 	 - nuclear factor κB
PMA 	 - 4β-phorbol-12β-myristate-α13-acetate
RLU 	 - relative luminescence units
Introduction
Reactive oxygen and nitrogen species, produced by
neutrophils and other inflammatory cells, participate
actively in the initiation and development of many
pathological states (Witko-Sarsat et al. 2000; Cascao
et al. 2010). In rheumatoid arthritis, the oxidants can
induce cartilage degradation, depolymerise hyaluronan
and decrease its lubricative properties, they can reduce
the protective antioxidant and antiproteinase capacity
of synovial fluid and participate in this way in joint erosion
(Edwards & Hallett 1997; Cross et al. 2006; Cascao
et al. 2009). These facts focussed attention on antioxidative
and neutrophil targeting substances as they may
increase the effectiveness and minimise unwanted side
effects of disease-modifying antirheumatic therapy.
Several compounds of plant or microbial origin were
found to be promising from this perspective (Drábiková
et al. 2009; Jančinová et al. 2009a; Rovenský et al. 2009).
Pinosylvin (3,5-dihydroxystilbene), one of the naturally
occurring resveratrol (3,4’,5-trihydroxystilbene)
analogues, is formed constitutively and after UV irradiation
or microbial attack in the wood and needles of
Pinus species. The majority of the available data characterises
antifungal, antibacterial and anticancer activities
of pinosylvin (Lee et al. 2005; Roupe et al. 2006a; Simard
et al. 2008), yet little is known about its antioxidant and
antiinflammatory effects (Park et al. 2004; Adams et al.
2005; Lee et al. 2006). Previously, we found that incubation
of human neutrophils with this stilbene reduced
production of reactive oxygen species and this effect
involved the inhibition of protein kinase C isoforms
α and βΙΙ (Perečko et al. 2008; Jančinová et al. 2009b).
In the present study, its interference with neutrophils
was further tested under in vivo conditions – in rats
with adjuvant arthritis, and formation of the inflammatory
mediator nitric oxide in macrophages was also
analysed.
Materials and methods
Materials
Pinosylvin was synthesised at the Institute of Organic
Chemistry and Biochemistry AS CR (Praha, Czech
Republic), methotrexate was from Pharmachemie
(Haarlem, Netherlands), Griess’ reagent, lipopolysaccharide
from Escherichia coli, luminol, and PMA from
Sigma-Aldrich Chemie (Deisenhofen, Germany),
murine RAW 264.7 macrophage cell line was obtained
from the American Type Culture Collection (ATCC,
Manassas, Virginia, USA).
Formation of reactive oxygen species – effects of pinosylvin
in arthritis
Adjuvant arthritis was induced in male Lewis rats by
a single intradermal injection of heat-killed Mycobacterium
butyricum (Poništ et al. 2010). The study was
performed in compliance with Principles of Laboratory
Animal Care and was approved by the local Ethics Committee
and by the State Veterinary and Food Administration
of the Slovak Republic. Pinosylvin (50 mg/kg,
daily, p.o.) and methotrexate (0.4 mg/kg, twice a week,
p.o.) were applied separately or in a combination over
a period of 28 days after arthritis induction. Then neutrophil
count and concentration of reactive oxygen species
in blood were assessed. The production of oxidants
(spontaneous or stimulated with 0.05 µmol/l PMA) was
estimated on the basis of luminol-enhanced chemiluminescence
and presented as the mean integral values
over 3600 s (Nosáľ et al. 2007; Jančinová et al. 2009a).
Formation of nitric oxide – effects of pinosylvin in vitro
Generation of reactive nitrogen species was determined
indirectly as the accumulation of nitrites – end products
of nitric oxide (NO) metabolism. The detection was
performed spectrophotometrically by Griess‘ method
in the supernatant of murine macrophages RAW 264.7,
which were pre-incubated for 1 h with pinosylvin and
after that stimulated for 24 h with lipopolysaccharide
(LPS). The concentrations of nitrites were derived by
regression analysis using serial dilutions of sodium
nitrite as a standard (Králová et al. 2008). The cell fractions
of these samples were used for the detection of
inducible NO synthase expression by Western blot.
Relative protein levels were quantified by scanning densitometry
using the Image J programme (Ambrožová et
al. 2010). The NO scavenging activity of pinosylvin was
measured amperometrically using an ISO-NO Mark II
NO meter and NO standard solutions. Changes in electrical
current were recorded and the index of scavenging
was calculated by dividing the maximum height and
width of the obtained curves (Číž et al. 2008).
Statistical analysis
Statistical significance of differences between means
was established by one-way analysis of variance
(ANOVA); p-values below 0.05 were considered statistically
significant.
Results
Adjuvant arthritis was accompanied by a significantly
increased number of neutrophils and by massive formation
of oxidants, which was manifested by elevated
chemiluminescence of blood (Table 1). Stimulated chemiluminescence
was increased seven times and it rose
more rapidly than the other parameters tested. This
indicates that neutrophils of arthritic rats responded
to PMA by an excessive production of radicals, similarly
as found by hyper-reactive „primed“ neutrophils
81Neuroendocrinology Letters  Vol. 31  Suppl. 2  2010  •  Article available online: http://node.nel.edu
Pinosylvin, neutrophils and macrophages
of patients with rheumatoid arthritis (Fairhurst et al.
2007). In rats treated with methotrexate, all the arthritis-induced
changes were significantly reduced and this
inhibition became more pronounced when methotrexate
was applied along with pinosylvin. Methotrexate
alone decreased neutrophil count, spontaneous and
stimulated chemiluminescence by 28%, 41% and 43%,
respectively, whereas in combination with pinosylvin,
it inhibited these parameters by 59%, 69% and 63%.
The monotherapy with pinosylvin failed to induce any
detectable changes either in the number of neutrophils
or in oxidant concentration.
Considering the fact that overproduction of nitric
oxide is involved in rheumatoid arthritis, and with the
aim to find further mechanism(s) potentially involved
in the antiinflammatory effect of pinosylvin, the interference
of this stilbene with macrophage activity and
with NO formation was analysed (Figures 1 and 2).
Pinosylvin significantly reduced the quantity of NO
produced by stimulated RAW 264.7 macrophages, as
evidenced by the decreased accumulation of nitrites in
supernatants of these cells. The reduced expression of
inducible NO synthase and the absence of NO scavenging
indicated that this inhibition resulted from diminished
generation of nitric oxide.
Discussion
The recently confirmed active involvement of neutrophils
in the initiation and development of rheumatoid
arthritis (Cascao et al. 2009 and 2010; Wright et al.
2010) focussed attention on substances which are able
to repress the activity of these cells both in vitro (Adams
Tab. 1. Effect of pinosylvin (PIN) and methotrexate (MTX), applied
separately or in combination, on spontaneous and stimulated
chemiluminescence and neutrophil count in blood of arthritic rats.
Chemiluminescence (RLU*s)
Neutrophil
count
Spontaneous PMA stimulated in 1 µl of blood
Healthy
control
41 802±2 452 150 789±9 159 12 174±747
Arthritis 168 203±12 815 1 165 603±94 470 40 260±3 325
Arthritis
+ PIN
190 157±32 580 1 132 747±104 750 44 002±4 398
Arthritis
+ MTX
98 806±8 136** 664 618±68 695** 29 180±1 903*
Arthritis
+ PIN + MTX
51 739±3 437++ 429 813±45 494++ 16 635±2 157++
Mean ± SEM, n = 8-10, *p<0.05, **p<0.01 (vs Arthritis) ++p<0.01 (vs
Arthritis + MTX), PMA – 4β-phorbol-12β-myristate-α13-acetate; RLU
– relative luminescence units
0
20
40
60
80
100
120
140
1 10 100 1 10 100
Pinosylvin (µmol/l)
%ofcontrol
Nitrite production NO scavenging
**
**
0
50
100
150
Ctrl- Ctrl+ 10 100
%ofcontrol
Pinosylvin (µmol/l)
42 kDa
130 kDa
β-actin
iNOS
**
Fig. 1. Nitrite production and nitric oxide (NO) scavenging assessed in the
presence of pinosylvin.
Concentration of nitrites was measured spectrophotometrically in
supernatants of RAW 264.7 cells stimulated with LPS; the NO scavenging
effect of pinosylvin was determined amperometrically in a cell-free chemical
system. Pinosylvin-induced changes in nitrite concentration or in the
scavenging index were expressed as percentage of controls (i.e. samples
incubated in the absence of pinosylvin). Mean ± SEM, n = 3, ** p<0.01 (vs
Control).
Fig. 2. Effect of pinosylvin on the expression of inducible
nitric oxide synthase (iNOS).
Densitometric analysis and representative Western blot
of iNOS protein expression in RAW 264.7 cells treated
with 10 and 100 µmol/l pinosylvin and stimulated
with LPS. Unstimulated (Ctrl–) and LPS-stimulated
(Ctrl+) control cells were used to express effects of
the stimulus and of pinosylvin, respectively. The
immunoblotting of β-actin verified the equal loading
of proteins. Mean ± SEM, n = 3, ** p<0.01 (vs Ctrl+).
et al. 2005; Perečko et al. 2008; Jančinová et al. 2009b)
and in vivo (Nosáľ et al. 2007; Drábiková et al. 2009;
Jančinová et al. 2009a; Rovenský et al. 2009; Poništ et al.
2010). Elimination of the inflammatory reaction caused
by neutrophils and resulting in tissue damage may significantly
potentiate the effectiveness of antirheumatic
therapy. Moreover, co-application of natural medicines
which are able to reduce harmful effects of neutrophils
could lower the dosage of antirheumatic drugs and thus
decrease their toxicity.
82 Copyright © 2010  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
Viera Jančinová, Rado Nosáľ, Antonín Lojek, Milan Číž, Gabriela Ambrožová, Danica Mihalová, Katarína Bauerová, Juraj Harmatha, Tomáš Perečko
The presented results indicate that pinosylvin may
be considered a candidate for the combined therapy of
rheumatoid arthritis. This stilbene potentiated the suppressive
effect of methotrexate on neutrophil activity
and concentration of oxidants in blood. Moreover, the
rats treated with the combined therapy showed reduced
hind paw swelling in comparison with those treated
with methotrexate alone (Bauerová et al. 2010). The
antiinflammatory activity of methotrexate arises from
its ability to inhibit T cell proliferation and cytotoxicity,
to decrease recruitment of monocytes and other cells
to the inflamed joint and from the increased release of
the endogenous anti-inflammatory mediator adenosine
(Cronstein 2005). All these alterations could lead to a
decreased number and activity of neutrophils and to
reduced whole blood chemiluminescence. The interference
of pinosylvin with inflammation seems to be
related to other mechanisms. As found for resveratrol
– a compound structurally similar to pinosylvin, the
stilbene-induced downregulation of the inflammatory
response involves reduced synthesis and release of proinflammatory
mediators, modified eicosanoid synthesis,
decreased activity of immune cells and suppressed
activation of nuclear factor κB – NFκB (Alarcón de
la Lastra & Villegas 2005; Khanna et al. 2007). Moreover,
resveratrol or its oligomeric derivative α-viniferin
induced apoptosis of human rheumatoid arthritis synovial
cells (Nakayama et al. 2010) and suppressed tissue
destruction in model arthritis (Lee et al. 2004; Elmali
et al. 2005). Some of these effects have been already
observed in the presence of pinosylvin – e.g. reduced
production of pro-inflammatory mediators (tumour
necrosis factor α, interleukin-8, prostaglandin E2 and
leukotriene B4) in neutrophils and macrophages as well
as the suppressed cyclooxygenase-2 protein and gene
expression resulting from inhibition of NFκB activation
(Park et al. 2004 and 2005; Adams et al. 2005; Lee
et al. 2006). Contrary to in vitro experiments (Perečko
et al. 2008; Jančinová et al. 2009b), orally administered
pinosylvin did not alter the activity of neutrophils when
applied in monotherapy. All the beneficial activities of
pinosylvin might intensify the anti-inflammatory activity
of methotrexate, although by itself it was not sufficient
to induce any detectable changes. This might
be due to the lower oral bioavailability of pinosylvin
(Roupe et al. 2006b) and/or to the period of administration
– not sufficient to achieve an effective concentration
in neutrophils.
Besides neutrophils and reactive oxygen species,
the benefit of pinosylvin applied in combined therapy
might involve interference of this stilbene with other
inflammatory cells and mediators. Under in vitro conditions,
pinosylvin decreased the activity of stimulated
RAW 264.7 macrophages and reduced the concentration
of nitric oxide in the supernatant of these cells.
Šmidrkal et al. (2010) observed similar effects in murine
peritoneal macrophages. In the present study, this inhibition
was in parallel with the decreased expression of
inducible NO synthase protein, indicating an interference
of pinosylvin with nitric oxide formation. The
mechanism of this effect may be related to the ability
of this stilbene to suppress NFκB activation and iNOS
gene expression, documented in cancer cells (Park et al.
2005). In contrast to flavonoids (Číž et al. 2008), pinosylvin
did not possess any NO scavenging activity, as
confirmed by amperometrical measurement in cell-free
system. Considering the fact that in the presence of
neutrophils and superoxide, nitric oxide is transformed
into highly reactive peroxynitrite which activates proinflammatory
signalling (Gao 2010) and contributes
to the pathogenesis of arthritis (Abramson 2008), the
ability of pinosylvin to decrease the concentration of
reactive oxygen and nitrogen species can be considered
beneficial and may be involved in the antiinflammatory
activity of this prospective natural substance.
Acknowledgements
The study was supported by grants APVV-0315-07,
APVV-SK-CZ-0034-09, VEGA-2/0003/10, GAČR-
203/07/1227 and ITMS 26240220005.
Reference
1 	 Abramson SB (2008). Nitric oxide in inflammation and pain
associated with osteoarthritis. Arthritis Res Ther. 10: - published
online DOI 10.1186/ar2463.
2 	 Adams M, Pacher T, Greger H, Bauer R (2005). Inhibition of leukotriene
biosynthesis by stilbenoids from Stemona species. J Nat
Prod. 68: 83–85.
3 	 Alarcón de la Lastra C, Villegas I (2005). Resveratrol as an antiinflammatory
and anti-aging agent: Mechanisms and clinical
implications. Mol Nutr Food Res. 49: 405–430.
4 	 Ambrožová G, Pekarová M, Lojek A (2010). Effect of polyunsaturated
fatty acids on the reactive oxygen and nitrogen species
production by raw 264.7 macrophages. Eur J Nutr. 49: 133–139.
5 	 Bauerová K, Poništ S, Dráfi F, Mihalová D, Paulovičová E,
Jančinová V, Nosáľ R (2010). Study of combination of pinosylvin
and methotrexate in the model of adjuvant arthritis: beneficial
effect on clinical and non-clinical parameters. Interdisc Toxicol.
3: A32–A33.
6 	 Cascao R, Rosário HS, Fonseca JE (2009). Neutrophils: warriors
and commanders in immune mediated inflammatory diseases.
Acta Reumatol Port. 34: 313–326.
7 	 Cascao R, Rosário HS, Souto-Carneiro MM, Fonseca JE (2010).
Neutrophils in rheumatoid arthritis: More than simple final effectors.
Autoimmun Rev. 9: 531–535.
8 	 Cronstein BN (2005). Low-dose methotrexate: a mainstay in the
treatment of rheumatoid arthritis. Pharmacol Rev. 57: 163–172.
9 	 Cross A, Barnes T, Bucknall RC, Edwards SW, Moots RJ (2006).
Neutrophil apoptosis in rheumatoid arthritis is regulated by
local oxygen tensions within joints. J Leukoc Biol. 80: 521–528.
10 	Číž M, Pavelková M, Gallová L, Králová J, Kubala L, Lojek A (2008).
The influence of wine polyphenols on reactive oxygen and nitrogen
species production by murine macrophages RAW 264.7.
Physiol Res. 57: 393–402.
11 	Drábiková K, Perečko T, Nosáľ R, Bauerová K, Poništ S, Mihalová D,
Kogan G, Jančinová V (2009). Glucomannan reduces neutrophil
free radical production in vitro and in rats with adjuvant arthritis.
Pharmacol Res. 59: 399–403.
12 	Edwards SW, Hallett MB (1997). Seeing the wood for the trees:
the forgotten role of neutrophils in rheumatoid arthritis. Immunol
Today 18: 320–324.
83Neuroendocrinology Letters  Vol. 31  Suppl. 2  2010  •  Article available online: http://node.nel.edu
Pinosylvin, neutrophils and macrophages
13 	Elmali N, Esenkaya I, Harma A, Ertem K, Turkoz Y, Mizrak B (2005).
Effect of resveratrol in experimental osteoarthritis in rabbits.
Inflamm res. 54: 158–162.
14 	Fairhurst AM, Wallace PK, Jawad ASM, Goulding NJ (2007). Rheumatoid
peripheral blood phagocytes are primed for activation
but have impaired Fc-mediated generation of reactive oxygen
species. Arthritis Res Ther. 9: - published online DOI 10.1186/
ar2144.
15 	Gao Y (2010). The multiple actions of NO. Eur J Physiol. 459:
829–839.
16 	Jančinová V, Perečko T, Nosáľ R, Košťálová D, Bauerová K,
Drábiková K (2009a). Decreased activity of neutrophils in the
presence of diferuloylmethane (curcumin) involves protein
kinase C inhibition. Eur J Pharmacol. 612: 161–166.
17 	Jančinová V, Perečko T, Drábiková K, Nosáľ R, Harmatha J,
Šmidrkal J, Cupaníková D (2009b). Pinosylvin inhibits formation
of reactive oxygen species in human neutrophils. Interdisc Toxicol.
2: 109.
18 	Khanna D, Sethi G, Ahn KS, Pandey MK, Kunnumakkara AB, Sung
B, Aggarwal A, Aggarwal BB (2007). Natural products as a gold
mine for arthritis treatment. Curr Opin Pharmacol. 7: 344–351.
19 	Králová J, Pekarová M, Drábiková K, Jančinová V, Nosáľ R, Číž M,
Lojek A (2008). The effects of dithiaden on nitric oxide production
by RAW 264.7 cells. Interdisc Toxicol. 1: 214–217.
20 	Lee JY, Kim JH, Kang SS, Bae CS, Choi SH (2004). The effects of
alpha-viniferin on adjuvant-induced arthritis in rats. Am J Chin
Med. 32: 521–530. 
21 	Lee SK, Lee HJ, Min HY, Park EJ, Lee KM, Ahn YH, Cho YJ, Pyee
JH (2005). Antibacterial and antifungal activity of pinosylvin, a
constituent of pine. Fitoterapia 76: 258–260.
22 	Lee J, Jung E, Lim J, Lee J, Hur S, Kim SS, Lim S, Hyun CG, Kim
YS, Park D (2006). Involvement of nuclear factor-kappaB in the
inhibition of pro-inflammatory mediators by pinosylvin. Planta
Med. 72: 801–806.
23 	Nakayama H, Yaguchi T, Yoshiya S, Nishizaki T (2010). Resveratrol
induces apoptosis MH7A human rheumatoid arthritis synovial
cells in a sirtuin 1-dependent manner. Rheumatol Int. - published
online DOI 10.1007/s00296-010-1598-8.
24 	Nosáľ R, Jančinová V, Petríková M, Poništ S, Bauerová K (2007).
Suppression of oxidative burst of neutrophils with methotrexate
in rat adjuvant arthritis. Chem listy 101: 243–244.
25 	Park EJ, Min HY, Ahn YH, Bae CM, Pyee JH, Lee SK (2004). Synthesis
and inhibitory effects of pinosylvin derivatives on prostaglandin
E2 production in lipopolysaccharide-induced mouse
macrophage cells. Bioorg Med Chem Lett. 14: 5895–5898.
26 	Park EJ, Ahn YH, Pyee JH, Park HJ, Chung HJ, Min HY, Hong JY,
Kang YJ, Bae IK, Lee SK (2005). Suppressive effects of pinosylvin,
a natural stilbenoid, on cyclooxygenase-2 and inducible nitric
oxide synthase and the growth inhibition of cancer cells. Proc
Amer Assoc Cancer Res. 46: 176.
27 	Perečko T, Jančinová V, Drábiková K, Nosáľ R, Harmatha J (2008).
Structure-efficiency relationship in derivatives of stilbene. Comparison
of resveratrol, pinosylvin and pterostilbene. Neuroendocrinol
Lett. 29: 802–805.
28 	Poništ S, Mihalová D, Jančinová V, Šnirc V, Ondrejičková O, Mascia
C, Poli G, Stančíková M, Nosáľ R, Bauerová K (2010). Reduction of
oxidative stress in adjuvant arthritis. Comparison of efficacy of
two pyridoindoles: stobadine dipalmitate and SMe1.2HCl. Acta
Biochim Pol. 57: 223–228.
29 	Roupe KA, Remsberg CM, Yáňez JA, Davies NM (2006a). Pharmacometrics
of stilbenes: Seguing towards the clinic. Curr Clin
Pharmacol. 1: 81–101.
30 	Roupe KA, Yáňez JA, Teng XW, Davies NM (2006b). Pharmacokinetics
of selected stilbenes: rhapontigenin, piceatannol and
pinosylvin in rats. J Pharm Pharmacol. 58: 1443–1450.
31 	Rovenský J, Stančíková M, Švík K, Bauerová K, Jurčovičová J
(2009). The effects of β-glucan isolated from Pleurotus ostreatus
on methotrexate treatment in rats with adjuvant arthritis. Rheumatol
Int. - published online DOI 10.1007/s00296-009-1258-z 
32 	Simard F, Legault J, Lavoie S, Mshvildadze V, Pichette A (2008).
Isolation and identification of cytotoxic compounds from the
wood of Pinus resinosa. Phytother Res. 22: 919–922.
33 	Šmidrkal J, Harmatha J, Buděšínský M, Vokáč K, Zídek Z,
Kmoníčková E, Merkl R, Filip V (2010). Modified approach for
preparing (E)-Stilbenes related to resveratrol, and evaluation of
their potential immunobiological effects. Collect Czech Chem
Commun. 75: 175–186.
34 	Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli
L (2000). Neutrophils: molecules, functions and
pathophysiological aspects. Lab Invest. 80: 617–653.
35 	Wright HL, Moots RJ, Bucknall RC, Edwards SW (2010). Neutrophil
function in inflammation and inflammatory diseases. Rheumatology
49: 1618–1631.
To cite this article: Neuroendocrinol Lett 2010; 31(Suppl.2):84–90
ORIGINALARTICLE Neuroendocrinology Letters  Volume 31  Suppl. 2  2010
Molecular targets of the natural antioxidant
pterostilbene: effect on protein kinase C, caspase-3
and apoptosis in human neutrophils in vitro
Tomáš Perečko 1, Katarína Drábiková 1, Lucia Račková 1, Milan Číž 4,
Martina Podborská 4, Antonín Lojek 4, Juraj Harmatha 2, Jan Šmidrkal 3,
Radomír Nosáľ 1, Viera Jančinová 1
1 	Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava,
Slovak Republic
2 	Institute of Organic Chemistry and Biochemistry, AS CR, Praha, Czech Republic
3 	Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Praha,
Czech Republic
4 	Institute of Biophysics, Department of Free Radical Pathophysiology, AS CR, Brno, Czech Republic
Correspondence to: Tomáš Perečko, PhD.
Institute of Experimental Pharmacology and Toxicology,
Slovak Academy of Sciences,
Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic.
tel: +421-2-59410672; fax: +421-2-54775928; e-mail: tomas.perecko@savba.sk
Submitted: 2010-09-19	 Accepted: 2010-11-24	 Published online: 2010-00-00
Key words: pterostilbene; reactive oxygen species; neutrophils; protein kinase C;
caspase-3; apoptosis
Neuroendocrinol Lett 2010; 31(Suppl.2):84–90  PMID: 21187824  NEL31S210A14  © 2010 Neuroendocrinology Letters • www.nel.edu
Abstract OBJECTIVE: Pterostilbene, a naturally occurring phenolic derivative, exhibits
various pharmacological effects, e.g. anti-cancerous, antioxidant, anti-inflammatory
and anti-diabetic. Based on our previous study, we assessed the cellular and
molecular effects of pterostilbene on human neutrophils and in cell free systems.
Experimental and theoretical molecular descriptors of stilbene derivatives were
also determined.
Methods: We assessed the antioxidant properties of pterostilbene using cell free
system and computational methods. The effect of pterostilbene on protein kinase
C activation/phosphorylation was detected by special anti-phospho protein
kinase C antibodies. Membrane associated changes determining the life span of
neutrophils and human recombinant caspase-3 assay were examined.
Results: Pterostilbene possessed comparable antioxidant properties as resveratrol
in cell free system. Computational methods were used to establish the
molecular characteristics of stilbene derivatives. The values of electronic parameters
suggest a slight enhancement of electron donor properties of pterostilbene
compared to resveratrol. Phosphorylation and thus activation of protein kinase C
alpha/beta II in activated neutrophils was not decreased by pterostilbene. Pterostilbene
in concentrations of 10–100 μM was found to inhibit the activity of human
caspase-3 purified enzyme and did not influence cell viability significantly.
Conclusion: Pterostilbene, an analog of resveratrol, was identified as a good
natural antioxidant compound. However, reducing the oxidative burst of human
neutrophils during their activation in vitro with pterostilbene does not include
protein kinase C phosphorylation pathway. Pterostilbene showed dose dependent
activation/inhibition of caspase-3 enzyme activity.
85Neuroendocrinology Letters Vol. 31 Suppl. 2 2010 • Article available online: http://node.nel.edu
Fig. 1. Pterostilbene. (A) Structure of pterostilbene. (B) Spin density isosurface map with a contour value of 0.002 of the optimal
conformer of pterostilbene in the form of radical derived from the only position (ΔH = 37.2 kcal/mol).
Pterostilbene & neutrophils
iNTRoDuCTioN
Knowledge that consumption of red wine leads to lower
cardiovascular risk opened the scientific scene for resveratrol
from stilbene type polyphenols (Fauconneau
et al. 1997; Šmidrkal et al. 2001). Chemical similarity
in the stilbene group made us switch from resveratrol
(trans-3,4´,5–trihydroxy-stilbene) to pterostilbene, i.e.
trans-3,5-dimethoxy-4´-hydroxy-stilbene (Figure 1.A).
Both chemical entities exist in cis/trans conformation,
but naturally they occur mostly in the trans form,
which is also more effective as to antioxidant properties
than the cis form (Šmidrkal et al. 2001).
Pterostilbene is of natural origin, contained in leaves
and grapes of Vitis vinifera, berries of Vaccinium spp.,
Pterocarpus marsupianum, etc. (Grover et al. 2005; Paul
et al. 1999). Pterostilbene was reported to scavenge 1,
1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and
to inhibit lipid peroxidation in rat liver microsomes
and there are reports about growth inhibition, antiproliferative
effects and induction of apoptosis by cell cycle
arrest induced by derivatives of stilbene in a number of
normal and cancer cell lines (Pan et al. 2008; Rimando
and Suh 2008; Stivala et al. 2001). Pterostilbene is
assumed to possess anti-inflammatory, antioxidant,
anti-diabetic, antifungal and anti-cancerous effects
(Remsberg et al. 2008; Roupe et al. 2006).
The physiological activity of human neutrophils
plays an important role in fighting intruding microorganisms
or foreign particles (Bissonnette et al. 2008).
Neutrophils may be called “first line defense system”.
In the process of oxidative burst, neutrophils by using
the enzyme NADPH-oxidase produce a  variety of
cytotoxic products, e.g. superoxide, hydrogen peroxide
and hypochloric acid (El-Benna et al. 2008). All
three substances are known as reactive oxygen species
(ROS) and are effective in killing microorganisms.
The whole process of NADPH-oxidase activation is
strictly controlled by various regulating/phosphorylating
enzymes, such as p21-activated kinase, phosphatidyl
inositol 3-kinase and nuclear factor κB (El-Benna
et al. 1996; Myhre et al. 2009; Sheppard et al. 2001).
A key enzyme in NADPH-oxidase activation is protein
kinase C (PKC) (Dekker et al. 2000). Increased production
of ROS during inflammation could be harmful to
surrounding tissues. This has been observed in chronic
inflammatory diseases, e.g. rheumatoid arthritis (Cross
et al. 2006). The importance of the regulation of activated
human neutrophils is the subject of our study.
Activated neutrophils represent a good model for testing
inflammation.
The present study is based on our previous research
on the generation of ROS by activated neutrophils by
using chemiluminescence assay (Perečko et al. 2008).
To evaluate the antioxidant capacity of pterostilbene,
we used oxygen radical absorbance capacity (ORAC)
and hydroxyl radical averting capacity (HORAC) assays
(Číž et al. 2010). Moreover, we examined the effect of
pterostilbene on the life-span of isolated human neutrophils.
Delayed apoptosis of neutrophils may play
a role in the injury of surrounding tissue in chronic
inflammatory diseases, e.g. rheumatoid arthritis (Wong
et al. 2009). Finally, we investigated the effect of pterostilbene
on phosphorylation of PKC of neutrophils
pre-activated with phorbol-myristate-acetate, which
activates cells via PKC (Klink et al. 2009). For better
understanding the biological effects of pterostilbene, its
molecular descriptors were estimated.
MATeRiALs AND MeTHoDs
Chemicals
Pterostilbene and resveratrol were prepared by targeted
regioselective synthesis, both compounds purely as
trans isomers (Šmidrkal et al. 2010). Phorbol-myristateacetate
(PMA) was purchased from Sigma (Steinheim,
Germany). Polyclonal antibody against phosphorylated-PKC-alpha/betaII
(Thr638/641) was purchased
from Cell Signaling (Danvers, MA, USA), secondary
anti-rabbit antibody and Lumigen Detection Reagent
were supplied by GE Healthcare Life Sciences (former
Amersham), Little Chalfont, UK. Human AnnexinV/
O
O
OH
CH3
H3C
A B
86 Copyright © 2010  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
T. Perečko, K. Drábiková, L. Račková, M. Číž, M. Podborská, A. Lojek, J. Harmatha, J. Šmidrkal, R. Nosáľ, V. Jančinová
FITC Kit was purchased from Bender MedSystems,
Vienna, Austria. Caspase-Glo 3/7 Assay was from Promega
(Madison, WI, USA), human purified caspase-3
was from Enzo Life Sciences, Lausen, Switzerland.
All other products are available commercially or their
origin is mentioned in the text.
Computational methods for pterostilbene and resveratrol
The lowest energy molecular conformations of the
compounds tested were calculated using Conformational
Search module in HyperChem molecular modeling
software (Račková et al. 2005) using Austin model
1, and Polak-Ribiere conjugate gradient algorithm
with 0.01 convergence limit in vacuum. For optimal
conformers of antioxidants, the heat of formation was
calculated. The ΔH values were calculated as described
previously (Račková et al. 2005). Further calculated
parameters of the compounds tested included energy
values of the highest occupied molecular orbital
(HOMO), energy of the lowest unoccupied molecular
orbital (LUMO), HOMO–LUMO energy gap (Δε)
and spin density (SD) belonging to the oxygen radical
derived from OH groups, which characterizes the distribution
of the electron spin and therefore decides the
stability of radicals (Cao et al. 2003).
Determination of Rm values
The lipophilicity parameters represented by Rm values
were measured by reversed-phase thin layer chromatography.
The mobile phase consisted of diluted acetic
acid (pH 2.5) mixture with acetonitril (20:80, v:v)
(Király-Véghely et al. 2004). The stationary phase was
obtained by impregnation of the layer of Silica gel G
F254 plates with 5% solution of liquid paraffin in ether.
The compounds were dissolved in methanol and about
1 μg of the compound was spotted onto the plates. A
migration of 10 cm was obtained by spotting the compound
on a line 2 cm from the lower edge of the plate.
The developed plates were dried and the compounds
were detected in UV light at 254 nm. The Rm values
were calculated by the formula: Rm=log(1/RF–1).
Antioxidant assays
The protective effects of pterostilbene and resveratrol
(0.01–100 μM) were measured by comparing the area
under curve of the sample to that of a known antioxidant,
trolox for ORAC assay and gallic acid for
HORAC assay. The loss of fluorescein fluorescence
indicated the formation of peroxyl radicals (ORAC)
or hydroxyl radicals (HORAC) in the sample. Production
of peroxyl radicals was induced by 2, 2´-azobis-(2amidino-propane)-dihydrochloride
(AAPH) at 37 °C.
The HORAC assay reveals the metal-chelating activity
of polyphenols, and thus the protective action against
hydroxyl radical formation. Complex Co (II) was used
as an initiator. High ORAC and HORAC values indicated
that the sample tested possessed a high potency
of antioxidant activity (Číž et al. 2010). The values of
ORAC are expressed as relative trolox equivalent (TE),
HORAC as relative gallic acid equivalent (GAE). Results
are expressed as means of triplicates in three independent
measurements.
PKC blotting
Human neutrophils were isolated as described previously
(Jančinová et al. 2009). Neutrophil suspension
(100 μl) containing 5×106 cells was preincubated at
37°C for 60 seconds with different concentrations of
pterostilbene (10 or 100 μM) prior to addition of PMA
(0.15 μM). Incubation with PMA (60 s) was stopped by
using a lysing medium containing protease and phosphatase
inhibitors (Na3VO4 and NaF). The suspension
was sonicated at 4°C for 20 minutes and centrifuged at
18 625×g at 4°C for 5 min. Finally, the supernatant was
taken for blotting assay. Total protein was measured
using Bradford Dye Reagent detection kit from Bio-Rad.
For the assay, the supernatant was boiled in sample
buffer (0.05 M Tris, 2% SDS, 2.5% mercaptoethanol)
containing 0.01% bromphenol-blue. The samples (20 μl
with 20 μg of protein fraction) were loaded on 10%
polyacrylamide gel. Separated proteins were transferred
onto a polyvinylidene difluoride membrane (Millipore).
The blot containing the transferred proteins was
blocked in 1% BSA (Sigma-Aldrich) buffer followed
by incubation with primary anti-phospho-PKC-alpha/
beta II (Thr638/641) antibody (1:8 000) and secondary
anti-rabbit antibody (1:10 000). After washing in TBS,
the proteins were detected with Lumigen DetectionReagent
kit, scanned and measured densitometrically
using free ImageJ program.
Analysis of apoptosis
Citrated whole human blood was collected from healthy
male volunteers. Dextrane (3%) was added (blood 2:1
dextrane) and centrifugated at 10×g at room temperature.
One ml of buffy coat containing mainly leukocytes
was collected and stored on ice before use. The
cells were counted on hemocytometer (Coulter Counter),
focusing on granulocytes. The cell suspension was
then dissolved to reach 200 000 neutrophils per sample.
Three different concentrations of pterostilbene (1, 10
and 100 μM) and a control sample were incubated at
37°C for 10 min. The cells were stained with Annexin-
V  conjugated with FITC (fluorescein isothiocyanate)
(BenderMedSystems) in dark at 4°C for 10 min, followed
by staining with propidium iodide (1μg/ml) and
analyzed immediately by Beckman Coulter Cytomics
FC500 cytometer. All samples were analyzed under the
same conditions (gains, volts). From the granulocytic
area, 5 000 cells were gated and analyzed.
Caspase-3 activity
Following caspase cleavage of the Z-DEVD-aminoluciferin
substrate, resulting in the reaction of luciferase
with amino-luciferin, the measurement of light production
was made on the Luminometer Immunotech
87Neuroendocrinology Letters  Vol. 31  Suppl. 2  2010  •  Article available online: http://node.nel.edu
Pterostilbene & neutrophils
Tab. 1. Electronic and physicochemical characteristics of
pterostilbene and resveratrol.
Pterostilbene Resveratrol
ΔH (kcal/mol) 37.17 37.39
εHOMO (eV) –8.45 –8.53
εLUMO (eV) –0.417 –0.517
Δe (eV) 8.033 8.013
Rm 1.45 0.21
ΔH – parameter of 4´-OH bond strength (corresponding to
minimum value of DH with regard to values of 3 and 5 –OH bonds
in resveratrol), εHOMO – energy of highest occupied molecular
orbital, εLUMO – energy of lowest unoccupied molecular orbital,
Δe - energy difference between the HOMO and LUMO, Rm –
lipophilicity index.
Tab. 2. Antioxidant profile of pterostilbene and resveratrol.
Pterostilbene (μM) ORAC μM TE HORAC μM GAE
100 14.75 ± 2.58 85.48 ± 15.65
10 5.45 ± 2.16 20.58 ± 1.30
1 0.94 ± 0.62 n.d.
Resveratrol (μM) ORAC μM TE HORAC μM GAE
100 17.98 ± 0.36 113.92 ± 23.18
10 10.71 ± 2.63 88.37 ± 13.66
1 2.84 ± 1.36 2.41 ± 1.50
ORAC (peroxyl radical quenching) is measured as trolox equivalent
(TE), HORAC (hydroxyl radical quenching) as gallic acid equivalent
(GAE). Results are expressed as Mean±SEM of triplicates in three
independent measurements. n.d. for not detectable.
LM-01T. According to the manufacturers´ instructions,
10 μl of 0.1 IU caspase was added to 20 μl aliquots of
pterostilbene and buffer solution, finally 50 μl of Caspase-Glo
3/7 Reagent was added, the mixture was measured
for 60 minutes and the activity of caspase-3 (EC
3.4.22.56) was identified. The vehiculum solution for
pterostilbene i.e. competent concentrations of NaOH,
were also tested.
Statistics
Data are expressed as mean±SEM, unless stated otherwise.
Statistical analysis was performed using Student´s
t-test to examine differences between treatments and
control. Differences were considered to be statistically
significant when p<0.05 (*) or p<0.01 (**).
Results
Computer assisted evaluation of pterostilbene and resveratrol
properties and determination of Rm
With regard to its relation to the potential of radical
scavenging effect, we computed the parameter energy of
HOMO for pterostilbene and resveratrol. The εHOMO
expresses the easiness of abstraction of an electron
from the neutral molecule, which may be involved in
the mechanism of reaction with free radicals (Cao et
al. 2003; Velkov et al. 2007). The value of εHOMO for
pterostilbene was –8.45 eV. Compared to resveratrol,
a typical representative of natural stilbene derivatives
(εHOMO = –8.53 eV), pterostilbene is more effective
in electron-donating processes. Furthermore, the value
of parameter of 4´–OH bond strength, ΔH (Korzekwa
et al. 1990), was slightly lower for pterostilbene than for
resveratrol molecule (37.17 kcal/mol vs 37.39 kcal/mol,
respectively) (Table 1).
However, the values of HOMO-LUMO energy difference
(a characteristic of the molecule excitability)
for both molecules differed just by 0.02 eV (8.033 eV
for pterostilbene vs 8.013 eV for resveratrol), indicating
similar chemical reactivity of both compounds (Table 1).
0.0
20.0
40.0
60.0
80.0
100.0
Density(AU)
spo PMA PMA PMA
–– PTE 10 PTE 100
PKCα/βII
beta actin
0
25
50
75
100
125
150
1 3 10 30 100
Concentration (μM)
%ofcontrolsample
**
**
**
+
Fig. 2. Effect of pterostilbene on PKC phosphorylation.
Densitometric evaluation of protein kinase C phosphorylation
from control, PMA (0.15 μM) and pterostilbene (10 and 100 μM)
treated isolated human neutrophils. (Mean±SEM, n=4)
(spo – control, PTE – pterostilbene, PMA – phorbol-myristateacetate,
AU – Arbitrary Units).
Fig. 3. Effect of pterostilbene on human recombinant caspase-3
activity. Effect of different concentrations of pterostilbene
(1–100 μM) on human recombinant caspase-3 activity shown
as percentage of control sample. (Mean±SEM, n=5). +p<0.05
for activation, *p<0.05 and ** p<0.01 for inhibition (versus
vehiculum).
88 Copyright © 2010  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
T. Perečko, K. Drábiková, L. Račková, M. Číž, M. Podborská, A. Lojek, J. Harmatha, J. Šmidrkal, R. Nosáľ, V. Jančinová
The spin density isosurface map shows the efficient
delocalization of the radical resulting from the only
4´-OH group through the extended conjugation system
of the stilbenoid structure (Figure 1.B). Correspondingly
spin density (SD) belonging to the oxygen radical derived
from 4`-OH groups showed the same values for the
molecules of both resveratrol and pterostilbene (0.101).
Due to substitution by two methyl-groups, pterostilbene
showed the higher value of lipophilicity parameter
(Rm = 1.45) than resveratrol (R m = 0.21) (Table 1).
Antioxidant activity assays
The ORAC assay provides data to measure the quenching
of peroxyl radicals expressed as trolox equivalent.
HORAC measures quenching of hydroxyl radicals (due
to metal-chelating activity) expressed as gallic acid
equivalent. Results on pterostilbene and resveratrol
antioxidant properties are expressed in Table 2. The
antioxidant capacity of 10 μM pterostilbene is equal to
that of 5.45 μM trolox and 20.58 μM gallic acid standards.
Pterostilbene of 100 μM concentration possessed
less antioxidant capacity in comparisson with trolox
(ORAC), but using HORAC method pterostilbene
was as equal as gallic acid standard (85.48 μM). However,
resveratrol in concentration of 1, 10 and 100 μM
is better antioxidant than gallic acid (HORAC), using
ORAC, the antioxidant effect diminished.
PKC response to incubation with pterostilbene
To get further insight into molecular mechanisms
underlying inhibition of ROS generation in whole
human blood and isolated neutrophils by pterostilbene,
we examined the effect of pterostilbene on the phosphorylation
of PKC α/β II. No significant changes in
PKC α/β II (Thr638/641) phosphorylation after PMA
stimulus were achieved by pterostilbene in either concentration
(10 and 100 μM) (Figure 2).
Effect of pterostilbene on life-span of human neutrophils
The percentage of viable and apoptotic cells was calculated
by flow cytometer measurement after staining the
cells with FITC conjugated Annexin-V plus propidium
iodide (PI). Only Annexin positive cells were considered
for apoptotic (also mentioned as pre-apoptotic) cells
and double positive cells (Annexin+/PI+) were considered
for late-apoptotic or dead cells. Pterostilbene in any
concentration tested (1, 10 and 100 μM) did not change
neutrophil viability significantly (data not shown).
The role of pterostilbene on caspase-3 activity
Figure 3 shows biphasic concentration-dependent effect
of pterostilbene on the human recombinant caspase-3
activity: in the concentration of 1 μM, pterostilbene
induces the activity of caspase-3 significantly, in concentrations
of 10, 30 and 100 μM, pterostilbene inhibits
the activity of the enzyme. The effect of appropriate
vehiculum concentrations (4–400 μM NaOH) did not
significantly influence the activity of caspase-3.
Discussion
The antioxidant properties of pterostilbene have, over
the years, attracted the interest of many researcher
groups however the effect of pterostilbene on human
neutrophils has not yet been fully investigated. Pterostilbene
is a dimethylether analogue of the well-known
stilbene derivative resveratrol. These compounds are
mainly synthetized in plants after stress stimuli (e.g.
irradiation, microorganism infection) (Keller et al.
2003) and act as phytoalexins (Breuil et al. 1999). But
there are few reports on pterostilbene effects on human
cells. Pterostilbene has been widely discussed because
of its anti-cancer and chemopreventive properties in
different in vivo and in vitro cancer models and cultured
cell lines (Cichocki et al. 2008; Rimando and Suh
2008; Tolomeo et al. 2005). Pterostilbene exhibits also
immunomodulatory (Šmidrkal et al. 2010) and antiinflammatory
effects (Cichocki et al. 2008). Based on
this information and our previous studies, we investigated
antioxidant effects of pterostilbene in cell free
systems and focused also on the effects of pterostilbene
on activated human neutrophils in vitro. Electronic
parameters for the minimum energy conformers of
pterostilbene in comparison to resveratrol were calculated.
Lipophilicity parameters for the compounds
tested were evaluated as well.
The polyphenolic structure of stilbenes, and thus of
pterostilbene, justifies its antioxidant testing. Pterostilbene
has been suggested to be a good antioxidant in different
cell free system methods, e.g. based on formation
of DPPH. In the study of Stivala et al. (2001) pterostilbene
gave a value comparable to trans-resveratrol
in antioxidant testing, using DPPH assay. Our experiments
with the ORAC and HORAC method describe
the antioxidant effects against peroxyl (ORAC) and
hydroxyl (HORAC) radicals in cell free system. In the
concentrations of 1, 10 and 100 μM, pterostilbene was
less effective in comparison to trolox standard. Remsberg
et al. (2008) showed better prevention of oxidation
in the lower concentration of pterostilbene. This may be
due to the pro-oxidant activity in higher concentrations
that is in agreement with its higher value of εLUMO
(–0.417 eV) compared to resveratrol (–0.517 eV)
(Table 1). Our study suggests that substitution of both
3 and 5 hydroxy-groups with methoxy-groups does not
decrease the antioxidant properties of pterostilbene in
cell free system dramatically (compared to resveratrol),
though resveratrol is showing better antioxidant properties
in HORAC assay (Table 2). The 4´-hydroxy group
in pterostilbene is able to assure sufficient antioxidant
activity. However 4´-methoxy derivative was less effective
in the study of Stivala et al. (2001). We may hypothesize
that the 4´-hydroxy group may play a crucial role
in antioxidant effects.
For better understanding of the antioxidant effects
of pterostilbene, we calculated the molecule parameters
for the minimum energy conformers of this molecule
89Neuroendocrinology Letters  Vol. 31  Suppl. 2  2010  •  Article available online: http://node.nel.edu
Pterostilbene & neutrophils
in comparison to the well-known stilbene representative
resveratrol. The structure of pterostilbene (Figure
1.A) enables only one proton subtraction in 4´-position,
thus forming a phenoxy-radical. The distribution
of spin density can have a decisive effect on the stability
of the phenoxyl radicals derived from stilbenoid structure
(Cao et al. 2003). Comparably to resveratrol, the
abstraction of proton from OH in para-position to the
double-bond of ethene group allows efficient electron
delocalization of the generated radical through the
pterostilbene molecule, as shown in the spin density
map (Figure 1.B). Moreover, the spin densities (SD)
belonging to the oxygen radical derived from 4´-OH
group for both stilbene analogues showed the same
value. Thus, the methylation of the free OH groups at
meta-position of ring A in pterostilbene can not significantly
reduce the scavenging activity compared to resveratrol.
In general, both pterostilbene and resveratrol
showed comparable values of parameters εHOMO, ΔH
and Δε (HOMO-LUMO energy gap), suggesting similar
free radical scavenging properties of both stilbene
derivatives. This is in accordance with the results of
Stivala et al. (2001), showing comparable DPPH radical
scavenging activity of both compounds in a chemical
system.
As discussed in the two paragraphs above, the physicochemical
characteristics of both derivatives tested
are conformable, but according to our results with chemiluminescence
assay (Perečko et al. 2008), the stilbenes
tested vary in biological medium. Substitution of
two hydroxyl groups with methoxy-groups increased
lipophilicity parameter Rm of resveratrol from 0.21 to
1.45 for pterostilbene. This may enhance the bioavailability
of pterostilbene in contrast to the low bioavailability
of resveratrol (Cichocki et al. 2008). On the
other side lipophilicity may influence the transition of
substances through cell membrane into cytosol. Also
the number and position of hydroxyl groups play a
role in the antioxidant effects of polyphenols in different
cell assays. Taking into consideration the effects
of different resveratrol derivatives on the production
of thiobarbituric acid reactive substances (TBARS) in
normal human fibroblasts (Stivala et al. 2001), pterostilbene
was as good as resveratrol. Its 4´-methoxy
derivative as well as 3, 4´, 5-trimethoxystilbene did
not exert a significant inhibition of TBARS production
(Stivala et al. 2001). These results support our findings
with chemiluminescence assay in whole blood. The 3,
5-methoxy groups increased the antioxidant properties
of pterostilbene compared to resveratrol in whole
human blood. However, inhibition of extracellular and
intracellular chemiluminescence with pterostilbene
was lower than with resveratrol and the difference
was more significant for intracellular compartment
(Perečko et al. 2008). In agreement with the biological
activity-lipophilicity parabolic findings (Hansch
and Clayton 1973) during transportation through the
membrane system, the entrapment of more hydrophobic
pterostilbene in the lipid phase of outer membrane
could diminish its inhibitory effect on intracellular
ROS production. Thus, free hydroxy-groups of resveratrol
may be appropriate for antioxidant activity in
respect of its intracellular availability directed by lipophilicity
parameter.
In the attempt to elucidate the molecular mechanism
of reducing the production of reactive oxygen
species, we examined the effect of pterostilbene on
activation/phosphorylation of PKC. Phosphorylation
of PKC is an activation marker and antecedent step of
phosphorylation of NADPH-oxidase subunits. Pterostilbene
is known to affect e.g. the p38-phosphorylation
pathway in breast cancer cell line (Pan et al. 2009), but
the effect on human neutrophil PKC phosphorylation
has not been established. In our study, pterostilbene
did not change the phosphorylation of PKC α/β II of
isolated human neutrophils activated by PMA, which is
known to activate cells just via activation of PKC (Klink
et al. 2009). We are suggesting that other mechanisms
in reducing of ROS production may be involved, e. g.
inhibition of NADPH-oxidase.
The in vitro Annexin-V/propidium iodide staining
assay revealed no significant effects of pterostilbene in
concentrations tested on human neutrophil cell death.
This is in agreement with other studies (Stivala et al.
2001). Remsberg et al. (2008) showed apoptotic effect
of pterostilbene on different cancer cell lines, suggested
also by other studies (Tolomeo et al. 2005), yet pointing
out that pterostilbene, and thus other stilbene derivatives,
was not cytotoxic against e.g. normal human
umbilical vein epithelial cells. Generally, pterostilbene,
comparable to resveratrol, was found to have antiproliferative,
cytostatic activity on different cell lines, but it
did not exhibit cytotoxic activity in the concentrations
tested (Stivala et al. 2001). This was acknowledged by
our study using ATP-cytotoxicity test (Perečko et al.
2008). According to caspase-3 activity assay, Pan et al.
(2007) showed activation of caspase-2,-3,-8 and -9 by
pterostilbene in human gastric carcinoma AGC cells.
In our experiment, pterostilbene in concentration of
1 μM showed significant increase of activity of caspase-3,
while in concentrations of 10, 30 and 100 μM
it inhibited caspase-3 activity. This result is in agreement
with no apoptotic changes caused by pterostilbene
in the concentrations tested on human neutrophil
viability studied by flow cytometry. Nevertheless caspase
transduction is not the only apoptotic signaling
pathway.
Taking into account the above presented results and
discussion, pterostilbene represents a derivative with
significant antioxidant effect. The structure-activity
relationship analysis may be used to advantage in rational
design and synthesis of derivatives with desired
antioxidant, anti-inflammatory and anti-proliferative
properties.
90 Copyright © 2010  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
T. Perečko, K. Drábiková, L. Račková, M. Číž, M. Podborská, A. Lojek, J. Harmatha, J. Šmidrkal, R. Nosáľ, V. Jančinová
Acknowledgements
This study was supported by grants VEGA 2/0003/10,
APVV-0315-07, GAČR 203/07/1227, OC08058 (MEYS,
CR) and APVV-SK-CZ-0034-09. Authors are thankful
to Prof. Magda Kouřilová for language corrections.
References
1 	 Bissonnette SA, Glazier CM, Stewart MQ, Brown GE, Ellson CD,
Yaffe MB (2008). Phosphatidylinositol 3-phosphate-dependent
and -independent functions of p40phox in activation of the
neutrophil NADPH oxidase. Journal of Biological Chemistry. 283:
2108–2119.
2 	 Breuil AC, Jeandet P, Adrian M, Chopin F, Pirio N, Meunier P, Bessis
R (1999). Characterization of a pterostilbene dehydrodimer producedbylaccaseofBotrytiscinerea.Phytopathology.
89:298–302.
3 	 Cao H, Pan X, Li C, Zhou C, Deng F, Li T (2003). Density functional
theory calculations for resveratrol. Bioorganic and Medicinal
Chemistry Letters. 13: 1869–1871.
4 	 Cichocki M, Paluszczak J, Szaefer H, Piechowiak A, Rimando AM,
Baer-Dubowska W (2008). Pterostilbene is equally potent as
resveratrol in inhibiting 12-O-tetradecanoylphorbol-13-acetate
activated NFκB, AP-1, COX-2, and iNOS in mouse epidermis.
Molecular Nutrition & Food Research. 52: 62–70.
5 	 Cross A, Barnes T, Bucknall RC, Edwards SW, Moots RJ (2006).
Neutrophil apoptosis in rheumatoid arthritis is regulated by local
oxygen tensions within joints. Journal of Leukocyte Biology. 80:
521–528.
6 	 Číž M, Čížová H, Denev P, Kratchanova M, Slavov A, Lojek A (2010).
Different methods for control and comparison of the antioxidant
properties of vegetables. Food Control. 21: 518–523.Dekker LV,
Leitges M, Altschuler G, Mistry N, McDermott A, Roes J, Segal
AW (2000). Protein kinase C-beta contributes to NADPH oxidase
activation in neutrophils. Biochemical Journal. 347: 285–289.
7 	 El-Benna J, Dang PM, Gougerot-Pocidalo MA (2008). Priming of
the neutrophil NADPH oxidase activation: role of p47phox phosphorylation
and NOX2 mobilization to the plasma membrane.
Seminars in Immunopathology. 30: 279–289.
8 	 El-Benna J, Han J, Park JW, Schmid E, Ulevitch RJ, Babior BM
(1996). Activation of p38 in stimulated human neutrophils: phosphorylation
of the oxidase component p47phox by p38 and ERK
but not by JNK. Archives of Biochemistry and Biophysics. 334:
395–400.
9 	 Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A,
Merillon JM (1997). Comparative study of radical scavenger and
antioxidant properties of phenolic compounds from Vitis vinifera
cell cultures using in vitro tests. Life Sciences. 61: 2103–2110.
10 	Grover JK, Vats V, Yadav SS (2005). Pterocarpus marsupium
extract (Vijayasar) prevented the alteration in metabolic patterns
induced in the normal rat by feeding an adequate diet containing
fructose as sole carbohydrate. Diabetes, Obesity and Metabolism.
7: 414–420.
11 	Hansch C and Clayton JM (1973). Lipophilic character and biological
activity of drugs II: The parabolic case. Journal of Pharmaceutical
Sciences. 62: 1–21.
12 	Jančinová V, Perečko T, Nosáľ R, Košťálová D, Bauerová K,
Drábiková K (2009). Decreased activity of neutrophils in the presence
of diferuloylmethane (curcumin) involves protein kinase C
inhibition. European Journal of Pharmacology. 612: 161–166.
13 	Keller M, Viret O, Cole FM (2003). Botrytis cinerea infection in
grape flowers: Defense reaction, latency, and disease expression.
Phytopathology. 93: 316–322.
14 	Király-Véghely Z, Kátay G, Tyihák E, Merillon JM (2004). Separation
of stilbene isomers from red wine by overpressured-layer chromatography.
Journal of Planar Chromatography. 17: 4–8.
15 	Klink M, Jastrzembska K, Bednarska K, Banasik M, Sulowska Z
(2009). Effect of nitric oxide donors on NADPH oxidase signaling
pathway in human neutrophils in vitro. Immunobiology. 214:
692–702.
16 	Korzekwa KR, Jones JP, Gillette JR (1990). Theoretical studies on
cytochrome P-450 mediated hydroxylation: A predictive model
for hydrogen atom abstractions. Journal of the American Chemical
Society. 112: 7042–7046.
17 	Myhre O, Mariussen E, Reistad T, Voie OA, Aarnes H, Fonnum F
(2009). Effects of polychlorinated biphenyls on the neutrophil
NADPH oxidase system. Toxicology Letters. 187: 144–148.
18 	Pan MH, Chang YH, Badmaev V, Nagabhushanam K, Ho CT (2007).
Pterostilbene induces apoptosis and cell cycle arrest in human
gastric carcinoma cells. Journal of Agricultural and Food Chemistry.
55: 7777–7785.
19 	Pan MH, Lin YT, Lin CL, Wei CS, Ho CT, Chen WJ (2009). Suppression
of Heregulin-{beta}1/HER2-Modulated Invasive and
Aggressive Phenotype of Breast Carcinoma by Pterostilbene via
Inhibition of Matrix Metalloproteinase-9, p38 Kinase Cascade and
Akt Activation. Evidence-based Complementary and Alternative
Medicine. doi:10.1093/ecam/nep093.
20 	Pan Z, Agarwal AK, Xu T, Feng Q, Baerson SR, Duke SO, Rimando
AM (2008). Identification of molecular pathways affected by
pterostilbene, a natural dimethylether analog of resveratrol. BMC
Medical Genomics. 1.
21 	Paul B, Masih I, Deopujari J, Charpentier C (1999). Occurrence
of resveratrol and pterostilbene in age-old darakchasava, an
ayurvedic medicine from India. Journal of Ethnopharmacology.
68: 71–76.
22 	Perečko T, Jančinová V, Drábiková K, Nosáľ R, Harmatha J (2008).
Structure-efficiency relationship in derivatives of stilbene. Comparison
of resveratrol, pinosylvin and pterostilbene. Neuroendocrinology
Letters. 29: 802–805.
23 	Račková L, Firaková S, Košťálová D, Štefek M, Sturdik E, Majeková
M (2005). Oxidation of liposomal membrane suppressed by flavonoids:
quantitative structure-activity relationship. Bioorganic &
Medicinal Chemistry. 13: 6477–6484.
24 	Rimando AM and Suh N (2008). Biological/chemopreventive
activity of stilbenes and their effect on colon cancer. Planta
Medica. 74: 1635–1643.
25 	Remsberg CM, Yańez JA, Ohgami Y, Vega-Villa KR, Rimando AM,
Davies NM (2008). Pharmacometrics of Pterostilbene: Preclinical
Pharmacokinetics and Metabolism, Anticancer, Antiinflammatory,
Antioxidant and Analgesic Activity. Phytotherapy Research.
22: 169–179.
26 	Roupe KA, Remsberg CM, Yańez JA, Davies NM (2006). Pharmacometrics
of Stilbenes: Seguing Towards the Clinic. Current Clinical
Pharmacology. 1: 81–101.
27 	Sheppard FR, Kelher MR, Moore EE, McLaughlin NJ, Banerjee A,
Silliman CC (2005). Structural organization of the neutrophil
NADPH oxidase: phosphorylation and translocation during priming
and activation. Journal of Leukocyte Biology. 78: 1025–1042.
28 	Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti
L, Pagnoni UM, Albini A, Prosperi E, Vannini V (2001). Specific
structural determinants are responsible for the antioxidant activity
and the cell cycle effects of resveratrol. Journal of Biological
Chemistry. 276: 22586–22594.
29 	Šmidrkal J, Filip V, Melzoch K, Hanzlíková I, Buckiová D, Křísa B
(2001). Resveratrol. Chemicke Listy. 95: 602–609.
30 	Šmidrkal J, Harmatha J, Buděšínský M, Vokáč K, Zídek Z,
Kmoníčková E, Merkl R, Filip V (2010). Modified approach for preparing
(E)-stilbenes related to resveratrol, and evaluation of their
potential immunobiological effects. Collection of Czechoslovak
Chemical Communications. 75: 175–186.
31 	Tolomeo M, Grimaudo S, Di Cristina A, Roberti M, Pizzirani D, Meli
M, Dusonchet L, Gebbia N, Abbadessa V, Crosta L, Barucchello
R, Grisolia G, Invidiata F, Simoni D (2005). Pterostilbene and
3´-hydroxypterostilbene are effective apoptosis-inducing agents
in MDR and BCR-ABL-expressing leukemia cells. International
Journal of Biochemistry and Cell Biology. 37: 1709–1726.
32 	Velkov ZA, Kolev MK, Tadjer AV (2007). Modeling and Statistical
Analysis of DPPH Scavenging Activity of Phenolics. Collection of
Czechoslovak Chemical Communications. 72: 1461–1471.
33 	Wong SH, Francis N, Chahal H, Raza K, Salmon M, Toellner D, Lord
JM (2009). Lactoferrin is a survival factor for neutrophils in rheumatoid
synovial fluid. Rheumatology (Oxford). 48: 39–44.
Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 106041, 7 pages
http://dx.doi.org/10.1155/2013/106041
Research Article
The Effects of Pterostilbene on Neutrophil Activity in
Experimental Model of Arthritis
Tomas Perecko,1,2
Katarina Drabikova,1
Antonin Lojek,2
Milan Ciz,2
Silvester Ponist,1
Katarina Bauerova,1
Radomir Nosal,1
Juraj Harmatha,3
and Viera Jancinova1
1
Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska Cesta 9,
841 04 Bratislava, Slovakia
2
Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Kralovopolska 135, 612 65 Brno, Czech Republic
3
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo Namesti 2,
166 10 Prague, Czech Republic
Correspondence should be addressed to Tomas Perecko; tomas.perecko@ibp.cz
Received 10 July 2013; Accepted 25 August 2013
Academic Editor: Fausto Catena
Copyright © 2013 Tomas Perecko et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
It has been demonstrated that pterostilbene inhibits reactive oxygen species production in neutrophils in vitro. However, little
is known about its effects on neutrophils during inflammation in vivo. In this study, the effect of pterostilbene on neutrophil
activity was investigated in experimental arthritis model. Lewis rats were injected by a single intradermal injection of heat-killed
Mycobacterium butyricum in Freund’s adjuvant to develop arthritis. Another group of arthritic animals received pterostilbene
30 mg/kg, daily, p.o. The number and activity of neutrophils in blood were measured on a weekly basis during the whole
experiment. Moreover, the total radical trapping potential in plasma was measured at the end of the experiment. In the pterostilbene
treated arthritic group, the treatment significantly lowered the number of neutrophils in blood on days 14 and 21 without
significant downregulation of neutrophil oxidative burst. Pterostilbene nonsignificantly increased total radical trapping potential
in arthritic animals. These results indicate that the promising effects of pterostilbene on reactive oxygen species operate by different
mechanisms in vitro and in the animal model of inflammation. In conclusion, the positive effects of pterostilbene in the model of
arthritis may be attributed to regulation of neutrophil number.
1. Introduction
Neutrophils are a crucial part of the innate immune system.
Once activated, they adhere to and migrate through the
endothelium to get to the inflamed tissue. Neutrophils contain
NADPH-oxidase which generates superoxide anion [1].
In response to infection or foreign particles, neutrophils produce
reactive oxygen species (ROS) to destroy the invading
pathogens, which is known as oxidative burst of neutrophils
[2]. The dark side of neutrophil activity is its contribution to
tissue damage. This is due to overproduction of ROS seen
in many inflammatory diseases, for example, rheumatoid
arthritis [3–5].
Rheumatoid arthritis is a chronic autoimmune inflammatory
disease characterised by bone erosion and cartilage
damage with synovial hyperplasia and pain [6]. Neutrophils
are present in the synovial fluid and on the pannus-cartilage
interface in arthritis [7, 8]. In addition, isolated neutrophils
from arthritic patients showed preactivation (priming) which
may be due to the presence of different cytokines in the
synovial fluid [3]. Thus by producing ROS, activated neutrophils
could contribute to joint destruction (Figure 1). This
highlights the importance of searching for therapeutic agents
capable of controlling the oxidative burst of neutrophils
in neutrophil-dominant inflammatory diseases. Recently,
significant research interest has been focused on resolution
as a means to treat inflammatory diseases [9]. Resolution was
thought to be a passive process caused by reduction of proinflammatory
chemokines. Now, however, it is believed to be
an active process with significant impact on neutrophil functionality
[10, 11]. During resolution, in particular, the activity
of neutrophils is downregulated [12]. To study the influence
2 BioMed Research International
Cartilage
Bone
Synovial fluid
Joint capsule
Synovial
membrane
Swollen joint capsule
Inflamed synovial
membrane
Cartilage and bone
destruction
Normal joint Arthritic joint
Invaded neutrophils
producing ROS
Figure 1: The role of neutrophils in pathogenesis of arthritis.
OH
CH3O
OCH3
Figure 2: Structure of pterostilbene.
of neutrophils on pathogenesis of rheumatoid arthritis and
to investigate the effects of new compounds on this process,
different animal models of arthritis are used. One of them is
the model of adjuvant arthritis induced by heat-inactivated
Mycobacterium butyricum in Freund’s adjuvant [13]. Mohr
et al. showed that under normal conditions, there were no
neutrophils in the articular capsule of nonarthritic animals,
while the inflamed synovial and capsular tissue of adjuvant
injected animals is heavily infiltrated with neutrophils [7].
We used this in vivo model of rat adjuvant arthritis to
study whether pterostilbene—a stilbene type polyphenol—
was capable of downregulating the activity of neutrophils.
Pterostilbene (trans-3,5-dimethoxy-4󸀠
-hydroxystilbene,
Figure 2) is a dimethyl derivative of resveratrol (trans-3,5,4󸀠
trihydroxystilbene).
The natural sources of pterostilbene are
various herbal drugs, for example, leaves and grapes of Vitis
vinifera, Vaccinium spp., and Pterocarpus marsupium [14–16].
Pterostilbene was found to have anti-inflammatory, antidiabetic,
antifungal, and anticancerous effects [17, 18]. It was
reported to inhibit lipopolysaccharide induced expression of
COX-2 [19]. In our previous experiments, pterostilbene was
the most effective resveratrol derivative tested in inhibiting
ROS production by human neutrophils in vitro. Moreover,
pterostilbene decreased the production of extracellular ROS,
similarly as did resveratrol. Yet on intracellular ROS production,
the effect of pterostilbene was lower in comparison with
resveratrol [20]. Extracellular ROS are responsible for the
tissue destruction, whereas intracellular ROS are involved in
killing pathogens and in intracellular signalling [21].
In this study we investigated the effects of pterostilbene on
the oxidative burst of neutrophils in arthritic rats. According
to our knowledge, no papers have been published on the
effects of pterostilbene on neutrophil activity in an in vivo
model of chronic inflammation.
2. Materials and Method
Pterostilbene (precisely specified as trans-pterostilbene in
this paper) was synthesised at the Institute of Chemical
Technology, Prague, Czech Republic, and structurally characterised
as (E)-4󸀠
-hydroxy-3,5-dimethoxystilbene in the
Institute of Organic Chemistry and Biochemistry, Prague,
Czech Republic [22]. TLC, RP-HPLC, and 1
H-NMR spectroscopy
were used for standardisation before and during the
experiments.
2,2-Azo-bis-2-amidinopropane dihydrochloride (AAPH),
horseradish peroxidase (HRP), luminol, phorbol myristate
acetate (PMA), trichloroacetic acid, and Trolox were
purchased from Sigma, Germany. Mycobacterium butyricum
obtained from Difco Laboratories (Detroit, MI, USA) was
suspended in Freund’s adjuvant. All other products are
available commercially or their origin is mentioned in the
text.
2.1. Model of Adjuvant Arthritis in Rats. The study was performed
in compliance with Principles of Laboratory Animal
Care and was approved by the institutional Ethics Committee
and by the State Veterinary and Food Administration of the
Slovak Republic (Ro-1668/09-221). Animals were kept in an
air-conditioned room with 12 hours day/night mode and
drinking water ad libitum.
Male Lewis rats (Dobra Voda, Slovaki) were injected by
a single intradermal injection of heat-killed Mycobacterium
BioMed Research International 3
butyricum in Freund’s adjuvant to develop arthritis [23].
Healthy control group and arthritic control group were
treated with the solvent agent-sunflower oil. The doses of
pterostilbene used in in vivo studies with rats range from
10 to 40 mg/kg [24–26]. In our experiment, pterostilbene
30 mg/kg, daily, p.o. in sunflower oil, was applied over a
period of 21 days after arthritis induction. There were 10
animals in each group. On days 0, 7, 14, and 21, whole
blood (10 𝜇L) from the tip of the tail was taken to citrated
pipette tip and immediately diluted in Tyrode solution. The
procedure was performed under local anaesthesia. The blood
was subjected to further analysis of chemiluminescence and
neutrophil numbers. After the experiment, the animals were
sacrificed by overdosing with ketamine/xylazine anaesthesia.
The blood was taken by cardiac puncture and plasma was
obtained by 2000 g/15 min centrifugation for further analysis
of total peroxyl radical trapping capacity (TRAP).
2.2. Evaluation of Neutrophil Number and ROS Production in
Rats. The number of neutrophils in rat blood was calculated
with a haemocytometer (Beckman Coulter). Spontaneous
and PMA-stimulated oxidative burst of neutrophils in rat
blood were determined by using chemiluminescence. Briefly
chemiluminescence of neutrophils in blood was measured in
a 96-well microplate luminometer (LM-01T Immunotech) at
37∘
C. Aliquots of Tyrode buffer and luminol (250 𝜇mol/L)
were added. To ensure sufficient concentration of extracellular
peroxidase in stimulated cells, we added horseradish
peroxidase (HRP) to the final concentration 8 U/mL. Finally,
50-times diluted blood was added and the reaction was
started by adding phorbol myristate acetate (PMA) to the
final concentrations of 0.005–0.5 𝜇mol/L [27]. The chemiluminescence
of the samples was recorded for 1 hour and
area under the curve was examined. Normalisation of the
oxidative burst of neutrophils in rat blood to the number of
neutrophils in rat blood was calculated by using
neutrophil activity
=
CL of sample
number of neutrophils in the sample
(1)
(CL: chemiluminescence).
2.3. Total Peroxyl Radical Trapping Capacity (TRAP) of Rat
Plasma. By using thermal decomposition of 2,2-azo-bis-2amidinopropane
dihydrochloride (AAPH), peroxyl radicals
were monitored by luminol-enhanced chemiluminescence
[28]. The reaction mixture consisted of 480 𝜇L of PBS and
50 𝜇L of 10 mmol/L luminol. Then 20 𝜇L of plasma samples
or Trolox was added directly into the cuvette and the samples
were preincubated for 10 min/37∘
C. Finally, 50 𝜇L of AAPH
was added directly into the cuvette. Time needed for a 50%
recovery of the original steady-state signal (so-called half
peak time) was identified for each sample. Trolox was used
as a standard inhibitor. The results obtained were expressed
as 𝜇mol of peroxyl radical trapped by one litre of plasma.
0 0.005 0.05 0.5
PMA ( 𝜇mol/L)
Control
AA
##
##
##
CL(RLU·s)
∗∗
∗∗
∗∗
∗∗
500,000
0
1,000,000
1,500,000
2,000,000
2,500,000
Figure 3: Priming of neutrophils in arthritic rats. Arthritic animals
(AA) showed significantly higher spontaneous (0) and PMAstimulated
ROS production ( ∗∗
𝑃 < 0.01 AA versus control).
PMA caused dose-dependent increase in ROS production which
was significantly higher in comparison with spontaneous ROS
production in arthritic rats ( ##
𝑃 < 0.01 AA-stimulated versus AAspontaneous).
Values are mean ± SEM, 𝑛 = 10. RLU∗s: relative light
units multiplied by time (seconds).
2.4. Statistics. Data were examined using the Student’s 𝑡-test,
and 𝑃 values below 0.05 and 0.01 were considered statistically
significant.
3. Results
3.1. Arthritis Caused Priming of Neutrophils. Arthritic animals
(rats with induced arthritis with solvent treatment only)
showed significantly ( ∗∗
𝑃 < 0.01) higher production of
ROS in blood in nonstimulated (spontaneous) and PMAstimulated
conditions in comparison with healthy controls
(rats with solvent treatment only) (Figure 3). In arthritic
animals the use of PMA increased significantly ( ##
𝑃 < 0.01)
the production of ROS in comparison with spontaneous ROS
production. The effect of PMA stimulation was concentration
dependent. The highest concentration of PMA used
(0.5 𝜇mol/L) did not induce much higher ROS production in
comparison with 0.05 𝜇mol/L PMA. In the further analysis,
we therefore discuss spontaneous and 0.05 𝜇mol/L PMAstimulated
samples.
3.2. Effect of Pterostilbene on Neutrophil ROS Production
in Arthritic Rats. Nonstimulated (spontaneous) neutrophil
ROS production in arthritic rats is evident already on day 7
and was significantly (𝑃 < 0.01) higher in comparison with
healthy controls during the whole experiment, reaching its
maximum on day 21 (Figure 4(a)). Although the arthritic rats
treated with pterostilbene exhibited lower values of spontaneous
ROS production, these changes were not significant
compared to the nontreated arthritic group.
The profile of PMA-stimulated neutrophil ROS production
was similar to the spontaneous one (Figure 4(b)), with a
4 BioMed Research International
0 7 14 21
Day
Control
AA
AA + PTE
CL(RLU·s)
∗∗
∗∗
∗∗
0
100,000
200,000
300,000
400,000
500,000
(a)
Control
AA
AA + PTE
0 7 14 21
Day
CL(RLU·s)
∗∗
∗∗
∗∗
500,000
0
1,000,000
1,500,000
2,000,000
2,500,000
(b)
Figure 4: Effect of pterostilbene on ROS production in arthritic rats. (a) Spontaneous and (b) PMA-stimulated (0.05 𝜇mol/L) whole blood
chemiluminescence (CL) of healthy (control), arthritic (AA), and pterostilbene treated arthritic rats (AA + PTE). Chemiluminescence was
measured at the beginning and every 7 days for 21 days of the experiment. Values are mean ± SEM, 𝑛 = 10, ∗∗
𝑃 < 0.01 AA versus control.
RLU∗s: relative light units multiplied by time (seconds).
0
20,000
40,000
60,000
80,000
0 7 14 21
Neutrophilsin1𝜇Lofratblood
Day
Control
AA
AA + PTE
##
#
∗∗
∗∗
∗∗
Figure 5: Pterostilbene downregulated the number of neutrophils in
arthritic rats. Number of neutrophils in 1 𝜇L of rat blood in healthy
(control), arthritic (AA), and pterostilbene treated arthritic rats (AA
+ PTE) measured at the beginning and every 7 days for 21 days of the
experiment. Values are mean ± SEM, 𝑛 = 10, ∗∗
𝑃 < 0.01 AA versus
control, #
𝑃 < 0.05, ##
𝑃 < 0.01 AA + PTE versus AA.
maximum value on day 14 and with no significant reduction
caused by pterostilbene.
3.3. Effect of Pterostilbene on Number of Neutrophils and
Neutrophil Activity in Arthritic Rats. Figure 5 shows the
change in the number of neutrophils in healthy, arthritic, and
in pterostilbene treated arthritic animals. Arthritic animals
demonstrated a significant increase (𝑃 < 0.01) in neutrophil
numbers from day 7 till the end of the experiment, with
a maximum value on day 21. A significant decrease was
0
50
100
150
200
250
300
0 7 14 21
Day
Control
AA
AA + PTE
##
++
CL(RLU·s)
∗∗
∗∗
∗∗
Figure 6: Effect of pterostilbene on neutrophil activity in arthritic
rats. PMA (0.05 𝜇mol/L) stimulated ROS production shown as
chemiluminescence (CL) per 1 neutrophil in whole blood of healthy
(control), arthritic (AA), and pterostilbene treated arthritic rats (AA
+ PTE) measured at the beginning and every 7 days for 21 days of
the experiment. Values are mean ± SEM, 𝑛 = 10, ∗∗
𝑃 < 0.01 AA
versus control, ##
𝑃 < 0.01 AA + PTE versus AA for inhibition,
++
𝑃 < 0.01 AA + PTE versus AA for elevation. RLU∗s: relative light
units multiplied by time (seconds).
observed within the arthritic group treated with pterostilbene
compared to the arthritic group on days 14 and 21 (𝑃 < 0.01
and 𝑃 < 0.05, resp.).
By normalisation of the oxidative burst to the number
of neutrophils (neutrophil activity), we found a significant
(𝑃 < 0.01) increase in PMA-stimulated neutrophil
activity in arthritic rats compared to healthy controls, with
a maximum value on day 7 (Figure 6). Neutrophils from
BioMed Research International 5
0
50
100
150
200
250
300
CO AA AA + PTE
TRAP(𝜇mol/LpermLofplasma)
∗∗
Figure 7: Effect of pterostilbene on TRAP in arthritic rats. Total
peroxyl radical trapping capacity (TRAP) in plasma of healthy (CO:
control), arthritic (AA), and pterostilbene treated arthritic rats (AA
+ PTE) measured at the end of the experiment. Values are mean
± SEM, 𝑛 = 10, ∗∗
𝑃 < 0.01 AA versus control. The values are
expressed as 𝜇mol of peroxyl radical trapped by one litre of rat
plasma.
pterostilbene treated arthritic animals showed significantly
decreased activity on day 7 compared to arthritic controls.
After day 14, however, neutrophils from pterostilbene
treated arthritic animals showed higher activity compared
to arthritic animals. No significant differences were seen
in spontaneous neutrophil activity in healthy, arthritic, or
pterostilbene treated arthritic rats (data not shown).
3.4. Effect of Pterostilbene Treatment on TRAP Levels in
Rat Plasma. The total peroxyl radical trapping capacity
of plasma (TRAP) was determined in the control,
arthritic, and pterostilbene treated arthritic groups. The
arthritic rats showed a significant (𝑃 < 0.01) decrease in
TRAP (94 𝜇mol/L) compared to the healthy control group
(267 𝜇mol/L). Pterostilbene treated arthritic rats showed
nonsignificant increase in the total peroxyl radical trapping
capacity of plasma (120 𝜇mol/L) compared to the arthritic
control group (Figure 7).
4. Discussion
The formation of reactive oxygen species (ROS) in neutrophils
is one of the essential microbicidal mechanisms in
an organism. When produced in high amounts, these highly
reactive substances may contribute to tissue injury. This
is seen in chronic inflammatory diseases (e.g., rheumatoid
arthritis) or ischaemic-reperfusion injury [3, 4, 29]. In this
work we examined the potential protective effects of pterostilbene
on neutrophil ROS production in adjuvant arthritis—
a model of neutrophil-dominant chronic autoinflammatory
disease. Pterostilbene was chosen because of its inhibitory
effects on neutrophil ROS production in vitro [20].
Adjuvant induced inflammation significantly increased
the production of ROS and the number of neutrophils in
arthritic animals. However, the increased spontaneous (nonstimulated)
ROS production in arthritic animals is due to the
increase of blood neutrophils rather than the increase in neutrophil
activity. This is because proinflammatory cytokines
involved in the development of rheumatoid arthritis cause
only weak ROS production in neutrophils [30]. Different
cytokines present in the synovial fluid induce priming of
the neutrophils [3]. By normalisation of ROS production
to the number of neutrophils (neutrophil activity), there
was no difference between the activities of nonstimulated
neutrophils from arthritic and healthy rats. But primed
neutrophils have upregulated ROS production when exposed
to a secondary stimulus such as PMA [30]. By using PMA,
the ROS production in whole blood of arthritic rats was
approximately 7 times higher than that in control animals,
and the neutrophil activity was significantly increased. Thus,
the increase in PMA-stimulated ROS production in arthritic
rats is due to an increase in neutrophil number and to an
increase in activity of primed neutrophils.
Administration of pterostilbene (30 mg/kg, daily, p.o.)
decreased significantly the number of neutrophils in arthritic
rats but pterostilbene had only limited effect on ROS
production and neutrophil activity. However, the effect of
pterostilbene on decreasing the number of neutrophils seems
to be more important than the effect on neutrophil oxidative
burst. This is because we found that the ROS production in
arthritic animals is due to the increase of blood neutrophils
rather than the increase of neutrophil activity. The higher
neutrophil activity in arthritic rats treated with pterostilbene
in comparison with nontreated arthritic rats on day 14
could be explained by the fact that pterostilbene on day 14
significantly decreased the number of neutrophils but the
ROS production was nearly the same as in arthritic rats. The
lower effect of pterostilbene in vivo is seen also with TRAP
assay, where pterostilbene did not increase significantly the
antioxidative capacity of arthritic rat plasma. These results
are in contrast to our findings with pterostilbene effects
on neutrophil activity in vitro and in cell free assays ([20]
and unpublished data), suggesting that the mild decrease in
ROS production in arthritic animals receiving pterostilbene
treatment may be attributed to pterostilbene downregulation
of neutrophilia in arthritic rats.
One of the limits of stilbene derivatives and polyphenols
is their low bioavailability. The bioavailability of stilbene
derivatives depends on the substitution of the hydroxyl
groups. Pterostilbene has a higher bioavailability in comparison
with resveratrol [31]. The doses of pterostilbene used in
in vivo studies with rats range from 10 to 40 mg/kg [24–26].
It is questionable whether increasing the pterostilbene dose
would increase its bioavailability and thus the effects in vivo.
Another stilbene derivative, pinosylvin (trans-3,5dihydroxy
stilbene), was more effective in the inhibition of
both spontaneous and PMA-stimulated ROS production
in arthritic rat blood and significantly increased TRAP
in plasma of arthritic animals [32]. Pinosylvin (30 mg/kg,
p.o., daily), though not pterostilbene, decreased the hind
paw volume (clinical symptom of adjuvant arthritis)
and myeloperoxidase (MPO) activity in hind paw joint
homogenates of arthritic rats [23, 33]. MPO may be used
to assess the infiltration of neutrophils in the joints [33].
Downregulation of the number of neutrophils in arthritic rats
was seen also with pinosylvin [32]. We suggest that stilbene
derivatives may interfere with cytokine signalling leading
6 BioMed Research International
to a decrease of neutrophils in arthritic rats. Resveratrol
was found to regulate different cytokines and intracellular
messengers involved in inflammation [34, 35]. Paul et
al. reported that pterostilbene decreased the levels of the
proinflammatory cytokines TNF-𝛼, IL-1𝛽, and IL-4 [36].
Protein kinase C (PKC) is important in the activation of
neutrophil NADPH-oxidase and thus in the production of
ROS [37]. Inhibition of PKC could be used as a strategy for
regulating various diseases involving PKC [38]. Derivatives
of resveratrol are able to attenuate the activity or activation
of PKC. Pinosylvin, but not pterostilbene, decreased the
activation of PKC in neutrophils in vitro [32, 39].
In conclusion, stilbene derivatives are effective in the
inhibition of neutrophil ROS production but this is structure
dependent. The structure influences also the bioavailability
of stilbene derivatives. Despite higher activity against
neutrophil ROS production in vitro, pterostilbene did not
decrease significantly the oxidative burst of neutrophils in
vivo. On the other hand, pterostilbene decreased the number
of neutrophils in arthritic rats. Our results contribute to
the knowledge of structure-dependent benefits of stilbene
derivatives in the management of chronic inflammatory
diseases where neutrophils play a role.
Conflict of Interests
The authors declare that there is no conflict of interests.
Acknowledgments
The authors wish to thank Ing. Danica Mihalova for her
kind assistance and Professor Magda Kourilova-Urbanczik
for English language correction of the paper. This study is
supported by the Grants APVV-0052-10 and 0315-07, VEGA-
2/0010/13 and 2/0045/11, and CZ.1.07/2.3.00/30.0030.
References
[1] J. K. Hurst, “What really happens in the neutrophil phagosome?”
Free Radical Biology and Medicine, vol. 53, no. 3, pp.
508–520, 2012.
[2] V. Witko-Sarsat, P. Rieu, B. Descamps-Latscha, P. Lesavre, and L.
Halbwachs-Mecarelli, “Neutrophils: molecules, functions and
pathophysiological aspects,” Laboratory Investigation, vol. 80,
no. 5, pp. 617–654, 2000.
[3] S. W. Edwards and M. B. Hallett, “Seeing the wood for the
trees: the forgotten role of neutrophils in rheumatoid arthritis,”
Immunology Today, vol. 18, no. 7, pp. 320–324, 1997.
[4] S. P. Loukogeorgakis, M. J. Van Den Berg, R. Sofat et al., “Role
of NADPH oxidase in endothelial ischemia/reperfusion injury
in humans,” Circulation, vol. 121, no. 21, pp. 2310–2316, 2010.
[5] R. Casc˜ao, H. S. Ros´ario, and J. E. Fonseca, “Neutrophils:
warriors and commanders in immune mediated inflammatory
diseases,” Acta Reumatol´ogica Portuguesa B, vol. 34, no. 2, pp.
313–326, 2009.
[6] H. Li and A. Wan, “Apoptosis of rheumatoid arthritis fibroblastlike
synoviocytes: possible roles of nitric oxide and the thioredoxin
1,” Mediators of Inflammation, vol. 2013, Article ID
953462, 8 pages, 2013.
[7] W. Mohr, A. Wild, and H. P. Wolf, “Role of polymorphs in
inflammatory cartilage destruction in adjuvant arthritis of rats,”
Annals of the Rheumatic Diseases, vol. 40, no. 2, pp. 171–176, 1981.
[8] W. Mohr, H. Westerhellweg, and D. Wessinghage, “Polymorphonuclear
granulocytes in rheumatic tissue destruction. III.
An electron microscopic study of PMNs at the pannus-cartilage
junction in rheumatoid arthritis,” Annals of the Rheumatic
Diseases, vol. 40, no. 4, pp. 396–399, 1981.
[9] S. Fox, A. E. Leitch, R. Duffin, C. Haslett, and A. G. Rossi, “Neutrophil
apoptosis: relevance to the innate immune response and
inflammatory disease,” Journal of Innate Immunity, vol. 2, no. 3,
pp. 216–227, 2010.
[10] P. Kohli and B. D. Levy, “Resolvins and protectins: mediating
solutions to inflammation,” British Journal of Pharmacology, vol.
158, no. 4, pp. 960–971, 2009.
[11] C. N. Serhan, “The resolution of inflammation: the devil in the
flask and in the details,” FASEB Journal, vol. 25, no. 5, pp. 1441–
1448, 2011.
[12] J. M. Hallett, A. E. Leitch, N. A. Riley, R. Duffin, C. Haslett,
and A. G. Rossi, “Novel pharmacological strategies for driving
inflammatory cell apoptosis and enhancing the resolution of
inflammation,” Trends in Pharmacological Sciences, vol. 29, no.
5, pp. 250–257, 2008.
[13] S. Ponist, D. Mihalova, V. Jancinova et al., “Reduction of
oxidative stress in adjuvant arthritis. Comparison of efficacy of
two pyridoindoles: stobadine dipalmitate and SMe1.2HCl,” Acta
Biochimica Polonica, vol. 57, no. 2, pp. 223–228, 2010.
[14] J. K. Grover, V. Vats, and S. S. Yadav, “Pterocarpus marsupium
extract (Vijayasar) prevented the alteration in metabolic patterns
induced in the normal rat by feeding an adequate diet
containing fructose as sole carbohydrate,” Diabetes, Obesity and
Metabolism, vol. 7, no. 4, pp. 414–420, 2005.
[15] P. Langcake, C. A. Cornford, and R. J. Pryce, “Identification
of pterostilbene as a phytoalexin from Vitis vinifera leaves,”
Phytochemistry, vol. 18, no. 6, pp. 1025–1027, 1979.
[16] B. Paul, I. Masih, J. Deopujari, and C. Charpentier, “Occurrence
of resveratrol and pterostilbene in age-old darakchasava, an
ayurvedic medicine from India,” Journal of Ethnopharmacology,
vol. 68, no. 1–3, pp. 71–76, 1999.
[17] C. M. Remsberg, J. A. Y´a˜nez, Y. Ohgami, K. R. Vega-Villa,
A. M. Rimando, and N. M. Davies, “Pharmacometrics of
pterostilbene: preclinical pharmacokinetics and metabolism,
anticancer, antiinflammatory, antioxidant and analgesic activity,”
Phytotherapy Research, vol. 22, no. 2, pp. 169–179, 2008.
[18] K. A. Roupe, C. M. Remsberg, J. A. Y´a˜nez, and N. M. Davies,
“Pharmacometrics of stilbenes: seguing towards the clinic,”
Current Clinical Pharmacology, vol. 1, no. 1, pp. 81–101, 2006.
[19] M. H. Pan, Y. H. Chang, M. L. Tsai et al., “Pterostilbene
suppressed lipopolysaccharide-induced up-expression of iNOS
and COX-2 in murine macrophages,” Journal of Agricultural and
Food Chemistry, vol. 56, no. 16, pp. 7502–7509, 2008.
[20] T. Pereˇcko, V. Janˇcinov´a, K. Dr´abikov´a, R. Nos´al’, and J. Harmatha,
“Structure-efficiency relationship in derivatives of stilbene.
Comparison of resveratrol, pinosylvin and pterostilbene,”
Neuroendocrinology Letters, vol. 29, no. 5, pp. 802–805, 2008.
[21] H. Miki and Y. Funato, “Regulation of intracellular signalling
through cysteine oxidation by reactive oxygen species,” Journal
of Biochemistry, vol. 151, no. 3, pp. 255–261, 2012.
[22] J. ˇSmidrkal, J. Harmatha, M. Bud`ıˇS´ınsk´y et al., “Modified
approach for preparing (E)-Stilbenes related to resveratrol,
and evaluation of their potential immunobiological effects,”
BioMed Research International 7
Collection of Czechoslovak Chemical Communications, vol. 75,
no. 2, pp. 175–186, 2010.
[23] K. Bauerova, S. Ponist, D. Mihalova, F. Drafi, and V. Kuncirova,
“Utilization of adjuvant arthritis model for evaluation of new
approaches in rheumatoid arthritis therapy focused on regulation
of immune processes and oxidative stress,” Interdisciplinary
Toxicology, vol. 4, no. 1, pp. 33–39, 2011.
[24] M. A. Satheesh and L. Pari, “The antioxidant role of pterostilbene
in streptozotocin-nicotinamide- induced type 2 diabetes
mellitus in Wistar rats,” Journal of Pharmacy and Pharmacology,
vol. 58, no. 11, pp. 1483–1490, 2006.
[25] M. F. Lee, M. L. Liu, A. C. Cheng et al., “Pterostilbene inhibits
dimethylnitrosamine-induced liver fibrosis in rats,” Food Chemistry,
vol. 138, no. 2-3, pp. 802–807, 2013.
[26] S. C. Yeo, P. C. Ho, and H. S. Lin, “Pharmacokinetics of pterostilbene
in Sprague-Dawley rats: the impacts of aqueous solubility,
fasting, dose escalation, and dosing route on bioavailability,”
Molecular Nutrition & Food Research, vol. 57, no. 6, pp. 1015–
1025, 2013.
[27] V. Jancinova, T. Perecko, R. Nosal, D. Kostalova, K. Bauerova,
and K. Drabikova, “Decreased activity of neutrophils in the
presence of diferuloylmethane (curcumin) involves protein
kinase C inhibition,” European Journal of Pharmacology, vol.
612, no. 1–3, pp. 161–166, 2009.
[28] I. Papezikova, A. Lojek, H. Cizova, and M. Ciz, “Alterations in
plasma antioxidants during reperfusion of the ischemic small
intestine in rats,” Research in Veterinary Science, vol. 81, no. 1,
pp. 140–147, 2006.
[29] G. B. Segel, M. W. Halterman, and M. A. Lichtman, “The
paradox of the neutrophil’s role in tissue injury,” Journal of
Leukocyte Biology, vol. 89, no. 3, pp. 359–372, 2011.
[30] J. Robinson, F. Watson, R. C. Bucknall, and S. W. Edwards, “Activation
of neutrophil reactive-oxidant production by synovial
fluid from patients with inflammatory joint disease: soluble
and insoluble immunoglobulin aggregates activate different
pathways in primed and unprimed cells,” Biochemical Journal,
vol. 286, no. 2, pp. 345–351, 1992.
[31] I. M. Kapetanovic, M. Muzzio, Z. Huang, T. N. Thompson, and
D. L. McCormick, “Pharmacokinetics, oral bioavailability, and
metabolic profile of resveratrol and its dimethylether analog,
pterostilbene, in rats,” Cancer Chemotherapy and Pharmacology,
vol. 68, no. 3, pp. 593–601, 2011.
[32] V. Jancinova, T. Perecko, R. Nosal, J. Harmatha, J. Smidrkal, and
K. Drabikova, “The natural stilbenoid pinosylvin and activated
neutrophils: effects on oxidative burst, protein kinase C, apoptosis
and efficiency in adjuvant arthritis,” Acta Pharmacologica
Sinica, vol. 33, pp. 1285–1292, 2012.
[33] T. Macickova, K. Drabikova, R. Nosal et al., “In vivo effect of
pinosylvin and pterostilbene in the animal model of adjuvant
arthritis,” Neuro Endocrinology Letters, vol. 31, supplement 2, pp.
91–95, 2010.
[34] X. Gao, Y. X. Xu, N. Janakiraman, R. A. Chapman, and
S. C. Gautam, “Immunomodulatory activity of resveratrol:
suppression of lymphocyte proliferation, development of cellmediated
cytotoxicity, and cytokine production,” Biochemical
Pharmacology, vol. 62, no. 9, pp. 1299–1308, 2001.
[35] N. Richard, D. Porath, A. Radspieler, and J. Schwager, “Effects of
resveratrol, piceatannol, triacetoxystilbene, and genistein on the
inflammatory response of human peripheral blood leukocytes,”
Molecular Nutrition and Food Research, vol. 49, no. 5, pp. 431–
442, 2005.
[36] S. Paul, A. J. DeCastro, H. J. Lee et al., “Dietary intake of pterostilbene,
a constituent of blueberries, inhibits the 𝛽-catenin/p65
downstream signaling pathway and colon carcinogenesis in
rats,” Carcinogenesis, vol. 31, no. 7, pp. 1272–1278, 2010.
[37] K. Makni-Maalej, M. Chiandotto, M. Hurtado-Nedelec et
al., “Zymosan induces NADPH oxidase activation in human
neutrophils by inducing the phosphorylation of p47phox and
the activation of Rac2: involvement of protein tyrosine kinases,
PI3Kinase, PKC, ERK1/2 and p38MAPkinase,” Biochemical
Pharmacology, vol. 85, no. 1, pp. 92–100, 2013.
[38] J. Das, S. Pany, and A. Majhi, “Chemical modifications of resveratrol
for improved protein kinase C alpha activity,” Bioorganic
and Medicinal Chemistry, vol. 19, no. 18, pp. 5321–5333, 2011.
[39] T. Perecko, K. Drabikova, L. Rackova et al., “Molecular targets
of the natural antioxidant pterostilbene: effect on protein kinase
C, caspase-3 and apoptosis in human neutrophils in vitro,”
Neuroendocrinology Letters, vol. 31, supplement 2, pp. 84–90,
2010.
Regular paper
Antioxidant, antimicrobial and neutrophil-modulating activities
of herb extracts
Petko Denev1, Maria Kratchanova1*
, Milan Ciz2, Antonin Lojek2, Ondrej Vasicek2, Denitsa
Blazheva3, Plamena Nedelcheva3, Libor Vojtek4 and Pavel Hyrsl4
1Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Laboratory of Biologicaly Active Substances,
Plovdiv, Bulgaria; 2Institute of Biophysics of the AS CR, Brno, Czech Republic; 3University of Food Technologies, Plovdiv, Bulgaria; 4Department of
Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic
The present study provides a comprehensive data on the
antioxidant, antimicrobial and neutrophil-modulating activities
of extracts from six medicinal plants — blackberry
(Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa)
leaves, hawthorn (Crataegus monogyna) leaves,
lady’s mantle (Alchemilla glabra) aerial parts, meadowsweet
(Filipendula ulmaria) aerial parts and raspberry
(Rubus idaeus) leaves. In order to analyze the antioxidant
activity of the herbs, several methods (ORAC, TRAP,
HORAC and inhibition of lipid peroxidation) were used.
Blackberry leaves and meadowsweet extracts revealed
the highest antioxidant activities via all methods. All extracts
studied blocked almost completely the opsonized
zymosan particle-activated ROS production by neutrophils
from human whole blood. On the other hand, the
effect of extracts on phorbol myristate acetate-activated
ROS production was much milder and even nonsignificant
in the case of chokeberry leaves. This latter result
suggests that extracts (apart from their antioxidative activity)
interfere with the signaling cascade of phagocyte
activation upstream of the protein kinase C activation.
The antimicrobial activity of the investigated extracts
against 11 human pathogens was investigated using
three different methods. Meadowsweet and blackberry
leaves extracts had the highest antimicrobial effect and
the lowest minimal inhibiting concentrations (MICs)
against the microorganisms tested.
Key words: herbs, polyphenols, antioxidant activity, antimicrobial activity,
phagocytes, reactive oxygen species
INTRODUCTION
Human gastrointestinal tract (GIT) is constantly exposed
to oxidizing agents of diverse origins. The diet
contains various prooxidants, including metals (copper
and free or heme-bound iron), lipid hydroperoxides, aldehydes
and nitrite, and consequently elevated levels of
lipid peroxides have been observed in the postprandial
state (Kanner & Lapidot, 2001). These prooxidants may
induce oxidative stress in the GIT, resulting in stomach
ulcer and stomach, colon, and rectal cancers. Besides
that, oxidative stress induced by microbial infections is
another major contributor to the development of gastro-intestinal
diseases. There is increasing evidence that
microbial pathogens induce oxidative stress in host cells,
resulting in considerable accumulation of inflammatory
cell, which could be related to the development of gastric
mucosal as well as neuromuscular disorders (Suzuki
et al., 2012). Recent studies have shown that many bacterial
pathogens can trigger apoptosis of infected host cells
(Krzyminska et al., 2011). It has been shown that phagocyte-derived
reactive oxygen species (ROS) and reactive
nitrogen species (RNS) are important factors inducing
apoptosis (Circu & Aw, 2010). The activation of phagocytes
in the gut may also increase the ROS and RNS
production, and gastric juice may promote lipid peroxidation
(Kanner & Lapidot, 2001). Phagocytic cells such
as macrophages and neutrophils play a key role in innate
immunity owing to their ability to recognize, ingest, and
destroy pathogens by oxidative and nonoxidative mechanisms.
In response to a variety of stimuli, NADPH oxidase
present in neutrophils is activated in a phenomenon
described as the respiratory burst, characterized by
the production of the superoxide anion which gives rise
to other forms of ROS. In addition nitric oxide (NO)
and other RNS produced mainly by macrophages are
among the major microbicidal agents of inflammation
during the fight against pathogenic microorganisms and
tumor cells. However, excessive or inappropriate ROS
and RNS production by phagocytes is associated with
oxidative damage to membrane lipids, DNA, proteins,
and lipoproteins, resulting in various autoimmune and
inflammatory diseases. Thus, the modulation of inflammation
and oxidative stress by natural substances can be
beneficial. Therefore, the role of antioxidants in the GIT
may be very important and antioxidants contained in
foods could suppress the oxidative stress and related diseases
in the gastrointestinal tract before being absorbed
(Halliwell et al., 2000) and thus could modulate the level
of oxidative stress by enhancing anti-inflammatory or
antioxidant capacity (Suzuki et al., 2012). In the recent
search for novel sources of anti-inflammatory and antioxidant
agents medicinal plants have attracted particular
attention. Since ancient times herbs have been used in
*
e-mail: lbas@plov.omega.bg
Abbreviations: AAPH, 2,2´-azobis (2-amidino-propane) dihydrochloride;
AUC, area under the curve; CL, chemiluminescence; DW,
dry weight; GAE, gallic acid equivalents; GIT, gastrointestinal tract;
HBSS, Hank‘s balanced salt solution; HORAC, hydroxyl radical averting
capacity; HPLC, high performance liquid chromatography; LPS,
lipopolysaccharide; MIC, minimal inhibiting concentration; ORAC,
oxygen radical absorbance capacity; OZP, opsonized zymosan particles;
PKC, protein kinase C; PLD, phospholipase D; PMA, phorbol
myristate acetate; RLU, relative light units; RNS, reactive nitrogen
species; ROS, reactive oxygen species; S.D., standard deviation; TE,
trolox equivalents; TRAP, total peroxyl-radical antioxidant parame-
ter
Received: 31 January, 2014; revised: 15 April, 2014; accepted:
08 May, 2014; available on-line: 18 June, 2014
Vol. 61, No 2/2014
359–367
on-line at: www.actabp.pl
360											 2014P. Denev and others
numerous areas, including nutrition, healing, cosmetics,
etc., without the exact mechanisms of their action being
known. In our recent study we observed that herbs are
a rich source of polyphenol compounds (Kratchanova et
al., 2010) and their antioxidant activities are several times
higher than those of vegetables (Ciz et al., 2010) and
fruits (Denev et al., 2013). There was also a good correlation
between the polyphenol content and the ORAC
antioxidant activity of the herbs investigated, indicating
that the polyphenol compounds are responsible for the
free-radical scavenging capacity. Bearing in mind the
great variety of medicinal plants and their potent antioxidant
properties, herbs appear to be a valuable but still
poorly-explored source of natural antioxidant and antimicrobial
agents. Therefore, the aim of the present study
was to investigate the polyphenol composition and content
of six less-investigated herbs and to determine their
antioxidant and antimicrobial activities, and their effect
on the production of ROS by activated neutrophils.
In order to obtain a broader view on their antioxidant
properties we used several antioxidant activity assays
(ORAC, TRAP, HORAC and inhibition of lipid peroxidation)
relying on different mechanisms. The antimicrobial
properties and the minimal inhibiting concentration
of the herb extracts were investigated against 11 human
pathogens and a genetically modified luminescent E. coli
was used for the first time to determine the antimicrobial
activity of natural products.
MATERIALS AND METHODS
Chemicals. Fluorescein disodium salt, 2,2-azobis-(2-amidino-propane)dihydrochloride
(AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid
(trolox), CoF2×4H2O, picolinic acid, linoleic acid and
HPLC reference compounds (gallic acid, 3,4-dihydroxybenzoic
acid, chlorogenic acid, caffeic acid, p-coumaric
acid, ferulic acid, ellagic acid, catechin, epicatechin, rutin,
naringin, myrecetin, quercetin, naringenin and kaempherol)
were purchased from Sigma-Aldrich (Steinheim, Germany).
Folin-Ciocalteu’s phenol reagent was purchased
from Merck (Darmstadt, Germany) and luminol from
Molecular Probes, (Eugene, Oregon, USA). All other reagents
used were of analytical grade and purchased from
local distributors.
Herb samples. All medicinal plants used were either
obtained from local pharmacies (Plovdiv, Bulgaria)
or collected from nature in 2012. The choice of the
plants investigated was based on the available data on
their biological activities. The following medicinal plants
were investigated: blackberry (Rubus fruticosus) — leaves,
chokeberry (Aronia melanocarpa) — leaves, hawthorn (Crataegus
monogyna) — leaves, lady’s mantle (Alchemilla glabra)
— aerial parts, meadowsweet (Filipendula ulmaria) — aerial
parts and raspberry (Rubus idaeus) — leaves. Freshly
collected plants were dried in the shade at room temperature
and all plant materials were inspected for contaminants.
1 kg of each herb was packed in paper bags
and stored at ambient temperature prior to extraction
and analysis.
Extraction. All plant materials were subjected to extraction
under the following conditions: 50 g of the dried
plant material was powdered in a laboratory mill. Then
5 g of the powder was transferred into extraction tubes
and mixed with 100 ml of the extragent (80% acetone
in 0.2% formic acid). Extraction was conducted on an
orbital shaker at room temperature for one hour. After
that, the samples were centrifuged (6 000×g) and supernatants
collected. Extracts were concentrated via rotary
evaporation to a volume of 15 ml in order to remove
the acetone. Then the volume was adjusted to 50 ml
with ultra clean water and the extracts were centrifuged.
Clear supernatants were used for all further analysis after
proper dilutions.
HPLC analysis of phenolic compounds. High
Performance Liquid Chromatography (HPLC) analysis
of phenolic components was performed on an Agilent
1220 HPLC system (Agilent Technology, Palo Alto,
USA) equipped with a binary pump and UV-Vis detector
(Agilent Technology) set at 280 nm. TC-C18 column
(5  μm, 4.6 mm×250 mm) was used at 25ºC. The mobile
phases were 0.5% acetic acid and 100% acetonitrile at a
flow rate of 0.8 ml/min. The eluent gradient started with
14% acetonitrile for 6 min, and linearly increased to 25%
acetonitrile in 24 minutes and to 50% acetonitrile in
10 min. Neochlorogenic acid content was calculated as
chlorogenic acid equivalents. Results are given as mean
values from three measurements of the same extract and
expressed as mg per 100 grams of dry weight (DW).
Total polyphenol compounds analysis. Total polyphenols
were determined according to the method of
Singleton & Rossi (1965) with Folin-Ciocalteu’s reagent.
Gallic acid was employed as a calibration standard and
the results were calculated as mean values from three
measurements of the same extract and expressed as gallic
acid equivalents (GAE) per 100 grams of dry weight.
ORAC assay. ORAC was measured according to
the method of Ou et al. (2001) with some modifications
(Ciz et al., 2010). The method measures the antioxidant
scavenging activity against peroxyl radical
generated by thermal decomposition of 2,2’-azobis
(2-amidino-propane) dihydrochloride (AAPH) at 37°C.
Fluorescein was used as the fluorescent probe. The
loss of fluorescence of fluorescein was an indication
of the extent of its oxidation through reaction with
the peroxyl radical. The protective effect of an antioxidant
was measured by assessing the fluorescence
area under the curve (AUC) plot relative to that of a
blank in which no antioxidant was present. Solutions
of AAPH, fluorescein and trolox were prepared in a
Na phosphate buffer (75 mmol/l, pH 7.4). Samples
were diluted in the phosphate buffer as well. Reaction
mixture (total volume 200 μl) contained fluorescein
(170 μl, final concentration 5.36×10–8 mol/l), AAPH
(20 μl, final concentration 51.51 mmol/l), and sample
— 10  μl. The fluorescein solution and sample were
incubated at 37°C for 20 min directly in a microplate
reader, and AAPH (dissolved in buffer at 37°C) was
added. The mixture was incubated for 30 s before the
initial fluorescence was measured. After that, fluorescence
readings were taken at the end of every cycle
(1  min) after shaking. For the blank, 10 μl of phosphate
buffer was used instead of the extract. Each
herb extract was analyzed in two runs with four replicates
and results are presented as mean value ± standard
deviation from the eight values. The antioxidant
activity is expressed in micromole trolox equivalents
(TE) per gram of dry weight (DW). Trolox solutions
(3.125, 6.25; 12.5; 25 and 50 μmol/l) were used for
defining the standard curve.
TRAP assay. Luminol-enhanced chemiluminescence
(CL) was used to follow the peroxyl radical reaction and
the principle was described previously in Cizova and
coworkers (2004). The CL signal is driven by the production
of luminal-derived radicals from thermal decomposition
of AAPH. The TRAP value is determined from
the duration of the time period (Tsample) during which the
Vol. 61 						 361Biological activities of herbs
sample quenched the CL signal due to the present antioxidants.
Trolox solution (8.0 nM) was used as a reference
inhibitor (Ttrolox) instead of the sample. The calculation
of the TRAP value followed the equation:
TRAP = 2 .0 [trolox]Tsample / f Ttrolox,
where 2.0 is the stoichiometric factor of trolox (the
number of peroxyl radicals trapped per one molecule
of trolox) and f is the dilution of the sample. All herb
extracts were analyzed in two runs with three replicates
each, and results are presented as mean ± standard deviation
from all six values.
HORAC assay. The HORAC assay developed by
Ou and coworkers (2002) measures the metal-chelating
activity of antioxidants in the conditions of Fenton-like
reactions employing a Co(II) complex and hence the
protecting ability against formation of hydroxyl radical.
Hydrogen peroxide solution of 0.55 M was prepared in
distilled water, and 4.6 mM Co(II) was prepared as follows:
15.7mg of CoF2×4H2O and 20mg of picolinic
acid were dissolved in 20 ml of distilled water. Fluorescein
(170 μl, final concentration 60 nM) and 10 µl of
sample were incubated at 37°C for 10 min directly in a
FLUOstar plate reader, then 10 μl of H2O2 (final concentration
27.5 mM) and 10 μl of Co(II) (final concentration
230 µM) solutions were added. Initial fluorescence
was measured immediately, then readings were taken every
minute after shaking. For the blank sample, phosphate
buffer (75 mM, pH=7.4) was used. Gallic acid solutions
(100, 200, 600, 800 and 1000 μM in phosphate buffer)
were used for standard curve. The AUC values were calculated
as they were for the ORAC assay. Each herb extract
was analyzed in two runs with four replicates and
results are presented as mean value ± standard deviation
from the eight values. ORAC and HORAC analyses were
carried out using a FLUOstar OPTIMA plate reader
(BMG Labtech, Germany) with excitation wavelength of
485 nm and emission wavelength of 520 nm.
Inhibition of lipid peroxidation assay. A modified
assay described by Anthon & Barrett (2001) and Kikuzaki
and coworkers (2002) was used. A mixture of 1 ml
sample, 1 ml of 83.5 mM linoleic acid in 99.5% ethanol,
2 ml of 0.05 M phosphate buffer (pH 7.0), and 1 mL
of water in a glass tube with a plastic stopper was incubated
at 40°C in the dark. Absorbance at 234 nm was
measured after one, three and six days of incubation on
Infinite M200 microplate reader (Tecan, Austria). Initial
values of absorbance (day zero before incubation) were
subtracted from these values. For conversion of absorbance
to the concentration of hydroperoxide, the extinction
coefficient 23 000 M–1cm–1 was used. Results are
presented as mean values from three independent measurements
each in duplicate.
Oxidative burst. Heparinized blood (50 IU/ml) from
healthy volunteers (n=7) was obtained by antecubital venipuncture.
The oxidative burst of phagocytes in whole
blood was measured chemiluminometrically using an
LM-01 microplate luminometer (Immunotech, Czech Republic).
The principle of the method was described previously
(Ciz et al., 2007). Each reaction mixture consisted
of 25 µl of whole blood diluted 10× in Hank’s balanced
salt solution (HBSS) pH 7.4, 2.5 µl of plant extract, 25 µl
of 1 mM luminol, and 25 µl of one of the activators —
62.5 μg/ml of opsonized zymosan particles (OZP; Sigma,
USA) or 0.81 μg/ml of phorbol-12-myristate-13-acetate
(PMA; Sigma, USA). HBSS was used to adjust the
total reaction volume to 250 μl. The assays were run in
duplicates. Measurements of spontaneous CL in samples
containing blood and other substances except any activator
were included in each assay. The CL activity of the
samples was measured immediately at 37°C. Light emission
expressed as relative light units (RLU) was recorded
continuously for 60 minutes to obtain kinetic curves. Integrals
of CL signal over 60 min were also calculated.
Results are presented as means ± standard error of the
mean from six measurements.
Determination of antimicrobial activity by agar
diffusion method. The agar diffusion method was used
according to Denev and coworkers (2014). Eleven human
pathogens (Escherichia coli ATCC 8739; Escherichia coli
ATCC 25922; Salmonella sp.; Salmonella enterica ssp. enterica
ATCC BAA-2162; Staphylococcus aureus ATCC 6538P;
Staphylococcus aureus ATCC 25923; Listeria monocytogenes;
Listeria monocytogenes I; Proteus vulgaris G; Pseudomonas aeruginosa
ATCC 9027; Klebsiella pneumoniae) were used. All
experiments were performed in duplicates and results are
expressed as mean values.
Determination of minimal inhibiting concentration
(MIC). The antimicrobial effects of the extracts
were determined by analyzing the MIC using a 96 well
microtiter plate method (broth dilution) according to the
modified protocol described by Gutierrez and coworkers
(2008). The first column of each plate contained 300
μl of nutrient broth (tryptone — 5.0 g/l, yeast extract
— 2.5 g/l, glucose — 1 g/l, рН 7.0) and 100 μl of the
tested extract. From the second to the twelfth column,
200 μl of broth were added and 200 μl of the mixture
from the first well was added to the second one and
so on (serially diluted two-fold) along each row, until
the eleventh column. The twelfth column was used for
growth control (positive control) and a row was used for
negative control at each dilution. Finally, 30 μl of working
culture of each test microorganism (1×107 cfu/ml)
was added to all the wells except the negative controls.
The positive control contained distilled water instead
of extract. Plates were incubated at 37ºC for 24 h and
then turbidity was determined at 600 nm in a microplate
spectrophotometer (SPECTROstarNano, BMG Labtech,
Germany). The lowest concentration of the tested extract
required to reduce the growth of the tested microorganism
to 50% of the positive control was designated
as the MIC. Results are presented as mean values from
three independent measurements.
Antimicrobial activity against genetically modified
Escherichia coli. In the following experiments
transformed E. coli K12 resistant to ampicillin carrying
the luxABCDEamp gene was used, expressing bacterial
luciferase and its substrate (Atosuo et al., 2013).
Bacteria were cultivated in LB medium with 100 µg/
ml ampicillin, washed twice in phosphate buffer (pH
7.0, 133  mM) and adjusted with the phosphate buffer
to the final concentration of approximately 330 000
cells per 100 µl before each measurement. The bioluminescence
signal of bacterial suspensions after
exposition to herb extracts was measured in relative
light units (RLU) for one hour on LM-01T plate luminometer
(Immunotech, Czech Republic) at 37°C.
The wells of microplates contained 180  µl of bacterial
suspension and 10 µl (5% concentration) of extracts,
buffer was used as a control. The light emission during
reaction is positively correlated with bacteria viability
(Atosuo et al., 2013). To determine antibacterial
properties of extracts, integrals (RLU*s) under kinetics
curves were calculated and expressed as percentage of
control. All samples were prepared in two independent
repetitions and each repetition was analysed three
times. Results are presented as mean values from the
six measurements.
362											 2014P. Denev and others
RESULTS AND DISCUSSION
Polyphenol composition and antioxidant
activity of herb extracts
Results of the HPLC analysis of herb extracts
are shown in Table 1. The analyzed extracts
showed substantial differences in their
polyphenol composition and content. Aronia
leaves are distinctive with a high content of
hydroxycinnamic acids (chlorogenic and neochlorogenic)
among polyphenol acids with a
total content of 3409 mg/100 g. Lady’s mantle
contains 3,4-hydroxybenzoic acid, chlorogenic
acid and p-coumaric acid totaling 429 mg/100
g. Among the investigated herbs, lady’s mantle
is the richest source of flavonoids, containing
catechin (250 mg/100 g), epicatechin (524
mg/100 g) and a significant amount of rutin
(1057 mg/100 g). The meadowsweet has the
highest content of total plyphenols (14596.2
mg/100 g) and is a balanced source of phenolic
acids (total content — 1531 mg/100 g)
and flavonoids — 1447 mg/100 g. Hawthorn
leaves contain 1080 mg/100 g of phenolic acids
and 1831 mg/100 g of flavonoids. Blackberry
leaves are a rich source of polyphenol
compounds — 12678 mg/100 g. They contain
3,4-dihydroxybenzoic acid (208 mg/100
g), ellagic acid (19 mg/100g) and myrecetin
(466 mg/100 g). Raspberry leaves contain
gallic, 3,4-dihydroxybenzoic, caffeic and ellagic
acids. Epicatechin and rutin are the most
abundant flavonoids in that herb. It is obvious
from the results in Table 1 that the sum of
the investigated phenolic acids and flavonoid
contents is less than the total polyphenol content
of the investigated herbs. We analyzed 16
of the most common polyphenol constituents
in plants but it could be expected that other
phenolic acids and flavonoids, not analyzed
in this work were also present. Smolyakova
et al. (2012) reported that lady’s mantle polyphenol
content is up to 9.6% of the dry herb
weight, which is closed to our result – 8.4%,
but luteolin and apigenin, and their glycosides
not analyzed in our study are the main flavonoids
in that herb. Besides flavonoids and
phenolic acids, medicinal plants are known to
be a rich source of tannins (condensed and
hydrolysable), which were not analyzed in the
current study either. For example, Duckstein
and coworkers (2013) identified 24 phenolic
compounds in lady’s mantle including pedunculagin
and agrimoniin, and other monomeric
and oligomeric ellagitannins, which constituted
the major phenolic fraction. Gudej and
Tomczyk (2004) reported that tannins are the
main polyphenol constituents in raspberry and
blackberry leaves. Fecka (2009) found 27 polyphenol
compounds in meadowsweet, including
substantial amounts of ellagitannins (up to
12500 mg/100 g DW) and rugosins A, B and
E were identified for the first time as meadowsweet
constituents. According to Fecka,
the total polyphenol content of meadowsweet
reached 16568  mg/100 g, which is similar to
our result — 14596 mg/100 g. The high con-
Table1.Contentofphenolicacidsandflavonoidsinherbs
Herb
gallicacid,
mg/100g
neochlorogenicacid,
mg/100g
3,4-dihydroxy-benzoicacid,
mg/100g
chlorogenicacid,
mg/100g
caffeicacid,
mg/100g
p-coumaric,
acidmg/100g
ferulicacid,
mg/100g
ellagicacid,
mg/100g
totalphenolicacids,
mg/100g*
Blackberryleaves––208––––19227
Chokeberryleaves–1350–2014–21–243409
Hawthornleaves–426–654––––1080
Lady’smantle63–13580151–––429
Meadowsweet261––232107728122811531
Raspberryleaves28–58–40––37163
catechin,
mg/100g
epicatechin,
mg/100g
rutin,
mg/100g
naringin,mg/100g
myrecetin,
mg/100g
quercetin,
mg/100g
naringenin,
mg/100g
totalflavonoids,
mg/100g*
totalpolyphenols,
mg/100g
Blackberryleaves––––466––46612678
Chokeberryleaves––22––––226111
Hawthornleaves53–843––68–9645283
Lady’smantle2505241057––––18318377
Meadowsweet––1374––6211144714596
Raspberryleaves–283118––––4013976
*Calculatedassumofindividualrepresentatives
Vol. 61 						 363Biological activities of herbs
tent of hydrolysable tannins in that herb was confirmed
by other studies (Barros et al., 2013). Chokeberry leaves’
polyphenols have not been sufficiently investigated and
data regarding their polyphenol composition and content
are scarce. A recent study found 12 polyphenol compounds
in chokeberry leaves and confirmed our results
that chlorogenic and neochlorogenic acids are the main
phenolic constituents (Lee et al., 2014). Beside that, dicaffeoylquinic
acid was detected in high amounts, along
with other minor components. Bearing in mind that
chokeberry fruits contain substantial quantity of condensed
tannins (Denev et al., 2012), we could expect
that their leaves also accumulates significant amounts of
these compounds.
The differences in the polyphenol content and composition
of the investigated herbs are reflected in their
biological activities and particularly antioxidant activity.
When investigating antioxidant properties of natural
antioxidants it is recommended to use more than one
antioxidant assay for better understanding the principles
of their antioxidant action (Ciz et al., 2010). Therefore,
we used several assays addressing various aspects
of the antioxidant action of polyphenols in our study –
ORAC, TRAP, HORAC and inhibition of lipid peroxidation.
The methods used embrace different aspects of
the antioxidant action and give a broader view on the
antioxidant potential of the herb extracts. The ORAC
and TRAP methods measure the ability of antioxidants
to scavenge peroxyl radicals via hydrogen atom transfer.
These radicals are physiologically the most important
ones and the hydrogen atom transfer is the most physiologically
relevant mechanism of antioxidant action. The
HORAC method measures the metal-chelating activity of
antioxidants under conditions of Fenton-like reactions
and hence indicates the protecting ability against formation
of hydroxyl radical. Results for the ORAC, TRAP
and HORAC antioxidant activities are shown in Ta-
ble  2. In our previous study, we reported ORAC antioxidant
activity of 25 herbs used traditionally in the Bulgarian
folk medicine (Kratchanova et al., 2010). Compared
to those results all the herbs investigated in the current
study showed relatively high ORAC antioxidant activities,
ranking among top ten herbs with the highest ORAC
values. Blackberry leaves revealed the highest antioxidant
activity using the three methods (ORAC — 1805.8 μmol
TE/g; TRAP — 2936.6 μmol TE/g; and HORAC —
2075.6 μmol GAE/g). Meadowsweet showed the second
highest ORAC and TRAP values — 1555.0 μmol TE/g
and 1992.7 μmol TE/g, respectively, indicating that this
herb is a rich source of chain-braking polyphenolic antioxidants.
Interestingly, lady’s mantle extract revealed the
second highest chelating ability expressed as a HORAC
value of 1999.4 μmol GAE/g.
The effect of the extracts on lipid peroxidation was
evaluated by assaying the autoxidation of linoleic acid
in ethanol-buffer system by measuring the formation of
linoleic acid hydroperoxides. This is a simple and reliable
method for evaluation of the effects of potential
antioxidants. As shown in Fig. 1A, the concentration
of hydroperoxides rises gradually during the six days
of linoleic acid autoxidation at 40°C. The formation of
hydroperoxides in the presence of the herb extracts is
shown in Fig. 1B. It is obvious that the autoxidation
of linoleic acid was very effectively inhibited, especially
by the extract from blackberry leaves. In this case, the
concentration of hydropexides was lowered to 2% of
the control of all time points. The second most effective
extract was that from meadowsweet. It inhibited the
generation of hydroperoxides to 22% of control at day
1 and to 2% at days 3 and 6. The third most effective
was the extract from lady’s mantle, followed by extracts
from chokeberry, hawthorn and raspberry leaves. For
the latter four extracts the inhibition of lipid peroxidation
increased from day 1 to day 6.
Effect of herb extracts on ROS production by
phagocytes
The effects of studied extracts on the production
of ROS by human whole blood phagocytes were studied
using luminol-enhanced CL. The results are shown
in Fig. 2 and expressed as kinetic curves of the CL response
after OZP activation (upper panel), PMA activation
(lower panel) and the integral values of the CL response.
It is obvious that all the extracts studied significantly
inhibited the ROS production, to the background
value in the case of OZP-activated phagocytes. On the
Table 2. ORAC, TRAP and HORAC antioxidant activity of herbs
ORAC,
μmol TE/g
TRAP,
μmol TE/g
HORAC,
μmol GAE/g
Blackberry leaves 1806±105 2937±139 2076±113
Chokeberry leaves 1363±69 1263±56 1120±53
Hawthorn leaves 1405±66 1301±27 882±203
Lady’s mantle 1337±68 1815±38 1999±70
Meadowsweet 1555±85 1993±102 1640±223
Raspberry leaves 1349±119 647±50 1257±82
Figure 1. Inhibition of lipid peroxidation by herb extracts.
Kinetics of hydroperoxide concentration in control samples without
herb extracts during six days of autoxidation of linoleic acid
(upper panel). Eeffect of herb extracts on hydroperoxide accumulation
at the end of the 1st, 3rd and 6th days of autoxidation of linoleic
acid (lower panel). Results are expressed as percentage of
control. A — blackberry leaves; B — chokeberry leaves; C — hawthorn
leaves; D — lady’s mantle; E — meadowsweet; F — raspberry
leaves.
364											 2014P. Denev and others
other hand, some differences were observed when the
effects were studied using PMA-activated human whole
blood phagocytes. The most effective was the extract
of meadowsweet (p≤0.01) followed by blackberry
leaves (p≤0.01), lady’s mantle (p≤0.01), raspberry leaves
(p≤0.05), hawthorn leaves (p≤0.05), chokeberry leaves.
The least effective extract of chokeberry leaves still inhibited
the PMA-activated CL response, but the effect
was statistically non-significant. The inhibitory effects of
the herb extracts on the PMA-activated CL response of
phagocytes and partially also on the OZP-activated CL
response of phagocytes could be caused by a direct ROS
scavenging activity of the extracts due to their total polyphenol
content. There was a strong negative correlation
between the PMA-activated CL response and the total
polyphenol content and a strong positive correlation between
the TRAP antioxidative parameter and total polyphenols
(not shown).
The effects of polyphenols as the basic constituents
of various plant extracts on the respiratory burst of neutrophils
have already been demonstrated. For example,
cocoa flavonoids have been found to moderate a subset
of signaling pathways derived from LPS stimulation of
neutrophils, mainly the neutrophil oxidative burst and
activation markers (Kenny et al., 2009). Those authors
hypothesized that flavonoids could decrease the impact
of LPS on the fMLP-primed neutrophil ability to generate
ROS by partially interfering in the activation of the
MAPK pathway. In another study, Ginkgo biloba extract
containing flavonoids slowed down the respiratory burst
of stimulated human neutrophils (Pincemail et al., 1987).
An inhibitory effect of pure flavonoids on the respiratory
burst of neutrophils has also been demonstrated
(Pagonis et al., 1986; Zielinska et al., 2000). Wang and
coworkers (2002) observed that cirsimaritin inhibited
superoxide anion radical generation and oxygen consumption
of neutrophils. On the other hand, cirsimaritin
slightly enhanced the superoxide anion radical generation
by PMA-activated NADPH oxidase. The results of those
authors indicate that it is likely that the inhibition of the
fMLP-induced respiratory burst by cirsimaritin in rat
neutrophils is mainly through the blockade of the phospholipase
D (PLD) signaling pathway. Selloum and coworkers
(2001) observed that myricetin, quercetin, kaempferol
and rutin inhibited the pholasin luminescence of
fMLP-stimulated neutrophils. Lee and coworkers (2010)
revealed that luteolin attenuated neutrophil respiratory
burst but had a negligible effect on superoxide anion
generation during PMA stimulation. Furthermore, luteolin
effectively blocked MAPK/ERK kinase 1/2 and Akt
phosphorylation in fMLP-stimulated neutrophils. Thus,
an inhibition of enzymes involved in signaling, rather
than scavenging of superoxide anion radicals dominated
in fMLP-stimulated neutrophils exposed to flavonoids in
those studies. fMLP used in the cited studies is a soluble
activator of neutrophils which binds to specific membrane
receptors. Similarly, OZP binds to surface opsonin
(either complement or Fc) receptors on phagocytes
and triggers a signaling cascade leading finally to the activation
of protein kinase C (PKC) with subsequent activation
of NADPH oxidase, producing the superoxide
anion — the first of the ROS produced by phagocytes.
In contrast, PMA activates directly PKC, the step before
NADPH oxidase activation. Our present results indicate
in accordance with the literature that the herb extracts
studied do not inhibit PKC or NADPH oxidase itself,
but interfere with the signaling cascade of phagocyte activation
upstream of the PKC activation.
Antimicrobial activity of herbs
The investigated plants are used in traditional medicine
in the treatment of different infections (Shmid &
Gorris, 2007), but their antimicrobial properties are
poorly investigated. Only few studies on antimicrobial
activities of their extracts have been published to date.
Gniewosz and coworkers (2014) determined the influence
of meadowsweet flower extract on three pathogens
and four saprophytes of apples, using fruit covered
with a pullulan film containing the extract. Martini and
coworkers (2009) analyzed the activity of blackberry
leaves extract against Helicobacter pylori. In our study initial
screening of the antimicrobial activity of the tested
extracts was performed against eleven foodborne pathogens
using the agar diffusion method. The antimicrobial
activity was classified according to Rota and coworkers
(2008) as weak (inhibition zone <12 mm), medium (inhibition
zone between 12 and 20 mm), and strong (inhibition
zone above 20 mm). The results are presented
in Table 3. The blackberry and raspberry leaves extracts
exhibited antimicrobial activity against all test microorganisms.
In some cases single colonies were present in
the inhibition zones (marked with an asterisk in Table
3). Some of the cells of E. coli ATCC 25922, both SalFigure
2. Effect of herb extracts on the production of reactive
oxygen species by phagocytes.
Kinetic curves of the CL response after OZP activation (upper panel)
and PMA activation (lower panel) are shown. A — blackberry
leaves; B — chokeberry leaves; C — hawthorn leaves; D — lady’s
mantle; E — meadowsweet; F — raspberry leaves. Integral values
of CL response (mean ± S.E.M.) are also included. Statistically significant
differences against control are shown by **p<0.01 and
*p<0.05.
Vol. 61 						 365Biological activities of herbs
monella strains, L. monocytogenes I and C.
albicans were less susceptible to the effect
of the blackberry leaves extract and were
able to grow in its presence. A number
of S. enterica ssp. enterica ATCC BAA-
2162, P. aeruginosa and C. albicans cells
were also more resistant to the activity
of the raspberry leaves extract than others.
The highest activity of the blackberry
extract was observed against P. aeruginosa
and P. vulgaris, where inhibition was detected
even for 2% extract. The raspberry
leaves extract produced the largest inhibition
zones (19 mm) against P. vulgaris.
Chokeberry and hawthorn leave extracts
had medium and weak antimicrobial activites,
respectively, against three of the
test strains — the two S. aureus strains
and P. vulgaris. The activity was observed
only for undiluted extracts. Lady’s mantle
extract did not inhibit the growth of
Salmonella, L. monocytogenes or P. aeroginosa,
but antimicrobial activity against S. aureus
ATCC 6538P, P. vulgaris, K. pneumoniae
and C. albicans was detected even
when the lowest extract concentration
was used. The activity against S. aureus
ATCC 25923 and E. coli was weaker —
an inhibitory effect was observed only
for 10% solutions. Meadowsweet extract
also demonstrated antimicrobial activity
against S. aureus strains, P. vulgaris, K.
pneumoniae and C. albicans even in a 2%
solution. Based on the obtained results
four extracts (blackberry, lady’s mantle,
meadowsweet and raspberry leaves) were
selected for further experiments.
The minimal inhibitory concentration
(MIC50) of the extracts was determined
in order to quantify their antimicrobial
activity (Table 4). The lowest MIC50
values were determined for the raspberry
leaves extract against S. aureus ATCC
25923 — 0.031% and the meadowsweet
extract against P. vulgaris and K. pneumoniae
– 0.08%. S. aureus ATCC 25923 was
the most susceptible to the activity of the
herb extracts. Its growth was inhibited at
concentrations lower than 0.3% of the
extracts in the medium. MIC50 values below
1% were found for: blackberry leaves
against Salmonellas sp., S. aureus ATCC
6538P, P. vulgaris and K. pneumoniae; Lady’s
mantle against P. vulgaris; meadowsweet
against P. vulgaris and K. pneumoniae;
raspberry leaves against Salmonella
sp. and Proteus vulgaris. For lady’s mantle
and raspberry leaves extracts the MICs
were 2.5% against S. aureus ATCC 6538P.
None of the extracts reduced the growth
of the L. monocytogenes strains below 50%
of their initial viable cell counts. They
also had low (MIC50=25%) or no activity
against S. enterica ssp. enterica ATCC
BAA-2162, P. aeruginosa or E. coli strains.
All six extracts showed an activity
against genetically modified E. coli. The
bioluminescence signal decreased within
few minutes after exposition of the bac-
Table3.Antimicrobialactivityofherbextractsagainsthumanpathogensmeasuredbyagardiffusionmethod
Test-microorganisms
Concentrationof
viablecellsx107,
cfu/cm3
BlackberryleavesChokeberryleavesHawthornleavesLady’smantleMeadowsweetRaspberryleaves
Extractconcentration,%
100102100102100102100102100102100102
Zoneofinhibition,mm
EscherichiacoliATCC87394.888–––––––1515––––1512–
EscherichiacoliATCC259227.717*12*–––––––1813––––1511–
Salmonellasp.3.216*12*8*––––––––––––1611–
Salmonellaentericassp.entericaATCC
BAA–2162
1.216*14*11*––––––––––––16*11*–
StaphylococcusaureusATCC6538P1.91711913––98–191292114101511–
StaphylococcusaureusATCC259232.71815815––98–2214–2415121612–
Listeriamonocytogenes4.18––––––––––––––1611–
ListeriamonocytogenesI2.524*18*11*––––––––––––1711–
ProteusvulgarisG2.62016813––12––2217112519151912–
PseudomonasaeruginosaATCC90274.9242211––––––––––––15*11*–
Klebsiellapneumoniae2.917128––––––151392011–1611–
CandidaalbicansATCC102311.324*14*10*––––––16141125191318*10*–
*singlecoloniespresentintheinhibitionzone
366											 2014P. Denev and others
teria to herb extracts and stayed low during the entire
experiment. Meadowsweet showed the highest antibacterial
activity (75% inhibition of control, Fig. 3) followed
by blackberry leaves (60%), lady’s mantle (56%), raspberry
leaves (52%) and hawthorn leaves (27%). A very
low activity was found for chokeberry leaves with only
4% growth inhibition. The antibacterial activity of all the
extracts supports the lipid peroxidation inhibition results
and those regarding production of ROS by phagocytes.
CONCLUSION
Results obtained in the present study indicate that all
herbs studied, especially blackberry leaves and meadowsweet,
are rich sources of chain-breaking, chelating
and lipid peroxidation inhibiting antioxidants, rendering
high antioxidant activity measured by several methods.
Besides that the herb extracts could prevent oxidative
stress in the gastrointestinal tract trough modulation of
neutrophils and an antimicrobial effect against a broad
spectrum of human pathogens.
Acknowledgement
This work was funded by project № BG161РО003-
1.1.05-0024-С0001 “Development of nutraceuticals with
antioxidant and immune-stimulating action” uder Operation
1.1.1: „Support for establishment and development
of start-up innovative enterprizes” of Operational Program
“Competitiveness” of the EU.
REFERENCES
Anthon GE, Barrett DM (2001) Colorimetric method for the determination
of lipoxygenase activity. J Agr Food Chem 49: 32–37.
Atosuo J, Lehtinen J, Vojtek L, Lilius EM (2013) Escherichia coli K-12
(pEGFPluxABCDEamp): a tool for analysis of bacterial killing by
antibacterial agents and human complement activities on a real-time
basis. Luminescence 28: 771–779.
Barros L, Alves CT, Duenas M, Silva S, Oliveira R, Carvalho AM,
Henriques M, Santos-Buelga C, Ferreira ICFR (2013) Characterization
of phenolic compounds in wild medicinal flowers from Portugal
by HPLC–DAD–ESI/MS and evaluation of antifungal properties.
Ind Crop Prod 44: 104–110.
Circu M, Aw TY (2010) Reactive oxygen species, cellular redox systems,
and apoptosis. Free Radical Bio Med 48: 749–762.
Ciz M, Cizova H, Denev P, Kratchanova M, Slavov A, Lojek A (2010)
Different methods for control and comparison of the antioxidant
properties of vegetables. Food Control 21: 518–523.
Ciz M, Komrskova D, Pracharova L, Okenkova K, Cizova H, Moravcova
A, Jancinosva V, Petrikova M, Lojek A, Nosal R (2007) Serotonin
modulates the oxidative burst of human phagocytes via various
mechanisms. Platelets 18: 583–590.
Cizova H, Lojek A, Kubala L, Ciz M (2004) The effect of intestinal
ischemia duration on changes in plasma antioxidant defence status
in rats. Physiol Res 53: 523–531.
Denev P, Kratchanov Ch, Ciz M, Lojek A, Kratchanova M (2012)
Bioavailability and antioxidant activity of black chokeberry (Aronia
melanocarpa) polyphenols: in vitro and in vivo evidences and possible
mechanisms of action. A review. Compr Rev Food Sci F 11: 471–489.
Denev P, Kratchanova M, Ciz M, Lojek A, Vasicek O, Nedelcheva P,
Blazheva D, Toshkova R, Gardeva E, Yossifova L, Hyrsl P, Vojtek
L (2014) Biological activities of selected polyphenol-rich fruits related
to immunity and gastrointestinal health. Food Chem 157: 37–44.
Denev P, Lojek A, Ciz M, Kratchanova M (2013) Antioxidant activity
and polyphenol content of Bulgarian fruits. Bulg J Agric Sci 19:
22–27.
Duckstein SM, Lotter EM, Meyer U, Lindequist U, Stintzing FC (2013)
Phenolic constituents from Alchemilla vulgaris L. and Alchemilla mollis
(Buser) Rothm. at different dates of harvest. Z Naturforsch C 68:
529–540.
Fecka I (2009) Qualitative and quantitative determination of hydrolysable
tannins and other polyphenols in herbal products from
meadowsweet and dog rose found rugosins A, B and E as a new
meadowsweet constituents. Phytochem Analysis 20: 177–190.
Gniewosz M, Synowiec A, Krasniewska K, Przyby1 J, Baczek K, Weglarz
Z (2014) The antimicrobial activity of pullulan film incorporatTable
4. Minimal inhibiting concentration (MIC50) of chosen herb extracts against human pathogens
Test microorganisms
Blackberry leaves Lady’s mantle Meadowsweet Raspberry leaves
MIC50, %
Escherichia coli ATCC 8739 25 – 25 –
Escherichia coli ATCC 25922 – – 25 –
Salmonella sp. 0.19 – – 0.78
Salmonella enterica ssp. enterica ATCC BAA-2162 25 – – –
Staphylococcus aureus ATCC 6538P 0.63 2.5 0.63 2.5
Staphylococcus aureus ATCC 25923 0.13 0.25 0.25 0.031
Listeria monocytogenes – – – –
Listeria monocytogenes I – – – –
Proteus vulgaris G 0.16 0.1 0.08 0.78
Pseudomonas aeruginosa ATCC 9027 25 – – –
Klebsiella pneumoniae 0.63 25 0.08 25
Figure 3. Luminometric measurement of antibacterial effect of
herb extracts on E. coli viability.
Results are expressed as percentage of control samples without
herb extracts. A — blackberry leaves; B — chokeberry leaves; C
— hawthorn leaves; D — lady’s mantle; E — meadowsweet; F —
raspberry leaves.
Vol. 61 						 367Biological activities of herbs
ed with meadowsweet flower extracts (Filipendulae ulmariae flos) on
postharvest quality of apples. Food Control 37: 351–361.
Gudej J, Tomczyk M (2004) Determination of flavonoids, tannins
and ellagic acid in leaves from Rubus L. species. Arch Pharm Res 11:
1114–1119.
Gutierrez J, Barry-Ryan C, Bourke P (2008) The antimicrobial efficacy
of plant essential oil combinations and interactions with food ingredients.
Int J Food Microbiol 124: 91–97.
Halliwell B, Zhao K, Whiteman M (2000) The gastrointestinal tract: a
major site of antioxidant action? Free Radical Res 33: 819–30.
Kanner J, Lapidot T (2001) The stomach as a bioreactor: dietary lipid
peroxidation in the gastric fluid and the effects of plant-derived antioxidants.
Free Radical Biol Med 31: 1388–1395.
Kenny TP, Shu SA, Moritoki Y, Keen CL, Gershwin ME (2009) Cocoa
flavanols and procyanidins can modulate the lipopolysaccharide
activation of polymorphonuclear cells in vitro. J Med Food 12: 1–7.
Kikuzaki H, Hisamoto M, Hirose K, Akiyama K, Taniguchi H (2002)
Antioxidant properties of ferulic acid and its related compounds. J
Agr Food Chem 50: 2161–2168.
Kratchanova M, Denev P, Ciz M, Lojek A, Mihailov A (2010) Evaluation
of the antioxidant activity of medicinal plants containing polyphenol
compounds. Comparison of two extraction systems. Acta
Biochim Pol 57: 229–234.
Krzymińska S, Tańska A, Kaznowski A (2013) Aeromonas spp. induce
apoptosis of epithelial cells through an oxidant-dependent activation
of the mitochondrial pathway. J Med Microbiol 60: 889–898.
Lee JE, Kim G-S, Park S, Kim Y-H, Kim M-B, Lee WS, Jeong SW,
Lee SJ, Jin JS, Shin SC (2014) Determination of chokeberry (Aronia
melanocarpa) polyphenol components using liquid chromatography–
tandem mass spectrometry: Overall contribution to antioxidant activity.
Food Chem 146: 1–5.
Lee JP, Li YC, Chen HY, Lin RH, Huang SS, Chen HL, Kuan PC,
Liao MF, Chen CJ, Kuan YH (2010) Protective effects of luteolin
against lipopolysaccharide-induced acute lung injury involves inhibition
of MEK/ERK and PI3K/Akt pathways in neutrophils. Acta
Pharm Sinia 31: 831–838.
Martini S, D’Addario C, Colacevich A, Focardi S, Borghini F, Santucci
A, Figura N, Rossi C. (2009) Antimicrobial activity against Helicobacter
pylori strains and antioxidant properties of blackberry leaves
(Rubus ulmifolius) and isolated compounds. Int J Antimicrob Ag 34:
50–59.
Ou B, Hampsch-Woodill M, Flanagan J, Deemer EK, Prior RL, Huang
DJ (2002) Novel fluorometric assay for hydroxyl radical prevention
capacity using fluorescein as the probe. J Agr Food Chem 50:
2772–2777.
Ou B, Hampsch-Woodill M, Prior RL (2001) Development and validation
of an improved oxygen radical absorbance capacity assay
using fluorescein as the fluorescence probe. J Agr Food Chem 49:
4619–4626.
Pagonis C, Tauber AI, Pavlotsky N, Simons ER (1986) Flavonoid impairment
of neutrophil response. Biochem Pharmacol 35: 237–245.
Pincemail J, Thirion A, Dupuis M, Braquet P, Drieu K, Deby C (1987)
Ginkgo biloba extracts inhibits oxygen species production generated
by phorbol myristate acetate stimulated human leukocytes. Experientia
43: 181–184.
Rota MC, Herrera A, Martinez RM, Sotomayor JA, Jordan MJ (2008)
Antimicrobial activity and chemical composition of Thymus vulgaris,
Thymus zygis and Thymus hyemalis essential oils. Food Control 19: 681–
687.
Selloum L, Reichl S, Muller M, Sebihi L, Arnhold J (2001) Effects of
flavonols on the generation of superoxide anion radicals by xanthine
oxidase and stimulated neutrophils. Arch Biochem Biophys 395:
49–56.
Shmid EJ, Gorris LGM (2007) Natural antimicrobials in food preservation.
In Handbook of food preservation. Rahman MS, ed. CRC Press,
Boca Raton.
Singleton V, Rossi J (1965) Colorimetry of total phenolic with phosphomolibdiphosphotungstic
acid reagents. Am J Enol Vitic 16: 144–
158.
Smolyakova IM, Andreeva VY, Kalinkina GI, Avdeenko SN, Shchetinin
PP (2012) Development of extraction techniques and standardization
methods for a common lady’s mantle (Alchemilla vulgaris) extract.
Pharm Chem J-USSR 45: 675–678.
Suzuki H, Nishizawa T, Tsugawa H, Mogami S, Hibi T (2012) Roles of
oxidative stress in stomach disorders. J Clin Biochem Nutr 50: 35–39.
Wang JP, Chang LC, Hsu MF, Chen SC, Kuo SC (2002) Inhibition of
formyl-methionyl-leucyl-phenylalaninestimulated respiratory burst by
cirsimaritin involves inhibition of phospholipase D signaling in rat
neutrophils. N-S Arch Pharmacol 366: 307–314.
Zielinska M, Kostrzewa A, Ignatowicz E (2000) Antioxidative activity
of flavonoids in stimulated human neutrophils. Folia Histochem Cyto
38: 25–30.
Biological activities of selected polyphenol-rich fruits related
to immunity and gastrointestinal health
Petko Denev a
, Maria Kratchanova a,⇑
, Milan Ciz b
, Antonin Lojek b
, Ondrej Vasicek b
,
Plamena Nedelcheva c
, Denitsa Blazheva c
, Reneta Toshkova d
, Elena Gardeva d
,
Liliya Yossifova d
, Pavel Hyrsl e
, Libor Vojtek e
a
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Laboratory of Biologically Active Substances, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria
b
Institute of Biophysics of the AS CR, Kralovopolska 135, 612 65 Brno, Czech Republic
c
University of Food Technologies, 26 Maritsa Blvd., 4002 Plovdiv, Bulgaria
d
Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 2, 1113 Sofia, Bulgaria
e
Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 10 October 2013
Received in revised form 7 January 2014
Accepted 5 February 2014
Available online 12 February 2014
Keywords:
Small fruits
Polyphenols
Antioxidant activity
Oxidative burst
Antimicrobial properties
Mitogenic activity
a b s t r a c t
Small fruits are a rich source of bioactive substances, including polyphenols, and are therefore suitable raw
materials for the production of functional foods. In the current work, we studied the antioxidative properties
of six fruits: rosehip, chokeberry, hawthorn, blackcurrant, blueberry and rowanberry via different methods
(ORAC, TRAP, HORAC and inhibition of lipid peroxidation). Their effect on the production of reactive oxygen
species (ROS) by phagocytes, antimicrobial properties against 11 human pathogens, and mitogenic effect on
hamster spleen lymphocytes were also tested. Rosehip extract showed the highest antioxidant activity via
ORAC, TRAP and HORAC assays, whereas blueberry extract was the most potent inhibitor of lipid peroxidation.
All extracts inhibited ROS production of opsonized zymosan-activated phagocytes, indicating that
extracts interfere with the signaling cascade of phagocyte activation upstream to the protein kinase C activation.
Chokeberry, blackcurrant and rowanberry extracts revealed strong antimicrobial properties against
a broad spectrum of microorganisms and also had the highest mitogenic activity.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
There are numerous studies demonstrating a correlation
between the consumption of fresh fruits and vegetables with the
prevention of socially significant diseases including cancer, cardiovascular
diseases and Alzheimer disease (Heber, 2004; Hertog et al.,
1996). A considerable amount of evidence indicates that increased
oxidative damage contributes to the development of these diseases
and several epidemiologic studies point out that the consumption of
polyphenol-rich foods and beverages is associated with a lower risk
of oxidative stress-related diseases (Ames, Shigenaga, & Hagen,
1993; Hertog et al., 1996). Polyphenols are a class of secondary plant
metabolites that are thought to exert beneficial health effects
through their antioxidant properties (Halliwell, Zhao, & Whiteman,
2000). Although there is a considerable volume of scientific data
regarding flavonoid health benefits, the majority of the mechanisms
of their bioactivity still remain unclear. Many of the polyphenol
compounds are barely, if not at all, absorbed in the gastrointestinal
tract (GIT). For example, anthocyanins which are broadly distributed
in many plants, appear to be poorly absorbed in the small
intestine. They are recovered in blood and urine in nanomolar concentrations,
so significant amounts probably pass into the large
intestine where bacterial degradation occurs. Similarly, proanthocyanidins,
which are referred to as polymeric flavonoids and are
strong antioxidants in vitro, have very limited absorption in the
GIT; oligomers larger than trimers are unlikely to be absorbed in
the small intestine in their native forms (Denev, Kratchanov, Ciz,
Lojek, & Kratchanova, 2012). Although some flavonoids can be
absorbed through the GIT, maximal plasma concentrations
achieved are very low, in part because of rapid metabolism by
human tissues and colonic bacteria (Halliwell, Rafter, & Jenner,
2005). Since absorption of phenolic compounds is incomplete, the
majority passes through the GIT and enters the colon. Their local
action may nevertheless be important because the intestine is
particularly exposed to oxidising agents and may be affected by
http://dx.doi.org/10.1016/j.foodchem.2014.02.022
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel./fax: +359 32 642759.
E-mail addresses: petkodenev@yahoo.com (P. Denev), lbas@plov.omega.bg (M.
Kratchanova), milanciz@ibp.cz (M. Ciz), alojek@ibp.cz (A. Lojek), ondrej.vasicek@
ibp.cz (O. Vasicek), pl.nedelcheva@gmail.com (P. Nedelcheva), dblazheva@gmail.
com (D. Blazheva), reneta_toshkova@yahoo.com (R. Toshkova), elena.gardeva@
gmail.com (E. Gardeva), lilyayosifova@gmail.com (L. Yossifova), hyrsl@sci.muni.cz
(P. Hyrsl), libor.vojtek@mail.muni.cz (L. Vojtek).
Food Chemistry 157 (2014) 37–44
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
inflammation and numerous diseases, such as cancer. The human
diet contains various pro-oxidants, including metals such as iron,
copper, lipid hydroperoxides, aldehydes and nitrite, and elevated
levels of lipid peroxides have been observed in the postprandial
state. These pro-oxidants may induce oxidative stress in the GIT, induce
stomach ulcers and the development of stomach, colon and
rectal cancers. In addition, there are vast numbers of phagocytic
cells that normally reside in the GIT. Phagocytic cells, such as
macrophages and neutrophils, play a key role in innate immunity
because of their ability to recognise, ingest and destroy pathogens
by oxidative and nonoxidative mechanisms. In response to a variety
of stimuli, NADPH oxidase present in neutrophils is activated in a
phenomenon described as ‘‘the respiratory burst,’’ characterised
by the production of superoxide anion, which gives rise to other
forms of reactive oxygen species (ROS). In addition to ROS, nitric
oxide (NO) and other reactive nitrogen species (RNS) produced
mainly by macrophages are one of the important microbicidal tools
in the process of inflammation during the fight against pathogenic
microorganisms, bacteria and tumor cells (Pekarova et al., 2009).
However, excessive or inappropriate ROS and RNS production by
phagocytes is associated with oxidative damage to membrane
lipids, DNA, proteins and lipoproteins, resulting in various autoimmune
and inflammatory diseases. Thus, the modulation of inflammation
and oxidative stress by natural substances can be
beneficial. The antioxidants contained in foods may suppress such
oxidative stress and related diseases in the gastrointestinal tract
before they are absorbed (Halliwell et al., 2000). Many of the phenolic
metabolites in the colon still have free –OH groups and maintain
antioxidant activity. Thus, in addition to the original phenolic compounds,
their metabolites also have to be considered to understand
the biological and antioxidant functions of foods. Polyphenol concentrations
in the colon can reach several hundred micromoles
per litre (Scalbert & Williamson, 2000) and together with a few
carotenoids, they constitute the only dietary antioxidants present
in the colon, because vitamins C and E are absorbed in the upper segments
of the intestine. Therefore, foods rich in flavonoids and other
polyphenols may protect against gastric, and possibly colon cancers.
In a recent comparative study, we observed that fruits such as
rosehip (Rosa canina), chokeberry (Aronia melanocarpa), hawthorn
(Crataegus monogyna), blackcurrant (Ribes nigrum), blueberry
(Vaccinium myrtillus) and rowanberry (Sorbus aucuparia) possess
the highest antioxidant activity measured by the ORAC method
among 26 kinds of fruits (Denev, Lojek, Ciz, & Kratchanova, 2013).
Based on these results we decided to perform more detailed studies
on the antioxidative properties of these fruits, their effect on the
production of ROS by phagocytes and their antimicrobial properties.
Therefore, the aim of the current study was to determine the
antioxidant properties of six fruit extracts with various methods
(ORAC, TRAP, HORAC and inhibition of lipid peroxidation), to investigate
their effect on the production of ROS from activated macrophages
and to determine their antimicrobial properties and the
minimal inhibiting concentration against 11 human pathogens.
The major polyphenol constituents of the extracts were also identified,
and their mitogenic effect on hamster spleen lymphocytes was
tested. Since some of the fruits such as chokeberry, rosehip, rowanberry
and hawthorn are not widely consumed fresh, the results of
the currents study will be used for the development of functional
foods directed to maintain immunity and gastrointestinal health.
2. Materials and methods
2.1. Plant materials
All fruit samples were collected from the region of Rhodopi
Mountain in the stage of full maturity in 2012. The following 6
kinds of fruits were investigated: rowanberry (S. aucuparia), hawthorn
(C. monogyna), rosehip (R. canina), chokeberry (A. melanocarpa),
blackcurrant (R. nigrum) and blueberry (V. myrtillus).
Chokeberries were cultivated, while all other fruits were widely
collected in nature. Fresh fruits were frozen immediately and
freeze dried in a Cryodos-50 laboratory freeze drier (Telstar Industrial,
Spain).
2.2. Extraction
All plant materials were subjected to extractions under the following
conditions: 50 g of the freeze dried fruits (edible part) were
powdered in a laboratory mill. Then 5 g of the powder was transferred
into extraction tubes and mixed with 100 ml of the extragent
(80% acetone in 0.2% formic acid). Extraction was conducted
on an orbital shaker at room temperature for one hour. After that,
the samples were centrifuged (6000g) and supernatants were
removed. Extracts were concentrated via rotary evaporation to a
volume of 15 ml in order to fully remove the acetone. Then the
volume was adjusted to 50 ml with ultra clean water and the
extracts were centrifuged. Clear supernatants were used for all
further analysis after proper dilutions.
2.3. HPLC analysis of phenolic compounds
High Performance Liquid Chromatography (HPLC) analyses of
phenolic components was performed on an Agilent 1220 HPLC system
(Agilent Technology, USA), equipped with a binary pump and
UV–vis detector. A wavelength of k = 280 nm was used. Separation
of phenolic compounds was performed using an Agilent TC-C18
column (5 lm, 4.6 Â 250 mm) at 25 °C. Mobile phases constituted
of 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of
0.8 ml/min. A gradient was used with 14% B, between 6 min and
30 linearly increased to 25% B and then 50% B at 40 min. The standard
compounds (gallic acid, 3,4-dihydroxy benzoic acid, chlorogenic
acid, caffeic acid, p-coumaric acid, ferulic acid, ellagic acid,
catechin, epicatechin, rutin, naringin, myrecetin, quercetin,
naringenin and kaempherol) were purchased from Sigma–Aldrich
(Steinheim, Germany).
2.4. Total polyphenol compound analysis
Total polyphenols were determined according to the method of
Singleton and Rossi (1965) with Folin–Ciocalteu’s reagent. Gallic
acid was employed as a calibration standard and results were
expressed as gallic acid equivalents (GAE) per litre of extract.
2.5. Total anthocyanins determination
Anthocyanins were determined by the pH-differential method
(Lee, 2005). The absorption of the sample was measured at pH
1.0 and pH 4.5 and the difference in absorbance is proportional
to the anthocyanin content. The total anthocyanin content was
expressed as mg cyanidin-3-glucoside equivalents per litre of
extract and calculated via the following formula:
Anthocyanin content ðmg=lÞ ¼
A Â MW Â DF Â 1000
e  L
;
where A = (A510nm pH 1.0 À A700nm pH 1.0) À (A510nm pH
4.5 À A700nm pH 4.5), MW = cyanidin-3-glucoside molecular weight
(449.2); DF = dilution factor; e = cyanidin-3-glucoside molar
absorbtivity (26,900); L = cell pathlength (usually 1 cm).
38 P. Denev et al. / Food Chemistry 157 (2014) 37–44
2.6. Antioxidant activity assays
2.6.1. Oxygen Radical Absorbance Capacity (ORAC) assay
ORAC was measured according to the method of Ou,
Hampsch-Woodill, and Prior (2001) with some modifications
described in details by Denev et al. (2010). The method measures
the antioxidant scavenging activity against peroxyl radical
generated by the thermal decomposition of 2,20
-azobis[2-methylpropionamidine]
dihydrochloride (AAPH) at 37 °C. Fluorescein
(FL) was used as the fluorescent probe. The loss of fluorescence
of FL was an indication of the extent of damage from its reaction
with the peroxyl radical. The protective effect of an antioxidant
was measured by assessing the area under the fluorescence decay
curve (AUC) relative to that of a blank, in which no antioxidant has
been present. Solutions of AAPH, fluorescein and trolox were prepared
in a phosphate buffer (75 mmol/l, pH 7.4). Samples were also
diluted in the phosphate buffer. The reaction mixtures (total volume
200 ll) contained FL – (170 ll, final concentration 5.36 Â 10À8
mol/
l), AAPH – (20 ll, final concentration 51.51 mmol/l), and sample –
(10 ll). The FL solution and sample were incubated at 37 °C for
20 min directly in a microplate reader and AAPH (dissolved in buffer
at 37 °C) was added. The mixture was incubated for 30 s before the
initial fluorescence was measured. After that, the fluorescence readings
were taken at the end of every cycle (1 min) after shaking. For
the blank, 10 ll of phosphate buffer was used instead of the extract.
The antioxidant activity was expressed in micromole trolox equivalents
(lmol TE) per litre of extract. Trolox solutions (6.25; 12.5; 25
and 50 lmol/l) were used for defining the standard curve. ORAC
and HORAC analyses were carried out using a FLUOstar OPTIMA
plate reader (BMG Labtech, Germany), with an excitation wavelength
of 485 nm and emission wavelength of 520 nm.
2.6.2. Total Peroxyl Radical Trapping Parameter (TRAP) assay
The luminol-enhanced chemiluminescence (CL) was used to follow
the peroxyl radical reaction and the principle of this method is
described by Cizova, Lojek, Kubala, and Ciz (2004). The CL signal is
driven by the production of luminol derived radicals from thermal
decomposition of AAPH. The TRAP value is determined from the
duration of the time period (Tsample) during which the sample
quenched the CL signal due to the present antioxidants. A known
quantity (8.0 nM) of trolox (a water-soluble analogue of tocopherol)
was used as a reference inhibitor (Ttrolox) instead of the sample.
The calculation of the TRAP value is represented by the
equation:
TRAP ¼ 2:0 ½TroloxŠ Tsample=f Ttrolox;
where 2.0 is the stoichiometric factor of trolox (the number of peroxyl
radicals trapped per one molecule of trolox) and f is the dilution
of the sample. The activity of the sample is expressed as
trolox equivalents (TE) per litre of extract. TRAP assay was conducted
on a Luminometer Orion II (Berthold Dection System GmbH,
Germany).
2.6.3. Hydroxyl Radical Averting Capacity (HORAC) assay
The HORAC assay developed by Ou et al. (2002) measures the
metal-chelating activity of antioxidants in the conditions of Fenton-like
reactions employing a Co(II) complex and, hence, measures
the protecting ability against formation of hydroxyl radical.
Hydrogen peroxide solution of 0.55 M was prepared in distilled
water. 4.6 mM Co(II) was prepared as follows: 15.7 mg of CoF2Á4H2O
and 20 mg of picolinic acid were dissolved in 20 ml of distilled
water. Fluorescein (170 ll, 60 nM final concentration) and 10 ll
of sample were incubated in 37 °C for 10 min directly in the FLUOstar
plate reader. After incubation, 10 ll of H2O2 (27.5 mM final
concentration) and 10 ll of Co(II) (230 lM final concentration)
solutions were added subsequently. The initial fluorescence was
measured after which the readings were taken every minute after
shaking. For the blank sample, phosphate buffer solution was used.
Gallic acid solutions (100, 200, 600, 800 and 1000 lM, in phosphate
buffer 75 mM, pH = 7.4) were used for building the standard
curve. The AUC were calculated the same way as the ORAC. The
results were expressed in micromole galic acid equivalents (lmol
GAE) per litre of extract.
2.6.4. Inhibition of lipid peroxidation assay
A modified assay described by Anthon and Barrett (2001) and
Kikuzaki, Hisamoto, Hirose, Akiyama, & Taniguchi (2002) was used
in the study. A mixture of 1 ml of a weighed test sample in 99.5%
ethanol, 1 ml of 83.5 mM linoleic acid in 99.5% ethanol, 2 ml of
0.05 M phosphate buffer (pH 7.0), and 1 ml of water in glass tube
with a plastic stopper was placed in a thermostat and incubated
at 40 °C in the dark. All samples were run in duplicates and the
absorbance at 234 nm was measured after 1, 3 and 6 days of incubation
using a microplate reader Infinite M200 (Tecan, Austria). For
conversion of absorbance to the concentration of hydroperoxide,
the extinction coefficient 23,000 MÀ1
cmÀ1
was used.
2.7. Oxidative burst
Heparinised blood (50 IU/ml) from healthy volunteers (n = 7)
was obtained by antecubital venipuncture. The oxidative burst of
phagocytes in whole blood was measured chemiluminometrically
using an LM-01 microplate luminometer (Immunotech, Czech
Republic). The principle of the method was described previously
(Ciz et al., 2007). Each reaction mixture consisted of 25 ll of whole
blood diluted 10Â in Hank’s balanced salt solution (HBSS), 2.5 ll of
one of the tested samples, 25 ll of 1 mM luminol and 25 ll of one
of the activators À62.5 lg/ml of opsonized zymosan particles
(OZP; Sigma, USA) or 0.81 lg/ml of phorbol-12-myristate-13-acetate
(PMA; Sigma, USA). HBSS was used to adjust the total reaction
volume to 250 ll. The assays were run in duplicates. Spontaneous
CL measurements in samples containing blood and other substances
except any activator were included in each assay. The
chemiluminescence (CL) activity of the samples was immediately
measured at 37 °C. Light emission expressed as relative light units
(RLU) was recorded continuously for 60 min to obtain kinetic
curves. Integrals of CL signal over 60 min were also calculated.
2.8. Mitogenic activity
The spleens from healthy hamsters were isolated and mechanically
dispersed, filtered over a fine mesh and layered on FicollPaque
at a ratio of 2:1. The tubes with spleen cell suspensions were
centrifuged at 1800 rpm for 30 min at room temperature. Cells at
interface-splenic lymphocytes (splenocytes) were collected and
washed three times with RPMI-1640 medium (without FCS).
Spleen cells were finally suspended in complete RPMI-1640 medium
(supplemented with 100 lg/ml penicillin–streptomycin and
10% FCS). Hamsters’ splenocytes were counted in a haemocytometer
and cell viability was determined using 0.2% trypan blue. Cell
concentration was adjusted to 2 Â 106
cells/ml in complete RPMI-
1640 medium.
The effect of fruit extracts on normal hamsters’ spleen lymphocytes
was assessed colorimetrically by the MTT assay as described
by Mossmann (1983). Cell number and viability were evaluated
by measuring the mitochondrial-dependent conversion of the yellow
tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to purple formazan crystals by
metabolically active cells. Cells were seeded in a 96-well microtiter
plate at a concentration of 2 Â 104
cells per well. After incubation
overnight at 37 °C in humidified air with 5% CO2 to allow cell
P. Denev et al. / Food Chemistry 157 (2014) 37–44 39
attachment, the medium was changed and cells were treated with
different concentrations of the tested compounds. Cells cultured in
only medium were used as negative control. After treatment for 24
and 48 h, the cells were washed twice with PBS (pH 7.4) and further
incubated with 100 ll of MTT working solution (Sigma–Aldrich,
Steinheim, Germany) at 37 °C for 3 h. Supernatants were aspirated
and 100 ll of lysing solution (dimethylsulfoxide:ethanol = 1:1, v/v)
an added to each well to dissolve the resulting formazan. MTT assay
was performed using an ELISA plate reader (Tecan, Austria). The
percentage of viable cells was calculated as follows:
Cell viability ð%Þ ¼ OD570 ðexperimentalÞ=OD570 ðcontrolÞ Â 100:
2.9. Ethical aspects
The animal tests were conducted in accordance with the principles
for laboratory animal use and care as found in the European
Community guidelines, Committee on Care of Laboratory Animal
resources, Commission on Life Sciences, National Research Council.
Hamsters were housed in the animal care facilities of the IEMPAM
Institute, which are fully accredited for Laboratory Animal Care.
2.10. Determination of the antimicrobial activity through agar
diffusion method
A suspension of each test microorganism (concentration
$1.107
cfu/cm3
) was prepared with saline solution. Melted PCA
(Scharlau) nutrient medium was inoculated through the addition
of 1 cm3
of microbial suspension and was poured in Petri dishes
(15 cm3
in each dish). Wells with 7 mm diameter were made in
the solidified and cooled agar medium. Tested extracts (50 ll)
were pipetted in the wells. The Petri dishes were incubated at
37 °C for 24–48 h. The inhibition zone was measured. Zones with
a diameter greater than 7 mm were considered zones of inhibition.
2.11. Determination of minimal inhibiting concentration (MIC)
The antimicrobial effects of the extracts were determined by
analysing the MIC using a 96 well microtiter plate method (broth
dilution) according to the modified protocol described by Gutierrez,
Barry-Ryan, and Bourke (2008). The first row of each plate contained
300 ll of nutrient broth (tripton – 5.0 g/dm3
, yeast extract
– 2.5 g/dm3
, glucose – 1 g/dm3
, pH 7.0) and 100 ll of the tested
extract. From the second to the twelfth row, 200 ll of broth was
added and wells containing each extract in the first row were then
diluted twofold along each column, up to the eleventh column. The
twelfth column was used for growth control (positive control) and a
row was used for control sample (negative control) for each dilution.
These additional controls contained distilled water and growth
media inoculated with each microorganism under investigation.
Finally, 30 ll of each working culture of the test microorganism
(1 Â 107
cfu/cm3
) was added to all the wells except the negative controls.
Plates were then incubated at 37 °C and after 24 h of incubation,
they were placed in a microplate spectrophotometer
(absorbance at 600 nm; SPECTROstarNano
, BMG Labtech, Germany)
to determine the presence or absence of turbidity. For each treatment,
the absence of turbidity in the wells compared to the control
was considered as an inhibitory effect. The lowest concentration of
the tested extract required to reduce the growth of the tested microorganism
to 50% of the positive control was designated as the MIC.
2.12. Statistical analysis
The results are expressed as the means from at least three
independent experiments ± standard error of the mean (SEM).
Experiments were conducted in duplicates or triplicates. Comparisons
with the control were performed by analysis of variance (ANOVA),
followed by the Newman–Keuls Post-hoc test. P values less
than 0.05 were considered significant.
3. Results and discussion
3.1. Major polyphenol constituents of fruit extracts
The results from the HPLC analysis of the fruit extracts are
shown in Table 1. The analysed extracts showed big differences
in the polyphenol composition and content. The total amount of
polyphenol compounds was the highest in chokeberry extract –
6274.8 mg/l, whereas blueberry extract was the richest source of
anthocyanins – 1242.4 mg/l. Rowanberry and chokeberry had the
highest content of phenolic acids. In rowanberry extract, the
hydroxycinnamic acids, chlorogenic and neochlorogenic acids,
were the only detected polyphenol compounds and their total
amount was 1133.1 mg/l. The total amount of these two acids in
aronia extract was 1112.8 mg/l. In hawthorn, rosehip and blackcurrant
flavonol and flavan-3-ol representatives were predominant
over the phenolic acids. From the investigated extracts, rosehip
had the highest rutin content at 107.3 mg/l, hawthorn the highest
epicatechin content – 119.8 mg/l and blackcurrant extract had the
highest amount of catechin – 144.5 mg/l. The differences in the
polyphenol profile of the investigated extracts are prerequisite
for different biological activity.
3.2. Antioxidant activity of fruit extracts
When assaying the antioxidant activity of natural antioxidants,
it is recommended to use more than one antioxidant assay for a
detailed understanding of the antioxidant properties of substances
(Ciz et al., 2010). Therefore, in our study we used several assays
expressing various aspects of the antioxidant action of polyphenols
– ORAC, TRAP, HORAC and inhibition of lipid peroxidation. The
methods used embrace different aspects of the antioxidant action
and give a broader view on the antioxidant potential of fruit
extracts. ORAC and TRAP methods measure the ability of the
antioxidant to scavenge peroxyl radicals via hydrogen atom transfer.
These radicals are physiologically the most important ones
and the hydrogen atom transfer is the most physiologically relevant
mechanism of antioxidant action. The HORAC method measures the
metal-chelating activity of antioxidants under the conditions of
Fenton-like reactions and hence indicates the compounds protecting
ability against formation of hydroxyl radical. Using the ORAC,
TRAP and HORAC assays rosehip extract had the highest antioxidant
activity (Fig. 1, ORAC – 93677.6 lmol TE/l; TRAP – 87109.4 lmol TE/l
and HORAC – 76069.4 lmol GAE/l). Hawthorn extract revealed the
second highest antioxidant activity with these three methods. The
results obtained for the antioxidant activities indicate that all fruits
tested and particularly rosehip and hawthorn, are suitable raw
materials for the production of functional foods with antioxidant
activity. These results are interesting because, with the exception
of blackcurrant and blueberry, all other fruits used are rarely consumed
in fresh form. The elaboration of antioxidant functional
foods from them will make them accessible for more people who
will benefit from their high antioxidant activity.
The effect of the tested samples on lipid peroxidation was
assayed using the autoxidation of linoleic acid in an ethanol-buffer
system and measuring the formation of linoleic acid hydroperoxides.
This is a simple and reliable method for the evaluation of
potential antioxidants (Kikuzaki et al., 2002). As shown in
Fig. 2A, the concentration of hydroperoxides rises gradually during
the six days of linoleic acid autoxidation at 40 °C. The formation of
40 P. Denev et al. / Food Chemistry 157 (2014) 37–44
hydroperoxides in the presence of the fruit extracts is shown in
Fig. 2B. It is obvious that the autoxidation of linoleic acid was very
effectively inhibited by extracts from blueberry, rowanberry and
blackcurrants at the end of the 1st day of linoleic acid autoxidation
(concentration of hydroperoxides 40%, 30%, and 27% of control,
resp.). The inhibition of hydroperoxide formation by these fruits
and, in addition by chokeberry, was observed also after the 3rd
day of linoleic acid autoxidation (less than 50% of control). And
finally, hydroperoxide formation was inhibited to more than 50%
of the control value by all extracts analysed after 6 days of linoleic
acid autoxidation. Extract from blueberry was the most effective
since it diminished the concentration of hydroperoxides to 8% of
the control value.
3.3. Effect on the production of ROS from phagocytes
The effects of studied extracts on the production of ROS in
human whole blood phagocytes was studied using luminolenhanced
CL. The results, depicted as kinetic curves and integral
values of the CL response, are shown on Fig. 3. It is obvious that
all studied extracts significantly inhibited ROS production in the
case of OZP-activated phagocytes. The most effective was the
extract of blackcurrants fruits and the effectiveness of all extracts
was in the following order: blackcurrants > blueberry > chokeberry
> rosehip > hawthorn > rowanberry. The weakest extract of
rowanberry still inhibited the OZP-activated CL response by more
than 50%. On the other hand, none of the studied extracts inhibited
the PMA-activated CL response of human whole blood phagocytes.
The inhibitory effect of flavonoids (kaempferol, morin, quercetin
and fisetin) on the respiratory burst of neutrophils was observed
by Pagonis, Tauber, Pavlotsky, and Simons (1986).
Similarly, Zielinska, Kostrzewa, and Ignatowicz (2000) approved
the inhibitory effects of natural flavonoids (quercetin, kaempferol
and isorhamnetin) against the respiratory burst of neutrophils
from healthy human donors in vitro, as measured by flow cytometry
using dichlorofluorescein diacetate and luminol-dependent
chemiluminescence. Wang, Chang, Hsu, Chen, and Kuo (2002)
investigated the cellular localisation of the inhibitory effect of a
natural flavonoid cirsimaritin against a formyl-methionyl-leucylphenylalanine
(fMLP)-induced respiratory burst in rat neutrophils.
Cirsimaritin inhibited the superoxide anion radical generation and
the oxygen consumption of neutrophils. On the other hand, cirsimaritin
did not reduce, but slightly enhanced, the superoxide anion
radical generation in PMA-activated NADPH oxidase. The results
from these authors indicate that it is likely that the inhibition of
the fMLP-induced respiratory burst by cirsimaritin in rat neutrophils
is mainly through the blockade of the phospholipase D
(PLD) signaling pathway.
Selloum, Reichl, Muller, Sebihi, and Arnhold (2001) compared
the effects of three aglycone flavonols (myricetin, quercetin and
kaempferol) and the natural glycoside rutin on superoxide anion
radical generation. All flavonols tested inhibited the pholasin
luminescence of fMLP-stimulated neutrophils. Lee et al. (2010)
observed that luteolin attenuated neutrophil respiratory burst
but had a negligible effect on superoxide anion generation during
PMA stimulation. Furthermore, luteolin effectively blocked
MAPK/ERK kinase 1/2 and Akt phosphorylation in fMLP-stimulated
neutrophils. Thus, an inhibition of enzymes involved in signaling,
rather than a scavenging of superoxide anion radicals dominates
in fMLP-stimulated neutrophils exposed to flavonoids in these particular
studies.
The effects of flavonoids from various plant extracts on the
respiratory burst of neutrophils were also studied. For example,
Kenny, Shu, Moritoki, Keen, and Gershwin (2009) demonstrated
that flavonoids isolated from cocoa could moderate a subset of signaling
pathways derived from the LPS stimulation of neutrophils,
Table 1
Major polyphenol and anthocyanin contents of fruit extracts.
Extract Gallic
acid
(mg/l)
Neochlorogenic
acid ( mg/l)
Chlorogenic
acid (mg/l)
p-Coumaric
acid (mg/l)
Ferulic
acid
(mg/l)
Ellagic
acid
(mg/l)
Catechin
(mg/l)
Epicatechin
(mg/l)
Rutin
(mg/l)
Quercetin
(mg/l)
Total
polyphenols
(mg/l)
Total
anthocyanins
(mg/l)
Rowanberry 427.4 705.7 – – – – – – – 2148.2 5.3
Hawthorn – 34.2 60.6 – 2.0 16.9 45.9 119.8 14.4 – 4025.8 13.0
Rosehip – – 44.4 – – – – – 107.3 14.7 5609.9 –
Chokeberry – 650.9 461.9 – – – 72.5 – – 14.8 6274.8 875.0
Blackcurrant 15.1 29.5 – 1.8 – – 144.5 – 25.8 6.9 4038.2 654.6
Blueberry – – 201.9 – 11.2 – – – 93.3 – 6012.5 1242.4
93,677.6
73,804.6
72,487.2
55,505.7
46,421.7
23,689.6
0 20000 40000 60000 80000 100000
Rosehip
Hawthorn
Blueberry
Chokeberry
Blackcurrants
Rowanberry
ORAC, μmol TE/l
(A) Oxygen Radical Absorbance Capacity
87,109.4
51,125.1
43,433.6
43,217.1
34,612.0
33,510.6
0 20000 40000 60000 80000 100000
Rosehip
Hawthorn
Blueberry
Chokeberry
Rowanberry
Blackcurrants
TRAP, μmol TE/l
(B) Total Peroxyl Radical Trapping Parameter
76,069.4
31,328.5
26,339.0
22,506.0
20,019.3
15,373.4
0 20000 40000 60000 80000 100000
Rosehip
Hawthorn
Blueberry
Chokeberry
Blackcurrants
Rowanberry
HORAC, μmol GAE/l
(C) Hydroxyl Radical AverƟng Capacity
Fig. 1. Antioxidant activity of fruit extracts measured by different assays: (A)
Oxygen Radical Absorbance Capacity (ORAC); (B) Total Peroxyl Radical Trapping
Parameter (TRAP); (C) Hydroxyl Radical Averting Capacity (HORAC).
P. Denev et al. / Food Chemistry 157 (2014) 37–44 41
mainly neutrophil oxidative bursts and activation markers. They
hypothesised that flavonoids could decrease the impact of LPS on
the fMLP-primed neutrophil ability to generate ROS by partially
interfering in the activation of the MAPK pathway. A Ginkgo biloba
extract containing flavonoids was tested by Pincemail et al. (1987)
for its effect on the release of ROS during the stimulation of human
neutrophils by a soluble agonist. The extract slowed down the oxygen
consumption (respiratory burst) of the stimulated cells. fMLP
used in the cited studies is a soluble activator of neutrophils which
binds to specific membrane receptors. Similarly, OZP binds to the
surface opsonin (either complement or Fc) receptors on phagocytes
and triggers the signaling cascade leading finally to the activation
of protein kinase C (PKC) with subsequent activation of NADPH
oxidase, the enzyme producing superoxide anion – the first of
ROS produced by phagocytes. On the contrary, PMA directly activates
the PKC, the step before NADPH oxidase activation. Our results
obtained are in accordance with the literature indicating
that all fruit extracts used do not inhibit either PKC or NADPH oxidase
itself, but interfere with the signaling cascade of phagocyte
activation upstream to the PKC activation.
3.4. Effects of fruit extracts on lymphocytes proliferation
Proliferation of hamsters’ lymphocytes, evaluated by MTT test,
was significantly stimulated by berry extracts in a dose-dependent
manner (p < 0.01) after a 48 h incubation period when compared to
the control group (Fig. 4). Mitogenic activity of fruit extracts on
lymphocyte cell proliferation was in the decreasing order: blackcurrants
> chokeberry > rowanberry > rosehip > hawthorn > blueberry,
and was several fold higher than the control, untreated cells.
The extracts of blackcurrants exhibited the highest proliferation
activity at a dose-dependent manner, followed by the extracts of
chokeberry and the higher used dose of rowanberry and rosehip
after 24 h incubation, but these extracts had a lower stimulatory
effect on lymphocytes than those observed after 48 h incubation.
In contrast, the extracts of blueberry, hawthorn and the lower used
dose of rowanberry and rosehip had no stimulatory effect on lymphocytes.
Many of the health benefits derived from fruit consumption
are due to their wide range of bioactive phytochemicals, the
most prominent of which are polyphenols, which are responsible
for many of their biological activities. The immunomodulatory role
of antioxidants has been shown in immune cell functions in ageing
and in an oxidative stress experimental model, namely endotoxic
shock, in which immune functions are altered in a way similar to
ageing (De la Fuente, 2002). Thus, since the immune system is a
health indicator and longevity predictor, the protection of this system
by an antioxidant diet supplementation may be useful for
health preservation.
3.5. Antimicrobial activity
The antimicrobial activity of the fruit extract was tested via two
assays and eleven foodborne pathogens were used as test microorganisms.
The initial screening of the antimicrobial activity was performed
using the agar diffusion method. The antimicrobial activity
was classified as weak (inhibition zone < 12 mm), medium (inhibition
zone between 12 and 20 mm) and strong (inhibition zone
above 20 mm) (Rota, Herrera, Martinez, Sotomayor, & Jordan,
Fig. 2. Inhibition of lipid peroxidation by fruit extracts. (A) The kinetics of
hydroperoxide concentration in the control samples without fruit extracts during
the 6 days of autoxidation of linoleic acid. (B) The effect of fruit extracts on
hydroperoxide accumulation at the end of 1, 3 and 6 days of autoxidation of linoleic
acid. The results are expressed as percentage of control samples without fruit
extracts. A – rowanberry; B – hawthorn; C – rosehip; D – chokeberry; E –
blackcurrants; F – blueberry.
Fig. 3. Effect of fruit extracts on the production of reactive oxygen species by
phagocytes expressed as the kinetic curves of the CL response after OZP activation
(upper panel) and PMA activation (lower panel). A – rowanberry; B – hawthorn; C –
rosehip; D – chokeberry; E – blackcurrants; F – blueberry. Integral values of the CL
response (RLU * min) are shown in the legend.
42 P. Denev et al. / Food Chemistry 157 (2014) 37–44
2008). The hawthorn and rosehip extracts displayed weak antimicrobial
activities, and the best results were obtained against
Staphylococcus aureus ATCC 6538P and Proteus vulgaris G (data
not shown). Only two Staphylococcus strains were sensitive against
the antimicrobial effect of the blueberry extract (data not shown).
Via this method, three extracts (rowanberry, chokeberry and blackcurrant)
showed the most significant antimicrobial activities
against broad spectrum of microorganisms. The rowanberry
extract exhibited highest activity against Salmonella enterica ssp.
enterica ATCC BAA-2162 and Pseudomonas aeruginosa ATCC 9027.
It also showed moderate activity against the two Listeria monocytogenes
strains and P. vulgaris G. The strongest antimicrobial activity
in the experiment was shown by the chokeberry extract against
Escherichia coli ATCC 25922, where an inhibition zone of 30 mm
was detected. The extract was moderately active against the Staphylococcus
strains. The blackcurrant extract exhibited medium antibacterial
activity against all test microorganisms, except Klebsiella
pneumonia. The highest activity was determined against S. enterica
ssp. enterica ATCC BAA-2162, where even a 10% concentration of
the extract caused inhibition of bacterial growth. Based upon the
obtained results rowanberry, chokeberry and blackcurrant extracts
were selected for further experiments and determination of
minimal inhibiting concentration (MIC) (Table 2).
The lowest MICs were determined for chokeberry extracts. It
presented antibacterial activity even at 0.02% concentration in
the medium. The most sensitive strains against its presence were
S. aureus ATCC 25923 and S. enterica ssp. enterica ATCC BAA-
2162. Two more strains (E. coli ATCC 25922 and Salmonella sp.)
were inhibited by the extract at concentrations below 2%. The rest
of the microorganisms were also inhibited but at concentration
higher than 3%. The strains E. coli ATCC 25922, Salmonella sp. and
S. aureus ATCC 25923 were the most susceptible to the antibacterial
action of chokeberry, rowanberry and blackcurrant. The latter
had very similar antimicrobial activity to chokeberry with the
same bacteria inhibited. Rowanberry, however, showed the lowest
activity compared to the other extracts. Only two pathogens from
the microbes tested were inhibited at concentrations <2% in the
medium, namely Salmonella sp. and S. aureus ATCC 25923. There
were strains (rowanberry against L. monocytogenes I; chokeberry
and blackcurrant against P. vulgaris G and K. pneumoniae) for which
MIC were not determined because reduction in bacterial growth
was less than 50%. In other cases, MIC were determined for extract
concentrations above 10%. These included: rowanberry against E.
coli ATCC 8739, L. monocytogenes, P. vulgaris G and K. pneumoniae;
chokeberry against L. monocytogenes and P. aeruginosa ATCC 9027;
blackcurrant against S. enterica ssp. enterica ATCC BAA-2162, L.
monocytogenes and P. aeruginosa ATCC 9027.
4. Conclusion
The current study assayed the potential of six edible fruits as
raw materials for the production of functional foods with antioxidant,
antimicrobial and mitogenic activities. The fruits investigated
Fig. 4. Effects of fruit extracts on the proliferation activity of lymphocytes from healthy hamsters.
Table 2
Antimicrobial activity of fruit extracts against human pathogens measured as zones of inhibition and minimal inhibiting concentrations.
Microorganism Rowanberry extract Chokeberry extract Blackcurrant extract
100% 10% MIC50 % 100% 10% MIC50 % 100% 10% MIC50 %
Zone of inhibition, mm Zone of inhibition, mm Zone of inhibition, mm
Escherichia coli ATCC 8739 12 – 12.5 11 – 3.125 11 – 3.125
Escherichia coli ATCC 25922 13 – – 30 – 1.56 13 – 1.56
Salmonella sp. 12 – 1.56 10 – 1.56 11 – 1.56
Salmonella enterica ssp. enterica ATCC BAA-2162 15 – 6.25 10 – 0.39 13 – 12.5
Staphylococcus aureus ATCC 6538P 9 – 6.25 13 – 6.25 15 9 3.125
Staphylococcus aureus ATCC 25923 11 – 0.1 13 – 0.02 12 – 0.02
Listeria monocytogenes 13 – 12.5 11 – 12.5 12 – 12.5
Listeria monocytogenes I 14 – – 11 – 6.25 13 – 3.125
Proteus vulgaris G 14 – 12.5 11 – – 14 – –
Pseudomonas aeruginosa ATCC 9027 15 – – 12 – 12.5 13 – 25
Klebsiella pneumoniae 12 – 25 10 – – – – –
P. Denev et al. / Food Chemistry 157 (2014) 37–44 43
are rich sources of several classes of polyphenol compounds and
differences in their polyphenol profile led to different biological
activities. The antioxidant activity data indicates that all the fruits
tested, and particularly rosehip and hawthorn, are very suitable
raw materials for the production of antioxidant foods. All extracts
studied and especially those from anthocyanin-rich fruits (blackcurrants,
chokeberry and blueberry) significantly inhibited the
ROS production of OZP-activated phagocytes, indicating that these
extracts interfere with the signaling cascade of phagocyte activation
upstream to the protein kinase C activation. Chokeberry,
blackcurrant and rowanberry extracts revealed strong antimicrobial
properties against a broad spectrum of microorganisms. These
extracts also had the highest mitogenic activity expressed as the
stimulating effect on hamster lymphocyte proliferation. These
results are interesting because with exception to blackcurrant
and blueberry, all the other fruits used are rarely consumed in
the fresh form. The elaboration of functional foods will make the
investigated fruits accessible for more people who will benefit
from their biological activities related to immunity and gastrointestinal
health.
Acknowledgements
The current study was supported by project DMU 03/74 – Bulgarian
Science Fund, Bulgarian Ministry of Education and Science,
and by the Joint Research Project between BAS and ASCR 2011–
2013, No. 11.
References
Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants and the
degenerative diseases of aging. Proceedings of the National Academy of Sciences of
the United States of America, 90, 7915–7922.
Anthon, G. E., & Barrett, D. M. (2001). Colorimetric method for the determination of
lipoxygenase activity. Journal of Agricultural and Food Chemistry, 49(1), 32–37.
Ciz, M., Cizova, H., Denev, P., Kratchanova, M., Slavov, A., & Lojek, A. (2010). Different
methods for control and comparison of the antioxidant properties of vegetables.
Food Control, 21, 518–523.
Ciz, M., Komrskova, D., Pracharova, L., Okenkova, K., Cizova, H., Moravcova, A., et al.
(2007). Serotonin modulates the oxidative burst of human phagocytes via
various mechanisms. Platelets, 18(8), 583–590.
Cizova, H., Lojek, A., Kubala, L., & Ciz, M. (2004). The effect of intestinal ischemia
duration on changes in plasma antioxidant defence status in rats. Physiological
Research, 53(5), 523–531.
De la Fuente, M. (2002). Effects of antioxidants on immune system ageing.
Europeran Journal of Clinical Nutrition, 56(Suppl. 3), S5–S8.
Denev, P., Ciz, M., Ambrozova, G., Lojek, A., Yanakieva, I., & Kratchanova, M. (2010).
Solid phase extraction of berries’ anthocyanins and evaluation of their
antioxidative properties. Food Chemistry, 123, 1055–1061.
Denev, P., Kratchanov, Ch., Ciz, M., Lojek, A., & Kratchanova, M. (2012).
Bioavailability and antioxidant activity of black chokeberry (Aronia
melanocarpa) polyphenols: In vitro and in vivo evidences and possible
mechanisms of action. A review. Comprehensive Reviews in Food Science and
Food Safety, 11(5), 471–489.
Denev, P., Lojek, A., Ciz, M., & Kratchanova, M. (2013). Antioxidant activity and
polyphenol content of Bulgarian fruits. Bulgarian Journal of Agricultural Science,
19(1), 22–27.
Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2008). The antimicrobial efficacy of plant
essential oil combinations and interactions with food ingredients. International
Journal of Food Microbiology, 124(1), 91–97.
Halliwell, B., Zhao, K., & Whiteman, M. (2000). The gastrointestinal tract: A major
site of antioxidant action? Free Radical Research, 33(6), 819–830.
Halliwell, B., Rafter, J., & Jenner, A. (2005). Health promotion by flavonoids,
tocopherols, tocotrienols, and other phenols: Direct or indirect effects?
Antioxidant or not? The American Journal of Clinical Nutrition, 81(Suppl. 1),
268S–276S.
Heber, D. (2004). Vegetables, fruits and phytoestrogens in the prevention of
diseases. Journal of Postgraduate Medicine, 50, 145–149.
Hertog, M. G. L., Bueno de Mesquita, H. B., Fehily, A. M., Sweetnam, P. M., Elwood, P.
C., Kromhout, D., et al. (1996). Fruit and vegetable consumption and cancer
mortality in the Caerphilly study. Cancer Epidemiology, Biomarkers and
Prevention, 5, 673–677.
Kenny, T. P., Shu, S. A., Moritoki, Y., Keen, C. L., & Gershwin, M. E. (2009). Cocoa
flavanols and procyanidins can modulate the lipopolysaccharide activation of
polymorphonuclear cells in vitro. Journal of Medicinal Food, 12(1), 1–7.
Kikuzaki, H., Hisamoto, M., Hirose, K., Akiyama, K., & Taniguchi, H. (2002).
Antioxidant properties of ferulic acid and its related compounds. Journal of
Agricultural and Food Chemistry, 50(7), 2161–2168.
Lee, J. (2005). Determination of total monomeric anthocyanin pigment content of
fruits juices, beverages, natural colorants and wines by the pH differential
method: Collaborative study. Journal of AOAC International, 88(5), 1269–1278.
Lee, J. P., Li, Y. C., Chen, H. Y., Lin, R. H., Huang, S. S., Chen, H. L., et al. (2010).
Protective effects of luteolin against lipopolysaccharide-induced acute lung
injury involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils.
Acta Pharmacologica Sinica, 31(7), 831–838.
Mossmann, T. (1983). Rapid colorimetric assay of cellular growth and survival:
Application to proliferation and cytotoxicity assays. Journal of Immunological
Methods, 65, 55–63.
Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescence probe. Journal of Agricultural and Food Chemistry, 49(10),
4619–4626.
Ou, B., Hampsch-Woodill, M., Flanagan, J., Deemer, E. K., Prior, R. L., & Huang, D. J.
(2002). Novel fluorometric assay for hydroxyl radical prevention capacity using
fluorescein as the probe. Journal of Agricultural and Food Chemistry, 50(10),
2772–2777.
Pagonis, C., Tauber, A. I., Pavlotsky, N., & Simons, E. R. (1986). Flavonoid impairment
of neutrophil response. Biochemical Pharmacology, 35(2), 237–245.
Pekarova, M., Kralova, J., Kubala, L., Ciz, M., Lojek, A., Gregor, C., et al. (2009).
Continuous electrochemical monitoring of nitric oxide production in murine
macrophage cell line RAW 264.7. Analytical and Bioanalytical Chemistry, 394(5),
1497–1504.
Pincemail, J., Thirion, A., Dupuis, M., Braquet, P., Drieu, K., & Deby, C. (1987). Ginkgo
biloba extracts inhibits oxygen species production generated by phorbol
myristate acetate stimulated human leukocytes. Experientia, 43(2), 181–184.
Rota, M. C., Herrera, A., Martinez, R. M., Sotomayor, J. A., & Jordan, M. J. (2008).
Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus
zygis and Thymus hyemalis essential oils. Food Control, 19, 681–687.
Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of
polyphenols. Journal of Nutrition, 130, 2073S–2085S.
Selloum, L., Reichl, S., Muller, M., Sebihi, L., & Arnhold, J. (2001). Effects of flavonols
on the generation of superoxide anion radicals by xanthine oxidase and
stimulated neutrophils. Archives of Biochemistry and Biophysics, 395(1), 49–56.
Singleton, V., & Rossi, J. (1965). Colorimetry of total phenolic with
phosphomolibdiphosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Wang, J. P., Chang, L. C., Hsu, M. F., Chen, S. C., & Kuo, S. C. (2002). Inhibition of
formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst by
cirsimaritin involves inhibition of phospholipase D signaling in rat
neutrophils. Naunyn-Schmiedeberg’s Archives of Pharmacology, 366(4), 307–314.
Zielinska, M., Kostrzewa, A., & Ignatowicz, E. (2000). Antioxidative activity of
flavonoids in stimulated human neutrophils. Folia Histochemica et Cytobiologica,
38(1), 25–30.
44 P. Denev et al. / Food Chemistry 157 (2014) 37–44
Research Article
On the Molecular Pharmacology of Resveratrol on Oxidative
Burst Inhibition in Professional Phagocytes
Radomír Nosá8,1
Katarína Drábiková,1
Viera JanIinová,1
Tomáš PereIko,1
Gabriela AmbroDová,2
Milan HíD,2
Antonín Lojek,2
Michaela Pekarová,2
Jan Šmidrkal,3
and Juraj Harmatha4
1
Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, D´ubravsk´a 9, 841 04 Bratislava, Slovakia
2
Institute of Biophysics AS CR v.v.i. Kr´alovopolsk´a 135, 612 65 Brno, Czech Republic
3
Institute of Chemical Technology, Faculty of Food and Biochemical Technology, Technick´a 5, 166 28 Praha 6, Czech Republic
4
Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Flemmingovo n´am. 2, 166 10 Praha 6, Czech Republic
Correspondence should be addressed to Radom´ır Nos´al’; radomir.nosal@savba.sk
Received 30 July 2013; Revised 12 December 2013; Accepted 17 December 2013; Published 28 January 2014
Academic Editor: Cristina Angeloni
Copyright © 2014 Radom´ır Nos´al’ et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Resveratrol—3,5,4󸀠
-trihydroxystilbene—possesses antioxidant activities in vitro. It dose-dependently inhibited the generation of
peroxyl, hydroxyl, peroxides, and lipid peroxidation products in cell free systems. Oxidative burst of whole human blood stimulated
with PMA, fMLP, OpZ, and A23187 was inhibited in a concentration-dependent way, indicating suppression of both receptor
and nonreceptor activated chemiluminescence by resveratrol. Results from isolated human neutrophils revealed that resveratrol
was active extracellularly as well as intracellularly in inhibiting the generation of reactive oxygen species. Liberation of ATP and
analysis of apoptosis showed that in the concentration of 100 𝜇M, resveratrol did not change the viability and integrity of isolated
neutrophils. Western blot analysis documented that resveratrol in concentrations of 10 and 100 𝜇M significantly decreased PMAinduced
phosphorylation of PKC 𝛼/𝛽II. Dose-dependent inhibition of nitrite production and iNOS protein expression in RAW
264.7 cells indicated possible interference of resveratrol with reactive nitrogen radical generation in professional phagocytes. The
results suggest that resveratrol represents an effective naturally occurring substance with potent pharmacological effect on oxidative
burst of human neutrophils and nitric oxide production by macrophages. It should be further investigated for its pharmacological
activity against oxidative stress in ischaemia reperfusion, inflammation, and other pathological conditions, particularly neoplasia.
1. Introduction
Neutrophils are present in high numbers in areas of inflammation,
where they constitute an important source of reactive
oxygen species (ROS). The massive production of antimicrobial
and tumoricidal ROS in an inflammatory environment
is called “oxidative burst” and plays an important role as
the first line of defense against environmental pathogens.
Paradoxically, however, neutrophils are also implicated in
tissue-damaging inflammatory reactions that underlie the
pathogenesis and exacerbation of many inflammatory diseases
[1, 2]. There are at least two signalling pathways
responsible for induction of neutrophil activation: one is the
protein kinase C (PKC) mediated pathway, which can be
driven by stimulation with phorbol-4𝛽-12𝛽-myristate-13𝛼acetate
(PMA), and the other is the Src family protein tyrosine
kinase mediated pathway [3].
Apoptosis is critical for the regulation of life span of
circulating as well as emigrated neutrophils. Accumulating
evidence indicates that neutrophil apoptosis is one of the
critical determinants of the outcome of the inflammatory
response and is a potential target for therapeutic interventions.
A delay of neutrophil apoptosis exacerbates and prolongs
inflammation or even prevents spontaneous resolution
of inflammation [4]. The apoptotic neutrophil and the process
of cell death exert anti-inflammatory effects that have been
shown to be of therapeutic value in inflammatory diseases
[5, 6].
Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2014,Article ID 706269, 9 pages
http://dx.doi.org/10.1155/2014/706269
2 Oxidative Medicine and Cellular Longevity
A wide array of phenolic substances, particularly those
present in edible and medicinal plants, have been reported
to possess substantial antioxidative, anticarcinogenic, and
antimutagenic activities by modulating important cellular
signalling processes [7–10]. Natural polyphenols suppressed
oxidative burst of stimulated human neutrophils by enhancing
their apoptosis and decreasing protein kinase C activation
[11–17].
Resveratrol (RES), a polyphenolic phytoalexin, is one of
the most extensively studied natural products, with wideranging
biological activities and tremendous clinical potential
[18]. RES has been shown to have antioxidant, antiinflammatory,
antiproliferative, and anti-angiogenic effects, while
those on oxidative stress are presumably the most important
[19].
In spite of the fact that almost 5000 papers are evidenced
in PubMed database, there is a lack of evidence about the
mechanism of the effect of resveratrol on oxidative burst
in human professional phagocytes at molecular level. In
this study, we investigated the effect of RES on the mechanism
of oxidative burst in human whole blood, isolated
neutrophils at extra- and intracellular level, activation of
protein kinase C, caspase-3 activity and cellular viability and
on free radical scavenging activity in cell free systems (oxygen
radical absorption capacity—ORAC, hydroxyl radical
averting capacity—HORAC, scavenging of ROS generation,
nitric oxide production, and inhibition of lipid peroxidation).
Moreover, we studied the effect of resveratrol on nitrite
production and iNOS protein expression in murine RAW
264.7 macrophage cell line.
2. Methods and Materials
2.1. Chemicals. Luminol, isoluminol, PMA (phorbol-4𝛽-12𝛽myristate-13𝛼-acetate),
Ca2+
-ionophore A23187, superoxide
dismutase, dextran (average MW 464,000), zymosan A
(from Saccharomyces cerevisiae), luciferase (from firefly
Photinus pyralis), and D-luciferin sodium salt from SigmaAldrich
Chemie (Deisenhofen, Germany). HRP (horseradish
peroxidase), catalase, and Folin-Ciocalteu’s phenol reagent
were purchased from Merck (Darmstadt, Germany)
and Lymphoprep (density 1.077 g/mL) from Nycomed
Pharma AS (Oslo Norway). 2,2󸀠
-Azobis(2-methylpropionamidine)
dihydrochloride (AAPH), 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic
acid (Trolox), fluorescein
disodium salt, cobalt(II) fluoride tetrahydrate, and gallic acid
were obtained from Sigma-Aldrich (Steinheim, Germany).
Picolinic acid was purchased from Fluka (Deisenhofen,
Germany), and human purified caspase-3 was from Enzo
Life Sciences, Lausen, Switzerland. All other chemicals used
were of analytical grade and obtained from commercial
sources.
ORAC and HORAC analyses were carried out on a FLUOstar
Galaxy plate reader (BMG Labtechnology, Offenburg,
Germany).
The phosphate buffered saline solution (PBS) used in
this study contained 136.9 mM NaCl, 2.7 mM KCl, 8.1 mM
Na2HPO4, 1.5 mM KH2PO4, 1.8 mM CaCl2, and 0.5 mM
MgCl2 × 6H2O and had a pH of 7.4. Tyrode’s solution
used in this study consisted of 136.9 mM NaCl, 2.7 mM
KCl, 11.9 mM NaH2CO3, 0.4 mM NaH2PO4 × 2H2O, 1 mM
MgCl2 × 6H2O, and 5.6 mM glucose, pH of 7.4.
Resveratrol (RES) was prepared by targeted regioselective
synthesis purely as transisomer [20] and was diluted in 1 : 50
(v/v) of 1 M NaOH in water.
2.2. Blood Collection and Neutrophil Separation. Fresh
human blood was obtained at the blood bank by venipuncture
from healthy male volunteers (20–50 years) who had
not received any medication for at least 7 days. It was
anticoagulated with 3.8% trisodium citrate (blood : citrate
ratio = 9 : 1). The Ethical Committee license for blood
sampling at the National Transfusion Service NTSKRA/2012/SVI
was registered. Human neutrophils were
isolated from whole blood, as described previously [21, 22].
The final suspension of neutrophils contained more than 96%
of viable cells, as evaluated by trypan blue exclusion and was
used within 2 h, as long as the control chemiluminescence
remained constant.
2.3. Chemiluminescence (CL) Assay of Whole Blood and
Isolated Neutrophils. The oxidative burst in whole blood was
stimulated with phorbol myristate acetate (PMA 0.05 𝜇M),
opsonized zymosan (OpZ; 0.5 mg/mL), fMLP (1 𝜇M), or
Ca ionophore A23187 (1 𝜇M). CL was measured in 250 𝜇L
samples consisting of 50 𝜇L aliquots that contained blood
(50× diluted), luminol (200 𝜇M), RES (0.01–100 𝜇M), and
phosphate buffer [11]. The effect of RES on extra- and
intracellular ROS production was measured in unstimulated
and PMA (0.05 𝜇M) stimulated neutrophils (5 × 105
per
sample) by isoluminol/luminol-enhanced CL. The CL of both
whole blood and isolated neutrophils was evaluated and
measured in a microplate luminometer Immunotech LM-01T
(Czech Republic) at 37∘
C [23].
2.4. Analysis of Apoptosis. Citrated whole human blood
was collected as described above. Dextran (3%) was added
(blood : dextran = 2 : 1) and centrifuged at 10 ×g at room
temperature [16]. Before use, 1 mL of buffy coat that contained
leukocytes was collected and stored on ice. The cells were
counted on the hemocytometer (Coulter Counter), which
focused on granulocytes. The cell suspension was adjusted
to get 2 × 105
neutrophils per sample. Three different
concentrations of RES (1, 10, and 100 𝜇M) were applied and
incubated with a control sample at 37∘
C for 10 min. The
cells were stained with Annexin V, conjugated with FITC
(BenderMedSystems) in the dark at 4∘
C for 10 min, followed
by staining with propidium iodide (1 𝜇g/mL), and then
analysed immediately by the Beckman Coulter Cytomics
FC500 cytometer (for details, see Pereˇcko et al. [16]).
2.5. Neutrophil Integrity. The cytotoxic effect of RES was
evaluated by means of ATP liberation by luciferin-luciferase
chemiluminescence [11]. The neutrophil suspension (30 𝜇L;
30 000 cells/sample) and 20 𝜇L of Tyrode’s solution were
incubated with 50 𝜇L of RES (1 to 100 𝜇M) for 15 min at
Oxidative Medicine and Cellular Longevity 3
37∘
C. The total ATP content was assessed immediately after
sonication of neutrophils for 10 s.
2.6. Recombinant Caspase-3 Activity. To determine the
caspase-3 activity, a modified method was applied [16]. The
final reaction with luciferase was detected by CL. The light
production was measured in the Luminometer Immunotech
LM-01T. The reagent was added and the mixture was measured
for 60 min to determine caspase-3 activity. The solvent
for RES, containing NaOH, was also evaluated.
2.7. Protein Kinase C Activation. Phosphorylation of protein
kinase (PKC) isoenzymes 𝛼 and 𝛽II was detected [11]. Isolated
human neutrophils (5 × 106
) were incubated at 37∘
C with
RES for 1 min, stimulated with PMA (0.15 𝜇M, 1 min), and
lysed by the addition of solubilisation buffer. After sonication
on ice, samples were centrifuged to remove unbroken cells,
the supernatant was boiled for 5 min with sample buffer,
and samples were loaded on 9.8% SDS polyacrylamide gels.
Membrane strips were blocked for 60 min with 1% bovine
serum albumin in Tris buffered saline. This was followed by
60 min incubation in the presence of the phospho-PKC 𝛼
and 𝛽II (Thr638/641) antibody (rabbit anti-human, 1 : 8000,
Cell Signaling Technology) or 𝛽-actin antibody (rabbit antihuman,
1 : 4000, Cell Signaling Technology, Danvers, MA,
USA). The membranes were subsequently washed six times
with TBS and incubated 60 min with the secondary antibody
conjugated to horseradish peroxidase (anti-rabbit from
donkey, 1 : 10,000, Amersham, UK). The optical density of
each PKC band was corrected by the optical density of the
corresponding 𝛽-actin band.
2.8. Cell Culture. Murine peritoneal macrophage cell line
RAW 264.7 (American Type Culture Collection, USA) was
cultivated in Dulbecco’s Modified Eagle Medium (PAN, Germany)
supplemented with 10% foetal bovine serum (PAN,
Germany) and 1% gentamycin (Sigma, USA). Cells were
maintained at 37∘
C and 5% CO2. After reaching confluence,
cells were harvested and washed. Cell numbers and viability
were determined by ATP test [24].
2.9. Formation of Nitric Oxide. Generation of reactive nitrogen
species was determined indirectly as the accumulation
of nitrites in the supernatant of murine macrophages RAW
264.7 [12]. Control cells were incubated with LPS without RES
treatment. At the end of the incubation period, culture media
were collected from wells and centrifuged at 5000 ×g and 4∘
C
for 5 min. Then 150 𝜇L of supernatant was mixed with equal
volume of Griess reagent (Sigma, USA) in a 96-well plate,
and the mixture was incubated at room temperature and in
the dark for 30 min. The cell fractions of these samples were
used for the detection of inducible NO synthase expression
by Western blot.
2.10. Western Blot Analysis of iNOS Expression. After removing
the supernatant for nitrite measurement, the remaining
cells were washed with cold phosphate buffered saline (PBS)
and lysed in the lysis buffer (1% sodium dodecyl sulphate—
SDS, 10−1
M Tris pH 7.4, 10% glycerol, 10−3
M sodium orthovanadate,
10−3
M phenylmethanesulfonyl fluoride). Protein
concentrations were determined by using BCATM protein
assay (Pierce, USA), with bovine serum albumin as standard.
Equal amounts of protein were then subjected to SDSpolyacrylamide
gel electrophoresis using 7.5% running gel.
The expression of iNOS protein was quantified by Western
blot analysis [25]. Relative protein levels were quantified by
scanning densitometry using the Image JTM programme,
and the individual band density value was expressed in
arbitrary units.
2.11. Antioxidant Activity Assays (Free Radical Scavenging
Activity in Cell Free Systems)
2.11.1. ORAC Assay. The ORAC method measures the antioxidant
scavenging activity against peroxyl radical induced
by 2,2󸀠
-Azobis(2-methylpropionamidine) dihydrochloride
(AAPH) at 37∘
C [26, 27]. Fluorescein (FL) was used as the
fluorescent probe. The protective effect of an antioxidant was
measured by assessing the area under the fluorescence decay
curve (AUC). The final ORAC values were calculated using
a regression equation between the Trolox concentration and
the net area under the curve.
2.11.2. HORAC Assay. HORAC measures the metal-chelating
activity of antioxidants under the conditions of Fenton-like
reactions employing a Co(II) complex and hence the protecting
ability against formation of hydroxyl radical [26, 27]. The
initial fluorescence was measured, after which the readings
were taken every minute after shaking in the presence of
100, 200, 400, 500, and 600 𝜇M gallic acid solutions (in
phosphate buffer 75 mM, pH 7.4). The final HORAC values
were calculated using a regression equation between gallic
acid concentration and the net area under the curve.
2.12. ROS Scavenging in Luminol-Horseradish Peroxidase
(HRP)-H2O2 Cell Free System. Aliquots of 50 𝜇L of RES
solutions, HRP (2 U/mL), and luminol (10 𝜇M) were mixed
in a 96-well luminescence plate to yield final concentrations
of RES 1, 10, and 100 𝜇M. The reaction was started by adding
hydrogen peroxide at the final concentration of 100 𝜇M
(final volume of the sample was 200 𝜇L). Chemiluminescence
was measured for 10 minutes at 37∘
C with Luminometer
Immunotech LM-01T (Beckman Coulter).
2.13. NO Scavenging Activity. The potential ability of extracts
to scavenge NO in chemical systems was tested by electrochemical
measurement of NO [28]. NO was measured using
three electrode systems: a porphyrinic microsensor working
electrode, counter electrode, and a reference electrode were
connected to the ISO-NO MARK II potentiostat (WPI, USA)
[28]. The injection of the 2 𝜇L NO-saturated water into
the measurement glass vial (final concentration of NO =
2.38 𝜇M) caused a rapid increase with subsequent gradual
decrease of the NO induced signal until it reached the
background current [29]. The scavenging properties of the
4 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
0 20 40 60
Time (min)
Control
Chemiluminescence(RLU)
100 𝜇mol/L0.1 𝜇mol/L
1 𝜇mol/L
10 𝜇mol/L
(a)
0
20
40
60
80
100
120
PMA OpZ A23187 fMLP
ROSproduction(%ofcontrol)
Stimuli
1 𝜇M
10 𝜇M
∗
∗∗
∗∗
∗∗ ∗∗
∗∗
(b)
Figure 1: (a) Resveratrol dose-dependently decreased luminol-enhanced representative chemiluminescence curves of human whole blood
stimulated with phorbol myristate acetate (PMA = 0.05 𝜇M) at 37∘
C. (b) Effect of resveratrol in 1 and 10 𝜇M concentration on PMA (0.05 𝜇M),
OpZ (0.5 mg/mL), fMLP (1 𝜇M), and A23187 (1 𝜇M) stimulated chemiluminescence. 𝑛 = 6–8, Mean ± SEM, ∗
𝑃 ≤ 0.05; ∗∗
𝑃 ≤ 0.01.
extracts tested were evaluated as the time needed for reaching
again the background current.
2.14. Lipid Peroxidation. The amount of 0.9 mL of 0.5 mM
𝛼-linolenic acid (Sigma-Aldrich, Steinheim, Germany) was
mixed with 0.1 mL sample. Then, a system generating
hydroxyl radical (0.1 mL Co(II) and 0.1 mL hydrogen peroxide
for details, see section HORAC) was added for the
induction of lipid peroxidation and the mixture was incubated
for 2 h in 37∘
C. The concentration of thiobarbituric
acid-reactive substances (TBARS) was measured as the index
of lipid peroxidation [30]. The absorbance of the upper layer
was measured at 532 nm. 1,1,3,3-Tetraethoxypropane (SigmaAldrich,
Steinheim, Germany) in the final concentration
of 0.1 𝜇M was used as standard. Lipid peroxidation was
expressed in nM of TBARS per 1 mL of the mixture 𝛼linolenic
acid/analysed sample.
2.15. Statistical Analysis. Data represent the mean ± SEM,
unless stated otherwise. Statistical analysis was performed
using the ANOVA paired test to examine differences between
the treatments and control. Differences were considered to be
statistically significant when 𝑃 < 0.05 (∗
) or 𝑃 < 0.01 (∗∗
).
3. Results
Figure 1(a) demonstrates representative dose-dependent CL
curves of whole blood treated with RES and stimulated with
PMA (0.05 𝜇M). Figure 1(b) shows the effect of RES in 1 and
10 𝜇M concentration on whole human blood CL stimulated
with PMA (0.05 𝜇M), OpZ (0.5 mg/mL), A23187 (1 𝜇M),
and fMLP (1 𝜇M). In 1 𝜇M concentration, RES significantly
decreased CL for PMA and fMLP stimuli to 19 and 30 per
cent of control value (=100%), respectively. RES in 10 𝜇M
concentration significantly inhibited CL with all stimuli
applied in the rank order of potency PMA = fMLP > OPZ
> A23187 demonstrating an evident difference in resveratrol
Table 1: Effect of resveratrol in concentrations of 1 to 100 𝜇M
on viability of isolated neutrophils. Cells were incubated with
resveratrol at 37∘
C for 10 min, stained with Annexin V, subsequently
conjugated with FITC in the dark at 4∘
C for 10 min, followed
by staining with propidium iodide (1 𝜇g/mL), and then analysed
immediately by the Beckman Coulter Cy. 𝑛 = 4–6, mean ± SEM.
Resveratrol (𝜇M) Live cells Apoptotic cells Dead cells
0 91.90 ± 1.02 7.90 ± 1.00 0.20 ± 0.04
1 92.20 ± 1.31 7.60 ± 1.30 0.20 ± 0.05
10 92.10 ± 1.16 7.80 ± 1.60 0.10 ± 0.03
100 81.20 ± 2.73∗∗
18.60 ± 2.70∗∗
0.20 ± 0.03
∗∗
𝑃 ≤ 0.01.
activity to decrease stimulated chemiluminescence of whole
blood.
The effect of RES on extra- and intracellular CL of
isolated neutrophils stimulated with PMA is demonstrated in
Figure 2. It is evident that RES dose-dependently decreased
extracellular CL, significantly starting at 0.1 𝜇M concentration.
At 100 𝜇M concentration (Figure 2(a)), there was complete
inhibition of stimulated CL due to PMA. At intracellular
level (Figure 2(b)), RES significantly decreased CL at 10 and
100 𝜇M concentrations to 26 and 0.33 percent of the control
value, respectively (Figure 2(c)).
Isolated intact neutrophils liberate 18 ± 3.8 nM ATP,
which represents 3.2% from the total ATP amount (548 ±
112 nM/3 × 104
cells). RES in any concentration used did not
liberate ATP from isolated neutrophils (results not shown),
indicating that RES did not disintegrate isolated neutrophils
in any concentration used.
Table 1 demonstrates the effect of RES in concentrations
of 1 to 100 𝜇M on viability of isolated neutrophils. In concentrations
of 1 and 10 𝜇M, RES did not change significantly
the amount of dead cells as compared with control cells.
In 100 𝜇M, concentration RES increased the number of
apoptotic cells from 7.9% (controls) to 18.6% of the total viable
Oxidative Medicine and Cellular Longevity 5
0
4
8
12
16
20
0 5 10 15 20 25 30
Time (min)
Chemiluminescence(RLU)
×103
Control
100 𝜇mol/L0.1 𝜇mol/L
1 𝜇mol/L
10 𝜇mol/L
(a)
0
200
400
600
800
0 10 20 30
Time (min)
Chemiluminescence(RLU)
Control
100 𝜇mol/L0.1 𝜇mol/L
1 𝜇mol/L
10 𝜇mol/L
(b)
0
20
40
60
80
100
120
0.1 1 10 100
ROSproduction(%ofcontrol)
Extracellular CL
Intracellular CL
Resveratrol (𝜇M)
∗∗
∗∗
∗∗
∗∗ ∗∗
(c)
Figure 2: Chemiluminescence of isolated neutrophils. (a) Dose-dependent extracellular representative chemiluminescence (luminol +
peroxidase) curves of human isolated neutrophils pretreated with resveratrol and stimulated with PMA (0.05 𝜇M). (b) Intracellular dosedependent
representative chemiluminescence (isoluminol + catalase + superoxide dismutase) curves of isolated human neutrophils pretreated
with resveratrol and stimulated with PMA (0.05 𝜇M). (c) Dose-dependent effect of resveratrol on PMA (0.05 𝜇M) stimulated extracellular
and intracellular chemiluminescence. 𝑛 = 6–8; mean ± SEM, ∗∗
𝑃 ≤ 0.01.
cells. The number of dead cells did not increase. These results
show that RES in the optimal concentrations used (1 and 10
𝜇M) did not change apoptosis of isolated human neutrophils.
Figure 3 shows the result of RES on radical scavenging
activity in cell free system. RES in concentrations 10 and
100 𝜇M increased hydroxyl scavenging activity (HORAC) to
88 and 114 𝜇M of gallic acid equivalents, respectively, and
peroxyl scavenging activity (ORAC) to 34 and 29 of Trolox
equivalents, respectively. This effect demonstrates effective
scavenging activity of RES on hydroxyl and peroxyl radicals
in vitro.
Figure 4 shows the inhibitory effects of different concentrations
(1, 10, and 100 𝜇M) of RES on production of
ROS in cell free system generated by means of luminol +
hydrogen peroxide + HRP. RES in 1 𝜇mol/L concentration
significantly (𝑃 < 0.01) inhibited and in 10 𝜇M concentration
totally blocked the chemiluminescence of 100 𝜇M hydrogen
peroxide in the samples.
Since lipids are very susceptible to lipid peroxidation, we
tested also the ability of RES to prevent the peroxidation of
polyunsaturated fatty acids induced by hydroxyl radical. It is
evident from Figure 5 that RES in all tested concentrations
significantly and dose-dependently inhibited lipid peroxida-
tion.
The activation of protein kinase C in isolated neutrophils
stimulated with PMA (0.15 𝜇M) in the presence of 10 and
100 𝜇M RES is demonstrated in Figure 6. In both concentrations
applied, RES reversed PMA stimulated PKC activation
6 Oxidative Medicine and Cellular Longevity
0
10
20
30
40
50
1 10 100
0
30
60
90
120
150
ORAC
HORAC
Resveratrol (𝜇M)
ORAC(𝜇MTroloxequivalents)
HORAC(𝜇Mgallicacidequivalents)
∗∗
∗∗
Figure 3: Effect of resveratrol on hydroxyl (HORAC) and peroxyl
(ORAC) radical scavenging activity in cell free system expressed as
gallic acid and Trolox equivalents, 𝑛 = 4, mean ± SEM, ∗∗
𝑃 ≥ 0.01.
0
20
40
60
80
100
120
1 10 100
ROSproduction(%ofcontrol)
Resveratrol (𝜇M)
∗∗
∗∗
∗∗
Figure 4: Dose-dependent effect of resveratrol on reactive oxygen
species generated in cell free system by means of luminol + hydrogen
peroxide + horseradish peroxidase. 𝑛 = 3, mean ± SEM, ∗∗
𝑃 ≥ 0.01.
to spontaneous (control) values indicating a suppressive
effect of RES on the activity of protein kinase C, one of the
essential regulatory enzymes in reactive oxygen generation.
The effect of RES on nitrite production and iNOS
expression in RAW 264.7 cell culture after LPS activation
is demonstrated in Figure 7. Figure 7(a) demonstrates that
RES in concentrations of 1, 10, and 100 M decreased nitrite
concentration in cell supernatants to 82, 65, and 6 percent of
the control value, respectively.
Figure 7(b) shows the effect of RES on iNOS expression
determined by Western blot analysis. In comparison with
the iNOS protein level in the control sample, iNOS protein
expression was significantly inhibited only by the highest
concentration (100 𝜇M) of RES to 60% of the control value.
0
3
6
9
12
15
0 1 10 100
TBARS(nM/mL)
∗∗
∗∗
∗∗
Resveratrol (𝜇M)
Figure 5: Effect of resveratrol on lipid peroxidation of 𝛼-linolenic
acid expressed as thiobarbituric acid-reactive substances (TBARS),
induced by hydroxyl radicals. 𝑛 = 6, mean ± SEM, ∗∗
𝑃 ≥ 0.01.
0
40
80
120
160
200
Spont. PMA 10.0 100.0
PKCactivation(%)
∗∗∗
Resveratrol (𝜇M)
𝛽-ActinPKC𝛼,𝛽II
Figure 6: Western blotting analysis of protein kinase C activation
in isolated human neutrophils pretreated with resveratrol (10 and
100 𝜇M) and stimulated with PMA (0.15 𝜇M). 𝑛 = 3-4, mean ± SEM;
∗
𝑃 ≤ 0.05.
4. Discussion
Resveratrol dose-dependently inhibited oxidative burst
in human whole blood stimulated with two membranebypassing
(PMA, A23187) and two membrane-operating
stimuli (OpZ, fMLP). There was no significant difference
between the stimuli applied and chemiluminescence decrease
of whole blood indicating that RES may not act only as an
extracellular scavenger but suppresses oxidative burst also
intracellularly. This suggestion was confirmed on isolated
neutrophils (Figure 2) stimulated with PMA demonstrating
that RES in a concentration dependent way inhibited not
only extracellularly determined chemiluminescence but
effectively suppressed formation of intracellularly generated
Oxidative Medicine and Cellular Longevity 7
0
20
40
60
80
100
120
1 10 100
Nitritesproduction(%ofcontrol)
Resveratrol (𝜇M)
∗∗
∗∗
∗∗
(a)
0
20
40
60
80
100
120
0 1 10 100
iNOSproteinexpression
(%ofcontrol=100)
Resveratrol (𝜇M)
iNOS
𝛽-Actin
∗∗
(b)
Figure 7: Nitrite production and iNOS expression in RAW 264.7 culture cells. Nitrite production (a) and densitometric analysis together
with representative Western blot of iNOS protein expression (b) in LPS stimulated RAW 264.7 cells treated with resveratrol. 𝑛 = 3, mean ±
SEM, ∗∗
𝑃 < 0.01.
ROS (Figure 2(b)). The difference was evident; RES started
to inhibit extracellular chemiluminescence at 0.1 𝜇M,
intracellularly at 10 𝜇M concentration (Figure 2(c)).
The stimulated generation of ROS in whole blood and
isolated neutrophils was decreased by many polyphenolic
compounds like curcumin, pterostilbene, pinosylvin, and Nferuloyl
serotonin [11, 14, 31]. Inhibition of fMLP-activated
human neutrophil chemiluminescence was accompanied by
inhibition of elastase and 𝛽-glucuronidase secretion and
production of 5-lipoxygenase metabolites leukotriene B4,
6-trans-LTB4, and 12-trans-epi-LTB4 after stimulation with
calcium ionophore, indicating that transresveratrol interferes
with the release of inflammatory mediators in activated
polymorphonuclear leukocytes [32].
Since RES did not liberate ATP from isolated neutrophils,
it is evident that even in higher concentrations used (up to
100 𝜇M) there was no disintegration of cells. Moreover, RES
dose-dependently decreased spontaneous ATP liberation.
RES did not change significantly the number of dead
cells even in the highest concentration used, decreasing the
number of live cells by 10.7% (Table 1). In contrast, RES
decreased the activity of recombinant caspase-3 activity in
cell free system, significantly at 10 𝜇M concentration. This
result has to be verified in the cellular model since apoptosis,
a programmed cell death, appears to be the most frequent
fate of cells treated with RES [33]. Moreover, RES induced
autophagy in human U251 glioma cells [34], decreased the
intracellular reactive oxygen species level, which correlated
with the induction of caspase-8 and caspase-3 cleavage in
human colon cancer cells [35] and induced apoptosis in
patients with chronic myeloid leukemia cells [36].
The antioxidant properties of RES were analysed via
five different methods: ORAC(peroxyl), HORAC(hydroxyl),
hydrogen peroxide-peroxidase dependent chemiluminescence,
NO scavenging, and lipid peroxidation inhibition. The
chosen methods embrace different aspects of the antioxidant
action and give a comprehensive view on the antioxidant
potential of the sample investigated.
In the following experiments, we tested also the scavenging
properties of RES against NO, using electrochemical
analysis which is considered to be a reliable method for
verifying NO scavenging. However, no scavenging properties
of resveratrol against NO were found in any concentration
used.
These observations confirmed previous findings that RES
is both a free radical scavenger and a potent antioxidant
because of its ability to promote the activities of a variety
of antioxidant enzymes [37]. The result of decreasing dosedependently
LDL oxidation in cell free system is supportive
of its effect on lipid peroxidation and atherosclerotic lesion
formation in animal hypercholesterolemic models [38].
RES decreased protein kinase C activation in PMAstimulated
neutrophils, indicating its interference with oxidative
burst in neutrophils. Similar results were demonstrated
in human gastric adenocarcinoma and CaSki cells [39, 40]
and for the polyphenolic compound N-feruloyl serotonin
in human neutrophils [14]. By inhibiting the activation of
PKC [41], RES may interfere with modulation of intracellular
signalling pathways involved in downregulation of COX-2
and iNOS expression and NF-𝜅B activation [10, 42]. Nitric
oxide, a member of reactive nitrogen species, is an important
molecule involved in the regulation of many physiological
and microbicidal processes. RES markedly inhibited NO
production by LPS stimulated macrophages. This finding
corresponds with the latest results of other authors [43,
44] who also reported suppression of inducible nitric oxide
synthase expression and NO production in macrophages
after RES administration. Our results showed that resveratrol
reduced nitrite accumulation more effectively than it reduced
iNOS protein expression in stimulated macrophages through
a mechanism which is at least partially independent of
the regulation of iNOS protein expression. Nevertheless, in
8 Oxidative Medicine and Cellular Longevity
electrochemical measurements we showed that RES was not
able to scavenge NO, suggesting that the direct scavenging
activity against NO resulting from the inhibitory action of
RES can be excluded.
In conclusion, RES possesses antioxidant activities in
vitro inhibiting generation of ROS in cell free systems,
oxidative burst of stimulated blood and isolated neutrophils
both at extracellular and intracellular level, as measured by
chemiluminescence.
Oxidative burst inhibition of human blood and isolated
neutrophils, suppression of free radical generation and NO
formation in cell free system confirmed the antioxidative
properties and supported the effort to enlarge clinical studies
with RES.
Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of the paper.
Acknowledgments
This work was supported in part by the Project APVV-0052-
10 of the Slovak Research and Development Agency and
by the Grant AV0Z50040702 of the Ministry of Education,
Youth and Sports of the Czech Republic. The authors wish to
thank Professor Magda Kourilova-Urbanczik for correcting
the English of the paper.
References
[1] W. Dr¨oge, “Free radicals in the physiological control of cell
function,” Physiological Reviews, vol. 82, no. 1, pp. 47–95, 2002.
[2] R. Casc˜ao, H. S. Ros´ario, and J. E. Fonseca, “Neutrophils:
warriors and commanders in immune mediated iflammatory
diseases,” Acta Reumatol´ogica Portuguesa, vol. 34, pp. 313–326,
2009.
[3] K. Suzuki, S. Kori, M. Morikawa, A. Takagi, and H. Namiki,
“Oxidative stress-mediated bimodal regulation of polymorphonuclear
leukocyte spreading by polyphenolic compounds,”
International Immunopharmacology, vol. 10, no. 11, pp. 1448–
1455, 2010.
[4] J. G. Filep and D. El Kebir, “Neutrophil apoptosis: a target for
enhancing the resolution of inflammation,” Journal of Cellular
Biochemistry, vol. 108, no. 5, pp. 1039–1046, 2009.
[5] D. El Kebir and J. G. Filep, “Role of neutrophil apoptosis in the
resolution of inflammation,” TheScientificWorldJOURNAL, vol.
10, pp. 1731–1748, 2010.
[6] S. K. Natarajan and D. F. Becker, “Role of apoptosis-inducing
factor, proline dehydrogenase, and NADPH oxidase in apoptosis
and oxidative stress,” Cell Health and Cytoskeleton, vol. 4, pp.
11–27, 2012.
[7] S. Kori, H. Namiki, and K. Suzuki, “Biphasic regulation of
polymorphonuclear leukocyte spreading by polyphenolic compounds
with pyrogallol moieties,” International Immunopharmacology,
vol. 9, no. 10, pp. 1159–1167, 2009.
[8] I. Rahman, S. K. Biswas, and P. A. Kirkham, “Regulation of
inflammation and redox signaling by dietary polyphenols,”
Biochemical Pharmacology, vol. 72, no. 11, pp. 1439–1452, 2006.
[9] P. Saiko, A. Szakmary, W. Jaeger, and T. Szekeres, “Resveratrol
and its analogs: defense against cancer, coronary disease and
neurodegenerative maladies or just a fad?” Mutation Research,
vol. 658, no. 1-2, pp. 68–94, 2008.
[10] Y.-J. Surh, K.-S. Chun, H.-H. Cha et al., “Molecular mechanisms
underlying chemopreventive activities of anti-inflammatory
phytochemicals: down-regulation of COX-2 and iNOS through
suppression of NF-𝜅B activation,” Mutation Research, vol. 480-
481, pp. 243–268, 2001.
[11] V. Janˇcinov´a, T. Pereˇcko, R. Nos´al’, D. Koˇst’´alov´a, K. Bauerov´a,
and K. Dr´abikov´a, “Decreased activity of neutrophils in the
presence of diferuloylmethane.(curcumin) involves protein
kinase C inhibition,” European Journal of Pharmacology, vol.
612, pp. 161–166, 2009.
[12] V. Janˇcinov´a, R. Nos´al’, A. Lojek et al., “Formation of reactive
oxygen and nitrogen species in the presence of pinosylvin—an
analogue of resveratrol,” Neuroendocrinology Letters, vol. 31, pp.
79–83, 2010.
[13] V. Janˇcinov´a, T. Pereˇcko, R. Nos´al’ et al., “Pinosylvin decreases
activity of neutrophils in vitro and in experimental arthritis,”
Acta Pharmacologica Sinica, vol. 10, pp. 1285–1292, 2012.
[14] R. Nos´al’, T. Pereˇcko, V. Janˇcinov´a, K. Dr´abikov´a, J. Harmatha,
and K. Svitekov´a, “Naturally appearing N-feruloylserotonin
suppress oxidative burst of human neutrophils at protein kinase
C level,” Pharmacological Reports, vol. 63, pp. 790–798, 2011.
[15] T. Pereˇcko, V. Janˇcinov´a, K. Dr´abikov´a, R. Nos´al’, and J.
Harmatha, “Structure-efficiency relationship in derivatives of
stilbene. Comparison of resveratrol, pinosylvin and pterostilbene,”
Neuroendocrinology Letters, vol. 29, pp. 802–805, 2008.
[16] T. Pereˇcko, K. Dr´abikov´a, L. Raˇckov´a et al., “Molecular targets
of the natural antioxidant pterostilbene: effect on proetin kinase
C, caspase-3, and apoptosis in human neutrophils in vitro,”
Neuroendocrinology Letters, vol. 31, pp. 101–107, 2010.
[17] T. Pereˇcko, K. Dr´abikov´a, R. Nos´al’, J. Harmatha, and V.
Janˇcinov´a, “Pharmacological modulation of activated neutrophils
by natural polyphenols,” Recent Research Developments
in Pharmacology, vol. 2, pp. 27–67, 2011.
[18] A. L. Holme and S. Pervaiz, “Resveratrol in cell fate decisions,”
Journal of Bioenergetics and Biomembranes, vol. 39, no. 1, pp. 59–
63, 2007.
[19] B. Catalgol, S. Batirel, Y. Taga, and N. Kartal Ozer, “Resveratrol:
French paradox revisited,” Frontiers in Pharmacology, vol. 3, pp.
1–18, 2012.
[20] J. ˇSmidrkal, J. Harmatha, M. Budˇeˇs´ınk´y et al., “Modified
approach for preparing (E)-stilbens related to resveratrol, and
evaluation of their potential immunobiological effects,” Collection
of Czechoslovak Chemical Communications, vol. 75, pp. 175–
186, 2010.
[21] K. Dr´abikov´a, V. Janˇcinov´a, R. Nos´al’, J. Peˇcivov´a, and T.
Maˇciˇckov´a, “Extra- and intracellular oxidant production in
phorbol myristate acetate stimulated human polymorphonuclear
leukocytes: modulation by histamine and H1- antagonist
loratadine,” Inflammation Research, vol. 55, pp. S19–S20, 2006.
[22] K. Dr´abikova, V. Janˇcinov´a, R. Nos´al’, J. Peˇcivov´a, T. Maˇciˇckov´a,
and P. Turˇc´ani, “Inhibitory effect of stobadine on FMLPinduced
chemiluminescence in human whole blood and isolated
polymorphonuclear leukocytes,” Luminescence, vol. 2, pp.
67–71, 2007.
[23] K. Dr´abikov´a, T. Pereˇcko, R. Nos´al’ et al., “Glucomanan reduces
neutrophil free radical production in vitro and in rats with
adjuvant arthritis,” Pharmacological Research, vol. 59, pp. 399–
403, 2009.
Oxidative Medicine and Cellular Longevity 9
[24] J. Kr´alov´a, M. Pekarov´a, K. Dr´abikov´a et al., “The effects of
dithiaden on nitric oxide production by RAW 264.7 cells,”
Interdisciplinary Toxicology, vol. 1, pp. 214–217, 2008.
[25] G. Ambroˇzov´a, M. Pekarov´a, and A. Lojek, “Effect of polyunsaturated
fatty acids on the reactive oxygen and nitrogen species
production by raw 264.7 macrophages,” European Journal of
Nutrition, vol. 49, no. 3, pp. 133–139, 2010.
[26] B. Ou, M. Hampsch-Woodill, and R. L. Prior, “Development and
validation of an improved oxygen radical absorbance capacity
assay using fluorescein as the fluorescent probe,” Journal of
Agricultural and Food Chemistry, vol. 49, no. 10, pp. 4619–4626,
2001.
[27] M. ˇC´ıˇz, M. Pavelkov´a, L. Gallov´a, J. Kr´alov´a, L. Kubala, and A.
Lojek, “The influence of wine polyphenols on reactive oxygen
and nitrogen species production by murine macrophages RAW
264.7,” Physiological Research, vol. 57, no. 3, pp. 393–402, 2008.
[28] J. Hrb´aˇc, C. Gregor, M. Machov´a et al., “Nitric oxide sensor
based on carbon fiber covered with nickel porphyrin layer
deposited using optimized electropolymerization procedure,”
Bioelectrochemistry, vol. 71, pp. 46–53, 2007.
[29] M. Pekarov´a, J. Kr´alov´a, L. Kubala et al., “Continuous electrochemical
monitoring of nitric oxide production in murine
macrophage cell line RAW 264.7,” Analytical and Bioanalytical
Chemistry, vol. 394, pp. 1497–1504, 2009.
[30] H. Slav´ıkov´a, A. Lojek, J. Hamar et al., “Total antioxidant
capacity of serum increased in early but not late period after
intestinal ischemia in rats,” Free Radical Biology & Medicine, vol.
25, pp. 9–18, 1998.
[31] K. Dr´abikov´a, T. Pereˇcko, R. Nos´al’, J. Harmatha, J. ˇSmidrkal,
and V. Janˇcinov´a, “Polyphenol derivatives—potential regulators
of neutrophil activity,” Interdisciplinary Toxicology, vol. 5, pp.
65–70, 2012.
[32] S. Rotondo, G. Rajtar, S. Manarini et al., “Effect of transresveratrol,
a natural polyphenolic compound, on human polymorphonuclear
leukocyte function,” British Journal of Pharmacology,
vol. 123, no. 8, pp. 1691–1699, 1998.
[33] V. Cucciolla, A. Borriello, A. Oliva, P. Galletti, V. Zappia, and
F. Della Ragione, “Resveratrol: from basic science to the clinic,”
Cell Cycle, vol. 6, no. 20, pp. 2495–2510, 2007.
[34] J. Li, Z. Qin, and Z. Liang, “The prosurvival role of autophagy in
Resveratrol-induced cytotoxicity in human U251 glioma cells,”
BMC Cancer, vol. 9, article 215, 2009.
[35] H. Miki, N. Uehara, A. Kimura et al., “Resveratrol induces
apoptosis via ROS-triggered autophagy in human colon cancer
cells,” International Journal of Oncology, vol. 40, no. 4, pp. 1020–
1028, 2012.
[36] G. Can, Z. Cakir, M. Kartal, U. Gunduz, and Y. Baran, “Apoptotic
effects of resveratrol, a grape polyphenol, on imatinibsensitive
and resistant K562 chronic myeloid leukemia cells,”
Anticancer Research, vol. 32, pp. 2673–2678, 2012.
[37] C. A. de la Lastra and I. Villegas, “Resveratrol as an antioxidant
and pro-oxidant agent: mechanisms and clinical implications,”
Biochemical Society Transactions, vol. 35, no. 5, pp. 1156–1160,
2007.
[38] M. P. Robich, L. M. Chu, M. Chaudray et al., “Anti-angiogenic
effect of high-dose resveratrol in a swine model of metabolic
syndrome,” Surgery, vol. 148, no. 2, pp. 453–462, 2010.
[39] M. J. Atten, B. M. Attar, T. Milson, and O. Holian, “Resveratrolinduced
inactivation of human gastric adenocarcinoma cells
through a protein kinase C-mediated mechanism,” Biochemical
Pharmacology, vol. 62, no. 10, pp. 1423–1432, 2001.
[40] J.-H. Woo, J. H. Lim, Y.-H. Kim et al., “Resveratrol inhibits
phorbol myristate acetate-induced matrix metalloproteinase-9
expression by inhibiting JNK and PKC 𝛿 signal transduction,”
Oncogene, vol. 23, no. 10, pp. 1845–1853, 2004.
[41] S. J. Slater, J. L. Seiz, A. C. Cook, B. A. Stagliano, and C. J. Buzas,
“Inhibition of protein kinase C by resveratrol,” Biochimica et
Biophysica Acta, vol. 1637, no. 1, pp. 59–69, 2003.
[42] J. K. Kundu, Y. K. Shin, and Y.-J. Surh, “Resveratrol modulates
phorbol ester-induced pro-inflammatory signal transduction
pathways in mouse skin in vivo: NF-𝜅B and AP-1 as prime
targets,” Biochemical Pharmacology, vol. 72, no. 11, pp. 1506–1515,
2006.
[43] M. H. Kim, D. S. Yoo, S. Y. Lee et al., “The TRIF/TBK1/IRF-3
activation pathway is the primary inhibitory target of resveratrol,
contributing to its broad-spectrum anti-inflammatory
effects,” Pharmazie, vol. 66, no. 4, pp. 293–300, 2011.
[44] A. A. Qureshi, X. Q. Guan, J. C. Reis et al., “Inhibition of nitric
oxide and inflammatory cytokines in LPS-stimulated murine
macrophages by resveratrol, a potent proteasome inhibitor,”
Lipids in Health and Disease, vol. 11, pp. 76–93, 2012.
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Black chokeberry (Aronia melanocarpa) polyphenols reveal different
antioxidant, antimicrobial and neutrophil-modulating activities
Petko Deneva,⁎
, Milan Čížb,c
, Maria Kratchanovaa
, Denica Blazhevad
a
Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000
Plovdiv, Bulgaria
b
Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, 612 65 Brno, Czech Republic
c
Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic
d
University of Food Technologies, 26 Maritsa Blvd., 4002 Plovdiv, Bulgaria
A R T I C L E I N F O
Keywords:
Black chokeberry (Aronia melanocarpa) fruits
Polyphenols
Antioxidant activity
Oxidative burst
Neutrophils
Antimicrobial properties
A B S T R A C T
The current study reports data on antioxidant, antimicrobial and neutrophil-modulating activities of different
polyphenolic preparations from black chokeberry fruits: crude extract, purified extract standardized to 20% and
40% anthocyanins, and proanthocyanidins; as well as pure compounds (chlorogenic acid, cyanidin-3-O-galactoside,
epicatechin, rutin and quercetin) present in black chokeberries. Minor phenolic components - quercetin
and epicatechin showed the highest ORAC and TRAP antioxidant activity. Given the amount of individual
phenolics in the fruits, proanthocyanidins are the major contributor to antioxidant activity of fresh black chokeberries.
Studied polyphenols and preparations had no effect on the spontaneous chemiluminescence (CL) of
human neutrophils and only mild effect on PMA-activated CL. Greater effects were observed on OZP-activated
CL, being statistically significant (p < 0.05) for quercetin and rutin. The antimicrobial activity test against 10
pathogens showed that black chokeberry proanthocyanidins are the most potent antimicrobial agents in the
fruit.
1. Introduction
In recent years, considerable attention has been devoted to medicinal
plants rich in polyphenols, with antioxidant and antimicrobial
properties. With over 8000 known representatives in the plant
kingdom, polyphenols are the most abundant dietary antioxidants (Ross
& Kasum, 2002). Natural polyphenols differ significantly in their
structure and vary from single molecules, such as phenolic acids to
polymers, such as tannins (Harborne & Simmonds, 1964). Phenolic
compounds are known to possess various pharmacological activities
and there is growing interest in polyphenol substances that are given to
humans through food, functional foods, nutraceuticals or even medi-
cines.
Black chokeberry (Aronia melanocarpa) fruits are particularly rich in
polyphenol compounds and are among the fruits with the highest antioxidant
activities. Their antioxidant action embraces radical scavenging,
suppression of the formation of reactive oxygen and nitrogen
species, restoration of antioxidant and inhibition of prooxidant enzymes
(Denev, Kratchanov, Číž, Lojek, & Kratchanova, 2012). In the last years,
there is an increasing research interest in black chokeberries, their
chemical composition (Denev et al., 2012; Kulling & Rawel, 2008),
health benefits (Denev et al., 2012; Valcheva-Kuzmanova & Belcheva,
2006) and clinical effectiveness (Chrubasik, Li, & Chrubasik, 2010).
Black chokeberry fruits (BCF) are very rich source of anthocyanins,
proanthocyanidins and hydroxycinnamic acids. Besides, quercetin,
quercetin glycosides and epicatechin are present in the fruits as minor
components (Denev et al., 2012; Kulling & Rawel, 2008). There is
strong scientific evidence for black chokeberry health benefits. Antidiabetic,
antimutagenic, cardioprotective, hepatoprotective and anticarcinogenic
effects of the fruits and their extracts have been reported
(Denev et al., 2012). Many of the studies were performed with complex
extracts, either crude or partly purified and it is not clear which particular
polyphenol components are responsible for the observed biological
effects. In our previous study, we observed that crude black chokeberry
extract possesses high antioxidant activity, measured via
different assays and inhibited reactive oxygen species (ROS) production
of opsonized zymosan-activated phagocytes (Denev et al., 2014). The
extract showed strong antimicrobial properties against broad spectrum
of microorganisms and had high mitogenic activity on hamster spleen
lymphocytes. Based on these results, we hypothesized that black
https://doi.org/10.1016/j.foodchem.2019.01.108
Received 25 July 2018; Received in revised form 16 January 2019; Accepted 16 January 2019
⁎
Corresponding author at: 139 Ruski Blvd., Plovdiv 4000, Bulgaria.
E-mail addresses: petkodenev@yahoo.com (P. Denev), milanciz@ibp.cz (M. Číž), lbas@plov.omega.bg (M. Kratchanova).
Food Chemistry 284 (2019) 108–117
Available online 23 January 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
T
chokeberry polyphenols (BCP) differ in their biological activity and
decided to perform detailed study on their antioxidant, antimicrobial
and neutrophil-modulating properties. Therefore, the aim of our study
was to determine the antioxidant properties of black chokeberry polyphenolic
preparations and pure phenolic compounds, and their effect
on spontaneous, opsonized zymosan particles (OZP)- and phorbolmyristate-acetate
(PMA)-activated human neutrophils. The antimicrobial
activity against 10 pathogens was investigated as well. The
results of the study shed light on the activity of different classes of
polyphenols in black chokeberry and could be used in activity-guided
development of functional foods and nutraceuticals with antioxidant,
antimicrobial and immuno-modulating properties.
2. Material and methods
2.1. Chemicals
Trolox, fluorescein disodium salt, AAPH, gallic acid, chlorogenic
acid, 3,4-dihydroxy benzoic acid, p-coumaric acid, caffeic acid, ferulic
acid, ellagic acid, naringin, myrecetin, quercetin, quercetin-3-glucoside,
rutin, naringenin, kaempherol, catechin, epicatechin, Amberlite XAD7
and Sephadeh LH20 were obtained from Sigma-Aldrich (Steinheim,
Germany). Folin-Ciocalteu’s reagent was delivered from Merck
(Darmstadt, Germany). Cyanidin-3-O-galactoside (Cy-3-gal) chloride
was purchased from Extrasynthese S.A. (Genay Cedex, France). All
other solvents used were of analytical grade and purchased from local
distributors.
2.2. Plant materials
Black chokeberry fruits were supplied from local growers in the
stage of full maturity, in august 2016. After collection, fresh fruits were
put in polyethylene bags, frozen immediately and stored at −18 °C
until their analysis. For analytical purposes 200 g berries were freeze
dried in Alpha 1–4 LDplus laboratory freeze drier (Martin Christ
Gefriertrocknungsanlagen GmbH, Germany) and extracted according to
(Denev et al., 2014). Extract was kept at −18 °C prior to analysis and
used for determination of total polyphenol, anthocyanin and proanthocyanidin
contents, as well as individual phenolic compounds via
HPLC.
2.3. Extraction, purification and isolation of black chokeberry preparations
The scheme for extraction and purification of black chokeberry
preparations is depicted on Fig. 1, and different steps are explained
bellow.
2.3.1. Extraction
100 g frozen fruits were defrosted at room temperature, mixed with
300 ml ultra-pure water and homogenized in a laboratory blender.
Homogenized sample was transferred into extraction tube and subjected
to extraction in thermostatic shaker water bath (NUVE, Turkey)
for one hour at 60 °C. After that, mixture was centrifuged (20 min,
6200×g) and the obtained supernatant was used for purification and
isolation of black chokeberry polyphenol preparations. 50 ml of the
extract were freeze dried in Alpha 1–4 LDplus laboratory freeze drier
and the dry extract was denoted as Crude Extract (CE). Extraction
procedure was repeated two times.
2.3.2. Column chromatography with Amberlite XAD7
Column chromatography was performed on SUPELCO VISIPER
system, connected to a vacuum pump (KNF lab) with 50 cm3
glass
cartridges. Amberlite XAD7 was soaked in ultra-pure water and then
loaded into the column. After that, the column was rinsed with ultrapure
water and crude extract (50 ml portion) was applied. The impurities
were eluted with ultra-pure water and 10 ml fractions were
collected until no soluble dry solids were detected. After that, black
chokeberry polyphenols were eluted with 200 ml 96% ethanol. Column
was washed with 100 ml water and another 50 ml portion of the extract
was applied and processed in the same way. The procedure was repeated
7 times in order to purify the whole amount of crude extract. All
polyphenol-containing ethanol fractions were combined, concentrated
via rotary evaporation at 50 °C and volume was adjusted to 100 ml with
ultra-pure water. After that, 20 ml of the concentrated extract containing
partially purified polyphenols were freeze dried to obtain
powdered dry product denoted as Purified Extract containing 20%
anthocyanins (PE20). PE20 was kept at −18 °C until its analysis. The
SPE procedure was repeated two times with both extracts obtained in
Section 2.3.1.
2.3.3. Column chromatography with Sephadex LH 20
Proanthocyanidins were further purified from the concentrated extract
containing partially purified polyphenols (described in Section
2.3.2) by the modified procedure of Howell et al. (2005). Briefly, 20 ml
of the concentrated extract were applied to 50 cm3
cartridge filled with
5 g of Sephadex LH-20 that was equilibrated overnight in ethanol:distilled
water (50:50) (v/v). After that, column was washed with 80 ml
ethanol:distilled water (50:50) (v/v) in order to remove low-molecular
polyphenols. Proanthocyanidin fraction was eluted with 80 ml 80%
acetone in distilled water (80:20) (v/v). The same procedure was repeated
four times in order to process the whole volume (80 ml) of the
partially purified polyphenol extract (see Section 2.3.2.). All ethanol:distilled
water (50:50) fractions were collected and combined and
ethanol was evaporated via rotary evaporation at 50 °C. Obtained water
extract was freeze dried in Alpha 1–4 LDplus laboratory freeze drier and
dry extract was denoted as Purified Extract containing 40% anthocyanins
(PE40). Aqueous acetone fractions were combined and acetone
evaporated via rotary evaporation at 40 °C. Obtained water extract was
freeze dried in Alpha 1–4 LDplus laboratory freeze drier and dry extract
was denoted as Proanthocyanidins (PACNs) fraction.
2.4. HPLC analysis of phenolic compounds
High Performance Liquid Chromatography (HPLC) analysis of phenolic
components was performed according to Denev et al. (2014), on
Agilent 1220 (Agilent Technology, USA), HPLC system with binary
pump and UV–Vis detector (Agilent Technology, USA). Wavelength of
280 nm was used. Phenolics separation was performed using Agilent
TC-C18 column (5 μm, 4.6 mm × 250 mm) at 25 °C. The results were
expressed in mg per 1 g preparation or fresh weight.
2.5. Total anthocyanins determination
Total anthocyanins content was determined by the pH-differential
method according to Lee, Durst, and Wrolstad (2005). The detailed
procedure is described in Denev et al. (2010). The results were expressed
in mg of cyanidin-3-O-glucoside equivalents per 1 g preparation
or fresh weight.
2.6. Total proanthocyanidin content analysis
Total proanthocyanidins were determined according to Sarneckis
et al. (2006). Proanthocyanidin content was calculated from a calibration
curve with catechin solutions and expressed in mg of catechin
P. Denev et al. Food Chemistry 284 (2019) 108–117
109
equivalents per 1 g preparation or fresh weight.
2.7. Oxygen Radical Absorbance Capacity (ORAC) assay
Oxygen Radical Absorbance Capacity was measured according to
the method of Ou, Hampsch-Woodill, and Prior (2001) with some
modifications described by Denev et al. (2010). ORAC analysis was
carried out using a plate reader FLUOstar OPTIMA (BMG Labtech,
Germany), excitation wavelength of 485 nm and emission wavelength
of 520 nm were used. ORAC values were expressed in micromole trolox
equivalents (µmol TE) per 1 g preparation or pure compound.
2.8. Total peroxyl radical trapping parameter (TRAP) assay
TRAP method was performed according to (Čížová, Lojek, Kubala, &
Číž, 2004). TRAP values are determined from the duration of the time
period (Tsample) during which the sample quenched the CL signal due to
the present antioxidants. TRAP analysis was performed on Luminometer
Orion II (Berthold Dection System GmbH, Germany) and activity
of the sample was expressed in micromole trolox equivalents
(µmol TE) per 1 g preparation or pure compound.
2.9. Oxidative burst
The oxidative burst of phagocytes in whole blood was measured
chemiluminometrically on LM-01 microplate luminometer
(Immunotech, Czech Republic). The principle of the method was described
previously (Číž et al., 2007) and the detailed procedure was
performed according to Denev et al. (2014).
2.10. Agar diffusion method
Determination was performed according to Denev et al. (2014).
Briefly, saline solution was used to prepare a suspension of each test
Fig. 1. Scheme for extraction and purification of aronia preparations.
P. Denev et al. Food Chemistry 284 (2019) 108–117
110
microorganism (concentration ∼1.107
cfu/ml). 1 ml of each microbial
suspension was used to inoculate melted PCA (Scharlau) nutrient
medium and 15 cm3
were poured in Petri dishes. 7 mm in diameter
wells were made in the solidified agar medium (after cooling) and 50 μl
of the samples were pipetted in the wells. Petri dishes were incubated
(37 °C, 24–48 h) and inhibition zones were measured. Zones with diameter
more than 7 mm were considered as zones of inhibition.
2.11. Minimal inhibiting concentration (MIC) determination
Minimal inhibiting concentrations of the extracts were analyzed as a
criterion for their antimicrobial effects, using 96 well microtiter plate
method (broth dilution), according to the modified procedure of
Gutierrez, Barry-Ryan, and Bourke (2008) and details are described by
Denev et al. (2014). MIC was the lowest concentration of the extract or
compound that reduced the growth of the tested microorganism to 50%
of the positive control.
2.12. Statistical analysis
The processing was repeated two times and the analysis performed
at least in triplicate. Results were expressed as mean values ± standard
deviations. In the case of chemical analysis and antioxidant activity,
one-way analysis of variance (ANOVA) and Student’s t-test were used to
evaluate the differences of the mean between groups. In the case of
effects of black chokeberry polyphenols and preparations on the production
of reactive oxygen species, the data were analyzed by F-test to
test the equality of two variances, followed by Student’s t-test to evaluate
the differences of the mean between individual samples and control.
P values less than 0.05 were considered to be significant. Microsoft
Excel, 2013 (Microsoft Corporation, Redmond, USA) was used in the
analyses.
3. Results and discussion
3.1. Purification and chemical composition of black chokeberry
preparations
For the extraction of anthocyanins and other phenolic compounds,
different extraction systems have been used (Nicoue, Savard, &
Belkacemi, 2007). Organic solvents (ethanol, methanol and acetone) or
their water solutions are usually used for extracting polyphenols.
Acetone and methanol are effective extragents, but their toxicity limits
their application in food processing. Ethanol is suitable for food application
but is more difficult to eliminate in the purification process.
Since the goal of our project is development of black chokeberry
functional foods we choose water as extragent for BCP. Common extraction
procedures including the one we used are non-selective and
yield solutions with impurities, such as sugars, sugar alcohols, organic
acids, amino acids, proteins, etc. It is known that besides polyphenols,
black chokeberries and crude extracts are rich source of other substances
such as sugars (sorbitol, fructose and glucose) and organic acids
(Denev et al., 2018; Kulling & Rawel, 2008). In the current study, we
applied several purification steps in order to obtain different black
chokeberry preparations: CE, PE20, PE40 and PACNs. Results for the
content of total polyphenols, anthocyanins and individual phenolic
compounds of these preparations are shown in Table 1.
As it is seen from Table 1, all preparations including the crude extract
contain several classes of phenolic compounds: hydroxycinnamic
acids (neochlorogenic acid and chlorogenic acid); flavan-3-ols (epicatechin);
flavonols (quercetin, rutin and quercetin-3-glucoside) and anthocyanins,
which is in agreement with the literature information
(Denev et al., 2012; Kulling & Rawel, 2008). The amount of polyphenolic
compounds in the crude extract is only 5.35% of which 1.48%
are anthocyanins. Crude extract was quite hygroscopic because of the
high content of monosaccharides and especially sorbitol, and required
further purification. Solid phase extraction using the absorbent Amberlite
XAD7 is a fast, simple and reliable method for purification of
anthocyanins and other phenolics from complex extracts, containing
impurities such as sugars, acids, proteins, pectin etc. (KraemerSchafhalter,
Fuchs, & Pfannhauser, 1998). In our previous study, we
enriched different crude berry extracts with anthocyanins and other
phenolics, which led to a significant increase in their antioxidant
properties (Denev et al., 2010). As it is evident from Table 1, application
of Amberlite XAD7 increased the total polyphenol content of the
crude extract more than 12-fold and the amount of anthocyanins
reached more than 200 mg/g extract. Sephadex LH 20 resin is broadly
used for purification and isolation of proanthocyanidins and other
biologically active compounds (steroids, terpenoids, lipids, low molecular
weight peptides, etc.) form plant matter. However, in our study
we used Sephadex LH 20 for simultaneous isolation of proanthocyanidins
and for further purification and enrichment of black chokeberry
low-molecular polyphenols. As a result, after elution of the resin with
50% ethanol and further freeze-drying of the extract, we obtained dry
black chokeberry extract containing more than 40% anthocyanins,
while the amount of total polyphenols reached 84%. In the same time,
this allowed us to isolate black chokeberry proanthocyanidins. The
purification scheme adopted by us could be easily produced in higher
scale, using simple two-step column purification, first with Amberlite
XAD 7 followed by Sephadex LH 20.
3.2. Antioxidant activity of black chokeberry polyphenols and polyphenolic
preparations
After the characterization of black chokeberry preparations by
means of their polyphenol content and composition (Section 3.1), we
Table 1
Content of total polyphenols, anthocyanins and individual phenolic compounds in black chokeberry preparations.
CE PE20 PE40 PACNs
Neochlorogenic acid 10.2c
± 2.1 130.4b
± 11.2 189.5 a
± 28.2 12.2c
± 3.1
Chlorogenic acid 8.3c
± 0.9 121.1b
± 8.1 177.1 a
± 15.4 11.9c
± 1.2
Epicatechin 2.3 d
± 0.3 29.3b
± 4.2 59.3 a
± 7.5 15.1c
± 0.9
Rutin 7.8c
± 0.5 45.5 a
± 5.6 35.3 ab
± 14.2 24.2b
± 0.6
Quercetin-3-glucoside 1.9b
± 0.1 23.4 a
± 2.1 20.1 a
± 3.5 18.2 a
± 0.4
Quercetin 2.1b
± 0.2 2.0 bc
± 0.9 1.4c
± 0.1 5.7 a
± 0.3
Anthocyanins 14.8c
± 0.9 213.5b
± 20.2 400.2 a
± 37.0 20.1c
± 5.4
Total polyphenols 53.5c
± 4.2 687.2b
± 35.6 840.5 a
± 62.1 910.2 a
± 46.3
Results are expressed as mean ± SD in mg/g preparation. There are no significant differences among values marked with the same superscript letters in individual
rows (p < 0.05).
P. Denev et al. Food Chemistry 284 (2019) 108–117
111
continued with determination of their antioxidant properties. In order
to better assess the antioxidant activity of black chokeberry polyphenols,
we additionally analyzed pure compounds found naturally in
these fruits. They belong to different classes of polyphenols and include:
cyanidin-3-O-galactoside (anthocyanin); chlorogenic acid (hydroxycinnamic
acid), epicatechin (flavan-3-ol), quercetin (flavonol aglycone)
and rutin (flavonol glycoside). To obtain more detailed information
about the antioxidant activity of black chokeberry
polyphenols and preparations, we used two complementary assays –
ORAC and TRAP. Both assays rely on hydrogen atom transfer and express
the ability of the sample to scavenge peroxyl radicals, which are
physiologically the most relevant. Moreover, these methods are performed
at physiological temperature and pH. Results are presented on
Fig. 2.
ORAC and TRAP results of studied compounds and preparations were
similar, giving the following rank order via ORAC: quercetin >
epicatechin > chlorogenic acid > rutin > PE20 > PE40 > cyanidin-
3-O-galactoside > proanthocyanidins > crude extract and via TRAP:
quercetin > epicatechin > chlorogenic acid > rutin > cyanidin-3-Ogalactoside
> PE20 > PE40 > proanthocyanidins > crude extract. Our
literature review revealed that there is only one study on the antioxidant
activity of black chokeberry polyphenols (Zheng & Wang, 2003). Authors of
the study reported that among them, quercetin glycosides possess the
highest ORAC values (13400–15800 µmol TE/g) without providing information
about the activity of quercetin and rutin. Our result for rutin –
11293 µmol TE/g is similar to those of other quercetin glycosides, but
quercetin itself revealed the highest antioxidant activity – 26228 µmol TE/g.
Our ORAC value for cyanidin-3-O-galactoside, which is the predominant
anthocyanin in black chokeberries is 10400 µmol TE/g which is slightly
lower than the result of (Zheng & Wang, 2003) – 11500 µmol TE/g. The
biggest discrepancy was obtained for the ORAC of chlorogenic acid –
12672 µmol TE/g in our study, compared to 7400 µmol TE/g in the abovementioned
work. TRAP values for pure polyphenols and black chokeberry
phenolic preparations further enrich the available data about the antioxidant
activity of BCP. As it could be expected, crude polyphenol extract
had the lowest activity via both methods. From the purified black chokeberry
fractions, proanthocyanidins revealed the lowest TRAP – 9261 µmol
TE/g and ORAC activity – 10245 µmol TE/g, despite the fact that they were
purified to more than 90% polyphenols. Interestingly, degree of purification
of anthocyanins did not affect their antioxidant activity and there was no
Fig. 2. Antioxidant activity of black chokeberry polyphenols and preparations measured by ORAC and TRAP assays. There are no significant differences among
values marked with the same symbols (p < 0.05).
P. Denev et al. Food Chemistry 284 (2019) 108–117
112
significant difference (p < 0.05) between antioxidant activity of PE20,
PE40 and pure cyanidin-3-O-galactoside. Antioxidant properties of phenolic
compounds are influenced by their structural characteristics, such as
number and arrangement of OH groups. In the case of flavonoids, the
presence of OH group at the 3th position, ortho-dihydroxy substitution in
the B ring and a 2,3 double bond conjugated with the 4-oxo function of the
C ring are another important structural features for strong antioxidant activity
(Bors, Heller, Michel, & Saran, 1990; Rice-Evans, Miller, & Paganga,
1996; Salah et al., 1995). Among black chokeberry polyphenols, quercetin
possesses all structural characteristics, necessary for potent antioxidant activity
and its high antioxidant activity is proved by our results. Zheng and
Wang (2003) estimated that anthocyanins contribute more than 52% to
ORAC antioxidant activity of black chokeberries, whereas flavonols and
caffeic acid (including its derivative), add additional 8.7% and 38.2%, respectively.
In order to calculate the contribution of individual phenolic
compounds to ORAC and TRAP activity of black chokeberry fruits, we
analyzed the content of anthocyanins and individual polyphenol compounds
in fresh berries. Results are presented in Table 2.
As it is evident from the table, proanthocyanidins are the major
antioxidant substances in BCF contributing to more than 38% of ORAC
value and 33% of TRAP value. This is an experimental evidence for our
previous assumption that about 40% of the in vitro antioxidant activity
of black chokeberry fruits is due to the potency of proanthocyanidins
(Denev et al., 2012). The second biggest contributor to antioxidant
activity of the berries are anthocyanins, followed by hydroxycinnamic
acids and epicatechin. Flavonols are characterized with very high antioxidant
activity, but due to their low quantity in fresh BCF, they
contribute to their antioxidant activity less than 10%. Plant extracts are
complex mixtures of many antioxidants acting simultaneously. Thus,
antioxidants could have additive effect in their action, but synergism
and antagonism are possible, as well. However, both TRAP and ORAC
assays could not account these effects, since they measure the common
antioxidant action of all antioxidants in the sample. Furthermore, plant
metabolites with low (i.e. ascorbic acid) or no (i.e. sugars, polysaccharides,
etc.) antioxidant activity could also interfere with the results.
Therefore, further studies with pure antioxidants model systems
could be very helpful in order to investigate the possible synergistic or
antagonistic effects in their action.
It should be noted that results presented in Table 2 are valid for the
particular batch of fruits used in the current study. Fresh black chokeberries
differ significantly in their content of individual polyphenols.
For example, their proanthocyanidin content vary in a broad range
(522–1002 mg/100 g fresh weight), but in all cases they are the most
abundant phenolic component in fresh black chokeberries (Denev et al.,
2018). Despite their high contribution to antioxidant activity of black
chokeberries, proanthocyanidins are barely absorbed in the gastrointestinal
tract (GIT), and the majority pass through and reach the colon
(Denev et al., 2012). Nevertheless, many of proanthocyanidins metabolites
in the colon are of phenolic nature, possess free –OH groups and
reveal antioxidant activity, as well. This could be very important, since
large numbers of phagocytic cells normally reside in the GIT. Thus, BCP
and in particular proanthocyanidins, and their metabolites could directly
modulate their function in vivo.
3.3. Effect of black chokeberry polyphenols and polyphenolic preparations
on the production of ROS from phagocytes
Knowledge of the immunomodulating effects of BCP is scarce. There
are just few studies on the effect of black chokeberry crude extracts or
isolated fractions mainly on murine macrophages. For example,
Ohgami et al. (2005) observed that crude polyphenolic extract from
black chokeberries reveal anti-inflammatory effect and suppress LPSinduced
iNOS and COX-2 protein expressions in RAW 264.7 cells in
vitro. In a more recent study, Ho et al. (2014) revealed that cyanidin,
procyanidin B2, B5 and C1 and proanthocyanidin-rich fractions from
black chokeberry were highly active in the complement-fixing assay. In
addition, the oligomeric procyanidins displayed inhibitory effects on
LPS-induced NO production in murine RAW 264.7 macrophages. As far
as we know, no studies exist on the immunomodulating effects of black
chokeberry polyphenolic extracts on human neutrophils, except our
earlier report for crude extract (Denev et al., 2014). We determined the
neutrophil-modulating activity of black chokeberry polyphenolic preparations
and pure phenolic compounds present in the fruits, as their
effect on spontaneous, OZP- and PMA-activated production of ROS by
human neutrophils. The oxidative burst of neutrophils in whole blood
was measured chemiluminometrically. The results are expressed as % of
control of the integrals of CL curves (the total production of ROS during
60 min), (Fig. 3). Any of the studied compounds and preparations had
no effects on the spontaneous chemiluminescence of human neutrophils
while only mild effects on PMA-activated neutrophils were observed. In
that case, the highest inhibitory effects were observed for quercetin,
cyanidin-3-O-galactoside and PE40. Greater effects of BCP were observed
in the case of OZP-activated neutrophils. The production of ROS
by OZP-activated neutrophils was inhibited most strongly by quercetin
and rutin, the effect being statistically significant (p < 0.05). This indicate
that probably black chokeberry flavonoids interfere with the
signaling cascade of phagocyte activation upstream to the protein kinase
C activation. Cyanidin-3-O-galactoside also inhibited greatly the
ROS production, but the effect was statistically insignificant
(p < 0.05).
Table 2
Content of different phenolics in fresh black chokeberries and their contribution to antioxidant activities of the fruit.
Content of phenolic components in
fresh berries, mg/g
Contribution to ORAC of fresh
berries, µmol TE/g
Contribution to TRAP of fresh
berries, µmol TE/g
% from total
ORAC
% from total
TRAP
Anthocyanins *
3.22 ± 0.17 33.5 ± 2.3 32.3 ± 1.2 24.1 22.1
Proanthocyanidins 5.22 ± 0.65 53.5 ± 1.0 48.3 ± 2.4 38.5 33.0
Neochlorogenic acid **
0.91 ± 0.05 11.5 ± 0.3 18.5 ± 0.6 8.4 12.8
Chlorogenic acid 1.02 ± 0.01 12.9 ± 0.3 20.8 ± 0.6 9.3 14.2
Epicatechin 0.65 ± 0.05 15.3 ± 0.1 13.6 ± 1.4 11.0 9.3
Rutin 0.5 ± 0.02 5.6 ± 0.1 5.5 ± 0.5 4.1 3.8
Quercetin-3-glucoside ***
0.27 ± 0.02 3.0 ± 0.1 3.0 ± 0.3 2.2 2.0
Quercetin 0.13 ± 0.03 3.4 ± 0.1 4.2 ± 0.2 2.4 2.8
Total 138.9 146.3 100.0 100.0
* Calculated as cyanidin-3-O-galactoside.
** Calculated as chlorogenic acid.
*** Calculated as rutin.
P. Denev et al. Food Chemistry 284 (2019) 108–117
113
There are several studies on the effects of flavonoids on the respiratory
burst of neutrophils. Kenny, Shu, Moritoki, Keen, and
Gershwin (2009) demonstrated that cocoa flavonoids moderate the
LPS-stimulated signaling pathways of neutrophils. They assumed that
flavonoids decrease the effect of LPS on the ability of fMLP-activated
neutrophills to generate ROS and hypothesized that the possible mechanism
is activation of the MAPK pathway. Pincemail et al. (1987)
revealed that flavonoid-containing Ginkgo biloba extract slowed down
the release of ROS from fMLP-stimulated human neutrophils. fMLP is a
soluble neutrophil activator that binds to specific receptors on the
Fig. 3. Effect of black chokeberry polyphenols and preparations on the production of reactive oxygen species from untreated (spontaneous); PMA-activated; and OZPactivated
human neutrophils. Values marked with asterisk are significantly different from the control (p < 0.05).
P. Denev et al. Food Chemistry 284 (2019) 108–117
114
membrane. Similarly, OZP bind to the surface opsonin receptors on
phagocytes and triggers a signaling cascade leading to the activation of
protein kinase C and subsequent NADPH oxidase activation. Wang,
Chang, Hsu, Chen, and Kuo (2002) investigated the inhibitory effect of
a cirsimaritin against respiratory burst in rat neutrophils and its cellular
localization and observed that it inhibited the generation of superoxide
radical and neutrophils oxygen consumption. Selloum, Reichl, Muller,
Sebihi, and Arnhold (2001) compared the effects of kaempferol, quercetin,
rutin and myricetin on the oxidative burst of neutrophils and
revealed that they inhibit the pholasin luminescence of fMLP-activate
neutrophils. Lee et al. (2010) showed that luteolin effectively blocked
MAPK/ERK kinase 1/2 and Akt phosphorylation in fMLP-stimulated
neutrophils, which is evidence that it is more likely for flavonoids to
inhibit the signaling enzymes, rather than to scavenge superoxide radicals
in fMLP-stimulated neutrophils. Furthermore, Qi, Feng, Li, Qi,
and Zhang (2017) observed that myricitrin exhibited anti-inflammatory
activity in LPS-activated RAW264.7 macrophages by blocking the activation
of JAKs and the downstream transcription factor STAT1, which
may result from the downregulation of NADH oxidase-dependent ROS
production mediated by myricitrin. However, myricitrin had no impact
on the MAPK signaling pathway. Myricitrin attenuated the generation
of intracellular ROS by inhibiting the assembly of components of the
gp91(phox) and p47(phox). Tian, Ding, Peng, and Lu (2017) showed
that (−)-epigallocatechin gallate, the main flavonoid present in green
tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro.
They reported that (−)-epigallocatechin gallate bound to the active site
of MPO and resulted in the accumulation of compound II, which was
unable to produce HOCl. This flavonoid also effectively inhibited HOCl
generation in activated neutrophils without influencing MPO and NADP
oxidase release and superoxide formation, suggesting that (−)-epigallocatechin
gallate specifically inhibited MPO but not NADPH oxidase
activity in activated neutrophils.
3.4. Antimicrobial activity
The antimicrobial activity of BCP (as phenolic preparations and
pure polyphenols) was tested against 10 human pathogens, via two
different methods – primary screening with the agar diffusion method
and determination of minimal inhibitory concentration. The broadspectrum
antibiotic ampicillin was used as a positive control. It should
be noted that the concentration of the extracts used in the current study
was significantly lower than that in our previous study (Denev et al.,
2014). In that concentration, crude black chokeberry extract showed no
antimicrobial activity against all pathogenic microorganisms included
in the study (Table 3).
As already stated, purification of crude extract with Amberlite XAD7
lead to a 12-fold increase in polyphenol content of the preparation. As a
result, the PE20 exhibited antimicrobial activity against Staphylococcus
aureus strains and Proteus vulgaris G. From the pure phenolic compounds,
only epicatechin and quercetin showed antimicrobial activities
against Candida albicans, but there was no activity against
Staphylococcus aureus strains and Proteus vulgaris G. In all other instances,
where inhibition zones were present, there were single colonies
in inhibition zones, indicating that a part of the microbial population
was more sensitive to the action of the tested pure substance and hence
no antimicrobial activity was attributed to those compounds.
Interestingly, the subsequent purification via Sephadex LH20 did not
result in further increase of the activity. Black chokeberry proanthocyanidins
that are partly present in PE20, exhibited inhibitory effect
against the same pathogens as PE20. The inhibition zones diameter
reached 11 mm for Proteus vulgaris and Staphylococcus aureus ATCC
6538P. The positive control - ampicillin exhibited strong inhibitory
effect against all bacterial pathogens.
In the second step, we determined minimal inhibitory concentrations
(MICs) of phenolic preparations and pure compounds, which
displayed antimicrobial activity in the agar-diffusion assay. Although
Table3
Antimicrobialactivityofblackchokeberrypolyphenolsandpreparations.
MicroorganismViablecellcountofmicroorganisminnutrientmedium,.107
cfu/cm3
CrudeextractPE20PE40PACNsChlorogenicacidEpicatechinQuercetinCy-3-galAmpicillin
Inhibitionzonesagainsthumanpathogens,mm
EscherichiacoliATCC87394.8–––––10*
9*
–28
Salmonellaentericassp.entericaATCCBAA-21621.2–––––12*
9*
–27
StaphylococcusaureusATCC6538P1.9–1010*
11–12*
10*
12*
30
StaphylococcusaureusATCC259232.7–910*
9–13*
11*
12*
30
Listeriamonocytogenes4.1––––––––22
ListeriamonocytogenesI2.5––––––––23
ProteusvulgarisG2.6–89*
11–––10*
31
PseudomonasaeruginosaATCC90274.9––––––––20
CandidaalbicansATCC102311.3–––––129––
MinimalInhibitingConcentration(MIC),mg/ml
EscherichiacoliATCC8739––––––––0.0125
Salmonellaentericassp.entericaATCCBAA-2162––––––––0.0063
StaphylococcusaureusATCC6538P–2.50–1.25––––0.0016
StaphylococcusaureusATCC25093–5.00–0.32––––0.0031
Listeriamonocytogenes––––––––0.0016
ListeriamonocytogenesI––––––––0.0008
ProteusvulgarisG–0.63–0.16––––0.0063
PseudomonasaeruginosaATCC9027––––––––0.1
CandidaalbicansATCC10231–––––1.252.50––
“-”–noantimicrobialeffect.
*Singlecoloniesintheinhibitionzone.
P. Denev et al. Food Chemistry 284 (2019) 108–117
115
PE40 contains more anthocyanins and other low-molecular phenolic
compounds, it does not contain proanthocyanidins, which could explain
the lack of antimicrobial activity. Interestingly, black chokeberry
proanthocyanidins revealed respectively 2- and 16-fold higher antimicrobial
activity towards Staphylococcus aureus strains in comparison
to PE20. Similar effect was observed for the antimicrobial activity
against Proteus vulgaris G. The lowest MIC (0.156 mg/ml) was found for
proanthocyanidins and it was four-fold lower than that of PE20.
Epicatechin and quercetin MICs of Candida albicans ATCC 10231 were
0.078 mg/ml and 2.5 mg/ml, respectively. Based on the results, it could
be concluded that antimicrobial effect of black chokeberries is mainly
due to the action of proanthocyanidins. Toxicity of tannins towards
microorganisms is well documented, but for the first time we demonstrate
that black chokeberry condensed tannins are the major antimicrobial
agents of the berry. The different mechanisms proposed so far
to explain tannin antimicrobial activity include inhibition of extracellular
microbial enzymes, deprivation of the substrates required for
microbial growth or direct action on microbial metabolism through
inhibition of oxidative phosphorylation (Scalbert, 1991). The study of
Taguri, Tanaka, and Kouno (2004) demonstrated that various types of
polyphenols including catechins and their oxidation products - proanthocyanidins
and hydrolyzable tannins, showed antibacterial activities
against four groups of food-borne bacteria. Authors concluded that the
sensitivity of bacteria to polyphenols depends on bacterial species and
polyphenol structure and showed the importance of 3,4,5-trihydroxyphenyl
groups for the observed antimicrobial activity. As it could be
expected, antibiotic positive control exhibited lower MICs than the
tested polyphenols and preparations against all sensitive pathogens.
The lowest MIC displayed by the proanthocyanidins against Proteus
vulgaris G. was 25 times higher than the activity of the positive control
ampicillin. However, it should be noted that there is no evidence that
BCP and in particular proanthocyanidins are toxic or have adverse effects,
characteristic for antibiotics. Therefore, condensed tannins from
black chokeberry fruits could exert their antimicrobial properties
against Staphylococcus aureus strains and Proteus vulgaris G in gastrointestinal
tract, more over their concentration in the GIT could be significant
(Scalbert & Williamson, 2000).
4. Conclusion
The current study confirmed our hypothesis that black chokeberry
polyphenols differ in their antioxidant, antimicrobial and neutrophilmodulating
activities. BCP have mild effect on PMA-activated and
greater effect on OZP-activated CL of human neutrophils. Quercetin and
epicatechin are the strongest antioxidants among BCP, but the amount
of proanthocyanidins makes them the major contributor to antioxidant
activity of fresh black chokeberries. Besides, we revealed that black
chokeberry proanthocyanidins are the most potent antimicrobial agents
in the fruit. Despite their high contribution to antioxidant activity of
Aronia melanocarpa berries, proanthocyanidins are barely absorbed in
the gastrointestinal tract and reach the colon. However, their action
may be very important, since numbers of phagocytic cells normally
reside in the gastrointestinal tract. Therefore, black chokeberry polyphenols
and in particular proanthocyanidins could directly modulate
their function and express their antioxidant and antimicrobial effects in
vivo.
Funding
This work was funded by the National Science Fund, Bulgarian
Ministry of Education and Science (Grant number DN 09/20 from
21.12.2016).
Conflict of interests
Authors declare no conflict of interests.
References
Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as antioxidants:
Determination of radical-scavenging efficiencies. Methods in Enzymology, 186,
343–355.
Chrubasik, C., Li, G., & Chrubasik, S. (2010). The clinical effectiveness of chokeberry: A
systematic review. Phytotherapy Research, 24(8), 1107–1114.
Číž, M., Komrsková, D., Prachařová, L., Okénková, K., Čížová, H., Moravcová, A., ... Nosá,
R. (2007). Serotonin modulates the oxidative burst of human phagocytes via various
mechanisms. Platelets, 18(8), 583–590.
Čížová, H., Lojek, A., Kubala, L., & Číž, M. (2004). The effect of intestinal ischemia
duration on changes in plasma antioxidant defense status in rats. Physiological
Research, 53(5), 523–531.
Denev, P., Číž, M., Ambrozova, G., Lojek, A., Yanakieva, I., & Kratchanova, M. (2010).
Solid-phase extraction of berries’ anthocyanins and evaluation of their antioxidative
properties. Food Chemistry, 123(4), 1055–1061.
Denev, P., Kratchanov, C., Číž, M., Lojek, A., & Kratchanova, M. (2012). Bioavailability
and antioxidant activity of black chokeberry (Aronia melanocarpa) polyphenols: In
vitro and in vivo evidences and possible mechanisms of action: A review.
Comprehensive Reviews in Food Science and Food Safety, 11(5), 471–489.
Denev, P., Kratchanova, M., Číž, M., Lojek, A., Vasicek, O., Nedelcheva, P., ... Vojtek, L.
(2014). Biological activities of selected polyphenol-rich fruits related to immunity
and gastrointestinal health. Food Chemistry, 157, 37–44.
Denev, P., Kratchanova, M., Petrova, I., Klisurova, D., Georgiev, Y., Ognyanov, M., &
Yanakieva, I. (2018). Black chokeberry (Aronia melanocarpa (Michx.) Elliot) fruits
and functional drinks differ significantly in their chemical composition and antioxidant
activity. Journal of Chemistry, 11.
Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2008). The antimicrobial efficacy of plant
essential oil combinations and interactions with food ingredients. International
Journal of Food Microbiology, 124(1), 91–97.
Harborne, J. B., & Simmonds, N. W. (1964). Biochemistry of phenolic compounds. London:
Academic Press.
Ho, G., Bräunlich, M., Austarheim, I., Wangensteen, H., Malterud, K. E., Slimestad, R., &
Barsett, H. (2014). Immunomodulating activity of Aronia melanocarpa polyphenols.
International Journal of Molecular Sciences, 15, 11626–11636.
Howell, A. B., Reed, J. D., Krueger, C. G., Winterbottom, R., Cunningham, D. G., & Leahy,
M. (2005). A-type cranberry proanthocyanidins and uropathogenic bacterial antiadhesion
activity. Phytochemistry, 66(18), 2281–2291.
Kenny, T. P., Shu, S. A., Moritoki, Y., Keen, C. L., & Gershwin, M. E. (2009). Cocoa flavanols
and procyanidins can modulate the lipopolysaccharide activation of polymorphonuclear
cells in vitro. Journal of Medicinal Food, 12, 1–7.
Kraemer-Schafhalter, A., Fuchs, H., & Pfannhauser, W. (1998). Solid-phase extraction
(SPE) - A comparison of 16 materials for the purification of anthocyanins from Aronia
melanocarpa var Nero. Journal of the Science of Food and Agriculture, 78(3), 435–440.
Kulling, S., & Rawel, H. (2008). Chokeberry (Aronia melanocarpa) – A review on the
characteristic components and potential health effects. Planta Medica, 74(13),
1625–1634.
Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric anthocyanin
pigment content of fruit juices, beverages, natural colorants, and wines by
the pH differential method: Collaborative study. Journal of AOAC International, 88(5),
1269–1278.
Lee, J. P., Li, Y. C., Chen, H. Y., Lin, R. H., Huang, S. S., Chen, H. L., ... Kuan, Y. H. (2010).
Protective effects of luteolin against lipopolysaccharide-induced acute lung injury
involves inhibition of MEK/ERK and PI3K/Akt pathways in neutrophils. Acta
Pharmaceutica Sinica B, 31, 831–838.
Nicoue, E. E., Savard, S., & Belkacemi, K. (2007). Anthocyanins in wild blueberries of
Quebec: Extraction and identification. Journal of Agricultural and Food Chemistry,
55(14), 5626–5635.
Ohgami, K., Ilieva, I., Shiratori, K., Koyama, Y., Jin, X., Yashida, K., ... Ohno, S. (2005).
Anti-inflammatory effects of Aronia extract on rat endotoxin-induced uiveitis.
Investigative Ophthalmology & Visual Science, 46, 275–281.
Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent
probe. Journal of Agricultural and Food Chemistry, 49(10), 4619–4626.
Pincemail, J., Thirion, A., Dupuis, M., Braquet, P., Drieu, K., & Deby, C. (1987). Ginkgo
biloba extracts inhibits oxygen species production generated by phorbol myristate
acetate stimulated human leukocytes. Experientia, 43, 181–184.
Qi, S., Feng, Z., Li, Q., Qi, Z., & Zhang, Y. (2017). Myricitrin modulates NADPH oxidasedependent
ROS production to inhibit endotoxin-mediated inflammation by blocking
the JAK/STAT1 and NOX2/p47(phox) pathways. Oxidative Medicine and Cellular
Longevity Article ID 9738745.
Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity relationships
of flavonoids and phenolic acids. Free Radical Biology & Medicine, 20(7),
933–956.
Ross, J. A., & Kasum, C. M. (2002). Dietary flavonoids: Bioavailability, metabolic effects,
and safety. Annual Review of Nutrition, 22(1), 19–34.
Salah, N., Miller, N. J., Paganga, G., Tijburg, L., Bolwell, G. P., & Rice-Evans, C. (1995).
Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking
antioxidants. Archives of Biochemistry and Biophysics, 322(2), 339–346.
Sarneckis, C. J., Dambergs, R. G., Jones, P., Mercurio, M., Herderich, M. J., & Smith, P. A.
(2006). Quantification of condensed tannins by precipitation with methyl cellulose:
Development and validation of an optimised tool for grape and wine analysis.
Australian Journal of Grape and Wine Research, 12(1), 39–49.
Scalbert, A. (1991). Antimicrobial properties of tannins. Phytochemistry, 30(12),
3875–3883.
P. Denev et al. Food Chemistry 284 (2019) 108–117
116
Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols.
Journal of Nutrition, 130, 2073S–2085S.
Selloum, L., Reichl, S., Muller, M., Sebihi, L., & Arnhold, J. (2001). Effects of flavonols on
the generation of superoxide anion radicals by xanthine oxidase and stimulated
neutrophils. Archives of Biochemistry and Biophysics, 395, 49–56.
Taguri, T., Tanaka, T., & Kouno, I. (2004). Antimicrobial activity of 10 different plant
polyphenols against bacteria causing food-borne disease. Biological and Pharmceutical
Bulletin, 27(12), 1965–1969.
Tian, R., Ding, Y., Peng, Y. Y., & Lu, N. (2017). Inhibition of myeloperoxidase- and
neutrophil-mediated hypochlorous acid formation in vitro and endothelial cell injury
by (-)-epigallocatechin gallate. Journal of Agricultural and Food Chemistry, 65(15),
3198–3203.
Valcheva-Kuzmanova, S. V., & Belcheva, A. (2006). Current knowledge of Aronia melanocarpa
as a medicinal plant. Folia Medica, 48(2), 11–17.
Wang, J. P., Chang, L. C., Hsu, M. F., Chen, S. C., & Kuo, S. C. (2002). Inhibition of ormylmethionyl-leucyl-phenylalaninestimulated
respiratory burst by cirsimaritin involves
inhibition of phospholipase D signaling in rat neutrophils. Naunyn-Schmiedeberg's
Archives of Pharmacology, 366, 307–314.
Zheng, W., & Wang, S. Y. (2003). Oxygen radical absorbing capacity of phenolics in
blueberries, cranberries, chokeberries, and lingonberries. Journal of Agricultural and
Food Chemistry, 51(2), 502–509.
P. Denev et al. Food Chemistry 284 (2019) 108–117
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Effects of Pectic Polysaccharides Isolated from Leek
on the Production of Reactive Oxygen and Nitrogen Species by Phagocytes
Mariana Nikolova,1
Gabriela Ambrozova,2
Maria Kratchanova,1
Petko Denev,1
Veselin Kussovski,3
Milan Ciz,2
and Antonin Lojek2
1
Center of Phytochemistry, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Plovdiv, Bulgaria.
2
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
3
Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
ABSTRACT The current survey investigates the effect of four polysaccharides isolated from fresh leek or alcohol insoluble
substances (AIS) of leek on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) from
phagocytes. The ability of the polysaccharides to activate serum complement was also investigated. Despite the lack of
antioxidant activity, the pectic polysaccharides significantly decreased the production of ROS by human neutrophils. Polysaccharides
isolated from AIS markedly activated RAW 264.7 macrophages for RNS production in a concentration-dependent
manner. The Western blot analysis revealed that this effect was due to the stimulation of the inducible nitric oxide synthase
protein expression of macrophages. The polysaccharides extracted from AIS with water showed the ability to fix serum
complement, especially through the alternative pathway. It was found that the polysaccharide that has the highest complement-fixing
effect is characterized by the highest content of uronic acids and the highest molecular weight.
KEY WORDS:  complement fixing  immunomodulatory activity  leek  macrophages  neutrophils  pectic
polysaccharides  reactive nitrogen species  reactive oxygen species
INTRODUCTION
Pectic substances are water-soluble heteropolysaccharides
with various applications in the food
industry.1
They are industrially produced mainly from apples
and citrus fruits, but are ubiquitous in the plant kingdom and are
of great importance for the structure of plant cells. There is an
increasing interest in pectic polysaccharides from various plant
sources, since they have revealed various biological activi-
ties.2,3
Recently, several articles reviewed the obtaining and
purifying of plant heteropolysaccharides rich in uronic acids
and investigated the relationship between their primary structure
and biological activity.4,5
Among the pharmacological
activities of these heteropolysaccharides, immunomodulating
activity seems to be the most important, and there is accumulating
evidence that they are involved in the modulation of
several components of the immune system.5,6
Innate immunity serves as an essential first-line of defense
against microbial pathogens and foreign substances.
Phagocytic cells such as macrophages and neutrophils play a
key role in innate immunity because of their ability to recognize,
ingest, and destroy pathogens by oxidative and
nonoxidative mechanisms. Thus, approaches designed to
enhance innate immune mechanisms nonspecifically could
increase the defense against microbial infections.7,8
In response
to a variety of stimuli, NADPH oxidase present in
neutrophils is activated in a phenomenon described as ‘‘the
respiratory burst,’’ characterized by the production of
superoxide anion, which gives rise to other forms of reactive
oxygen species (ROS). In addition to ROS, nitric oxide
(NO) and other reactive nitrogen species (RNS) produced
mainly by macrophages are one of the important microbicidal
tools in the process of inflammation during the fight
against pathogenic microorganisms, bacteria, and tumor
cells.9,10
However, excessive or inappropriate ROS and RNS
production by phagocytes is associated with oxidative
damage to membrane lipids, DNA, proteins, and lipoproteins,
resulting in various autoimmune and inflammatory
diseases. Thus, the modulation of inflammation and oxidative
stress by natural substances can be beneficial.
The complement cascade is another important part of the
innate immune defense. Therefore, an activation of the
complement system contributes to inflammatory responses
and immunological defense reactions. As reported before,
interaction with the complement system of polysaccharides
due to fixation could be a good therapeutic strategy for
treating inflammatory diseases.11,12
Manuscript received 14 September 2012. Revision accepted 3 May 2013.
Address correspondence to: Antonin Lojek, PhD; Institute of Biophysics, Academy
of Sciences of the Czech Republic Kralovopolska 135, Brno 612 65, Czech Republic,
E-mail: alojek@ibp.cz
JOURNAL OF MEDICINAL FOOD
J Med Food 16 (8) 2013, 711–718
# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2012.0234
711
Leek (along with onion and garlic) is part of the traditional
Balkan diet, and its medicinal action has been known
since ancient times. So far, there is scarce data about the
chemical composition of leek polysaccharides and their biological
activity,13–15
but it was established that some
polysaccharides are promising stimulators of some components
of the immune system. That provoked our interest to
conduct a more extensive study on the effects of leek pectic
polysaccharides obtained through different extraction
methods on the production of ROS and RNS by phagocytes.
Taken together, the complement system and phagocytes
are both recruited by activation processes. Therefore, the
main aim of the present study was to investigate the effects of
pectic polysaccharides isolated from leek on ROS and NO
production by phagocytes as well as on complement activity.
MATERIALS AND METHODS
Preparation of leek polysaccharides
Fresh leek was purchased from the local market in
Plovdiv, Bulgaria. Polysaccharides 1 and 2 (PS1 and PS2,
respectively) were obtained from fresh leek by the following
procedure: 500 g of leek was homogenized and mixed with
250 mL 0.5% HCl and 250 mL 0.5% citric acid (for PS1) or
500 mL 0.5% HCl (for PS2). The mixtures were incubated
with stirring for 1 h at 80°C and centrifuged (4400 g,
30 min). The supernatants were removed, coagulated with
96% ethanol (1/1.5; v/v), and left at room temperature for
1 h. The coagulated polysaccharides were then separated by
centrifugation (4400 g, 15 min). Separated polysaccharides
were washed consecutively with 70% ethanol and 96%
ethanol and dried at 60°C in a laboratory drier.
Polysaccharides 3 and 4 (PS3 and PS4, respectively) were
obtained by the following procedure: fresh leek (1kg) was cut
into small pieces of 8–10 mm and treated with 2500 mL 96%
ethanol preheated to 65°C. The obtained mixture was kept for
1 h at 65°C followed by 24h at room temperature, and then
filtered through cheese-cloth, after which, the insoluble material
was washed with 96% ethanol. The obtained alcohol
insoluble substances (AIS) were dried at 60°C. Fifty grams of
dried AIS was treated with 1250mL water at 80°C for 1 h and
then filtered. The filtrate was processed to obtain water extractable
pectic PS3. The residue was further extracted with
1000 mL 0.5% HCl for 1 h under the same conditions. The
mixture was filtered through cheese-cloth, and the filtrate
represents the crude acid extractable pectic PS4. The two
obtained aqueous filtrates were coagulated using an equal
volume of 96% ethanol and left for an hour. The coagulated
crude pectins were separated by filtration, washed with
100 mL 70% acidified ethanol (0.5% HCl), then with 70%
ethanol to a neutral pH, and finally with 100mL 96% ethanol.
They were dried at 60°C in a laboratory drier.
Determination of polyuronic content and degree
of esterification of pectic polysaccharides
The polyuronic content and degree of esterification were
determined by the method of Owens.16
Determination of molecular weight
Molecular weights of the polysaccharides were assayed
through high-performance size exclusion chromatography
(HPSEC) on a Waters (Millipore) system as described by
Kratchanova et al.15
Cell culture
The murine peritoneal macrophage cell line RAW 264.7
(American Type Culture Collection) was cultivated in
Dulbecco’s modified Eagle’s medium (PAN) and supplemented
with 10% of fetal bovine serum (PAN). Cells were
maintained at 37°C, 5% CO2.
Whole blood phagocyte-derived ROS production
Heparinized (50 IU/mL) blood samples were obtained
from the cubital vein of healthy human volunteers after
overnight fasting. The kinetics of ROS production by blood
phagocytes was analyzed by luminol-enhanced chemiluminescence
(CL) for a period of 60 min, using a microtiter plate
Luminometer LM-01T (Immunotech). For activation of ROS
production, opsonized zymosan particles (OZP) or phorbol
myristate acetate (PMA; Sigma-Aldrich) were used. The
detailed procedure is described elsewhere.17
Spontaneous CL
measurements (without any activator) were included in each
assay. The recorded values included the intensity of CL
emitted during the time interval studied (integral of the CL).
Measurement of nitrite concentration by Griess reaction
The accumulation of nitrites in a medium was measured
spectrophotometrically according to Pekarova et al.10
Cells
were incubated in 12-well plates at 1 · 106
cells/well for 24 h
without/with 0.1 lg/mL lipopolysaccharide (LPS, Escherichia
coli serotype 0111.B4; Sigma-Aldrich) and pectic polysaccharides
at 37°C, 5% CO2. At the end of the incubation period,
culture media were collected from wells and centrifuged at
5000 g, 4°C for 5 min. Then, 150 lL of supernatant was mixed
with an equal volume of Griess reagent (Sigma) in a 96-well
plate, and the mixture was incubated at room temperature and
in the dark for 30 min. The absorbance was measured at
546 nm. Sodium nitrite was used as the standard.
Western blot analysis of inducible nitric oxide synthase
expression
After removing the supernatant for the nitrite measurement,
the remaining cells were washed with cold phosphatebuffered
saline and lysed in the lysis buffer (1% sodium
dodecyl sulfate, 10- 1
M Tris pH 7.4, 10% glycerol, 10- 3
M
sodium ortho-vanadate, 10- 3
M phenylmethanesulfonyl
fluoride). Protein concentrations were determined using
BCAÔ protein assay (Pierce), with bovine serum albumin
as a standard. The equal amounts of protein were then
subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis, using 7.5% running gel. The expression of
inducible nitric oxide synthase (iNOS) protein was quantified
by Western blot analysis, as described previously.18
712 NIKOLOVA ET AL.
Anti–iNOS/NOS Type II mouse monoclonal antibody (BIORAD;
1:5000) and ECLÔ anti–mouse IgG horseradish
peroxidase linked whole antibody (from sheep; Biosciences;
1:2000) were used. The immunoreactive bands were detected
using an ECLÔ detection reagent kit (Pierce) and
exposed to radiographic film (AGFA). Equal loading of
proteins was confirmed by determination of b-actin. Relative
protein levels were quantified by scanning densitometry
using the ImageJÔ program, and the individual
band density value was expressed in arbitrary units.
Antioxidant activity determination
The antioxidant activity of the investigated polysaccharides
was investigated by total peroxyl radical-trapping antioxidant
parameter (TRAP) and oxygen radical absorbance
capacity (ORAC) assays.19,20
Complement fixation assay
The test is based on the colorimetric measurement of
hemoglobin released from target erythrocytes after incubation
with normal human serum.21
The detailed procedure is
described elsewhere.15
Statistical analysis
The results are presented as mean from at least three independent
experiments – standard error of mean (SEM).
Experiments were realized in duplicates or triplicates.
Comparisons with the control were performed by analysis of
variance (ANOVA), followed by the Newman–Keuls Post
hoc test. p values of less than .05 and .01 were considered
significant.
RESULTS
Characteristics of leek pectic polysaccharides,
isolated by different extraction methods
The yield and characteristics of the isolated pectic polysaccharides
from leek are presented in Table 1. It is evident
that extraction conditions lead to different characteristics of
the extracted pectins. Generally, pectic polysaccharides
extracted from dry AIS are distinctive, with a lower degree
of esterification when compared to the polysaccharides obtained
from fresh leek. Water extracted PS3 has the highest
polyuronic content - 72.8%, and the highest molecular
weight - 8.8 · 105
Da.
The effect of pectic polysaccharides on ROS production
Phagocytes play a key role in host defense against invading
pathogens, and play a crucial role in inflammatory
processes. In our study, the effect of pectic polysaccharides
on ROS production by whole blood neutrophils was analyzed
using luminol-enhanced CL. Typical kinetic curves of
spontaneous neutrophil CL and neutrophil CL activated
with receptor-bypassing (PMA) and receptor-operating
(OZP) stimuli are shown in Figure 1. Spontaneous CL
represents the basal production of ROS in nonactivated
neutrophils. On the other hand, the activation of neutrophils
with both PMA and OZP induced a significant increase in
ROS production. The pectic polysaccharides in our study
decreased both spontaneous and PMA- or OZP-activated
ROS production in a 0.1 mg/mL concentration (Table 2).
This effect was expressed even more significantly when a
concentration of 0.5 mg/mL was used.
The effect of pectic polysaccharides on nitrite production
and iNOS expression
In our previous experiments,18
the highest concentration
of nitrites in a medium was observed in control RAW 264.7
cells after their 1-day stimulation with LPS, and this
schedule was chosen for the experiments. Figure 2 demonstrates
that while PS1 and PS2, in both concentrations, do
not activate resting macrophages (not treated with LPS) to
produce nitrites, PS3 at the concentration of 0.1 mg/mL
markedly activated macrophages for nitrite production. This
effect became significant ( p < .01) when 0.5 mg/mL PS3
was used. PS4 was even more effective activating macrophages
very significantly ( p < .01) in a 0.1 mg/mL concentration,
with a further activation increase in a 0.5 mg/mL
concentration. Interestingly, none of the tested pectic polysaccharides
influenced the production of nitrites in macrophages
treated with LPS (data not shown). The possibility
Table 1. Yield and Characteristics of Pectic Polysaccharides from Leek Obtained by Different Extraction Methods
HPSEC fractionation of polysaccharides
Material Extragent
Polyuronic
content (%)
Degree of
esterification (%) % Molecular weight (Da) Average molecular weight (Da)
PS1 Fresh leek 0.5% HCl + 0.5%
citric acid
48.8 67.4 80.4 8.5 · 105
6.8 · 105
19.6 3.3 · 104
PS2 Fresh leek 0.5% HCl 49.0 73.1 71.4 8.4 · 105
6.0 · 105
28.6 2.2 · 104
PS3 AIS Water 72.8 56.3 100 8.8 · 105
8.8 · 105
28.7 8.8 · 105
PS4 AIS 0.5% HCl 49.3 52.2 52.0 1.2 · 105
3.2 · 105
19.3 3.8 · 104
HPSEC, high-performance size exclusion chromatography; PS1–PS4, polysaccharides 1–4.
EFFECTS OF LEEK POLYSACCHARIDES ON PHAGOCYTES 713
that the changes in nitrite concentration induced with PS3
and PS4 were associated with changes in iNOS protein expression
was determined using Western blot analysis (Fig.
3). In comparison with the iNOS protein level in the control
macrophages without LPS treatment, iNOS protein expression
was stimulated with PS3 and PS4 in a manner very
similar to the increase in nitrite concentrations in the cultured
media shown in Figure 2. The expression of iNOS
protein was not changed in LPS-treated macrophages, which
also corresponds with our data on NO production.
Scavenging properties against the peroxyl radical or NO
The results of some authors (e.g., Kaur et al.22
) suggest
that polysaccharides isolated from plants may play important
roles in free radical scavenging. Therefore, we analyzed
whether the antioxidant properties of polysaccharides
against ROS and NO can interfere with changes in phagocytederived
ROS and NO production. All assays used (TRAP,
ORAC, and direct amperometrical analysis) confirmed that
none of the tested pectic polysaccharides exerted significant
scavenging properties against the peroxyl radical or NO
(data not shown).
The effect of pectic polysaccharides on the complement
system
The polysaccharides were tested for their ability to fix
complement. As shown in Table 3, PS1, PS2, and PS4
showed a weak complement-fixing effect, whereas the addition
of pectic PS3 resulted in dose-dependent erythrocyte
hemolysis, indicating that this fraction exhibited potent
complement-fixing activity. The effect was more profound
via the alternative pathway of complement activation.
Therefore, the fixation of complement by this polysaccharide
can be proposed as a potential therapeutic strategy for
treating inflammatory diseases.
DISCUSSION
One of the most promising recent alternatives to classical
medical treatment is the use of immunomodulators for enhancing
host defense responses. In recent decades, polysaccharides,
including pectic substances isolated from plant
sources, have attracted a great deal of attention because of
their broad spectrum of therapeutic properties and relatively
low toxicity.4,5
While our understanding of the mechanism
of action of these substances is still developing, it appears
that one of the primary mechanisms involves modulation of
the phagocyte and the complement activity.23
In our study,
we investigated the effects of pectic polysaccharides isolated
from leek on ROS and NO production by neutrophils
and macrophages, as well as on the complement activity.
Only limited information exists on the macrophage immunomodulatory
activity of pectic polysaccharides; and, to
our knowledge, there are no reports regarding the effects of
FIG. 1. Kinetics of luminol-enhanced spontaneous and activated
chemiluminescence (CL) of human whole blood neutrophils. The
results are expressed in relative light units (RLU).
FIG. 2. The effect of pectic polysaccharides on NO production by
nonstimulated murine RAW 264.7 macrophages. Data are expressed
as mean – SEM from three independent experiments. **Indicates
significance at level P < .01 when compared with a relevant control.
PS1–4, polysaccharides 1–4; NO, nitric oxide; SEM, standard error of
the mean.
Table 2. Effect of Tested Polysaccharides on Spontaneous
and Activated Chemiluminescence of Human Neutrophils
Concentration
(mg/mL)
Spontaneous
CL
PMAactivated
CL
OZP-
activated
CL
PS1 0.1 76.0 – 11.0 75.3 – 8.8* 78.5 – 3.8**
0.5 60.7 – 11.2* 65.1 – 12.5* 75.2 – 3.1**
PS2 0.1 77.1 – 8.1* 75.9 – 3.6** 85.5 – 4.2*
0.5 59.3 – 10.4* 65.3 – 13.5* 76.5 – 3.4**
PS3 0.1 79.3 – 7.6 80.8 – 3.8** 78.7 – 4.0**
0.5 69.6 – 10.1* 79.9 – 7.0* 72.1 – 2.4**
PS4 0.1 78.6 – 12.4 80.8 – 6.2* 85.5 – 4.4*
0.5 69.0 – 10.9* 71.6 – 9.4* 85.5 – 4.4**
The data are expressed as % of respective controls and represent
mean – SEM, n = 3.
Asterisks indicate significant differences at **P £ .01 or *P £ .05 levels
when compared to respective control values (cells incubated without
lipopolysaccharide [LPS]).
PMA, phorbol myristate acetate; OZP, opsonized zymosan particles; CL,
chemiluminescence; SEM, standard error of the mean.
714 NIKOLOVA ET AL.
leek polysaccharides on neutrophils. However, in contrast
with studies by other authors, where the stimulation effects
of some polysaccharides on neutrophils is reported,8,24–26
the pectic polysaccharides in our study decreased both
spontaneous and PMA- or OZP-activated ROS production
in a concentration-dependent manner. The results of some
authors22
suggest that polysaccharides isolated from plants
may play an important role in ROS scavenging. Schepetkin
et al.27
found that Opuntia polyacantha polysaccharide
fractions exhibited ROS scavenging activity, with the high
molecular weight fractions being the most active. In our
study, both TRAP and ORAC assays confirmed that none of
the tested pectic polysaccharides exerted antioxidant properties
against peroxyl radicals, proving that the decrease in
neutrophil-derived CL is not caused by the antioxidant
properties of the tested polysaccharides.
It is well established that a wide range of plant polysaccharides
exhibit beneficial pharmacological effects via their
ability to modulate macrophage function, including the
production of NO by iNOS.23
The macrophage-modulating
activity of polysaccharides toward NO production has been
reported for polysaccharides isolated from different sour-
ces,8,25,26,28
but very little is known about the structural
characteristics of the polysaccharides that determine this
activity. Schepetkin et al.27
proposed that the macrophageimmunomodulatory
activity of Opuntia polysaccharides is
positively correlated with the average molecular weight of
the polysaccharides. The authors suggested that polysaccharides
may activate macrophages via receptor(s) or other
surface structures, although the nature of these surface targets
is currently unknown. Contrary to these observations, in
our study, polysaccharide PS4 exhibited the most potent
macrophage-modulatory properties, by activating macrophages
to produce NO, even when a concentration of
0.1 mg/mL was used (Fig. 2). HPSEC analysis showed that
this acid-extractable polysaccharide is heterogeneous, with
an average molecular weight of 3.2 · 105
Da, which is the
lowest, compared to other polysaccharides isolated from
leek. The homogeneous PS3 with the highest molecular
weight among leek pectic polysaccharides (8.8 · 105
Da)
also activated macrophages for NO production, although its
activity in a concentration of 0.1 mg/mL was lower compared
with PS4. At a concentration of 0.5 mg/mL, there was
no significant difference in the macrophage-modulatory
activity of the two polysaccharides tested. Our results indicate
that a high molecular weight is not the only prerequisite
for macrophage-immunomodulatory activity. There
are probably some structural characteristics that also determine
this activity, but further studies are necessary to understand
the relationship between the structure and
biological activity.
While interpreting results from an in vitro study, one has
to keep in mind that the absorption of plant polysaccharides
into the bloodstream after oral administration is not well
understood and is a disputable issue. Nevertheless, the
metabolic activity of neutrophils can be modulated by
polysaccharides directly in the intestinal lumen, especially
during inflammatory conditions. One of the challenges of
the immune system is to ensure, on one hand, a balance
between the protection of barrier surfaces from pathogens
FIG. 3. Densitometric analysis and representative
Western blot of iNOS protein expression in LPS-stimulated
RAW 264.7 cells treated with pectic polysaccharides.
The data represent mean – SEM from three
independent experiments. Asterisks indicate a significant
difference (**P < .01) when compared to the relevant
control value (cells incubated without LPS). iNOS,
inducible NO synthase; LPS, lipopolysaccharide.
Table 3. Complement-Fixing Activity of Polysaccharides
Inhibition of hemolysis [%]
Classical pathway Alternative pathway
Concentration of polysaccharides [lg/mL]
6000 3000 3000 1500 750
PS1 7.5 – 0.23a
0a
21.1 – 0.86a
5.4 – 0.46a
0a
PS2 6.8 – 0.69a
0a
8.5 – 0.34b
0b
0a
PS3 38.2 – 5.08b
0a
65.4 – 10.57c
54.9 – 8.32c
17.3 – 0.46b
PS4 21.4 – 2.6c
0a
14.8 – 4.97d
0b
0a
The data represent mean – SEM from three independent experiments.
abcd
Values marked with different superscript letters within individual
columns are significantly different (P £ .01).
EFFECTS OF LEEK POLYSACCHARIDES ON PHAGOCYTES 715
and, on the other, the establishment of a beneficial relationship
with commensal bacteria.29
In mucosal tissues, the
inflammatory response is central to effective host defense
against invading pathogens. The mucosal immune system
must be tightly regulated to prevent abnormal responses to
innocuous environmental antigens and commensal organisms,
which could result in an allergy or chronic inflammatory
diseases. Many intestinal inflammatory illnesses are
marked by neutrophil transepithelial migration and arrival in
the lumen, forming crypt abscesses. Here neutrophils not
only directly defend the surface, but also release biologically
active mediators and stimulate a secretory flush.30,31
Neutrophil migration across the intestinal epithelium and
their presence in the lumen enable pectins to interact directly
with the receptors on the surface of neutrophils.
The absorption of plant polysaccharides into the bloodstream
after oral administration is another possibility for
how pectins can influence the components of the immune
system, including neutrophils, monocytes/macrophages,
and the complement system. The majority of the intestine is
covered with a single layer of columnar epithelial cells,
which make up the absorptive surface for water and nutrient
absorption.30
While the intestinal barrier is permeable to
digested nutrients as well as fluids, it is, in general, impermeable
to macromolecules, particular antigens, and most
microorganisms. However, the immune system needs direct
contact with antigens or pathogens to generate specific
immune responses. For this purpose, the intestine provides
specialized epithelial cells, called M cells, which are responsible
for the uptake of antigens and microorganisms.
The most noticeable feature of M cells is their active
transport (by a phagocytic mode of transport that has yet to
be fully characterized) of a wide variety of inert material
from the gut lumen toward the follicles, from where particles
can migrate to the blood via the mesentery nodes and
the thoracic lymph duct.32
Because polysaccharides also
behave as hydrated nanoparticles in an aqueous solution, it
is postulated that plant polysaccharides become incorporated
into the lymphoid follicles of Peyer’s patches and
isolated lymphoid follicles by a mechanism similar to other
nanoparticles, resulting in their dissemination to systemic
circulation through the mesenteric lymph nodes and the
thoracic duct. Some experimental results suggest that oral
administration of certain immunostimulating polysaccharides
affects the gastric mucosal immune system through
Peyer’s patch cells. Otsuka et al.33
reported that peptide–
mannan complexes with a molecular weight of 60,000–
95,000, when orally administered to rats, reached the lymph
and the blood stream. Mitsuhashi et al.34
found that a-glucan
isolated from the liquid-cultured mycelium of Tricholoma
matsutakwith, with a molecular weight over 2.0 · 106
daltons administered as a 14
C-labeled derivative, was absorbed
in the digestive system and remained in the blood
stream even 168 h after administration. The peptide–mannan
complex, as well as a-glucan, was observed to accumulate
in the mesenteric lymph nodes and Peyer’s patches
as well as in the liver, spleen, and kidneys. Yunoki et al.35
reported the accumulation of the polysaccharides derived
from the basidiomycetes Coriolus versicolor in the liver of
mice after oral administration, whereas Sakurai et al.36
observed the same for the pectic polysaccharide bupleuran
2IIc, from Bupleurum falcatum, which was also accumulated
in Peyer’s patches. Otsuka et al.33
indicated that
peptide–mannan appeared in aortic blood more than 2 h
after it was detected in portal blood, suggesting that the
peptide–mannan, which was absorbed in the digestive system
was initially incorporated by the phagocytic system of
the liver, while the excess of peptide–mannan overflowed
into the systemic blood stream. It is assumed that the formation
of the immune complexes of the pectic polysaccharide-reacting
natural IgA antibody with the active pectic
polysaccharide in the intestinal fluid may not only participate
in effective incorporation of the active pectic polysaccharides
into Peyer’s patches, but may also activate
complement components that result in certain modulations
of the intestinal immune system.37
The complement can also meet pectins directly in the
epithelium or even in the intestinal lumen. In general, the
complement system plays an important role as a primary
defense against bacterial and viral infections, and appears to
be intrinsically associated with several immune reactions,
such as the chemotactic attraction of leukocytes, immune
adherence, the modulation of antibody production, and increased
local vascular permeability.37,38
According to the
study of Kiyohara et al.,37
the pectic polysaccharidereacting
natural and secretory IgA antibody exists in the
mucosal sites of the human intestine. Dimeric IgA has been
assumed to contribute to activating the alternative complement
pathway, producing some biologically active complement
fragments, and complement components such as C3
and C4, which are known to be produced by the epithelial
cells of the intestinal tract.39,40
It is also speculated that the
formation of immune complexes of the pectic polysaccharide-reacting
natural IgA antibody with the active pectic
polysaccharides in the intestinal fluid may activate complement
components in the fluid, resulting in certain modulations
of the intestinal immune system.
As reported by Gasque,41
complement component proteins
can be activated through three cascade pathways: the
classical pathway, the alternative pathway, and the antibody-independent
lectin pathway. In general, the alternative
and lectin pathways contribute to the early natural defense
mechanism of the host before production of the antibody. It
is known that some plant polysaccharides are good modulators
of the immune system, and their activity depends on
several factors, such as raw material, extraction conditions,
chemical composition, and the structure of the polysaccharide.
However, it is disputable which part of the pectic
macromolecule is responsible for their immunomodulating
properties. Most polysaccharides with immunomodulating
properties have been isolated from hot-water extracts of
medicinal herbs,5
and some authors suggest that a high
molecular weight and a higher content of galacturonic acids
are prerequisites for a higher complement-fixing ability.3
Our results confirm these observations, and show that PS3
obtained after water extraction of AIS reveals the highest
716 NIKOLOVA ET AL.
complement-fixing effect and is characterized by a high
content of uronic acids and a high molecular weight.
CONCLUSION
It is evident from the performed study that leek polysaccharides
modulate the immune system via different
mechanisms. Polysaccharides PS3 and PS4 isolated from
AIS exhibited potent macrophage-activating properties resulting
in increased generation of NO via the induction of
inducible NO synthase. Pectin PS3 obtained after water
extraction of AIS revealed the highest complement-fixing
effect. These findings make leek a valuable source of biologically
active polysaccharides, which could also be considered
for further medicinal applications. It was shown that
molecular weight is not the only thing that determines the
macrophage immunomodulatory activity of leek polysaccharides,
and further studies are required to investigate the
structural features in relation to the biological activity.
ACKNOWLEDGMENTS
This study was supported by grants IF-02-66/2007 (Bulgarian
Innovation Fund) and 524/08/1753 (Czech Science
Foundation).
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist. All authors report
no conflicts of interest.
REFERENCES
1. Voragen AGJ, Pilnik W, Thibault JF, Axelos MAV, Renard
CMCG: Pectins. In: Food Polysaccharides and Their Applications
(Stephen AM, ed.). Marcel Dekker Inc., New York/Basel/
HongKong, 1995, pp. 287–339.
2. Silva DC, Freitas AL, Pessoa CD, et al.: Pectin from Passiflora
edulis shows anti-inflammatory action as well as hypoglycemic
and hypotriglyceridemic properties in diabetic rats. J Med Food
2011;14:1118–1126.
3. Srivastava R, Kulshreshtha D: Bioactive polysaccharides from
plants. Phytochemistry 1989;28:2877–2883.
4. Paulsen BS: Biologically active polysaccharides as possible lead
compounds. Phytochem Rev 2002;1:379–387.
5. Yamada H, Kiyohara H: Immunomodulating activity of plant
polysaccharide structures. In: Comprehensive Glycoscience:
From Chemistry to Systems Biology (Kamerling JP, ed.). Elsevier,
Oxford, 2007, pp. 663–694.
6. Wang JH, Luo JP, Yang XF, Zha XQ: Structural analysis of a
rhamnoarabinogalactan from the stems of Dendrobium nobile
Lindl. Food Chem 2010;122:572–576.
7. Belska NV, Guriev AM, Danilets MG, et al.: Water-soluble
polysaccharide obtained from Acorus calamus L. classically activates
macrophages and stimulates Th1 response. Int Immunopharmacol
2010;10:933–942.
8. Xie G, Schepetkin IA, Quinn MT: Immunomodulatory activity of
acidic polysaccharides isolated from Tanacetum vulgare L. Int
Immunopharmacol 2007;7:1639–1650.
9. Teruya T, Takeda S, Tamaki Y, Tako M: Fucoidan isolated from
Laminaria angustata var. longissima induced macrophage activation.
Biosci Biotechnol Biochem 2010;74:1960–1962.
10. Pekarova M, Kralova J, Kubala L, et al.: Continuous electrochemical
monitoring of nitric oxide production in murine macrophage cell line
RAW 264.7. Anal Bioanal Chem 2009;394:1497–1504.
11. Nergard CS, Diallo D, Michaelsen TE, et al.: Isolation, partial
characterisation and immunomodulating activities of polysaccharides
from Vernonia kotschyana Sch. Bip. ex Walp. J Ethnopharmacol
2004;91:141–152.
12. Togola A, Inngjerdingen M, Diallo D, et al.: Polysaccharides
with complement fixing and macrophage stimulation activity
from Opilia celtidifolia, isolation and partial characterisation. J
Ethnopharmacol 2008;115:423–431.
13. Schols HA, Voragen AGJ: Occurrence of pectic hairy regions in
various plant cell wall materials and their degradability by
rhamnogalacturonase. Carbohyd Res 1994;256:83–95.
14. Kratchanova M, Gocheva M, Pavlova E, et al.: Characteristics of
pectic polysaccharides from leek obtained through consecutive
extraction with various reaction agents. Bul Chem Commun
2008;40:561–567.
15. Kratchanova M, Nikolova M, Pavlova E, Yanakieva I, Kussovski
V: Composition and properties of biologically active pectic
polysaccharides from leek (Allium porrum). J Sci Food Agric
2010;90:2046–2051.
16. Owens HS: Methods Used at Western Regional Research Laboratory
for Extraction and Analysis of Pectic Materials. Bureau
of Agricultural and Industrial Chemistry, Agricultural Research
Administration, U.S. Department of Agriculture, Sacramento, CA,
1952.
17. Lojek A, Kubala L, Cizova H, Ciz M: A comparison of whole
blood neutrophil chemiluminescence measured with cuvette and
microtitre plate luminometers. Luminescence 2002;17:1–4.
18. Ambrozova G, Pekarova M, Lojek A: The effect of lipid peroxidation
products on reactive oxygen species formation and
nitric oxide production in lipopolysaccharide-stimulated RAW
264.7 macrophages. Toxicol In Vitro 2011;25:145–152.
19. Ciz M, Cizova H, Denev P, Kratchanova M, Slavov A, Lojek A:
Different methods for control and comparison of the antioxidant
properties of vegetables. Food Control 2010;21:518–523.
20. Denev P, Ciz M, Ambrozova G, Lojek A, Yanakieva I, Kratchanova
M: Solid-phase extraction of berries’ anthocyanins and
evaluation of their antioxidative properties. Food Chem
2010;123:1055–1061.
21. Klerx JP, Beukelman CJ, Van Dijk H, Willers JM: Microassay
for colorimetric estimation of complement activity in guinea pig,
human and mouse serum. J Immunol Methods 1983;63:215–220.
22. Kaur G, Hamid H, Ali A, Alam MS, Athar M: Antiinflammatory
evaluation of alcoholic extract of galls of Quercus infectoria. J
Ethnopharmacol 2004;90:285–292.
23. Schepetkin IA, Quinn MT: Botanical polysaccharides: macrophage
immunomodulation and therapeutic potential. Int Immunopharmacol
2006;6:317–333.
24. Hajkova V, Svobodova A, Krejcova D, et al.: Soluble glucomannan
isolated from Candida utilis primes blood phagocytes.
Carbohyd Res 2009;344:2036–2041.
25. Schepetkin IA, Faulkner CL, Nelson-Overton LK, Wiley JA,
Quinn MT: Macrophage immunomodulatory activity of polysaccharides
isolated from Juniperus scopolorum. Int Immunopharmacol
2005;5:1783–1799.
EFFECTS OF LEEK POLYSACCHARIDES ON PHAGOCYTES 717
26. Xie G, Schepetkin IA, Siemsen DW, Kirpotina LN, Wiley JA,
Quinn MT: Fractionation and characterization of biologicallyactive
polysaccharides from Artemisia tripartita. Phytochemistry
2008;69:1359–1371.
27. Schepetkin IA, Xie G, Kirpotina LN, Klein RA, Jutila MA,
Quinn MT: Macrophage immunomodulatory activity of polysaccharides
isolated from Opuntia polyacantha. Int Immunopharmacol
2008;8:1455–1466.
28. Cheng A, Wan F, Wang J, Jin Z, Xu X: Macrophage immunomodulatory
activity of polysaccharides isolated from Glycyrrhiza
uralensis Fish. Int Immunopharmacol 2008;8:43–50.
29. Fritz JH, Le Bourhis L, Magalhaes JG, Philpott DJ: Innate immune
recognition at the epithelial barrier drives adaptive immunity:
APCs take the back seat. Trends Immunol 2008;29:41–49.
30. Kelsall BL: Innate and adaptive mechanisms to control [corrected]
pathological intestinal inflammation. J Pathol 2008;214:
242–259.
31. Chin AC, Parkos CA: Neutrophil transepithelial migration and
epithelial barrier function in IBD: potential targets for inhibiting
neutrophil trafficking. Ann NY Acad Sci 2006;1072:276–287.
32. Hussain N, Jaitley V, Florence AT: Recent advances in the understanding
of uptake of microparticulates across the gastrointestinal
lymphatics. Adv Drug Deliv Rev 2001;50:107–142.
33. Otsuka H, Maeda H, Yamashita A: Absorption of antitumor
peptidomannan KS-2, via lymphatic and portal vein after oral
administration. Jap J Cancer Chemother 1981;8:1570–1576.
34. Mitsuhashi S, Hoshi H, Matsunaga K: Absorption, distribution
and excretion of a functional foodstuff (Kureha M6271)
derived from mycelia of a basidiomycete Tricholoma matsutake,
after oral administration. J New Rem Clin 2003;52:
840–852.
35. Yunoki S, Tanaka N, Hizuta A, Orita K: Enhancement of antitumor
cytotoxicity of hepatic lymphocytes by oral administration
of PSK. Int J Immunopharmacol 1994;16:123–130.
36. Sakurai MH, Matsumoto T, Kiyohara H, Yamada H: Detection
and tissue distribution of anti-ulcer pectic polysaccharides from
Bupleurum falcatum by polyclonal antibody. Planta Med
1996;62:341–346.
37. Kiyohara H, Matsumoto T, Nagai T, Kim SJ, Yamada H: The
presence of natural human antibodies reactive against pharmacologically
active pectic polysaccharides from herbal medicines.
Phytomedicine 2006;13:494–500.
38. Inngjerdingen KT, Coulibaly A, Diallo D, Michaelsen TE,
Paulsen BS: A complement fixing polysaccharide from Biophytum
petersianum Klotzsch, a medicinal plant from Mali, West
Africa. Biomacromolecules 2006;7:48–53.
39. Andoh A, Fujiyama Y, Bamba T, Hosoda S: Differential cytokine
regulation of complement C3, C4, and factor B synthesis in
human intestinal epithelial cell line, Caco-2. J Immunol
1993;151:4239–4247.
40. Michael W, Russell MK, Michael EL: Biological activities of
IgA. In: Mucosal Immunology, second ed. (Ogra PL, Mestecky J,
Lamm ME, Strober W, Brienenstock J, McGhee JR, eds.).
Academic Press, New York, 1999, pp. 225–240.
41. Gasque P: Complement: a unique innate immune sensor for
danger signals. Mol Immunol 2004;41:1089–1098.
718 NIKOLOVA ET AL.
International Journal of Biological Macromolecules 105 (2017) 730–740
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Acidic polysaccharide complexes from purslane, silver linden and
lavender stimulate Peyer’s patch immune cells through innate and
adaptive mechanisms
Yordan N. Georgieva,b
, Manol H. Ognyanova,b
, Hiroaki Kiyoharac
,
Tsvetelina G. Batsalovad
, Balik M. Dzhambazovd
, Milan Cize
, Petko N. Deneva,b
,
Haruki Yamadac
, Berit S. Paulsenf
, Ondrej Vasiceke
, Antonin Lojeke
, Hilde Barsettf
,
Daniela Antonovag
, Maria G. Kratchanovaa,b,∗
a
Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139 Ruski
Blvd., 4000 Plovdiv, Bulgaria
b
Innovative-Technological Center Ltd., 20 Dr. G. M. Dimitrov Str., 4000 Plovdiv, Bulgaria
c
Department of Drug Discovery Science, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, 108-8641 Tokyo, Japan
d
Department of Developmental Biology, Plovdiv University Paisii Hilendarski, 24 Tsar Assen Str., 4000 Plovdiv, Bulgaria
e
Department of Free Radical Pathophysiology, Institute of Biophysics, Czech Academy of Sciences, 135 Kralovopolska, 612 65 Brno, Czech Republic
f
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway
g
Laboratory of Experimental Chromatography and Mass Spectrometry, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy
of Sciences, Acad. G. Bonchev Str. Bl. 9, 1113 Sofia, Bulgaria
a r t i c l e i n f o
Article history:
Received 9 May 2017
Received in revised form 13 July 2017
Accepted 14 July 2017
Available online 19 July 2017
Keywords:
Purslane
Lavender
Silver linden
Immunomodulating polysaccharides
Immune cells
Peyer’s patches
a b s t r a c t
Three polysaccharide complexes (PSCs) were isolated from the aerial parts of common purslane (Portulaca
oleracea L.), and the flowers of common lavender (Lavandula angustifolia Mill.) and silver linden
(Tilia tomentosa Moench) by boiling water extraction and ethanol precipitation. The chemical composition
and immunomodulating effects of isolated PSCs were characterized. The chemical characterization
revealed that the three samples contain mainly pectic polysaccharides. They exhibited ex vivo intestinal
immunomodulating activity through the murine Peyer’s patch-mediated bone marrow cell proliferation
test at 100 ␮g/ml concentration. At the same time, they stimulated ex vivo human blood T-cell populations
(CD4+
/CD25+
and CD8+
/CD25+
), phagocytic leukocytes (CD14+
and CD64+
cells) and induced IL-6
production from human white blood cells and Peyer’s patch cells. The herbal PSCs stimulated ex vivo ROS
production from whole blood phagocytes and showed unspecific in vitro anti-proliferative activity against
normal and A549, HeLa and LS180 tumor cells. This is the first report on immunomodulating studies of
linden flower pectins and chemical and biological activity characterization of lavender polysaccharides.
Our study demonstrates that similarly to purslane, lavender and silver linden herbal materials contain
immunomodulating polysaccharides that could be useful for support of compromised immune system.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
Herbal medicine is a symbol of human knowledge, because it has
been accompanying every nation through its development and people
will always prepare herbal infusions, decoctions and extracts
∗ Corresponding author at: Laboratory of Biologically Active Substances, Institute
of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,
139 Ruski Blvd., 4000 Plovdiv, Bulgaria.
E-mail address: lbas@plov.omega.bg (M.G. Kratchanova).
[1]. The thorough research in the last 30 years in the Chinese
and especially the Japanese Kampo medicine, traditional European,
African and Indian Ayurvedic medicines has shown that herbal pectic
polysaccharides contribute to the biological activity of widely
used herbs [2–5]. Already in 1988, polysaccharide hydrocolloids
were included in the German Rote Liste
®
as antitussive, antipyretic,
wound healing, nutraceutical, laxative, immune stimulating and
gastric agents [6]. Furthermore, examples of commercial products
containing immunomodulating polysaccharides are modified
citrus pectin, Bazoton
®
, Viscum album extract, Astragalus polysac-
http://dx.doi.org/10.1016/j.ijbiomac.2017.07.095
0141-8130/© 2017 Elsevier B.V. All rights reserved.
Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740 731
charide extract and Kampo medicine formulations Juzen-taiho-to
and Hochuekkito [7–10].
Generally, pectic polysaccharides contain three main building
blocks − unbranched homogalacturonan (HG), ramified rhamnogalacturonan
(RG) I and II [11]. The unbranched HG is composed
of an ˛-(1 → 4)-linked poly-d-GalpA chain, which COOH groups
are methyl-esterified to different extent and some OH groups
are acetylated at C-2 and/or C-3. The ramified RGI has a
backbone structure of the linear repeating fragment [→2)-˛-lRhap-(1
→ 4)-˛-d-GalpA-(1 → ]n. The main fragment carries side
chains of ˛-(1 → 5)-l-arabinans, ˇ-(1 → 4)-d-galactans, arabinogalactan
(AG) type I and II at the OH group of C-4 of the L-Rha
residues. The AGI is composed of a linear ˇ-d-(1 → 4)-linked galactan,
which is branched at O-3 mainly with ˛-l-(1 → 5)-linked Araf
residues. The AG II is composed of a ˇ-d-(1 → 3)-linked galactan
backbone with side chains at O-6 of ˇ-(1 → 6)-linked galactans. 1,3and
1,6-galactans are substituted mainly with ˛-l-(1 → 3)-linked
Araf residues. The glycosidic coupling of L-Ara in the AG structures
is highly diverse (1,2; 1,3; 1,5) [2,4,11].
The most important immune active pectic side chains are
AGs [2,4]. The immunomodulating effects of herbal pectins in
the gastrointestinal tract expand a lot the classical concept for
dietary fibers and human health. The pectic polysaccharides from
herbal infusions, traditional foods and drinks pass easily through
the gastrointestinal tract, where they can interact with mucosal
immune system. Stimulation of the mucosal immune system
through immunocompetent cells in Peyer’s patches by polysaccharides
has shown perspective application for supplementary
treatment of infectious diseases [10,12–14]. The Peyer’s patches
contain B-, T-cells and phagocytic leukocytes, such as macrophages,
dendritic cells and neutrophils, which can be affected by the
pectic polysaccharides [2,3,15]. The pectins activate oxidative
burst and phagocytosis of neutrophils and macrophages, thus
influencing the innate immune response [16]. Furthermore, the
activated macrophages and dendritic cells could stimulate adaptive
immune cells [17,18]. Direct or indirect immunomodulating effect
of polysaccharides on T-cell subpopulations can be used for prevention
and treatment of tumor and autoimmune diseases [19,20].
Silver linden (Tilia tomentosa) and common lavender (Lavandula
angustifolia), together with the edible weed common purslane
(Portulaca oleracea) are traditionally used in the herbal medicine
[21]. Bulgaria is a major exporter of wild and cultivated aromatic
medicinal plants and their derived products, which are raw materials
for the pharmaceutical, cosmetic and nutraceutical industries.
It is the biggest lavender oil producer in the world since 2011
and leading country in the export of Tiliae flos. Interestingly, T.
tomentosa forms coenoses only in the Balkans and although it is
widely used in the gemmotherapy, it is still poorly studied [22].
On the other hand, the medicinal use of lavender is focused on its
essential oil. The third herb, purslane is a very popular leaf vegetable
in the Indian and Mediterranean cuisine, which is named
the “global panacea” by WHO [23]. The immunomodulating activity
of the three herbs is related mainly to the lavender essential
oil, linden tiliroside, scopoletin and lignans, and only for purslane
to polysaccharides [24,25,23]. The purslane contains acidic pectic
polysaccharides and neutral AGs, arabinoglucomannans and starch
[26–28]. It is shown that purslane polysaccharides have potential
to be used in the treatment of tumor diseases because they
exhibit immune-enhancing activities, but studies on their intestinal
immunomodulating activity are missing [19,29,30]. Kram &
Franz [31] reported that small-leaved linden Tilia cordata flower
mucilage is rich in pectin, as later Schmidgall et al. [32] showed
that its raw polysaccharide complex expresses moderate bioadhesion
to epithelial tissue ex vivo. To our knowledge investigations
on the chemical composition and biological activity of silver linden
and lavender pectic polysaccharides are absent.
The aim of the current study was to isolate and characterize the
chemical composition and immunomodulating effects of polysaccharide
complexes from L. angustifolia and T. tomentosa, and to
compare their effects with the known bioactive polysaccharide
complex from P. oleracea.
2. Materials & methods
2.1. Plant material
Common purslane aerial parts were collected from natural habitats
in September 2013 in Plovdiv region, washed, cut into small
pieces, freeze-dried and stored in a desiccator until use. The plant
material was identified as Portulaca oleracea L. (Portulacaceae) by
Assoc. Prof. Dr. Ivanka Dimitrova-Dyulgerova (Plovdiv University
Paisii Hilendarski). Lavender flowers Lavandula angustifolia Miller
(syn. L. vera) sort “Sevtopolis” (Institute of Roses, Essential and Medical
Cultures, Kazanlak, Bulgaria) were provided dried by ET “Ve Pe
Pi – Vesko Pipev” (Velingrad, Bulgaria). Linden flowers were collected
in June 2013 in Plovdiv, air-dried for two weeks and stored
in a desiccator until use. The plant material was identified as silver
linden, Tilia tomentosa Moench (Tiliaceae), by Dr. Evgeny Tsavkov
(University of Forestry, Sofia). A voucher specimen with given number
SOA061426 was deposited in the Herbarium of the Agricultural
University of Plovdiv.
2.2. Isolation of the herbal PSCs
Preparation of the herbal PSCs was performed partly as the
Japanese Kampo medicine decoctions are prepared [33]. Thirty
grams of each milled herbal material were mixed with 600 ml
water and boiled until the mixture volume was reduced to half.
The extracts were filtered through a nylon cloth and the plant
residue was rinsed with 150 ml water and re-boiled with 600 ml
water again, following the same procedure. The collected extracts
and washes were centrifuged (4000 rpm, 20 min), filtered through
a Büchner funnel and concentrated four times in volume under
vacuum at 50 ◦C. The plant extracts were centrifuged again and
coagulated with 4 vol. 95% ethanol at 4 ◦C over night. The obtained
precipitates were collected by a Büchner funnel separation for
linden and purslane PSCs (linden PSC and purslane PSC) or centrifugation
(4000 rpm, 30 min, 4 ◦C) for lavender PSC. Then, the three
crude polysaccharides were re-dissolved in water, dialyzed for 72 h
against water at 4 ◦C (VISKING
®
, SERVA Electrophoresis, MWCO
12–14 kDa), centrifuged, freeze-dried and stored in a desiccator in
dark until use.
2.3. General methods
The polyuronic acid content (PUC) in the plant materials was
determined titrimetrically, according to Owens et al. [34]. The
anhydrouronic acid content (AUAC) and simultaneous determination
of GalA and GlcA (in the absence of 4-O-methyl-GlcA)
in the herbal PSCs were measured colorimetrically by the mhydroxydiphenyl
(GalA as a standard) and 3,5-dimethylphenol
(GalA and d-glucuronolactone as standards) methods [35,36].
Degree of methoxylation (DM) was analyzed with alcohol oxidase
(P. pastoris, Sigma-Aldrich) and Purpald
®
(Sigma-Aldrich) [37].
Degree of acetylation (DA) was assayed by the method of МcComb
& McCready [38], using ˇ-d-glucose pentaacetate as a standard.
Molecular weight was determined by HPSEC-RID (Waters) with
Shodex standard P-82 kit (pullulan standards, Showa DENKO,
Japan) [39]. Protein content (BSA standard), total phenolics (gallic
acid standard) and presence of total saponins were analyzed
according to Bradford [40], Singleton & Rossi [41] and Pasaribu et al.
732 Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740
[42], respectively. Only distilled water was used for all experiments.
All analyses were performed in duplicates (n = 6).
2.4. Monosaccharide composition
Polysaccharide samples (10 mg) with added myo-inositol as an
internal standard (1 mg) were pre-hydrolyzed with 72% (w/w)
H2SO4 for 1 h at 30 ◦C. Then, the samples were diluted with
water to 1 M H2SO4 and further hydrolyzed for 3 h at 100 ◦C.
The released monosaccharides were converted into their alditol
acetates, according to Selvendran et al. [43]. The formed alditol
acetates were analyzed by GC/MS on a HP 6890 Series Plus gas
chromatograph coupled with a HP 5973 mass selective detector
(Hewlett–Packard, Palo Alto, USA), on a Supelco SP-2380 column
(30 m × 0.25 mm ID × 0.25 ␮m). The monosaccharide composition
(including GalA and GlcA) of the three samples (1 mg) containing
100 ␮g myo-inositol was also determined by HPAEC-PAD after
methanolysis with anhydrous 2 M HCl in methanol for 16 h at 80 ◦C,
followed by hydrolysis with 2 M TFA for 1 h at 121 ◦C [44]. All samples
were analyzed in duplicates.
The presence of arabinogalactan type II (AGII) was detected by
precipitation with the ß-glucosyl Yariv reagent [45].
2.5. Fourier transform infrared spectroscopy (FTIR)
The lavender and linden PSCs were dissolved in water
(10 mg/ml) and coagulated several times with 95% ethanol with
final alcohol concentration of 70%. After each run the polysaccharides
were separated by centrifugation at 4000 rpm for 30 min at
20 ◦C, dissolved in water and freeze-dried. FTIR spectra of lavender
and linden PSCs (2 mg) after an exhaustive removing of alcoholsoluble
molecules were obtained on a NicoletTM Avatar (Thermo
Fisher Scientific, Waltham, USA) spectrometer in a KBr tablet. The
spectra were recorded in the range 4000 and 400 cm−1 and analyzed
by the SpectraGryph software (Friedrich Menges).
2.6. Immunomodulatory effect
2.6.1. Ethics
Blood sampling procedure was in accordance with the Helsinki
Declaration of 1975, as revised in 1983. All participants signed a
written informed consent prior to initiation of the study. The animal
experiments were approved by the Animal Research Committee
of the Kitasato University, and performed in accordance with the
Guidelines for Care and Use of Laboratory Animals at the Kitasato
University and the National Research Council Guide for the Care
and Use of Laboratory Animals in Japan.
2.6.2. Animal procedure and care
Female C3H/HeJ mice (6–8 weeks old) were purchased from
Japan SLC (Shizuoka, Japan), and male ICR mice (4 weeks old) were
from Charles River (Tokyo, Japan). The mice were housed in plastic
cages in an air-conditioned room at 23 ± 2 ◦C with a relative humidity
of 55 ± 10% under a 12 h-light-dark cycle, and fed a standard
laboratory diet with water given ad libitum.
2.6.3. Flow cytometric analyses
Peripheral venous blood was collected from healthy volunteers
(n = 3; gender = 2 men and 1 women, age range 25–39 years)
using Venoject
®
tubes (Terumo Corporation, Japan). The blood
samples were centrifuged at 1500g for 15 min. Then, the serum
was removed and the red blood cells were lysed using 0.84%
NH4Cl buffer. The samples were washed twice with phosphatebuffered
saline (PBS) buffer. The isolated white blood cells were
pooled, centrifuged at 1000 rpm for 10 min and the cell pellet was
re-suspended in a complete DMEM culture medium (Gibco, Invitrogen,
USA) supplemented with 10% fetal calf serum and antibiotics.
The cell suspension was seeded in four 25 cm2 culture flasks (TPP,
Switzerland), as one flask served as a control (cells cultured in
a standard growth medium), the cells in the other three culture
vessels were treated with 100 ␮g/ml purslane, lavender and linden
PSCs, respectively for 48 h in a humidified incubator at 37 ◦C
and 5% CO2 content. At the end of the incubation period, the control
and the PSC-treated cells were harvested and centrifuged at
1000 rpm for 10 min. The cell pellets were re-suspended in FACS
buffer (PBS containing 5% fetal calf serum and 0.05% NaN3). Then,
the cells were stained with fluorochrome labeled antibodies specific
for CD3, CD4, CD8, CD14, CD20, CD64 markers. The cell samples
were incubated with antibody mixes for 10 min at room temperature
in dark. After that, the cells were washed twice with the FACS
buffer and acquired on a FC500 flow cytometer (Beckman Coulter,
USA). The levels of different leukocytes were compared between
the control and PSC-treated cells.
2.6.4. Reactive oxygen species (ROS) production from human
whole blood phagocytes (WBP)
Heparinized (50 IU/ml) blood samples were obtained from the
cubital vein of healthy human volunteers after overnight fasting.
The kinetics of ROS production by WBP was analyzed by
luminol-enhanced chemiluminescence (CL) for a period of 60 min
at 37 ◦C, using 96-well white flat bottom culture plates on luminometer
(Orion II Berthold Detection Systems GmbH, Germany).
The principle of the method was previously described [46]. Briefly:
25 ␮l of ten times diluted blood with Hank’s buffered salt solution
(HBSS) were mixed with 25 ␮l 1 mM luminol (10 mM stock solution
in 0.2 M borate buffer) HBSS solution and incubated with HBSS
solutions of the three PSCs (10 and 100 ␮g/ml) for spontaneous
(without activators) CL. Furthermore, 12.5 ␮l or 5 ␮l of ten times
diluted blood mixed with 25 ␮l luminol were used for incubation
of the samples with 25 ␮l (0.5 ␮g/ml) phorbol myristate acetate
(PMA-activated, Sigma-Aldrich) or 25 ␮l (62.5 ␮g/ml) opsonized
zymosan particles (OZP-activated). The final volume for spontaneous,
PMA- and OZP-activated CL tests was 250 ␮l. The recorded
values included the intensity of CL emitted during the studied time
interval (integral of CL). The results are expressed as mean percentage
of the control ± SEM from six independent experiments.
2.6.5. Ex vivo immunomodulating activity against murine
Peyer’s patch (PP) cells
Immunomodulating activity was measured by the enhanced
production of bone marrow cell-proliferative cytokines from the PP
cellsofpathogen-freeC3H/HeJfemalemice(Tlr4Lps−d),accordingto
Hong et al. [47]. Suspensions (1.2 × 106 cells/ml, 180 ␮l) of PP cells
(5–9 patches obtained per mouse) from the small intestine of the
C3H/HeJ mice were cultured with H2O (negative control), AMOL-1
(pectic polysaccharide from Astragalus mongholics as a positive control,
Kiyohara et al. [48]) or 100 ␮g/ml of the three PSCs (20 ␮l) in a
96-well flat bottom culture plate (FALCON 3072) for 6 days at 37 ◦C
in a humidified atmosphere of 5% CO2–95% air. The resulting culture
supernatants (50 ␮l) were further cultured with a bone marrow cell
suspension (5.0 × 105 cells/ml) from male ICR mice for 6 days. The
numbers of proliferated bone marrow cells were measured by Alamar
BlueTM, as described previously [47]. IL-6 production from the
murine PP cells stimulated by the tested samples was measured in
the cell culture supernatants by ELISA, according to the company
manual (BD Pharmingen) and as described by Kiyohara et al. [48].
The results are expressed as mean ± SEM.
2.6.6. In vitro tumor cell growth inhibition
The growth inhibitory effect of the three PSCs was studied
against FL cell line (ATCC CCL 62, NBIMCC 94) derived from human
Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740 733
amniotic cells, normal human fibroblasts (isolated from testicular
tissue; cultures obtained following the 5th passage) and different
human tumor cell lines: A549 (ATCC CCL 185), LS180 (ATCC CL-
187) and HeLa (ATCC CCL-2). All cell lines were expanded in 75 cm2
culture flasks (TPP, Switzerland) in a DMEM growth medium, supplemented
with 10% fetal calf serum and antibiotics. The cells were
trypsinized and adjusted to a concentration of 2 × 105 cells/ml and
then seeded on 96-well plates (TPP, Switzerland) with a working
volume of 200 ␮l/well and cultured for 24 h at 37 ◦C, 5% CO2 and
high humidity. After that, the growth medium was replaced with
a fresh medium containing 2, 10, 50, 100 or 200 ␮g/ml polysaccharide
samples and the cells were cultured for 24, 48 and 72 h. At
the end of each test-period, MTT (3-(4,5-dimethylthiazol-2-yl)-2,4diphenyltetrazolium
bromide) (Sigma–Aldrich) solution in a final
concentration of 0.5 mg/ml was added to all wells. The cells were
incubated for 4 h in a humidified incubator at 37 ◦C and 5% CO2 content.
Eventually, the MTT-containing medium was removed; 100 ␮l
DMSO were pipetted to each test well and incubated for 15 min at
room temperature on a shaker, in order to dissolve the formazan
crystals accumulated in the cells. The absorbance was measured at
570 nm using a Synergy-2 spectrophotometer (BioTek, USA). The
percent inhibition of cell growth was calculated using the mean
absorbance units from each test sample and the mean data from
the control cells incubated in the absence of polysaccharides. All
samples were assayed in triplicates and the results are expressed
as IC50 ± SEM (48 h).
2.7. Statistical analysis
Statistical significance was analyzed by the Student’s t-test,
using the Statview software (SAS Institute, Inc.). The mean
results ± SEM were compared to those from the control. Values of
p < 0.05 were considered as statistically significant.
3. Results
3.1. Isolation and chemical characterization
In Table 1 are presented results from the chemical characterization
of the three crude PSCs. The polyuronide content in the initial
herbal materials ranges between 14.1–22.2%, which indicates the
presence of pectic type polysaccharides in all of them. The differences
between the polyuronides in the initial materials and the
residues after extraction reveal that about 77%, 57% and 71% of the
detected polyuronides in the used parts of purslane, lavender and
linden are recovered by the double extraction with boiling water.
The corresponding PSCs were obtained with yields of 8.6%, 3.2%
and 6.1%, respectively. The three PSCs contain >55% total sugars.
The AUAC in the samples is in the range 37.0–45.9%. The most of
the AUAC in the purslane and lavender PSCs is represented by GalA
according to the 3,5-dimethylphenol method (Table 1). Interestingly
for the linden PSC we found 33.6% GalA and 14.3% GlcA by the
3,5-dimethylphenol, which in total is in agreement with the AUAC
result (45.9%). The three PSCs contain methoxyl and acetyl esters
in different extents, which are normally located in the GalA units in
the pectins and rarely found in the neutral sugars [49]. The linden
PSC has the lowest methoxyl (2.5%) and the highest acetyl content
(3.3%) in comparison with the other two samples. The lavender and
purslane PSCs have also relatively high acetyl contents. The AUAC
and methoxyl-ester content of the linden PSC are in a good agreement
with the data obtained by Yakovlev [50], however he did not
mention the presence of acetyl groups. According to the HPSECRID
analysis, linden PSC is characterized by two main populations
with molecular weights 1.23 × 106 and 4.72 × 104 g/mol. The other
Table 1
Chemical characterization of the three herbal PSCs. The results are presented as
mean (w/w% DS).
Purslane
PSC
Lavender
PSC
Linden PSC
Polyuronidesa
, initial
herb [%]
14.1 15.5 22.2
Polyuronidesa
, residue
after extraction [%]
3.3 6.6 6.5
Yield [%] 8.6 3.2 6.1
Total sugarsb
[%] 82.5 64.9 56.0
AUAC [%] 43.2 37.0 45.9
GalAc
40.1 35.8 33.6
DMd
[mol%]
(Methoxyl content [%])
68.2
(5.0)
70.8
(4.6)
40.5
(2.5)
DAd
[mol%]
(Acetyl content [%])
16.1
(1.6)
25.7
(2.3)
40.4
(3.3)
Molecular weight
distribution [g/mol]
5.3 × 104
(49.0)
1.7 × 104
(51.0)
1.6 × 106
(2.5)
1.5 × 104
(97.5)
1.23 × 106
(67.5)
4.72 × 104
(32.5)
Monosaccharide composition [%]
Ara 9.5e
(11.0)f
5.7 (5.4) 4.4 (4.8)
Rha 3.8 (1.1) 2.5 (0.8) 11.5 (1.2)
Fuc trace 0.2 (0.1) trace
Xyl 1.4 (1.0) 1.6 (1.0) 1.2 (0.8)
Man 0.7 (0.5) 2.3 (1.0) (0.1)
Gal 11.3 (10.8) 6.3 (5.2) 3.3 (2.8)
Glc 13.2 (10.1) 5.1 (3.1) 1.0 (0.5)
GalA 39.1 35.8 24.0
GlcA 3.5 5.4 10.6
Protein content [%] 3.3 2.2g
2.2
Total phenolics [%] 0.8 11.0 2.7
Presence of saponins + + +
a
Determined titrimetrically according to Owens et al. [34].
b
Sum of the monosacharides determined by the HPAEC-PAD analysis.
c
Determined on the basis of the differentiation between GalA and GlcA with the
3,5-dimethylphenol method.
d
Calculated on the GalA basis, moles of methoxyl or acetyl groups per 100 mol of
GalA.
e
Monosaccharide composition determined by HPAEC-PAD analysis.
f
Neutral monosaccharide composition determined as alditol-acetates.
g
Determined after an exhaustive treatment with 95% ethanol.
two samples are characterized by predominant populations with
molecular weights in the range 1.5 × 104–5.3 × 104 g/mol.
Because of the proposed complex polysaccharide composition
of the samples, two different techniques for determination of the
monosaccharides were used. When we compared the results from
both monosaccharide composition analyses, we found that the neutral
monosaccharides and especially Rha are detected in lower
amounts after H2SO4 hydrolysis, as was already described [31,44]
(Table 1). For the comments presented below and for estimation
of the total amount of sugars are used the data from the HPAECPAD
analysis. The monosaccharide composition confirmed that the
three samples contain pectic polysaccharides with predominant
GalA and the neutral monosaccharides Ara, Gal and Rha. They also
have Glc, Xyl, Man and Fuc, as the latter is related to RGII structures
[11]. The purslane PSC is characterized by the highest GalA
content within the three PSCs, which indicates that the HG fragments
in the purslane pectins are in considerable amounts. It has
also the highest Glc content and relatively comparable amounts of
Gal (11.3%) and Ara (9.5%). The sum GalA + GlcA in this sample is
42.6%, which is in agreement with the determined AUAC (43.2%).
The ratio Gal/Ara in the complex is 1.2 and it is comparable to the
ratio of 1.1 found by Amin & El-Deeb [26] in mucilage B from the
leaves of P. oleracea. It was found that P. oleracea contains an AG
polysaccharide with a low GlcA content that was elucidated to be
AGII [27,28]. The high Glc content in the PSC could be attributed to
the presence of different glucans, as previously found [27,28].
734 Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740
Similarly, the lavender PSC is characterized by high Gal (6.3%),
Ara (5.7%) and Glc (5.1%) contents. To best of our knowledge, there
are not studies on the polysaccharides from lavender described
in the literature so far. The considerable amount of GalA and the
low Rha content indicate that the HG segments are predominantly
presented in the lavender polysaccharides. The ratio Gal/Ara in
the lavender PSC is 1.1, suggesting the presence of AG structures
similarly to the purslane PSC. Furthermore, the detected Fuc is
indicative for presence of RGII structures. Interestingly, it has relatively
high Man (2.3%) and Glc (5.1%) contents. Both purslane and
lavender PSCs contain low amounts of GlcA, which is in agreement
with other studies on herbal polysaccharides [13,48]. The results
for GalA of both PSCs are in a good agreement with the colorimetric
data (Table 1).
The linden PSC is characterized by high Rha and GlcA contents,
as the latter finding is confirmed by the colorimetric quantification
of GalA and GlcA. The high Rha content, in addition to the high GalA
amount suggest the existence of a RGI fragment in a representable
proportion in the linden PSC. The GlcA is present in a surprisingly
high content, comparable to the amount of Rha. We calculated that
GlcA is 30%-31% from uronides in the linden PSC on the basis of the
colorimetric and chromatographic determination of both uronic
acids. Similarly, Kram & Franz [31] and Yakovlev [50] found high
Rha content in the crude linden polysaccharide, and the first group
mentioned the simultaneous presence of GalA and GlcA, but they
did not provide any quantitative data for both uronic acids. The linden
PSC had the lowest Gal content, but we found by the Yariv’s test
that the sample contains the immune active AGII structures (data
not shown).
The purslane and linden PSCs are also characterized by low
protein and polyphenol contents. The lavender PSC has a high
polyphenol content (11%), which interferes with the quantification
of total proteins, as was observed by Kiyohara et al. [9]. After
an exhaustive removing of the alcohol-soluble molecules from the
lavender PSC, it was determined that it contains 2.2% proteins by
the Bradford method. The presence of saponins was evaluated only
qualitatively by the vanillin-sulfuric acid method because of a possible
Rha interference. The qualitative and quantitative analyses
of the non-carbohydrate constituents were necessary for a better
explanation of the observed immunomodulatory effects.
3.2. FTIR spectroscopy
After an exhaustive removing of alcohol-soluble molecules from
the lavender and linden PSCs, the recovered PSCs were analyzed by
FTIR spectroscopy (Fig. 1). Both spectra contain the typical bands for
polysaccharides and pectins: 3446, 2920 (2929), 1751, 1734, 1635
and 1420, 1146 (1151) cm−1, arising from O-H of hydroxyl groups,
C-H of CH2, C=O of methoxyl ester group (COOCH3), C=O of acetyl
ester group (OCOCH3), C=O of ionized (free) carboxyl group and CO-C
of the glycosidic bond (or within the ring), respectively [51]. For
the linden PSC the signal at 1751 cm−1 is negligible and the peak
at 1734 cm−1 is with equal intensity to the signal at 1716 cm−1.
The latter is proposed to arise only from (C=O) of the acetyl group
[51]. The absorption band at 1255 or 1244 cm−1 is from (C-O-C)
of the acetyl group [51]. The absence of a characteristic peak at
1751 cm−1, the higher intensities of the two signals at 1255 and
1716 cm−1 in the spectrum of linden PSC than in the lavender PSC
spectrum confirm its lower DM and higher DA. Furthermore, the
signal at 1373 (1375) cm−1 originates from ıs(CH3) of ester groups.
It is clearly seen that the intensity of the band at 1635 cm−1 for
the linden PSC is higher than this in the spectrum of lavender PSC,
which is related to the higher AUAC and free COOH derived from the
GalA and GlcA in the first sample. The region of 1200–1000 cm−1 is
the so called “fingerprinting region” for each pectin and it contains
mainly the following skeletal ring vibrations: C-OH, C-C and C-O-C
glycosidic bond vibrations [52]. We found five signals in this region
for the lavender PSC − 1146, 1101 ( C-O ring), 1076, 1051 and 1016
( C-OH, C-C) cm−1 and three signals for the linden PSC − 1151,
1070 and 1041 cm−1. The bands at 1101 and 1019 cm−1 are the
strongest for the lavender PSC, indicating the predominance of the
HG structure in it. Conversely, for the linden PSC the signals at 1070
and 1041 cm−1 show that it contains a considerable amount of the
RGI structure [53]. In both spectra are found bands in the anomeric
region (C1-H, ring) at 822 or 833 cm−1 and at 908 (893) cm−1 for
(C-OH)ring characteristic for ˛- and ˇ-linked aldopyranoses [51].
In the region 1600–1500 cm−1 are expected signals derived from
different functional groups, such as COOH, NH2 and aromatic ring
vibrations.
3.3. Immunomodulating effects
3.3.1. Effect on human white blood cells
The effect of the three PSCs on various human white blood cells
was studied at a concentration of 100 ␮g/ml by a flow cytometric
immunophenotyping analysis (Fig. 2). The purslane PSC stimulated
CD4+ cells, whereas the lavender PSC stimulated CD8+ cells. The
three samples did not express stimulating activity on CD20+ cells,
representing B-cell population. They activated to different extent
CD25+ cells, which in addition to the B-cells are also related to
T-cells. The linden and lavender PSCs even showed inhibitory activity
on CD20+ B-cells, which could be partly associated with the
non-carbohydrate constituents. The purslane and lavender PSCs
exhibited activity on CD4+/CD25+ and CD8+/CD25+ T-cells, and
the stimulatory effect of the linden PSC was weak. In addition to
the effect on the T-cell subpopulations the herbal polysaccharides
can activate phagocytes and thus again exhibit immunostimulating
activity. The three PSCs showed activity on CD14+ cells or
monocytes. The linden and lavender PSCs had activity on CD64+
cells, representing mainly macrophages. Conversely, the effect of
purslane PSC on the CD14+ cells was not observed on the CD64+
cells, which could be difficultly explained. We also found that all
samples increased IL-6 production from white blood cells, which
was in agreement with the stimulation of monocytes and T-cells
(data not included).
3.3.2. Effect on ROS formation by human WBP
The three herbal PSCs stimulated to different extent the oxidative
burst in WBP at 10 and 100 ␮g/ml, which was expressed by
the increase in spontaneous CL response, as a measure of ROS generation
(Fig. 3). The highest activity at 100 ␮g/ml was observed
for the purslane PSC and then for the linden PSC. Both samples
have some important differences in the monosaccharide composition
in respect to Gal, Ara, Glc, GalA, Rha and GlcA contents
and in their molecular weights. The purslane PSC is characterized
with higher Gal, Ara and Glc, which are indicators for presence of
immune active AGs and glucans. On the other hand, the lavender
PSC expressed a higher stimulating activity at 10 ␮g/ml than at
100 ␮g/ml, which was expected because of the mimic antioxidant
effect from polyphenols in it. As can be seen from Fig. 3 the control
values for PMA- and OZP-activated CL were much higher than those
for the spontaneous CL response of the polysaccharide-treated
cells. Interestingly, the three PSCs did not increase CL response
of the PMA-activated phagocytes. The lavender PSC decreased
the PMA-activated CL at 100 ␮g/ml, expressing a suppressive or
protective-like effect on the activated cells. This mode of action
was related to polyphenols, as reviewed by Lojek et al. [16]. Similarly,
the lavender PSC induced a decrease of the OZP-stimulated
CL at 100 ␮g/ml. Contrary, purslane and linden PSCs increased the
CL response in the OZP-treated cells at both studied concentra-
Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740 735
Fig. 1. FTIR spectra of selected herbal PSCs.
tions, which was probably in agreement with the spontaneous ROS
generation.
3.3.3. Intestinal immunomodulating activity
In the current study the three PSCs exhibited ex vivo intestinal
immunomodulating activity through the PP-mediated bone marrow
cell proliferation test and increased IL-6 production from the
PP cells in comparison with the positive control AMOL-1 (Fig. 4).
The PP cells were isolated from the LPS low responder (Tlr4Lps−d)
C3H/HeJ mice, thus it was considered that the observed activity
was not a result of possible LPS contamination. The bone-marrow
cell proliferation was measured to estimate the relative production
of hemopoietic growth factors from the polysaccharide-stimulated
PP cells. The highest stimulating activity was expressed by the
purslane PSC, followed by the linden and lavender PSCs, as in the
same order was IL-6 production. The purslane PSC even induced a
higher IL-6 production than the positive control. Therefore, the IL-6
production from the white blood cells induced by the three herbal
PSCs was also confirmed by the animal immune cells. The control
of IL-6 production in the PP cells is important because this cytokine
can induce IgA antibody production through the B-cell proliferation
in the patches.
3.3.4. Anti-tumor activity in vitro
In Table 2 are presented IC50 values of the three PSC samples
against the growth of different human tumor cell lines after
48 h of incubation. Normal human fibroblasts (NF) and amniotic
cells (FL) were used as controls. The linden PSC was the
most active sample, expressing inhibitory activities against A549,
HeLa and LS180 cells with IC50 values after 48 h of incubation as
follows: 92.6 ± 2.7 ␮g/ml, 96.7 ± 1.6 ␮g/ml and 185.9 ± 6.3 ␮g/ml,
respectively. The IC50 values for A549 and HeLa cell lines after
48 h of culturing with the lavender PSC were 162 ± 2.6 ␮g/ml and
170 ± 2.5 ␮g/ml, and for the purslane PSC were 169.9 ± 9.2 ␮g/ml
736 Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740
Fig. 2. Flow cytometric analysis for immunophenotyping of human white blood cells treated with 100 ␮g/ml purslane, lavender and linden PSCs.
Fig. 3. Effect of purslane (PSCI), lavender (PSCII) and linden PSCs (PSCIII) on ROS production from non-stimulated (spontaneous), PMA- and OZP-activated whole blood
phagocytes. 10 (10 ␮g/ml) and 100 (100 ␮g/ml). The asterisks indicate statistical difference (**p < 0.01, ***p < 0.001) vs. control.
Fig. 4. Enhancement of production of bone marrow cell-proliferating cytokines from Peyer’s patch cells cultured in presence or absence of purslane, lavender and linden PSCs.
(A). Enhancement of production of IL-6 from Peyer’s patch immunocompetent cells cultured in presence or absence of polysaccharides (100 ␮g/ml) (B). The symbols indicate
statistical difference (*p < 0.05, **p < 0.01, ***p < 0.001) vs. water control or among samples (#p < 0.05, ##p < 0.01, ###p < 0.001). AMOL-1 is an active pectic polysaccharide
isolated from A. mongholics, containing AGII.
Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740 737
Table 2
IC50 values (± SEM) of the three herbal PSCs on the growth of different cell lines after
48 h of treatment.
Polysaccharide complex Cell line IC50 [␮g/ml]
Purslane PSC NFa
>200
FLb
173.9 ± 1.6
A549c
169.9 ± 9.2
HeLad
195.4 ± 0.9
LS180e
>200
Lavender PSC NF >200
FL 183.7 ± 5.4
A549 162.0 ± 2.6
HeLa 170 ± 2.5
LS180 >200
Linden PSC NF >200
FL 167.5 ± 17.2
A549 92.6 ± 2.7
HeLa 96.7 ± 1.6
LS180 185.9 ± 6.3
a
NF (normal fibroblasts).
b
FL (normal amniotic cells).
c
A549 (lung adenocarcinoma).
d
HeLa (cervical adenocarcinoma).
e
LS180 (colon adenocarciona cells).
and 195.4 ± 0.9 ␮g/ml. The three PSCs were the most effective
against the lung adenocarcinoma A549 cells. It was found that
only the linden PSC expressed anti-proliferative activity against the
growth of colon adenocarcinoma LS180 cells with IC50 value under
200 ␮g/ml. However, the results from the control treatment of NF
and FL cells with the three PSCs showed that control cells were
inhibited in a similar manner like abnormal cells. This means that
the three samples exhibited non-specific inhibitory activity on the
basis of the chosen control cell lines.
4. Discussion
Only on the basis of the monosaccharide composition data and
the ester content (Table 1) could be suggested that the purslane
and lavender PSCs contain methoxylated and acetylated pectic
polysaccharides with predominant HG fragments, followed by AG
structures most probably attached in the RGI fragments. The chemical
features of the purslane PSC, such as the highest Gal, Ara and
Glc contents among the three PSCs must be important for the
potent activity of this sample in most of the observed effects. Similarly,
it is proposed that the linden PSC contains high molecular
weight pectins with considerable DA and low DM and with high
RGI and GlcA contents, which could also influence the observed
activities. From the three studied PSCs only polysaccharides of
the purslane are investigated previously for expression of in vitro
and in vivo immunomodulating activity [19,29]. Our findings on
the stimulation of human white blood cells (Fig. 2) showed that
the purslane PSC elevated the CD4+ and CD25+ cells, CD4+/CD25+
and CD8+/CD25+ T-cells, and CD14+ monocytes. The lavender PSC
stimulated the CD8+ and CD25+ cells, T-cells and phagocytes. The
linden PSC expressed activity mainly against the CD14+ and CD64+
cells. CD14 and CD64 proteins related to innate immune response
are expressed on phagocytes, whereas CD4 and CD8 are related
mainly to T-cells and adaptive immunity. CD64 or Fc␥RI receptor
is expressed on monocytes, macrophages, neutrophils and
other granulocytes and it interacts with antibody-coated antigens
(IgG-Ag), thus triggering cell activation, such as phagocytosis and
antibody-dependent cell-mediated cytotoxicity [54]. Its increased
CD64 expression in treated with the studied herbal PSCs blood cells
could mean Ig interaction with polysaccharides or other molecules
in them [55]. When herbal pectins and probably their AG side chains
interact with neutrophils through different receptors, they serve
as antigens and similarly to macrophages activate phagocytosis
with production of ROS [2]. The neutrophils comprise between 50
and 70% of all leukocytes in the blood and they are considered as
main contributor to the total luminol CL [16]. Furthermore, the
neutrophils exhibit more rapid rates of phagocytosis and a more
intense oxidative response than the macrophages. Only the lavender
PSC showed inhibitory effects on the PMA- and OZP-activated
ROS generation (Fig. 3). Unlike the lavender, purslane and linden
PSCs have low phenolic content and did not show any prominent
antioxidant activity by the ORAC method (data not included). Similar
effects to these of the lavender PSC on the spontaneous, PMAand
OZP-activated WBP were observed by Nikolova et al. [56] for
leek pectic polysaccharides, which also showed antioxidant activity
through the ORAC method. PMA is a potent inflammatory activator
of protein kinase C, which could induce NADPH-oxidase activity
and further the neutrophil degranulation and death [57]. OZP are
opsonized zymosan particles that are coated with immunoglobulins
and some complement factors which could be recognized
through Fc and CR3 on neutrophils or other phagocytic leukocytes
[58]. The stimulation of WBP by the three studied PSCs was
in agreement with the elevated levels of CD14+ and CD64+ cells
(Fig. 2). Therefore, the studied herbal PSCs could activate phagocytic
leukocytes, which are involved in the first innate mechanism
for immune defense against different pathogens and in inflammation
processes. Interestingly, polysaccharides can affect positively
the immune potentiation and negatively the inflammation [3]. This
dual mode of action makes them perspective candidates for development
of polysaccharide-derived healthcare products.
It was proposed that the studied herbal PSCs stimulated Tregs
cells, represented most probably by the CD4+/CD25+ T-cells. The
CD4+/CD25+ and CD8+/CD25+ T-cell subpopulations are responsible
for regulation of the immune response in autoimmune, tumor
diseases and immune homeostasis. The stimulatory effect of herbal
polysaccharides on T-cells could be useful in the supplementary
therapy of infectious and tumor diseases [59]. For example, Shen
et al. [19] demonstrated that polysaccharide POP from P. oleracea
exhibited a potent anti-tumor activity in vivo and increased the
animal’s immune responses, including increase in the number of
white blood cells and CD4+ T-cells, as well as the ratio of CD4+/CD8+
cells. Similarly, Shu et al. [60] showed that polysaccharide PSSC
from Salvia chinensis expressed an anti-tumor immunostimulatory
activity in vivo and elevated CD4+ T-cells in both spleen and lymph
nodes and the cytotoxic activity of NK cells and CD8+ T-cells.
Herbal polysaccharides are mainly administrated in the human
organism by herbal infusions, foods or other formulations. They can
express intestinal immunomodulating activity through interaction
with immunocompetent cells in the PPs localized in the small intestine
[2]. Although, polysaccharides are little absorbed by intestinal
epithelial cells, their communication with the epithelium could also
lead to immunomodulation of the underlying immunocompetent
cells [10]. The AGII, together with the AGI structures in pectins
are important for expression of the intestinal immunomodulating
activity through the PPs [2]. It was found by the Yariv’s test that the
linden PSC contains AGII. Similarly, literature data indicate that the
purslane pectins also contain AGII [28]. Therefore, the purslane PSC
logically showed the most potent activity because it had the highest
amount of Gal, which was assumed to be mostly linked in the AGII
and AGI structures. It also contained a considerable amount of Glc,
derived probably from starch that activates PP cells as well [61]. Our
results were in a good agreement with those obtained for a lignincarbohydrate
complex from the Kampo extract preparation TJ48
(Juzen-Taiho-To), BP-II pectic fraction from Biophytum petersianum
and RP-1 pectic fraction from Rosa damascena [9,13,62].
Murine PPs contain about 60% B-cells (B220+), 25% T-cells
(CD3+), 10% dendritic cells (CD11c+), and less than 5% macrophages
(F4/80+) or polymorphonuclear neutrophils (Ly-6G+). Among the
738 Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740
T-cells 45% are CD4+, 35% are CD8+ and 20% are CD4−/CD8−
T-cells. Among the CD4+ T-cells, 85% are memory T-cells
(CD25−CD45RB1o), 10% are Naïve (CD25−CD45RBhi) and 5% are
regulatory T-cells (CD25+CD45RB1o). Furthermore, bone marrow
contains T-cells, B-cells, neutrophils, dendritic cells and monocytes,
as it has 8%–20% lymphocytes with T/B-cell ratio of 5:1. The
mouse bone marrow contains 1–5% CD3+, 1.5% CD4+, 2.0%–2.5%
CD8+ T-cells. Importantly, CD4+/CD25+ and CD8+/CD25+ T-cells are
found to be about 10% and 5% in the human PPs and 5–30% of the
CD4+ T-cells in the mouse bone marrow are CD4+/CD25+ Tregs
cells [63,64]. Therefore, on the basis of our results with human
white blood cells (Fig. 2) and WBP (Fig. 3), it could be proposed
that the studied polysaccharides expressed activity against the PP
mediated-bone marrow cell proliferation through the phagocytic
leukocytes and T-lymphocytes. Additionally, the three samples
induced IL-6 production from PP cells and human white blood cells,
which is produced by T-cells and macrophages [2]. Administration
of herbal polysaccharides active against the intestinal immune system
could support the compromised immune health. For example,
Hou et al. [59] found that treatment (i.p.) of mice suffering from
polymicrobial sepsis with 100 and 200 mg/kg Astragalus polysaccharide
APS increased the percentage of activated Th cells in the
PPs.
When it was found that the herbal PSCs expressed immunostimulating
activity against innate and adaptive human immune cells
and intestinal murine PP cells, it was also important to be tested
their effect on abnormal cells. This was necessary in respect to
the perspective application of the herbal PSCs and related products
in the prophylaxis and prevention of infectious, autoimmune and
tumor diseases. Only the anti-tumor activity of purslane polysaccharides
was investigated, so far [29,30]. Zhao et al. [30] obtained
IC50 = 489 ␮g/ml after 48 h treatment of HeLa cells with polysaccharide
fraction POL-P3b isolated from P. oleracea, which was 2.5
fold higher than those we obtained for the purslane PSC (Table 2).
The purslane PSC expressed a higher in vitro activity, but this
could be related primarily to the presence of a mixture of purslane
neutral and pectic polysaccharides, as well as to the supplementary
effect of smaller molecules, which were expected to be <18%
(Table 1). Our results with the A549 cells for the purslane and
lavender PSCs were in agreement with those obtained for crude
polysaccharides extracted from the fruits of Cyphomandra betacea,
which showed IC50 = 170 ␮g/ml after 48 h with A549 cells [65].
Even though, the combinatorial effects of different compounds in
herbal extracts are rather complex, it cannot be excluded that the
polysaccharides and some of the non-carbohydrate constituents
act in a synergistic manner. For example, it was found that supplementation
of breast cancer patients undergoing postoperative
radiation therapy with aronia juice enriched with apple pectin
led to an immune system stimulation expressed by a significant
increase of the CD4+ and CD8+ T-cells levels [66]. Although, studies
of the polysaccharide effect on LS180 cells are still scarce,
the anti-tumor activity of the herbal polysaccharides against gastrointestinal
tumors expands their immunomodulating potential
and deserves more studies for their protective and suppressive
effects. However, our results showed that the studied herbal PSCs
expressed non-specific in vitro anti-proliferative activity against
normal and tumor cells. This effect needs to be further studied with
the purified polysaccharide fractions, otherwise it seems rather
unspecific and not promising.
Further immunomodulating characterization of the purslane
PSC is necessary because of the increasing popularity of purslane
as a food and its use in the functional and dietary nutrition [23].
The immunomodulating studies on the lavender and linden PSCs
are important primarily due to the traditional use of these herbs
and their water extracts in the folk medicine and cosmetics. The
increasing lavender oil production implies better utilization of the
bioactive compounds, including the pectins in the distilled residue
into secondary value-added products, such as water extracts for
nutraceuticals and cosmetics. We found that the polyuronides, representing
pectins in industrially distilled lavender flowers are 18.9%
with a very close value of 19.1% for the stalks, thus the obtained byproduct
could be a source of bioactive polysaccharides in addition
to smaller molecules.
For further explanation of the biological results it was necessary
to differentiate between the herbal polysaccharides and the other
constituents in the samples. This was done for linden and lavender
PSCs in subsequent studies, confirming the polysaccharide role in
the observed immunomodulating activities and will be published.
5. Conclusion
We demonstrated that similarly to the edible herb purslane, the
other two widely used medicinal plants − common lavender and
silver linden contain also immune active acidic PSCs rich in pectic
polysaccharides. Interestingly, GlcA is about 31% of the uronides
(HPAEC-PAD analysis basis) in the linden PSC, which is also characterized
by high Rha content in addition to high GalA amount,
suggesting the existence of a considerable amount of a RGI fragment
in this sample. The three PSCs expressed ex vivo stimulating
activity against different blood T-cell populations and phagocytic
leukocytes. These herbal PSCs also stimulated ex vivo ROS
production from WBP and inhibited non-specifically the growth
of different tumor cell lines in vitro. Furthermore, they showed
ex vivo intestinal immunomodulating activity against PP cells at
100 ␮g/ml. It was proposed that the studied herbal PSCs can modulate
the PP immunocompetent cells through the CD4+/CD25+ and
CD8+/CD25+ T-cells and phagocytes, because they are also found in
the patches and in the bone marrow. The purslane PSC expressed
the highest stimulating activity against normal immune cells and
the linden PSC showed the highest inhibitory activity against tumor
cells with an IC50 value of 93 ␮g/ml for A549 cells. Our results
suggest that similarly to the purslane polysaccharides, these isolated
from lavender and silver linden have also perspectives to be
studied their supplementary role in treatment of health conditions
characterized with compromised immune system.
Acknowledgements
This work was supported by the European Regional Development
Fund, EU [Grant № BG161PO0003-1.1.05-0024-C0001,
2012-2014], European Social Fund, EU [Grant № BG051PO001/3.3-
05-0001, 2014] and EEA grants. The first two authors are also
thankful to Prof. Henk A. Schols for the access to the Laboratory of
Food Chemistry of the Wageningen University (The Netherlands)
for realization of the HPAEC-PAD analysis.
References
[1] A. Nedelcheva, Medicinal plants from an old Bulgarian medical book, J. Med.
Plants Res. 6 (2012) 2324–2339.
[2] H. Yamada, H. Kiyohara, Immunomodulating activity of plant polysaccharide
structures, in: J.P. Kamerling (Ed.), Comprehensive Glycoscience – from Chemistry
to Systems Biology, Elsevier, Oxford, 2007, pp. 663–694.
[3] S.V. Popov, Y.S. Ovodov, Polypotency of the immunomodulatory effect of
pectins, Biochem. Mosc. 78 (2013) 823–835.
[4] H. Wangensteen, D. Diallo, B.S. Paulsen, Medicinal plants from Mali: chemistry
and biology, J. Ethnopharmacol. 176 (2015) 429–437.
[5] M. Thakur, A. Weng, H. Fuchs, V. Sharma, C.S. Bhargava, N.S. Chauhan, V.K. Dixit,
S. Bhargava, Rasayana properties of Ayurvedic herbs: are polysaccharides a
major contributor, Carbohydr. Polym. 87 (2012) 3–15.
[6] G. Franz, Polysaccharides in pharmacy: current applications and future concepts,
Planta Med. 55 (1989) 493–497.
[7] M. Azémar, B. Hildenbrand, B. Hearing, M.E. Heim, C. Unger, Clinical benefit
in patients with advanced solid tumors treated with modified citrus pectin: a
prospective pilot study, Clin. Med. Oncol. I (2007) 73–80.
Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740 739
[8] J.E. Chrubasik, B.D. Roufogalis, H. Wagner, S. Chrubasik, A comprehensive
review on the stining nettle effect and efficacy profiles. Part II: Urticae radix,
Phytomedicine 14 (2007) 568–579.
[9] H. Kiyohara, T. Matsumoto, H. Yamada, Lignin-carbohydrate complexes:
intestinal immune system modulating ingredients in Kampo (Japanese hebral)
medicine Juzen-Taiho-To, Planta Med. 66 (2000) 20–24.
[10] H. Kiyohara, K. Nonaka, M. Sekiya, T. Matsumoto, T. Nagai, Y. Tabuchi, H.
Yamada, Polysaccharide-containing macromolecules in a Kampo (Traditional
Japanese Herbal) medicine, Hochuekkito: dual active ingredients for modulation
of immune functions on intestinal Peyer’s patches and epithelial cells,
Evid. Based Complement. Alterat. Nat. Med. (2011), 492691, http://dx.doi.org/
10.1093/ecam/nep193, 13 pages.
[11] H.A. Schols, A.G.J. Voragen, The chemical structure of pectins, in: G.B. Seymour,
J.P. Knox (Eds.), Pectins and Their Manipulation, Blackwell Publishing,
CRC Press, Oxford, 2002, pp. 1–29.
[12] N. Inagaki, Y. Komatsu, H. Sasaki, H. Kiyohara, H. Yamada, H. Ishibashi, S. Tansho,
H. Yamaguchi, S. Abe, Acidic polysaccharides from rhizomes of Atractylodes
lancea as protective principle in Candida-Infected Mice, Planta Med. 67 (5)
(2001) 428–431.
[13] T.E. Grønhaug, H. Kiyohara, A. Sveaass, D. Diallo, H. Yamada, B.S. Paulsen,
Beta-D-(1 → 4)-galactan-containing side chains in RG-I regions of pectic
polysaccharides from Biophytum petersianum Klotzsch. contribute to expression
of immunomodulating activity against intestinal Peyer’s patch cells and
macrophages, Phytochemistry 72 (2011) 2139–2147.
[14] K.-W. Yu, H. Kiyohara, T. Matsumoto, H.-C. Yang, H. Yamada, Characterization of
pectic polysaccharides having intestinal immune system modulating activity
from rhizomes of Atractylodes lancea DC, Carbohydr. Polym. 46 (2001) 125–134.
[15] M. Inngjerdingen, K.T. Inngjerdingen, T.R. Patel, S. Allen, X. Chen, B. Rolstad,
G.A. Morris, S.E. Harding, T.E. Michaelsen, D. Diallo, B.S. Paulsen, Pectic
polysaccharides from Biophytum petersianum Klotzsch, and their activation of
macrophages and dendritic cells, Glycobiology 18 (12) (2008) 1074–1084.
[16] A. Lojek, P. Denev, M. Ciz, O. Vasicek, M. Kratchanova, The effects of biologically
active substances in medicinal plants on the metabolic activity of neutrophils,
Phytochem. Rev. 13 (2014) 499–510.
[17] X.R. Zhang, W.X. Zhou, Y.X. Zhang, C.H. Qi, H. Yan, Z.F. Wang, B. Wang,
Macrophages, rather than T and B cells are principal immunostimulatory target
cells of Lycium barbarum L. polysaccharide LBPF4-OL, J. Ethnopharmacol. 136
(3) (2011) 465–472.
[18] Q.-Y. Liu, Y.-M. Yao, S.-W. Zhang, Z.-Y. Sheng, Astragalus polysaccharides
regulate T cell-mediated immunity via CD11chigh
CD45RBlow
DCs in vitro, J.
Ethnopharmacol. 136 (3) (2011) 457–464.
[19] H. Shen, G. Tang, G. Zeng, Y. Yang, X. Cai, D. Li, H. Liu, N. Zhou, Purification
and characterization of an antitumor polysaccharide from Portulaca oleracea L,
Carbohydr. Polym. 93 (2013) 395–400.
[20] K. Kouakou, I.A. Schepetkin, S. Jun, L.N. Kirpotina, A. Yapi, D.S. Khramova, D.W.
Pascual, Y.S. Ovodov, M.A. Jutila, M.T. Quinn, Immunomodulatory activity of
polysaccharides isolated from Clerodendrum splendens: beneficial effects in
experimental autoimmune encephalomyelitis, BMC Complement. Altern. Med.
13 (2013) 149.
[21] M.L. Leporatti, S. Ivancheva, Preliminary comparative analysis of medicinal
plants used in the traditional medicine of Bulgaria and Italy, J. Ethnopharmacol.
87 (2003) 123–142.
[22] A. Allio, C. Calorio, C. Franchino, D. Gavello, E. Carbone, A. Marcantoni, Bud
extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing
through GABAA and benzodiazepine receptors activation, J. Ethnopharmacol.
172 (2015) 288–296.
[23] Y.-X. Zhou, H.-L. Xin, K. Rahman, S.-J. Wang, C. Peng, H. Zhang, Portulaca oleracea
L.: a review of phytochemistry and pharmacological effects, BioMed Res. Int.
(2015), 925631, http://dx.doi.org/10.1155/2015/925631.
[24] G. Laekeman, Assessment report on Lavandula angustifolia Miller, aetheroleum
and Lavandula angustifolia Miller, flos, European Medicines Agency, 2012,
143183/2010.
[25] I. Chinou, Assessment report on Tilia cordata Miller, Tilia platyphyllos Scop.,
Tilia x vulgaris Heyne or their mixtures, flos, European Medicines Agency, 2012,
337067/2011.
[26] E.S. Amin, S.M. El-Deeb, Isolation of Portulaca oleracea (REGLA) mucilage and
identification of its structure, Carbohydr. Res. 56 (1977) 123–128.
[27] G.E. Wenzel, J.D. Fontana, J.B.C. Correa, The viscous mucilage from the weed
Portulaca oleracea L, Appl. Biochem. Biotechnol. 24 (25) (1990) 341–353.
[28] C.-X. Dong, K. Hayashi, J.-B. Lee, T. Hayashi, Characterization of structures and
antiviral effects of polysaccharides from Portulaca oleracea L, Chem. Pharm. Bull.
58 (4) (2010) 507–510.
[29] Y. Li, Y. Hu, S. Shi, L. Jiang, Evaluation of antioxidant and immune-enhancing
activities of Purslane polysaccharides in gastric cancer rats, Int. J. Biol. Macromol.
68 (2014) 113–116.
[30] R. Zhao, X. Gao, Y. Cai, X. Shao, G. Jia, Y. Huang, X. Qin, J. Wang, X. Zheng, Antitumor
activity of Portulaca oleracea L. polysaccharides against cervical carcinoma
in vitro and in vivo, Carbohydr. Polym. 96 (2013) 376–383.
[31] G. Kram, G. Franz, Untersuchungen über die Schleimpolysaccharide aus Lindenblüten,
Planta Med. 49 (1983) 149–153.
[32] J. Schmidgall, E. Schnetz, A. Hensel, Evidence for bioadhesive effects of polysaccharides
and polysaccharide-containing herbs in an ex vivo bioadhesion assay
on buccal membranes, Planta Med. 66 (2000) 48–53.
[33] H. Kiyohara, T. Matsumoto, H. Yamada, Combination effects of herbs in a multiherbal
formula: expression of Juzen-taiho-to’s immuno-modulatory activity
on the intestinal immune system, Evid. Based Complement. Alternat. Med. 1
(2004) 82–91.
[34] H.S. Owens, R.M. McCready, A.D. Shepheral, T.H. Schultz, E.L. Pippen, N.A.
Swenson, J.C. Miers, F.R. Erlander and W.D. Maclay, Methods used at Western
Regional Research Laboratory for extraction and analysis of pectic materials,
U.S. Dept. Agr. Bull. AIC-340, 1952.
[35] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination
of uronic acids, Anal. Biochem. 54 (1973) 484–489.
[36] R. Scott, Colorimetric determination of hexuronic acids in plant materials, Anal.
Chem. 51 (7) (1979) 936–941.
[37] G.E. Anthon, D.M. Barrett, Combined enzymatic and colorimetric method for
determining the uronic acid and methyl ester content of pectin: application to
tomato products, Food Chem. 110 (2008) 239–247.
[38] E.A. МcComb, R.M. McCready, Determination of acetyl in pectin and in acetylated
carbohydrate polymers (hydroxamic acid reaction), Anal. Chem. 29 (1957)
819–821.
[39] Y. Georgiev, M. Ognyanov, I. Yanakieva, V. Kussovski, M. Kratchanova, Isolation,
characterization and modification of citrus pectin, J. BioSci. Biotech. 1 (3) (2012)
223–233.
[40] М.М. Bradford, A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248–254.
[41] V. Singleton, J. Rossi, Colorimetry of total phenolic with phosphomolibdiphosphotungstic
acid reagents, Am. J. Enol. Vitic. 16 (1965) 144–158.
[42] T. Pasaribu, D.A. Astuti, E. Wina, A. Sumiati, Saponin content of Sapindus rarak
affected by particle size and type of solvent, its biological activity on Eimeria
tenella Oocysts, Int. J. Poult. Sci. 13 (6) (2014) 347–352.
[43] R.R. Selvendran, J.F. March, S.G. Ring, Determination of aldoses and uronic acid
content of vegetable fiber, Anal. Biochem. 96 (1979) 282–292.
[44] G.A. De Ruiter, H.A. Schols, A.G.J. Voragen, F.M. Rombouts, Carbohydrate
analysis of water-soluble uronic acid-containing polysaccharides with highperformance
anion-exchange chromatography using methanolysis combined
with TFA hydrolysis is superior to four other methods, Anal. Biochem. 207 (1)
(1992) 176–185.
[45] G.-J. Van Holst, A.E. Clarke, Quantification of arabinogalactan-protein in plant
extracts by single radial gel diffusion, Anal. Biochem. 148 (2) (1985) 446–450.
[46] O. Vasicek, A. Lojek, V. Jancinova, R. Nosal, M. Ciz, Role of histamine receptors in
the effects of histamine on the production of reactive oxygen species by whole
blood phagocytes, Life Sci. 100 (1) (2014) 67–72.
[47] T. Hong, T. Matsumoto, H. Kiyohara, H. Yamada, Enhanced production of
hematopoietic growth factors through T cell activation in Peyer’s patches by
oral administration of Kampo (Japanese herbal) medicine, Juzen-Taiho-To, Phytomedicine
5 (1998) 353–360.
[48] H. Kiyohara, T. Uchida, M. Takakiwa, T. Matsuzaki, N. Hada, T. Takeda, T. Shibata,
H. Yamada, Different contributions of side-chains in ˇ-D-(1 → 3, 6)-galactans
on intestinal Peyer’s patch-imunomodulation by polysaccharides from Astragalus
mongholics Bunge, Phytochemistry 71 (2010) 280–293.
[49] A. Togola, K.H. Naess, D. Diallo, H. Barsett, T.E. Michaelsen, B.S. Paulsen, A
polysaccharide with 40% mono-O-methylated monosaccharides from the bark
of Cola cordifolia (Sterculiaceae), a medicinal tree from Mali (West Africa), Carbohydr.
Polym. 73 (2008) 280–288.
[50] A.I. Yakovlev, Polysaccharides of the inflorescenes of Tilia cordata, Chem. Nat.
Compd. 21 (1) (1985) 114–115.
[51] A. Synytsya, J. ˇCopíková, P. Mat˘ejka, V. Machoviˇc, Fourier transform Raman and
infrared spectroscopy of pectins, Carbohydr. Polym. 54 (2003) 97–106.
[52] D. Murdzheva, N. Petkova, I. Vasileva, M. Todorova, M. Ognyanov, I. Ivanov,
P. Denev, Accelerated modification of low-methoxylated celery pectin, in: Proceedings
of the 12th International Conference on Polysaccharide-Glycoscience,
19th–21th October, Prague, 2016, pp. 213–217.
[53] M. Kaˇcuráková, P. Capek, V. Sasinková, N. Wellner, A. Ebringerová, FT-IR study of
plant cell wall model compounds: pectic polysaccharides and hemicelluloses,
Carbohydr. Polym. 43 (2000) 195–203.
[54] T. Matsumoto, H. Yamada, Regulation of immune complexes binding of
macrophages by pectic polysaccharide from Bupleurum falcatum L.: pharmacological
evidence for the requirement of intracellular calcium/calmodulin on
Fc receptor up-regulation by bupleuran 2IIb, J. Pharm. Pharmacol. 47 (1995)
152–156.
[55] H. Kiyohara, T. Matsumoto, T. Nagai, S.-J. Kim, H. Yamada, The presence of
natural human antibodies reactive against pharmacologically active pectic
polysaccharides from herbal medicines, Phytomedicine 13 (2006) 494–500.
[56] M. Nikolova, G. Ambrozova, M. Kratchanova, P. Denev, V. Kussovski, M. Ciz, A.
Lojek, Effects of pectic polysaccharides isolated from leek on the production
of reactive oxygen and nitrogen species by phagocytes, J. Med. Food 16 (2013)
711–718.
[57] T.A. Fuchs, U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, Y. Weinrauch,
V. Brinkmann, A. Zychlinsky, Novel cell death program leads to neutrophil
extracellular traps, J. Cell Biol. 176 (2) (2007) 231–241.
[58] A. Sobota, A. Strzelecka-Kiliszek, K. Gładkowska, K. Mrozi ´nska, K. Kwiatkowska,
Binding of IgG-opsonized particles to Fc␥R is an active stage of phagocytosis
that involves receptor clustering and phosphorylation, J. Immunol. 175 (2005)
4450–4457.
[59] Y.-C. Hou, J.-M. Wu, M.-Y. Wang, M.-H. Wu, K.-Y. Chen, S.-L. Yeh, M.-T. Lin,
Modulatory effects of Astragalus polysaccharides on T-cell polarization in mice
with polymicrobial sepsis, Mediators Inflamm. (2015), 826319, http://dx.doi.
org/10.1155/2015/826319.
740 Y.N. Georgiev et al. / International Journal of Biological Macromolecules 105 (2017) 730–740
[60] G. Shu, W. Zhao, L. Yue, H. Su, M. Xiang, Antitumor immunostimulatory activity
of polysaccharides from Salvia chinensis Benth, J. Ethnopharmacol. 168 (2015)
237–247.
[61] L. Stertman, E. Lundgren, I. Sjöholm, Starch microparticles as a vaccine adjuvant:
only uptake in Peyer’s patches decides the profile of the immune
response, Vaccine 24 (17) (2006) 3661–3668.
[62] A. Slavov, H. Kiyohara, H. Yamada, Immunomodulating pectic polysaccharides
from waste rose petals of Rosa damascena Mill, Int. J. Biol. Macromol. 59 (2013)
192–200.
[63] C. Jung, J.-P. Hugot, F. Barreau, Peyer’s patches: the immune sensors of the intestine,
Int. J. Inflam. (2010), 823710, http://dx.doi.org/10.4061/2010/823710.
[64] E. Zhao, H. Xu, L. Wang, I. Kryczek, K. Wu, Y. Hu, G. Wang, W. Zou, Bone marrow
and the control of immunity, Cell Mol. Immunol. 9 (2012) 11–19.
[65] C.S. Kumar, M. Sivakumar, K. Ruckmani, Microwave-assisted extraction of
polysaccharides from Cyphomandra betacea and its biological activities, Int. J.
Biol. Macromol. 92 (2016) 682–693.
[66] M.P. Yaneva, A.D. Botushanova, L.A. Grigorov, J.L. Kokov, E.P. Todorova,
M.G. Krachanova, Evaluation of the immunomodulatory activity of aronia in
combination with apple pectin in patients with breast cancer undergoing postoperative
radiation therapy, Folia Med. 44 (2002) 22–25.
Carbohydrate Polymers 174 (2017) 948–959
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
The common lavender (Lavandula angustifolia Mill.) pectic
polysaccharides modulate phagocytic leukocytes and intestinal
Peyer’s patch cells
Yordan N. Georgieva,b
, Berit S. Paulsenc
, Hiroaki Kiyoharad
, Milan Cize
,
Manol H. Ognyanova,b
, Ondrej Vasiceke,i
, Frode Risef
, Petko N. Deneva,b
,
Haruki Yamadad
, Antonin Lojeke
, Vesselin Kussovskig
, Hilde Barsettc
,
Albert I. Krastanovh
, Irina Z. Yanakievaa,b
, Maria G. Kratchanovaa,b,∗
a
Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139 Ruski
Blvd., BG-4000 Plovdiv, Bulgaria
b
Innovative-Technological Center Ltd., 20 Dr. G. M. Dimitrov Str., BG-4000 Plovdiv, Bulgaria
c
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, NO-0316 Oslo, Norway
d
Department of Drug Discovery Science, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, JP-108-8641 Tokyo, Japan
e
Department of Free Radical Pathophysiology, Institute of Biophysics, Czech Academy of Sciences, 135 Kralovopolska, CZ-612 65 Brno, Czech Republic
f
Department of Chemistry, University of Oslo, P.O. Box 1033 Blindern, NO-0315 Oslo, Norway
g
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 26, BG-1113 Sofia, Bulgaria
h
Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., BG-4002 Plovdiv, Bulgaria
i
International Clinical Research Center − Center of Biomolecular and Cellular Engineering, St. Anne’s University Hospital Brno, 53 Pekarska, CZ-656 91
Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 29 May 2017
Received in revised form 27 June 2017
Accepted 5 July 2017
Available online 8 July 2017
Keywords:
Lavender
Pectin
Immunomodulation
Neutrophils
Macrophages
Peyer’s patches
a b s t r a c t
Two pectic (chPS-L1, chPS-L2) and one polyphenolic (chPP-L) fractions were obtained from lavender flowers
after boiling water extraction, exhaustive removing of alcohol-soluble molecules and SEC. chPS-L1
(52.4 kDa) contains mainly low-acetylated and high-methoxylated homogalacturonans (HG), and smaller
rhamnogalacturonan (RG) I backbone fragments rich in 1,3,5-branched arabinan and arabinogalactan
(AG) II side chains. chPS-L2 (21.8 kDa) contains predominantly similarly esterified HG, followed by RGI
with AGII structures and RGII. The prevalence of catechin and epicatechin in chPP-L indicates that they
form weak interactions with pectins. chPS-L1 and chPS-L2 enhanced ß2-integrin expression on neutrophils,
inducing ROS generation and macrophage NO production. Both the effects on ß2-integrin and
high complement fixation activity of chPS-L1 were proposed for its inhibitory action against PMA- and
OZP-activated ROS formation. This, together with suppression of NO generation after co-stimulation
with chPS-L1 and LPS, suggested anti-inflammatory activity of studied pectins. Lavender polysaccharides
expressed intestinal Peyer’s patch immunomodulating activity.
© 2017 Elsevier Ltd. All rights reserved.
Abbreviations: AGII, arabinogalactan type II; AUAC, anhydrouronic acid content; BSA, bovine serum albumin; CD18, marker for ˇ2integrin; CD62L, marker for L-selectin;
chPP-L, polyphenolic fraction obtained after SEC from lavender PSC2; chPS-L1, first fraction of SEC purified lavender pectic polysaccharides; chPS-L2, second fraction of
SEC purified lavender pectic polysaccharides; CL, chemiluminescence; CR3, omplement receptor 3; DA, degree of acetylation; DM, degree of methoxylation; DSS, sodium
4,4-dimethyl-4-silapentane-sulfonate; fMLP, N-formylmethionyl-leucyl-phenylalanine; FTIR, Fourier transform infrared spectroscopy; HBSS, Hank’s buffered salt solution;
HG, homogalacturonan; IC50, a concentration giving 50% hemolysis of erythrocytes; iNOS, inducible nitric oxide synthase; KDO, 3-deoxy-d-manno-2-octulosic acid; Lavender
PSC, crude pectic polysaccharide complex from Lavandula angustifolia; Lavender PSC2, obtained from lavender PSC after exhaustive ethanol treatment; LDH, lactate dehydrogenase;
LPS, lipopolysaccharides; NMR, Nuclear magnetic resonance spectroscopy; NO, nitric oxide; OZP, opsonized zymosan particles; PBS, phosphate-buffered saline;
PMA, phorbol 12-myristate 13-acetate; PP, Peyer’s patches; RGI, rhamnogalacturonan type I; ROS, reactive oxygen species; SEC, size-exclusion chromatography; WBP, whole
blood phagocytes.
∗ Corresponding author at: Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139
Ruski Blvd., 4000 Plovdiv, Bulgaria. Tel./fax.: +359 32 64 27 59.
E-mail address: lbas@plov.omega.bg (M.G. Kratchanova).
http://dx.doi.org/10.1016/j.carbpol.2017.07.011
0144-8617/© 2017 Elsevier Ltd. All rights reserved.
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 949
1. Introduction
In Bulgaria common lavender, Lavandula angustifolia Miller
(Lamiaceae) is cultivated predominantly in the Rose valley and
since 2011 the country is the biggest lavender oil producer worldwide.
Already Dioscorides (c. 40–90 AD) documented that lavender
has laxative and invigorating properties and advised its tea for chest
complaints (Castle & Lis-Balchin, 2002). The German Commission E
has approved L. angustifoliatea for treatment of restlessness,insomnia
and nerve disorders of the intestines. The lavender flowers
express also anti-microbial, repellent, anti-inflammatory, antioxidant
and diuretic activities, which are mainly attributed to the
essential oils (Laekeman, 2012). However, the lavender contains
also non-volatile biologically active constituents, such as triterpenoids,
flavonoids and lignans (Harborne & Williams, 2002).
In a previous study, it was shown that a crude pectic polysaccharide
complex (lavender PSC) from L. angustifolia expresses
ex vivo immunomodulating activity against phagocytic leukocytes,
T-lymphocytes and intestinal Peyer’s patch cells (Georgiev,
Ognyanov, Kiyohara et al., 2017). Pectic polysaccharides express
different bioactivities outside the plants when they are properly
extracted. Pectins contain three main building blocks − HG, RG type
I and II and substituted galacturonans (Voragen, Coenen, Verhoef,
& Schols, 2009). Generally, the immunomodulating activity of pectic
polysaccharides is explained by the presence of immune active
AG type I and II structures O-4 attached to Rha of the RGI domain
(Yamada & Kiyohara, 2007; Wangensteen, Diallo, & Paulsen, 2015).
Additionally, in a recent study we found that highly glucuronidated
RGI isolated from Tilia tomentosa also exhibits immunomodulating
activity (Georgiev, Paulsen et al., 2017). Immune cells express
signaling receptors, such as complement receptor 3 (CR3), Fc receptor,
toll-like receptors and scavenger receptors that can interact
with polysaccharides to initiate up- or down-regulation of numerous
immunological responses (Leung, Liu, Koon, & Fung, 2006).
The immunomodulating properties of pectins are shown by exhibition
of moderate complement fixation activity, activation and
regulation of phagocytic leukocytes, B- and T-lymphocytes, thrombocytes,
suppression of tumor cells, etc (Yamada & Kiyohara, 2007;
Wangensteen et al., 2015). Because of these effects, pectins are
included in in vivo studies and some clinical trials for their supplementary
role for treatment of socially significant diseases, as
reviewed by Georgiev, Ognyanov, Denev et al. (2017). Furthermore,
together with smaller molecules, pectic polysaccharides are
partly extracted during preparation of lavender teas or decoctions.
In this way, polysaccharides are coupled with naturally
occurring compounds like polyphenols, forming bioactive mixtures
with combinatorial effects (Jakobek, 2015). At the same time, the
increasing rate of lavender cultivation for perfume and healthcare
industries expands development of different lavender-derived
products and needs a proper utilization of the waste after distilla-
tion.
The aim of the current study is to characterize the chemical
structure and immunomodulating properties of L. angustifolia pectic
polysaccharides purified from the immune active crude lavender
PSC obtained with boiling water (Georgiev, Ognyanov, Kiyohara
et al., 2017).
2. Materials & methods
2.1. Plant material
Lavender flowers from Lavandula angustifolia Miller (syn. L. vera
DC) sort “Sevtopolis” (Institute of Roses, Essential and Medical Cultures,
Kazanlak, Bulgaria) were provided dried by ET “Ve Pe Pi −
Vesko Pipev” (Velingrad, Bulgaria).
2.2. Isolation and purification of lavender flower polysaccharides
The crude lavender polysaccharide complex (lavender PSC) was
extracted with boiling water, as previously described (Georgiev,
Ognyanov, Kiyohara et al., 2017).
Lavender PSC was dissolved in water (10 mg/mL) and precipitated
five times (each for 24 h) with three volumes of
95% ethanol. This step was for partly removing of ethanolsoluble
molecules prior to size exclusion chromatography (SEC).
After each run, the polysaccharide-containing precipitates were
obtained by centrifugation at 4000 rpm for 30 min at 20 ◦C. Then,
the recovered polysaccharide-containing fraction (lavender PSC2)
was dissolved in water, centrifuged and the supernatant was
applied to a Sephacryl
®
S-200 Superfine (GE Healthcare Life Sciences)
column (100 × 1.5 cm) coupled with a FPLC BioLogic
®
LP
system (BioRad). The sample was eluted with 0.2 M NaCl at
0.2 mL/min flow rate and 5 mL fractions were collected. Two pectic
fractions chPS-L1 and chPS-L2, and one polyphenolic fraction
chPP-L were obtained. The carbohydrate peaks were visualized
by the m-hydroxydiphenyl and phenol-sulfuric acid methods,
and polyphenols by the Folin-Ciocalteu reagent, and UV–Vis fingerprinting
(200–800 nm). The chPP-L fraction was concentrated
under vacuum to 50 mL and loaded on an Amberlite
®
XAD7HP
(Sigma-Aldrich) column (12 × 3 cm), followed by column elution
with 200 mL water and the polyphenols were eluted with 150 mL
95% ethanol. The polyphenolic fraction was evaporated to dryness
under vacuum, dissolved in water and freeze-dried. chPS-L1 and
chPS-L2 were concentrated under vacuum, dialyzed against water
(MWCO 12–14 kDa, 72 h), filtered through 0.2 ␮m cellulose nitrate
filters (Sartorius) and freeze-dried. The three samples were stored
in a desiccator in dark.
2.3. General methods
Anhydrouronic acid content (AUAC) was determined by the
m-hydroxydiphenyl method in Skalar San++ autoanalyzer (Analytical
BV, Breda, The Netherlands), using galacturonic acid as a
standard (Blumenkrantz & Asboe-Hansen, 1973). Total amount
of sugars was quantified by the phenol-sulfuric acid method,
using 1:1 mixture of glucose and galacturonic acid as a standard
(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Degree of
methoxylation (DM) was determined by alcohol oxidase (P. pastoris,
Sigma-Aldrich) and Purpald
®
(Sigma-Aldrich) (Anthon & Barrett,
2008). Degree of acetylation (DA) was measured according to
МcComb and McCready (1957), using ˇ-d-glucose pentaacetate as a
standard. The relative molecular weight was determined by HPSECRID
(Waters) with Shodex standard P-82 kit (pullulan standards,
Showa DENKO, Tokyo, Japan) (Georgiev, Ognyanov, Yanakieva,
Kussovski, & Kratchanova, 2012). Total phenolics (gallic acid standard)
and protein content (BSA standard) were analyzed according
to Singleton and Rossi (1965) and Bradford (1976), respectively.
2.4. Monosaccharide composition and glycosidic linkage
determination
Polysaccharide samples (1 mg) with added 100 ␮g mannitol as
an internal standard were subjected to methanolysis with anhydrous
3 M HCl in methanol for 24 h at 80 ◦C. The formed methyl
glycosides of different monosaccharides were converted into their
corresponding TMS derivatives and analyzed by GC-FID on a Focus
GC system (Thermo Scientific, Milan, Italy), according to Chambers
& Clamp (1971) and Barsett, Paulsen, & Habte (1992). The presence
of KDO (3-deoxy-d-manno-2-octulosic acid) was detected by
the thiobarbituric acid assay (York, Darvill, McNeil, & Albersheim,
1985).
950 Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959
The glycosidic linkage composition of chPS-L1 (2 mg) was examined
after methylation. Prior to methylation uronic acids in the
sample were reduced with NaBD4 to 6,6 -dideuterio-sugars that
could be differentiated from the neutral sugars (Kim & Carpita,
1992). The procedure was completed by methylation, acid hydrolysis
and preparation of alditol acetates, partly as described by
Ciucanu & Kerek (1984). The hydrolysis was performed with 2.5 M
TFA for 2 h at 100 ◦C. The partly methylated alditol acetates were
analyzed by a GC-EI/MS-QP2010 system (Shimadzu, Kyoto, Japan),
coupled with a Restek Rxi-5MS column (30 m, 0.25 mm i.d., 0.25 ␮m
film) in a Scan mode (40–450 ions), as described by Ho, Zou,
Aslaksen, Wangensteen, and Barsett (2016). The detection and
quantification of the relative amounts of each glycosidic linkage
type was based on the chromatographic behavior (RT), ionization
patterns and results from the monosaccharide composition.
The presence of AGII was detected by precipitation with the
Yariv ß-glucosyl reagent (Van Holst & Clarke, 1985).
2.5. Contamination with lipopolysaccharides (LPS)
Possible LPS contamination was checked by the detection of 3hydroxy
fatty acids as their specific chemical markers in the form of
acetylated fatty acid methyl esters on the GC-EI/MS-QP2010 system
(Shimadzu, Kyoto, Japan), coupled with a Restek Rxi-5MS column
(30 m, 0.25 mm i.d., 0.25 ␮m film) in a SIM mode (257 ion intensity
for calculation), as described by de Santana-Filho et al. (2012) and
Ho et al. (2016).
2.6. Fourier transform infrared spectroscopy (FTIR)
Infrared (IR) spectra of chPS-L1 (2 mg) were obtained on a
NicoletTM Avatar (Thermo Fisher Scientific, Waltham, USA) spectrometer
in a KBr tablet. The spectra were recorded in the range
4000 and 400 cm−1 and analyzed by the SpectraGryph software
(Friedrich Menges).
2.7. Nuclear magnetic resonance (NMR) spectroscopy
chPS-L1 (12 mg) was dissolved in D2O, freeze-dried and redissolved
in 0.7 mL D2O. 0.5 mL of chPS-L1 was investigated by the
following NMR experiments. 1D 1H, 13C, DEPT 135 and 2D 1H/13C
HSQC, 1H/1H COSY, 1H/1H TOCSY, 1H/1H ROESY, 1H/1H NOESY,
13C/1H HMBC experiments were performed on a Bruker Avance
III HD Ascend 800 MHz spectrometer operating at 800.03 MHz
(Bruker, Fällanden, Switzerland) with the Bruker TopSpinTM 3.5
software at a temperature of 60 ◦C. The chemical shifts were
referenced to the internal standard DSS (sodium 4,4-dimethyl-4-
silapentane-sulfonate).
2.8. Immunomodulatory effect
2.8.1. Ethics
Blood sampling procedure was in accordance with the Helsinki
Declaration of 1975, as revised in 1983. Animal experiments were
approved by the Animal Research Committee of the Kitasato University,
and performed in accordance with the Guidelines for Care
and Use of Laboratory Animals at the Kitasato University and the
National Research Council Guide for the Care and Use of Laboratory
Animals in Japan.
2.8.2. Animal procedure and care
Female C3H/HeJ mice (6–8 weeks old) were purchased from
Japan SLC (Shizuoka, Japan), and male ICR mice (4 weeks old) were
from Charles River (Tokyo, Japan). Mice were housed in plastic
cages in an air-conditioned room at 23 ± 2 ◦C with a relative humidity
of 55 ± 10% under a 12 h-light-dark cycle, and fed a standard
laboratory diet with water given ad libitum.
2.8.3. Complement fixation assay
The inhibition of complement hemolysis (complement fixation)
of target antibody sensitized sheep red blood cells (classical
pathway) or rabbit erythrocytes (alternative pathway) by normal
porcine serum pre-treated with chPS-L1, chPS-L2 or chPP-L
was studied. The analyses were performed according to Klerx,
Beukelman, Dijk, and Willers (1983) and Klerx et al. (1985), as
described by Georgiev et al. (2012). The complement fixation activity
induced by the test samples was calculated on the basis of
a colorimetric measurement of hemoglobin released from lysed
cells after incubation with PS-treated normal porcine serum by the
formula: [Acontrol−Atest]/Acontrol × 100%. From these data a doseresponse
curve was constructed and the concentration of test
sample giving 50% inhibition of hemolysis (IC50) was calculated
(Fig. S2). The samples were analyzed in triplicates.
2.8.4. Reactive oxygen species (ROS) production from human
whole blood phagocytes (WBP) and isolated neutrophils
Heparinized (50 IU/mL) blood samples were obtained from the
cubital vein of healthy human volunteers after overnight fasting.
The isolation of human neutrophils from venous blood was accomplished
according to the procedure of Nauseef (2014). The kinetics
of ROS production by WBP and isolated neutrophils was analyzed
by luminol-enhanced chemiluminescence (CL) for a period
of 60 min at 37 ◦C, using 96-well white flat bottom culture plates
on luminometer (Orion II Berthold Detection Systems GmbH,
Germany). The principle of the method was previously described
(Vasicek, Lojek, Jancinova, Nosal, & Ciz, 2014). Briefly: 25 ␮L of ten
times diluted blood with Hank’s buffered salt solution (HBSS) or
0.2 × 106 cells/well of isolated neutrophils in phosphate-buffered
saline (PBS) were mixed with 25 ␮L 1 mM luminol (10 mM stock
solution in 0.2 M borate buffer) HBSS solution and incubated with
HBSS solutions of chPS-L1, chPS-L2 or chPP-L (50 and 100 ␮g/mL)
for spontaneous (non-stimulated with known activators) CL. Furthermore,
12.5 ␮L or 5 ␮L of ten times diluted blood (or 0.2 × 106
cells/well) mixed with 25 ␮L luminol were used for incubation of
each fraction with 25 ␮L (0.5 ␮g/mL) phorbol myristate acetate
(PMA-activated, Sigma-Aldrich) or 25 ␮L (62.5 ␮g/mL) opsonized
zymosan particles (OZP-activated). The final volume for spontaneous,
PMA- and OZP-activated CL tests was 250 ␮L. The recorded
values included the intensity of CL emitted during the studied time
interval (integral of CL). The results are expressed as percentage of
the control (mean ± SEM) from five independent experiments.
2.8.5. Determination of the cell surface expression of adhesion
molecules
The measurements were performed according to the method
described by Gallova, Kubala, Ciz, and Lojek (2004). Briefly, isolated
human neutrophils (1 × 105 cells) were incubated in plastic
tubes (Falcon, USA) with chPS-L1 or chPS-L2 (100 ␮g/mL) for 1 h
and with the activators fMLP (N-formyl-met-leu-phe, 0.1 ␮M) and
PMA (0.01 ␮g/mL) for 10 min. After incubation anti-CD18 (anti-ˇ2integrin)
and anti-CD62L (anti-l-selectin) monoclonal antibodies
(5 ␮L of each) were added. The samples were incubated at 4 ◦C
for 15 min, subsequently centrifuged, re-suspended in PBS and
analyzed by a flow cytometer FACSVerseTM (Becton Dickinson,
USA). The median fluorescence intensity was determined and corrected
for unspecific staining by subtracting the fluorescence of
cells stained with the control antibody (isotype control). The data
are expressed as CD62L−/CD18+ population (mean% of total leukocytes
± SEM) from five independent experiments.
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 951
2.8.6. Nitric oxide (NO) production from murine macrophages
Murine macrophage cell line RAW 264.7 (ATCC, USA) was
incubated in 12-well flat bottom culture plates at 1 × 105
cells/well with chPS-L1 or chPS-L2 (50 and 100 ␮g/mL) alone
in DMEM-high Glc supplemented with 10% (v/v) FBS and 1%
(v/v) penicillin/streptomycin (500 ␮L final volume) (HyCloneTM, GE
Healthcare Life Sciences) for 24 and 48 h at 37 ◦C and 5% CO2. The
cells were also cultured by combination of polysaccharides with
10 ng/mL LPS (E. coli/026:B6, Sigma-Aldrich). The generation of NO
was determined indirectly as the accumulation of nitrites in the
cell culture supernatants. At the end of the incubation period the
culture media were collected and centrifuged at 16 000 × g, 4 ◦C
for 10 min. Then, 150 ␮L of supernatants were mixed with an equal
volume of Griess reagent (Sigma-Aldrich) in a 96-well plate and the
mixture was incubated at room temperature for 30 min in dark. The
absorbance was measured at 546 nm and NaNO2 (0–52 ␮M) was
used as a standard (Lojek et al., 2011). The results are expressed as
mean ± SEM from three independent experiments.
2.8.7. Western blot analysis of inducible nitric oxide synthase
(iNOS)
RAW 264.7 cells, which were obtained as described in 2.8.6.,
were lysed in the lysis buffer (1% SDS, 0.1 M Tris pH 7.4, 10%
glycerol, 0.001 M sodium o-vanadate, 0.001 M PMSF). The protein
concentrations were determined by using the BCATM protein assay
(Pierce, USA), with BSA as a standard. Equal amounts of protein
(18 ␮g) were then subjected to SDS-PAGE, using 10% separating
gel. The expression of iNOS protein was quantified by a Western
blot analysis (Pekarova et al., 2011). Anti-iNOS/NOS Type II
mouse monoclonal antibody (Cell signaling Technology, 1:5000)
and ECLTM anti-rabbit IgG horseradish peroxidase linked whole
antibody (from goat; Cell Signaling Technology, 1:2000) were used.
The immunoreactive bands were detected using an ECLTM detection
kit (Pierce) and exposed to a radiographic film (AGFA). The relative
protein levels were quantified by scanning densitometry, using the
ImageJTM programme, and the individual band density value was
expressed in arbitrary units. The results are shown as mean ± SEM
from three independent experiments.
2.8.8. Cytotoxicity assay
Cytotoxicity was estimated by lactate dehydrogenase (LDH)
activity in the cell culture supernatants of RAW 264.7 cells, as
obtained in 2.8.6., using the LDH Cytotoxicity Detection KitPLUS
(Roche Applied Science, Switzerland). The results are expressed as
mean ± SEM from three independent experiments (Fig. S4).
2.8.9. Immunomodulating activity against murine Peyer’s patch
(PP) cells
Immunomodulating activity was measured by the enhanced
production of bone marrow cell-proliferative cytokines from PP
cells of pathogen-free C3H/HeJ mice (Tlr4Lps−d), according to Hong,
Matsumoto, Kiyohara, and Yamada (1998). Suspensions (1.2 × 106
cells/mL, 180 ␮L) of PP cells (5–9 patches obtained per mouse)
from the small intestine of C3H/HeJ mice were cultured with H2O
(negative control), AMOL-1 (pectic polysaccharide from Astragalus
mongholics as a positive control, Kiyohara et al., 2010) or 100 ␮g/mL
of the pectic fractions (20 ␮L) in a 96-well flat bottom culture plate
(FALCON 3072) for 6 days at 37 ◦C in a humidified atmosphere of
5% CO2–95% air. The resulting culture supernatants (50 ␮L) were
further cultured with a bone marrow cell suspension (5.0 × 105
cells/mL) from male ICR mice for 6 days. The numbers of proliferated
bone marrow cells were measured by Alamar BlueTM, as
described previously (Hong et al., 1998). The results are shown as
mean ± SEM.
2.9. Statistical analysis
The statistical significance was analyzed by the Student’s ttest,
using the Statview software (SAS Institute, Inc.). The mean
results ± SEM were compared to those from the control. Values of
p < 0.05 were considered as statistically significant.
3. Results
3.1. Purification and structural characterization of lavender
pectic polysaccharides
3.1.1. Purification and chemical composition of lavender fractions
After the ethanol pretreatment of lavender PSC, the obtained
lavender PSC2 having 41% AUAC and low protein content (2.9%)
was subjected to SEC (Fig. 1(A)). The presence of non-carbohydrate
compounds with diene and aromatic structure (e.g. polyphenols,
fatty acids, proteins) in all fractionation tubes was estimated by
reading of the absorbance at 254/280 nm. This in addition to the
UV–Vis fingerprinting of selected tubes helped for determination
of two polysaccharide and one polyphenolic fractions. As can be
seen from Fig. 1(B) the three lavender fractions are distinguished
by the intensity of two domains at 201–202 nm and around 280 nm.
The first domain originates from different ␲-bonds, such as C O of
carboxyl group in pectin (202 nm) and other compounds and the
second has ␭max=276 nm typical for catechin and tannins.
The total yield of the two polysaccharide fractions in dried lavender
flowers is 1.6% (w/w). The monosaccharide composition of
these fractions shows that they contain mainly pectic polymers
with the highest content of GalA, followed by Gal, Ara and Rha
(Table 1). This result is in agreement with the relatively high AUAC
between 41 and 45%. chPS-L1 and chPS-L2 give a cross-reaction
with the ß-glucosyl Yariv reagent, which indicates that both fractions
contain 3,6-linked galactans or AGII. The reaction ring for
chPS-L1 is more intensive, thus it has higher AGII content than chPSL2
(gels not shown). Furthermore, the presence of small amounts
of GlcA, Xyl and Fuc, which are specific for some pectic side chains
are detected. The presence of the rare monosaccharides 2-OMeXyl
and 2-OMeFuc in chPS-L2 indicated that it contains RGII, which
was further confirmed by the presence of KDO. Additionally, negligible
contamination with other polysaccharides containing Man
and Glc is also found. Both polysaccharides are classified as high
methoxylated pectins with DA <20%. As was expected chPS-L2
(2.18 × 104 Da) has lower molecular weight (relative to pullulan)
than chPS-L1 (5.24 × 104 Da) and higher total polyphenol content
(see Fig. 1(A) & (B)).
Direct SEC fractionation of lavender PSC2 was chosen because
of the interest for identification of some polyphenols retained associated
to pectins after an exhaustive extraction of ethanol-soluble
molecules from lavender PSC. The yield of chPP-L is 9.2% from lavender
PSC2. Interestingly, 10 phenolic acids and 8 flavonoids were
identified in minor amounts in chPP-L (Table S1). Ferulic acid, protocatechuic
acid, quercetin, catechin and epicatechin are presented
in the highest concentrations. For comparison, in the alcoholsoluble
fraction obtained from lavender PSC, only the catechin
(385.4 ␮g/100 mg) and epicatechin (497.7 ␮g/100 mg) contents are
much lower than in chPP-L based on the identical polyphenols
detected in the two samples. Both flavonoids are ethanol-soluble,
thus weak interactions with pectic polysaccharides might occur
and they could be broken during the saline SEC fractionation.
3.1.2. Glycosidic linkage determination
The quantitative glycosidic linkage analysis of chPS-L1 indicated
that it contains mainly 1,4-linked GalpA (47.3 mol%) with
considerably lower amounts of 1,2- and 1,2,4-linked Rhap. It is
952 Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959
Fig. 1. Purification and fractionation of lavender pectic polysaccharides. (A) Elution profile of lavender PSC2 on Sephacryl S-200. (B) UV–Vis fingerprinting analysis of obtained
pectic (chPS-L1 and L2) and polyphenolic (chPP-L) fractions. TS (total sugars), TPP (total polyphenols).
rich in AGII structures (26.7 mol%), shown by 1,3,6-linked Galp
(6.9 mol%), T-Araf (5.6 mol%) and 1,5-linked arabinan (3.3 mol%)
(Table 2). chPS-L1 is a pectic type polymer composed mainly of HG
with a smaller RGI backbone fragment, containing 1,3,5-branched
arabinan and highly branched AGII side chains. The molar ratio
1,2:1,2,4-linked Rhap in chPS-L1 is 41:59, indicating a high degree
of branching on the Rhap present in RGI. The 3,6-linked Gal and 1,5arabinans
in the Rha side chains are 25.8 and 12.4 mol% calculated
from the total galactan and arabinan structures. The predominance
of 3,6-linked Gal is in a good agreement with the Yariv’s test results.
chPS-L1 is also characterized with a minor amount of 1,3,4-linked
GalpA, which probably indicates the presence of some substituted
galacturonan, such as RGII or others. The other uronic acid in the
fraction − GlcA is T-, 1,2- and 1,2,4-linked most probably in AG side
chains (Yamada & Kiyohara, 2007). Additionally, the methylation
analysis showed that Glc in chPS-L1 is T- and 1,4-linked probably
in an independent glucan polymer, such as starch. Concluding,
HG (GalA − Rha) and RGI (2Rha + Ara + Gal) content in chPS-L1 are
roughly estimated to be 47.6 mol% and 39.1 mol%, respectively
(Table 1) (Denman & Morris, 2015).
3.1.3. FTIR analysis
The IR spectrum of chPS-L1 (Fig. S1) contains the typical bands
for polysaccharides and pectins, as follows: 3408, 2935, 1743, 1616,
1331, 1146 and 1101 cm−1, arising from: O H of hydroxyl groups,
C H of CH2, C O of methoxyl ester group (COOCH3), C O of ionized
(free) carboxyl group, C H in the ring, C O C of the glycosidic
bond (or within the ring) and C C or C O in the ring, respectively
(Synytsya, ˇCopíková, Matˇejka, & Machoviˇc, 2003). According to the
formula: [A1743/(A1743 + A1616)] × 100, where A is the area of the
corresponding signal, the DM of chPS-L1 is 61.4%. This result is in
agreement with the already determined DM of 63.4% (Table 1).
Additionally, the signal at 1373 cm−1 is attributed to ıs(CH3) of
methoxyl ester and two overlapped bands at 1242–1259 cm−1 originate
from (C O C) of acetyl esters (Synytsya, ˇCopíková, Matˇejka
et al., 2003). The signals at 1146, 1101 and 1018 cm−1, as well as the
peaks at 833 (C1-OH, ˛-anomer), 764, 638 and 536 cm−1 confirm
the predominance of the HG structure in the studied polysaccharide
(Kaˇcuráková, Capek, Sasinková, Wellner, Ebringerová, 2000).
The presence of arabinogalactorhamnoglycan structure or the hairy
region in chPS-L1 is demonstrated by the characteristic signals at
1078, 1050 and 914 cm−1 (Kaˇcuráková et al., 2000).
3.1.4. NMR structural studies
In Table 3 are presented 13C and 1H assignments for the
identified structural fragments of chPS-L1 found from specific
correlations in the HSQC spectrum, as well as from the COSY,
TOCSY, ROESY and HMBC spectra. The NMR result interpretation
was done with the help of literature data and in accordance
with the monosaccharide and glycosidic linkage compositions
(Shakhmatov, Atukmaev, & Makarova, 2016). The absolute configurations
were assumed to be the same as already found for pectins
(McNeil, Darvill, Albersheim, 1980). The detailed NMR study confirmed
that chPS-L1 is a pectic polysaccharide with a predominant
HG fragment with existence of methoxyl and acetyl esters, followed
by a RGI branched with arabinans and AGII.
The presence of a HG fragment, revealing the 1,4-linked ˛d-polygalacturonic
acid in chPS-L1 is shown by the correlations
H1/H4 of ˛-d-GalpA-6-OMe units at 4.95/4.45 ppm (A) and H1/H4
of ˛-d-GalpA units at 5.10 (5.05)/4.45 ppm (B) in the ROESY spectrum
(Fig. 2). This was also proved by through bond correlations
in the COSY spectrum. The C/H correlations at 55.5/3.81 ppm for
methyl carbon and proton of CH3O group, at 22.8/2.08 ppm and
22.5/2.17 ppm for methyl carbon and proton of CH3CO group in the
HSQC spectrum show the presence of methoxyl and acetyl esters in
chPS-L1. The intensive through space correlations between the CO
signal at 173.4 ppm and the CH3O group at 3.81 ppm, as well as the
cross-peaks at 173.4/5.05 ppm and 173.4/5.10 ppm in the HMBC
spectrum indicate that the methoxyl groups are located at C6 of
˛-d-GalpA units in the HG. The calculated DM by NMR is 62%, using
the introduced by Synytsya, ˇCopíková, and Brus (2003) formula:
DM = ACOOCH3
/AC-6 tot. × 100 (%) (1)
where A is the peak area and AC-6 tot. includes the combined area
of the C6 peak regions of GalA. The ratio between the integral
intensities of the proton signals of CH3CO- at 2.08 and 2.17 ppm
is 69:31. According to the literature data the peak at 2.08 ppm is
attributed to O-3 acetylation and this at 2.17 ppm to O-2 acetylation
of GalA in the HG (Perrone et al., 2002). The CO signal of the acetyl
group attached at O-3 of GalA was determined with the help of a
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 953
Fig. 2. 1
H/1
H ROESY spectrum of chPS-L1 pectic fraction. Each letter (see Section 3.1.4.) corresponds to important correlations in the HG fragment (A–D); RGI backbone
(E–G); 1,3-Gal chain (H–J); 1,6-Gal (N–P); 1,3,6-Gal (K–M); 1,5-Ara (Q–R); 1,3,5-Ara (S–U).
Table 1
Yields and chemical characterization of lavender pectic fractions obtained after SEC
of lavender PSC2.
chPS-L1 chPS-L2
Yield [% of lavender
PSC]
26.9 19.6
Yield [% of lavender
PSC2]
35.2 25.6
Yield [% of dried
flowers]
0.9 0.7
Total sugars [%] 73.7 58.5
AUAC [%] 40.8 44.9
DMa
[mol%]
(Methoxyl
content [%])
63.4
(4.7)
61.0
(5.0)
DAa
[mol%]
(Acetyl content
[%])
16.8
(1.7)
15.3
(1.7)
Protein content [%] 1.1 4.2
Total polyphenols
[%]
0.7 2.1
LPS [%] n.f. n.f.
Monosaccharide composition [w/w%]
Ara 10.1b
(12.2)c
4.6 (5.7)
Rha 5.6 (6.2) 3.7 (4.2)
Fuc 0.7 (0.8) 0.9 (1.0)
Xyl 2.8 (3.4) 2.1 (2.6)
Man 1.7 (1.7) 0.5 (0.5)
Gal 14.3 (14.5) 6.5 (6.7)
Glc 4.7 (4.7) 3.7 (3.8)
GlcA 2.9 (2.8) 1.5 (1.4)
GalA 57.3 (53.8) 76.6 (73.9)
2-OMeXyl n.f. +
2-OMeFuc n.f. +
Kdo n.d. ++
AGII ++ +
Relative molecular
weight [Da]
5.24 × 104
2.18 × 104
n.d. − not determined. n.f. − not found.
a
Calculated on AUAC basis, moles of methoxyl or acetyl groups per 100 mol of
GalA.
b
ww%, calculated on total carbohydrate content basis.
c
mol% of total carbohydrate content.
Table 2
Glycosidic linkage composition of chPS-L1 fraction.
Glycosidic linkages chPS-L1
T-Araf 4.6a
(5.6)b
1,2-Araf trace
1,3-Araf 1.4 (1.7)
1,5-Araf 2.8 (3.3)
1,3,5-Araf 1.3 (1.5)
T-Rhap 1.4 (1.6)
1,2-Rhap 1.7 (1.8)
1,4-Rhap 0.1 (0.2)
1,2,4-Rhap 2.4 (2.6)
T-Galp 3.3 (3.4)
1,3- Galp 2.0 (2.1)
1,6-Galp 2.1 (2.2)
1,3,6-Galp 6.8 (6.9)
T-Glcp 1.9 (1.9)
1,4-Glcp 2.8 (2.8)
T-GlcpA 0.7 (0.6)
1,4-GlcpA 1.7 (1.7)
1,2,4-GlcpA 0.5 (0.5)
T-GalpA 4.7 (4.5)
1,4-GalpA 50.4 (47.3)
1,3,4-GalpA 2.2 (2.0)
a
w/w%, calculated on total carbohydrate content basis.
b
mol% of total carbohydrate content.
correlation at 176.2/2.08 ppm in the HMBC spectrum. The proton
sequence within ˛-d-GalpA-3-OAc unit was determined according
to Perrone et al. (2002) by the following correlations: 2.08/4.52 ppm
(C) and 2.08/4.72 ppm (D) in the ROESY spectrum, revealing interactions
between the CH3CO- with H4 and H5 of GalA, respectively;
176.2/4.09 ppm and 176.2/4.52 ppm in the HMBC spectrum, showing
through space interaction between the CH3CO- with H2 and H4
of GalA; finding of the adjacent protons in the COSY spectrum and
checking the correlations in the TOCSY spectrum. The gross calculation
of DA of chPS-L1 by NMR is 15.4%, using the formula (Synytsya,
ˇCopíková, Matˇejka et al., 2003):
DA = AOCOCH3
/AC- 6 tot. × 100 (%) (2)
954 Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959
Table 3
13
C and 1
H NMR chemical shift assignment (ı in ppm) of chPS-L1 pectic fraction, referenced to DSS.
Residue C-1 C-2 C-3 C-4 C-5 C-6 CH3COb
H-1 H-2 H-3 H-4 H-5:5 H-6:6
→4)-˛-GalpA-6-OMe-(1→ 102.7 70.6 71.4 81.5 73.2 173.4a
4.95 3.72 4.00 4.45 5.10; 5.05
→4)-˛-GalpA-3-OAc-(1→ n.d. 71.6 73.5 79.3 74.0 177.2 22.8
5.31 4.09 5.14 4.52 4.72 2.08
→4)-˛-GalpA-(1→ 102.0 70.6 71.4 81.5 74.0 177.2
5.10 3.75 4.00 4.45 4.72
→4)-˛-GalpA-(1 → 2 100.3 71.4 n.d. 81.5 74.4 177.4
5.02 3.93 4.13 4.45 4.69
→2)-˛-Rhap-(1 → 101.7 79.3 71.9 74.7 71.9 19.2
5.25 4.13 4.00 3.44 3.72 1.25
→2,4)-˛-Rhap-(1 → 101.7 79.3 72.6 82.8 72.1 19.4
5.25 4.13 4.09 3.67 3.82 1.30
˛-Araf-(1→ 111.8 84.0 79.4 86.7 64.1 –
5.25 4.21 3.98 4.09 3.72; 3.81
→3,5)-˛-Araf-(1→ 110.2 n.d. 85.1 83.6 69.6 –
5.10 4.28 4.13 4.29 3.81; 3.93
→5)-˛-Araf-(1→ 110.2 84.0 79.4 85.1 68.4 –
5.11 4.13 4.03 4.21 3.81; 3.87
→3)-ˇ-Galp-(1→ 106.1 72.9 84.8 71.1 n.d. 63.9
4.69 3.78 3.87 4.24 3.73 3.81
→6)-ˇ-Galp-(1→ 106.1 74.7 75.5 71.3 n.d. 63.9
4.45 3.54 3.67 3.98 3.93
→3,6)-ˇ-Galp-(1→ 106.1 73.2 83.6 71.4 76.4 72.6
4.51 3.67 4.03 4.13 3.93 3.93; 4.03
a
-OCH3 ı at 55.5/3.81 ppm.
b
CH3CO- ı at 176.2 ppm.
where AOCOCH3
presents the area of the peak at 176.2 ppm, as found
in the HMBC spectrum. This result is in agreement with the colorimetric
determination of DA (16.8%).
The following important through space cross-peaks are also
found in the ROESY spectrum (Fig. 2): H1 of 1,2(,4)-˛-l-Rhap and
H4 of 1,4-˛-d-GalpA residues at 5.25/4.45 ppm (E); H2 of ˛-l-Rhap
and H1 of ˛-d-GalpA residues at 4.13/5.02 ppm (F); H1 of ˛-l-Rhap
with H3 of ˛-d-GalpA units at 5.25/4.13 ppm (G). These correlations
confirm the presence of the RGI backbone structure [→4)-˛-dGalpA-(1→2)-˛-l-Rhap-(1→4)-˛-d-GalpA-(1
→ ]n in chPS-L1. The
ratio between the integral intensities of H6 of 1,2:1,2,4-˛-l-Rhap
gives 45:55, showing the prevalence of O-4 substituted Rha in
agreement with the glycosidic linkage results.
The anomeric proton at 4.69 ppm shows through bond
correlation peaks between H1/H3 (4.69/3.87 ppm) (H), H1/H2
(4.69/3.78 ppm) (I) and H1/H5 (4.69/3.73 ppm) (J) within ˇ-dGalp
residues in the ROESY spectrum confirming the presence
of [→3)-ˇ-d-Galp-(1→3)-ˇ-d-Galp-(1→]n fragment (Makarova,
Patova, Shakhmatov, Kuznetsov, & Ovodov, 2013). Additionally,
the through space interaction between C1 and H6 of ˇ-d-Galp
units at 106.1/3.93 ppm in the HMBC spectrum demonstrates 1,6glycosylation
in the galactan chains. The presence of 3,6-galactan
structure was elucidated by the COSY correlations between the
adjacent protons and the following interactions in the ROESY
spectrum: H1/H3 (4.51/4.03 ppm) (K), H1/H2 (4.51/3.67 ppm) (L)
and H1/H6 (4.51/3.93 ppm) (M) of the ˇ-d-Galp units. Similarly,
the found adjacent proton correlations for 1,6-ˇ-d-Galp units
in the COSY spectrum and H1/H2 (4.45/3.54 ppm) (N), H1/H4
(4.45/3.98 ppm) (O) and H1/H6 (4.45/3.93 ppm) (P) of the same
units in the ROESY spectrum demonstrate the presence of [→6)-ˇd-Galp-(1→6)-ˇ-d-Galp-(1→]n
fragment (Shakhmatov, Toukach,
Kuznetsov, & Makarova, 2014). Generally, the 3,6-galactans in
pectins are linked to O-4 of ˛-l-1,2,4-Rhap through the 1,3galactan
backbone (Voragen et al., 2009). The ROESY spectrum
also shows the presence of [→5)-˛-l-Araf-(1→5)-˛-l-Araf-(1 →]n
structure by the cross-peaks 5.25/3.81;3.72 ppm (Q) for H1/H5 of
the terminal Araf residues and 5.10/3.87;3.81 ppm (R) for 1,5-Araf
residues (Shakhmatov et al., 2014). Furthermore, the correlations
H1/H2 (5.10/4.28 ppm) (S); H1/H3 (5.10/4.13 ppm) (T) and H1/H4
(5.10/4.29 ppm) (U) for 1,3,5-substituted Araf units are found in
the ROESY spectrum in addition to the respective COSY correlations
(Makarova et al., 2013). This confirms the presence of
1,3,5-branching of the Araf units most probably presenting the
1,3,5-arabinans attached to O-4 of ˛-l-1,2,4-Rhap units (Voragen
et al., 2009). The presence of terminal ˛-l-Araf units linked to O-
6 and/or O-3 of ˇ-d-Galp residues within the 3,6-galactans is also
suggested because of the positive reaction of chPS-L1 with the ßglucosyl
Yariv reagent.
3.2. Immunomodulatory effect
chPS-L1 and chPS-L2 were tested for presence of LPS, which
are considered to be contaminants that might influence the biological
activity of studied compounds. It appears that there is no
LPS contamination in the two fractions because the LPS marker
3-hydroxy tetradecanoic acid (3-OH-C14:0) was not detected (de
Santana-Filho et al., 2012).
3.2.1. Complement fixation activity
The IC50 values of the pectic fractions chPS-L1 and chPS-L2 via
the classical pathway were 94.0 and 456.3 ␮g/mL, revealing the
higher activity of the first fraction (Fig. S2). The IC50 values of
chPS-L1 and chPS-L2 through the alternative pathway were 38.9
and 689 ␮g/mL, as LPS were not detected in both fractions. It was
important to be tested the complement fixation activity of the
polyphenolic fraction chPP-L, because it practically accompanies
polysaccharides in tea and water extracts. The IC50 values of chPP-L
were 86.9 and 539.4 ␮g/mL via the classical and alternative pathways,
respectively. In this case polyphenols expressed a similar
effect to the most active chPS-L1, which means that they could
also contribute to the observed polysaccharide activity in a proper
combined formulation.
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 955
Fig. 3. Effect of lavender pectic (chPS-L1 and chPS-L2) and polyphenolic (chPP-L, only in A) fractions on A) ROS production from non-stimulated (spontaneous), PMA- and
OZP-activated human whole blood phagocytes. B) ROS production from isolated neutrophils. C) Proportion of CD62L−
/CD18+
population of total human neutrophils treated
with lavender pectins. The asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) vs. control. 50 (50 ␮g/mL) and 100 (100 ␮g/mL).
3.2.2. Effects on phagocytic leukocytes
3.2.2.1. Human whole blood and isolated neutrophils.
The effect of lavender fractions on WBP and isolated neutrophils
was evaluated ex vivo by the change of CL emitted by the cells in
response to ROS formation. From both studied concentrations of
the three samples, only chPS-L1 enhanced the spontaneous ROS
generation from WBP at the higher dose of 100 ␮g/mL (Fig. 3A). Its
effect was negligible in comparison with the independent PMA and
OZP treatments alone. Under the co-stimulation with PMA, chPPL
expressed an inhibitory activity against the ROS production in
comparison with the PMA control. This inhibition was observed
also in the absence of activator and it could be explained by the
ROS-scavenging properties of the polyphenols. Interestingly, under
the co-stimulation with OZP, the three lavender samples showed an
inhibitory activity against the ROS formation, as it was statistically
significant for chPS-L2 and chPP-L at 100 ␮g/mL.
At the higher concentration, chPS-L1 and chPS-L2 slightly
increased the CL response of isolated human neutrophils, but again
their effect was much lower in comparison with the effect of PMA
and OZP activators (Fig. 3B). Because the activity of chPP-L on generation
of free radicals was predictable, it was not investigated
further. Both pectic fractions did not increase the CL response of
PMA-activated cells and chPS-L1 showed an evident inhibitory
activity. The observed inhibitory effect of chPS-L2 on the OZPtreated
WBP was also seen with isolated neutrophils, but it was not
so clearly expressed like in the blood environment. Furthermore,
both pectic fractions exhibited a statistically significant induction of
the neutrophil activity, which was accompanied with an increased
expression of CD18 and a decreased expression of CD62L (Figs. 3 C &
S3). Interestingly, the effect of chPS-L2 was much higher than those
of fMLP. It was proposed that both polysaccharide fractions could
interact with CD18. Our findings suggest that lavender pectins are
active ingredients in the crude lavender PSC, affecting ROS production
of human WBP and neutrophils.
3.2.2.2. Murine macrophages.
The effect of the lavender pectins on macrophages was estimated
in vitro by measurement the production of NO from treated cells.
The pectin behavior in a simulated macrophage infection by a costimulation
with LPS was also studied. Firstly, both polysaccharide
fractions increased in a similar manner the expression of iNOS
after 24 h of incubation (Fig. 4A). The enzyme expression in the
growth medium control was negligible, which showed that the
cells were not artificially activated by the plate surface. As was proposed,
under the co-stimulation with LPS the induction was higher
than this for pectin-treated cells. Contrary to chPS-L1, chPS-L2 did
not increase the iNOS levels of the independent polysaccharide
and combined with LPS treatments at the higher concentration of
100 ␮g/mL. After 48 h of incubation, the effects on the iNOS expression
of both fractions alone or in the combinations with LPS were
comparable (Fig. 4A). chPS-L2 again did not increase its induction
ability at 100 ␮g/mL, as the iNOS level was the highest at 50 ␮g/mL
with LPS. The enzyme expression with this fraction at the two border
points − 50 ␮g/mL alone and under the co-stimulation with LPS
at 100 ␮g/mL was almost identical.
There was no a significant difference between the activity of
the two lavender polysaccharides, concerning stimulation of NO
production after 24 or 48 h. However, chPS-L2 showed a negligible
prevalence after 24 h (Fig. 4B). Furthermore, the enhancement of
NO production induced by chPS-L1 or chPS-L2 at 100 ␮g/mL after
48 h of culturing were 9 or 6 fold (on mean value basis) higher
than the corresponding NO release after 24 h. These results demon-
956 Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959
Fig. 4. Effect of lavender pectic fractions on A) iNOS protein expression in murine macrophages Raw 264.7 cells cultured with samples alone or co-stimulated with LPS. B)
NO production from murine macrophages cultured with samples alone or co-stimulated with LPS. LPS from E. coli were used as a positive control. The asterisks indicate
statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) vs. control − untreated cells (growth medium) and (#p < 0.05, ##p < 0.01, ###p < 0.001) vs. LPS.
strate that lavender pectins similarly to other already investigated
polysaccharides could unlock macrophage activation. Additionally,
lavender pectins were not toxic for the macrophages (Fig. S4).
Under the conditions of co-stimulation with LPS and both fractions,
the NO production increased at the higher concentration of pectins
used after 24 h. After 48 h, the macrophage stimulation with chPSL1
did not lead to the same effect, as at 100 ␮g/mL the NO level
was decreased in the presence of LPS and elevated in the absence of
LPS. Generally, it was observed that combining 100 ␮g/mL lavender
pectins with 10 ng/mL LPS did not lead to a synergistic elevation of
the iNOS expression and NO production after 48 h. Interestingly, the
results from the LDH test for co-stimulation of macrophages with
100 ␮g/mL pectins and 10 ng/mL LPS after 48 h gave absorbance
values close to that from the control cells (Fig. S4).
3.2.3. Intestinal immunomodulating activity
Normally, polysaccharides are administered orally (e.g. tea,
folk remedies and food) and they can interact with the intestinal
immune system through different immunocompetent cells localized
in the PP, including the phagocytes. We investigated the
intestinal immunomodulating activity of the lavender polysaccharides
by the PP-mediated bone marrow cell proliferation test
(Fig. 5). It was found that both lavender fractions express intestinal
immunomodulating activity (p < 0.01) comparable to that of
the positive control AMOL-1. This result confirmed that lavender
pectins are involved in the exhibited PP stimulating activity of the
crude lavender PSC (Georgiev, Ognyanov, Kiyohara et al., 2017).
4. Discussion
The lavender pectic polysaccharides are chemically characterized
with predominant HG fragments that are acetylated (<20%)
and highly methoxylated (>60%), followed by smaller RGI domains,
which are extremely branched with arabinans and AGII structures.
chPS-L1 has a higher Mw and AGII content than chPS-L2,
which could explain its higher complement fixation and intestinal
immunomodulating activities (Yamada & Kiyohara, 1999; 2007).
chPS-L2 is immune active on PP cells and phagocytes and it has a
lower Mw than chPS-L1 and contains monosaccharides characteristic
for RGII. It exhibited very potent stimulating activity against
CD18 expression (Figs. 3 C & S3).
Interestingly, chPS-L1 expressed a high complement fixation
activity through both tested pathways, but it was more potent
through the alternative pathway. Therefore, it was suggested that
the complement activation by chPS-L1 was not only antibodydependant,
but it could be a result of a direct polysaccharide (AGII)
interaction with C3 component (C3b), initiating the alternative
pathway (Michaelsen, Gilje, Samuelsen, Høgåsen, & Paulsen, 2000;
Kiyohara, Matsumoto, Nagai, Kim, & Yamada, 2006). In fact, Wang
et al. (2016) found that pectic fraction Fs-8-ba2 from Forsythia suspensa
selectively interacts with C1q, C1r, C1s, C2, C3 and C9, but not
with C4 and C5 complement proteins.
It was supposed that the high complement fixation activity
of chPS-L1 would reflect on the phagocyte immune response,
because the fraction could activate antibody-initiated and direct
C3-initiated complement system (Leung et al., 2006). chPS-L1 and
chPS-L2 induced CD18 expression on isolated neutrophils (Fig. 3C).
Thus, the inhibitory effect of lavender pectins on the OZP-activated
ROS production from WBP (Fig. 3A) could be due to their direct
interaction with CD18 and the complement fixation activity developed
in the tested blood environment. The CD18 is a constructive
unit of CR3, CR4 and LFA-1 receptor expressed on leukocytes.
CR3 is expressed on phagocytes and involved in the phagocytosis
via iC3b-bound antigens. Zymosan is a complement activator
via the alternative pathway by binding to (i)C3b and it can interact
with CR3 on neutrophils inducing phagocytosis and superoxide
generation (Fearon & Austen, 1977; Ross & Vˇetviˇcka, 1993). OZP
(62.5 ␮g/mL) and the pectins (50 and 100 ␮g/mL) could compete
for the neutrophil receptor recognition. The inhibitory effect of
lavender pectins on the OZP-activated ROS generation from isolated
neutrophils was less pronounced probably because of the
lack of blood environment containing complement components
and immunoglobulins(Fig. 3B). Pectins (100 ␮g/mL) induced higher
CL responses with WBP than with isolated neutrophils. Conversely,
chPS-L1 expressed a better inhibitory effect against the PMAactivated
ROS generation with isolated neutrophils. PMA induced
the CD18 expression, as it can bind to CR3, initiating phagocytosis
and oxidative burst (Ross & Vˇetviˇcka, 1993). Thus, chPS-L1 could
compete with PMA for a direct CD18 binding.
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 957
Fig. 5. Enhancement of production of bone marrow cell-proliferating cytokines from Peyer’s patch cells cultured in presence or absence of lavender pectic fractions. The
asterisks indicate statistical significance (*p < 0.05, **p < 0.01) vs. water control. AMOL-1 is an active pectic polysaccharide from A. mongholics used as a positive control. (A)
is a picture of a Peyer’s patch.
The elevation of iNOS levels and NO production induced by
the lavender pectins confirmed that they could stimulate the
macrophage activation. Therefore, the lavender polysaccharides
are at least partly responsible for the observed enhancement
of CD14+ monocytes and CD64+ cell population (phagocytes)
provoked by the lavender PSC (Georgiev, Ognyanov, Kiyohara
et al., 2017). CD64 is a marker for Fc-␥ receptor 1 expressed
on macrophages, which can be activated by the pectic ramified
RGI regions, which are also present in the studied pectins
(Matsumoto et al., 1993). In a previous study it was found that T.
tomentosa pectins exhibit suppression-like effect on LPS-induced
iNOS expression and NO production from macrophages (Georgiev,
Paulsen et al., 2017). chPS-L1 (100 ␮g/mL) showed a clear reduction
of the NO generation under the co-stimulation with LPS in
comparison with its separate action after 48 h. According to Chen
et al. (2006), the observed phenomenon could be a result from
a pectin-LPS interaction or a competition for the receptor binding
(TLR4), but not a pectin inhibition of the LPS-signaling protein
activities in the macrophages. Gallet et al. (2009) found that pectic
hairy regions inhibiting the LPS-induced NO production in J774.2
macrophages reduced the TNF-˛ production stimulated by LPS.
Therefore, chPS-L1 may exhibit anti-inflammatory effects in the
simulated microbial infection, regulating the synthesis of inflammatory
molecules. This regulation could result in a reduction of the
NO generation, which was initially induced by chPS-L1 alone.
Pectins are little absorbed by the intestinal epithelium, but
they may interact with it and most importantly with the mucosal
immune system through immunocompetent cells in the PP. Functionally
modulated immunocompetent cells by polysaccharides are
proposed to result in modulations of several mucosal and systemic
immune systems (Yamada & Kiyohara, 2007). Undoubtedly,
the complement fixation activity and the stimulatory effect on the
neutrophils and macrophages of lavender pectins contributed to
the PP-mediated bone-marrow cell proliferation. However, lavender
PSC stimulated not only phagocytes, but also CD4+/CD25+ and
CD8+/CD25+ T cells that constitute about 10% and 5% of PP cells, as
5–30% of CD4+ T-cells in the mouse bone marrow are CD4+/CD25+
Treg (Georgiev, Ognyanov, Kiyohara et al., 2017; Jung, Hugot,
& Barreau, 2010; Zhao et al., 2012). Therefore, in the intestinal
immunomodulating activity of lavender pectins might be involved
also T-cells. And as well, the CD18 expression, which was found to
be induced by the lavender pectins is important for establishment
of the intestinal mucosal T-cell compartment (Huleatt & Lefranc¸ ois,
1996).
Investigation of the type of attached polyphenols to pectin
enables a better understanding of the chemistry of the polyphenolpolysaccharide
complexes and the polysaccharide affinity to
different bioactive polyphenols. In a previous study, it was found
that pectic polysaccharides in rosehip are at least partly involved
in the observed synergism in ORAC antioxidant activity after combining
rosehip with other herbal extracts (Kratchanova, Denev, &
Kratchanov, 2014). The physico-chemically attached polyphenols
to pectin have valuable physiological activities in the human gastrointestinal
tract (Jakobek, 2015). From a technological point of
view, the observed affinity of catechin and epichatechin to the studied
polysaccharides reveals the pectin potential for reduction of
catechin astringency in drinks. Interestingly, Slavov, Kiyohara, and
Yamada, (2013) showed that distilled rose petals from Rosa damascena
contain pectins with intestinal immunomodulating activity.
Méndez-Tovar, Herrero, Pérez-Magari˜no, Pereira, and Asensio-S.Manzanera
(2015) reported that a distilled lavender waste could
serve for recovery of polyphenolic antioxidants. Therefore, the
lavender by-product could be subjected to a further extraction
of bioactive non-volatile compounds for production of infusion
formulations and nutraceuticals with immunomodulating and
antioxidant activities.
5. Conclusion
The detailed chemical analysis revealed that chPS-L1 and chPSL2
are acetylated and highly methoxylated pectic type polymers
with a predominant HG fragment, followed by a RGI with AGII
structural motifs. Our findings with chPS-L1 and chPS-L2 confirmed
that lavender pectins are at least partly responsible for
the observed ex vivo and in vitro effects for lavender PSC. The
lavender pectins activated innate and adaptive immune response
through the complement system, WBP, neutrophils, macrophages
and PP immunocompetent cells. Therefore, they could be considered
as immunomodulating compounds in the lavender flowers.
The observed inhibitory effects of chPS-L1 against the PMA- and
OZP-activated ROS production, and the suppression of NO generation
under the co-stimulation between chPS-L1 and LPS suggested
anti-inflammatory activity of the studied pectins. chPP-L showed
a high complement fixation activity via the classical pathway and
was effective against the PMA and OZP inflammatory activation
of WBP. The proper formulation or individual use of lavender
polysaccharides and polyphenols could be of benefit in the supplementary
treatment of a compromised immune system and control
of inflammation. Furthermore, different immunomodulating and
antioxidant nutraceutical formulations could be developed on the
basis of the polysaccharide and polyphenol interaction.
958 Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959
Acknowledgements
This work was supported by the European Regional Development
Fund, EU [Grant No BG161PO0003-1.1.05-0024-C0001,
2012–2014], European Social Fund, EU [Grant No BG051PO001/3.3-
05-0001, 2014], Erasmus+ Programme and EEA grants. The
Research Council of Norway is acknowledged for the financial support
through the Norwegian NMR Platform, NNP (226244/F50).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.07.
011.
References
МcComb, E. A., & McCready, R. M. (1957). Determination of acetyl in pectin and in
acetylated carbohydrate polymers (Hydroxamic acid reaction). Analytical Chemistry,
29, 819–821.
Anthon, G. E., & Barrett, D. M. (2008). Combined enzymatic and colorimetric method
for determining the uronic acid and methyl ester content of pectin: Application
to tomato products. Food Chemistry, 110, 239–247.
Barsett, H., Paulsen, B. S., & Habte, Y. (1992). Further characterization of polysaccharides
in seeds from Ulmus glabra Huds. Carbohydrate Polymers, 18(2), 125–130.
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination
of uronic acids. Analytical Biochemistry, 54, 484–489.
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72, 248–254.
Castle, J., & Lis-Balchin, M. (2002). History of usage of Lavandula species. In M.
Lis-Balchin (Ed.), Lavender. The genus Lavandula (pp. 35–50). London: Taylor &
Francis.
Chambers, R. E., & Clamp, J. R. (1971). An assessment of methanolysis and other
factors used in the analysis of carbohydrate-containing materials. Biochemical
Journal, 125, 1009–1018.
Chen, C.-H., Sheu, M.-T., Chen, T.-F., Wang, Y.-C., Hou, W.-C., Liu, D.-Z., et al.
(2006). Suppression of endotoxin-induced proinflammatory responses by citrus
pectin through blocking LPS signaling pathways. Biochemical Pharmacology,
72, 1001–1009.
Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of
carbohydrates. Carbohydrate Research, 131(2), 209–217.
Denman, L. J., & Morris, G. (2015). An experimental design approach to the chemical
characterisation of pectin polysaccharides extracted from Cucumis melo
Inodorus. Carbohydrate Polymers, 117, 364–369.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chemistry,
28(3), 350–356.
Fearon, D. T., & Austen, K. F. (1977). Activation of the alternative complement
pathway due to resistance of zymosan-bound amplification convertase to
endogenous regulatory mechanisms. Proceedings of the National Academy of Sciences
United States of America, 74(4), 1683–1687.
Gallet, M., Vayssade, M., Morra, M., Verhoef, R., Perrone, S., Cascardo, G., et al. (2009).
Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by
immobilized enzymatically tailored pectins. Acta Biomaterialia, 5, 2618–2622.
Gallova, L., Kubala, L., Ciz, M., & Lojek, A. (2004). IL-10 does not affect oxidative burst
and expression of selected surface antigen on human blood phagocytes in vitro.
Physiology Research, 53, 199–208.
Georgiev, Y., Ognyanov, M., Yanakieva, I., Kussovski, V., & Kratchanova, M. (2012).
Isolation, characterization and modification of citrus pectin. Journal of BioScience
and Biotechnology, 1(3), 223–233.
Georgiev, Y. N., Ognyanov, M. H., Denev, P. N., & Kratchanova, M. G. (2017). Chapter
X. Perspective therapeutic effects of immunomodulating acidic herbal heteropolysaccharides
and their complexes in functional and dietary nutrition. In A.
M. Holban, & A. M. Grumezescu (Eds.), Handbook of Food Bioengineering, Volume
VIII: Therapeutic Foods. San Diego: Elsevier. ISBN: 9780128115176 [in press]
Georgiev, Y. N., Ognyanov, M. H., Kiyohara, H., Batsalova, T. G., Dzhambazov,
B. M., Ciz, M., et al. (2017). Acidic polysaccharide complexes from common
purslane, silver linden and lavender stimulate Peyer’s patch immune cells
through innate and adaptive mechanisms, International Journal of Biological
Macromolecules. [in press] https://authors.elsevier.com/tracking/article/
details.do?aid=7903&jid=BIOMAC&surname=Kratchanova.
Georgiev, Y.N., Paulsen, B.S., Kiyohara, H., Ciz, M., Ognyanov, M.H., Vasicek, O.,
et al., (2017). Tilia tomentosa pectins exhibit dual mode of action on phagocytes
ˇ-glucuronic acid monomers are abundant in their rhamnogalacturonans
I, [Submitted (Carbohydrate Polymers)].
Harborne, J. B., & Williams, C. A. (2002). Phytochemistry of the genus Lavandula. In
M. Lis-Balchin (Ed.), Lavender. The genus Lavandula (pp. 86–99). London: Taylor
& Francis.
Ho, G. T. T., Zou, Y.-F., Aslaksen, T. H., Wangensteen, H., & Barsett, H. (2016). Structural
characterization of bioactive pectic polysaccharides from elderflowers (Sambuci
flos). Carbohydrate Polymers, 135, 128–137.
Hong, T., Matsumoto, T., Kiyohara, H., & Yamada, H. (1998). Enhanced production
of hematopoietic growth factors through T-cell activation in Peyer’s patches
by oral administration of Kampo (Japanese herbal) medicine, Juzen-Taiho-To.
Phytomedicine, 5(5), 353–360.
Huleatt, J. W., & Lefranc¸ ois, L. (1996). ˇ2 integrins and ICAM-1 are involved in establishment
of the intestinal mucosal T cell compartment. Immunity, 5(3), 263–273.
Jakobek, L. (2015). Interactions of polyphenols with carbohydrates lipids and proteins.
Food Chemistry, 175, 556–567.
Jung, C., Hugot, J.-P., & Barreau, F. (2010). Peyer’s patches: The immune sensors of the
intestine. International Journal of Inflammation, http://dx.doi.org/10.4061/2010/
823710. Article ID 823710
Kaˇcuráková, M., Capek, P., Sasinková, V., Wellner, N., & Ebringerová, E. (2000). FT-IR
study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses.
Carbohydrate Polymers, 43, 195–203.
Kim, J., & Carpita, N. C. (1992). Changes in esterification of the uronic acid groups of
cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiology,
98(2), 646–653.
Kiyohara, H., Matsumoto, T., Nagai, T., Kim, S.-J., & Yamada, H. (2006). The presence
of natural human antibodies reactive against pharmacologically active pectic
polysaccharides from herbal medicines. Phytomedicine, 13, 494–500.
Kiyohara, H., Uchida, T., Takakiwa, M., Matsuzaki, T., Hada, N., Takeda, T., et al.
(2010). Different contributions of side-chains in ˇ-D-(1 → 3, 6)-galactans on
intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus
mongholics Bunge. Phytochemistry, 71, 280–293.
Klerx, J. P. A. M., Beukelman, C. J., Dijk, V. H., & Willers, J. M. N. (1983). Microassay
for colorimetric estimation of complement activity in guinea pig: Human and
mouse serum. Journal of Immunological Methods, 63, 215–220.
Klerx, J. P. A. M., Beukelman, C. J., Dijk, H. V., Van Overveld, F. J., Van der Maaden,
W. J., & Willers, J. M. N. (1985). Increased local complement levels upon
intraperitoneal injection of mice with Listeria monocytogenes and regulation by
polyanions. Infection and Immunity, 49, 841–843.
Kratchanova, M. G., Denev, P. N., & Kratchanov, C. G. (2014). Rose hip extract synergistically
increase antioxidant activity of fruit and herb extracts. Bulgarian
Chemical Communications, 46(A), 59–64.
Laekeman, G. (2012). Assessment report on Lavandula angustifolia Miller,
aetheroleum and Lavandula angustifolia Miller, flos. European Medicines
Agency. HMPC/143183/2010.
Leung, M. Y. K., Liu, C., Koon, J. C. M., & Fung, K. P. (2006). Polysaccharide biological
response modifiers. Immunology Letters, 105, 101–114.
Lojek, A., Ciz, M., Pekarova, M., Ambrozova, G., Vasicek, O., Moravcova, J., et al. (2011).
Modulation of metabolic activity of phagocytes by antihistamines. Interdisciplinary
Toxicology, 4(1), 15–19. http://dx.doi.org/10.2478/v10102-011-0004-z
Méndez-Tovar, I., Herrero, B., Pérez-Magari˜no, S., Pereira, J. A., & Asensio-S.Manzanera,
M. C. (2015). Journal of Food and Drug Analysis, 23, 225–233.
Makarova, E. N., Patova, O. A., Shakhmatov, E. G., Kuznetsov, S. P., & Ovodov, Y. S.
(2013). Structural studies of the pectic polysaccharide from Siberian fir (Abies
sibirica Ledeb.). Carbohydrate Polymers, 92, 1817–1826.
Matsumoto, T., Cyong, J.-C., Kiyohara, H., Matsui, H., Abe, A., Hirano, M., et al. (1993).
The pectic polysaccharide from Bupleurum falcatum L. enhances immunecomplexes
binding to peritoneal macrophages through Fc receptor expression.
International Journal of Immunopharmacology, 15(6), 683–693.
McNeil, M., Darvill, A. G., & Albersheim, P. (1980). X. Rhamnogalacturonan I, a structurally
complex pectic polysaccharide in the cell walls of suspension-cultured
sycamore cells. Plant Physiology, 66(6), 1128–1134.
Michaelsen, T. E., Gilje, A., Samuelsen, A. B., Høgåsen, K., & Paulsen, B. S. (2000). Interaction
between human complement and a pectin type polysaccharide fraction,
PMII: From the leaves of Plantago major L. Scandinavian Journal of Immunology,
52, 483–490.
Nauseef, W. M. (2014). Isolation of human neutrophils from venous blood. In M. T.
Quinn, & F. R. DeLeo (Eds.), Methods in molecular biology, neutrophil methods and
protocols (Vol. 1124) (2nd ed., Vol. 1124, pp. 13–18). New York: Humana Press
Inc.
Pekarova, M., Lojek, A., Martiskova, H., Vasicek, O., Bino, L., Klinke, A., et al. (2011).
New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived
superoxide anion production in Raw 264.7 macrophages. The Scientific World
Journal, 11, 2443–2457.
Perrone, P., Hewage, C. M., Thomson, A. R., Bailey, K., Sadler, I. H., & Fry, S. C. (2002).
Patterns of methyl and O-acetyl esterification in spinach pectins: New complexity.
Phytochemistry, 60, 67–77.
Ross, G. D., & Vˇetviˇcka, V. (1993). CR3 (CD11b: CD18): A phagocyte and NK cell
membrane receptor with multiple ligand specificities and functions. Clinical and
Experimental Immunology, 92, 181–184.
Shakhmatov, E. G., Toukach, P. V., Kuznetsov, S. P., & Makarova, E. N. (2014).
Structural characteristics of water-soluble polysaccharides from Heracleum sosnowskyi
Manden. Carbohydrate Polymers, 102, 521–528.
Shakhmatov, E. G., Atukmaev, K. V., & Makarova, E. N. (2016). Structural characteristics
of pectic polysaccharides and arabinogalactan proteins from Heracleum
sosnowskyi Manden. Carbohydrate Polymers, 136, 1358–1369.
Singleton, V., & Rossi, J. (1965). Colorimetry of total phenolic with phosphomolibdiphosphotungstic
acid reagents. American Journal of Enology and Viticulture, 16,
144–158.
Y.N. Georgiev et al. / Carbohydrate Polymers 174 (2017) 948–959 959
Slavov, A., Kiyohara, H., & Yamada, H. (2013). Immunomodulating pectic polysaccharides
from waste rose petals of Rosa damascena Mill. International Journal of
Biological Macromolecules, 59, 192–200.
Synytsya, A., ˇCopíková, J., & Brus, J. (2003). 13
C CP/MAS NMR spectra of pectins: A
peak-fitting analysis in the C-6 region. Czech Journal of Food Sciences, 21(1), 1–12.
Synytsya, A., ˇCopíková, J., Matˇejka, P., & Machoviˇc, V. (2003). Fourier transform
Raman and infrared spectroscopy of pectins. Carbohydrate Polymers, 54, 97–106.
Van Holst, G.-J., & Clarke, A. E. (1985). Quantification of arabinogalactan-protein
in plant extracts by single radial gel diffusion. Analytical Biochemistry, 148(2),
446–450.
Vasicek, O., Lojek, A., Jancinova, V., Nosal, R., & Ciz, M. (2014). Role of histamine
receptors in the effects of histamine on the production of reactive oxygen species
by whole blood phagocytes. Life Sciences, 100(1), 67–72.
Voragen, A. G. J., Coenen, G.-J., Verhoef, R. P., & Schols, H. A. (2009). Pectin a versatile
polysaccharide present in plant cell walls. Journal of Structural Chemistry, 20,
263–275.
Wang, S., Shi, S., Lian, H., Zhu, C., Wang, H., Liu, R., et al. (2016). Structural features
and anti-complementary activity of an acidic polysaccharide from Forsythia suspensa.
Journal of Glycomics and Lipidomics, 6(2), 138. http://dx.doi.org/10.4172/
2153-0637.1000138
Wangensteen, H., Diallo, D., & Paulsen, B. S. (2015). Medicinal plants from Mali:
Chemistry and biology. Journal of Ethnopharmacology, 176, 429–437.
Yamada, H., & Kiyohara, H. (1999). Complement-activating polysaccharides from
medicinal herbs. In H. Wagner (Ed.), Immunomodulatory agents from plants (pp.
161–202). Basel AG: Springer.
Yamada, H., & Kiyohara, H. (2007). Immunomodulating activity of plant polysaccharide
structures. In J. P. Kamerling (Ed.), Comprehensive glycoscience − From
Chemistry to Systems Biology (pp. 663–694). Oxford: Elsevier.
York, W. S., Darvill, A. G., McNeil, M., & Albersheim, P. (1985). 3-deoxy-D-manno-
2-octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic
polysaccharide in the primary cell walls of plants. Carbohydrate Research, 138(1),
109–126.
Zhao, E., Xu, H., Wang, L., Kryczek, I., Wu, K., Hu, Y., et al. (2012). Bone marrow and
the control of immunity. Cellular & Molecular Immunology, 9, 11–19.
de Santana-Filho, A. P., Noleto, G. R., Gorin, P. A. J., de Souza, L. M., Iacomini, M., &
Sassaki, G. L. (2012). GC–MS detection and quantification of lipopolysaccharides
in polysaccharides through 3-O-acetyl fatty acid methyl esters. Carbohydrate
Polymers, 87(4), 2730–2734.
Carbohydrate Polymers 175 (2017) 178–191
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Tilia tomentosa pectins exhibit dual mode of action on phagocytes as
ˇ-glucuronic acid monomers are abundant in their
rhamnogalacturonans I
Yordan N. Georgieva,b
, Berit S. Paulsenc
, Hiroaki Kiyoharad
, Milan Cize
,
Manol H. Ognyanova,b
, Ondrej Vasiceke,i
, Frode Risef
, Petko N. Deneva,b
, Antonin Lojeke
,
Tsvetelina G. Batsalovag
, Balik M. Dzhambazovg
, Haruki Yamadad
, Reidar Lundf
,
Hilde Barsettc
, Albert I. Krastanovh
, Irina Z. Yanakievaa,b
, Maria G. Kratchanovaa,b,∗
a
Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139 Ruski
Blvd., BG-4000, Plovdiv, Bulgaria
b
Innovative-Technological Center Ltd., 20 Dr. G. M. Dimitrov Str., BG-4000, Plovdiv, Bulgaria
c
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, NO-0316, Oslo, Norway
d
Department of Drug Discovery Science, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, JP-108-8641, Tokyo, Japan
e
Department of Free Radical Pathophysiology, Institute of Biophysics, Czech Academy of Sciences, 135 Kralovopolska, CZ-612 65, Brno, Czech Republic
f
Department of Chemistry, University of Oslo, P.O. Box 1033 Blindern, NO-0315, Oslo, Norway
g
Department of Developmental Biology, Plovdiv University Paisii Hilendarski, 24 Tsar Assen Str., BG-4000, Plovdiv, Bulgaria
h
Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., BG-4002, Plovdiv, Bulgaria
i
International Clinical Research Center − Center of Biomolecular and Cellular Engineering, St. Anne’s University Hospital Brno, 53 Pekarska, CZ-656 91,
Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 1 June 2017
Received in revised form 21 July 2017
Accepted 24 July 2017
Available online 25 July 2017
Keywords:
Tilia flowers
Pectin
Immunomodulating polysaccharides
Anti-inflammatory activity
Neutrophils
Peyer’s patches
a b s t r a c t
Silver linden flowers contain different pectins (PSI-PSIII) with immunomodulating properties. PSI is a
low-esterified pectic polysaccharide with predominant homogalacturonan region, followed by rhamnogalacturonan
I (RGI) with arabinogalactan II and RGII (traces) domains. PSII and PSIII are unusual
glucuronidated RGI polymers. PSIII is a unique high molecular weight RGI, having almost completely O-3
glucuronidated GalA units with >30% O-3 acetylation at the Rha units. Linden pectins induced reactive
oxygen species (ROS) and NO generation from non-stimulated whole blood phagocytes and macrophages,
resp., but suppressed OZP-(opsonized zymosan particles)-activated ROS generation, LPS-induced iNOS
expression and NO production. This dual mode of action suggests their anti-inflammatory activity, which
is known for silver linden extracts. PSI expressed the highest complement fixation and macrophagestimulating
activities and was active on intestinal Peyer’s patch cells. PSIII was active on non-stimulated
neutrophils, as it induced ß2-integrin expression, revealing that acetylated and highly glucuronidated
RGI exhibits immunomodulating properties via phagocytes.
© 2017 Elsevier Ltd. All rights reserved.
Abbreviations: AGII, arabinogalactan type II; AUAC, anhydrouronic acid content; BSA, bovine serum albumin; CD18, marker for ˇ2-integrin; CD62L, marker for L-selectin;
CL, chemiluminescence; DA, degree of acetylation; DM, degree of methoxylation; DSS, sodium 4,4-dimethyl-4-silapentane-sulfonate; fMLP, N-Formylmethionyl-leucylphenylalanine;
FTIR, Fourier transform infrared; GlcA-RGI, glucuronidated RGI; HBSS, Hank’s buffered salt solution; HG, homogalacturonan; IC50, a concentration giving 50%
hemolysis of erythrocytes; iNOS, inducible nitric oxide synthase; KDO, 3-deoxy-d-manno-2-octulosic acid; LDH, lactate dehydrogenase; Linden PSC, crude polysaccharide
complex from Tilia tomentosa; Linden PSin70, obtained from linden PSC after an exhaustive ethanol treatment; LPS, lipopolysaccharides; NMR, Nuclear magnetic resonance;
NO, nitric oxide; OZP, opsonized zymosan particles; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; PP, Peyer’s patches; PSI-PSIII, polysaccharide
fractions I, II, and III obtained after anion-exchange chromatography from linden PSin70; RGI and II, rhamnogalacturonan type I and II; ROS, reactive oxygen species; SRBC,
sheep red blood cells; WBP, whole blood phagocytes.
∗ Corresponding author at: Laboratory of Biologically Active Substances, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 139
Ruski Blvd., BG-4000, Plovdiv, Bulgaria. Tel./Fax.: +359 32 64 27 59.
E-mail address: lbas@plov.omega.bg (M.G. Kratchanova).
http://dx.doi.org/10.1016/j.carbpol.2017.07.073
0144-8617/© 2017 Elsevier Ltd. All rights reserved.
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 179
1. Introduction
Tilia genus (Tiliaceae) comprises around 45 species of deciduous
trees, which are native to the Northern hemisphere, as
silver linden (T. tomentosa, syn. T. argentea) forms coenoses only
in the Balkans (Hritcu & Cioanca, 2016; Radoglou, Dobrowolska,
Spyroglou & Nicolescu, 2009). Linden (lime tree, basswood or “lipa”
in Slavic) is famous in the European mythology, literature, folklore
and medicine. Since the middle ages, the linden flower tea
has been used as a diaphoretic to promote perspiration. The traditional
use of the linden blossom is for common cold, feverish
colds, cold-related coughs, catarrhs, mental stress, high blood pressure,
headache, indigestion, diarrhea, skin ailments, etc (Chinou,
2012a; WHO, 2010). Tilia flos (T. cordata, T. platyphyllos, T. vulgaris)
is included in the European Pharmacopeia and approved by the
German Commission E for internal use for colds and cold-related
coughs. It is listed by the Council of Europe as a natural source of
food flavouring and with GRAS status from FDA. T. tomentosa buds
are popular and used worldwide for preparation of tea and dietary
supplements, such as the commercially available “silver linden bud
extract” used in gemmotherapy (Allio et al., 2015). The largest suppliers
of the vegetal product now are China, Bulgaria, Poland, Russia,
Romania and Turkey (Hritcu & Cioanca, 2016).
The active constituents in the linden inflorescences are
flavonoids, phenolic acids, tannins, essential oils, mucilage, triterpenes
and sterols (Hritcu & Cioanca, 2016; Kosakowska et al.,
2015). There are not many studies on the biologically active constituents
of T. tomentosa blossom, except for its flavonoids having
anti-inflammatory activity (Chinou, 2012b; Matsuda, Ninomiya,
Shimoda, & Yoshikawa, 2002; Viola et al., 1994). It is found that
the mucilage is the main component of the linden flower bracts
and it is also detected in the subglandular parenchyma tissue of
sepals (Hritcu & Cioanca, 2016). The traditional use of the linden
mucilage is related to its emollient properties and antitussive activity,
but many of its immunomodulating compounds, such as the
anti-inflammatory constituents are unknown (Hritcu & Cioanca,
2016). It has been reported that Tilia flower mucilage is rich in
pectic polysaccharides, however their structural diversity and biological
activity are little studied. Kram and Franz (1983, 1985),
and Yakovlev (1985) found that pectins are abundant in Tilia
cordata blossoms. Earlier, Simson and Timell (1978) found that
a cambial tissue from basswood, T. americana, contains 50–60%
pectin. The immunomodulating activity of the linden blossom is
attributed to its flavonoids (tiliroside, rutin), coumarins (scopoletin)
and monoterpenes, but there are not such studies on the
linden pectins (Arcos et al., 2006; Manuele, Ferraro, & Anesini, 2008;
Matsuda et al., 2002). Interestingly, aqueous extracts from linden
flowers, which should contain pectins, have shown in vitro stimulatory
effects on lymphocyte proliferation and antitumor activity
(Brizi, Marrassini, Zettler, Ferraro, & Anesini, 2012). In search of
the mucilaginous effect on irritated buccal membranes, Schmidgall,
Schnetz, and Hensel (2000) showed that a raw polysaccharide
complex from the flowers of T. cordata expresses a moderate bioadhesion
to epithelial tissue ex vivo.
Pectic polysaccharides contain three main building blocks covalently
linked together − unbranched homogalacturonan (HG),
ramified RGI and II. The linear HG is methyl-esterified and Oacetylated
to different extent. RGI is also acetylated but branched
at C-4 of the L-Rha residues with side chains of ˛-(1 → 5)-larabinans,
ˇ-(1 → 4)-d-galactans, arabinogalactan (AG) type I and
II (Ridley, O’Neill, & Mohnen, 2001; Schols & Voragen, 2002). The
AG structural motifs are extremely important for the expression
of immunomodulating activity. This could clearly be seen from
the works of Yamada & Kiyohara on pectic polysaccharides isolated
from the Japanese Kampo medicine formulations and Paulsen
and coworkers on pectins from Malian medicinal plants (Paulsen
& Barsett, 2005; Wangensteen, Diallo, & Paulsen, 2015; Yamada
& Kiyohara, 2007). RGII is composed of a partly methyl-esterified
galacturonan backbone, carrying four oligosaccharide side chains
rich in rare sugars (2-Me-Fuc, 2-Me-Xyl, Api, Kdo and Dha) (Ridley
et al., 2001). Pectins have beneficial effects on the immune, digestive,
excretory, cardiovascular, endocrine, nervous and respiratory
systems (Groudeva, Kratchanova, Panchev, & Kratchanov, 1997;
Fan et al., 2012; Liu, Dong, Yang, & Pan, 2016; ˇSutovská, Capek,
Fraˇnová, Pawlaczyk, & Gancarz, 2012; Xie, Zhang, & Zhang, 2015;
Yamada & Kiyohara, 2007). The pectic polysaccharides exhibit
moderate immunomodulating effects against T-cells, B-cells, NKcells,
neutrophils, macrophages, dendritic cells, thrombocytes and
abnormal tumor cells (Bijak et al., 2013; Holderness et al., 2011;
Inngjerdingen et al., 2007; Inngjerdingen et al., 2008; Ma et al.,
2013; Yamada & Kiyohara, 2007). These actions combined with
their regulatory effects on immune signaling proteins, complement
system and probiotic bacteria could be useful for prevention and
supplementary therapy of infectious, tumor, gastrointestinal and
inflammatory diseases.
In our preliminarily study a crude polysaccharide complex
from T. tomentosa (linden PSC) expressed ex vivo immunomodulating
activity against human phagocytic leukocytes, T-cells, murine
Peyer’s patch cells and anti-tumor activity in vitro (Georgiev et al.,
2017). The aim of the current study is to characterize the chemical
diversity and immunomodulating properties of purified pectic
polysaccharides from T. tomentosa Moench blossoms. This will help
in elucidation of the structure-activity relationship of these possible
pharmacologically active linden constituents.
2. Materials & methods
2.1. Plant material
Linden flowers were collected in June 2013 in Plovdiv (Bulgaria),
air-dried for two weeks and stored in a desiccator until use.
The plant material was identified as silver linden, Tilia tomentosa
Moench (Tiliaceae), by Dr. Evgeny Tsavkov from the Department of
Dendrology in the University of Forestry, Sofia. A voucher specimen
(SOA061426) was deposited in the Herbarium of the Agricultural
University of Plovdiv.
2.2. Isolation and purification of silver linden flower
polysaccharides
Linden PSC was extracted with boiling water as previously
described (Georgiev et al., 2017).
The linden PSC was dissolved in water (10 mg/ml) and precipitated
twice with 95% ethanol (24 h each) with a final alcohol
concentration of 70%. This step was for partly removing of
smaller molecules. After each run polysaccharide precipitates
were separated by centrifugation at 4000 rpm for 30 min at
20 ◦C. Then, the recovered highest in yield (47.1% on linden PSC
basis) polysaccharide-containing fraction insoluble in 70% ethanol
(linden PSin70) was applied to a DEAE-Sepharose FF column
(1.5 × 50 cm) in HCO−
3 form, coupled with a FPLC BioLogic
®
LP
system (BioRad). The column was eluted successively with 170 ml
water, then with 300 ml of: 0.35 M NH4HCO3; 0.45 M NH4HCO3 and
1.0 M NH4HCO3, at 1 ml/min flow rate. Three pectic polysaccharide
fractions PSI-PSIII were obtained, as 7 ml fractions were collected
and peaks were visualized by the phenol-sulfuric acid (not shown)
and m-hydroxydiphenyl methods (Dubois, Gilles, Hamilton, Rebers,
& Smith, 1956; Blumenkrantz & Asboe-Hansen, 1973). The samples
were concentrated under vacuum, dialyzed against water (MWCO
12–14 kDa, 72 h at 4 ◦C), filtered through 0.2 ␮m cellulose nitrate
filters (Sartorius), freeze-dried and stored in a desiccator in dark.
180 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
2.3. General methods
Total amount of sugars was quantified by the phenol-sulfuric
acid method, using 1:1 mixture of glucose and galacturonic acid
as a standard (Dubois et al., 1956). Anhydrouronic acid content
(AUAC) was determined by the m-hydroxydiphenyl method,
using galacturonic acid as a standard (Blumenkrantz & AsboeHansen,
1973). Degree of methoxylation (DM) was determined
after methanol release with 0.5 M NaOH and its further oxidation
to formaldehyde by alcohol oxidase (P. pastoris, Sigma-Aldrich),
and measuring of the absorbance of the obtained colored product
with Purpald
®
(Sigma-Aldrich) at ␭ = 550 nm (Anthon & Barrett,
2008). Degree of acetylation (DA) was measured according to
МcComb and McCready (1957), using ˇ-d-glucose pentaacetate
as a standard. Molecular weight was determined by HPSEC-RID
(Waters) with Shodex standard P-82 kit (pullulan standards, Showa
DENKO, Tokyo, Japan) (Georgiev, Ognyanov, Yanakieva, Kussovski,
& Kratchanova, 2012). The elution profile of PSIII (1.9 mg/1 ml) was
also analyzed by FPLC on a Sephacryl S300 column (100 × 1.5 cm)
at a flow rate of 0.5 ml/min 0.2 M NaCl and 5 ml fractions were collected.
Protein content (BSA standard) was analyzed according to
Bradford (1976). Only distilled water was used for all experiments.
2.4. Monosaccharide composition and glycosidic linkage
determination
Polysaccharide samples (1 mg) with added 100 ␮g mannitol as
an internal standard were subjected to methanolysis with anhydrous
3 M HCl in methanol for 24 h at 80 ◦C. The formed methyl
glycosides of different monosaccharides were converted into their
corresponding TMS derivatives and analyzed by GC-FID on a Focus
GC system (Thermo Scientific, Milan, Italy), according to Chambers
and Clamp (1971) and Barsett, Paulsen, and Habte (1992). The presence
of KDO (3-deoxy-d-manno-2-octulosic acid) was detected by
the thiobarbituric acid assay, as modified by York, Darvill, McNeil,
and Albersheim (1985).
Glycosidic linkage composition of PSI and PSIII (2 mg) were
examined by the methylation approach. Prior to methylation uronic
acids in the polysaccharides were reduced with NaBD4 to 6,6 dideuterio-sugars
that could be differentiated from the neutral
sugars (Kim & Carpita, 1992). The procedure was completed by
methylation, acid hydrolysis and preparation of alditol acetates,
partly as described by Ciucanu and Kerek (1984). First hydrolysis
was performed with 2.5 M TFA for 2 h at 100 ◦C. Secondly, only for
PSIII (2 mg) formolysis with 90% HCOOH (1 ml) for 6 h at 120 ◦C
and a further hydrolysis by addition of 5 ml water for 1 h at 100 ◦C
was carried out on the methylated polymer. This was necessary
for the complete hydrolysis of the glycosidic bonds in PSIII. The
partly methylated alditol acetates were analyzed by a GC-EI/MSQP2010
system (Shimadzu, Kyoto, Japan), coupled with a Restek
Rxi-5MS column (30 m, 0.25 mm i.d., 0.25 ␮m film) in a Scan mode
(40–450 ions), as described by Ho, Zou, Aslaksen, Wangensteen,
and Barsett (2016). The detection and quantification of the relative
amounts of each glycosidic linkage type was based on the chromatographic
behavior (RT), ionization patterns and results from
the monosaccharide composition.
The presence of AGII was detected by precipitation with the ßglucosyl
Yariv reagent (Van Holst & Clarke, 1985).
2.5. Contamination with microbial lipopolysaccharides (LPS)
Eventual LPS contamination was checked by a possible detection
of 3-hydroxy fatty acids as their specific chemical markers in the
form of acetylated fatty acid methyl esters on the GC-EI/MS-QP2010
system (Shimadzu, Kyoto, Japan), coupled with the Restek Rxi-5MS
column (30 m, 0.25 mm i.d., 0.25 ␮m film) in a SIM mode (257 ion
intensity for calculation), as described by de Santana-Filho et al.
(2012) and Ho et al. (2016).
2.6. Nuclear magnetic resonance (NMR) spectroscopy
PSIII (12 mg) was dissolved in D2O, freeze-dried, re-dissolved
in 700 ␮l D2O and 500 ␮l sample was used. 1D 1H, 13C and 2D
13C/1H HSQC, 1H/1H COSY 90, 1H/1H TOCSY, 1H/1H ROESY, 13C/1H
HMBC, 13C/1H HSQC-NOESY experiments were performed on a
Bruker Avance III HD Ascend 800 MHz spectrometer operating at
800.03 MHz (Bruker, Fällanden, Switzerland) with the software
Bruker TopSpinTM 3.5 at a temperature of 333.15 K. The chemical
shifts were referenced to an internal standard DSS (sodium
4,4-dimethyl-4-silapentane-sulfonate).
2.7. Fourier transform infrared (FTIR) spectroscopy
FTIR spectra of PSIII before and after saponification with NaOH
were obtained on a NicoletTM FTIR spectrometer (Thermo Fischer
Scientific, USA) controlled by the OMNIC 3.2 software. The sample
was pressed onto the attenuated total reflectance (ATR) crystal
using a high pressure tower to provide consistent results. No more
than 2 mm3 of the sample was used to cover the ATR diamond cell.
2.8. Small-angle X-ray scattering (SAXS)
SAXS experiments of PSIII aqueous solutions in different
concentrations and at different temperatures were carried out
using a Bruker Nanostar instrument (RECX-Norway, Cu-K␣ radiation
␭= 1.54 Å). The instrument setting allows to access a
Q-range between 0.009 Å−1 to 0.2 Å−1, where Q is defined by
Q = 4␲sin(␪/2)/␭, as ␪ is the scattering angle and ␭ the wavelength.
The samples were measured in a Quartz cell of 1 mm path length.
The data were analysed on an absolute scale (water as a primary
standard), using a coexistence model of a linear polymer chain:
I (Q) = Icluster (Q) + I(Q)chain
where the contribution from the dissolved polymer chains can be
written as:
I(Q)chain =
ϕMw
dp
( p − 0)2
P(Q)chain
Here ␸ is the volume fraction of a polymer, Mw is the weight
average molecular weight, dp = 1.2 g/ml is the density for typical
hydrogen-bonding polymers (Sommer, Pedersen, & Stein, 2004), ␳
is the electron density for the solvent, ␳0 = 9.4 × 1010 cm−2 and the
polymer (␳p = 1.1 × 1011 cm−2). P(Q)chain is the form factor derived
by Beaucage (1996):
P(Q)chain= exp −
Q2R2
g
3
+
df
QRg
df
df
2
·
⎛
⎜
⎝
erf Q · Rg
√
6
3
Q
⎞
⎟
⎠
df
where Rg is the radius of gyration, df is the fractal dimension
(df = 2.0, 1.7 for ␪ and a good solvent, respectively). (x) is the
gamma function and erf(x) is the error function. The cluster contribution
can be taken into account by the term:
I(Q)blob =
C
(1 + Q2 2)
2
where ␰ is the characteristic size of the cluster (correlation length),
and C is a numerical constant.
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 181
2.9. Immunomodulatory effect
2.9.1. Ethics
Blood sampling procedure was in accordance with the Helsinki
Declaration of 1975, as revised in 1983. Animal experiments were
approved by the Animal Research Committee of the Kitasato University,
and performed in accordance with the Guidelines for Care
and Use of Laboratory Animals at the Kitasato University and the
National Research Council Guide for the Care and Use of Laboratory
Animals in Japan.
2.9.2. Animal procedure and care
Female C3H/HeJ mice (6–8 weeks old) were purchased from
Japan SLC (Shizuoka, Japan), and male ICR mice (4 weeks old) were
from Charles River (Tokyo, Japan). The mice were housed in plastic
cages in an air-conditioned room at 23 ± 2 ◦C with a relative humidity
of 55 ± 10% under a 12 h-light-dark cycle, and fed a standard
laboratory diet with water given ad libitum.
2.9.3. Complement fixation assay
The inhibition of complement hemolysis (complement fixation)
of target antibody sensitized sheep red blood cells (SRBC) by a normal
human serum pre-treated with PSI, II or III was performed
according to Michaelsen, Gilje, Samuelsen, Høåsen, and Paulsen
(2000) (Method A). A highly active pectic polysaccharide BP-II from
the aerial parts of Biophytum petersianum was used as a positive
control (Grønhaug et al., 2011). The complement fixation activity
induced by the test samples was calculated on the basis of a colorimetric
measurement of hemoglobin released from lysed SRBC after
incubation with a normal human serum pretreated with PSI, II or
III by the formula: [Acontrol − Atest]/Acontrol × 100%. From these data
a dose-response curve was constructed and the concentration of a
test sample giving 50% inhibition of lysis (IC50) was calculated. The
results are obtained from two independent experiments.
2.9.4. ROS production from human whole blood phagocytes
(WBP) and isolated neutrophils
Heparinized (50 IU/ml) blood samples were obtained from the
cubital vein of healthy human volunteers after overnight fasting.
The isolation of human neutrophils from venous blood was accomplished
according to the procedure of Nauseef (2014). Kinetics
of ROS production by WBP and isolated neutrophils was analyzed
by luminol-enhanced chemiluminescence (CL) for a period
of 60 min at 37 ◦C, using 96-well white flat bottom culture plates
on a luminometer (Orion II Berthold Detection Systems GmbH,
Germany). The principle of the method was previously described
(Vasicek, Lojek, Jancinova, Nosal, & Ciz, 2014). Briefly: 25 ␮l of ten
times diluted blood with Hank’s buffered salt solution (HBSS) or
0.2 × 106 cells/well of isolated neutrophils in phosphate-buffered
saline (PBS) were mixed with 25 ␮l 1 mM luminol (10 mM stock
solution in 0.2 M borate buffer) HBSS solution and incubated with
HBSS solutions of PSI, II or III (50 and 100 ␮g/ml) for spontaneous
(non-stimulated with known activators) CL. Furthermore,
12.5 ␮l or 5 ␮l of ten times diluted blood (or 0.2 × 106 cells/well)
mixed with 25 ␮l luminol were used for incubation of each linden
polysaccharide with 25 ␮l (0.5 ␮g/ml) phorbol 12-myristate
13-acetate (PMA-activated, Sigma-Aldrich) or 25 ␮l (62.5 ␮g/ml)
OZP (OZP-activated). The final volume for spontaneous, PMA- and
OZP-activated CL tests was 250 ␮l. PMA and OZP were used as ROS
production activators. The recorded values included the intensity
of CL emitted during the time interval studied (integral of CL). The
results are expressed as percentage of the control (mean ± SEM)
from five independent experiments.
2.9.5. Determination of the cell surface expression of adhesion
molecules
The measurements were performed according to the method
previously described (Gallova, Kubala, Ciz, & Lojek, 2004). Briefly,
human neutrophils (1 × 105 cells) were incubated in plastic tubes
(Falcon, USA) with PSI, II or III (100 ␮g/ml) for 1 h and with
the activators fMLP (N-formyl-met-leu-phe, 0.1 ␮M) and PMA
(0.01 ␮g/ml) for 10 min. After the incubation anti-CD18 (anti-ˇ2integrin)
and anti-CD62L (anti-l-selectin) monoclonal antibodies
(5 ␮l of each) were added. The samples were incubated at 4 ◦C for
15 min, subsequently centrifuged, re-suspended in PBS and analyzed
by a flow cytometer FACSVerseTM (Becton Dickinson, USA).
The median fluorescence intensity was determined and corrected
for unspecific staining by subtracting the fluorescence of cells
stained with the control antibody (isotype control). The data are
expressed as CD62L−/CD18+ population (mean% of total leukocytes
± SEM) from five independent experiments.
2.9.6. Nitric oxide (NO) production from murine macrophages
Murine macrophages cell line RAW 264.7 (ATCC, USA) were
incubated in 12-well flat bottom culture plates at 1 × 105 cells/well
with PSI, II or III (50 and 100 ␮g/ml) alone in DMEM-high Glc supplemented
with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin
(500 ␮l final volume) (HyCloneTM, GE Healthcare Life Sciences)
growth medium for 24 and 48 h at 37 ◦C and 5% CO2. The cells
were also incubated with polysaccharides in combination with
10 ng/ml LPS (E. coli/026:B6, Sigma-Aldrich). The generation of NO
was determined indirectly as the accumulation of nitrites in the cell
culture supernatant. At the end of the incubation period, the culture
media were collected and centrifuged at 16 000×g, 4 ◦C for 10 min.
Then, 150 ␮l of supernatants were mixed with equal volumes of
the Griess reagent (Sigma-Aldrich) in a 96-well plate and the mixtures
were incubated at room temperature for 30 min in dark. The
absorbances were measured at 546 nm and NaNO2 (0–52 ␮M) was
used as a standard (Lojek et al., 2011). The results are expressed as
mean ± SEM from three independent experiments.
2.9.7. Western blot analysis of inducible nitric oxide synthase
(iNOS)
RAW 264.7 cells, which were obtained as described in 2.9.6.,
were lysed in the lysis buffer (1% SDS, 0.1 M Tris pH 7.4, 10%
glycerol, 0.001 M sodium o-vanadate, 0.001 M PMSF). The protein
concentrations were determined by using the BCATM protein assay
(Pierce, USA), with BSA as a standard. Equal amounts of protein
(18 ␮g) were then subjected to SDS-PAGE using a 10% separating
gel. The expression of iNOS protein was quantified by a Western
blot analysis (Pekarova et al., 2011). Anti-iNOS/NOS Type II
mouse monoclonal antibody (Cell signaling Technology, 1:5000)
and ECLTM anti-rabbit IgG horseradish peroxidase linked whole
antibody (from goat; Cell Signaling Technology, 1:2000) were used.
The immunoreactive bands were detected using the ECLTM detection
kit (Pierce) and exposed to a radiographic film (AGFA). The
relative protein levels were measured by scanning densitometry
using the ImageJTM programme, and the individual band density
value was expressed in arbitrary units. The results are expressed as
mean ± SEM from three independent experiments.
2.9.8. Cytotoxicity assay
Cytotoxicity was estimated by lactate dehydrogenase (LDH)
activity in the cell culture supernatant of RAW 264.7 cells, as
obtained in 2.9.6., using the LDH Cytotoxicity Detection KitPLUS
(Roche Applied Science, Switzerland). The results are expressed as
mean ± SEM from three independent experiments.
182 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
2.9.9. Immunomodulating activity against murine Peyer’s patch
(PP) cells
Immunomodulating activity was measured by the enhanced
production of bone marrow cell-proliferative cytokines from PP
cells of specific pathogen-free C3H/HeJ mice (Tlr4Lps−d) according
to Hong, Matsumoto, Kiyohara, & Yamada (1998). Suspensions
(1.2 × 106 cells/ml, 180 ␮l) of the PP cells (5–9 patches obtained
per mouse) from the small intestine of C3H/HeJ mice were cultured
with H2O (negative control), AMOL-1 (pectic polysaccharide from
Astragalus mongholics as a positive control, Kiyohara et al., 2010) or
100 ␮g/ml of the polysaccharide fractions (PSI, II or III, 20 ␮l) in a
96-well flat bottom culture plate (FALCON 3072) for 6 days at 37 ◦C
in a humidified atmosphere of 5% CO2–95% air. The resulting culture
supernatants (50 ␮l) were further cultured with a bone marrow cell
suspension (5.0 × 105 cells/ml) from male ICR mice for 6 days. The
number of proliferated bone marrow cells was measured by Alamar
BlueTM, as described previously (Hong et al., 1998). The results
are shown as mean ± SEM.
2.9.10. Tumor cell growth inhibition in vitro
Growth inhibitory effect of some of the linden polysaccharides
was studied against FL cell line (ATCC CCL 62, NBIMCC 94) derived
from normal human amniotic cells and different human tumor cell
lines: A549 (ATCC CCL 185), LS180 (ATCC CL-187), HT29 (ATCC HTB-
38), HeLa (ATCC CCL-2), CaOV (NBIMCC 1108), Jurkat (ATCC, TIB-
152) and MOLT-4 (ATCC, CRL-1582). The studied cell lines were
treated with PSI or III (50 and 25 ␮g/ml) for 24, 48 and 72 h, and the
cell viability was tested with the MTT (3-(4,5-dimethylthiazol-2yl)-2,4-diphenyltetrazolium
bromide) (Sigma-Aldrich) reagent as
previously described (Georgiev et al., 2017). The percent inhibition
of the cell growth was calculated using the mean absorbance units
from each test sample and the mean data from the control cells
incubated in the absence of linden polysaccharides. The samples
were analyzed in triplicates.
2.10. Statistical analysis
The statistical significance was analyzed by the Student’s t-test,
using the Statview software (SAS Institute, Inc.). The mean ± SEM
were compared to those from the control. Values of p < 0.05 were
considered as statistically significant.
3. Results
3.1. Purification and structural characterization of the linden
polysaccharides
3.1.1. Purification and chemical composition of the linden
polysaccharide fractions
The linden PSin70 fraction (AUAC = 43.4%) was subjected
to anion-exchange chromatography on a DEAE-Sepharose FF
(−HCO3
−) column and three defined acidic polysaccharide fractions
PSI-III were obtained by a stepwise elution with 0–1 M
NH4HCO3 (Fig. S1). After sample loading the column was eluted
with 170 ml water, but this gave no neutral fractions. We did not
find any further carbohydrate-containing fractions when the column
was successively washed with 2 M NaCl after the stepwise
elution. In Table 1 results from the chemical characterization of
the obtained three pectic fractions are presented. The combined
yield of the studied samples is 42.7% and it is 20.2% and 1.25% from
the linden PSC and dried T. tomentosa flowers, respectively. The
results from the AUAC analysis (39.9-46.8%) of the samples show
that the isolated polysaccharides have acidic character. Kram and
Franz (1983) reported between 10 and 63% AUAC in the obtained
four acidic fractions from T. cordata flowers. Methoxyl esters are
detected only in PSI, which is found to be a low methoxylated pectin
with DM = 13.1%. The total acetyl content in the three fractions
varies between 0.6-3.3%, as PSII and PSIII are characterized with
higher acetyl content than PSI, which indicates that these fractions
are considerably acetylated. The three polysaccharide samples contain
all monosaccharides typical for pectins and also small amounts
of Glc and Man. Interestingly, the Rha content is high in all fractions,
originating from the high Rha content (11.5%) in the linden
PSC for which was shown by a FTIR analysis that it is rich in RGI
backbone structures (Georgiev et al., 2017). The values for Rha and
GalA in PSII and III are in the same range. Theoretically, the ratio
GalA:Rha in the RGI backbone tends to be 1:1, as this ratio for PSI,
II and III is 6.2, 1.3 and 1.1, respectively. According to Denman and
Morris (2015) the difference between GalA and Rha could give the
approximate content of HG (%) in pectins. For PSI the HG content is
estimated to be 57%, following 8.3% for PSII and 2.0% for PSIII. It was
calculated that GlcA is 30–31% from the uronides in the linden PSC
on the basis of colorimetric and chromatographic determination
of both uronic acids (Georgiev et al., 2017). Interestingly, the GlcA
content is high in PSII and III, compared to the other monosaccharides
present. Generally, GlcA and/or 4-O-Me-GlcA are presented
in minor amounts linked to the AG side chains, thus expected to
be lower than Gal + Ara (Ridley et al., 2001). This is the case in PSI
but it is not the same for PSII and III. Additionally, GlcA:Rha ratio is
0.7 and 1.0 for II and III, which shows that the uronic acid is not a
rare substituent in our RGI. PSI appears to be a typical pectic polymer
with high HG content, PSII and III are unusual RGsI, due to the
high content of GlcA. The gross calculation for the glucuronic acidcontaining
polymers indicates that they are presented in more than
0.7% in the dried flowers.
PSI has a considerably lower Mw (28.2 kDa) than PSII and III −
949 and 382 kDa, respectively. Furthermore, PSIII was eluted as a
single peak with 0.2 M NaCl on a Sephacryl S300 column and its
molecular weight was determined to be 352 kDa, using pullulan
calibration standards (0.59 × 104–40.4 × 104 Da). This value is in
the range between Mw and the number average molecular weight
Mn calculated for PSIII from the HPSEC-RID results (Table 1). The
polydispersity index Mw/Mn for PSII and PSIII is 1.39 and 1.22,
respectively. The thiobarbituric acid assay shows the presence of
KDO in PSI, which is used as an indicator for presence of RGII.
The specific immunodiffusion with the ß-glucosyl Yariv reagent for
detection of 3,6-galactans or AGII is positive only for PSI and II. PSI
is distinctive with the highest content of Ara and Gal, followed by
PSII. The isolated linden pectins are also characterized by a very low
protein content <1.0%.
The three purified pectic fractions (PSI-PSIII) were tested for the
presence of LPS, which are considered to be contaminants during
the purification procedure by detection of some characteristic LPS
fatty acid constituents using GC–MS analysis. It appears that there
is no LPS contaminants in these polysaccharide fractions because
the LPS marker 3-hydroxy tetradecanoic acid (3-OH-C14:0) was not
detected (de Santana-Filho et al., 2012).
3.1.2. Linkage determination
In Table 2 are shown the results for estimation of the linkage
composition in PSI and III. Based on the quantitative linkage
results, we found that PSI is composed predominantly of 1,4-linked
GalpA (59.4%) and 1,2-linked Rhap (8.4%) with a minor amount
of 1,2,4-linked Rhap (1.4%) most probably substituted with arabinans
and highly branched AGs with AGII structural characteristics.
This means that PSI contains mainly HG and RGI structures. These
findings are in a good agreement with the calculated HG content
according to Denman and Morris (2015) and the Yariv’s test for
AGII. It is estimated that the 1,2,4-linked Rhap is 12.7% from the
total Rha in PSI (Table 2). The presence of 1,3,4-linked GalpA units
in both samples is not unusual and it could be an indicator for RGII
structures and other substituted galacturonans, such as xylogalac-
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 183
Table 1
Yields and chemical characterization of silver linden pectic fractions obtained after anion-exchange chromatography of linden PSin70.
PSI PSII PSIII
Yield [% of linden PSC] 9.0 2.6 8.6
Yield [% of linden PSin70] 19.1 5.4 18.2
Yield [% of dried flowers] 0.56 0.16 0.53
Total sugarsa
[%] 55.2 67.5 64.8
AUAC [%] 46.8 39.7 39.9
DMb
[mol%] 13.1 n.f. n.f.
(Methoxyl content [%]) (1.1)
Acetyl content [%] 0.6 3.3 2.9
Protein content [%] 0.4 n.d. 0.5
LPS [%] n.f. n.f. n.f.
Monosaccharide composition [w/w%]
Ara 7.5c
(9.2)d
2.3 (2.7) 1.9 (2.2)
Rha 11.0 (12.3) 29.2 (32.3) 29.8 (33.1)
Fuc trace trace trace
Xyl 1.9 (2.3) 0.9 (1.1) 1.0 (1.2)
Man 0.1 (0.1) 0.4 (0.4) 0.2 (0.2)
Gal 6.7 (6.8) 4.7 (4.8) 3.4 (3.5)
Glc 3.3 (3.4) 3.6 (3.6) 2.2 (2.3)
GlcA 1.6 (1.5) 21.4 (20.0) 29.6 (27.8)
GalA 67.9 (64.3) 37.5 (35.1) 31.8 (29.8)
Kdo + n.d. n.d.
AGII + + n.f.
Molecular weight [Da] 2.82 × 104 9.73 × 105
(68.0%) 3.91 × 105
(78.3%)
1.31 × 105
(14.7%) 5.05 × 104
(15.4%)
(Mw)e
0.14 × 104
(17.3%) 0.14 × 104
(6.3%)
(Mn)f
(9.49 × 105
) (3.82 × 105
)
(6.81 × 105
) (3.14 × 105
)
n.f. − not found; n.d. − not determined.
a
Mixture of 1:1 Glc:GalA was used as a standard.
b
Calculated on the AUAC basis, moles of methoxyl groups per 100 mol of GalA.
c
w/w%, calculated on the total carbohydrate content basis.
d
mol% of the total carbohydrate content.
e
Weight average molecular weight.
f
Number average molecular weight.
Table 2
Glycosidic linkage composition of two linden pectic fractions obtained after anionexchange
chromatography of linden PSin70.
Glycosidic linkages PSIc
PSIIId
T-Araf 3.4a
(4.2)b
–
1,3-Araf 0.3 (0.4) –
1,5-Araf 3.0 (3.6) –
1,3,5-Araf 0.8 (1.0) –
T-Rhap 1.2 (1.4) –
1,2-Rhap 8.4 (9.4) 29.8 (33.1)
1,2,4-Rhap 1.4 (1.6) –
1,3-Fucp trace –
1,4-Xylp 1.9 (2.3) –
T-Galp 2.6 (2.6) –
1,3- Galp 0.9 (0.9) –
1,6-Galp 1.1 (1.1) –
1,3,6-Galp 2.1 (2.2) –
1,4-Glcp 3.3 (3.4) 2.2 (2.3)
T-GlcpA 1.6 (1.5) 29.6 (27.8)
T-GalpA 5.3 (5.0) –
1,4-GalpA 59.4 (56.2) 1.8 (1.7)
1,3,4-GalpA 3.3 (3.1) 30.0 (28.1)
a
w/w%, calculated on the total carbohydrate content.
b
mol% of the total carbohydrate content.
c
2.5 M TFA for 2 h at 100 ◦
C.
d
90% HCOOH for 6 h at 120 ◦
C and further hydrolysis by addition of 5 ml d. water
for 1 h at 100 ◦
C.
turonans (Schols & Voragen, 2002). Normally, these fragments do
not occupy high proportion in the initial not enzymatically treated
pectins (Grønhaug et al., 2011). It could be stated that the 1,3,4linked
GalpA in PSI might be related to RGII structures because this
sample is KDO positive.
The methylated PSIII was subjected to formolysis in order to
get a complete hydrolysis of the strong glycosidic bonds in this
polysaccharide. Similar approaches have been applied for studying
of other acidic polymers (Ohtani, Okai, Yamashita, Yuasa, &
Misaki, 1995). After formolysis of the methylated PSIII, 30% of the
1,3,4-linked GalpA units are obtained, which is in a comparable
amount to that of the 1,2-linked Rhap (29.8%) and T-linked GlcpA
(29.6%). The obtained 1:1:1 ratio of 1,2-Rhap:1,3,4-GalpA:T-GlcpA
reveals that the GlcpA monomers are O-3-bound to the GalpA
units in RGI. The ratio 1,4-linked GalpA:(1,4+1,3,4)-linked GalpA
for PSIII from Table 2 gives a value of 5.7%, which shows that
a minor amount of the GalA units are not O-3 glucuronidated.
These GalA units could originate from HG structures. According to
the polysaccharide classification, the glucuronidated RGI could be
named a glucurono-rhamnogalacturonan I (GlcA-RGI) or an acetylated
glucurono-rhamnogalacturonan I in this case.
Because of the low yield of PSII, it was not possible to be performed
a linkage determination of this sample. PSII is characterized
by a similar acetyl content to PSIII and a high GlcA content of 21.4%,
therefore it is assumed that the GlcpA units might be also O-3 linked
to the GalpA.
3.1.3. NMR structural studies
On the basis of the monosaccharide, glycosidic linkage composition
data and yield, PSIII was chosen for further detailed NMR
analyses. One- and two-dimensional homo- and heteronuclear
NMR experiments on a high-resolution 800 MHz spectrometer
were performed in order to elucidate the structural features on
the substitution of the GalA with GlcA and to localize the acetyl
esters in PSIII. In Table 3 are presented 13C and 1H assignments
found from COSY, TOCSY, ROESY, HSQC, HMBC and HSQC-NOESY
spectra of PSIII, identified according to literature data and in
accordance with the monosaccharide and glycosidic linkage compositions
(Ravenscroft et al., 2015; Renard, Crépeau, & Thibault,
1999; Shakhmatov, Atukmaev, & Makarova, 2016; Sengkhamparn
184 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
Table 3
13
C and 1
H NMR chemical shift assignment (ı in ppm) of PSIII − acetylated glucurono-rhamnogalacturonan I, referenced to DSS.
Residue C-1 C-2 C-3 C-4 C-5 C-6 CH3CO
H-1 H-2 H-3 H-4 H-5 H-6
→4)-˛-GalpA-3-OGlcpA-(1 → 2 100.3 70.1 81.3 80.3 73.5 176.2
5.02 4.10 4.20 4.66 4.77
→2)-˛-Rhap-(1→ 101.3 79.3 72.2 74.7 71.8 19.2
5.25 4.11 3.87 3.42 3.71 1.24
→2)-˛-Rhap-3-OAc-(1→ 101.3 70.4 – 78.2 72.8 19.2 22.9
5.37 4.20 5.09 3.61 4.11 1.28 2.09
ˇ-GlcpA-(1→ 106.6 76.4 78.4 74.3 78.2 177.1
4.71 3.49 3.52 3.61 3.81
Fig. 1. 2D NMR analysis of linden pectic fraction PSIII. A) H1
/H1
ROESY spectrum. Each letter (see section 3.1.3.) corresponds to important correlations. B) Partial 13
C/H1
HSQC-NOESY spectrum. R3-R5 are C3-C5 of the Rha residues.
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 185
et al., 2009; Shimizu & Tomoda, 1985). The absolute configurations
were assumed to be the same as already found for RGI (McNeil,
Darvill, & Albersheim, 1980). The detailed NMR study confirmed
that PSIII is a GlcA-RGI, which is predominantly acetylated at O-3
of the ˛-l-Rhap units. The ROESY spectrum(Fig. 1A) reveals the following
cross-peaks: H1 of ˛-l-Rhap and H1 of ˛-d-GalpA residues
at 5.25/5.02 ppm (A); H1 of internal ˛-l-Rhap with H4 of ˛-d-GalpA
units at 5.37/4.66 ppm (B); H1 of ˛-l-Rhap with H3 of ˛-d-GalpA
residues at 5.37/4.20 ppm (C); H2 of ˛-l-Rhap with H1 of ˛-dGalpA
units at 4.11/5.02 (5.09) ppm (D). These correlations confirm
the presence of the RGI backbone structure [→4)-˛-d-GalpA(1
→ 2)-˛-l-Rhap-(1 → 4)-˛-d-GalpA-(1 → ]n in PSIII. The absence
of a correlation between H1 and H4 of ˛-d-GalpA units confirms
that the HG is really a minor part of the polymer.
More intriguing is a the through space cross-peak between
H1 of terminally linked ˇ-d-GlcpA units and H3 of 1,4-˛-d-GalpA
residues at 4.71/4.20 ppm (E) in the ROESY spectrum (Fig. 1A).
A correlation between C1 of ˇ-d-GlcpA and H3 of 1,4-˛-d-GalpA
at 106.6/4.20 ppm in the HSQC-NOESY spectrum is also found
(Fig. 1B). This NMR information reveals that 1,4-˛-d-GalpA units in
the RGI are O-3 substituted with ˇ-d-GlcpA monomers. In fact, the
chemical shifts for H2 (4.10 ppm), H3 (4.20 ppm) and H4 (4.66 ppm)
of ˛-d-GalpA, and H1 of 1,2-˛-l-Rhap show markedly movement to
the downfield region in comparison with literature data for pectins
(Shakhmatov et al., 2016). This was also observed by Renard et al.
(1999) and it indicates that ˛-d-GalpA is substituted out of the RGI
backbone and that the substitution influences the nearly located
Rha. Beta anomeric configuration of GlcpA is suggested directly by
the identified chemical shift of the H1 proton at 4.71 ppm (Renard
et al., 1999).
The cross-peaks in the HSQC and HSQC-NOESY spectra for carbon
and proton signals from methyl group of acetic acid ester at
22.9/2.09 ppm and 22.9/2.15 ppm are found, which confirms the
presence of ester-linked acetic acid in PSIII. Correlations between
the acetyl methyl protons and acetyl carbonyl carbons are observed
in the C6 region of uronic acids in the HMBC spectrum. C6 signals
of both uronic acid residues and CH3CO group are overlapped. The
presence of two peaks for −CH3CO group at 2.09 ppm and 2.15 ppm
suggests O-3 and O-2 acetylation in PSIII. The signal at 2.09 ppm is
more intensive (integral ratio 2.09:2.15 ppm 3,8:1) and according
to literature data attributed to O-3 acetylation of GalA in the HG,
as well as of the Rha in RGI (Renard & Jarvis, 1999; Sengkhamparn
et al., 2009). The proton methyl signal at 2.09 ppm is correlated with
the peaks at 1.28 ppm (F), 3.61 ppm (G), 4.71 ppm (H) and 5.09 ppm
(I) in the ROESY spectrum (Fig. 1A). The correlation G is the most
intensive, supporting the closeness between the proton at 3.61 ppm
with the acetyl protons. The correlation F indicates that the acetyl
groups are within the Rha ring. The detailed analysis of the COSY,
TOCSY, ROESY, HMBC and HSQC-NOESY spectra is in agreement
with the predominant localization of the acetyl groups at O-3 of 1,2˛-l-Rhap
units (Fig. 2). This is demonstrated by the downfield shift
of H2 (4.20 pm), H3 (5.09 ppm), H4 (3.61 ppm) and H5 (4.11 ppm) of
the 1,2-˛-l-Rhap residues. By calculating the DA of PSIII on Rha basis
instead of GalA basis, it is estimated that 33.3% of the Rha units are
acetylated. The ratio between integral intensities of H6 of 1,2,-˛l-Rhap:1,2-˛-l-Rhap-3OAc
is 2.16:1, indicating that approximately
32% of the Rha units are acetylated. Similarly, Sengkhamparn et al.
(2009) observed downfield shift of H2 (4.22 ppm), H3 (5.24 ppm)
and H5 (3.91 ppm) of the O-3 acetylated 1,2,4-˛-l-Rhap in pectin
isolated from okra. Interestingly, A. esculentus (okra) belongs to
Malvaceae family, where the linden was classified initially. The correlation
between the resonances at 2.09 ppm and 4.71 ppm in the
ROESY spectrum suggests that the proton at 4.71 ppm is conformationally
close to the acetyl group. The monosaccharide composition
and preliminarily glycosidic linkage analysis of PSIII indicate that it
contains a small amount of Gal that might be linked to O-4 of 1,2-˛l-Rhap-3OAc.
Therefore, it is proposed that the signal at 4.71 ppm
contains the anomeric proton of T-ˇ-d-Galp units (Shakhmatov,
Udoratina, Atukmaev, & Makarova, 2015). This is supported by the
finding that the proton at 4.71 ppm (106.6 ppm) is adjacent to the
proton at 3.52 ppm (74.3 ppm) in the COSY spectrum and by one
cross-peak between 106.6 ppm and 3.61 ppm in the HSQC-NOESY
spectrum (Fig. 1B).
The assignment of carboxyl carbonyl carbons C6 of both uronic
acids was done from the HMBC spectrum, where the following
information revealed, the proton H5 of 1,3,4-˛-d-GalpA at 4.77 ppm
is correlated with the carbons at 176.2 ppm. Similarly, the H5 proton
of ˇ-d-GlcpA resonating at 3.81 ppm is connected with the
carbons at 177.1 ppm. The absence of a signal at 53–55 ppm arising
from −OCH3 of methyl ester group confirmed that PSIII is with very
low DM or not methyl-esterified (Shakhmatov et al., 2016). From
additional analysis of the COSY, TOCSY, ROESY and HMBC spectra,
it was suggested that the carbon and proton resonances respectively
at 183.3 ppm and 1.35-1.36 ppm arise from −CO and −CH3
of fatty acids (RCOOH), which were detected in the initial linden PSC
and probably partly left in PSIII after the alcohol pretreatment and
anion-exchange chromatography of the linden PSin70 (not shown).
A model structure of PSIII with some important H/H and C/H interactions
from the NMR analyses are presented on Fig. 2.
3.1.4. FTIR analysis
The presence of acetyl esters is also confirmed by the FTIR
study (Fig. S2), where bands with similar intensities at 1248 cm−1
(C O C)Ac and 1721 cm−1 (C O)Ac are detected and disappear
when the sample is saponified with NaOH (Paulsen, Fagerheim, &
Øverbye, 1978). The bands at 3347 cm−1, 2935 cm−1, 1600 cm−1
and 1417 cm−1 for (OH), (CH), as(COO−) and s(COO−) are also
observed (Filippov, Luknár, & Kohn, 1978). The presence of the signals
at about 1151, 1037, 991, 958 and 819 cm−1 are also found in
the linden PSC (Georgiev et al., 2017). Most of these signals, in addition
to those at 916 and 844 cm−1 registered in the FTIR spectrum of
PSIII are related to the RGI backbone structure according to the literature
data (Kaˇcuráková, Capek, Sasinková, Wellner, Ebringerová,
2000).
3.1.5. Small-angle X-ray scattering studies
In order to investigate the nanostructure of PSIII in water, SAXS
experiments were performed. The absolute macroscopic scattering
cross-section d /d (Q) as a function of modulus of the scattering
vector Q (Q = 4 sin Â
2
/ , where ␭ = 1.54 Å and ␪ is the scattering
angle) is depicted on Fig. 3. The function represents a typical
polymer-like scattering at an intermediate and a high Q, while
at a lower Q a significant upturn is observed with a close to Q−4
power-law characteristic for large clusters. This indicates the presence
of larger aggregates (> 100 nm) in coexistence with chain-like
molecules. Interestingly, the structure is essentially independent of
concentration and temperature, suggesting rather long-lived stable
aggregates. More quantitatively, the data can be described using a
simple coexistence model of clusters and single chain (see section
2.8.). The fits provide an average molecular weight of 332 kDa for
the dissolved chain, Rg = 4.6 nm and df = 1,7. The clusters are larger
than what can be resolved by SAXS (> 100 nm) and could not be
analyzed in details. The aggregation of pectins in aqueous solutions
could be expected, thus different salts were used in the HPSEC and
preparative SEC analyses to prevent it. Despite of the aggregation,
the calculated Mw is in a good agreement with the determined values
from HPSEC (382 kDa) and preparative SEC analyses (352 kDa).
3.2. Immunomodulating studies
We have already found that the linden PSC possesses ex vivo
immunomodulating potential against human phagocytic leuko-
186 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
Fig. 2. Chemical structure of linden pectic fraction PSIII, elucidated by GC–MS glycosidic linkage analysis and 2D NMR spectroscopy. The most important H/H and C/H
interactions are shown.
cytes, T-cells, murine PP cells and anti-tumor activity in vitro
(Georgiev et al., 2017). In order to investigate the role of linden
pectins in the observed immunomodulating activity of the initial
linden PSC and to evaluate immune potency of each purified linden
pectic fraction were performed some important biological experiments.
In the following sections are presented the results from
these experiments with purified linden pectic fractions.
3.2.1. Complement fixation activity
The three linden pectic fractions were tested against inhibition
of human complement hemolysis of target SRBC in vitro (Fig. S3).
PSI expressed the highest complement fixation activity with IC50
of 154.5 ␮g/ml, which was 4.7 fold higher than the IC50 value of
the positive control, the highly active pectic polysaccharide BP-II
from B. petersianum (Grønhaug et al., 2011). PSII and PSIII expressed
lower activities, as IC50 value of PSIII was 491.6 ␮g/ml. Most probably
the prevalence of the complement fixation activity of PSI was
related to the presence of AGII structures active on the human
complement system (Ho et al., 2016).
3.2.2. Phagocyte immune-response
3.2.2.1. Human WBP and isolated neutrophils
The typical pectic polysaccharide (PSI) and the two GlcA-RGsI
(PSII and PSIII) increased ROS production from WBP at 100 ␮g/ml
and only PSIII increased CL response from isolated neutrophils
(Fig. 4A and B). Meanwhile, with co-stimulation by PMA, PSI and
PSIII did not increase the ROS production from WBP, however
PSII slightly enhanced the ROS formation at a concentration of
100 ␮g/ml (Fig. 4A). Interestingly, under the condition on a costimulation
with OZP, PSI and PSII clearly decreased the ROS
production at the higher concentration of 100 ␮g/ml in comparison
with the control of the OZP stimulation alone (Fig. 4A). However, all
polysaccharide fractions did not show a clear enhancing activity on
the ROS formation from the isolated neutrophils in the presence of
PMA and OZP (Fig. 4B). PMA and OZP significantly increased the CL
signal of both WBP and isolated neutrophils. In the case of spontaneous
ROS formation the CL signal caused by the linden pectins at
the higher concentration of 100 ␮g/ml was much lower than those
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 187
Fig. 3. Small-angle X-ray scattering data showing the absolute macroscopic scattering
cross-section d /d (Q) as a function of modulus of the scattering vector, Q
for PSIII aqueous solutions. a) at different concentrations and b) at different tem-
peratures.
in the presence of PMA or OZP (Fig. 4A and B). When the effect of the
polysaccharide fractions on the expression of CD18 and CD62L on
isolated neutrophils was analyzed, only PSIII showed a weak effect
on CD18 expression (Fig. S4). This was in agreement with the results
from the spontaneous ROS production from both WBP and isolated
neutrophils. It was proposed that the unique glucuronidated RGI
structure of PSIII might be involved in the observed effects.
3.2.2.2. Murine macrophages
Macrophage activation can be evaluated by the release of
antimicrobial NO under the catalytic action of iNOS. The purified
linden pectins did not have a cytotoxic effect on the RAW 264.7
cells on the basis of the LDH test (Fig. S5). When the iNOS induction
was analyzed by western blotting, the stimulation with all
linden pectic fractions for 24 h increased the iNOS protein level of
the RAW 264.7 cells. The co-stimulation with LPS (10 ng/ml) in the
presence of the polysaccharides significantly enhanced the iNOS
protein level by comparison with the stimulating condition with
the purified pectins alone (Fig. 5A). Otherwise, the simultaneous
treatment of the macrophages with LPS and linden polysaccharides
led to statistically significant lower iNOS levels at 100 ␮g/ml than
at 50 ␮g/ml for PSI and PSIII. This effect was similar to the reduction
of the ROS formation from WBP co-stimulated with OZP and PSI or
PSII (Fig. 4A). When the RAW 264.7 cells were stimulated with the
linden pectic fractions for 48 h, the iNOS production reached almost
to the same level as that observed in the co-stimulation condition
with LPS and linden fractions (Fig. 5A). Similarly to the results from
24 h, the iNOS levels in the cells co-treated with LPS and PSI or PSIII
at 100 ␮g/ml after 48 h were slightly decreased in comparison with
the pectic fractions alone. But unlike the results from 24 h, where
there was not observed any clear difference in the iNOS expression
induced by three samples alone, after 48 h PSI exhibited a higher
induction activity at the both studied concentrations.
Meanwhile, the stimulation effect of polysaccharide fractions,
alone or in a combination with LPS was analyzed on the NO production
from RAW 264.7 cells (Fig. 5B). The NO production was
dramatically increased by the stimulation with all fractions for 24 h
at concentrations of 50 and 100 ␮g/ml compared with the growth
medium control. The NO release in the control medium was negligible,
therefore the adherent cells were not activated by the plate
surface. The stimulating activity of PSII seemed to be weaker than
those of PSI and PSIII, and there was no a significant difference
between the stimulatory activities of PSI and PSIII. However, the
combinatorial effect of linden polysaccharides with LPS was tested,
and the NO production induced by all linden pectins at 100 ␮g/ml
were lower in comparison with the production in the stimulation
with the polysaccharide fractions alone (Fig. 5B). When the stimulation
was prolonged for 48 h, PSI induced more than 2 fold higher
NO production than PSIII, which could be explained by the different
structural features of both samples (p < 0.001). This result was
in agreement with the iNOS expression after 48 h of incubation
(Fig. 5A). After 48 h NO production in the co-stimulation condition
was reduced again at 100 ␮g/ml in comparison with the separate
polysaccharide treatments (p < 0.001) (Fig. 5B). This effect was in
agreement with the iNOS expression levels after 24 h and 48 h of
macrophage stimulation. From the cytotoxicity data, combinations
with LPS showed relative absorbance units close to the control,
which was another confirmation for the suppression-like effect on
the LPS-induced inflammatory response in macrophages (Fig. S5).
For elucidation of this phenomenon more studies on the effect of
linden polysaccharides on the iNOS expression with higher doses
are needed.
3.2.3. Intestinal immunomodulating activity
It has been found that the linden PSC exhibited ex vivo intestinal
immunomodulating activity through the PP-mediated bone marFig.
4. Effect of linden pectic fractions (PSI-PSIII) on A) ROS production from non-stimulated (spontaneous), PMA- and OZP-activated human whole blood phagocytes. B)
ROS production from isolated neutrophils. 50 (50 ␮g/ml) and 100 (100 ␮g/ml). The asterisks indicate statistical significance (*p < 0.05, **p < 0.01) vs. control.
188 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
Fig. 5. Effect of linden pectic fractions on A) iNOS expression in RAW 264.7 cells cultured with samples alone or co-stimulated with LPS. B) NO production from RAW 264.7
cells cultured with samples alone or co-stimulated with LPS. LPS from E. coli was used as a positive control. The asterisks indicate statistical significance (*p < 0.05, **p < 0.01,
***p < 0.001) vs. control − growth medium (untreated cells) and (#p < 0.05, ##p < 0.01, ###p < 0.001) vs. LPS.
row cell proliferation test and increased IL-6 production from the
PP cells (Georgiev et al., 2017). In order to clarify whether partially
purified polysaccharides (PSI-PSIII) also show activity, their
stimulatory potential was investigated as well. However, only PSI
expressed a weak activity (p < 0.01) and PSII and PSIII were inactive
(Fig. S6).
3.2.4. Anti-tumor activity
When we found that the linden pectins exhibited immunomodulating
properties against phagocytes, we decided to test their
effect on abnormal cells. In a previous study, the linden PSC
expressed a non-specific inhibitory activity against different tumor
cell lines and normal amniotic cells and fibroblasts in vitro
(Georgiev et al., 2017). On the basis of our preliminarily screening
results with PSI and PSIII, we found that in addition to the tumor
cells, polysaccharides inhibited in a similar degree the growth of
the studied FL cells (Fig. S7).
4. Discussion
The need of a further characterization of the silver linden pectins
was provoked by elucidation of the role of different pectic fractions
as immunomodulating compounds in Tilia flowers and also due to
the traditional use of the linden tea and mucilage. It was found that
PSI is a low-esterified typical pectic polysaccharide with HG and
RGI, which is O-4 substituted at the Rha units with 1,3,5-arabinans
and highly branched bioactive AGII structures. Furthermore, it was
suggested that PSI contains trace amounts of RGII on the basis of
the KDO test, the presence of 1,3,4-linked GalA residues and the
trace amounts of Fuc. PSII and PSIII are two unusual high molecular
weight free RGI polymers that are highly acetylated and highly glucuronidated,
almost lacking neutral sugar side chains. Renard et al.
(1999) estimated that one GalA unit out of 72 are O-3 substituted
with GlcA in sugar beet RGI. Sengkhamparn et al. (2009) found for
the first time that Rha in okra pectic RGI can be O-3 acetylated. Later,
Leijdekkers, Huang, Bakx, Gruppen, and Schols (2015) reported
that a single GalA unit in the RGI from sugar beet pectin could be
both acetylated and substituted with GlcA. In the present study
it was demonstrated that silver linden flowers contain a unique
RGI (PSIII) in which almost every GalA unit is O-3 glucuronidated
and at the same time significantly acetylated (>30%) at O-3 of the
Rha residues. The O-3 glucuronidated RGI core structure was found
in the mucilage of the inner bark of Hydrangea paniculata Sieb. in
1976–1977 (Tomoda & Satoh, 1977). The structure is not found
often in plants and to our knowledge, it has not been isolated from
flowering plant organs (Shimizu & Tomoda, 1985).
Phagocytic leukocytes, such as macrophages and neutrophils,
producing ROS and NO play a key role in the innate immunity. They
are also involved in the acute inflammatory diseases, thus a proper
modulation of their activation is often needed. Our findings suggest
that the three purified polysaccharide fractions (PSI-PSIII) are
active ingredients in the crude linden PSC for enhancing activity on
the ROS production of human WBP and neutrophils (Georgiev et al.,
2017). Prevalence in the activity was observed for PSII and PSIII, as
only PSIII increased the ROS production from isolated neutrophils. It
was suggested that the unique glucuronidated RGI structure of PSIII
was important for expression of the activity, which was supported
by the weak increase of the surface ß2-integrin levels on neutrophils
induced by the sample (Fig. S4). However, the effect of the
three polysaccharides was negligible in comparison with the potent
activators PMA, OZP and fMLP (Figs. 4 & S4). Furthermore, the effect
of PSI-III against the spontaneous ROS generation from WBP at
100 ␮g/ml was lower than this for the crude linden PSC (420 ± 60%),
which could be explained by the combinatorial action of different
active compounds in the complex (Georgiev et al., 2017). On the
other hand, PSI and PSIII expressed a suppression-like activity on
the OZP-induced activation of WBP at the higher concentration of
100 ␮g/ml, which was not observed with the linden PSC (Georgiev
et al., 2017). This effect was also not observed in the experiments
with isolated neutrophils. Kiyohara, Matsumoto, Nagai, Kim, &
Yamada (2006) showed a significant positive correlation between
the reactivity of pectic polysaccharides with IgG and the degree of
complement-activating ability. PSI expressed a high complement
fixation activity, which supposed that the linden polysaccharides
could interact with some blood serum components like antibodies
and then act in such a manner to negatively influence the OZPinitiated
oxidative burst. Interestingly, Popov, Ovodova, Popova,
Nikitina, and Ovodov (2005) reported that a pectic polysaccharide
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 189
(100 ␮g/ml) from Comarum palustre does not influence the OZPactivated
ROS generation from human neutrophils, but it could
reduce the PMA-induced ROS production. The crude linden PSC
also enhanced the spontaneous ROS production from WBP and
did not increase the CL signal under the co-stimulation with PMA
(Georgiev et al., 2017). The three pectic fractions did not contribute
to a synergistic increase of the ROS production from the PMA- and
OZP-treated neutrophils.
The induction of NO production by the linden pectins did not
lead to severe cytotoxic effects under the studied conditions (Fig.
S5). PSI expressed a higher macrophage stimulating activity than
PSII and PSIII, as probably the RGI AG side chains are important
for the macrophage activation, as described in the literature
(Grønhaug et al., 2010). Generally, our results from the NO production
demonstrated that linden pectins could activate macrophages
and this was in agreement with the increased human CD14+ and
CD64+ white blood cells and induction of IL-6 production by the
linden PSC (Georgiev et al., 2017). The linden pectins exhibited
suppression-like effect on the LPS-induced inflammatory response
in macrophages, shown by reduction of the iNOS expression and
NO production. However, both LPS and linden polysaccharides
alone were inductors of the iNOS and NO production. Therefore,
there was not a synergism between the microbial antigen and the
plant antigen-mimetic, as was expected and even observed by Zhu
et al. (2012) for a combination between LPS and a polysaccharide
from Chaenomeles speciosa fruit. The observed effect suggests
that the linden pectins have a potential for stimulation of the
innate immunity and suppression of the LPS-induced macrophage
inflammatory process similarly to the reduction of the OZP-induced
ROS generation from WBP. This could be explained by the proposed
polysaccharide anti-inflammatory behavior, which could be
useful in supplementation during treatment of autoimmune and
infectious diseases. In fact, T. tomentosa is famous with its antiinflammatory
action which is attributed mainly to its flavonoids,
but probably polysaccharides also contribute to this effect (Allio
et al., 2015; Matsuda et al., 2002; Viola et al., 1994). Similar to the
dual effect observed in our study was also reported for fucoidan,
which stimulated the NO release from RAW 264.7 cells at 100 ␮g/ml
alone, but in a combination with LPS at concentrations higher
than 10 ␮g/ml it inhibited the LPS-stimulated NO release and iNOS
expression (Nakamura, Suzuki, Wada, Kodama, & Doi, 2006; Yang,
Yoon, Oh, Kim, & Kang, 2006). Authors concluded that the fucoidan
may not act on the extracellular receptors, but on the intracellular
signaling machinery, such as the process of transcription factor activation.
Chen et al. (2006) reported that the LPS-induced (100 ng/ml)
iNOS expression level in RAW 264.7 cells was similar to those
obtained by a co-stimulation with LPS and DE90 citrus pectin, but
increasing of the pectin concentration resulted in a decrement of
the iNOS protein expression. They found that DE90 pectin had a
potential to inhibit the downstream signals of LPS, and proposed
that the inhibition was not a result of the DE90 directly inhibiting
the activities of signal proteins, but from the pectin-LPS interactions
or different affinity to cell-surface receptors.
The role of GlcA in plant polysaccharides and its impact on
the biological activity are intriguing but still not fully elucidated
(Grønhaug et al., 2011; Kiyohara et al., 2010). Interestingly, Duan,
Chen, Dong, Ding, and Fang (2010) showed that pectin from D. kaki
structurally similar to PSIII could inhibit the LPS-induced B-cell proliferation
and had no effect on the Concanavalin A-induced T-cell
proliferation. This, together with our results also suggests that GlcA
might be involved in the suppression effect of the linden pectins
on the LPS-induced and OZP-induced inflammatory response in
macrophages and WBP.
Normally, herbal polysaccharides are contained in infusions,
extracts, traditional foods and are administered orally from these
formulations. Pectins are little absorbed by the intestinal epithelium,
but they may interact with the mucosal immune system
through immunocompetent cells in the PP localized in the small
intestine. By this mode of action of the active polysaccharides,
functionally modulated immunocompetent cells are proposed to
result in modulations of several mucosal and systemic immune systems
(Yamada & Kiyohara, 2007). Only PSI showed a weak activity
(p < 0.01) probably related to RGI AG side chains as found previously
(Grønhaug et al., 2011; Kiyohara et al., 2010). Maybe acetylated RGI
backbone and attached GlcA in PSIII were not enough for expression
of activity.
The neutrophils and macrophages are involved in the regulation
of both innate and adaptive immunity in various inflammatory
processes including tumor diseases. The phagocytes present in the
tumor microenvironment can promote or inhibit cancer formation
and development (Mantovani, 2014). Brizi et al. (2012) reported
that aqueous and ethanol extracts from the flowers of Tilia x viridis
(T. tomentosa and T. americana hybrid) exhibited anti-proliferative
effects on BW 5147 cells (T-cell lymphoma) and normal Concanavalin
A-stimulated lymphocytes in vitro, as they related the
activity to polyphenols. Our preliminarily screening study with
the MTT test demonstrated that linden pectins expressed a nonselective
growth inhibitory activity against tumor cells and the
studied normal FL cells (Fig. S7). However, PSI-PSIII did not show
cytotoxic effects (LDH test) on murine macrophages (Fig. S5) and
they did not inhibit the proliferation of murine PP immunocompetent
cells (Fig. S6). The difference between the polysaccharide effect
on the FL cells and the studied immune cells could be explained
primarily by their different functions in the organism. Generally,
the immune cells could be activated by complex pectins unlike the
tumor cells and probably FL cells.
5. Conclusions
In the current paper, we show that the silver linden blossoms
contain two structurally different pectic type polysaccharides −
a low-esterified typical pectic polysaccharide PSI (Mw = 28.2 kDa)
and unusual considerably acetylated (2.9-3.3%) and glucuronidated
RGI polymers PSII (Mw = 949 kDa) and PSIII (Mw = 382 kDa). Furthermore,
PSIII is a unique RGI macromolecule, which GalA residues
are almost completely O-3 glucuronidated and >30% acetylated at
O-3 of the Rha units. The three pectic fractions could stimulate the
innate immune response through induction of the ROS and NO
generation by neutrophils and macrophages in a non-aggressive
manner. At the same time, silver linden pectins suppressed the OZPactivated
ROS generation and the LPS-induced iNOS expression
and NO production by WBP and murine macrophages, respectively.
This dual mode of action on the phagocytes revealed
the anti-inflammatory potential of the silver linden polysaccharides.
Furthermore, PSI showed the highest complement fixation
(IC50 = 154.5 ␮g/ml) and macrophage stimulating (almost 18 ␮M
NO production after 48 h) activities and at the same time, it was
the only sample expressing intestinal immunomodulating activity
against PP cells. It was shown for the first time that acetylated
and highly glucuronidated RGI may exhibit immunomodulating
properties via neutrophil and macrophage activation, and suppression
of viability of different tumor cell lines. On the basis of our
results, we conclude that pectins in T. tomentosa inflorescences are
immune-active compounds. The silver linden pectins deserve to be
included in the supplementary therapy research for prevention and
treatment of immune related diseases like inflammatory diseases.
Acknowledgements
We would like to thank Dr. Mariana Nikolova (Plovdiv University
Paisii Hilendarski) for kindly providing the herbal material.
190 Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191
We are also indebted to Dr. Veronika Sivova for performing the
complement assay. This work was supported by the European
Regional Development Fund, EU [Grant № BG161PO0003-1.1.05-
0024-C0001, 2012-2014], European Social Fund, EU [Grant no
BG051PO001/3.3-05-0001, 2014], the Ministry of Education, Youth
and Sport of the Czech Republic [Grant no LQ1605 NPU II; project
MSM0021622430], Erasmus+ Programme and EEA grants. The
Research Council of Norway is acknowledged for financial support
through the Norwegian NMR Platform, NNP (226244/F50).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.07.
073.
References
Allio, A., Calorio, C., Franchino, C., Gavello, D., Carbone, E., & Marcantoni, A. (2015).
Bud extracts from Tilia tomentosa Moench inhibit hippocampal neuronal firing
through GABAA and benzodiazepine receptors activation. Journal of Ethnopharmacology,
172, 288–296.
Anthon, G. E., & Barrett, D. M. (2008). Combined enzymatic and colorimetric method
for determining the uronic acid and methyl ester content of pectin: Application
to tomato products. Food Chemistry, 110, 239–247.
Arcos, M. L. B., Cremaschi, G., Werner, S., Coussio, J., Ferraro, G., & Anesini, C. (2006).
Tilia cordata Mill. extracts and scopoletin (isolated compound): Differential cell
growth effects on lymphocytes. Phytotherapy Research, 20(1), 34–40.
Barsett, H., Paulsen, B. S., & Habte, Y. (1992). Further characterization of polysaccharides
in seeds from Ulmus glabra Huds. Carbohydrate Polymers, 18(2), 125–130.
Beaucage, G. (1996). Small-angle scattering from polymeric mass fractals of arbitrary
mass-fractal dimension. Journal of Applied Crystallography, 29, 134–146.
Bijak, M., Saluk, J., Tsirigotis-Maniecka, M., Komorowska, H., Wachowicz, B.,
Zaczy ´nska, E., et al. (2013). The influence of conjugates isolated from Matricaria
chamomila L. on platelets activity and cytotoxicity. International Journal of
Biological Macromolecules, 61, 218–229.
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination
of uronic acids. Analytical Biochemistry, 54, 484–489.
Bradford, М. М. (1976). A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72, 248–254.
Brizi, M. R., Marrassini, C., Zettler, G., Ferraro, G., & Anesini, C. (2012). Comparative
antiproliferative action of two extracts from Tilia × viridis on normal and tumoral
lymphocytes: Relationship with antioxidant activity. Chinese Medicine, 3, 20–29.
Chambers, R. E., & Clamp, J. R. (1971). An assessment of methanolysis and other
factors used in the analysis of carbohydrate-containing materials. Biochemical
Journal, 125, 1009–1018.
Chen, C. H., Sheu, M. T., Chen, T. F., Wang, Y. C., Hou, W. C., Liu, D. Z., et al.
(2006). Suppression of endotoxin-induced proinflammatory responses by citrus
pectin through blocking LPS signaling pathways. Biochemical Pharmacology,
72(8), 1001–1009.
Chinou, I. (2012a). Assessment report on Tilia cordata Miller, Tilia platyphyllos Scop.,
Tilia x vulgaris Heyne or their mixtures, flos. European Medicines Agency, Committee
on Herbal Medicinal Products., 337067/2011
Chinou, I. (2012b). Assessment report on Tilia tomentosa Moench, flos. European
Medicines Agency, Committee on Herbal Medicinal Products., 346780/2011.
Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of
carbohydrates. Carbohydrate Research, 131(2), 209–217.
de Santana-Filho, A. P., Noleto, G. R., Gorin, P. A. J., de Souza, L. M., Iacomini, M., &
Sassaki, G. L. (2012). GC–MS detection and quantification of lipopolysaccharides
in polysaccharides through 3-O-acetyl fatty acid methyl esters. Carbohydrate
Polymers, 87, 2730–2734.
Denman, L. J., & Morris, G. (2015). An experimental design approach to the chemical
characterisation of pectin polysaccharides extracted from Cucumis melo
Inodorus. Carbohydrate Polymers, 117, 364–369.
Duan, J., Chen, V. L., Dong, Q., Ding, K., & Fang, J. (2010). Chemical structure and
immunoinhibitory activity of a pectic polysaccharide containing glucuronic
acid from the leaves of Diospyros kaki. International Journal of Biological Macromolecules,
46, 465–470.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, A., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chemistry,
28(3), 350–356.
Fan, Y., Sun, C., Gao, X., Wang, F., Li, X., Kassim, R. M., et al. (2012). Neuroprotective
effects of ginseng pectin through the activation of ERK/MAPK and Akt survival
signaling pathways. Molecular Medicine Reports, 5(5), 1185–1190.
Filippov, M. P., Luknár, O., & Kohn, R. (1978). Infrared spectra of the acetyl derivatives
of pectin. I. Potassium salts of the citrus pectic acid with different degrees of
acetylation. Chemical Papers, 32(2), 211–217.
Gallova, L., Kubala, L., Ciz, M., & Lojek, A. (2004). IL-10 does not affect oxidative burst
and expression of selected surface antigen on human blood phagocytes in vitro.
Physiological Research, 53, 199–208.
Georgiev, Y., Ognyanov, M., Yanakieva, Y., Kussovski, V., & Kratchanova, M. (2012).
Isolation, characterization and modification of citrus pectin. Journal of BioScience
and Biotechnology, 1, 223–233.
Georgiev, Y. N., Ognyanov, M. H., Kiyohara, H., Batsalova, T. G., Dzhambazov, B. M., Ciz,
M., et al. (2017). Acidic polysaccharide complexes from purslane, silver linden
and lavender stimulate Peyer’s patch immune cells through innate and adaptive
mechanisms. International Journal of Biological Macromolecules, http://dx.
doi.org/10.1016/j.ijbiomac.2017.07.095
Grønhaug, T. E., Ghildyal, P., Barsett, H., Michaelsen, T. E., Morris, G., Diallo, D., et al.
(2010). Bioactive arabinogalactan from the leaves of Opilia celtidifolia Endl. ex
Walp. (Opiliaceae). Glycobiology, 20(12), 1654–1664.
Grønhaug, T. E., Kiyohara, H., Sveaass, A., Diallo, D., Yamada, H., & Paulsen, B. S.
(2011). Beta-D-(1 → 4)-galactan-containing side chains in RG-I regions of pectic
polysaccharides from Biophytum petersianum Klotzsch. contribute to expression
of immunomodulating activity against intestinal Peyer’s patch cells and
macrophages. Phytochemistry, 72, 2139–2147.
Groudeva, J., Kratchanova, M. G., Panchev, I. N., & Kratchanov, C. G. (1997). Application
of granulated apple pectin in the treatment of hyperlipoproteinaemia.
Zeitschrift für Lebensmittel-Untersuchung und −Forschung, 204, 374–378.
Ho, G. T. T., Zou, Y.-F., Aslaksen, T. H., Wangensteen, H., & Barsett, H. (2016). Structural
characterization of bioactive pectic polysaccharides from elderflowers (Sambuci
flos). Carbohydrate Polymers, 135, 128–137.
Holderness, J., Schepetkin, I. A., Freedman, B., Kirpotina, L. N., Quinn, M. T., Hedges, J.
F., et al. (2011). Polysaccharides isolated from ac¸ aí fruit induce innate immune
responses. PLOS ONE, 6(2), e17301.
Hong, T., Matsumoto, T., Kiyohara, H., & Yamada, H. (1998). Enhanced production
of hematopoietic growth factors through T-cell activation in Peyer’s patches
by oral administration of Kampo (Japanese herbal) medicine, Juzen-Taiho-To.
Phytomedicine, 5, 353–360.
Hritcu, L., & Cioanca, O. (2016). Prevalence of use of herbal medicines and complementary
and alternative medicine in Europe. In C. Grosso (Ed.), Herbal medicine
in depression (pp. 135–182). Switzerland: Springer International Publishing.
Inngjerdingen, K. T., Kiyohara, H., Matsumoto, T., Petersen, D., Michaelsen, T. E.,
Diallo, D., et al. (2007). An immunomodulating pectic polymer from Glinus
oppositifolius. Phytochemistry, 68, 1046–1058.
Inngjerdingen, M., Inngjerdingen, K. T., Patel, T. R., Allen, S., Chen, X., Rolstad, B., et al.
(2008). Pectic polysaccharides from Biophytum petersianum Klotzsch, and their
activation of macrophages and dendritic cells. Glycobiology, 18(12), 1074–1084.
Kaˇcuráková, M., Capek, P., Sasinková, V., Wellner, N., & Ebringerová, E. (2000). FT-IR
study of plant cell wall model compounds: Pectic polysaccharides and hemicelluloses.
Carbohydrate Polymers, 43, 195–203.
Kim, J., & Carpita, N. C. (1992). Changes in esterification of the uronic acid groups of
cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiology,
98(2), 646–653.
Kiyohara, H., Matsumoto, T., Nagai, T., Kim, J.-S., & Yamada, H. (2006). The presence
of natural human antibodies reactive against pharmacologically active pectic
polysaccharides from herbal medicines. Phytomedicine, 13, 494–500.
Kiyohara, H., Uchida, T., Takakiwa, M., Matsuzaki, T., Hada, N., Takeda, T., et al.
(2010). Different contributions of side-chains in ˇ-D-(1 → 3, 6)-galactans on
intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus
mongholics Bunge. Phytochemistry, 71, 280–293.
Kosakowska, O. K., B˛aczeka, K., Przybył, J. L., Ejdys, M., Ku´zma, P., Obiedzi ´nski, M.,
et al. (2015). Intraspecific variability in the content of phenolic compounds,
essential oil and mucilage of small-leaved lime (Tilia cordata Mill.) from Poland.
Industrial Crops and Products, 78, 58–65.
Kram, G., & Franz, G. (1983). Untersuchungen über die Schleimpolysaccharide aus
Lindenblüten. Planta Medica, 49, 149–153.
Kram, G., & Franz, G. (1985). Structural investigations on the watersoluble polysaccharides
of lime tree flowers (Tilia cordata L.). Pharmazie, 40, 501–502.
Leijdekkers, A. G. M., Huang, J.-H., Bakx, E. J., Gruppen, H., & Schols, H. A. (2015). Identification
of novel isomeric pectic oligosaccharides using hydrophilic interaction
chromatography coupled to travelling-wave ion mobility mass spectrometry.
Carbohydrate Research, 404, 1–8.
Liu, Y., Dong, M., Yang, Z., & Pan, S. (2016). Anti-diabetic effect of citrus pectin in diabetic
rats and potential mechanism via PI3K/Akt signaling pathway. International
Journal of Biological Macromolecules, 89, 484–488.
Lojek, A., Ciz, M., Pekarova, M., Ambrozova, G., Vasicek, O., Moravcova, J., et al. (2011).
Modulation of metabolic activity of phagocytes by antihistamines. Interdisciplinary
Toxicology, 4(1), 15–19.
Ma, L., Qin, C., Wang, M., Gan, D., Cao, L., Ye, H., et al. (2013). Preparation, preliminary
characterization and inhibitory effect on human colon cancer HT-29 cells of an
acidic polysaccharide fraction from Stachys floridana Schuttl. ex Benth. Food and
Chemical Toxicology, 60, 269–276.
МcComb, E. A., & McCready, R. M. (1957). Determination of acetyl in pectin and in
acetylated carbohydrate polymers (Hydroxamic acid reaction). Analytical Chemistry,
29, 819–821.
Mantovani, A. (2014). Macrophages, neutrophils, and cancer: A double edged sword.
New Journal of Science, 271940 http://dx.doi.org/10.1155/2014/271940
Manuele, M. G., Ferraro, G., & Anesini, C. (2008). Effect of Tilia × viridis flower
extract on the proliferation of a lymphoma cell line and on normal murine
lymphocytes: Contribution of monoterpenes, especially limonene. Phytotherapy
Research, 22(11), 1520–1526.
Y.N. Georgiev et al. / Carbohydrate Polymers 175 (2017) 178–191 191
Matsuda, H., Ninomiya, K., Shimoda, H., & Yoshikawa, M. (2002). Hepatoprotective
principles from the flowers of Tilia argentea (Linden): Structure requirements
of tiliroside and mechanisms of action. Bioorganic and Medicinal Chemistry, 10,
707–712.
McNeil, M., Darvill, A. G., & Albersheim, P. (1980). X. Rhamnogalacturonan I, a structurally
complex pectic polysaccharide in the cell walls of suspension-cultured
sycamore cells. Plant Physiology, 66, 1128–1134.
Michaelsen, T. E., Gilje, A., Samuelsen, A. B., Høåsen, K., & Paulsen, B. S. (2000). Interaction
between human complement and a pectin type polysaccharide fraction,
PMII, from the leaves of Plantago major L. Scandinavian Journal of Immunology,
52, 483–490.
Nakamura, T., Suzuki, H., Wada, Y., Kodama, T., & Doi, T. (2006). Fucoidan induces
nitric oxide production via p38 mitogen-activated protein kinase and NFkB-dependent
signaling pathways through macrophage scavenger receptors.
Biochemical and Biophysical Research Communications, 343(1), 286–294.
Nauseef, W. M. (2014). Isolation of human neutrophils from venous blood. In M. T.
Quinn, & F. R. DeLeo (Eds.), Methods in molecular biology 1124, Neutrophil methods
and protocols (pp. 13–18). New York: Humana Press Inc.
Ohtani, K., Okai, K., Yamashita, U., Yuasa, I., & Misaki, A. (1995). Characterization of an
acidic polysaccharide isolated from the leaves of Corchorus olitorius (Moroheiya).
Bioscience, Biotechnology, and Biochemistry, 59(3), 378–381.
Paulsen, B. S., & Barsett, H. (2005). Bioactive pectic polysaccharides. In T. Heinze (Ed.),
Advances in polymer science 186, polysaccharides I. structure, characterization and
use (pp. 69–101). Berlin Heidelberg: Springer-Verlag.
Paulsen, B. S., Fagerheim, E., & Øverbye, E. (1978). Structural studies of the polysaccharide
from Aloe plicatilis Miller. Carbohydrate Research, 60, 345–351.
Pekarova, M., Lojek, A., Martiskova, H., Vasicek, O., Bino, L., Klinke, A., et al. (2011).
New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived
superoxide anion production in Raw 264.7 macrophages. The Scientific World
Journal, 11, 2443–2457.
Popov, S. V., Ovodova, R. G., Popova, G. Y., Nikitina, I. R., & Ovodov, Y. S. (2005). Adhesion
of human neutrophils to fibronectin is inhibited by comaruman, pectin
of marsh cinquefoil Comarum palustre L., and by its fragments. Biochemistry
(Moscow), 70(1), 133–138.
Radoglou, K., Dobrowolska, D., Spyroglou, G., & Nicolescu, V.-N. (2009). A review on
the ecology and silviculture of limes (Tilia cordata Mill., Tilia platyphyllos Scop.
and Tilia tomentosa Moench.) in Europe. Die Bodenkultur, 60(3), 9–19.
Ravenscroft, N., Cescutti, P., Gavini, M., Stefanetti, G., MacLennan, C. A., Martin, L.
B., et al. (2015). Structural analysis of the O-acetylated O-polysaccharide isolated
from Salmonella paratyphi A and used for vaccine preparation. Carbohydrate
Research, 404, 108–116.
Renard, C. M. G. C., & Jarvis, M. C. (1999). Acetylation and methylation of homogalacturonans
1: Optimization of the reaction and characterization of the products.
Carbohydrate Polymers, 39, 201–207.
Renard, C. M. G. C., Crépeau, M.-J., & Thibault, J.-F. (1999). Glucuronic acid directly
linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.
European Journal of Biochemistry, 266, 566–574.
Ridley, B. L., O’Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and
oligogalacturonide-related signaling. Phytochemistry, 57, 929–967.
Schmidgall, J., Schnetz, E., & Hensel, A. (2000). Evidence for bioadhesive effects of
polysaccharides and polysaccharide-containing herbs in an ex vivo bioadhesion
assay on buccal membranes. Planta Medica, 66, 48–53.
Schols, H. A., & Voragen, A. G. J. (2002). The chemical structure of pectins. In G. B.
Seymour, & J. P. Knox (Eds.), Pectins and their manipulation (pp. 1–29). Oxford:
Blackwell Publishing, CRC Press.
Sengkhamparn, N., Bakx, E. J., Verhoef, R., Schols, H. A., Sajjaanantakul, T., & Voragen,
A. G. J. (2009). Okra pectin contains an unusual substituion of its rhamnosyl
residues with acetyl and alpha-linked galactosyl groups. Carbohydrate Research,
344, 1842–1851.
Shakhmatov, E. G., Udoratina, E. V., Atukmaev, K. V., & Makarova, E. N. (2015). Extraction
and structural characteristics of pectic polysaccharides from Abies sibirica
L. Carbohydrate Polymers, 123, 228–236.
Shakhmatov, E. G., Atukmaev, K. V., & Makarova, E. N. (2016). Structural characteristics
of pectic polysaccharides and arabinogalactan proteins from Heracleum
sosnowskyi Manden. Carbohydrate Polymers, 136, 1358–1369.
Shimizu, N., & Tomoda, M. (1985). Carbon-13 nuclear magnetic resonance spectra
of alditol-form oligosaccharides having the fundamental structural units
of the Malvaceae plant mucilages and a related polysaccharide. Chemical and
Pharmaceutical Bulletin, 33(12), 5539–5542.
Simson, B. W., & Timell, T. E. (1978). Polysaccharides in cambial tissues of Populus
tremuloides and Tilia americana. IV 4-O-methylglucuronoxylan and pectin.
Cellulose Chemistry and Technology, 12, 79–84.
Sommer, C., Pedersen, J. S., & Stein, P. C. (2004). Apparent specific volume measurements
of poly(ethylene oxide), poly(butylene oxide), poly(propylene oxide), and
octadecyl chains in the micellar state as a function of temperature. The Journal
of Physical Chemistry B, 108(20), 6242–6249.
ˇSutovská, M., Capek, P., Fraˇnová, S., Pawlaczyk, I., & Gancarz, R. (2012). Antitussive
and bronchodilatory effects of Lythrum salicaria polysaccharide-polyphenolic
conjugate. International Journal of Biological Macromolecules, 51, 794–799.
Tomoda, M., & Satoh, N. (1977). Plant mucilages XVII. Partial hydrolysis and a
possible structure of paniculatan. Chemical and Pharmaceutical Bulletin, 25(11),
2910–2916.
Van Holst, G.-J., & Clarke, A. E. (1985). Quantification of arabinogalactan-protein
in plant extracts by single radial gel diffusion. Analytical Biochemistry, 148(2),
446–450.
Vasicek, O., Lojek, A., Jancinova, V., Nosal, R., & Ciz, M. (2014). Role of histamine
receptors in the effects of histamine on the production of reactive oxygen species
by whole blood phagocytes. Life Sciences, 100, 67–72.
Viola, H., Wolfman, C., Levi de Stein, M., Wasowski, C., Pe˜na, C., Medina, J. H., et al.
(1994). Isolation of pharmacologically active benzodiazepine receptor ligands
from Tilia tomentosa. Journal of Ethnopharmacology, 44(1), 47–53.
WHO. (2010). Flos Tiliae. In A. Zakaryan, & A. Arbor (Eds.), WHO monographs on
medicinal plants commonly used in the Newly Independent States (pp. 393–406).
Geneva: WHO Press.
Wangensteen, H., Diallo, D., & Paulsen, B. S. (2015). Medicinal plants from Mali:
Chemistry and biology. Journal of Ethnopharmacology, 176, 429–437.
Xie, Y., Zhang, B., & Zhang, Y. (2015). Protective effects of Acanthopanax polysaccharides
on cerebral ischemia-reperfusion injury and its mechanisms. International
Journal of Biological Macromolecules, 72, 946–950.
Yakovlev, A. I. (1985). Polysaccharides of the inflorescences of Tilia cordata. Chemistry
of Natural Compounds, 21(1), 114–115.
Yamada, H., & Kiyohara, H. (2007). Immunomodulating activity of plant polysaccharide
structures. In J. P. Kamerling (Ed.), Comprehensive glycoscience − from
chemistry to systems biology (pp. 663–694). Oxford: Elsevier.
Yang, J. W., Yoon, S. Y., Oh, S. J., Kim, S. K., & Kang, K. W. (2006). Bifunctional effects
of fucoidan on the expression of inducible nitric oxide synthase. Biochemical and
Biophysical Research Communications, 346(1), 345–350.
York, W. S., Darvill, A. G., McNeil, M., & Albersheim, P. (1985). 3-deoxy-D-manno-
2-octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic
polysaccharide in the primary cell walls of plants. Carbohydrate Research, 138(1),
109–126.
Zhu, Q., Liao, C., Liu, Y., Wang, P., Guo, W., He, M., et al. (2012). Ethanolic extract
and water-soluble polysaccharide from Chaenomeles speciosa fruit modulate
lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage
cells. Journal of Ethnopharmacology, 144, 441–447.