Pharmacognosy lab exercise 1 What is Pharmacognosy? What is a drug? How can we identify drugs? How to prepare microscopic preparations? How to obtain course credits •100% participation in practical lessons – in case of one-time absence you will write a short paper on a given topic (2-3 standard pages) • • •At least 60% of two tests (12 points of 20 max.) • Test 1 – 6th week of semester • Test 2 – 10th week of semester • • •Reports from lab exercises – sketches of microscopic preparations, with designations of typical structures, possibly denote the magnification used for observation, sketches are made during the practical class and are handed in at the end of the that class Pharmacognosy •Science about drugs of natural origin – plant, animal, microbial, which are used for treatment, prevention of diseases, and diagnostics and affection of physiological functions • •Pharmacognosy relates to other pharmaceutical disciplines (pharmacology, medicinal chemistry, galenic pharmacy) • •Starting material for isolation of natural medicinal products are drugs Drug usually means a medicine, an active compound. In pharmacognosy, the word drug has another meaning Drug •Word origin: droog waere (flemish word for dry spices and herbs) • •Modified or non-modified preserved plants or their parts; animals or their parts; or products of metabolism of plants, animals, or microbials (starch, mucilage, honey) • •Drugs according to structure: • with organized body structure (cellular structure, Malvae flos) – amorphous (products of metabolism, Balsamum peruvianum) – •Drugs according to origin: plant-derived • animal-derived (whole animal – Cantharis, animal organs – glands, products – waxes, hormones, enzymes) • •Conservation stops the decomposition of plant material after it has been removed from the mother plant • - freezing, freeze-drying • - preferred way of conservation – drying Historically, drugs were used directly for treatment or preparation of healing tinctures and extracts. Nowadays, drugs are merely a raw material for isolation of their active compounds, which are then used for treatment. Drugs according to usage: drugs for treatment, drugs for isolation of active compounds Amorphous drugs can be physiological products – natural products of plant/animal body; or pathological products – these are produced after an external intervention, for example an injury such as cutting the bark of a tree. Mother plant ( mother organism) – plant (organism), from which the drug is derived Drying §direct sunlight is unsuitable §in shade and well-ventilated rooms §properly dried plant material contains most of its original constituents in the same amounts as before drying §drying temperature, duration and method of drying depend on the constituents of the dried plant – plants containing volatile essential oils up to 40°C, • – easily hydrolysed constituents (glycosides) – fast drying with a higher temp. (enzyme inactivation) and then drying with usual drying temp. 60-70°C • •Change of colour during the drying process: §Plants with neutral cellular fluid – almost no colour change §Plants with acidic cellular fluid get dark §Flowers with anthocyans content change colour from pinkish or red to purple or blue § •We need to know: • Drying temperature • Way of drying • Length of drying • Storage parameters (no direct sunlight, 5-15°C) • Right labelling § Drying speed depends on the temperature and the relative air humidity – it occurs faster in higher temp. and with better air circulation above the dried material. When the drying process is finished, the material has to cool down prior to packaging (residual water vapor). In some drugs, the desired compounds are synthesized even after harvest. This is known as post-mortal biosynthesis (Digitalis – foxglove). Other drugs have to undergo fermentation, during which desired compounds are enzymatically created (black tea). Drugs with stark effects (Opium) should be kept separately, also those with sharp smell. Systematic classification of drugs •Pharmaco-botanical – according to the phylogenetic development of mother plants • •Pharmaco-chemical – according to the chemical structure of content compounds • •Pharmacological – according to the therapeutic effect of content compounds • •Biogenetic – according to the biosynthetic origin of content compounds Pharmacochemical system: drugs with content of alkaloids, phenolic glycosides, essential oils Pharmacological system: drugs with effect on the digestive system Nomenclature of Drugs •Binomic – first part of name expresses mother plant (genus or species), second part expresses name of corresponding plant organ • Lini semen, Belladonnae radix, Conii fructus • •One name - Lycopodium, Mel • •Exceptions - Liquiritiae radix, Cynosbati fructus • •It is necessary to state the mother plant and family each time Mother plant (organism) – plant (organism), from which the drug is derived Glycyrrhiza glabra = liquorice Rosa canina = dog rose Drugs from aerial plant parts •Folium (leaf) – plant leaves • • •Herba (herb) – whole aerial plant part • • •Flos (flower) - whole flowers or their parts •(floral leaf petals), or whole inflorescence Drugs from aerial plant parts •Fructus (fruit) – whole fruit or infructescence, •or fruit imitations • • •Pericarpium – pericarps of Rutacae family • • •Semen (seed) – seed or its part (germ) • • Drugs from aerial plant parts •Cortex (bark) – all kinds of plant tissues •upon the cambium • • •Lignum (wood) – woody parts of plant •only (xylem) • 124-2442_IMG Drugs from aerial plant parts •Stipes – drug contains peduncle only • •Gemma - drug contains eye only • •Sporae – drug contains spores only • •Glandulae - drug contains glands only • •Caulis - drug contains footstalk (stem) only • •Strobilus - drug contains strobiles only Less frequent drugs Peduncle - a stalk supporting an inflorescence Strobiles – seed cones (hops flowers) Drugs derived from underground plant organs •Radix – used for drug made of rhizomes, roots, bulbs and tubers Nomenclature of plant products and most often used adjectives •Amylum - starch •Balsamum - balm •Resina – resin/oleoresin •Gummi - mucilage •Gummiresina – gum resin • (bdellium) •Cera - wax •Oleum - oil, volatile oil •naturalis - natural •pulvis - powdered •mundata - peeled •maturus - ripened •imaturus - unripened •amari - bitter •dulcis - sweet • Review of traditional pharmacological effects of drugs •astringent (adstringent) •amarum •anabolic •analgesic •anthelmintic •antianemic •antiarrhytmic •antiasthmatic •antidiabetic •antidiarrhoic •antidysmenoreic •antiphlogistic •antihemoroidal •antihydrotic •antirheumatic •antisclerotic •aromatic •balneologic Adstringent – shrinks or constricts body tissues Amarum – contains bitter substances that promote secretion of digestive juices Anabolic – promotes the synthesis of compounds Anthelmintic - expels parasitic worms (helminths) and other internal parasites from the body Antiasthmatic – eases respiratory distress in asthma Antidiabetic – lowers glycaemia Antidysmenoreic – against painful menstruation Antihemoroidal - Th of piles Antihydrotic – lowers the secretion of sweat glands and lowers perspiration Antirheumatic – acts anti-inflammatory Antisclerotic – against atherosclerosis Aromatic - drug containing aromatic substances, which positively influences the fragrance of the herbal preparation Balneologic – additive substance to bath •cardioactive •cardiotonic •carminative •cholagogue •cholekinetic •choleretic •corigens •cytostatic •derivans •dermatic •desinficiens •diaphoretic •diuretic •emenagogue •emetic •expectorant •gargarism •gynecologic •hypotensive Review of traditional pharmacological effects of drugs Cardioactive – acts on myocard Cardiotonic - enhances contractions of myocard and thus boosts heart function Carminative – against meteorism, flatulence Cholagogue – increases the secretion of bile into small intestine Cholekinetic - boosts the contractions of gallbladder Choleretic – increases the production of bile Corigens – modifies the unpleasant taste, smell or look Cytostatic – stops the growth and multiplication of tumor cells Derivans – locally increases the amount of blood flowing through the skin, making it red and warm, also increases inflammation development and edema absorption Dermatic – for treatment of skin diseases Desinficiens – destroys germs Diaphoretic – promotes sweating Diuretic – promotes urine production Emenagogue – for irregular menstruation Emetic – causes vomiting Expectorant – eases coughing up of sputum or mucus Gargarism – used for gargling Hypotensive – lowers blood pressure •lactagogue •laxative •metabolic •nervine •roborans •sedative •spasmolytic •stimulans •stomachic •urologic •venotonic Review of traditional pharmacological effects of drugs Lactagogue – stimulates production and secretion of mother´s milk Laxative – against constipation Metabolic – stimulates metabolism Nervine – soothes the nervous system, calms ruffled nerves Roborans – strengthens the organism Sedative – lowers CNS activity Spasmolytic – acts against painful contractions of smooth muscles Stimulans – stimulating effect, enhances psychological and physiological functions for a short period of time Stomachic – stimulates metabolism, enhances appetite Urologic – used in treatment uf urinary tract diseases Venotonic – enhances the tension of veins, lowers edema formation Drug analysis •Using the demands of pharmacopaeia or other mandatory rules •Identification •Purity checking •Content determination • •Important – sample collection and preparation Identification •Sensoric (organoleptic) assays – using our natural sensors –Smell –Taste –Vision –Touch – •Microscopic identification – directed on characteristic signs (shape of skin cells, presence of typical structures/organelles containing essential oils, mucilages, starch grains, vascular bundles) • •Physico-chemical methods – microsublimation, fluorescence, simple chemical reaction or thin layer chromatography Smell can point to deterioration of plant material or to a plant rich in volatile essential oil. Taste can point to characteristically tasting compounds such as bitter substances, mucilages, tannins. Using vision we macroscopically characterize the plant, shape of its leaves, shape of leaves' edges, and thickness of petiole, amount of trichomes and their shape. Chemical reactions are carried out after extraction of constituents from the drug. Usually the extraction is done by water, or by an organic solvent such as methanol, ethanol. We use two types of reactions: color reactions = result is a change of color, and precipitation reaction = precipitate forms Purity checking •Foreign matter, impurities •Loss on drying •Determination of total ash •Determination of HCl-insoluble ash •Optical rotatory power •Index of refraction • • • Foreign matter can be sand, other plant parts. Loss on drying does not ​qual the loss of water. A drug dried at high temperatures such as 105•C loses also volatile substances such as essential oils. Determination of HCl-insoluble ash gives information about different minerals present. Content determination •Direct / indirect determination of compounds • •Colorimetric methods •Gravimetric methods •Titration methods •Biological methods •HPLC, GC Biological methods are used when the amount of constituents measured by chemical methods does not match the effect of the drug (tannins and their adstringent effect) Division of plant metabolites •primary metabolites - saccharides, amino acids, low-molecular carboxylic acids – mevalonate, pyruvic acid, shikimic acid •secondary metabolites - polysaccharides, alkaloids, terpenoids, phenolic glycosides, flavonoids, coumarines, anthocyanins, tannins, anthraglycosides… Primary metabolites are simple and universal substances which occur in all organisms. They have several precursors. Secondary metabolites are synthesized from primary metabolites by specific, genetically controlled and enzymatically catalyzed reactions. Secondary metabolites are usually referred to as natural drugs. Preparation of microscopic slides •Temporary microscopic slides •Permanent microscopic slides •Surface specimens - leaves, flowers •Cuts - barks, wood, root –transversal –tangential –radial •Ways of cutting –manual –microtome cut thickness: 50 nm- 100 μm •Sledge-type microtome •Rotary microtome •Cryomicrotome •Laser microtome contact free slicing 124-2438_IMG 124-2439_IMG Obrázok, na ktorom je text Automaticky generovaný popis Procedure of permanent slide preparation •Softening and conservation –Alcohol-glycerol mixture, chlorine dioxide in acetic acid, formaldehyde – •Fixation –Immediate termination of biochemical reactions –Fixation mixtures – FAA (formaldehyde/acetic acid/alcohol), chromic acid – •Clearing –Chloral hydrate, hydrogen peroxide, glycerol •Fastening of object –Encapsulation into paraffin •it is necessary to remove water by treatment with ethanol (ethanol series) –Encapsulation into gelatin –Freezing – •Cutting and adhesion –Mixture of albumin and glycerol 1:1 – •Staining –Before using stains paraffin must be removed by hydration with alcohol series –Staining according to the procedure •progressive, regressive, succedaneous, simultaneous –Staining according to the result •diffusive, differentiative –phloroglucine, picric acid, iodine solution, saphranine, fuchsine, ferric chloride, congo red barveni1 barveni Progressive staining – up to a certain hue Regressive staining – we apply a dye and then we remove the color to the desired hue Succedaneous – uses different dyes, one after another Simultaneous – uses different dyes combined in one solution Diffusive staining – all parts of tissue are dyed with the same color Differentiative – different dyes to highlight certain structures •Mounting –Necessary to dehydrate –Canada balsam, resins, liquid paraffin – •Enframement –lanoline-colophony glue, albumin, colorless nail polish • The most important information sources about natural products •Text books for Pharmacognosy –Bruneton J.: Pharmacognosy, phytochemistry –Evans W.C.: Pharmacognosy –Dewick: Natural products –Wichtl M.: Herbal drugs and phytopharmaceuticals –European Pharmacopoeia • •internet –Web of Science –Science Direct –Belstein Crossfire –American Chemical Society –SciFinder