MODULE 1 - Pharmacognosy and Plant Chemistry - PDF

Pharmacognosy and Plant Chemistry Laboratory MODULE 1 EVALUATION OF CRUDE DRUGS  EXERCISE 1 THE PHYTOCHEMICAL INVESTIGATION PHARMACOGNOSY Application of scientific disciplines with the object of acquiring knowledge of drug from every point of view – Fluckiger Applied s...

Pharmacognosy and Plant Chemistry Laboratory MODULE 1 EVALUATION OF CRUDE DRUGS  EXERCISE 1 THE PHYTOCHEMICAL INVESTIGATION PHARMACOGNOSY Application of scientific disciplines with the object of acquiring knowledge of drug from every point of view – Fluckiger Applied science that deals with the biological and economic features of natural drugs and their constituent - Tyler PLANT CHEMISTRY Art of resolving plants into its chemical constituents Isolating the most important components in the form of pure chemical compounds PHYTOCHEMISTRY Study of the composition of plant principle, their extraction, biosynthesis, and identification PLANT SOURCES Geographic source - region in which the plant or animal yielding the drug grows Indigenous - Plant grows in their native countries Naturalized - plant grows in foreign land aside from their country of origin CLASSIFICATION OF DRUGS Morphology grounded according to plant part Taxonomy natural relationship or Phylogeny Therapeutic application Pharmacologic activity Chemical Constituent active / inert FAMILY NAMES OLD NAME NEW NAME EXAMPLES Cool Cruciferae Big Brassicaceae Many Mustard Girls Graminae Pandas Poaceae Green Grass Love Labiatae love Laminaceae Mints Mint Pink Palmae Amazing Arecaceae Paint Palm Umbrellas Umbelliferae Animals Apicaceae Pretty Parsley Like Leguminosae Furry Fabaceae Flowers Pea Guttiferae cats Clusiaceae stunning St. John’s Wort Gorgeous wouds Compositae Always Asteraceae Daisies Daisy CRUDE DRUG Vegetable or animal drugs that consist of natural substances that have undergone only the process of collection and drying PLANT METABOLITES PRIMARY METABOLITES relatively more abundant in plants, but are of lower value carbohydrates, proteins, lipids SECONDARY METABOLITES present in small quantities but are of high value unique to a particular species may have biological role (anti-oxidant, antimicrobial, anti-inflammatory properties, etc.) PREPARATION OF CRUDE DRUGS  small scale 1. Collection: part of plant which may have the highest content of active constituent Rules Rhizomes/Roots – after the vegetative process Stem/barks- before the vegetative process Leaves – when the photosynthesis is active Flower – When they are about to bloom Fruits – when ripe Seeds – when matured  large scale 2. Harvesting – hand labor and mechanical device 3. Drying – Removal of moisture To ensure good keeping qualities – prevent molding Prevent the action of enzymes Prevent action of bacteria and chemical or other possible changes It fixes the constituents Facilitates grinding and milling Methods of Drying Natural – air dying , sun drying and shade drying Artificial – oven at 40-60°C. RULE: Leaves, herbs and flower 20-40°C, Barks and Roots 30-60°C Rapid drying Retains the color of flower and leaves Retains the aroma of aromatic plants 4. Garbling – removal of extraneous matters such as plant part, dirt and added adulterant 5. Packaging, Storage and Preservation PROPER STORAGE OF PLANT EXTRACT as a standard precaution, all plant extracts properly labelled should be stored in the cold, at temperatures between 0-5C if kept at room temperature, the extract can be treated with a trace of chloroform (CAUTION: carcinogenic) or toluene (CAUTION: toxic fumes) to prevent fungal growth FACTORS FOR DETERIORATION Moisture – activate enzymes present in the leaves and bring about decomposition of the glycosides Containerized Shipment- due to excessive condensation of moisture to the inner metal walls Increase in temperature - leads to loss of volatile constituents Direct Sunlight - decomposition of certain constituent Oxygen - assist resinification of volatile oils and rancidity of fixed oil PHYTOCHEMICAL SCREENING PHYTOCHEMICAL SCREENING It refers to the extraction, screening and identification of the medicinally active substances found in plants. Advantages: 1.Simple & rapid 2.designed for a minimum of equipment; 3.reasonably selective for the class of compounds under study; 4.quantitative in so far as having a knowledge of the lower limit of detection is concerned; and if possible 5.should give additional information as to the presence or absence of specific members of the group being evaluated. PHYTOCHEMICAL SCREENING Extraction is mext ru is the separation of medicinally active portions of plant/animal tissues using selected solvents through standard procedures. METHODS OF EXTRACTION Selected solvent macerate using not Hz 0 - 24-48 hus heat gentle Boil for 5 mins pressure in put Basic parameters that may influence the quality of the extracts 1.plant part used as extracting material 2.solvent used for extraction 3.extraction procedure What are the common variation in extraction methods? 1.length of the extraction period 2.solvent used 3.pH of the solvent 4.temperature 5.particle size 6.Solvent-to-sample ratio Solvents for Commonly Used for Extraction Petroleum Ether Fats, Fixed oils, waxes, pigments, resin Ether and Chloroform Alkaloids, resins and Glycosides 1% HCl Alkaloids, salts of veg. acids 5%NaOH Pentosans and hemicellulose  80% alcohol visible secondary metabolite preferred for phytochemical screening 95% Alcohol Glycosides, tannins, saponin, resin Water glycosides, sugar, salt, gum mucin, proteins Acetone Hydrophilic & lipophilic components, phenolics, tannins Dichloromethanol – selective extraction of terpenoids The effect of extracted phytochemicals depends on: 1.Nature of plant material 2.Its origin 3.Degrees of processing 4.Moisture content 5.Particle size The variations in different extraction methods that will affect the quantity and secondary metabolite composition of an extract depend upon: 1. Type of extraction 2.Time of extraction 3.Temperature 4.Nature of solvent 5.Solvent concentration 6.Polarity METHODS FOR SEPARATION 1. Paper Chromatography – water soluble constituents like carbohydrates, and amino acids. METHODS FOR SEPARATION 2. Thin layer Chromatography - lipid soluble constituents like fats, fixed oils, waxes, and chlorophyll. METHODS FOR SEPARATION 3. Gas Liquid Chromatography - volatile compounds like HC, terpenes, and fatty acids. METHODS FOR SEPARATION 4. High-Pressure Liquid Chromatography LABORATORY PROCEDURES (MANUAL) EXTRACTION OF SAMPLES EXTRACTION OF SAMPLES EXTRACTION OF SAMPLES after ma-blender PHYTOCHEMICAL TESTS ALKALOIDS basic nitrogenated compounds comprising the largest class of secondary plant metabolites physiologically active usually occur in plants as their water-soluble salts form double salts with Hg, Au, Pt, and other heavy metals; these salts are obtained as precipitates which are micro- crystallographic MAYER’S TEST (+) cream color precipitate - Mayer’s reagent (Potassium mercuric iodide TS) DRAGENDORFF’S TEST ALKALOIDS (+) orange color precipitate Dragendorff’s reagent (Potassium bismuth iodide TS) WAGNER’S TEST (+) reddish brown color precipitate Wagner’s reagent (Iodine in potassium iodide TS) VALSER’S TEST ALKALOIDS (+) white color precipitate Valser’s reagent (Mercuric iodide TS) HAGER’S TEST (+) yellow color precipitate Hager’s reagent (Saturated solution of picric acid) ALKALOIDS GLYCOSIDES plant constituents which produces sugars as products of hydrolysis sugar ethers hydrolyzed by boiling mineral acids regulatory, protective and sanitary compounds GLYCOSIDES GLYCONE AGLYCONE sugar component non-sugar component common basis of denominator classification inactive active CARDENOLIDE & BUFADIENOLIDE steroidal glycosides also known as “cardiac glycosides” Digoxin have a prominent effect on the heart muscle steroid structure: cyclopentanoperhydrophenanthrene nucleus (CPPP nucleus) CARDENOLIDE & BUFADIENOLIDE CARDENOLIDES BUFADIENOLIDES less common (from toad skin of more common Bufo spp.) optimum activity less activity C-23, , -unsaturated C-24, doubly unsaturated 5 membered lactone ring 6 membered lactone ring CARDENOLIDE & BUFADIENOLIDE KELLER-KILIANI’S TEST STEROIDAL GLYCOSIDES test for 2-deoxysugars reagent: Ferric chloride TS (3 mL 1% FeCl3 in 50 mL glacial acetic acid) positive result: reddish-brown color, which may turn blue or purple LIEBERMANN-BURCHARD TEST STEROIDAL GLYCOSIDES test for unsaturated steroids and triterpenes reagents: dichloromethane, acetic anhydride, conc. H2SO4 positive result: colors ranging from blue to green, red, pink, purple or violet KEDDE’S TEST STEROIDAL GLYCOSIDES test for unsaturated lactones reagent: Kedde’s reagent (1 g 3.5-dinitrobenzoic acid in 50 mL methanol and 50 mL 2M KOH mixture) positive result: blue-violet color positive standard: 2 mL 0.025% digitoxin in methanol SALKOWSKI’S TEST STEROIDAL GLYCOSIDES test for sterols reagent: conc. H2SO4 positive result: red layer in junction ANTHRAQUINONE GLYCOSIDES largest group of naturally occurring quinone pigments occur as hydroxylated, methylated, or carboxylated derivatives of anthraquinones, anthrones, anthranal, or dianthrone O-glycosides or C-glycosides, or as their aglycones  / BORNTRÄGER’S TEST ANTHRAQUINONE GLYCOSIDES reagent: ammonia solution (35.7 mL of 28% ammonia in 64.3 mL d. H2O) positive result: red coloration in the lower ammoniacal layer MODIFIED BORNTRÄGER’S TEST reagents: 0.5M KOH, 5% H2O2, glacial acetic acid, benzene, ammonia positive result: pink color FLAVONOIDS phenolic plant pigments containing γ-benzopyrone nucleus include anthocyanins, leucoanthocyanins, cathechins, aurones, chalcones have reported anti-viral, anti-fungal, anti- inflammatory and cytotoxic activities BATE-SMITH AND METCALF METHOD test for leucoanthocyanins reagents: conc. hydrochloric acid FLAVONOIDS positive result: strong red or violet WILSTATTER “CYANIDIN” TEST test for γ-benzopyrone nucleus reagent: conc. hydrochloric acid, magnesium turnings, octanol FLAVONOIDS positive result: colors ranging from orange to red, to crimson and magenta, and occasionally to green or blue SAPONINS toxic for cold-blooded animals steroidal/triterpenoid glycosides characterized by their ability to froth when agitated exert a powerful hemolytic action on RBCs and are highly toxic when injected into the bloodstream has interesting spermicidal & molluscicidal activity SAPONINS has two types: steroidal – monocot (Dioscoreaceae) triterpenoid – dicot (Agavaceae) other plants with saponins: Sarsaparilla – a beverage (root beer) Gugo – used as shampoo Ginseng – “drug for longevity”, adaptogen, tonic FROTH TEST formation of persistent honeycomb froth upon shaking positive result: the honeycomb froth is greater than 2 cm height from the surface of the liquid persists after 10 minutes for plant extracts with poor frothing effects, add a little 5% SAPONINS Na2CO3 to basify the extract; the formation of a stable and dense froth indicates presence of free fatty acids AGAR CUP HEMOLYTIC TEST Blood agar plate 100 mL 0.9% NaCl sol’n + 1.8 g agar powder, let it stand for 30 mins. heat to 80C on a water bath with stirring, then cool to 40 C add 1 mL defibrinated cow’s blood & stir then pour 10 Ml in each Petri dish and allow to set Bore cleanly three equidistant cups of 10 mm diameter into the agar SAPONINS Fill one agar cup with the extract, one with the gugo extract (positive control) and one with distilled water (negative control) positive result: clear zones of hemolysis (hemolytic halos) TANNINS AND POLYPHENOLS astringent first applied to plant constituents capable of transforming raw animal skin into leather precipitates proteins, alkaloids and glycosides tannic acid: antidote for alkaloid poisoning acidic, sharp-puckering taste TANNINS AND POLYPHENOLS HYDROLYZABLE NON- HYDROLYZABLE other name “condensed” structure related to pyrogallol related to catechol + HCl phenolic acid + sugars phlobaphenes (red dye) + FeCl3 bluish black brownish green leather type bloom tanner’s red TANNINS AND POLYPHENOLS uses: tanning industry, treatment of burns, potential value as cytotoxic and/or antineoplastic agents, astringent properties polyphenols have similarities to tannin structures; they are used as additives in tanning agents in the leather industry GELATIN TEST reagent: gelatin-salt reagent (equal amount of 1% gelatin sol’n & 10% NaCl sol’n) - positive result: formation of jelly- precipitate - TANNINS FERRIC CHLORIDE TEST reagent: iron (III) chloride, FeCl3 (3 mL 1% FeCl3 sol’n in 100 mL d. H2O) positive result: bluish black for hydrolysable tannins while brownish green for condensed TANNINS tannins CYANOGENIC GLYCOSIDES In Cyanide often found in legumes, root crops and grasses usually found in plants together with hydrolytic enzymes most cyanogenic glycosides are lost after plant harvest due to spontaneous hydrolysis CYANOGENIC GLYCOSIDES the roots of Manihot esculenta Crantz (cassava), causes poisoning when eaten if tubers are half-cooked or cooked with intact root peelings due to HCN released by enzymatic hydrolysis of cyanogenic glycosides Prunus amydalus (almonds) are also known to contain cyanogenic glycosides; emulsin hydrolyses glycosides GUIGNARD’S TEST CYANOGENIC GLYCOSIDES Picrate paper dip strips of filter paper into freshly prepared sodium picrate sol’n drip dry and then finally dry between sheets of newsprints positive result: appearance of various shades of red within 15 mins when the tube is warmed (yellow to brick red) CARBOHYDRATES polyhydroxy ketones or aldehydes most abundant group of organic molecules provide significant amount of energy; storage form of energy; cell component; structural component building blocks: monosaccharides FEHLING’S TEST test for reducing sugars (presence of aldehydes but not ketones) CARBOHYDRATES reagents: Fehling’s A (blue aqueous solution of copper II sufate) and Fehling’s B (clear colorless solution of aqueous potassium sodium tartrate and a strong alkali) positive result: brick red precipitate MOLISCH TEST furfural hydroxy test for pentoses and hexoses reagent: 10% a-naphthol positive result: bluish violet ring at the junction (furfural CARBOHYDRATES & hydroxy methyl furfural derivatives of pentoses and hexoses respectively) BENEDICT’S TEST test for reducing sugars (monosaccharide or disaccharide containing hemiacetal or hemiketal group) reagent: Benedict’s solution (copper II sulfate) CARBOHYDRATES positive result: brick red precipitate PROTEINS group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids building blocks: amino acids MILLON’S TEST reagent: Millon’s reagent positive result: flesh precipitate XANTHOPROTEIC TEST PROTEINS reagent: nitric acid positive result: yellow precipitate LIPIDS any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides, that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch GREASY SPOT TEST extract dissolved in petroleum ether or hexane few drops on paper positive result: greasy spot on paper LIPIDS SUMMARY: PLANT SAMPLE dried, grounded, and macerated in 80% ethanol PHYTOCHEMICAL SCREENING ANTHRAQUINONE CYANOGENIC ALKALOIDS SAPONINS PROTEINS GLYCOSIDES GLYCOSIDES STEROIDAL FLAVONOIDS TANNINS CARBOHYDRATES LIPIDS GLYCOSIDES Bornträger Guignard ModifiedBornträger Greasyspot Keller-Kiliani Gelatin FeCl3 Liebermann-Burchard Millon Kedde Xanthoproteic Froth Salkowski Agar-cuphemolytic Fehling Preliminarytest Molisch Bate-Smith&Metcalf Confirmatorytest Benedict Wilstatter“cyanidin” TestforQuaternaryamines THIN LAYER CHROMATOGRAPHY (TLC) Methods of Identification and Evaluation organs Organoleptic Evaluation Microscopic Evaluation Phytochem Chemical Evaluation Physical Evaluation solubility Biological Evaluation rat rabbit ,  EXERCISE 2 MOISTURE CONTENT DETERMINATION OF CRUDE DRUGS MOISTURE CONTENT moisture is simply water diffused in a relatively small quantity water content is difficult to measure because of the complex intermolecular bonding properties within the substance matrix Moisture Content Determination The moisture content of a drug should be minimized in order to prevent decomposition of crude drug either due to chemical change or due to microbial contamination to EXCESSIVE MOISTURE Is considered as Adulterant for it causes Added weight Promotion of mold and bacterial growth Deterioration and spoilage of the drug 5% moisture is not considered as excessive  MOISTURE CONTENT influences the physical properties of a substance: presence of moisture ↑ weight of sample presence of moisture ✓weight ↑ density ✓density presence of moisture ✓viscosity ↑ viscosity presence of moisture ↑ refractive index ✓refractive index ✓electrical conductivity presence of moisture promotes electrical ✓etc conductance TWO TYPE OF WATER PRESENT IN DRUGS water of crystallization water in the adsorbed form METHODS OF MOISTURE DETERMINATION Thermogravimetric method Calculation of loss of weight Heating of sample at 105 °C Volumetric method Calculation of volume Uses azeotropic distillation and toluene Titrimetric (Karl Fischer) Method quantitative reaction of water with iodine and sulfur dioxide in the presence of a low molecular weight alcohol (e.g. methanol) and an organic base (e.g. pyridine) LABORATORY PROCEDURES (MANUAL) Loss on Drying: Drying Oven and Balance Method Loss on Drying: IR Moisture Analyzer  EXERCISE 3 ASH CONTENT DETERMINATION OF CRUDE DRUGS ASH CONTENT why measure the ash content? 1. provides a measure of the total amount of minerals within a sample 2. furnishes a basis for judging the identity and cleanliness of a drug 3. gives information relative to its adulteration with inorganic matter ASH DETERMINATION Used to determine quality and purity of crude drug Ash contains inorganic radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium Total ash: carbon and organic matter present in the drug is converted to ash at a temperature of 450º C. It mostly contains carbonates, phosphates, silicates and silica. Acid insoluble ash: Total ash may be treated with 2N HCl, which removes many inorganic salts to yield mainly silica in the residue of acid insoluble ash. Water soluble ash is produced by separating the water-soluble material from the total ash. In this case most of the water-insoluble salts that may contribute in total ash are removed to find water-soluble ash content acid- insoluble CRUCIBLE & COVER used to contain compounds when heated to extremely high temperatures QUARTZ CRUCIBLE & COVER satisfies high- temperature requirements up to 1050°C (1922°F) CRUCIBLE TONGS used to grasp a hot crucible or evaporating dishes ASH CONTENT DETERMINATION careful control of temperature is the most important analytical factor to be regulated in making ash determinations careful selection of crucible is important the ash consists largely of C a C O 3 formed from Ca(C 2 HO 4 ) 2 contained in the drug when incineration is carried out as a: dull red heat – Ca(C 2 HO 4 ) 2 is converted to C a C O 3 bright red heat – varying amounts of the C a C O 3 are converted to the C a O with consequent variable results METHODS OF ASH CONTENT DETERMINATION Direct incineration Muffle furnace LABORATORY PROCEDURES (MANUAL) Determination of the Total Ash Content Determination of the Acid-insoluble ash END OF MODULE 1 ANY QUESTIONS?