Crude Drugs: Pharmacognostic Investigation

Crude Drugs Pharmacognostic InvestigationIntroduction microscopic examination and pharmacognostic rating of phyto drug may not app bently stand out each direct co-relation with pharmacological and phytochemical evaluations. One should always remember that botanic identity of the phyto drug is an inwrought pre-requisite for undertaking the analysis of medicinal properties of any plant. If botanical identity of drug happens to be doubtful the entire phytochemical and pharmacological work on the plant becomes invalid. Thus the botanical identity of a tender drug threshold in the process of pharmacological investigations.Pharmacognostic paygradeA systematic pharmacognostic study was carried out on the herbal drugs selected, to outline them more scientifically and to identify specific characteristics, if any, which forget be useful in the quality assurance and standardization of these plant drugs.Leaf Constants finale of stomatal IndexStomatal index is the percentage which the arrive of stomata conformity to the total calculate of epidermal cells, each stoma being counted as one cell. Stomatal index was be measured by employ the following equation.I = S X nose candyE+SI=Stomatal index,S= zero(prenominal) of stomata per unit range,E=No. of epidermal cells in the same unit country.Middle part of the leafage was cle atomic number 18d by boiling with chloral hydrate final result. The lower epidermis was rude(a) by means of forceps and mounted on the slide with glycerine piss. television camera lucida and drawing room were arranged for making drawings to scale. A squ be of 1mm was draw by means of stage micrometer. The slide with cle atomic number 18d leaf (epidermis) was located on the stage. The epidermal cells and stomata were traced out. The number of stomata give birth in 1sq mm bea was counted. (Stomatal Number). The result for each of the ten fields was recorded and the average number of stomata per sq.mm was careful.The stomatal inde x was determined victimization the above formula.The slides were wide-awake for Gynandropsis gynandra,(fig.2). The Stomatal number and Stomatal index determine are given in Table.2.Determination of Vein-Islet NumberVein-islet is the humiliated area of light-green tissue touch by the stainlets. The vein-islet number is the average number of vein-islet per square millimeter of a leaf surface. It is determined by find the number of vein-islets in an area of 4 sq.mm of the central part of the leaf between the midrib and the margin.A portion of leaf was cleaned by boiling in chloral hydrate solution for about thirty proceeding and slide was prepared. Camera lucida and drawing display panel were arranged for making drawings to scale. Stage micrometer was arranged on the microscope and employ 16 mm objective, a line was drawn equivalent to 1 mm as seen through the microscope. A square was constructed on this line. The spiel was moved so that the square is seen in the eye mo, in the centre of the field. The slide with the cleared leaf epidermis was brandd on the stage. The veins which are let ind within the square were traced off, completing the outlines of those islets which overlap adjacent cheeks of the square. The number of vein islets in 1sq mm was counted. (The slides were prepared for Gynandropsis gynandra,(fig.5)). The average number of vein islets in the four-spot adjoining squares gave. The Vein islet number.(Table -3)Determination of circumvent RatioPalisade ratio is the average number of palisade cells to a lower place one epidermal cell of a leaf. It is determined by counting the palisade cells beneath four continuous epidermal cells.A piece of the leaf was cleared by boiling in chloral hydrate solution for about thirty minutes and slide was prepared. Camera lucida and drawing board were arranged for making drawings to scale. Using 4mm objective, the outlines of four cells of the epidermis were traced off. The palisade spirit level was f ocussed and sufficient cells were traced off to cover the tracings of the epidermal cells. The outlines of those palisade cells which are intersected by the epidermal walls, were completed. The palisade cells under the four epidermal cells were counted. The average number of cells beneath a single epidermal cell was calculated. (The slides were prepared for Gynandropsis gynandra,(fig.8). The closing was repeated for fin groups of four epidermal cells from disparate separate of the leaf. The average of the results gave the palisade ratio. (Table-4)Histology of Gynandropsis gynandraMidrib of LeafThe transverse section of midrib of Gynandropsis gynandra Linn comprises of the epidermis, cortex, endodermis and vascular bundles. (fig.13)Upper epidermisComprises of barrel shaped cells which are fast jammed, devoid of chloroplast and possess glandular trichomes.CortexBelow the epidermis floors of cortical cells are endue which are do up of polygonal parenchymatous cells.EndodermisEn dodermis is made up of rectangular barrel shaped cells with casparian thickenings.PericycleBelow the endodermis three shape pericycle is present which is made up of parenchymatous cells.Vascular BundlesA four to five layered phloem tissue is present that is made up of thinwalled phloem parenchymatous cells and phloem confrere cells.Xylem tissue is made up of xylem elements, xylem parenchyma and xylem companion cells. reject EpidermisIs made up of polygonal cells which are closely packed together.2.4.2 StemTransverse section of Gynandropsis gynandra Linn stem comprises of epidermis, exodermis, cortex, endodermis and vascular bundles. (fig.14)Epidermis outside layer with tightly joined cells that are devoid of stomata. This layer is commonly termed as rhizodermis. It is also known as epiblema. This layer with cove glory trichomes dries and its place is taken by typical secondary boundary tissue called exodermis having glandular trichomes.ExodermisThis layer is present below the e pidermis and is often regarded as a protective layer. The walls of the cells become suberized.Eames, in 1947, regarded this as hypodermis Foster and Guttenberg, in 1943, gave it the light upon exodermis because of the carriage of suberin in its walls. The suberin lamella develop on the inner side of the primary wall. They differ from cork cells since they contain protoplasmic contents.CortexThe cortex is relatively simple in histology and is generally composed of thin walled cells with lots of intercellular spaces. The cells are arranged in concentric layers with cells in each layer alternating with others.EndodermisIt is a distinct layer of cells differentiated from the innermost layer of cortex. The layer is uniseriate, made up of barrel shaped cells. Casparian strips are present radially.PericycleBelow the endodermis, a few layers of parenchymatous cells are present which make up the pericycle.Vascular BundlesThe stem exhibits secondary growth, hence a complete ring of cambium is formed. A distinct secondary phloem is visible on the outer(a) side. T present is outer fascicular cambium which is made of parenchymatous cells. The phloem consists of phloem fibres, sieve tubes and companion cells. The secondary xylem shows distinct vessels and forms a continuous band interrupted here and there by narrow rays which are uniseriate.The secondary xylem constitutes a largish portion of the bundles it is present on the inner side and consists of vessels with simple pierce tracheids with a few simple pits on radial walls and whatever xylem parenchyma. shopping mallThin walled or thick walled cells filled with tannin and crystals of gypsum constitute the small pith.StomataAnisocytic or cruciferous (unequal) type of stomata which occurs in Capparadaceae family. The stoma is usually encircled by three or four subsidiary cells, one of which is markedly smaller than the others. (fig.15)Physico Chemical Evaluation of Crude DrugsExtractive ValuesExtractive fosters are useful for evaluation of jolting drugs and give an idea about the constitution of chemical constituents present in them. The amount of kindleive a drug yields to a given firmness of purpose is often an approximate measure of a certain(p) constituent or group of link constituents the drug contains. In some cases the amount of a certain constituent or group of related constituents the drug contains, in some cases the amount of drug dissolvable in a given outcome is an index of its purity. The resultant role used for root should be in a position to dissolve quantities of substances desired.Determination of inebriant Soluble Extractive5 g of macerated and air- dry out coarse powder of drug was mixed with 100 ml of 95% alcohol in a closed flask and kept for 24 hours, shaking frequently during the premiere 6 hours and therefore allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precautions against handout of the solvent. About 25 ml of the tense was ev aporated to dryness in a tared, flat-bottomed shallow dish, modify at 105o C and weighed. The percentage of alcohol- disintegrable exceptive was calculated with rootage to the air-dried drug.Determination of piddle Soluble ExtractiveProceeded as enjoin for the determination of alcohol soluble extractive, using chloroform pee I.P. as a solvent.Determination of put under Soluble ExtractiveProceeded as directed for the determination of alcohol soluble extractive, using chloroform as solvent.Determination of vegetable oil Ether Soluble ExtractiveProceeded as directed for the determination of alcohol soluble extractive, using petroleum ether as a solvent. (Table 6)Loss On DryingAbout 5 g of powder was accurately weighed, placed in a petri-dish and dried in hot-air oven at 110 C for four hours. After cooling, it was placed in a desiccator. The loss in pack was recorded. This was repeated till aeonian charge was obtained and % Loss on Drying was calculated with reference to the air-dried drug. (Table 7)Determination of modify ValuesAsh cherishs are helpful in determining the quality and purity of crude drugs in fine-grained form. Ashing involves an oxidation of the components of the product. The total modify usually consists of inorganic radicals like changes, phosphates, silicates and silica of sodium, potassium, magnesium and calcium. A high ash tree value is indicative of contamination, substitution or adulteration.Sometimes, inorganic variables like calcium oxalate, silica, carbonate content of crude drug affects total ash values such variables are then removed by treating with doseulous (as they are soluble in hydrochloric acid) and then acid- water-insoluble ash value is determined. Ash insoluble in hydrochloric acid is the resi due obtained after extracting the total ash with hydrochloric acid. This acid-insoluble ash value particularly indicates contamination with silicious materials like earth or sand. soluble ash is that part of the tota l ash content which is soluble in water. It is a good indicator of either previous extraction of water soluble salts in the drug or incorrect preparation.For the determination of unhomogeneous ash values viz. total ash, acid-insoluble ash, water-soluble ash, the shade dried parts of the selected plant materials were powderize and passed through sieve no40 and studies were carried out. The values vary within fairly wide limits and is therefore an significant parameter for the purpose of evaluation of crude drugs.Determination of fall AshA flat, thin porcelain crucible was weighed and ignited. About 2 g of the powdered drug was taken into the crucible. The crucible was incinerated at temperatures not exceeding 4500C, until shrive from carbon.The crucible was cooled in a desiccator and weighed. The procedure was repeated to get unvarying charge.The percentage of total ash was calculated with reference to the air dried drug. (Table No.8)Determination of Acid-insoluble AshThe tota l ash obtained was boiled with 25 ml of 2 M hydrochloric acid for 5mins. The insoluble ash was collected on an ashless filter penning and washed with hot water. The insoluble ash was transferred to a pre-weighed silica crucible, ignited, cooled, weighed and procedure was repeated to get constant weight. The percentage of Acid-insoluble ash of the crude drug was calculated with reference to the air-dried prove of the crude drug. (Table No.9)Determination of water-soluble AshThe total ash obtained was boiled in 25 ml chloroform water for five minutes. The insoluble ash was collected on an ashless filter paper and washed with hot water. The insoluble ash was transferred into pre-weighed silica crucible, ignited for 15 minutes at a temperature not exceeding 450o C. The crucible was cooled, weighed and the procedure was repeated to get constant weight .Weight of the insoluble matter was subtracted from the weight of the total ash. The difference of weight was considered as the water-s oluble ash. The percentage of water-soluble ash was determined with reference to the air-dried drug. (Table No.10)Fluorescence analysis of the crude drugsMany crude drugs show fluorescence when the sample is exposed to ultra purplish radiation. Evaluation of crude drugs based on fluorescence in daylight is not much used, as it is usually unreliable due to the weakness of the fluorescence effect. Fluorescence lamps (366 nm) are fitted with suitable filters, which eliminates visible radiation from the lamp and transmits ultraviolet radiation of definite wavelength. Several crude drugs show characteristic fluorescence useful for their evaluation. (Table No.11) summate Solid ContentAbout 5 g of extract was accurately weighed in a petri-dish and kept in a hot-air oven and maintained at 110C for four hours. After cooling, the loss in weight was recorded. This procedure was repeated till constant weight was obtained. (Table No. 12)Total solid content (%) = Loss in weight x 100/WW = Weight of the extract in gramsExtraction emaciationThe powdered materials were extracted with alcohol (95%) by cold maceration method.Weighed quantity of powdered crude drugs were taken into round bottom flasks with alcohol, in the drug to solvent ratio 13 and kept for maceration for a limit of 7 days. Finally the flask was left undisturbed for 12 hrs and then the contents were shaker and filtered through Whatman filter paper No.1. The marc was re-extracted with drug solvent ratio of 12. The extracts were combined and concentrated in a rotary flash evaporator, till innocuous from solvent. The extracts, thus obtained were stored in a icebox at 40C until used. (Table No.13)Qualitative Phytochemical ScreeningA spectrum of natural compounds like alkaloids, glycosides, tannis, essential oils and other similar secondary metabolites which exert physiological activity are synthesized in the plant, in addition to the carbohydrates, proteins and lipids utilized by man as food articles.A systema tic and complete study of crude drugs should include a thorough investigation of both primary and secondary metabolites derived as a result of plant metabolism. The different qualitative chemical attempts are to be performed for establishing profile of a given extract/fraction for its record of chemical composition.The following tests were carried out on the extracts to detect various phytoconstituents present in them. detecting of AlkaloidsAbout 50 mg of solvent free extract was stirred with little quantity of dilute hydrochloric acid and filtered. The filtrate was time-tested carefully with various alkaloid tests viz., Mayers try, Wagners Test, Hagers Test, Dragendroffs TestDetection of CarbohydratesAbout 100mg of the extract was fade out in 5 ml of distilled water and filtered. The presence of carbohydrates were tested by Molischs Test, Fehlings Test, Barfoeds Test and Benedicts TestDetection of GlycosidesFor detection of glycosides, about 50 mg of extract was hydrolyzed wi th concentrated hydrochloric acid for 2 hrs on a water bath, filtered and the hydrolysate was subjected to the Glycoside testa viz., Borntragers Test, Legals Test,Detection of SaponinsFoam or Froth TestDetection of Proteins and Amino AcidsAbout 100 mg of extract was dissolved in 10 ml of distilled water and filtered through Whatmann No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. Viz., Millons Test, Biuret Test, Ninhydrin TestDetection of Phytosterols and triterpenoidsTested by Libermann Burchards and Salkwoski testDetection of Phenolic Compounds and TanninsTested by Ferric chloride test, Gelatin test, locomote ethanoate test, Alkaline reagents, and Shinoda test or Magnesium Hydrochloric acid declineThin form ChromatographyThin Layer Chromatography of extracts was done by using standard procedures and is mainly used for the detection of the nature of phytoconstituents present.Thin Layer Chromatography is a very effective technique for the separation of chemical constituents of an extract and for their identification. The history of tender loving care has been reviewed by various authors. A major find in this field was the commercial availability of convenient pre cover abodes in the other(a) 70s Pharmacopoeias are increasingly employing this technique for assessing the quality and purity of compounds of both celluloid and natural origin. TLC profiles developed for an extract from a define solvent system and other parameters could be used as a fingerprint in comparative qualitative evaluation of herbal drugs. The trend of evaluation by this method is becoming popular in view of its restraint and reproducibility.TLC is an important analytical tool in the separation, identification and thought of different classes of natural products. In this technique, the different components are separated by the differential migration of solute between two phases a stationary phase and a mobile phase. Here, the principle of s eparation is adsorption and the stationary phase acts as an adsorbent. Depending on the particular type of stationary phase, its preparation and use with different solvents, separation can be achieved on the basis of partition or a combination of partition and adsorption.Preparation of Plates100 g of silicon dioxide gel-G was weighed and made into a homogenous suspension with 200 ml of distilled water to form aslurry. The slurry was poured into a TLC applicator, which was adjusted to 0.25 mm thickness on flat glass plate of different dimensions (10 x 2, 10 x 5, 20 x 5, 20 x 10 cm etc.). The coated plates were allowed to dry in air, followed by heating at 100 105o C for 1 hour, cooled and stored in a dry atmosphere to protect from moisture. in the first place using, the plates were activated by heating at 100o C for 10 minutes.Detection of Steroids / Triterpenoids and their GlycosidesSolvent systems usedEthyl ethanoate Methanol Water 81 11 8Ethyl acetate Methanol Water 75 15 10Chloroform Methanol Water 70 30 4Chloroform Methanol Water 64 50 10n-Butanol Acetic acid Water 4 1 5 (upper phase) benzine Ethyl acetate 90 10, 80 20, 50 50Chloroform Methanol 95 5, 90 10, 80 20Ethyl acetate Methanol 90 10, 80 20, 50 50Spray Reagents1) Vanillin sulphuric acid (VS) reagent solvent I 5% ethanolic sulphuric acidSolution II 1% ethanolic vanillinThe developed TLC plate was sprayed with 10 ml of solution I, followed immediately by 5-10 ml of solution II, then heate for 5-10 minutes at 100o C under observation. steroids / triterpenoids and their glycosides give blue, blue violet or tap colored spots.2) Vanillin Phosphoric acid (VPA) reagentSolution a 1 gm vanillin dissolved in 100 ml of 50% phosphoric acidSolution b 2 parts 24 % phosphoric acid and 8 parts 2% ethanolic Vanillic acidAfter nebuliser with either solution a or b, the plate was heated for 10 minutes at 100o C Red Violet colour indicates the presence of steroids / triterpenoids an d their glycosides.3) Antimony (III) chloride reagent20% solution of antimony (III) chlorideThe developed TLC plate was sprayed with reagent and then heated for 5-6 minutes at 100o CRed violet color in visible light red violet, blue and green fluorescence in UV at 365 nm indicates the presence of steroids / triterpenoids and their glycosides.4) Anisaldehyde sulphuric acid reagent0.5 ml of anisaldehyde was mixed with 10 ml glacial acetic acid, followed by 85 ml of methanol and 5 ml of concentrated sulphuric acid, in that order. The developed TLC plate was sprayed with reagent, heated at 100o C for 5 10 minutes. steroids / triterpenoids and their glycosides give blue, blue violet or pink coloured spots.Detection of Flavonoids and their GlycosidesSolvent systems usedChloroform Methanol 8020, 7030, 5050Ethyl acetate Methanol Water 81118n- Butanol Acetic acid Water 4 1 5 (upper phase)Ethyl acetate formic acid Glacial acetic acid water100111127Ethyl acetate Formic acid Glacial acetic acid Ethyl methyl ketone Water 50733010DetectionThe developed TLC plate was detect in visible light and in UV at 365 nm. Flavonoids and their glycosides come in as yellow, dark blue, orange zones / spots. The color gets intensified on moving-picture show of the plates to ammonia vapors.Detection of AlkaloidsSolvent systems usedBenzene Ethyl acetate Diethylamine 631Toluene Ethyl acetate Formic acid 541DetectionDragendorffs reagentThe developed TLC plate was sprayed with reagent and then heated for 5-6 minutes at 1000C, spot will be developed.