Crude Drugs: Pharmacognostic Investigation

Crude Drugs Pharmacognostic InvestigationIntroductionMicroscopical examination and pharmacognostic military rating of phyto drug may not appargonntly bear any direct co-relation with pharmacological and phytochemical substance evaluations. One should always remember that botanical identity of the phyto drug is an essential 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 unadulterated drug threshold in the bear upon of pharmacological investigations.Pharmacognostic EvaluationA systematic pharmacognostic study was carried out on the herbal drugs selected, to describe them more scientifically and to identify specific characteristics, if any, which will be helpful in the quality assurance and behaveardization of these plant drugs.Leaf Constants tendency of Stomatal IndexStomatal index is t he dowery which the number of stomata act to the total number of cutaneous cells, each stoma being counted as one cell. Stomatal index was be calculated by using the pastime equation.I = S X ascorbic vitriolicE+SI=Stomatal index,S=No. of stomata per unit area,E=No. of epidermal cells in the same unit area.Middle part of the leaf was cleared by boiling with chloral hydrate solution. The lower shield was peeled by means of forceps and mount on the microscope slide with glycerine water. Camera lucida and drawing board were arrange for making drawings to scale. A square of 1mm was drawn by means of stage micrometer. The slide with cleared leaf (epidermis) was placed on the stage. The epidermal cells and stomata were traced out. The number of stomata indicate in 1sq mm area was counted. (Stomatal Number). The result for each of the ten line of merchandises was enter and the bonnie number of stomata per sq.mm was calculated.The stomatal index was determined using the above fo rmula.The slides were prepared for Gynandropsis gynandra,(fig.2). The Stomatal number and Stomatal index values are wedded in Table.2.Determination of Vein-Islet NumberVein-islet is the small area of jet tissue surrounded by the veinlets. The vein-islet number is the average number of vein-islet per square millimeter of a leaf surface. It is determined by counting 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 voice of leaf was cleaned 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. Stage micrometer was arranged on the microscope and using 16 mm objective, a line was drawn equivalent to 1 mm as seen through with(predicate) the microscope. A square was constructed on this line. The patter was moved so that the square is seen in the eye hang on, in the centre of the field. The slide with the cleare d leaf epidermis was placed on the stage. The veins which are included within the square were traced off, completing the outlines of those islets which overlap adjacent sides 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 quadsome adjoining squares gave. The Vein islet number.(Table -3)Determination of Palisade RatioPalisade ratio is the average number of palisade cells beneath 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 shape was focussed and sufficient cells were traced off to cover the tracings of th e 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 determination was repeated for quint groups of four epidermal cells from contrary parts 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 brake drum shaped cells which are closely packed, devoid of chloroplast and possess glandular trichomes.CortexBelow the epidermis layers of cortical cells are present which are do up of polygonal parenchymatous cells.EndodermisEndodermis is made up of rectangular barrel shaped cells with cas parian thickheadedenings.PericycleBelow the endodermis three layered 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 companion cells.Xylem tissue is made up of xylem elements, xylem parenchyma and xylem companion cells. inflict 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)EpidermisExternal layer with tightly joined cells that are devoid of stomata. This layer is usually termed as rhizodermis. It is also known as epiblema. This layer with cove donut trichomes dries and its place is taken by typical alternate boundary tissue called exodermis having glandular trichomes.ExodermisThis layer is present below the epidermis and is often regarded as a protective layer. Th e walls of the cells become suberized.Eames, in 1947, regarded this as hypodermis Foster and Guttenberg, in 1943, gave it the name exodermis because of the presence of suberin in its walls. The suberin lamella develop on the inner side of the primary wall. They differ from cork cells since they consume protoplasmic contents.CortexThe cortex is comparatively 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 out er side. There 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 large portion of the bundles it is present on the inner side and consists of vessels with simple perforated tracheids with a few simple pits on radial walls and some xylem parenchyma.PithThin 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 surrounded by three or four subsidiary cells, one of which is markedly smaller than the others. (fig.15)Physico Chemical Evaluation of Crude DrugsExtractive ValuesExtractive values are useful for evaluation of cancel drugs and give an root about the nature of chemica l constituents present in them. The amount of take awayive a drug yields to a given solvent is often an approximate measure of a certain constituent or group of related to 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 soluble in a given solvent is an index of its purity. The solvent used for extraction should be in a position to dissolve quantities of substances desired.Determination of Alcohol Soluble Extractive5 g of macerated and air-dried 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 first 6 hours and then allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precautions against loss of the solvent. About 25 ml of the filtrate was evaporated to dryness in a tared, flat-bottomed shallow dish, dried at 105o C and weighed. The percentage of alcohol-soluble e xtractive was calculated with reference to the air-dried drug.Determination of Water Soluble ExtractiveProceeded as directed for the determination of alcohol soluble extractive, using trichloromethane water I.P. as a solvent.Determination of chloroform Soluble ExtractiveProceeded as directed for the determination of alcohol soluble extractive, using chloroform as solvent.Determination of Petroleum 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. later cooling, it was placed in a desiccator. The loss in pack was recorded. This was repeated till constant weight was obtained and % Loss on Drying was calculated with reference to the air-dried drug. (Table 7)Determination of Ash ValuesAsh values are helpful in determining the quality and purity of crude drugs in powdered form. Ashing involves an oxidation of the components of the product. The total change usually consists of inorganic radicals like carbonates, phosphates, silicates and silica of sodium, potassium, magnesium and calcium. A high modify value is indicative of contamination, substitution or adulteration.Sometimes, inorganic variables like calcium oxalate, silica, carbonate content of crude drug affects total ash tree values such variables are then removed by treating with back breaker (as they are soluble in hydrochloric sultry) and then pungent-insoluble ash value is determined. Ash insoluble in hydrochloric sour is the residue obtained after extracting the total ash with hydrochloric acid. This acid-insoluble ash value particularly indicates contamination with silicious materials like earth or sand. Water-soluble ash is that part of the total ash content which is soluble in water. It is a good indicator of either previous extraction of water soluble salts in the drug o r incorrect preparation.For the determination of various ash values viz. total ash, acid-insoluble ash, water-soluble ash, the shade dried parts of the selected plant materials were powdered and passed through sieve no40 and studies were carried out. The values take off within fairly wide limits and is therefore an important parameter for the purpose of evaluation of crude drugs.Determination of Total AshA flat, thin porcelain melting pot was weighed and ignited. About 2 g of the powdered drug was taken into the crucible. The crucible was incinerated at temperatures not portentous 4500C, until free from carbon.The crucible was cooled in a desiccator and weighed. The surgical process was repeated to get constant weight.The percentage of total ash was calculated with reference to the air dried drug. (Table No.8)Determination of Acid-insoluble AshThe total ash obtained was boiled with 25 ml of 2 M hydrochloric acid for 5mins. The insoluble ash was sedate on an ashless filter paper 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 sample 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-soluble 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 ultraviolet beam of light. 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)Total 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 gramsExtractionMacerationThe powdered materials were extracted with alcohol (95%) by cold maceration method.Weighed qu antity 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 period of 7 days. Finally the flask was left undisturbed for 12 hrs and then the contents were shaker and filtered through What human race filter paper No.1. The marc was re-extracted with drug solvent ratio of 12. The extracts were combined and turn in a rotary flash evaporator, till free from solvent. The extracts, thus obtained were stored in a refrigerator 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 systematic 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 tests are to be performed for establishing profile of a given extract/fraction for its nature of chemical composition.The following tests were carried out on the extracts to detect various phytoconstituents present in them.Detection of AlkaloidsAbout 50 mg of solvent free extract was steamy with little quantity of dilute hydrochloric acid and filtered. The filtrate was tested carefully with various alkaloid tests viz., Mayers Test, Wagners Test, Hagers Test, Dragendroffs TestDetection of CarbohydratesAbout 100mg of the extract was dissolved 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 with 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, Lead acetate test, Alkaline reagents, and Shinoda test or Magnesium Hydrochloric acid diminutionThin Layer 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 breakthrough in this field was the commercial availability of convenient precoated habitations in the early 70s Pharmacopoeias are increasingly employing this technique for assessing the quality and purity of compounds of both synthetic and natural origin. TLC profiles real 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 simplicity and reproducibility.TLC is an important analytical tool in the separation, identification and estimation of different classes of natural products. In this technique, the different components are separated by the differential migration of solute between two degrees a stationary phase and a mobile phase. Here, the principle of separation 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 Silica 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 looking glass denture 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. Before using, the plates were activated by heating at 100o C for 10 minutes.Detection of Steroids / Triterpenoids and their GlycosidesSolvent systems usedethyl acetate wood alcohol 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)Benzene 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) reagentSolution I 5% ethanolic sulphuric acidSolution II 1% ethanolic vanillinThe developed TLC plate was sprayed with 10 ml of solution I, followed forthwith 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 pink 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 spraying 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 and 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 observed in visible light and in UV at 365 nm. Flavonoids and their glycosides appear as yellow, dark blue, orange zones / spots. The color gets intensified on motion-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.