Determination of Stomatal Index

Determination of Stomatal Index The Plant material of Viscum capitellatum Smith. parasitism on Dendrophthoe falcata which is itself parasitic on M. indica was collected from Amba Ghat, Kolhapur, Western Ghat region of Maharashtra from India in November 2009. The collection are lies [Latitude 16o 58 0.59N and Longitude 73 ° 48 36.61E at altitude 1100m]. The plant specimen (Voucher no. 550) was authenticated by Dr. Vinay Raole, Reader, Department of Botany, M.S. University, Baroda, India. Pharmacognostical Study Macroscopical Study[68] It includes the shape, size, colour, texture, surface and odour of the drug in crude or powered form and often sufficient to enable to identify the whole drugs. Microscopical Study Histochemistry It gives the idea about the colour reaction of specific chemical reagent towards plant tissues [68]. Microscopical images are given in Figure no. 2. Quantitative Microscopy [66-69] Transverse sections of scale and stems were obtained by means of a microtome and stained with different staining reagents as per standard procedures [66, 70-71]. All observations were performed using Motic Digital Photomicroscope. Histological study of leaves and stem were performed by reported method [69]. Leaves were boiled in a 5% aqueous solution of NaOH for 5 min while stems were boiled with 10% aqueous solution of NaOH for 10 min. After cooling and washing with water, pieces were treated with a 25% aqueous solution of chromic acid for 30 min at room temperature. Washed pieces of both leaf and stem were pressed in between two slides and slides coves. Determination of Stomatal Number The average number of stomata per square millimeter of epidermis is termed the stomatal number. Determination of Stomatal Index The percentage proportional to the ultimate divisions of the epidermis of a leaf, which has been converted into stomata, is termed the stomatal index. SI = S ÃÆ'- 100 E + S Where SI = Stomatal index, S = number of stomata per unit area and E = number of ordinary epidermal cells in the same unit area. Procedure [68] Pieces of leaf between margin or midrib was cleared and mounted, and the lower surface examined by means of a microscope with a 4mm objective and an eyepiece containing a 5mm square micrometer disc. Counts were made of the numbers of the epidermal cells and of stomata within a square grid, a cell being counted if at least half of its area lies within the grid. The stomata index was determined for both leaf surfaces. Results pertaining to quantitative microscopical study are given in table no. 8. Analytical Study Ash Value 1.1 Total ash Total ash gives the idea about the residue obtained after ignition. It consist of physiological ash obtain by ignition of plant tissues and non physiological ash obtain by ignition of extraneous matter adhering to the surface of Plant. 2 gm of accurately weighed air dried powdered drug was taken in silica crucible. This silica crucible with drug material was kept in muffle furnace and ignited at temperature 4500C. The material was heated till the white coloured ash and constant weight is obtained. The procedure was performed in triplicate. Result is given in table No. 9. The total ash was calculated by subtracting the weight of crucible with ash of drug after ignition from weight of crucible with drug powder before ignition. Percentage of total ash was calculated with reference to air-dried drug. Acid insoluble ash Acid insoluble ash gives the idea about the presence of inorganic material such as calcium oxalate present in plant material. The ash obtained in the total ash method was boiled with 25 ml of 2N hydrochloric acid for 5 min. Insoluble matter was collected on ash less filter paper (Whatman paper) and washed with hot water. The material retained on filter paper and along with filter paper, was further ignited and weighed. Percentage of acid insoluble ash was calculated with reference to air dried material. Result is given in table No. 9. Water soluble ash The ash obtained from total ash was boiled with 25 ml water for 5 min. All insoluble matter was collected on ash less filter paper, washed with hot water and ignited for 15 min at the temperature not exceeding 4500C. The percentage of water soluble ash was calculated by subtracting weight of insoluble matter from weight of total ash. The difference between weights represents water soluble ash. Percentage of water soluble ash was calculated with reference to air dried drug. Result is given in table No. 9. Extractive Value Extraction by cold maceration It is the process of extraction of crude drugs with solvents with several daily shakings or stirring at room temperature.1 kg of powdered plant was extracted with 5 lit of methanol by cold maceration method. The extract was concentrated on rotary vacuum evaporator (Roteva Equitron, Mumbai) and further dried in vacuum dryer [73]. Successive extraction by using Soxhlet apparatus Weighed accurately 200gm of dried, powered crude drug and kept in a filter paper cover which was already placed in thimble. Then the solvent was slowly poured onto it. The solvent from thimble goes to lower round bottom flask via siphon tube due to the siphoning or syphon cycle. Such 2-3 cycles of solvent were performed and then drug powder was kept for 12 hours with solvent for imbibitions. After 12 hours imbibitions, solvent from flask heated to form vapors. Due to heat the solvent from RBF gets converted into its vapors, and then these vapors pass via side tube into the condenser where it gets condensed. This solvent dripped again on to drug material, which was placed in thimble. This process was continued till thimble gets filled with solvent and when level of solvent reaches to syphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several times and drug material gets extracted continuously with fresh solvent. This process was performed for 3 days and when syphon solution showed negative test for phytoconstituents, extraction was completed. Then the heating was stopped and the mixture was collected and cooled. Then this mixture was filtered and concentrated by using rotary flash vacuum evaporator. The extract was dried in vacuum dryer and was stored in freeze. Then this marc obtained after pet ether extraction and subjected again to extraction by following solvents (Table 10) [73]. Moisture content by Loss on Drying 2 g of air powdered drug was placed in a silica crucible. Before that, crucible was cleaned and dried and weight of empty crucible was taken. The powder was spread in a thin uniform layer. The crucible was then placed in the oven at 1050C. The powder was dried for 4 h and cooled in a desiccator to room temperature and weight of the cooled crucible plus powder was noted. Result is given in table no. 9. Analysis of inorganic constituents (Elemental analysis) Ash of drug material was prepared and adds 50% v/v HCl or 50% v/v HNO3 to ash. Keep it for 1 hour. Filtered and with the filtrate performed the test as per method reported [74]. The results of analysis of inorganic constituents are given in (Table 11). Test for calcium a) Add dil. NH4OH and saturated ammonium oxalate solution to filtrate. White ppt of calcium oxalate forms which is soluble in HCl. Calcium present. b) Add ammonium carbonate to filtrate. White ppt which is insoluble in NH4Cl. Calcium present. Tests for iron a) Add 2% potassium ferricyanide to filtrate. Dark blue coloration. Iron present. b) To filtrate, add 5% ammonium thiocyanate. Blood red color. Iron present. c) To filtrate, add dil. HCl and sol. of KMnO4. Pink color. Iron present. Tests for magnesium a) To filtrate add NaOH. White ppt. Magnesium present. b) To filtrate add (NH4)2CO3. White ppt, redissolve in NH4Cl. Magnesium present. Tests for potassium a) Add sodium cobalt nitrite to filtrate. Yellow ppt. Potassium present. b) Flame test. Violet color to flame. Potassium present. Tests for sodium a) Add uranyl zinc acetate to filtrate, shake well. Yellow crystalline ppt. Sodium present. Tests for carbonate a) Add HgCl2 to filtrate. Brownish red ppt. Carbonate present. b) Add dil. Acid to the filtrate. Effervescence of CO2 Carbonate present. c) Add MgSO4 to filtrate. White ppt. Carbonate present. Tests for Sulphate a) Add BaCl2 to filtrate. White crystalline ppt Sulphate present. b) Add filtrate to lead acetate sol. White ppt. Sulphate present. Tests for phosphate a) Add HNO3 and ammonium molybdate to filtrate, heat 10 min. cool. b) Add silver ammonium- nitrate to filtrate Yellow crystalline ppt. Light yellow ppt Phosphate present. Phosphate present. Tests for chloride a) Add AgNO3 to filtrate. b) To filtrate, add manganese dioxide and H2SO4 White curd ppt, soluble in dil. NH3. Odour of chlorine Chloride present. Chloride present. Tests for nitrate a) Add water to filtrate, add H2SO4 from side of test tube. b) Add H2SO4 and copper to filtrate, warm Brown color at junction of two liquid Liberation of red fumes Nitrate present. Nitrate present. Determination of Type of Starch Grains The shape of starch grains present was determined according to the reported method [68]. Size of starch grains were measured with the help of calibrated Photomicroscope using Motic software. Starch grains were identified by staining with Iodine solution. The Motic digital Photomicroscope was calibrated with images obtained with various magnifications (10x, 40x and 100x) by using standard slide in 1.3 software. The images obtained in triplicate and average figures calculated from 20 readings in each parameter (Table no. 12). Crude Fiber Content Pre-weighed dried powder material was extracted with Petroleum ether (b.p. 40- 600C) using soxhlet apparatus for 8 h. The marc obtained after extraction was utilized for determination of Crude Fiber Content. Crude fiber was investigated by acid-base digestion with H2SO4 (1.25%) and of NaOH (1.25%) solution. The marc after extraction was taken into a 500ml beaker and 200ml of boiling H2SO4 added. The content was boiled for 30 minutes, cooled, filtered and the residue washed three times with 50ml of boiling water. The washed residue was further boiled in 200ml of NaOH for 30 minutes. The digest was filtered to obtain residue. This was washed three times with 50ml of boiling water and lastly with 25ml of ethanol. The washed residue was dried in an oven at 1250C to constant weight and cooled in dessicator. The residue was scraped into a pre-weighed porcelain crucible, weighed, ashed at 5500C for 2 hours, cooled in a dessicator and weighed. Crude fiber content was expressed as percentage loss in weight on ignition. Result is given in table No. 13. Phyto-chemical Analysis Extracts Petroleum ether, benzene, chloroform, acetone and methanol extract obtained by successive extraction method and aqueous extract by maceration method [68, 95]. Qualitative analysis All the extracts were subjected to proximate chemical analysis and its result is given in table no. 14. Tests for Acidic compounds: a) To the test solution add sodium bi-carbonate b) Test solution treated with warm water and filter. Test the filtrate with litmus paper. Tests for Alkaloids: a) Dragendorffs Test: Test solution treated with Dragendorffs reagent (potassium bismuth iodide) b) Mayers Test: Test solution treated with Mayers reagent (Potassium mercuric iodide). c) Wagners Test: Test solution treated with Wagners reagent (Iodine in potassium iodide). d) Hagers Test: To the test solution add gives with Hagers reagent (Saturated picric acid solution). e) Tannic acid test: Test solution treated with Tannic acid solution. f) Picrolonic acid test: Test solution treated with Picrolonic acid. Test for amino acids: a) Millions Test: Test solution treated with Millions reagent and heated on a water bath. b) Ninhydrin Test: Test solution boiled with Ninhydrin reagent. Test for Carbohydrates: a) Molischs Test: To the test solution add with few drops of Molischs reagent (Alcoholicà ¯Ã‚ Ã‚ ¡-naphthol) and 2ml of conc. sulphuric acid is added slowly from the sides of the test tube. b) Barfords Test: Test solution heated with Barfords reagent on water bath. c) Selivanoffs test (Test for Ketones): To the test solution add crystals of resorcinol and equal volumes of concentrated hydrochloric acid and heat on a water bath. d) Test for pentose: To the test solution add equal volumes of hydrochloric acid containing small amount of Phloroglucinol and heat. e) Osazone formation test: Heat the test solution with the solution of phenyl hydrazine hydrochloride, sodium acetate, and acetic acid. Test for Flavonoids: a) Shinoda Test: Test solution treated with fragments of magnesium ribbon and conc. Hydrochloric acid. b) Alkaline Reagent Test: Test solution treated with sodium hydroxide solution c) Zinc-Hydrochloride test: Treat test solution with zinc dust and few drops of HCL Test for glycosides: General test: Extract 200 mg of drug with 5 ml of dilute sulphuric acid by warming on a water bath, filter it, and neutralize the acid extract with 5 % solution of sodium hydroxide. Add 0.1 ml of Fehlings solution A and B until it becomes alkaline (test with pH paper) and heat on water bath for 2 minutes. Test B: Repeat Test A procedure by using 5 ml of water instead of dilute sulphuric acid. Note the quantity of red precipitate formed. Chemical tests for specific glycosides: Tests for Anthraquinone glycosides: a) Borntragers test: Boil the test material with 1ml of sulphuric acid for 5minutes. Filter while hot. Cool the filtrate; shake with equal volume of dichloromethane or chloroform. Separate the lower layer of dichloromethane or chloroform; shake it with half of its volume of dilute ammonia. b) Modified Borntragers test: Boil 200 mg of test material with 2ml of sulphuric acid. Treat with 2 ml of 5 % aqueous ferric chloride solution (freshly prepared) for 5 minutes, shake it with equal volume of chloroform and continue the test as above. c) Test for hydroxy anthraquinones: treat the sample with potassium hydroxide solution. Tests for cardiac glycosides: a) Keddes test: Extract the drug with chloroform, evaporate to dryness. Add one drop of 90 % alcohol and 2 drops of 2 % sodium hydroxide solution. b) Keller-Killiani Test: (Test for deoxy sugars) Extract the drug with chloroform and evaporate it to dryness. Add 0.4 ml of glacial acetic acid containing ferric chloride, add carefully 0.5 ml of conc. sulphuric acid by the side of test tube. c) Raymonds number: treat the test solution with hot methanolic alkali. d) Baljets Test: The test solution treated with sodium picrate or picric acid. e) Legals Test: Test solution treated with pyridine [made alkaline by adding sodium nitroprusside solution]. f) Tests for coumarins glycosides: Place small amount of sample in test tube and covered it with a filter paper, moistened with dilute sodium hydroxide solution. Placed the covered test tube on water bath for several minutes. Remove the paper and expose it to ultraviolet (UV) light. Cynogentic glycosides: Place 200 mg of drug in conical flask and moisten with few drops of water.( Flask should be completely dry because hydrogen cyanide produced will dissolve in the water rather than come off as gas to react with paper) moisten a piece of picric acid paper with 5% aqueous sodium carbonate solution and suspended in neck of flask. Warm gently at about 37oC. Observe the change in color. Saponin glycosides: Froth test: Place 2 ml solution of drug in water in a test tube, shake well. Tests for steroids and triterpenoids: a) Liebermann Burchard Test: Treat the extract with few drops of acetic anhydride, boil and cool, add conc. sulphuric acid from the sides of test tube. b) Salkowski test: Treat the extract with few drops of conc. sulphuric acid. c) Sulfur powder test: Add small amount of sulfur powder to the test solution. d) Tests for inulin: To the test solution add the solution of à ¯Ã‚ Ã‚ ¡-naphthol and sulphuric acid. e) Tests for Lignin: Treat the sample with hydrochloric acid and Phloroglucinol. Tests for Mucilage: Treat the sample with thionine solution. After 15 min wash with alcohol Tests for tannins: a) Ferric-Chloride Test: Treat test solution with few drops of ferric chloride solution. b) Gelatin test: To the test solution add 1 % gelatin solution containing 10 % sodium chloride. Tests for proteins: a) Heat test: Heat the test solution in boiling water bath. b) Biuret Test: Test solution treated with Biuret reagent (40% sodium hydroxide and dilute copper sulfate solution). c) Xanthoproteic test: To the test solution, add 1 ml of conc. nitric acid and boil yellow precipitate is formed. After cooling it, add 40 % sodium hydroxide solution. d) Test for starch: To the test solution, add weak aqueous iodine solution. Blue color indicates presence of starch, which disappears on heating and reappears on cooling. Effervescence produces Litmus paper turns blue Gives reddish brown colored precipitate Gives cream colored precipitate Gives reddish brown colored precipitate Gives yellow colored precipitate Gives buff colored precipitate Gives yellow colored precipitate White colored precipitate Gives violet color Purple to violet ring appears at the junction of two liquids If red cupric oxide is formed Rose color is produced Red color produced. Yellow crystals formed. Observe under microscope. Shows pink scarlet, crimson red or occasionally green to blue color after few minutes. Shows increase in the intensity of yellow color on addition of few drops of dilute acid. Shows red color after few minutes. Red Precipitate formed compared with precipitate of test A A rose pink to red color is produced in ammonical layer. A rose pink to red color is produced in ammonical layer. Red color produced Purple color is produced. Acetic acid layer shows blue colour. Violet colour produced Gives yellow to orange color Gives blood red color Paper shows green fluorescence. Reddish purple color Stable froth (foam) formed Brown ring is formed at the junction of two layers, If upper layer turns green If upper layer turns deep red Red color at lower layer Yellow color at lower layer It sinks at the bottom Brownish red color formed Pink color formed Mucilage turns violet red. Gives dark blue color Green color appears Precipitate formed Proteins gets coagulated Gives violet color Orange color formed Blue color, which disappears on heating and reappears on cooling Acidic compounds present Acidic compounds present Alkaloids present Alkaloids present Alkaloids present Alkaloids present Alkaloids present Alkaloids present Amino acids present Amino acids present Carbohydrates present Monosaccharides are present. Carbohydrates present Carbohydrates present Carbohydrates present Flavonoids present Flavonoids present Flavonoids present If the precipitate in Test A is greater than in Test B then glycoside may be present. Anthraquinone glycosides present Anthraquinone glycosides present Hydroxy anthraquinones present Cardiac glycosides present Cardiac glycosides present Cardiac glycosides present Cardiac glycosides present Cardiac glycosides present Coumarins glycosides present Cynogentic glycosides present Saponin glycosides Present Steroids present Triterpenoids present Steroids present Triterpenoids present Steroids present Inulin Present Lignin Present Mucilage present Hydrolysable tannins Condensed tannins Tannins present Proteins present Proteins present Proteins present Starch present Floroscence Analysis of various extracts Petroleum ether, Benzene, Chloroform, Acetone, Methanol and Aqueous extracts were screened for fluorescence characteristic. The observation pertaining to their colour in day light and under ultra-violet light were noticed and represented in table. Many substances for example quinine in solution in dilute sulphuric acid when suitably illuminated emit light of a different wavelength or colour from that which falls on them. This emitted light (fluorescence) ceases when the exciting light is removed [68].Results given in Table No. 15. HPLC Analysis of sample drug The chromatographic pattern of plant was obtained as per report with some modifications for which the HPLC conditions are as follows. Extract: The methanol extract diluted with HPLC grade methanol and filtered through whatman filter paper and used for analysis Instrument: Shimadzu LC-20AT with UV/visible detector Stationary Phase: Bonda- pack C-18 column with 250ÃÆ'-4mm Mobile Phase: Methanol (80): Water (20) Detection wave length: 350 nm Flow Rate: 2 ml/min. HPLC Chromatogram is given in Fig. 3 and its retention time is given in Table no. 16 HPTLC Analysis of sample drug The chromatographic pattern of plant was obtained as per report with some modifications for which the HPTLC conditions are as follows. Extract: Methanolic Extract Instrument: HPTLC (Camag, Switzerland) Stationary Phase: pre-coated silica gel plates Mobile Phase: Ethyl acetate: Formic acid: Glacial acetic acid: water (100:05:10:20) Spraying Reagent: Natural Product Reagent (NP reagent) Detection: 365 nm. HPTLC Chromatogram is given in Fig. 4 and its retention time is given in Table no. 17. Isolation and characterization of chemical principle Compound I The methanol extract was dissolved in water and partitioned with ethyl acetate and n- butanol. The ethyl acetate fraction was subjected to column chromatography for isolation of compounds. Column chromatography: The separation of extract constituents was done by column chromatography. The clean and dried glass column was used. The silica gel for column chromatography (#60-120) was activated at 1100c.The column was filled with silica gel and mobile phase without formation of any air bubbles. The silica gel was then allowed to stabilize in the column. Mixture of two or three compounds was isolated from the ethyl acetate fraction of methanol extract of the plant with following experimental conditions [73]. Height of column: 20 cm Diameter of column: 3.5 cm. Stationary phase: Silica gel (#60-120). Mobile phase: Benzene† Ã¢â‚¬â„¢ Chloroform † Ã¢â‚¬â„¢ Ethyl acetate† Ã¢â‚¬â„¢ Methanol with variant Proportions Elution: Gradient elution. Fraction quantity: 25 ml Preparative TLC: 20 X 20 glass plates were coated with the thick layer of silica gel or any other adsorbent material. The plates were then activated at 1100c.The sample-containing mixture of two or more compounds were applied in the form of thin band on the plate. The plate was then developed. The different bands separated on the plate were scratched and recovered with methanol. Purity of dried sample was checked by TLC method. One single compound was isolated with the help of preparative chromatography from fractions 54- 58. The compound is given for spectral analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 5, 6 and 7 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 18 and 19 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 8. Compound II Petroleum ether extract obtained is processed for separation of the unsaponifiable and saponifiable matter. Extract is allowed to saponify using alcoholic KOH with reflux and then it is extracted with solvent ether for separation of unsaponifiable matter. The aqueous phase is acidified with concentrated H2SO4 and then again extracted with the solvent ether for separation of the saponifiable matter [73]. Fractionation of unsaponifiable matter Experimental: Height of column: 25 cm Diameter of column: 3.5 cm. Stationary phase: Silica gel for column chromatography (#60-120). Mobile phase: Benzene† Ã¢â‚¬â„¢ Ethyl acetate Elution: Gradient elution. Fraction quantity: 30 ml Fractions No. 24-27 were subjected for thin layer chromatography with following experimental conditions. Stationary phase: Silica gel H Mobile phase: Ethyl acetate: Benzene (1: 9) Detection: Vanilin-sulphuric acid reagent Identification: Whitish Purple colour Fraction was concentrated and single band was applied. After plate development; developed band was scraped (Rf. 0.62). After separation of single compound from the silica, it is dried. This sample was further given for spectroscopic analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 9, 10 and 11 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 20 and 21 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 12. Biochemical Estimations a) Estimation of Total carbohydrate content The estimation of carbohydrate was done using the method acid base digestion. Principle: In hot acidic media glucose is converted to hydroxy methyl furfural by dehydration. This forms a green colour product with phenol. Procedure: 100mg of the aqueous extract was taken and it was hydrolyzed by keeping it on water bath for 3 hours with 5 ml of HCl (2.5N) and cooled at room temperature. Neutralized it with sodium carbonate and volume was made up to 100 ml and from this centrifuge 10 ml of the solution. Then 0.2, 0.4, 0.6, 0.8 and 1ml of working standard was pipetted out into a series of test tube and in separate test tubes 0.1 and 0.2 ml of sample solution was pipetted out and the volume was make up to 1ml with water. The blank was prepared with 1 ml distilled water. Then 1ml phenol solution and 5ml of sulphuric acid (96%) was added to each test tube and shaken well. After 10 min the test tube was placed in water bath at 25-30à ¯Ã¢â‚¬Å¡Ã‚ °C for 20 min. The absorbance was read at 490 nm. And the amount of total carbohydrate present was calculated in the sample using standard graph. Result pertaining to Total carbohydrate content is given in Table no. 22 and Calibration curve of standard glucose dilutions are gi ven in Fig. No. 13. Estimation of Bitterness value The bitterness value of plant material was compared with diluted solution of Quinine hydrochloride. Preparation of Solutions Preparation of Quinine hydrochloride solution The stock solution of 100 µg/ml was prepared from which a series of dilutions 42, 44, 46, 48, 50, 52, 54, 56 and 58  µg/ml were prepared. Preparation of Sample Preparation Form the stack solution of 1000  µg/ml, 100, 200, 300 and 400 µg/ml dilutions were prepared. Method Tasted all the dilutions of sample and Quinine sulphate by taking the solution in mouth and swirled it for 30 secs in mouth mainly near to the tongue. After tasting each dilution the mouth wash rinsed thoroughly with drinking water and taken the interval of 10 mins. Until the bitter sensation of previous dilution was no more remain. Then compared the dilution of sample which produced the same bitterness equivalent to the dilution of Quinine sulphate. Then bitterness value was calculated according to following formula. Bitterness value in units per gram = 2000 ÃÆ'- A B ÃÆ'- C Where A= quantity of Quinine sulphate (mg) having higher bitterness B= the concentration of stock solution (mg/ml) C= Volume of sample in ml having higher bitterness Result pertaining to estimation of bitterness value is given in Table no. 22 Total Phenolic content The total phenolic content of methanol extract of V. capitellatum Smith. (VCM) was estimated using Folin-Ciocalteu reagent. In this method, the blue colour formed due to the polyphenol was measured at 760 nm using UV spectrophotometer. Chemicals Folin- Ciocalteu reagent (Merck Co.) Gallic acid (Sigma Ltd., USA) Sodium carbonate (SISCO Research Laboratory Pvt. Ltd., Mumbai, India) Reagent preparation Folin-Ciocalteu (phenol) reagent The reagent was prepared by diluting 1ml with 5ml of distilled water. Sodium carbonate 15% solution was prepared in distilled water. Gallic acid solution The stock solution was prepared by dissolving 1mg gallic acid in 10ml of water from which different concentrations (20-100 µg/ml) were prepared. Sample preparation Sample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100  µg/ml) solution. Procedure 0.1ml of extract was mixed with the 0.2ml of Folin-Ciocalteu reagent, 2 ml water and 1 ml of sodium carbonate solution, and absorbance was measured at 760 nm after 10 min incubation at 50 0C. The total phenolic was expressed as  µg gallic acid equivalent. Result pertaining to Total phenolic content is given in Table no. 22 and Calibration curve of standard gallic acid dilutions are given in Fig. No. 14. Total Flavonoid Content Total flavonoid content of VCM was determined using method reported [79].