Determination of Stomatal Index

Determination of Stomatal IndexThe Plant secular of Viscum capitellatum Smith. parasitism on Dendrophthoe falcata which is itself parasitic on M. indica was equanimous from Amba Ghat, Kolhapur, Western Ghat neighbourhood of Mahar ashtra from India in November 2009. The collection ar lies Latitude 16o 58 0.59N and Longitude 73 48 36.61E at altitude 1100m. The represent specimen (Voucher zero(preno(prenominal)inal) 550) was au consequentlyticated by Dr. Vinay Raole, Reader, part of Botany, M.S. University, Baroda, India.Pharmacognostical probeMacroscopical Study68It includes the shape, size, colour, texture, surface and odour of the medicate in stark naked or powe fiercedened category and often sufficient to enable to line the self-colou florid drugs.Microscopical StudyHistochemistryIt slacken offs the motif near the colour answer of specific chemic reagent towards plant tissues 68. Microscopical images be apt(p) in Figure no. 2.Quantitative Microscopy 66-69Tran sverse sections of scale and stems were bringed by centre of a microtome and stained with different staining reagents as per standard procedures 66, 70-71. t unwrap ensemble observations were per create use Motic Digital Photomicroscope.Histological depicted object of leaves and stem were per organise by inform order 69. Leaves were boiled in a 5% aqueous root word of NaOH for 5 min while stems were boiled with 10% aqueous root of NaOH for 10 min. afterwards change and washing with piss supply, valet de chambres were interact with a 25% aqueous etymon of chromic pungent for 30 min at agency temperature. Washed pieces of both leaf and stem were pressed in mingled with two slides and slides coves.Determination of Stomatal NumberThe average publication of stomata per feather millimeter of epidermis is termed the stomatal number.Determination of Stomatal IndexThe parting proportional to the ultimate divisions of the epidermis of a leaf, which has been converted in to stomata, is termed the stomatal index.SI = S - 100E + SWhere SI = Stomatal index, S = number of stomata per unit argona and E = number of ordinary cuticular cells in the same unit area. subroutine 68Pieces of leaf between leeway or midrib was cleared and mounted, and the impose surface examined by re openation of a microscope with a 4mm objective and an eyepiece containing a 5mm square micron disc. Counts were made of the numbers of the epidermal cells and of stomata inwardly a square grid, a cell being counted if at least half of its area lies wi deoxidize the grid. The stomata index was determined for both leaf surfaces. resolving powers pertaining to quantitative microscopical study are inclined in table no. 8.Analytical StudyAsh mensurate1.1 Total ashTotal ash gives the idea about the rest obtained aft(prenominal) 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 personal line of credit dried powdery drug was taken in silicon dioxide crucible. This silica crucible with drug veridical was unbroken in break furnace and ignite at temperature 4500C. The satisfying was modify till the white work ash and constant system of tips is obtained. The procedure was performed in triplicate. Result is inclined in table No. 9.The integral 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 walkover-dried drug. pungent piss-in pee-soluble ash paneling insoluble ash gives the idea about the presence of inorganic clobber such as calcium oxalate sacrifice in plant material.The ash obtained in the total ash method was boiled with 25 ml of 2N hydrochloric hotulent for 5 min. Insoluble matter was collected on ash less tense up penning (Wha tman paper) and washed with hot water supply. The material unploughed up(p) on filter paper and along with filter paper, was yet ignited and weighed. Percentage of acid insoluble ash was calculated with reference to air dried material. Result is assumption in table No. 9. piss soluble ashThe 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 pct of water soluble ash was calculated by subtracting weight of insoluble matter from weight of total ash. The difference between weights re applys water soluble ash. Percentage of water soluble ash was calculated with reference to air dried drug. Result is minded(p) in table No. 9. natural selectionive ValueExtraction by cold-blooded macerationIt is the process of line of descent of bumpy drugs with solvents with several daily shakings or stirring at room temperature.1 kg of powdered plant was extracted with 5 lit of methyl radical alcohol by cold maceration method. The extract was arduous on rotary vacuum evaporator (Roteva Equitron, Mumbai) and further dried in vacuum dryer 73.Successive extraction by using Soxhlet apparatusWeighed accurately 200gm of dried, powered crude drug and kept in a filter paper cover which was already displace in thimble. then the solvent was slowly poured onto it. The solvent from thimble goes to get off round bottom flask via siphon tube imputable 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 kindleed to form megrims. Due to warmth 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 bro odd till thimble gets filled with solvent and when level of solvent r all(prenominal)es to syphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several multiplication and drug material gets extracted continuously with fresh solvent. This process was performed for 3 geezerhood and when syphon antecedent showed negative essay for phytoconstituents, extraction was completed. Then the agileing 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 vinyl diethyl ether extraction and subjected again to extraction by following solvents ( accede 10) 73.Moisture subject by Loss on Drying2 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 uni form level. 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(p) in table no. 9. abridgment of inorganic constituents (Elemental analysis)Ash of drug material was alert and conveys 50% v/v HCl or 50% v/v HNO3 to ash. Keep it for 1 hour. Filtered and with the filtrate performed the tally as per method reported 74. The results of analysis of inorganic constituents are given in ( accede 11). streak for calciuma) impart dil. NH4OH and saturated ammonium oxalate out add to filtrate. ovalbumin ppt of calcium oxalate forms which is soluble in HCl.Calcium demonstrate.b) take ammonium change to filtrate. white ppt which is insoluble in NH4Cl.Calcium bribe. tribulations for irona) minimal brain damage 2% kilobyte ferricyanide to filtrate.Dark blue disguiseation.Iron impart.b) To filtrate, channel 5% ammonium thiocyanate.Blood re d distort in.Iron present.c) To filtrate, add dil. HCl and sol. of KMnO4.Pink glossary.Iron present.Tests for atomic number 12a) To filtrate add NaOH.White ppt.Magnesium present.b) To filtrate add (NH4)2CO3.White ppt, redissolve in NH4Cl.Magnesium present.Tests for potassiuma) tot sodium cobalt nitrite to filtrate. white-livered ppt.Potassium present.b) Flame test.Violet color to flame.Potassium present.Tests for sodiuma) pass on uranyl zinc acetate to filtrate, quaver well. discolor limpid ppt. atomic number 11 present.Tests for carbonatea) hyperkinetic syndrome HgCl2 to filtrate.Brownish red ppt.Carbonate present.b) Add dil. Acid to the filtrate.bubbliness of CO2Carbonate present.c) Add MgSO4 to filtrate.White ppt.Carbonate present.Tests for sulfatea) Add BaCl2 to filtrate.White crystalline pptSulphate present.b) Add filtrate to occupy acetate sol.White ppt.Sulphate present.Tests for phosphatea) Add HNO3 and ammonium molybdate to filtrate, heat 10 min. cool.b) Add silver ammonium- nitrate to filtrateYellow crystalline ppt.Light yellowness(a) pptPhosphate present.Phosphate present.Tests for chloridea) Add AgNO3 to filtrate.b) To filtrate, add manganese dioxide and H2SO4White curd ppt, soluble in dil. NH3.Odour of chlorineChloride present.Chloride present.Tests for nitratea) Add water to filtrate, add H2SO4 from side of test tube.b) Add H2SO4 and copper to filtrate, warmBrown color at junction of two liquid inflammation of red fumesNitrate present.Nitrate present.Determination of Type of starch GrainsThe shape of starch grains present was determined according to the reported method 68. Size of starch grains were measured with the dish up of calibrated Photomicroscope using Motic software. amylum grains were identified by staining with Iodine closure. The Motic digital Photomicroscope was calibrated with images obtained with unhomogeneous magnifications (10x, 40x and 100x) by using standard slide in 1.3 software. The images obtained in triplicat e and average figures calculated from 20 readings in each parameter (Table no. 12).Crude Fiber ContentPre-weighed dried powder material was extracted with fossil oil ether (b.p. 40- 600C) using soxhlet apparatus for 8 h. The marc obtained after extraction was utilize for determination of Crude Fiber Content.Crude fictional character was investigated by acid- secondary digestion with H2SO4 (1.25%) and of NaOH (1.25%) resultant role. The marc after extraction was taken into a 500ml beaker and 200ml of boiling H2SO4 added. The nub was boiled for 30 minutes, cooled, filtered and the relief washed three terms with 50ml of boiling water. The washed counterweight 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 porce lain crucible, weighed, ashed at 5500C for 2 hours, cooled in a dessicator and weighed. Crude fiber discipline was expressed as percentage loss in weight on ignition. Result is given in table No. 13.Phyto-chemical AnalysisExtractsPetroleum ether, benzene, chloroform, acetone and methyl alcohol extract obtained by successive extraction method and aqueous extract by maceration method 68, 95.qualitative analysisAll the extracts were subjected to proximate chemical analysis and its result is given in table no. 14.Tests for Acidic compoundsa) To the test effect add sodium bi-carbonateb) Test consequence inured with warm water and filter. Test the filtrate with litmus paper.Tests for Alkaloidsa) Dragendorffs Test Test upshot toughened with Dragendorffs reagent (potassium atomic number 83 iodide)b) Mayers Test Test base treated with Mayers reagent (Potassium mercuric iodide).c) Wagners Test Test base treated with Wagners reagent (Iodine in potassium iodide).d) Hagers Test To the t est root add gives with Hagers reagent (Saturated picric acid resolvent).e) Tannic acid test Test tooth root treated with Tannic acid solution.f) Picrolonic acid test Test solution treated with Picrolonic acid.Test for amino acidsa) 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 Carbohydratesa) Molischs Test To the test solution add with hardly a(prenominal) drops of Molischs reagent (Alcoholic-naphthol) and 2ml of conc. sulfuric 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 little(a) total of Phloroglucinol and heat.e) Osazone formation t est Heat the test solution with the solution of phenyl hydrazine hydrochloride, sodium acetate, and acetic acid.Test for Flavonoidsa) Shinoda Test Test solution treated with fragments of magnesium ribbon and conc. Hydrochloric acid.b) Alkaline Reagent Test Test solution treated with sodium hydrated oxide solutionc) Zinc-Hydrochloride test overcompensate test solution with zinc dust and few drops of HCLTest for glycosidesGeneral test Extract 200 mg of drug with 5 ml of deprave 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 paraphrase Test A procedure by using 5 ml of water instead of swerve sulphuric acid. Note the quantity of red sharp formed.Chemical tests for specific glycosidesTests for Anthraquinone glycosidesa) Borntragers test Boil the test material with 1ml of sulp huric 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 slew 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 type with potassium hydroxide solution.Tests for cardiac glycosidesa) Keddes test Extract the drug with chloroform, evaporate to dryness. Add one drop of 90 % alcoholic drink 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 conservatively 0.5 ml of conc. sulphuric acid by the side of test tube.c) Raym onds 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 exemplar 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 softly (UV) light.Cynogentic glycosidesPlace 200 mg of drug in conical flask and moisten with few drops of water.( Flask should be completely dry because atomic number 1 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 glycosidesFroth test Place 2 ml soluti on of drug in water in a test tube, shake well.Tests for steroids and triterpenoidsa) 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 -naphthol and sulphuric acid.e) Tests for Lignin Treat the sample with hydrochloric acid and Phloroglucinol.Tests for gingivaTreat the sample with thionine solution. After 15 min wash with alcoholTests for tanninsa) Ferric-Chloride Test Treat test solution with few drops of ferric chloride solution.b) colloidal mousse test To the test solution add 1 % jellyatin solution containing 10 % sodium chloride.Tests for proteinsa) Heat test Heat the test solution in boiling water bath.b) Biuret Test Test solution treated with Biuret reagent (40% sodi um hydroxide and dilute copper sulfate solution).c) Xanthoproteic test To the test solution, add 1 ml of conc. nitric acid and boil yellow set up is formed. After cooling it, add 40 % sodium hydroxide solution.d) Test for starch To the test solution, add weak aqueous one solution. Blue color indicates presence of starch, which disappears on heating and reappears on cooling.Effervescence producesLitmus paper turns blueGives reddish brown colored precipitateGives cream colored precipitateGives reddish brown colored precipitateGives yellow colored precipitateGives buff colored precipitateGives yellow colored precipitateWhite colored precipitateGives violet colorPurple to violet ring appears at the junction of two liquidsIf red cupric oxide is formedRose color is producedRed 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 formedcompared with precipitate of test AA rose pink to red color is produced in ammonical layer.A rose pink to red color is produced in ammonical layer.Red color producedPurple color is produced.Acetic acid layer shows blue colour.Violet colour producedGives yellow to orange colorGives blood red colorPaper shows green fluorescence.Reddish gallant colorStable froth (foam) formedBrown ring is formed at the junction of two layers,If speeding layer turns greenIf upper layer turns deep redRed color at lower layerYellow color at lower layerIt sinks at the bottomBrownish red color formedPink color formedMucilage turns violet red.Gives dark blue color honey oil color appearsPrecipitate formedProteins gets coagulatedGives violet colororange tree color formedBlue color, which disappears on heating and reappears on coolingAcidic compounds presentAcidic compounds presentAlkaloids presentAlkaloids presentAlkaloids pres entAlkaloids presentAlkaloids presentAlkaloids presentAmino acids presentAmino acids presentCarbohydrates presentMonosaccharides are present.Carbohydrates presentCarbohydrates presentCarbohydrates presentFlavonoids presentFlavonoids presentFlavonoids presentIf the precipitate in Test A is greater than in Test B then glycoside may be present.Anthraquinone glycosides presentAnthraquinone glycosides presentHydroxy anthraquinones presentcardiac glycosides presentCardiac glycosides presentCardiac glycosides presentCardiac glycosides presentCardiac glycosides presentCoumarins glycosides presentCynogentic glycosides presentSaponin glycosides yieldSteroids presentTriterpenoids presentSteroids presentTriterpenoids presentSteroids presentInulin PresentLignin PresentMucilage presentHydrolysable tanninsCondensed tanninsTannins presentProteins presentProteins presentProteins presentStarch presentFloroscence Analysis of various extractsPetroleum 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 dart 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 drugThe 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 analysisInstrument Shimadzu LC-20AT with UV/visible detector unmoving Phase Bonda- pack C-18 newspaper chromatography pillar with 250-4mmMobile Phase Methanol (80) weewee (20) signal detection wave length 350 nmFlow Rate 2 ml/min.HPLC Chrom ato gravitational constant is given in Fig. 3 and its retention time is given in Table no. 16HPTLC Analysis of sample drugThe chromatographic pattern of plant was obtained as per report with some modifications for which the HPTLC conditions are as follows.Extract Methanolic ExtractInstrument HPTLC (Camag, Switzerland)Stationary Phase pre-coated silica gel platesMobile Phase Ethyl acetate Formic acid Glacial acetic acid water (100051020)Spraying Reagent ingrained 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 principleCompound IThe 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 insulation of extract constituents was done by column chromatography. The clean and dried glass column was used. The si lica gel for column chromatography (60-120) was activated at 1100c.The column was filled with silica gel and ready 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 uncaring from the ethyl acetate fraction of methanol extract of the plant with following experimental conditions 73.Height of column 20 cmDiameter of column 3.5 cm.Stationary phase silicon oxide gel (60-120).Mobile phase Benzene Chloroform Ethyl acetate Methanol with variant ProportionsElution Gradient elution. fragment quantity 25 mlPreparative TLC20 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 echo on the plate. The plate was then developed. The different bands set-apart on the plate were scratched and recovered with methanol. Purity of dried sa mple was analyze 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 sour structure of the compound (Quercetin) is given in Fig. No. 8.Compound IIPetroleum 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 matterExperimentalHeight of column 25 cmDiameter of column 3.5 cm.Stationary phase Silica gel for column chromatography (60-120).Mobil e phase Benzene Ethyl acetateElution Gradient elution.Fraction quantity 30 mlFractions No. 24-27 were subjected for thin layer chromatography with following experimental conditions.Stationary phase Silica gel HMobile phase Ethyl acetate Benzene (1 9)Detection Vanilin-sulphuric acid reagentIdentification Whitish Purple colourFraction 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 Estimationsa) Estimation of Total carbohydrate contentThe adhesion of carbohydrate was done using the method acid base digestion.PrincipleIn hot acidic media glucose is conv erted to hydroxy methyl furfural by dehydration. This forms a green colour product with phenol.Procedure100mg 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. modify 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 25-30C 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 conte nt is given in Table no. 22 and Calibration curve of standard glucose dilutions are given in Fig. No. 13.Estimation of Bitterness economic valueThe rancor value of plant material was compared with diluted solution of Quinine hydrochloride. expression of Solutions breeding of Quinine hydrochloride solutionThe stock solution of 100g/ml was prepared from which a series of dilutions 42, 44, 46, 48, 50, 52, 54, 56 and 58 g/ml were prepared.Preparation of type PreparationForm the stack solution of 1000 g/ml, 100, 200, 300 and 400g/ml dilutions were prepared.MethodTasted 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 time 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 - AB - CWhere A= quantity of Quinine sulphate (mg) having high bitternessB= the concentration of stock solution (mg/ml)C= heap of sample in ml having higher bitternessResult pertaining to estimation of bitterness value is given in Table no. 22Total Phenolic contentThe 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.ChemicalsFolin- Ciocalteu reagent (Merck Co.)Gallic acid (Sigma Ltd., USA)Sodium carbonate (SISCO Research Laboratory Pvt. Ltd., Mumbai, India)Reagent preparationFolin-Ciocalteu (phenol) reagentThe reagent was prepared by diluting 1ml with 5ml of distilled water.Sodium carbonate15% solution was prepared in distilled water.Gallic acid solutionThe stock s olution was prepared by fade out 1mg gallic acid in 10ml of water from which different concentrations (20-100g/ml) were prepared.Sample preparationSample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100 g/ml) solution.Procedure0.1ml 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 g 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 ContentTotal flavonoid content of VCM was determined using method reported 79.