Stem Barks of Bauhinia Acuminata | Analysis

Stem Barks of Bauhinia Acuminata epitomeMaterials and MethodsPlant collectionThe stem barks of Bauhinia acuminata L. were collected from Rajshahi university campus, shungladesh, in the month of September, 2013. The plant was au accordinglyticated by a taxonomist of Department of Botany, University of Rajshahi. A voucher specimen (Voucher No. MN-13) was deposited to the herbarium in the Department of Botany, University of Rajshahi. The stem barks were then washed separately with fresh water to remove dirty materials and were shade dried for several days with occasional sun drying. The dried barks were then grunge into coarse powder by grinding machine and the materials were stored in dark at room temperature for future use.Extract preparationThe stateion was performed according to order described by Alam el al. (Alam et al., 2002). About 400 gm of dried powdered stem bark were taken in an amber colored reagent bottleful (2.5-liter capacity) and the materials were soaked in 2.0 l iter of coulomb% methanol. The bottle with its sums were sealed and kept for a period of about 7 days with occasional trembling and stirring. The whole commixture was then filtered through cotton and then through Whatman No.1 filters paper and were concentrated with a rotary evaporator (Bibby Sterlin Ltd, UK) under reduced pressure at 45C temperature to afford crude extract of the bark. The crude extract was assorted with 90% methanol in water to obtain a slurry of satisfying volume of 100ml. The slurry was taken in a separating funnel and added equal amount of antithetical partitioning solvent sequentially as like n-hexane, chloroform, ethyl acetate and finally water. The funnel was shaken vigorously and allowed to stand for a few minutes for separation of the compounds according to their partition co-efficient and thus extracts were prepared for the experiment purpose. The butt on was repeated three times. At last, the different segmentated parts were evaporated using rot ary evaporator at 400C to obtain n-hexane fraction (NHF, 1.12 gm), chloroform fraction (CHF, 1.33 gm), ethyl acetate fraction (EAF, 2.27 gm) and aqueous fraction (AQF, 8.86 gm) respectively. chemicals1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium ferricyanide, catechin (CA), ferrous ammonium sulphate, butylated hydroxytoluene (BHT), gallic acid (GA), ascorbic acid (AA), AlCl3, trichloro acetic acid (TCA), sodium phosphate, sodium nitrate, ammonium molybdate, 2-deoxy ribose, sodium hydroxide, EDTA and FeCl3 were purchased from Sigma Chemical Co. (St. Louis, MO, USA) potassium acetate, phosphate buffer, thiobarbituric acid(TBA), HCl, H2SO4, H2O2were purchased from Sigma-Aldrich, vinblastine sulphate (VBS) from Cipla India, folin-ciocalteuss phenol reagent and sodium carbonate were obtained from Merck (Dam-stadt, Germany).Determination of total phenolicsTotal phenolic contents in the extracts were determined by the Folin-Ciocalteu method described by Singleton et al. (Slinkard Single ton, 1977). 40 l of the extract/fractions (250g/ml) were taken in test tubes and to each 3.16 ml of water was added to make up the volume 3.2ml. 200 l of folin-Ciocalteu (Undiluted) reagent etymon was added into the test tubes and kept for 5-8min. 600 l of sodium carbonate (20%) solution was added into the test tubes and shake to mix. The test tubes were incubated for 2 hours at 20C to complete the reception. Then the absorbance of the solution was measured at 765 nm using a spectrophotometer (Shimadzu, USA) against blank solution. A typical blank solution contains all reagents except plant extract or standard solution. Standard Gallic acid solutions (50-250g /ml) concentrations were also treated as above. The total content of phenolic compounds in plant methanol extract and in different fractions was expressed as Gallic acid equivalent (GAE)/gm of dry extract in respect to standard gallic acid curve equation (y = 0.0008x 0.005, R = 0.975).Determination of total flavonoidsTotal f lavonoids were estimated using aluminum chloride colorimetric arrest described by Zhishen et al. (Zhishen, Mengcheng, Jianming, 1999). To 0.5 ml of samples/standard, 150 l of 5% sodium nitrate and 2.5 ml of distilled water were added. After 5 min, 0.3 ml of 10% AlCl3 was added. At 6 min, 1 ml of 0.001M NaOH and 0.55 ml distilled water was added to the mixture and left at RT for 15 min. Absorbance of the mixtures was measured at 510 nm. Total flavonoid contents were expressed in terms of catechin equivalent, CAE /gm of dry extract in respect to standard curve equation (y = 0.0178x+0.0524, R = 0.9862).Determination of free motif scavenging activityDPPH radical scavenging activityFree radical scavenging ability of the extracts was tested by DPPH radical scavenging assay (DRSA) as described by Braca et al. (Braca et al., 2001). Aliquots of 2.5mL of methanolic solution containing sample at different concentration was mixed with 2.5 ml of 0.008% DPPH solution in methanol. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 minutes. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as reference. Percentage DPPH radical scavenging activity (% DRSA) was calculated by the following equation,% DRSA = (A0-A1/A0) x 100Where,A0 = Absorbance of control andA1 = Absorbance of sample.IC50 set denote the concentration of sample required to scavenge 50% DPPH free radicals.Hydrogen peroxide scavenging activityHydrogen peroxide scavenging activity of extract was assessed by the method described by Zhang et al. (Zhang He). Aliquot of 1.0 ml of 0.1mmol/L H2O2 and 1.0 ml of various concentrations of extracts were mixed. Followed by 2 drops of 3% ammonium molybdate, 10 ml of 2M H2SO4 and 7.0 ml of 1.8 mol/L KI. The mixed solution was titrated with 5.09mmol/L Na2S2O3 until yellow color disappeared. The extent of scavenging of hydrogen peroxide was calculated as% scavenging of hydrogen peroxid e = (V0-V1) / V0 - 100Where,V0 = Volume of Na2S2O3 solution used to titrate the control sample in the front man of hydrogen peroxide (without sample),V1 = Volume of Na2S2O3 solution used in the presence of samples.Hydroxyl radical scavenging activityHydroxyl radical scavenging activity (HRSA) of the extracts was determined by the method of Halliwell et al. (Halliwell, Gutteridge, Aruoma, 1987) with a slight modification. 100 l of various concentrations of extracts were mixed with 100 l of hydrogen peroxide (10mmol/L). To this 200 l premixed FeCl3 (100mmol/L) and EDTA (100mmol/L) solution (11v/v) was added. Followed by addition of 500 l of 2.8mmol/L 2-deoxyribose in phosphate buffer (PH7.4) and finally the reaction was triggered by adding 100 l ascorbate (300mmol/L). Then the reaction mixture was incubated at 37.5C for 1 hour. To the above reaction mixture 2 ml of TCA (2.8% w/v aqueous solution) and 2 ml of TBA (1% w/v aqueous solution) was added. The final reaction mixture was he t for 15min in boiling water bath, cooled and absorbance was taken at 532nm using a spectrophotometer.The hydroxyl radical radical scavenging capacity was evaluated with the inhibition percentage of 2-deoxyribose oxidation on hydroxyl radicals. The percentage of hydroxyl radical scavenging (%HRSA) activity was calculated according to the following formula% HRSA = A0 (A1-A2 -100/A0where A0 is the absorbance of the control without a sample. A1 is the absorbance after adding the sample and 2-deoxyribose. A2 is the absorbance of the sample without 2-deoxyribose.REFERENCESAlam, A., Rahman, M., Baki, M., Rashid, M., Bhuyan, M., Sadik, G. (2002). Antidiarrhoeal principle of Achyranthes ferruginea Roxb. and their cytotoxicity. Ban Pharm J, 12, 1-4.Braca, A., De Tommasi, N., Di Bari, L., Pizza, C., Politi, M., Morelli, I. (2001). 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