The plants namely Persicaria hydropiper were collected from Village: Naya Dingi, Thana: Doulatpur, District: Manikgong, Bangladesh and the plants namely Andrographis paniculata, Hemidesmus indicus and Myristica fragrans were collected from Jahangirnagar University, Savar, Dhaka area, Bangladesh in March 2009 and were identified at the Bangladesh National Herbarium, Mirpur, Dhaka where the Voucher specimens have been deposited for future reference.
Drugs and Chemicals Ascorbic acid & sodium nitroprusside, sodium phosphate, sulphanilamide, phosphoric acid, naphthyl ethelene diamine were obtained from SD Fine Chem. Ltd. India, ammonium molybdate from Merck, Germany, ferric chloride & neocuproine were obtained from Sigma Chemical Co. USA.
Preparation of extracts The collected and identified plant parts were dried (after cutting and slicing where necessary) in the sun and finally in a mechanical dryer at 60 – 700C. The dried samples were ground to coarse powder with a Grinding Mill and powdered samples were kept in clean closed glass containers pending extraction. During grinding of sample, the grinder was thoroughly cleaned to avoid contamination with any remnant of previously ground material or other foreign matter deposited on the grinder. Then hundred (100) grams of dry powder of each plant were taken in each separate glass jar and soaked in 1 litre of 97% methanol for 3-5 days with intermittent shaking. At the end of extraction, it was passed through Whatman filter paper No.1 (Whatman Ltd., England). These methanolic filtrates were concentrated at 50ºC under reduced pressure using vacuum pump rotary evaporator (STUART RF3022C, UK) to obtain the extracts.
Phytochemical screening: Phytochemical screening of the prepared extracts was conducted with various qualitative tests to identify the presence of chemical constituents. To perform the tests the following chemicals and reagents were used: Carbohydrates with Molisch’s test, glycoside with water and sodium hydroxide solution, saponins with the capability of producing suds, steroids with chloroform and sulphuric acid, flavonoids with Mg and HCl, tannins with ferric chloride solution. Alkaloids were tested with Mayer’s reagent, Hager’s reagent and Dagendorff’s reagent. These were identified by characteristic color changes using standard procedures.
Determination of Total Phenol The content of total phenolic compounds in plant methanolic extracts was determined by Folin–Ciocalteu Reagent (FCR) using UV spectrophotometer (UV–1501PC SHIMADZU, Japan) described by the method McDonald et al. 0.5 ml of diluted plant extract and standard of different concentrations solution were taken in the test tube followed by adding 5 ml of Folin – ciocalteu (Diluted 10 fold with water) & 4 ml of Sodium carbonate (1 M) respectively. Solutions were then incubated for 15 minutes at 450C in the water bath. The absorbance was measured colorimetrically at 765 nm to determine the total phenol content. Various concentrations of gallic acid (25, 50, 100, 200 µg/ml) were used to prepare the standard curve and the total content of phenolic compounds in the crude extracts was calculated according to the following formula:
C = (c x V)/m where C is the total content of phenolic compounds in mg/g plant extract; c is the concentration of gallic acid established from the calibration curve in mg/ ml; V is the volume of extract in ml and m = the weight of methanolic extract in g. The value of total content of phenolic compounds is expressed as GAE (Gallic Acid Equivalent) in mg/g extract.
Determination of Flavonoid Content The total flavonoid in the crude methanolic extract was measured using the Aluminum Chloride Colorimetric Method as described by Chang et al. To 1 ml of plant extract or standard of different concentrations 3 ml methanol, 0.2 ml of 10% aluminium chloride, 0.2 ml potassium acetate (1M) and 5.6 ml of distilled water were added. Then the solution was incubated for 30 minutes at room temperature. The absorbance was measured at 415 nm using UV spectrophotometer against a blank. Standard curve was prepared using quercetin by dissolving it in methanol followed by serial dilution to 25, 50, 100, 200 µg/ml.
Total Antioxidant CapacityAssessment The determination of antioxidant capacity in the plant extract was assessed by the phosphomolybdenum method as described by Prieto et al. The method is based on the reduction of Mo (VI)–Mo (V) by means of the extract followed by the formation of a green phosphate/Mo (V) complex at acid pH. A 0.3 ml of crude methanol extract and 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) were mixed together. The resulting solution was incubated at 950C for 90 min. Then the absorbance of the solution was measured at 695 nm using UV spectrophotometer against a blank after cooling to room temperature. Methanol (0.3 ml) instead of plant extract is used as the blank. The antioxidant activity is expressed as the number of equivalents of ascorbic acid (AAE) and was calculated by the following formula/ equation:
A = (c x V)/m Where A is the total content of antioxidant compounds, mg/g plant extract, in Ascorbic acid; c represents the concentration of Ascorbic acid established from the calibration curve, mg/ml; V is the volume of extract in ml and m is the weight of pure plant methanolic extracting.