2.1 Sample Collection Fresh leaves of Spondias pinnata plant were collected from a farmland area in Faridpur, Bangladesh. Authentication was done at Bangladesh National Herbarium, where voucher specimen (Accession No.35357) was deposited.
2.2 Extract Preparation The leaves were separated from undesirable materials and washed thoroughly with distilled water to remove debris and dust particles. Cleaned leaves were air-dried under shade at room temperature to constant weight. Dried samples were then powdered using a laboratory scale mill and blender. Ground material (100 g) was extracted independently with 500 ml of hexane, ethyl acetate and ethanol. The extraction was done for 5 days with occasional shaking. The obtained extracts were filtered using filter paper (Whatman No. 1) and each filtrate was then evaporated to dryness in a rotary evaporator. All the extracts were kept stored at 4ºC in a refrigerator until required for further analyses.
2.3 Phytochemical Screening To identify the chemical constituents present in the extracts, standard phytochemical screening was carried out. Alkaloids test was performed with Wagner’s reagent, flavonoids with conc. HCl, gum and carbohydrate with Molisch’s reagent, saponin by frothing test, tannins by FeCl3 and terpenoids by Salkowski test.
2.4 Determination of Total Phenolic Content: Total phenolic content (TPC) in the extracts was determined spectrophotometrically according to the Folin–Ciocalteu procedure. Briefly, 1.5 ml Folin–Ciocalteu’s reagent (diluted 1:10) and 1.2 ml of 7.5% (w/v) Na2CO3 were added to 0.3 ml of the extract. After incubation at room temperature for 1 h in dark, the absorbance was measured at 765 nm against blank. The total phenolic content was evaluated from a gallic acid standard curve and the results were expressed as gallic acid equivalents (GAE) in milligrams per gram extract.
2.5 Determination of Total Flavonoid Content: Total flavonoid content of the extracts was determined by aluminum chloride colorimetric method [19]. 1.5 ml of methanol, 0.1 ml of aluminium chloride (10%), 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water were mixed with 0.5 ml of extract solution. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with spectrophotometer. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. Quercetin was used to make the calibration curve and the results were expressed as quercetin equivalents (QE) in milligrams per gram extract.
2.6 Antioxidant Activity
2.6.1 DPPH radical scavenging assay The free radical scavenging activity of the extracts was measured in terms of hydrogen donating or radical scavenging ability of the stable 1, 1-diphenyl-2- picrylhydrazyl (DPPH) free radical [20]. Briefly, 0.1 ml of plant extract at various concentrations (0-200 µg/ml) was added to 3 ml of a 0.002% methanolic solution of DPPH. The reaction mixtures were incubated for 30 min at room temperature and the absorbance at 517 nm was measured against a blank. A low absorbance of the reaction mixture indicated a high free radical scavenging activity. Ascorbic acid was used as standard in the experiment. The radical scavenging activity was calculated using the following formula: Percentage of inhibition = [(Abscontrol - Abssample) / Abscontrol ] x 100
2.6.2 Nitric oxide radical scavenging activity Nitric oxide radicals generated in aqueous sodium nitroprusside solution at physiological pH interact with oxygen to produce nitrite ions, which were measured by the Griess Illosvoy reaction. Briefly, 3 ml of the reaction mixture containing 10 mM sodium nitroprusside and the extract solutions (0 –200 μg/ml) in phosphate buffer were incubated at 25°C for 150 min. This was followed by addition of 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) to 0.5 ml of the incubated solution. The mixture was allowed to stand for 5 min followed by mixing with 1 ml of 0.1% naphthyl ethylene diamine dihydrochloride (NED) and incubation at 25°C for 30 min. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with NED was measured spectrophotometrically at 540 nm against the corresponding blank solutions. Ascorbic acid was used as a standard. The ability to scavenge the nitric oxide radical is expressed as % inhibition and calculated using the following equation: Percentage of inhibition = [(Abscontrol - Abssample) / Abscontrol ] x 100
2.6.3 Scavenging of superoxide anions The superoxide anion radical scavenging activity of the extracts was assayed by the inhibition of nitro blue tetrazolium (NBT) reduction by NADH in the presence of phenazine methosulfate (PMS) [22]. Reaction mixtures containing 73 mM NADH, 15 mM PMS, 50 mM NBT in 20 mM phosphate buffer, pH 7.4 and the samples at various concentrations were incubated for 5 min at room temperature. Finally, absorbance at 560 nm was measured against blank samples. Quercetin was used as a positive control. Decreased absorbance of the reaction mixture indicates increased superoxide radical scavenging activity. The % inhibition of superoxide radical generation was calculated using the following formula: Percentage of inhibition = [(Abscontrol - Abssample) / Abscontrol ] x 100
2.6.4 Total reducing power Total reducing capacity of the prepared extracts was determined according to the method of Oyaizu. Briefly, 1ml of the extract at different concentrations was added with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% (w/v) solution of potassium ferricyanide. The resulting mixtures were incubated in a water bath at 50°C for 20 min followed by the addition of 2.5 ml of trichloroacetic acid (10% w/v). The mixture was centrifuged at 3000 rpm for 10 min. A 2.5 ml aliquot of the upper layer was combined with 2.5 ml of distilled water and 0.5 ml of a 0.1% (w/v) solution of ferric chloride. The absorbance was measured at 700 nm with a spectrophotometer. Ascorbic acid was used as positive control. The higher the absorbance of the reaction mixture the greater is the reducing power. All tests were carried out in triplicates.