5 The leaves, dried at room temperature, were grounded to fine powder and stored at 4 °C for further

analysis. Dried leaf powder (10 g) was mixed with 25 ml methanol (ME), ethyl acetate (EA), n-butanol (n-B), acetone/water (AW) (3:2) and water (aqueous/WE), separately. The leaf extract was stirred continuously for 24 h and then filtered. The filtrate was centrifuged at 10,000 rpm for 10 min and the supernatant, was stored at 4 °C prior to use (within 2 days). Total phenolic and flavonoid contents were determined by Folin–Ciocalteu’s and aluminum chloride calorimetric methods, click here respectively6 and 7 following quantification on the basis of standard curve of gallic acid and quercetin. Results are presented in milligrams (mg) gallic acid (GAE) and quercetin (QE) equivalent, respectively, per gram of leaf sample on dry weight basis. Total antioxidant activity was measured by ABTS, DPPH and FRAP assays following methods of Cai et al8 and Amarowicz et al9 and 10 Standard curve of a range of concentrations of ascorbic acid was prepared for

quantification of antioxidant potential. Results were expressed in milligram (mg) ascorbic acid equivalent (AAE) per gram of leaf sample on dry weight basis. Determination of total phenolic and flavonoid contents and antioxidant CP-673451 mouse capacity by ABTS, DPPH and reducing power assay was conducted in triplicates. The value for each sample was calculated as the mean ± SD. Factorial analysis of variance and significant difference among means were tested by two way ANOVA in replication. Correlation coefficients were calculated using Microsoft Excel 2007. Significant variations (p < 0.05) were observed in phytochemicals and antioxidants in leaf extracts of different

locations in different solvents. In ME and AW, GB2 gave higher phenolic content, while lower values were recorded in EA extracts of GB3 and GB4, respectively. In WE, maximum content was for GB4 and minimum for GB1. GB3 gave Mephenoxalone maximum value for n-B and GB5 for EA for total phenolic content ( Fig. 1A). Total flavonoids were higher in GB3 in ME and n-B, respectively, in comparison to GB2 and GB4. Higher flavonoid content was in EA for GB4 and in WE for GB5 ( Fig. 1A). Antioxidant activity in ABTS was higher in ME and WE for GB2, respectively. Subsequently, GB1 gave higher antioxidant activity in EA and AW, respectively, while GB3 showed maximum antioxidants in n-B. Based on DPPH assay, GB3 exhibited highest values for antioxidants in n-B, AW and WE, respectively. For GB1 and GB5, highest values were recorded in EA and ME, respectively. In FRAP assay, GB5 showed higher activity in AW and WE, respectively; GB3 in n-B; GB2 in EA and GB1 in ME ( Fig. 1B). Variations in phytochemicals arise due to the specific environmental conditions, including both biotic and abiotic.