Antioxidant activity assay

Antioxidant activity of leaf and bark extracts were estimated by using DPPH free radical scavenging bioassay that is described as Brand-Williams et al. with a little changes [32]. Based on this protocol, stock solution of experimental extracts (1 mg/ml concentration) and methanolic solution of DPPH (1 mg/25 ml concentration) were prepared. Then, ten different concentrations (25, 50, 75, 100, 125, 150, 175, 200, 225 and 250 µg/ml) of stock solution were taken into ten different test tubes and made volume up to 1 ml by adding absolute methanol. After that, 1.5 ml of DPPH solution was added in each test tube and kept them in a dark place at room temperature to complete the reaction. After 30 min, the optical density of each solution was measured by using a spectrophotometer (GENESYS 10S UV–Vis, Thermo Scientific, USA) at 517 nm. The BHT, methanol, and DPPH solution were used as standard, blank, and control respectively. The scavenging percentage of DPPH was evaluated by using the following formula:

where A₀ = absorbance of the control; A₁ = absorbance of the leaf and bark extracts.

The 50% inhibition concentration (IC₅₀) values was calculated by using regression line of gained scavenging percentages and corresponding concentrations.