Scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals
This assay was performed by following the standard method of Bloise (1958) with several modifications. DPPH solution was prepared by dissolving 5 mg DPPH powder in 2 ml of ethanol, sealed in aluminium foil and kept in a fridge. Accurately 100 µl of test samples (0.6-20.0 mg/ml) in ethanol was added with 5 µl DPPH solution in 96-well microtitter plates. Mixture was incubated in the dark for 30 min. Absorbance was measured at 517 nm (SpectraMax Plus384, United States) and IC50value (concentrations of each sample required to give 50% of the optical density shown by control) was calculated. Inhibition of DPPH free radicals was calculated by using following formula: