Determination of antioxidant activity by DPPH assay

The antioxidant activity of extracts was determined by DPPH assay using Blois's method with some modifications.15 Each extract was prepared in various concentrations. The extract (2 mL) was added to 2 mL of DPPH solution (50 μg/mL) to initiate the reaction for obtaining a calibration curve. The absorbance at 515 nm was measured after incubation for 30 min by using an ultraviolet (UV)–Vis spectrophotometer (Beckman Coulter DU 720). DPPH (50 μg/mL) was used as the control, ascorbic acid as the standard, and methanol as the blank. Analysis was conducted in triplicate for the standard and each extract. The antioxidant activity was revealed as IC₅₀ of DPPH scavenging activity by observing the 50% inhibitory concentration for each extract using the calibration curve.