2.6. 1,1-Diphenyl-2-picryl hydrazyl (DPPH) Assay

Free radical scavenging activity of 2-APB and its analogues was measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH; Sigma-Aldrich, St. Louis, MO, USA) as described previously [22] with small modifications. Briefly, 160 μL of DPPH (41.25 μM) in ethanol was mixed with 40 μL of various concentrations of indicated compounds in ethanol. The mixture was shaken vigorously, and then allowed to stand at RT for 30 min. The absorbance was measured at 517 nm using spectrophotometer (Multiskan EX; Thermo Scientific, Rockford, IL, USA). Ascorbic acid was used as a positive control for the DPPH assay. The percentage of DPPH scavenging effect was calculated by the formula (A₀ – A₁)/A₀ × 100%, where A₀ is the absorbance of control reaction and A₁ is the absorbance in presence of test or standard sample.