DPPH Radical Scavenging Assay Protocol

DPPH radical scavenging assay was performed according to the previously described method with minor modification [3 (link),15 (link)]. The samples and a positive control, ascorbic acid, were dissolved in DMSO with final concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL. DPPH was dissolved in anhydrous ethanol (EtOH) with a concentration of 0.04 mg/mL. Tested samples (50 µL) were added to 50 µL of fresh DPPH, then kept in room temperature in the dark for 30 min. The optical density (OD) was measured by an AMR-100 microplate reader (Hangzhou Allsheng Instruments, Hangzhou, China) at 517 nm. The EtOH and DMSO were used as a blank and negative control, respectively. The IC₅₀ values were determined by the software of GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) [3 (link)].