DPPH Radical Scavenging Assay Protocol

The DPPH radical scavenging assay (RSC) was performed according to a previously described method [42 (link)]. Briefly, 1 mL of freshly prepared ethanolic solution of DPPH radical (100 μM) was mixed with the tested extract solution in EtOH at various concentrations (0.5, 1.0, 2.0, and 4.0 mg/mL). The mixture was then vortexed and incubated at room temperature in the dark for 30 min, followed by absorbance measurement at 517 nm on the reader Infinite^® 200 PRO series (Tecan Group Ltd., Männedorf, Switzerland). Experiments were performed in triplicate for each sample and twice in total. Gallic acid was used as a positive control at a concentration of 30 μM. A negative control with 10 μL DMSO and 190 μL DPPH was performed each time. The blank solution contained 190 μL EtOH and 10 μL sample. The radical scavenging activity percentage (AA%) was calculated as follows: AA% = [1 − ((A_sample − A_blank)/A_control)] × 100, where A_control is the absorbance of the negative control, A_sample is the absorbance after the reaction of samples with DPPH, and A_blank is the absorbance of sample with EtOH instead of DPPH. Moreover, the IC₅₀ value indicating the extract amount that caused 50% scavenging of the DPPH radical was calculated.