DPPH Radical Scavenging Assay Protocol

The DPPH radical scavenging assay was carried out using an EL800 microplate reader (Bio-Tek Instruments, Winooski, VT, USA) [56 (link)]. Briefly, 180 μL of freshly prepared DPPH solution (0.32 mM in methanol) was mixed with 20 μL of sample (dissolved in methanol, extract 250 µg/mL; compounds 62.5–1000 µM) in a 96-well plate. The mixture was incubated for 20 min in the dark at 25 °C, and then, the absorbance was measured at 570 nm. Trolox was used as a positive control, which showed a linear radical inhibition curve within final concentrations of 1.56 µg/mL (6.25 µM) and 25 µg/mL (100 µM). Results are expressed in µg Trolox equivalents/µg extract or µM Trolox equivalents/µM compound. The DPPH radical scavenging activity (%) is calculated using Equation (1): $\%inhibition=\left(1-\frac{A_{sample}-A_{blank1}}{A_{control}-A_{blank2}}\right)\times 100\%,$
where A_sample is the absorbance of DPPH solution with a sample, A_blank1 is the absorbance of the test sample without DPPH, A_control is the absorbance of DPPH solution without a sample, A_blank2 is the absorbance of methanol, without DPPH or a sample.