DPPH Antioxidant Assay Protocol

The DPPH assay was performed according to [58 (link)] by mixing 500 µL of the DPPH solution (Sigma Aldrich, St. Louis, MO, USA) with 500 µL of the antioxidant solution (PSC/HC/ ascorbic acid). The mixture was reacted for 30 min in the dark at room temperature, and absorbance was measured at λ = 517 nm using a UV-VIS spectrophotometer (Hitachi, Japan). All the experiments were performed in triplicate, and the scavenging activity (%) was calculated as 100% × (As−Ax)/Ac, where As is the absorbance of the sample + DPPH, Ax is the absorbance of the sample + DPPH solvent, and Ac is the absorbance of the DPPH solvent + DPPH. The IC50 value expresses antioxidant activity, representing the concentration of the test sample that provides a 50% reduction of free radicals from the equation generated by linear regression of percent inhibition.