2.1.4. DPPH assay

Radical-scavenging properties of polysubstituted 3-hydroxy-3-pyrroline-2-ones were evaluated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.^44–48 DPPH solution (1 mM) was prepared in methanol and solutions of each polysubstituted 3-hydroxy-3-pyrroline-2-one were prepared in DMSO at various concentrations (128, 32 and 8 µg mL⁻¹). Then, 200 µL DPPH solution was added to 1.28 µL samples at each tested concentration and the free radical scavenging reactions were carried out on a 96-well plate at 37 °C for 30 minutes. The absorbance was measured at 517 nm wavelength by a BioTek Epoch 2 Microplate Spectrophotometer. The percentage of free radical scavenging was calculated as SP(%) = [(OD₀ − OD₁)/OD₀] × 100, where OD₀ was defined as the final absorbance of the control reaction with quercetin as the reference antioxidant, and OD₁ stands for the absorbance in the presence of the sample. Each experiment was repeated three times and quercetin was used as the positive control.