2.5. DPPH Radical Scavenging Assay

The stable free radical DPPH•, shows a deep violet color in solutions and has a strong absorption at 517 nm. When an odd electron is paired off by an antioxidant, the deep violet color disappears. The decrease in absorption is a measure for antioxidant activity [20]. The procedure was modified from Brand-Williams et al. [21]. In a 96-well plate, 100 μL of 0.2 mM DPPH• in methanol was added to 100 μL serial-diluted plant extracts and allowed to react for 30 min in darkness at ambient temperature. Ascorbic acid and EGCG were used as positive controls. The absorption was read spectrophotometrically at 517 nm with a Tecan Nano Quant infinite M200 PRO Plate Reader (Tecan, Männedorf, Switzerland). Results are expressed as EC₅₀ (the concentration where 50% of the DPPH radical is inhibited). The calculation equation is:

where AB and AE are the absorptions in the absence and presence, respectively, of antioxidant substances (plant extracts).