2.3. 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) Radical Scavenging Assay

The 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging assay was conducted following the description by Okoh et al. using vitamin C as the positive control [28]. Here, 0.1 mL of 0.135 mM DPPH in methanol was mixed with 1.0 mL of the solution prepared in methanol containing 0.025–0.50 mg/mL of DMLEs, CGA, rutin, and vitamin C. The reaction mixture was vortexed thoroughly, left in the dark at 25°C for 30 min, and measured at 517 nm using a spectrophotometer. The DPPH radical scavenging effect was calculated using the equation below:

Here, A_c is the absorbance of the control reacted with methanol (50 μL) and DPPH working solution (1 mL); and A_s is the absorbance of the samples.

The sample concentration required for inhibiting 50% DPPH radicals (IC₅₀ DPPH values) was obtained by extrapolating the regression analysis. Antioxidant activity was evaluated based on this IC₅₀ value.