4.7. Analysis of DPPH Free Radical Scavenging Potential

Determination of antioxidant activity of tested compounds was assessed with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay. DPPH radical solution (7.1 × 10⁻⁵ M) in methanol was freshly prepared prior to the experiment. In a 96-well plate, 20 μL of extracts, eugenol, and ergosterol (all dissolved in methanol; concentration of eugenol and extracts 1 and 2 was equal to 0.039% v/v; concertation of ergosterol, trolox, and H₂O₂ was 400, 500 μg/mL, and 0.0125% v/v, respectively) were added to the wells, followed by the addition of 180 μL of DPPH solution. Subsequently, the plate was incubated for 90 min (37 °C, 200 rpm), and at specific time points (0, 10, 60, and 90 min), absorbance at λ = 517 nm was recorded using the Spark multimode microplate reader (Tecan, Männedorf, Switzerland). To determine the scavenging activity of compounds against DPPH radicals, the reduction in absorbance at 517 nm was monitored (with methanol absorbance subtracted as background), and the percentage inhibition was calculated according to the control (DPPH alone). All the measurements were performed in three independent replicates.