4.3. In Vitro DPPH• Assay

The free radical scavenging activity of the 4-hydroxycoumarin was measured using the stable DPPH• radical, according to Blois’s method [35]. Briefly, 3 mL of each sample solution was prepared in methanol at different concentrations (150–750 µg/mL) and was added, individually, 1 mL of DPPH• solution (0.1 mM). The mixture was shaken vigorously and left in the dark for 30 min. Then, the absorbance was measured in a Spectrophotometer (Thermo Scientific™ GENESYS 10S UV-Vis, Waltham, MA, USA) at 517 nm. BHT was used as reference in the experimental assay. This procedure was repeated three times for reproducibility. The capability to scavenge the DPPH• radical was expressed as IC₅₀ (concentration of antioxidant that produces 50% of absorbance inhibition).