2.4.3. DPPH Radical Scavenging Activity

DPPH (2,2-DiPhenyl-1-PicrylHydrazyl, Sigma USA) radical scavenging activity was determined as recommended by Sharma and Bhat (2009). Extracts were first dissolved in 70% methanol and centrifuged at 2500× *g* for 5 min. Then, 150 μL of the supernatant was collected and mixed with 50 μL DPPH in methanol. The blank contained 150 μL of 70% ethanol solution instead of supernatant, while the control was prepared with 50 μL of 70% ethanol instead of DPPH solution. L-ascorbic acid was used as reference standard. Absorbance (A) was measured for 30 min at 520 nm with a microplate reader (POLARstar Optima BMG labtech, O ffenburg, Germany). The scavenging e ffect is expressed as:

#### [(Ablank - Acontrol) − (Asample − Acontrol)]/(Ablank−Acontrol) × 100

Increasing concentrations of extracts were used to construct a linear curve, calculated plotting extract concentrations against percentage scavenging e ffects, and deduce the half maximal inhibitory concentration (IC50) of the extracts: the concentration that was able to quench 50% of the DPPH radical.
