2.6. Determination of antioxidant activity by DPPH and Superoxide radical scavenging assays

DPPH and superoxide free radical scavenging efficacies of AEFC was assesed as per the standard protocols described by Kumar et al. [20]. The DPPH radical assay system contained 187 μL DPPH reagent (0.3 mM in methanol) and different concentrations of extract (10–100 μg/mL) in a final volume of 1 mL. The solution was immediately mixed and incubated at 37 °C for 20 min in dark condition. The reduction in absorbance of the test and experimental tubes was measured using UV/Vis spectrophotometer at 517 nm. DPPH solution (0.3 mM) was taken as blank. The percentage (%) radical scavenging was calculated by the formula.

Where Ac = Absorbance of control at 517 nm; As = Absorbance of the sample.

Superoxide scavenging activity of AEFC was determined by Chun et al. [21] with slight modifications. The superoxide radical scavenging assay system contained Nitro blue tetrazolium (0.18 mM), riboflavin (0.12μM), NaCN/EDTA (0.3 mM NaCN in 0.1 M EDTA), phosphate buffer (0.06 M, pH 7.8) and the drug at different concentrations (10–100 μg/mL) in a volume of 3 mL. The tubes were illuminated for 15 min. The absorbance at 560 nm was measured before and following illumination for 15 min. The % inhibition and IC₅₀ was calculated according to the formula;

Where Ac-Absorbance of control, As- Absorbance of the test. Unit activity of SOD is referred to as the amount of enzyme required to produce 50% inhibition of photo reduction of NBT.