3.7. Antioxidant Capacity Determinations

The free radical-scavenging potential of the extract was determined using the Brand–Williams methods with minor modification [53]. Three milliliters of freshly prepared methanol DPPH solution (6 × 10⁻⁵ M) were added to 100 μL of each sample separately and the mixtures incubated in dark for 15 min at room temperature, and then the sample absorption (A sample) was recorded at 515 nm. A blank sample absorbance (A blank) containing 100 μL of methanol was also measured. Vitamin C was used as positive controls and methanol as negative control. The triplicate experiment was executed and their scavenging capacity was determined using the following equation:

where A blank = absorbance of the blank, and A sample = absorbance of the sample extract.

The ABTS radical scavenging potential was determined on the basis of measuring the degree of absorbance reduction of the radical ABTS+• ion by the tested sample extracts [43]. In addition, 10 μL of sample extract and 20 μL solution of peroxidase were mixed with 170 μL solution of ABTS and allowed to react for 6 min in the dark, after which the absorption was measured using a 405 nm UV-visible spectrophotometer (A sample). By mixing 10 μL of distilled water with 20 μL solution of peroxidase and 170 μL solution of ABTS, a blank sample was prepared, and the absorbance of the blank sample was measured (A blank). Vitamin C was used as positive controls and methanol as negative. Determinations were performed in triplicate. The ABTS+• inhibition percentage was determined using the above Equation (1).

Ferric reduction capability of extracts was determined using a modified version of the FRAP assay [54]. By adding 20 mL of acetate buffer (300 mM, pH 3.6,) and 2 mL of TPTZ (10 mM) in 40 mM hydrochloric acid and 2 mL of ferric chloride (20 mM), the working solution of the FRAP reagent was prepared. Then, the mixture was allowed to incubate at 37 °C for 30 min in a water bath. In addition, 180 μL of FRAP reagent were added to 5 μL of sample extract, and the sample absorption was estimated at 593 nm. Absorbance of a blank without a sample extract was also measured, and analyses were executed in triplicate. In order to measure the FRAP value, the difference was calculated between the sample absorbance and the blank absorbance. On the day of preparation, all solutions were used. A standard curve of ferrous sulphate (0.15–1.5 mmol/L) was used, and the results were reported in mmol ferrous ion equivalents per gram dry weight of the sample (Fe²⁺/g DW).