3.7. Preparation of Antioxidant Activities Tests

The free-radical-scavenging activity of the extracts of A. seyal was measured using an improved DPPH assay [38]. The extract solution with concentration of 0.3 mL was mixed with a solution of 0.2 mmol/L DPPH in methanol (2.7 mL). The mixture was mixed vigorously and then left to stand for 1 h at room temperature before measuring the absorbance value at 517 nm. The percent of inhibition rate of radical scavenging activity was calculated using the following Equation (2):

where “As” is the absorbance of DPPH alone, and “Ai” is the absorbance of DPPH in the presence of various extracts. The concentrations of Trolox and Vitamin C (VC) identical to the experimental samples were used as reference.

The ability of the extract to scavenge ABTS radical was determined according to a previously published method [39]. ABTS was dissolved in deionized water at 7 mmol/L concentration, and potassium persulfate with a concentration of 2.45 mmol/L was added afterward. The reaction mixture was kept in the dark at room temperature for 16 h. The mixture was then diluted with 80% ethanol to obtain an absorbance value of 0.700 ± 0.005 at 734 nm. Test substances (0.3 mL) at various concentrations were incubated with ABTS + solution (2.7 mL) in a 30 1C water bath for 30 min in the dark. The absorbance at 734 nm was immediately recorded. Samples of BHT and VC at the same concentrations were used as references. The level of radical scavenging was calculated using the aforementioned equation for DPPH.

The FRAP assay was carried out according to [40] with slight modification. Briefly, the FRAP reagent was prepared from acetate buffer (pH 3.6), 10 mmol TPTZ solution in 40 mmol HCl and 20 mmol iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. The FRAP reagent was prepared fresh daily and was warmed to 37 C in a water bath prior to use. Fifty microliters of sample were added to 1.5 ml of the FRAP reagent. The absorbance of the reaction mixture was then recorded at 593 nm after 4 min. The standard curve was constructed using Vitamin C solution (0.065–33.3 µg/ml), and the results were expressed as µmol VCE/g dry weight of plant material. All the measurements were taken in triplicate and the mean values were calculated.