2.8. Antioxidant activities

A protocol reported by [22] was slightly modified to evaluate the DPPH radical scavenging assay. Briefly, 20 μL of the diluted samples was added to 135 μL of 0.1 mM DPPH solution and kept in a dark place at room temperature for 30 min. The absorbance of the mixture was determined at 517 nm. Trolox was used as a standard and the values were presented as mg of Trolox equivalent antioxidant capacity (TEAC) per gram of sample. The radical scavenging activity of the DPPH assay was evaluated as follows:

DPPH radical scavenging activity (%) = [(absorbance of control – absorbance of sample)/ absorbance of control] × 100.

The method stated by [22] was modified to evaluate the ABTS assay. 7 mM ABTS and 2.45 mM potassium persulfate were mixed to prepare ABTS^•+ stock solution and incubated in the dark at room temperature for 16 h. The working solution was prepared by mixing 5 mL of stock solution of ABTS^•+ and 75 mL of deionized water. 160 μL of ABTS^•+ working solution was mixed with 10 μL of the diluted sample or Trolox (as a standard solution) and incubated in the dark at room temperature for 30 min. The absorbance of the mixture was determined at 743 nm. Trolox was used as a standard and the values were represented as mg of Trolox equivalent antioxidant capacity (TEAC) per gram of sample. The radical scavenging activity of the ABTS assay was calculated as follows:

ABTS radical scavenging activity (%) = [(absorbance of control – absorbance of sample)/ absorbance of control] × 100.

A previous protocol stated by [24] with minor modifications was used to evaluate the FRAP assay. The FRAP reagent was prepared by mixing 30 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl₃·6H₂O solution in the ratio of 10:1:1. The FRAP solution was freshly prepared before use. 10 μL of the diluted sample was mixed with 180 μL of the FRAP solution and kept at room temperature for 4 min. The absorbance of the mixture was determined at 593 nm. FeSO₄ was used as a standard and the values of the FRAP assay were presented as mg of FeSO₄ per gram of sample.