2.7. Antioxidant Activity

The ferric-reducing antioxidant power (FRAP) assay was used to measure the antioxidant activity of the extracts. The reagents were prepared for the FRAP assay as follows: To prepare the 300 mM acetate buffer, 3.1 g of C₂H₃NaO₂·3H₂O (sodium acetate trihydrate) was added to 16 mL of C₂H₄O₂ (glacial acetic acid), with distilled water (DW) added to bring the total volume to 1 L. The 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) solution (10 mM) was prepared by adding 3.1233 g of TPTZ to 1 L of 40 mM HCl, and the FeCl₃·6H₂O solution was prepared by adding 1 L of DW to 5.406 g of FeCl₃·6H₂O. The working FRAP reagent was made by mixing the 300 mM acetate buffer, 10 mM TPTZ, and FeCl₃·6H₂O solution at a ratio of 10:1:1, and then preheated at 37 °C. The sample (10 μL) and 300 μL of working FRAP reagent were reacted on a 96-well plate and incubated for 5 min at 37 °C in the dark. The absorbance was measured at 593 nm. Trolox was used to obtain a standard curve, and the antioxidant activity was expressed in milligrams of trolox equivalent (TE) per gram fresh weight (FW).