4.4. Antioxidant Activity Assays

In this study, DPPH, FRAP and ABTS assays were used for in vitro antioxidant capacity evaluation. The DPPH assay was performed according to the method described by Barreca et al. [53]. An aliquot of each sample (0.5 mL) was mixed with 75 μM (3.5 mL) of DPPH in methanol to a final volume of 4.0 mL. After reacting for 30 min without light, the absorption of the mixture was detected at a wavelength of 517 nm. The inhibition percentage of radical scavenging activity was the DPPH value. The FRAP assay was carried out according to Hungder et al. [54] with some modifications. A 0.2 mL aliquot of the sample was mixed with 3.8 mL of FRAP reagent (0.3 mol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ solution, and 20 mmol/L ferric chloride (FeCl₃) were mixed (10:1:1, volume ratio)). After 30 min, the absorbance was detected at wavelength of 593 nm. And the ABTS assay was followed the method of Mnb et al. [55] with few modifications. The ABTS radical cation (ABTS •+) was generated by reaction of 176 μL of potassium persulfate solution (140 mM) and 10 mL aqueous ABTS solution (7 mM) under the condition of no light for 12–16 h. Then it was diluted with methanol to an absorption value of 0.7 ± 0.02 units at 734 nm. 0.1 mL sample was added to 4.9 mL ABTS reagent. The absorbance was measured at a wavelength of 734 nm after 10 min reaction. All absorbance values were determined by using the UV–VIS spectrophotometer (PerkinElmer Lambda 25 UV/VIS, Waltham, MA, USA). Antioxidant values were calculated by standard curve method and expressed as trolox equivalents (TE mg/g DW).