**2.8. Total antioxidant content**

The TAC was determined by ferric reducing antioxidant power (FRAP) according to the method of Benzie and Strain [28]. Briefly, the FRAP reagent was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl<sup>3</sup> •6H<sup>2</sup> O at the ratio of 10:1:1 (v/v/v). Then add 2.4 mL FRAP reagent and 80 μl of diluted sample, and the plate was incubated at 37°C for 4 min. The absorbance readings were taken at 593 nm using Agilent 8453 spectrophotometer (Agilent Technologies, Waldbronn, Germany). The TAC value was calculated on the basis of Fe<sup>2</sup> SO<sup>4</sup> ∙7H<sup>2</sup> O and expressed as micromoles of Fe<sup>2</sup> SO<sup>4</sup> ∙7H<sup>2</sup> O equivalent per gram of fresh frozen weight.
