2.6. Determination of Total Phenolic Compounds, ABTS, and FRAP Assay

One gram of each sample (GP, GP0, GP5, GP10) was extracted for 24 h, at room temperature, with 15 mL of MeOH:HCl (97:3) under continuous stirring in the dark [22]. After centrifugation at 3500 g for 10 min at 4 °C, the supernatant was recovered and utilized for assessing the total phenolic content (TPC), ABTS and FRAP radical scavenging activities. The TPC was determined according to Singleton and Rossi [23] with slight modifications. In detail, 0.2 mL of the extract was mixed with 0.2 mL of Folin-Ciocalteau reagent. After 5 min, 4 mL of Na2CO3 solution (0.7 M) was added and the final volume was brought to 10 mL using Milli-Q water. After 1 h, the absorbance of the solution was measured at 725 nm (ATi Unicam UV2, Akribis Scientific, Cambridge, UK). The TPC was expressed as mg of gallic acid equivalent (GAE)/g of dry matter (DM). The ABTS assay was performed according to the method proposed by Del Pino-García et al. [22]. A stock solution of the radical cation (ABTS^•+) was prepared by incubating in the dark for 12 h, at room temperature, with ABTS (7 mM) and K2S2O8 (2.45 mM) (1:1 ratio). Subsequently, 0.2 mL of the methanolic extract obtained as described were added to 9.8 mL of ABTS^•+ working solution and incubated at room temperature, in the dark, for 30 min. Absorbance was measured spectrophotometrically at 734 nm. The results were expressed as the µM of Trolox equivalent (TE)/g of DM, using a Trolox calibration curve. The FRAP assay was performed according to Benzie and Strain [24]. The FRAP reagent was obtained by mixing in a volume ratio of 10:1:1, a sodium acetate buffer (300 mM, pH 3.6), TPTZ solution (10 mM) in HCl (40 mM), and a FeCl3.6H2O solution (20 mM). A total of 10 μL of the methanolic extract were mixed with 1 mL of MilliQ water and 1.8 mL of FRAP reagent. The absorbance was then measured at 593 nm and the results expressed as µM of TE/g of DM, using a Trolox calibration curve.