*2.5. Analytical Techniques*

In order to have a more complete characterization of the antioxidant activity of the extracts, various methods were used, namely, the DPPH and ABTS assays based on the capacity to scavenge free radicals and the FRAP assay that measures the capacity to reduce a metal ion.

The DPPH radical scavenging ability of the extracts was determined following the method proposed by Barreira et al. [13] modified as described in Vázquez et al. [11]. The results were expressed as mmol Trolox equivalent (TRE) per g extract d.b. and as the EC50 value, or extract concentration necessary to achieve a 50% DPPH radical inhibition. ABTS scavenging activity was determined according to the method of Re et al. [14], and the results expressed as mmol Trolox equivalent (TRE) per g extract d.b. and as the EC50 value. The FRAP assay was done according to Szöllösi and Szöllösi-Varga [15]. The results were expressed as nmol ascorbic acid equivalent (AAE) per mg extract d.b.

Phenolic compounds in the extract selected as the optimum were determined by UPLC/ESI-QTOF-MS using a Bruker Elute UHPLC (Billerica, MA, USA) and a Bruker TimsTOF (Billerica, MA, USA). Separations were performed using a Bruker Intensity Solo C18 2 μm (2.1 mm × 100 mm) column (Billerica, MA, USA) and a binary gradient of 0.1% aqueous formic acid for mobile phase A and 0.1% formic acid in methanol for mobile phase B at a flow rate of 0.25 mL/min. The LC gradient was 5% B from 0 to 0.4 min, from 5% to 35% B from 0.4 to 0.5 min, from 35% to 100% B from 0.5 to 7 min, 100% B from 7 to 12 min, from 100% to 5% B from 12 to 12.1 min and 5% B from 12.1 to 15 min. Table 3 shows the regression equation for each standard compound analyzed. As observed, all the compounds showed good linearity in a relatively wide concentration range.

**Table 3.** Calibration curves for the standard compounds analyzed by ultra-performance liquid chromatography coupled with electrospray ionization and time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS).

