3.4. Determination of Total Phenolic and Flavonoid Content and Antioxidant Activity

To evaluate the extraction efficiency, the TPC, TFC, DPPH-RSA and FRAP assays were performed for all the obtained extracts. Absorbance measurements were made on a Synergy HT microplate reader (BioTek Instruments, USA) using the Gen5 2.00 program, and all the analyses were performed in triplicate.

TPC assay, based on the Singleton and Rossi original method [42], was performed as described by Paz et al. [43]. In this assay, the Folin–Ciocalteau’s reagent composed of tungsten and molybdenum changes its color from yellow to blue after reacting with reducing species, under alkaline conditions [36]. Results were expressed as mg gallic acid equivalents (GAE) per g of DW of milled vine-canes (mg GAE/g DW). TFC method measures the formation of the flavonoid-aluminium compound [44]. The assay was performed according to Paz et al. [43]. Results were expressed as mg epicatechin equivalents (EE) per g of DW of milled vine-canes (mg EE/g DW).

DPPH-RSA was performed following the protocol described by Paz et al. [43] using Trolox^® to perform the calibration curve. Results were expressed in mg of Trolox^® equivalents (TE) per g of DW of milled vine-canes (mg TE/g DW).

The original FRAP assay developed by Benzie and Strain [45] was performed with some modifications as described by Paz et al. [43]. Results were expressed as mg of ascorbic acid equivalents (AAE) per g of DW of milled vine-canes (mg AAE/g DW).