2.4.1. Free Polyphenol Content (FPC) and Antioxidant Capacity (AC)

Extracts for FPC and AC were obtained from 250 µL of frozen samples mixed in plastic tubes with 750 µL of methanol: water (80:20, v/v). The extraction was carried out in triplicate with an orbital shaker (Stuart, Stone, UK) for 1 h at 200 rpm in darkness at 4 °C. Finally, the extracts were centrifuged at 3220× g for 10 min at 4 °C, with the supernatant being used for analysis. FPC determination was carried out according to Singleton et al. [33] with some modifications. Briefly, 19 µL of the supernatant extract was dispensed into a 96-well plate, followed by the addition of 29 μL of 1 N Folin–Ciocalteu reagent. The plate was then incubated in darkness at room temperature (20 °C) for 3 min. Then, 192 μL of Na₂CO₃ (0.4%) and NaOH (2%) were added, and the mix was incubated at room temperature (in darkness) for 1 h. Finally, each sample was spectrophotometrically measured at a wavelength of 750 nm in a microplate reader (Infinite PRO 2000, Tecan Trading AG, Männedorf, Switzerland). The FPC was calculated using a gallic acid standard and expressed as mg of gallic acid equivalent per L of sample.

The determination of AC was carried out following the DPPH method [4] and the iron reduction power assay (FRAP) [34]. For the DPPH assay, 194 μL of DPPH solution were added to 21 μL of extract in a 96-well plate. The mixture was incubated for 30 min at room temperature (20 °C) in darkness. The absorbance was measured by changes at 515 nm. For the FRAP method, 198 μL of daily FRAP solution were added to 6 μL of extract in a 96-well plate. The mixture was incubated for 30 min at room temperature (20 °C) in darkness, and the absorbance was measured by changes at 493 nm. For both methods, the antioxidant capacity was calculated using a Trolox standard and expressed as mg of Trolox equivalents per L of sample.