2.5. In Vitro Antioxidant Capacity

The obtained bilberry pomace extracts’ total phenolic content was estimated as previously described in the literature [23]. Briefly, 150 μL of sample or blank was mixed with 750 μL of Folin–Ciocalteu’s reagent (1:9, v/v) followed by 600 μL Na₂CO₃ solution (75 g/L). Samples were kept in the dark for two h. The absorbance of optically clear supernatants was measured at 760 nm with a GENESYS 150 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The TPC was expressed as gallic acid equivalents (mg GAE/g extract or recalculated per g DW of bilberry pomace residue after SFE-CO2 taking into consideration the extraction yield; mean values ± standard deviation, n = 4), employing a dose–response curve (0–80 μg/mL) for gallic acid.

The ABTS^•+ assay was carried as described elsewhere [24]. Briefly, 50 mL of ABTS^•+ (2 mmol/L PBS) solution was prepared and mixed with 200 μL K₂S₂O₈ (70 mmol/L). The stock mixture was allowed to stand in the dark at room temperature for 15–16 h prior to dilution. The working solution was then prepared by diluting the stock solution with PBS until a final absorbance of AU 0.700 ± 0.010 at 734 nm was reached. Afterward, 25 μL of extract or blank control sample were mixed with 1500 μL of working radical solution. Then mixtures were vortexed for 15 s and then shaken at 250 rpm in the dark. After two h, samples were centrifuged (1960× g for 5 min), and the absorbance of optically clear supernatant was measured. TEAC_ABTS was calculated through a dose–response curve for Trolox (0–1500 μmol/L MeOH).

ORAC of bilberry extracts was measured following previously described procedures [25]. The method utilizes fluorescein as a fluorescent probe. Briefly, 25 μL of the sample or distilled H₂O used as a blank control sample were mixed with 150 μL of fluorescein solution (14 μmol/L) in a 96-well black opaque microplate. Samples were then preincubated for 15 min at 37 °C. Then, 25 μL of AAPH solution (240 mmol/L) used as a peroxyl radical generator was added. A total of 150 cycles were recorded using a FLUOstar Omega plate reader (BMG Labtech, Offenburg, Germany). Results were expressed as Trolox equivalent antioxidant capacity (TEAC, mg sample) by means of the dose–response curve for Trolox (0–500 μmol/L PBS).

CUPRAC of bilberry extracts was measured following a previously described procedure with slight modifications [26]. Briefly, 0.6 mL of Cu(II), neocuproine, and NH₄Ac buffer solutions were added to a test tube. Then, 0.6 mL of extract (or standard) was added to the initial mixture to make the final volume 2.4 mL. The tubes were stoppered, and after 30 min, the absorbance was recorded at 450 nm (760 nm with a GENESYS 150 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The CUPRAC antioxidant capacity was expressed as Trolox equivalents (either as mg TE/g extract or per g DW of bilberry pomace residue after SFE-CO₂, taking into consideration the extraction yield; mean values ± standard deviation, n = 4), employing a dose–response curve for Trolox.