2.6. Determination of Total Phenols, Flavonoids, Non-Flavonoids, and Total Antioxidant Capacity

The ratio of scallop tissue and the selected solvent (water, 90% methanol, or 70% ethanol) was fixed at 1:3. Roughly 0.5 g of the bivalve tissue samples was mixed with 1.5 mL of distilled water, 90% methanol, or 70% ethanol. The solvents for this work were chosen based on their polarities and they ensured different extractions of the compounds, based on which the AC was compared [58]. The mixture was then homogenized using an OV5 Homogenizer (VELP Scientifica Srl, Usmate Velate, Italy) for 5 min. The homogenate was then centrifuged (10,000 rpm, 30 min at 4 °C) with a 5430 R-High-Speed Centrifuge (Eppendorf SE, Hamburg, Germany).

TP was determined spectrophotometrically, as previously described by Singleton et al. [62]. Briefly, by adding the Folin–Ciocalteu reagent to the reaction mixture, it reacted with the phenoxide ion from the sample, forming a blue color solution. The absorbance was read at 765 nm two hours after adding the reagent to the reaction. TF were determined according to the method of Martins et al. [45]. Briefly, by adding aluminum chloride to the reaction mixture, a red color was formed, which was read at 510 nm. For determining the content of TNF, the method of Ough and Amerine [63] was used. Briefly, flavonoids were first removed from the samples using formaldehyde. Formaldehyde reacts with flavonoids in an acidic medium and causes their sedimentation, after which the same method described above for TP was applied. The results were expressed in mg gallic acid equivalent per 100 g of shell tissue (mg GAE/100 g of tissue) and in mg of catechin equivalents per 100 g of shell tissue (mg CE/100 g of tissue). The results of TP and TNF were calculated according to the calibration curve for gallic acid in water (y = 0.001x, y = absorbance at 765 nm, x = concentration of gallic acid mg/L, R² = 0.9995), 70% ethanol (y = 0.0004x, y = absorbance at 765 nm, x = concentration of gallic acid mg/L, R² = 0.9915), and 90% methanol (y = 0.0009x, y = absorbance at 765 nm, x = concentration of gallic acid mg/L, R² = 0.9952). The results of TF were calculated according to the calibration curve for catechin in water (y = 0.0036x, y = absorbance at 510 nm, x = concentration of catechin mg/L, R² = 0.9999), 70% ethanol (y = 0.0024x, y = absorbance at 510 nm, x = concentration of catechin mg/L, R² = 0.9933), and 90% methanol (y = 0.0025x, y = absorbance at 510 nm, x = concentration of catechin mg/L, R² = 0.9991).

The AC of bivalve tissue was determined as previously described by Poljuha et al. [60] using three methods. The results were expressed in µM ascorbic acid equivalent per g of shell tissue (µM AA/g of tissue). The ABTS method was carried out on the principle that the blue–green ABTS radical cation and potassium persulfate are added to the solutions in order to cause oxidation. The reduction of the ABTS was measured at 734 nm as the disappearance of the color. The results of antioxidant capacity measured by the ABTS assay were calculated according to the calibration curve for ascorbic acid in water (y = 52.665x, y = percentage of inhibition of ABTS radical, x = concentration of ascorbic acid mmol/L, R² = 0.9968), 70% ethanol (y = 42.657x, y = percentage of inhibition of ABTS radical, x = concentration of ascorbic acid mmol/L, R² = 0.9958), and 90% methanol (y = 43.11x, y = percentage of inhibition of ABTS radical, x = concentration of ascorbic acid mmol/L, R² = 0.9969). The experiments were carried out in triplicate. The FRAP method is based on the reduction of iron as a part of the Fe (TPTZ)³⁺ complex, into the ferrous form of Fe (TPTZ)²⁺ in the presence of antioxidants, resulting in an intense blue color for which absorbance was measured at 593 nm. The reaction took place in an acidic medium (pH = 3.6) in order to preserve the solubility of iron and increase the redox potential. The results of antioxidant capacity measured with FRAP were calculated according to the calibration curve for ascorbic acid in water (y = 1.6851x, y = absorbance at 593 nm, x = concentration of ascorbic acid mmol/L, R² = 0.9822), 70% ethanol (y = 1.8485x, y = absorbance at 593 nm, x = concentration of ascorbic acid mmol/L, R² = 0.9543), and 90% methanol (y = 1.6927x, y = absorbance at 593 nm, x = concentration of ascorbic acid mmol/L, R² = 0.9822). The experiments were carried out in triplicate. The DPPH method is based on the reduction of the DPPH radical in the presence of antioxidants, during which the purple color changes to yellow. The absorbance color was measured at 517 nm after 60 min. The results of antioxidant capacity measured by the DPPH assay were calculated according to the calibration curve for ascorbic acid in water (y = 59.688x, y = percentage of inhibition of DPPH radical, x = concentration of ascorbic acid mmol/L, R² = 0.9812), 70% ethanol (y = 48.506x, y = percentage of inhibition of DPPH radical, x = concentration of ascorbic acid mmol/L, R² = 0.9943), and 90% methanol (y = 60.757x, y = percentage of inhibition of DPPH radical, x = concentration of ascorbic acid mmol/L, R² = 0.997). The experiments were carried out in triplicate.

All measurements were performed using a NanoPhotometer P300 spectrophotometer (Implen GmbH, München, Germany).