Synergy ht gens5

FRAP assay was determined according to Ferreira et al. (2023) [36 (link)]. In a microplate, aliquots of 35 µL of each extract (Section 3.6) were mixed with 265 µL of the FRAP reagent: acetate buffer (0.3 M), TPTZ solution (10 mM), and FeCl₃ (20 mM). The microplate was incubated (37 °C, 30 min), light-protected, and the absorbance was measured in a microplate reader at 595 nm (Synergy HT GENS5, BioTek Instruments, Inc., Winooski, VT, USA). A calibration curve with ferrous sulphate (y = 0.002x + 0.080, R² = 0.999, 25–500 mg/L) was used for quantification. Results were expressed as g of FSE/100 g of sample in fw and dw.
DPPH^●-SA assay was determined according to Ferreira et al. (2023) [36 (link)]. In a microplate, aliquots of 30 µL of each extract (Section 3.6) were mixed with 270 µL of fresh DPPH^● solution in ethanol (6 × 10⁻² mM). Absorbance (525 nm) was measured in a microplate reader (Synergy HT GENS5, BioTek Instruments, Inc., Winooski, VT, USA) every 2 min until reaction endpoint at 20 min to assess the kinetics of the reaction. A Trolox calibration curve was obtained (y = −0.007x + 0.540, R² = 0.999, 5.62–175.34 mg/L) and results presented in g of TE/100 g of sample in fw and dw.

For the extraction of phenolic compounds, the mass/volume ratio was optimized using milled olive leaves and 80/20 methanol/water (V/V) as solvent. The mixture was agitated in a magnetic stirrer (MS-H-S10 magnetic stirrer, ChemLand, Usługowa, Stargard, Poland) at a constant temperature (40 °C) and agitation (600 rpm) for 1 h, according to Melo et al. (2021) [127 (link)]. The total phenolic content (TPC) was determined by a spectrophotometric method with the Folin–Ciocalteu reagent at room temperature using an absorbance reading of 765 nm in a microplate reader (BioTek Instruments, Synergy HT GENS5, Winooski, VT, USA) following Melo et al. (2021) [127 (link)]. Results are reported in g of gallic acid equivalents (GAE)/100 g of sample fresh weight.

TPC was determined according to Ferreira et al. (2023) [36 (link)]. In a microplate, 30 µL of each extract (Section 3.6) was mixed with 150 µL of Folin–Ciocalteu’s reagent (1:10) and 120 µL of 7.5% (m/V) Na₂CO₃. The microplate was incubated at 45 °C for 15 min, followed by 30 min at room temperature (RT), light-protected. The sample’s absorbance was measured in a microplate reader (Synergy HT GENS5, BioTek Instruments, Inc., Winooski, VT, USA) at 765 nm. A gallic acid calibration curve (y = 0.009x + 0.006, R² = 0.999, 5–100 mg/L) was used for quantification. Results are expressed in g of GAE/100 g of sample in fw and dw.