3.3.5. Copper Reduction Assay (CUPRAC)

The cupric ion-reducing antioxidant capacity (CUPRAC) assay is based on measuring the use of copper (II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) [Cu (II)-Nc] as a chromogenic oxidizing agent. The determinations were performed by modifying the method proposed by Apak et al. [46] for 96-well plates. Nine dilutions of extracts were used for analysis. Samples were prepared by mixing 40 μL of test extract, 50 μL of 10⁻² M copper (II) chloride dihydrate, 50 μL of 7.5 × 10⁻³ methanolic neocuproine solution, and 60 μL of ammonium acetate buffer (pH 7.0) in each well. After 30 min of incubation at room temperature (25 °C), the absorbance of the prepared mixtures was measured at =450 nm using an Epoch microplate spectrophotometer with a control station and Gen5 Data Analysis Software, Version 3.08 (BioTek Instruments, Winooski, VT, USA). For each dilution of the extract, 3 replicates (n-3) were performed. Based on the results, a standard curve was drawn for the standard Trolox (y = 1.9695x + 0.05). The equivalent antioxidant capacity of Trolox was calculated. using the formula:

where c—sample concentration read from the calibration curve (mg/mL); V—solvent volume (mL); sample mass (mg).