3.4.3. CUPRAC Method

The CUPRAC method is based on the reduction of the cupric neocuproine complex (Cu(II)-Nc) to the cuprous form (Cu(I)-Nc) by examined antioxidants [27]. In this method, the applied CUPRAC test solution is usually prepared using CuCl₂ (final concentration of Cu(II) in the solution was 10 mM), neocuproine in absolute ethanol (final concentration 7.5 mM), and 1.0 M CH₃COOH/CH₃COONH₄ buffer solution at pH = 7.0. The reagents are measured as follows: 500 µL of Cu(II) solution+ 500 µL of Nc solution + 500 µL of buffer + 100 µL of tested plant extracts dissolved in a mixture containing hexane: ethyl acetate (in the ratio 4:1, v/v). + 450 µL of water. The obtained mixture was vigorously shaken for 30 s and left in the dark for 60 min. Then, it was poured into an optical glass cuvette and immediately placed in a spectrophotometer to measure the increase in absorbance at 450 nm at room temperature.

A mixture containing all reagents and 100 µL of extraction solvent (n-hexane: ethyl acetate (4:1, v/v)) was used to zero the spectrophotometer. The antioxidant properties were expressed as the absorbance values of the colored complex formed during the reaction after the reduction of Cu (II) by antioxidants.