*3.1.4. CUPric Reducing Antioxidant Capacity (CUPRAC) assay*

The CUPRAC assay, developed by Turkish researchers from Istanbul University [28], has undergone many modifications by which it has been adapted to wider applications [29, 30]. The mechanism of monitoring the antioxidant activity of the sample has remained unchanged. The assay is based on a coloured reaction during which copper ions in the CUPRAC reagent, Cu(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline (Nc)), are reduced by antioxidants contained in the analysed sample. Chelates Cu(I)-Nc formed during the reaction have the maximum light absorption at the wavelength of 450 nm. The reaction runs at pH 7, which – as the authors of the method have pointed out – is closer to the natural physiological environment, unlike in the FRAP assay (pH 3.6) and the Folin-Ciocalteu assay (pH 10) [29, 30]. Those same authors have pointed out the low cost of the method, its simplicity, response to thiol groups of antioxidants and, importantly, its flexibility, which enables using it – by changing a solvent – to examine both lipophilic and hydrophilic antioxidants. The reaction of Cu(II)-Nc with an antioxidant runs vigorously at 37ºC, but certain compounds, such as naringine, require previous acidic hydrolysis.
