Cupric-reducing antioxidant capacity (CUPRAC) assay

CUPRAC method described by Apak et al. (2004) was used to determine the antioxidant capacity of methanol extract, ethyl acetate fraction and butanol fraction from P. erinaceus roots. In each well of 96-well microplate 50 μL of extract, fraction or standard control were mixed with 50 μL of distilled water and copper (II) chloride (CuCl2, 17.05 mg/mL, 50 μL), ammonium acetate buffer (NH4Ac, 77.08 mg/mL, 50 μL), neocuproine (1.56 mg/mL, 50 μL). After 1 hour of incubation, the absorbance was recorded at □450 nm using Multiskan EX (Thermo Electron Corporation^®) spectrophotometer equipped with Ascent Software 2.6. The cupric ion reducing capacity of extract or fractions was expressed as trolox equivalent.