Xanthine Oxidase Inhibition Assay

To determine xanthine oxidase inhibitory activity, measuring the production of uric acid from xanthine substrate was the method of choice by most researchers and was chosen by us with slight modification (31, 40). The assay mixture consisted of 70 μl of 120 mmol/l phosphate buffer (pH = 7.5), 500 μl of 150 μmol/l xanthine (pH = 7.5), 400 μl of procyanidins, and solution of phenolic acids diluted to corresponding concentration in phosphate buffer and 30 μl of enzyme solution (0.5 units/ml in buffer). The reaction was initiated by the addition of enzyme and inhibition was evaluated after 2 min. Measurement of each sample was performed in triplicate and the absorbance was detected at 295 nm using a UV-1700 spectrophotometer (Shimadzu Co., Kyoto, Japan). Xanthine oxidase inhibitory activity was expressed as the percentage inhibition of xanthine oxidase in the aforementioned assay system, calculated according to the following equation:

where A_i and A_j were the activities of the test sample with and without xanthine oxidase, A_o was the activity of enzyme without test sample, and A_k was the control of A_o without test sample and enzyme. Allopurinol, a known inhibitor of xanthine oxidase, was used as a positive control. IC₅₀ values were calculated from the mean values of data.