Mak078

Serum UA concentration was determined by enzymatic-colorimetric method, using a standard diagnostic kit (MAK077, Sigma-Aldrich, USA) according to manufacturer's instructions.
XO levels in serum and liver were determined using a standard diagnostic kit (MAK078, Sigma-Aldrich, USA) according to manufacturer's instructions.

The method opted to evaluate the inhibitory potential of hesperitin derivatives was a modified protocol of Sigma, done by UV-spectrophotometric method by using xanthine oxidase activity assay kit purchased from sigma (MAK078, sigma-aldrich.co, USA). The colorimetric product obtained in the form of hydrogen peroxide generated during the oxidation of XO was determined by a coupled enzyme technique, measured at 570 nm in a 96-well plate, using the plate reader EPOCH™ “MICROPLATE READER (BIOTEK)”. One unit of XO is defined as the amount of enzyme that catalyzes the oxidation of xanthine substrate, yielding 1.0 µmol of uric acid and hydrogen peroxide per minute at 25 °C. Reagents used were 44 µL of xanthine oxidase assay buffer, 2 µL xanthine substrate solution and 2 µL of xanthine oxidase enzyme solution. All the solutions mentioned above were mixed to prepare reaction mixture. The different concentrations of synthesized derivatives having final volume 50 µL were prepared in dimethyl sulfoxide (DMSO) and added to 96 well plate. To each well 50 µL of reaction mix was added and mixed well. After 2–3 min initial measurement was taken. The plates were incubated at 25 °C taking measurements at every 5 min. Allopurinol served as positive control. Absorbance at different time intervals was noted for further statistical analysis.