2.4.1. Xanthine Oxidase Assay

The test sample (Ag NPs) inhibitory potential against xanthine oxidase was determined by the hydroxylation reaction of xanthine (substrate) in which uric acid (colorless) formed as an end product. The uric acid shows adsorption at 296 nm. The reaction mixture, which was used in this protocol, consists of sample, phosphate buffer, xanthine, and xanthine oxidase enzyme. First 1 mmol/L solution of pure sample was prepared and then 10 μL of this was dissolved in Dimethyl sulfoxide (DMSO). 0.003 units of XO enzyme were dissolved in 20 μL of buffer (phosphate buffer). 20 μL of xanthine (0.1 mmol L⁻¹) was used as substrate. When XO was added then this mixture was incubated for 10 min at room temperature. After incubation the mixture was first analyzed in the UV region (λ max 295 nm). Then substrate was added to the mixture, and continues reading for 15 min at an interval of 1 min was observed (Spectra MAX-340, Molecular Devices, San Jose, CA, USA). The % inhibition of test sample (AgNPs) was calculated by using the formulas:

where A is absorbance and L is the thickness of the sample and then absorption is calculated by the following formula:

where aλ is absorptivity coefficient which depend upon λ, b is path length and c is concentration of analyte. By using EZ-Fit windows-based software version 5.03 (Perrella Scientific Inc. Amherst, MA, USA) IC₅₀ values of the compounds was calculated. Allopurinol was used as positive control (standard). The inhibitory activities of the test samples were then compared with the standard. The reaction for each compound was performed in triplicate [23].