Xanthine oxidase inhibition assay and kinetics study

Xanthine oxidase activity was measured spectrophotometrically at 295 nm by continuously measuring uric acid formation, according to the protocol of Kong et al. [28]. The reaction mixture is composed of 250 μL of test solution, 300 μL of 70 mM phosphate buffer (pH 7.5) and 300 μL of substrate solution (150 μM xanthine in the same buffer). After preincubation at 25 °C during 15 min, the reaction was triggered by the addition of 150 μL enzyme solution (0.1 units/mL in 70 mM phosphate buffer (pH 7.5) freshly prepared before use. Pure control having 100% enzyme activity was conducted by replacing the essential oil with DMSO. Blank for pure control having 0% enzyme activity was conducted with DMSO and by replacing the enzyme with buffer. Allopurinol was used as a positive control. The RaEO was tested for xanthine oxidase inhibitory activity at various concentrations. Each sample was carried out in triplicate. The inhibitory activity was determined by IC₅₀, which was obtained from percent inhibition calculated by the following equation:

Percent inhibition (%) = (1 − (ΔOD_sample/ΔOD_control)) × 100.

The IC₅₀, which is the concentration of the sample required to inhibit 50% of the enzyme was determined for each sample.

For the kinetics study, the reaction mixture was as described above, except that the substrate concentration increased from 37.5 to 300 μM, and in the presence of different concentrations of RaEO (2.5, 3.75 and 5 μg/mL). The reaction was initiated by the addition of enzyme, and monitored at 295 nm and at 5 min intervals during 30 min. The obtained results were used to construct Lineweaver–Burk plots to determine the inhibition mode, Michaelis-Menten constant (K_m) and maximum velocity (V_max) values. The inhibition constant (Ki) value was calculated from the secondary plot constructed using Y-intercepts of Lineweaver–Burk plots. (Ki) expresses the equilibrium constant for the binding of RaEO to xanthine oxidase.

For a general analysis of non-competitive inhibition, the Lineweaver-Burk equation can be written in double-reciprocal form:

Secondary plots can be constructed from

The secondary replot of Y-intercept vs. [I] is linearly fitted assuming a single inhibition site or a sing class of inhibition sites.