Novel mechanistic insights into Luteolin and its analogues as potent inhibitors of xanthine oxidase activity: Inhibition thermodynamics, kinetics and docking simulation

The utilization of potent flavonoid inhibitors for systematically investigating their inhibitory mechanisms on Xanthine oxidase (XOD) offers significant potential for the development of functional foods from a novel perspective. The deprotonation of Luteolin and its analogues into corresponding phenolates has been confirmed as effective inhibitory agents, exhibiting differential effects on superoxide production originating from XOD. However, the Stern-Volmer plots of F₀/F versus [Q] at various temperatures failed to demonstrate polynomial-linear fitting, instead approaching the X-axis to varying extents, which deviates from the anticipated good linearity. This indicates that the thermodynamics of inhibition may be concentration-dependent. At lower inhibitor concentrations, the formation of the ground-state complex is characterized by static fluorescence quenching within an accessible interaction region, as evidenced by linear-fitting plots. As the concentration increases, inaccessible interactions are observed, indicated by curves in the Stern-Volmer plots that approached the X-axis, suggesting a reduction in fluorescence quenching intensity. Furthermore, in the inhibition kinetics, Luteolin and its analogues exhibit mixed-type inhibition (both non-competitive and competitive) at higher concentrations, contrasting with non-competitive inhibition at lower concentrations. Simultaneously, the binding interaction between the inhibitor and enzyme is validated through synchronous fluorescence analysis and molecular docking simulations. Consequently, this study successfully redefines the novel inhibition mechanism of flavonoids from a new perspective.