Abstract: This study aims to investigate the inhibitory effects of luteolin, quercetin, morin, and fisetin against xanthine oxidase (XOD) and to elucidate the mechanism of the effect of structural differences among the four flavonoids on their XOD inhibitory activity. Enzyme kinetics analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and molecular docking were used in this study. The results showed that all four compounds significantly inhibited XOD activity. Luteolin and quercetin, with half maximal inhibitory concentration (IC50) of 1.68 and 1.77 μmol/L, respectively, exhibited stronger inhibitory effects than morin, fisetin, and the positive control allopurinol (IC50 = 20.29 μmol/L). Inhibition kinetics revealed a mixed-type inhibition mechanism, and each compound bound to a single site on the enzyme (n ≈ 1). Luteolin showed the highest binding affinity with an inhibition constant (Ki) of 2.21 nmol/L. CD spectroscopy indicated that the flavonoids disrupted the conformation of the active site of XOD, increasing the α-helix content from 24.8% to 34.6%–45.4%. Fluorescence spectroscopy confirmed that the flavonoids statically quenched the intrinsic fluorescence of XOD, with the binding process being spontaneous and exothermic (ΔH < 0, ΔG < 0), primarily driven by hydrogen bonds and van der Waals forces. Molecular docking simulations suggested that the ortho-dihydroxyl group on the B ring is a key binding site, forming a hydrogen bond network with residues in the XOD active pocket; the 5,7-dihydroxyl group on the A ring maintains the planarity of the flavonoid skeleton via intramolecular hydrogen bonding, resulting in enhancing binding stability, while differences in hydroxyl position and spatial arrangement affect the binding affinity.

Key words: xanthine oxidase; uric acid; molecular docking; inhibition mechanism