Unveiling the synergistic inhibition mechanism of polyphenols in Flos Sophorae Immaturus tea on xanthine oxidase by multi-spectroscopy, molecular docking and dynamic simulation methods

Flos Sophorae Immaturus tea (FSIt) was rich in polyphenols and exhibited significant xanthine oxidase (XOD) inhibitory activity. Herein, the synergistic inhibitory effect and structure–activity relationship of chlorogenic acid (CA) with quercetin (QU), isorhamnetin (IS), and kaempferol (KA) in FSIt on XOD was evaluated. The mechanism of synergistic effect was further clarified through inhibition type, ultraviolet–visible spectrum, circular dichroism, fluorescence quenching, thermodynamic parameters, molecular docking and simulation analysis. Results showed that QU-CA, KA-CA, and IS-CA groups at the ratio of 1:1 exhibited significant synergistic inhibitory effect with the combination indexes of 1.17, 1.35, and 1.30, respectively. Meanwhile, the combination of CA and QU, KA, or IS induced a mixed inhibition type, thus enhancing the binding with XOD-xanthine complexes. Moreover, QU-CA, KA-CA, and IS-CA groups increased the ultraviolet–visible peak intensity of xanthine and decreased that of uric acid. CA also strengthened the effect of flavones on the secondary structure of XOD. The combination of polyphenols and XOD was endothermic and spontaneous. Interestingly, CA caused a red shift in the fluorescence spectra of XOD and promoted the binding ability of flavones with XOD. Furthermore, CA also improved the stability of XOD-polyphenols complexes by increasing the hydrogen bonds. QU-CA, KA-CA, and IS-CA groups generated a more stable and denser structure with XOD compared with the single polyphenols.