Synthesis, characterization, toxicity evaluation and inhibitory effect of hesperitin-copper (Ⅱ) complex on xanthine oxidase

In this study, a non-toxic hesperitin (Hsp)-Cu(Ⅱ) complex consisting of two Hsp and one Cu2+ was synthesized. The complex showed a significantly higher inhibitory activity against xanthine oxidase (XO) [IC50: (1.07 ± 0.08) × 10−6 mol/L] than Hsp [IC50: (1.17 ± 0.12) × 10−5 mol/L] and Cu2+ [IC50: (4.93 ± 0.21) × 10−6 mol/L], and a decreased antioxidant activity. As a mixed type inhibitor, the complex bound to XO or xanthine-XO complex to inhibit XO activity compared to the competitive inhibitor Hsp that only bound to XO. Both Hsp and Hsp-Cu(Ⅱ) complex statically quenched the endogenous fluorescence of XO, which was driven by hydrophobic interaction and hydrogen bond, and the complex exhibited stronger binding affinity with XO than Hsp. Hsp bound to the molybdopterin (Mo-pt) domain of XO, which tightened the structure of XO and hindered the binding of substrate xanthine to XO, whereas Hsp-Cu(Ⅱ) complex bound near the center of flavin adenine dinucleotide (FAD) with high steric hindrance, resulting in partial unfolding of XO structure. In addition, Cu2+, acting as a bridge for electron transfer, absorbed electrons in the complex, hindered the substrate O2 from gaining enough electrons, and reduces the formation of O2– radicals, thus slowing down the enzymatic reaction and decreasing the activity of XO. These findings may provide new insights into the complex as a promising inhibitor of XO for the prevention and treatment of hyperuricemia.