Study On Inhibition Mechanism Of Xanthine Oxidase Activity By Black Rice Anthocyanins

Xanthine Oxidase（XO）exists widely in organisms and is a key enzyme in the uric acid synthesis pathway.Excessive uric acid is prone to arthritis,kidney failure and other diseases.The existing methods for lowering uric acid mainly focus on inhibiting uric acid synthesis and promoting uric acid excretion.Black rice anthocyanins（ATC）is a kind of natural edible functional pigment which widely exists in the seed coat of black rice with various physiological activity.In this study,the inhibitory effect of black rice ATC on XO in vitro was systematically analyzed,the main monomers in black rice ATC were identified by HPLC,and the inhibitory effect and inhibitory mechanism of the monomers on XO were discussed.The main results were as follows:（1）Black rice ATC had a certain inhibitory effect on XO in vitro,with IC₅₀=0.613mg/m L.Six monomers were identified in black rice ATC by HPLC,among which C3G was the main component.（2）In vitro inhibition results showed that C3G reversibly inhibited XO activity with IC₅₀=0.485 mg/m L,which was a good XO inhibitor.The C3G-XO binding was systematically studied by multispectral method combined with molecular docking technology.The results show that C3G could effectively quench the endogenous fluorescence of XO,which was a static quenching effect,and the hydrophobic interaction was the main driving force for the binding between them.With the increase of C3G concentration,the fluorescence peak intensity of tyrosine and tryptophan residues in XO gradually decreased,the tyrosine peak type did not change significantly,and the tryptophan fluorescence peak appeared blue shift.（3）Circular dichroism showed thatα-helical structure was the main secondary structural unit in XO.With the increase of C3G:XO molar ratio,α-helical structure content gradually increased,whileβ-angle ratio decreased,which limited the binding and catalysis between substrate and XO to a certain extent.（4）The 3D diagram of molecular docking showed that C3G could occupy the active cavity channel leading to the active center of XO,and the glycoside structure covered the hydrophobic channel,which hindered the entry of the substrate.The 2D docking diagram showed that C3G did not directly interact with the active center Mo,but acted on amino acid residues near the active site.This process mainly took hydrophobic interaction as the main driving force and hydrogen bond as the auxiliary force to induce the conformational change of XO.