Inhibitory Actions Of Catechins On Xanthine Oxidase And The Effects Of Processing Conditions On Its Activity

Xanthine oxidase（XO）exists in the human liver and kidney,it can continuously oxidize hypoxanthine and xanthine to generate uric acid,and the catalytic process is also accompanied by the formation of superoxide anions（O₂^-）and peroxides.Sustained high level of uric acid in the blood will induce hyperuricemia.In severe case,supersaturated uric acid will precipitate into sodium salts and deposit in peripheral joints and tissues,eventually leading to gout.Simultaneously,it is also at risk of developing complications such as hyperlipidemia,hypertension,diabetes and atherosclerosis.In recent years,foods with high purine have gradually been favored by people,which has led to a rapid increase in the incidence of gout and has become a multifocal and serious metabolic disease.At present,common uric acid-lowering drugs in clinical practice,such as allopurinol and febuxostat,have been reported to have side effects such as fever,allergy,diarrhea,liver and kidney damage.Therefore,it is of great practical significance to discover XO inhibitors with high activity,safety and non-toxicity.As important active components in tea,catechins have excellent pharmacological activities such as antioxidant,anti-inflammatory,anti-cancer and hypolipidemic activities.Thus,catechins have become an important resource for the research and development of anti-diabetic,anti-hyperglycemic and anti-hyperuricemia medicines.In this paper,the three most representative catechins,namely epigallocatechin gallate（EGCG）,gallocatechin gallate（GCG）and epigallocatechin gallate（ECG）,were selected to systematically explore the inhibitory effects and molecular mechanisms of the above three catechins on XO,their binding properties with XO and the effects on the XO conformation through multi-spectroscopic methods and computer simulation techniques.The inhibitory modes of three catechins on the formation of O₂^-in the catalytic system of XO were discussed,and their combined inhibitory effects with allopurinol were studied based on dose-normalized isobologram theory.In addition,the effects of p H,heating and microwave pretreatment on the antioxidation and XO inhibitory activity of ECG were studied.A simulated digestion model was established in vitro to determine the bioaccessibility of ECG.The main results are as follows:EGCG,GCG and ECG all have excellent inhibitory activity on XO with half inhibition concentration(IC₅₀)values of 40.50±0.32μM,33.60±0.53μM and19.33±0.45μM,respectively,and the order of inhibitory ability was ECG>GCG>EGCG.The inhibition constants of EGCG,GCG and ECG were 6.68±0.04μM,5.10±0.13μM and 5.27±0.24μM,respectively,the apparent coefficient values were all greater than 1,indicating that the three catechins were more likely to bind to free XO.All the three catechins can significantly quench the endogenous fluorescence of XO,and the quenching mechanism is single static state.The binding constants of three catechins at room temperature were in the order of magnitude of 10⁴L mol^-1the n values were close to 1,indicating that the three catechins had moderate binding affinity with XO and there was only one class of binding site in XO,among which the binding of GCG and XO was the most stable.The bindings of catechins with XO were driven by hydrogen bonds and van der Waals forces.The results of molecular simulation showed that all the three catechins inserted into the flexible region near flavin adenine dinucleotide（FAD）and formed complexes with the surrounding amino acid residues through hydrogen bonds,and the conformation of catechins was distorted after binding.The results of MM-PBSA free energy decomposition showed that the polar solvation free energy contributed the most to the total free energy.Molecular dynamics results revealed that the presence of the three catechins enhanced the structural compactness and stability of XO and increased the surface hydrophobicity.The results of synchronous and three-dimensional fluorescence displayed that EGCG,GCG and ECG basically did not affect the microenvironment of tryptophan and tyrosine residues,but destroyed the stability of the polypeptide backbone of XO.The results of circular dichroism spectra and inverted fluorescence microscopy images showed that catechins induced the contraction of XO structure and the increase ofα-helix andβ-sheet contents.In contrast,ECG and GCG had significantly greater effects on XO structure than EGCG.Combining these results,it is possible to infer the inhibitory mechanism of three catechins on XO:EGCG,GCG and ECG bound to the highly flexible region near FAD,interacting with the surrounding key amino acid residues through hydrogen bonds,and leading to the formation of more compact conformation of XO,thereby hindering the entry of the substrate and the release of the catalytic product,finally the XO activity was inhibited.EGCG,GCG and ECG can effectively inhibit the production of O₂^-radical by XO in the catalytic process,which mainly achieved indirectly through inhibiting the production of uric acid catalyzed by XO and reducing XO molecule to form H₂O₂.In addition,all the three catechins with allopurinol have synergistic inhibitory effects on XO,among which GCG and allopurinol have the best synergistic effects.Combined with the results of molecular docking,the possible synergistic mechanism is that catechins and allopurinol bound to different active sites in XO respectively,enhancing the binding affinity between them and enzyme,and finally showing synergistic inhibitory effect.The research results of the effects of p H value,heating and microwave treatment on the antioxidant activity and the inhibitory activity on XO of ECG showed that ECG was relatively stable in acidic environment,and the scavenging rate on 1,1-Diphenyl-2-picrylhydrazyl（DPPH）radical and the inhibitory rate on XO were also higher in low p H conditions.Microwave treatment and low temperature heating can improve the antioxidant and XO inhibitory activities of ECG,but high temperature heating will greatly reduce the inhibitory rate on XO of ECG.Under two processing conditions,ECG still quenched the intrinsic fluorescence of XO in a static manner.The binding affinity of ECG to XO after heating and microwave treatments showed a weakening and increasing trend,respectively,and the binding driving force was still hydrogen bonds and van der Waals forces.Heating and microwave treatments strengthened the effects of ECG on the secondary structure of XO,resulting in a more compact structure of XO.The degradation mechanism of ECG under heating and microwave conditions was explored by HPLC technique,and the results showed that ECG would be transformed into catechin gallate（CG）,epigallocatechin（EGC）,epicatechin（EC）and gallic acid（GA）,there may be synergistic effects between these degradation products and ECG,thus the systems exhibited higher antioxidant activity and inhibitory activity against XO.The bioaccessibility of ECG after simulated digestion in vitro was calculated,and the order was untreated>microwave treated>heating treated ECG.The promotion effects of heating and microwave treatments on the scavenging rate of DPPH radical of ECG was weakened after digestion.In contrast,the microwave-treated ECG still had good XO inhibitory activity after digestion.