| Recently,the incidence of gout has showed an increasing trend,and become one of common diseases to threaten the people health.Gouty arthritis is attributed to purine metabolic disorder or abnormal changes in uric acid excretion,which is often accompanied by related diseases,such as hypertension,diabetes,uric acid nephrolithiasis,etc.Repeated gout attacks have a direct impact on people’s quality of life and even results in kidney failure.Xanthine oxidase（XOD）,a key enzyme of purine metabolism in body,maintains the balance of uric acid in the blood,and is frequently deemed as the main molecular target for the drugs.Compared to the chemical-based medicine for gout in clinic,the natural active constituents extracted from plants exert therapeutic properties and low toxicity.Therefore,it is of great importance to find efficient and less toxic XOD inhibitors and investigate the inhibitory mechanism between them.However,flavonoids compounds are susceptible to external condition,including light,temperature,acidity,etc.The solubility and stability of flavonoids compounds is low,which have an effect on the bioavailability and limit its clinical application.How to enhance the stability and bioavailability is a major subject in the field of flavonoids application.The inhibitory mechanism between XOD and several flavonols compounds were investigated with the application of multiple spectroscopy methods including ultraviolet-visible（UV–vis）absorption,fluorescence and circular dichroism（CD）combined with molecular modeling approach.The aim of this work was to determine the inhibitory kinetics,antioxidant effect,binding characteristics of flavonols compounds with XOD and analyze conformational changes of XOD induced by flavonols compounds.In order to protect the bioactivity and improve the bioavailability of quercetin,BSA NPs was made to encapsulate quercetin.These investigations have important theoretical significance in understanding the pharmacological mechanism,and can provide useful information for the development and application of novel XOD inhibitors.The main contents of these investigations were summarized as follows:1.The inhibition of galangin,quercetin and myricetin on XOD was investigated in Tris–HCl buffer（pH 7.4）.The result showed that galangin,quercetin and myricetin had the ability of XOD inhibiton,the concentrations of galangin,quercetin and myricetin resulting in a loss of 50% enzyme activity（IC50）were determined to be（3.38 ± 0.04）× 10-6,（2.74 ± 0.04）× 10-6 and（8.66 ± 0.03）× 10-6 mol L–1,respectively.The result of enzyme kinetics experiments indicated that galangin acted as a competitive inhibitor to compete with xanthine for Mo center,and quercetin and myricetin was a reversible mixed-type inhibitor of XOD.Moreover,the catalytic process of XOD is a two-substrate reaction,which conforms to the ping-pong mechanism.Base on above-mentioned mechanism,the effect of galangin,quercetin and myricetin on the superoxide anion（O2-）generated by XOD was studied.The result indicated that galangin,quercetin and myricetin inhibited O2-generated by XOD,the IC50（loss of 50% enzymatic activity）value of galangin,quercetin and myricetin was（6.31 ± 0.02）× 10-6,（2.90 ± 0.03）× 10-6 and（4.55 ± 0.02）× 10-6 mol L–1,respectively.Further studies found that galangin competitively inhibit the formation of uric acid generated by XOD,and this inhibition suppresses the generation of both O2-and hydrogen peroxide（H2O2）.The inhibition of myricetin on O2-was attributed to the reduced form of XOD,then the substituted enzyme intermediate（EII）only catalyzes the formation of H2O2.Both the inhibition of quercetin on the formation of uric acid and the reduced form of XOD played an important role in inhibiting O2-.2.The results of fluorescence titration that galangin,quercetin and myricetin could interact with XOD and quench the intrinsic fluorescence of XOD,and the binding constants at 298 K were（3.60 ± 0.01）× 104,（4.28 ± 0.02）× 104 and（1.87 ± 0.02）× 104 L mol-1,respectively.The calculated thermodynamic parameters indicated that hydrogen bond and hydrophobic interaction were the main driving forces in the binding of galangin to XOD on account of the negative enthalpy and positive entropy changes,and the binding process of quercetin and myricetin to XOD were mainly driven by van der Waals forces and hydrogen bonds.Molecular simulation was used to predict the visible combination models between flavonols compounds and XOD.It was found that galangin inserted into the active cavity,where the active site with molybdenum（Mo）domain lies in.Galangin was adjacent to some hydrophobic residues（Leu648,Phe649,Phe883,Phe914,Phe1009,Leu1014 and Pro1076）in the active center of XOD,suggesting the presence of hydrophobic interactions between galangin and XOD.Furthermore,there was one hydrogen bond formed between the oxygen atom（C-4 ketone group of galangin）and the hydrogen atom on Asn768 of XOD with distance of 2.049 ?.Quercetin and myricetin had easy access to the active cavity region,where the flavin adenine dinucleotide（FAD）reaction site locates in.Two hydrogen bonds were formed between hydroxyl groups of quercetin and the hydrogen atoms on amino acid residues of XOD,and the hydroxyl group at position 5 and 7 in A ring of quercetin was connected with Glu263 and Arg394 by 2.162 and 1.749 ? hydrogen bonds,respectively,suggesting that hydrogen bonding may play a key role in the process of the quercetin-XOD interaction.Two hydrogen bonds were formed between the oxygen atoms（50-OH on the B ring and 3-OH on the C ring of myricetin）and the hydrogen atoms on Arg394 and Ser1225 of XOD,and their bond lengths were 2.107 and 2.208 ?,respectively,suggesting the involvement of hydrogen bonding in the myricetin–XOD interaction.3.The conformational changes of XOD induced by flavonols compounds were detected by synchronous fluorescence and circular dichroism（CD）.Results from synchronous fluorescence and CD spectral analysis showed that the binding of quercetin to XOD resulted in the increasing hydrophobicity around the tyrosine residue and the decreasing hydrophobicity around the tryptophan residue.Galangin and myricetin had little effect on the microenvironment of the tyrosine residue,while the hydrophobicity decreased and the polarity increased around the tryptophan residue.RSFQ analysis indicated that tryptophan residue may make a better contribution to the intrinsic fluorescence quenching and the binding site of flavonols compounds to XOD was nearer to tryptophan than tyrosine residue.Galangin,quercetin and myricetin the conformational changes with increases in a-helix contents and decreases in β-sheet structure of XOD,indicating the increasing compactness of XOD structure in the presence of flavonols compounds,and thus may have an impact on the function of the enzyme as a catalyst.4.According to the experimental results,the inhibitory mechanism of flavonols compounds on XOD was concluded: galangin competitively bound to the site in the Mo center to block the entrance of the substrate xanthine.The oxidation of xanthine was restrained at Mo center,resulting in the failure of intramolecular electron transfer to FAD center.Thus,the generation of O2-was inhibited due to the substantially lower reduction potential for the FADH·/FADH2 couple.Quercetin and myricetin may bind to the site around isoalloxazine ring in the FAD domain and block the diffusion of O2-out of the FAD site,leading to the transfer of another electron from FADH2 to O2-to form H2O2.The binding of quercetin or myricetin to XOD induced the conformational changes with increases in α-helix and decreases in β-sheet structure of XOD,and resulted in the increasing compactness of XOD structure in the presence of quercetin or myricetin,thus hamper the formation of active center or prevent substrate binding,which may have an impact on the function of the enzyme as a catalyst.5.The average size and size distribution index of Que-BSA NPs were（382.7 ± 5.2）nm and 0.047,respectively.The encapsulation efficiency and loading efficiency achieved were（69.7 ± 1.4）% and（8.96 ± 1.2）%,respectively.Zeta potential of Que-BSA NPs was（-46.0 ± 1.2）mV.Fresh Que-BSA NPs were uniform on size and remained stable in a week.The particles exhibited an approximately spherical shape in SEM imaging.Furthermore,quercetin released from Que-BSA NPs could inhibit XOD in the simulated gastrointestinal digestion,indicating that BSA NPs protected the activity of quercetin to some extent. |
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