Study On The Structure-activity Mechanism Of Targeting Inhibition Of Xanthine Oxidase By Uric Acid-lowering Peptides Derived From Walnut

Hyperuricemia,a metabolic disease,is closely related to uric acid,however,the drugs allopurinol and febuxostat,etc,present adverse effects such as allergic reactions,skin rashes,fever and nephropathy.Walnut is a human diet with high protein nutritional value and bioactivities.In this paper,degreased walnut meal was investigated for its uric acid lowering efficacy by the animal model with hyperuricemia,and the isolation,purification,structure identification and functional evaluation of walnut-derived peptides were processed based on the xanthine oxidase(XO)inhibitory activity.Simultaneously,high-throughput screening and structure-activity relationship analysis of XO-inhibitory peptides were performed on 20 kinds of amino acids,400 dipeptides,and 8000 tripeptides using molecular docking technology.The structure of walnut-derived peptides was further modified and designed,and then the XO-inhibitory and antioxidant activities of newly peptides were evaluated again by correlation analysis and structure-activity analysis.In addition,the in vitro digestion and in vivo absorption function of walnut-derived peptides were also carried out.The results were shown as follows:Both degreased walnut meal hydrolysates(WMH)and dephenolized walnut meal hydrolysates(DWMH)showed certain uric acid-lowering efficacy,and protected renal tissue from the damage caused by excessive uric acid when evaluating the uric acid-lowering effect on hyperuricemia rats in vivo.Among them,DWMH was more marked than WMH in reducing uric acid in vivo,inhibiting XO in vitro,decreasing urea nitrogen and creatinine.After isolation and purification,seven kinds of walnut-derived peptides Trp-Asp-Asp(WDD),His-Cys-Pro-Phe(HCPF),Trp-Asp-Gln-Trp(WDQW),Pro-Pro-Lys-Asn-Trp(PPKNW),Trp-Pro-Pro-Lys-Asn(WPPKN),Ala-Asp-Ile-Tyr-Thr-Glu(ADIYTE)and Trp-Ser-Arg-Glu-Glu-Gln-Glu(WSREEQE)from DWMH were identified by LC-MS/MS.The molecular docking revealed that aromatic amino acids,including Trp,Tyr,Phe,and His,got higher Vina scores.Therefore,these amino acids might promote the XO-inhibitory activity of peptides.In particular,Trp,which had similar effect to allopurinol,showed relatively higher score by interacting with important XO-residues such as Glu802,Leu873,Ser876,Arg880,Phe914,Phe1009,Thr1010,Val1011,Leu1014,Ala1078 and Ala1079.In general,aromatic amino acid residues at N-terminal position could improve the Vina score,while in the middle,they were not as effective as at N-terminal position.Thus it was predicted that aromatic amino acid residues at middle position couldn’t effectively inhibit XO.The walnut-derived peptides WDD、WDQW、PPKNW、WPPKN and WSREEQE,and especially peptide WDQW,have both strong XO-inhibitory and antioxidant activities in vitro.Besides,Trp and Cys residues in peptides significantly increased XO-inhibitory activity and DPPH radical scavenging activity,respectively.Trp and Tyr residues were responsible for higher ORAC activity,and Trp residue also improved the DPPH radical scavenging activity of peptides more profoundly at the C-terminal than at the N-terminal.Both hydrogen donor amino acid residue content and the number of Trp residue of peptide could distinctively enhance the XO-inhibitory activity,ORAC and DPPH radical scavenging activity of the peptides,and strong correlation existed between these activities.After structural modification based on peptide WDQW,the peptides Trp-Asp-Gln-Trp-Trp,Trp-Trp-Trp-Asp-Gln-Trp,Trp-Asp-Gln-Trp-Trp-Trp and Trp-Asp-Gln-Trp-Cys-Ile-Trp showed significant enhancement in XO-inhibitory and antioxidant activity,as compared to peptide WDQW.In vitro digestion simulation showed that peptide PPKNW had not been completely degraded after 4 h exposure to pepsin and trypsin,suggesting a certain degree of stability.After intragastric administration with peptides PPKNW and WPPKN,the XO-inhibitory activity of mice serum improved significantly.And the XO-inhibitory activity of peptide PPKNW was shown in concentration-dependent manner.In addition,the ORAC activity of mice serum significantly increased after intragastric administration with high doses of PPKNW and low doses of WPPKN.This study showed that degreased walnut meal had certain uric acid-lowering efficacy.Moreover,some walnut-derived peptides were confirmed having both XO-inhibitory and antioxidant activities,providing experimental and theoretical basis for utilizing walnut-derived peptides and structure-modified peptides as potential uric acid-lowering peptides.Present work expanded the potential application of walnut-derived peptides and increased the additional value of degreased walnut meal.In short,the further development of uric acid lowering functional food using walnut-derived peptides can make great contribution to the improvement of hyperuricemia,gout,and oxidative stress-related diseases.