Preparation,Isolation,Structural Characterization,and The Effect Mechanism Of Uric Acid-lowering Peptides Derived From Bonito

Hyperuricemia has become the second largest metabolic disease after diabetes.However,the uric acid-lowering drugs applied in clinic is known to cause allergic reactions and severe damage of liver and kidney.Therefore,it is important to seek more safe and effective natural products with uric acid-lowering activity.In this study,the purified fraction Frc2 was devided from bonito hydrolysates,and the XO inhibitory activity in vitro and the structural characterization of Frc2 were investigated.The interactional mechanism of peptides identified from Frc2 with XO were analyzed,and the hyperuricemic cell model were established to evaluate the uric-acid-lowering activity of peptides.Eight hydrolysates with different temperature,pH,and amounts of enzyme were prepared from bonito with papain.The protein recovery rat,XO inhibitory activity in vitro,molecular weight distribution,amino acid cotent,and the correlation between different test indexes was analyzed.The results showed that the protein recovery rate of bonito hydrolysate was highest when pH was 7.0,temperature was 55 ?,and enzyme content was 1%,while hydrolysates with pH was 7.0,temperature was 55 ?,and enzyme content was 0.35% exhibited greatest XO inhibitory activity.Peptides in bonito hydrolysates were mainly oligopeptides with molecular weight less than 1000 Da,and the molecular weight distribution of different bonito hydrolysates was not correlated with XO inhibitory activities,while the content of aliphatic amino acids was negatively correlated with the XO inhibitory activities.The purified fractions were selected from bonito hydrolysates with the index of XO inhibitory activity.After the purification of the ion-exchange colum chromatographic and size exclusion of gel filtration chromatography,Frc2 was selected and exhibited greatest XO inhibitory activity at the concentration of 4 mg/mL(32.33% ± 6.38).The structure of Frc2 was analyzed by HPLC-MS/MS.Four peptides were identified as Pro-Gly-Ala-Cys-Ser-Asn(PGACSN),Trp-Met-Leu(WML),Ala-Met-Pro-Phe(AMPF),and Phe-Gly-Val-Gly(FGVG).The interactional mechanism of peptides with XO molecule was simulated by molecular docking.No hydrogen bond was observed between peptides AMPF with XO,and FGVG with XO.The hydrophilic hexapeptide PGACSN and the hydrophobic tripeptide WML embedded into the hydrophobic channel.In especial.WML is almost completely embedded into the XO hydrophobic channel.These two peptides were bind to XO with hydrogen bond,and their binding energy were-4.51 kcal/mol(PGACSN)and-7.34 kcal/mol(WML),respectively.Peptides PGACSN and WML were synthesized and showed great XO inhibitory activity,and the XO inhibitory activity at the concentration of 20 mmol/L WML was comparable to that of 40 ?mol/L allopurinol.Circular dichroism(CD)were further was used to analyze the mechanism of peptides and XO.The result was consistent with that of molecular docking analysis.The secondary structure of XOD was variated by those two peptides,and proved the interaction happened between amino acid residues of peptides and the amino acid residues of XO,especially those hydrophobic amino acids in WML.In order to study the biological uric-acid-lowering activity of peptides,adenosine was used to induce HK2 cell for set up a huperuricemic cell model.The metabolites in cell supernatant and cell lysate were monitored by HPLC.The supernatant hypoxanthine in adenosine-induced cell was significantly increase as compared with control group,but no uric acid was detected.Besides,the expression of XO in HK2 cells was far below that in tissues.The pcDNA3.1 XDH was transfected into HK2 cell to enhance the expression of XO in cell.Adenosine was further used to induce hyperuricemia,the level of metabolites in transfected cell were consistent with those in untransfected cell,and uric acid was still not detected.We further compared the uric acid levels and XO activities of cell and tissue.Only uric acid at concentration of 0.5 ?mol/L was measured in 10 mg liver tissue,while that in cells was too low to be detected.The XO activity was not observed in cell lysates.Finally,the hyperuricemia cell model was successfully established by the combination of exogenous XO and adenosine.Hypoxanthine was completely converted into uric acid when we added exogenous XO in cell supernatant.Then,the cell model induced by the combination of exogenous XO and adenosine was used to evaluate the uric-acid-lowering activity of peptides PGACSN and WML.Both PGACSN and WML showed significant uric-acid-lowering activity at a concentration of 2.5 mmol/L.As result,bonito bioactive peptides can develop as supplementary ingredients in functional food and drugs for alleviated hyperuricemia and high uric acid-related diseases.