Preparation Of Anti-inflammatory Peptides From Walnut Dregs And Research On Its Anti-ulcerative Colitis Activity

Ulcerative colitis(UC)is an immune-mediated inflammatory disease of the intestine,whose incidence and prevalence are on the rise worldwide.However,the traditional therapeutic drugs have certain toxic side effects and there is still a need for healthier drugs or food-derived functional nutrition factors to assist in the treatment and relief of UC.The walnut dreg is a high quality plant protein resource and the peptides prepared using walnut dreg are rich in physiological activity and have greater use and edible value.In this work,the structural characteristics and functional properties of four protein fractions were investigated by graded extraction of walnut protein from walnut dreg.The highly anti-inflammatory active peptides were prepared by enzymatic hydrolysis,and the highly anti-inflammatory active fractions were further isolated and purified by in vitro anti-inflammatory activity and cyclooxygenase-2(COX-2)inhibitory activity,and their amino acid sequences were determined.Network pharmacology and bioinformatics were used to predict the mechanism of anti-UC effect of walnut peptides,and molecular docking techniques were used to virtually screen walnut peptides for their anti-UC effect.The screened walnut peptides were used to evaluate their in vivo anti-UC effects.The effects of walnut antiinflammatory peptide on intestinal microorganisms of colitis were evaluated by gut microbiome technique.The results showed that:(1)Walnut dreg albumin,globulin,prolamin and glutenin were prepared by extraction using different extraction solvents,with glutenin being the main protein in walnut protein.The surface microstructures of the four walnut protein fractions were different.The FTIR results showed that albumin,globulin and glutenin had the highest β-Sheet content and prolamin had the highest Turn content.All four isolated proteins had β-Sheet structures,and glutenin had diffraction peaks of α-Helix structures.Glutamic acid,isoleucine,leucine,prolamin,arginine and aspartic acid were the main amino acids in the four walnut isolates,and hydrophobic amino acids dominated in the four walnut proteins,especially in the glutenin.(2)Walnut dreg protein hydrolysates were prepared using six proteases,of which alkaline protease and neutral protease hydrolysates showed the highest degree of hydrolysis and could effectively cleave walnut proteins.The alkaline protease and papain hydrolysates could effectively inhibit COX-2 and LPSinduced Nitric Oxide(NO)production by macrophages.The alkaline protease hydrolysate was selected for subsequent separation experiments.After ultrafiltration separation,fractions with molecular weights<1 KDa showed the best COX-2 and NO inhibition activity.1262 unique peptides were identified by nano-LC-MS/MS and these peptide sequences may have a strong anti-inflammatory potential.(3)A total of 602 "peptide-targets" were obtained by searching the Swiss ADME and Swiss Target Prediction databases.The Disgenet,Gene Cards,Drugbank and OMIM databases yielded 1646 ulcerative colitis-related targets.23 key targets were obtained by protein interaction network analysis.GO analysis showed that the key targets were mainly involved in inflammatory and immune-related biological processes.KEGG enrichment analysis showed that the key targets were involved in multiple signalling pathways related to inflammation and colon disease,with TNF signalling pathway and MAPK signalling pathway being the most significantly enriched signalling pathways.The target-pathway network showed that MAPK1,JUN,AKT1 and FOS were the core targets of walnut peptides for the treatment of UC.Molecular docking techniques were used to high-throughput screen potential anti-UC active peptides to obtain SHTLP,HYNLN and LGTYP,which form stable bindings with the receptor proteins MAPK1,JUN,AKT1 and FOS via hydrogen bonding,hydrophobic interactions and electrostatic interactions.(4)Walnut dreg protein-derived bioactive peptides SHTLP,HYNLN and LGTYP(WPPs)can improve the pathological phenotypes in colitis mice,including alleviating DSS-induced weight loss,elevated DAI scores,rectal bleeding,and poor mental status,etc.WPPs can improve colonic mucosal damage in colitis mice,reduce inflammation in colitis mice,decrease macrophage and neutrophil infiltration,improved intestinal physical and chemical barrier function;and inhibited MAPK signaling pathway.(5)WPPs intervened to increase the abundance of Firmicutes,decrease the relative abundance of Bacteroidota and regulate the structure of the intestinal flora.WPPs reduced the harmful bacteria Bacteroidaceae,Peptostreptococcaceae and Enterobacteriaceae,Bacteroidota,Helicobacter,Enterococcus and increased the beneficial bacteria Lachnospiraceae,Ruminococcaceae and Echinococcus,Ruminococcaceae and Erysipelotrichaceae,Candidatus＿Saccharimonas in abundance to restore the homeostasis of intestinal microorganisms.Correlation analysis showed that alterations in intestinal flora were closely associated with the production of colitis-related indicators.In conclusion,this study demonstrates the reliability and validity of applying virtual screening,bioinformatics and network pharmacology methods to the screening of bioactive peptides,providing new ideas for effective screening of bioactive peptides.In addition,walnut dreg protein is a high-quality plant protein resource and its derived peptides(SHTLP,HYNLN and LGTYP)are functional nutritional factors with great potential in the management of ulcerative colitis,which are important for improving colitisrelated diseases.This study also provides theoretical support for the further development of antiinflammatory functional foods using walnut dreg protein peptides,and is of great significance in realizing the comprehensive utilization of walnut dreg and improving the added value of by-products.