Preparation Of Ethanol-soluble Polypeptide From Sturgeon Cartilage And Anti-inflammatory Activity

Bioactive peptides have many physiological functions,such as immune regulation,antibacterial,antihypertensive,anticancer and so on.Animal by-products are important sources of active peptides.Sturgeon cartilage,as a kind of processing by-product with high yield,is rich in protein,chondroitin sulfate and other potential bioactive substances.Ethanol-soluble cartilage hydrolysate of sturgeon(ESCH)was prepared by hot-pressure,two-step enzymatic hydrolysis and ethanol extraction.ESCH was purified,sequenced and its activity was verified.The anti-inflammatory activity of ESCH was evaluated by LPS induced RAW264.7 macrophages,and its possible anti-inflammatory mechanism was explored at transcription and translation levels.The main research contents and results are as follows:(1)The characteristics of ESCH were analyzed by agarose gel electrophoresis,ultraviolet spectrum,FT infrared spectroscopy and circular two chromatography.The potential anti-inflammatory activity was evaluated by nitric oxide(NO)release.The results showed that chondroitin sulfate was not detected in ESCH,and the molecular weight of the peptide was mainly below 1 k Da(94.71%).Amino acid analysis showed that there were a lot of hydrophobic amino acids,which may be the mixed hydrolysate of extracellular matrix.ESCH at the concentration of 12.5-800 ?g/m L was non-toxic.Except 800 ?g/m L,the release of NO in macrophages was significantly reduced at other concentrations(P<0.001).(2)Sephadex G-15 gel chromatography was used to separate ESCH.The antiinflammatory and antioxidant activities of the components were evaluated by NO release of macrophages and free radical scavenging activity,and the possible antiinflammatory mechanism was explored.The results showed that the inhibitory rates of the three crude components on NO production were F1: 5.95-6.37%,F2: 6.27-9.38%,F3: 14.39-22.17%.F3 was selected to further study: F3 significantly inhibited the secretion of IL-6 and TGF-?,and increased the secretion of IL-10.After treatment with F3,the phosphorylation of MAPKs protein decreased.It is speculated that the inhibition of inflammation by F3 may be related to the release of cytokines and inflammatory mediators mediated by MAPK signaling pathway.In addition,F3 also showed good antioxidant activity,and the scavenging rates of DPPH and ABTS were 28.71% and99.75% respectively.(3)F3 was further purified by hydrophobic chromatography(ODS)and identified by LC-MS/MS.The peptide sequence was synthesized and verified by NCBI database.The results showed that: five anti-inflammatory peptides were obtained,which were LTGP(Leu-Thr-Gly-Pro,LP4),LLLE(Leu-Leu-Leu-Glu,LE4),LLEL(Leu-Leu-GluLeu,LL4),VGPAGPAGP(Val-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro,VP9)and FSGLDGAKGD(Phe-Ser-Gly-Leu-Asp-Gly-Ala-Lys-Gly-Asp,FD10).Except LP4,other peptides were found for the first time.All of the five peptides could significantly inhibit the release of NO under the condition of inflammation in the dose range without cytotoxicity.LP4,LE4,LL4 and VP9 could significantly reduce the release of IL-6 and increase the secretion of IL-10.The transcriptome study of LL4 on RAW264.7macrophages showed that LL4 reduced the level of inflammation in vitro and regulated the signaling pathways of inflammation and inflammation-related diseases,which may be bound up with the down-regulation of Cda,Ccl6,Stfa3,Mrc1,S100a8 and Mmp3 genes,partially ascribe to the suppression of MAPK and i NOS signal pathways.