Study On Anti Rheumatoid Arthritis Peptide Of Deer Sinew

Objective: Rheumatoid Arthritis(RA)is an autoimmune disease characterized by synovitis.Traditional Chinese medicine has certain advantages and characteristics in the treatment of RA.Firstly,the purpose of this paper is to screen the anti RA active components in the peptide components of deer sinew protein;Secondly,based on the the theory of thermal oscillation of peptide bond and enzyme engineering technology,the dynamic changes of the active components of deer sinew before and after processing and enzymatic hydrolysis were analyzed to obtain the anti RA active components;Finally,the anti RA active components of deer sinew were analyzed by liquid chromatography-mass spectrometry.The chemical composition of the active components can provide a reference for the further development of the anti RA effect of deer sinew.Methods: 60 ng/m L tumor necrosis factor-α(TNF-α)was used to induce MH7 A cell in vitro.MTT method was used to determine the effect of staghorn protein extracted from different polar parts(petroleum ether,dichloromethane,ethyl acetate,n-butanol,70% ethanol,water)on the proliferation inhibition activity of MH7 A cell,and the best part with the best proliferation inhibition activity was selected.Response surface methodology was used to optimized the technology of deer sinew natural protein(DSNP),deer sinew processed protein(DSPP)and deer sinew enzymolysis protein(DSEP).The total extract was further divided into three parts by ultrafiltration device according to different molecular weight:total extract,> 10 k Da,3-10 k Da,1-3 k Da and <1k Da.The ultrafiltration classification was carried out,and the proliferation inhibitory activity of different molecular weight ultrafiltration components of DSNP,DSPP and DSPP was detected by MTT method.The components with the best proliferation inhibitory activity were selected,and the effect of the active components on the secretion of MH7 A cell was determined.The anti-inflammatory activity before and after processing and enzymolysis were compared.The difference between DSNP and DSPP was analyzed through the correlation analysis of the dynamic changes of the activity components of deer sinew before and after processing;The difference between DSNP and DSEP was analyzed through the correlation analysis of the dynamic changes of activity components of deer sinew before and after enzymatic hydrolysis.The best components were separated and purified.The anti RA active components in DSNP,DSPP and DSEP were analyzed and identified by liquid chromatography-mass spectrometry.Results: The components extracted from different polar parts of deer sinew that water was the most active.The optimal extraction condition of DSNP was solid-liquid ratio 1:21 g/m L,extraction temperature 88 ℃,extraction time 8.6 h,and the protein content was 12.53%.The optimum processing condition of DSPP was processing temperature 260 ℃,processing time10.5 min,feed ratio 1:37.Under this condition,the protein content was 9.17%.The result showed that the optimum enzymatic hydrolysis conditions of DSEP were p H 9.4,hydrolysis time 5 h and hydrolysis temperature 53 ℃.Under this condition,the degree of hydrolysis was31.67%.Among the different ultrafiltration components of DSNP,DSPP and DSEP,each component had certain inhibitory effect on MH7 A cell.Components with molecular weight less than 1k Da(DSNP-5,DSPP-5 and DSEP-5)had the strongest inhibitory effect on MH7 A cell proliferation(P<0.05),and they could inhibit the secretion of inflammatory cytokines NO,IL-6.The comparison of anti-inflammatory activities of DSNP-5 and DSPP-5 before and after processing showed that the mass concentration was 25-200 μg/m L.DSPP-5 had stronger inhibitory effect on MH7 A cell release of NO and IL-6 than DSNP-5(P< 0.05).The correlation analysis of the dynamic changes of the activity and components of deer sinew before and after processing showed that the chemical which had the greatest impact on the activity were Pro and Arg-Cys.The comparison of anti-inflammatory activities of deer sinew before and after enzymatic hydrolysis showed that the mass concentration was 25-200μg/m L.DSNP-5 had stronger inhibitory effect on MH7 A cell release of NO and IL-6 than DSEP-5(P< 0.05).The correlation analysis between the activity and composition of deer sinew after enzymatic hydrolysis showed that the chemical which had the greatest impact on the activity were Gly and Leu-Phe.The results of separation and purification showed that four compounds were Phe-Leu/Ile,Phe,Arg-Cys,Pro-Gly-Gln/Lys(DSNP-5).Five compounds were Leu/Ile,Arg-Cys,Ser-Tyr,Leu/Ile-Asn,Gly-Phe(DSPP-5).And two compounds were Leu-Phe,Pro-Thr-Ala(DSEP-5).Conclusion: DSNP,DSPP and DSEP have anti RA activity based on less than 1 k Da components.Based on multivariate statistical analysis,high performance liquid chromatography(HPLC)fingerprint and the theory of thermal oscillation of peptide bond,the correlation between processing and inhibition of MH7 A cell secreting inflammatory factors was analyzed to further clarify the processing mechanism of deer sinew;Based on multivariate statistical analysis,HPLC fingerprint and enzyme engineering,the correlation between processing and inhibition of MH7 A cell secreting inflammatory factors was discussed to explore the effect of enzymolysis on the anti RA activity of deer sinew;DSNP-5-3,DSPP-5-3,DSEP-5-3 were isolated and purified.The structure of the compounds were determined by liquid chromatography-mass spectrometry.The results of this study provide a reliable scientific basis for the development and application of deer sinew in the treatment of RA.