Studies On The Preparation And Immunoregulatory Effect Of Peptides From Nibea Japonica

Nibea japonica,which is Mainly distributed in the East China Sea and the southern sea of Japan,is a kind of edible fish belongs to Nibea.Nibea japonica is delicious and nutritious,but few studies have been reported on its use as a protein source for the extraction and preparation of immunoactive peptides.In this paper,the Nibea japonica is used as raw material to extract the crude peptides of Nibea japonicar by enzymatic hydrolysis.The crude peptides were subjected to ultrafiltration to compare the immunological activity of each of the retained components,and the component with the highest immunological activity of <1 kDa was named Nibea japonica peptides(NJP).In this study,the immunomodulatory effect of NJP in vitro and in vivo were studied.Finally,NJP was further separated and purified by DEAE Sepharose Fast Flow anion column,Sephadex G25 gel column chromatography and RP-HPLC.(1)The Papain was selected as the best enzyme.The optimum condition for the extraction of Nibea japonica peptides were found to be hydrolysis at 59 �C for 5.4 h with a material/solvent ratio of 1:11(g/mL)at pH 6.Under these conditions,the proliferation rate of RAW264.7 cells was 62.3%.(2)To investigate the immunoregulatory effect of NJP on RAW 264.7 macrophages.NJP has no obvious toxicity to RAW 264.7 macrophages,and can significantly improve the proliferation activity,phagocytosis ability,NO release and cytokine(IL-6?IL-1??TNF-?)secretion level of macrophages.The results of flow cytometry analysis showed that NJP could promote the growth and proliferation of RAW 264.7 macrophages,mainly in G0/G1 phase.(3)To further explore the immunoregulatory effect of NJP in vivo,and to study the effect of NJP on the antioxidant index of cyclophosphamide immunosuppressed mice.NJP can improve the immune organ index of mice,enhance the phagocytosis ability of peritoneal macrophages,increase the proliferation of spleen lymphocyte,enhance the degree of DTH,increase the content of IL-2?IFN-??TNF-? in serum and the levels of immunoglobulin IgA,IgM and IgG in mice,and also have certain antioxidant activity.(4)In order to further explore the active components of NJP immunological activity,three immunologically active peptides were obtained by ultrafiltration,DEAE Sepharose Fast Flow anion column,Sephadex G25 gel column chromatography and reversed-phase high performance liquid chromatography.Using the N-terminal amino acid sequence,three peptide chains Asp-Val-Ala-Asn,Tyr-Thr-Asn,Leu-Tyr-Gly-Asn were determined.In this study,the immunoregulatory activity of Nibea japonica peptides were expPlored in vitro and in vivo,which could provide a theoretical basis and technical support for the high value utilization of the material and industrialization development of immunomodulating peptide health-care product.