Separation,structure Identification,immunoactivity And Application Of Polysaccharide From Sea Cucumber Viscera

Sea cucumber visceral polysaccharide is one of the main active ingredient of sea cucumber viscera,and has a variety of physiological activities.However,in the process of sea cucumber,the sea cucumber viscera are usually directly discarded.At present,the research on sea cucumber visceral polysaccharide is limited to the simple biochemical index evaluation,but there is no systematic study on the structural characteristics and related active mechanism of sea cucumber visceral polysaccharide.In this study,sea cucumber viscera were used as raw material.The main components of sea cucumber visceral polysaccharides were separated and purified,the primary and advanced structure were identified,and their immune activities in vitro and vivo were systematically studied.The application of sea cucumber visceral polysaccharide/lysozyme nanocomplex as a curcumin delivery carrier was discussed.Below are main research findings:(1)Separation of polysaccharides from sea cucumber viscera: The crude polysaccharide of sea cucumber viscera was obtained by enzymolysis,quaternary ammonium salt and alcohol precipitation,with an average yield of 4.90%.The crude polysaccharide of sea cucumber viscera was separated and purified by DEAE-52 column chromatography.The two main polysaccharide components were obtained by activity pre-screening,and named as SCVP-1 and SCVP-2,respectively.After further separation and purification with Sephadex G-200,purified SCVP-1 and SCVP-2 were obtained,with purity of 92.51% and 96.87%,and molecular weights of 180.8 kDa and 209.1 kDa,respectively.(2)Structural characterization of sea cucumber visceral polysaccharides: HPLC analysis acid,galactosamine,glucose,galactose and fucose,with a molar ratio of 1.00:1.41:0.88:2.14:1.90:1.12:1.24.SCVP-2 was composed of glucosamine,galactosamine,glucose,galactose and fucose,with a molar ratio of 1.61:1.00:4.79:1.32:3.29.Based on IR and NMR analysis,it was inferred thatSCVP-1 was a glycosaminoglycan with chondroitin sulfate structure,and its main skeleton structure was ?4)GlcUA?(1?3)GalNAc?(1?.Fuc2,4s and Fuc3,4s were connected with O-3 position of GlcUA through 1?3 glycoside bond.SCVP-2 was a fucoidan sulfate with the main skeleton structure of [?3)Fuc2S?(1?3)Fuc4S?(1?3)Fuc0s?(1?]n.Advanced structure analysis showed that the surface of SCVP-1 was rough and had a loose and irregular flaky structure,and the surface of SCVP-2wasinterlaced and extended with fibrous network.These two components both had crystal structure and good thermal stability.The thermal decomposition temperatures were 239.54 ? and 236.11 ?,respectively.SCVP-1 exhibit spherical chain conformation in solution,and SCVP-2 exhibit random coil chain conformation in solution.The particle size distribution of the two polysaccharide molecules was relatively dispersed,and aggregation behavior existed in solution.The effective diameters were 1112.4 nm and 1316.0 nm,respectively.The two sea cucumber visceral polysaccharides were both anionic polysaccharides.(3)Evaluation of immunoregulatory activity in vitro of sea cucumber visceral polysaccharides: SCVP-1 could promote the secretion of NO,IL-1?,IL-6 and TNF-?by RAW264.7 macrophages and enhance the phagocytosis of macrophages.TLR4 receptor was the target of SCVP-1 to activate macrophages.SCVP-1 activated macrophages through MAPKs and NF-?B signal transduction pathways.SCVP-2 could regulate MAPKs and NF-?B signal transduction path ways to affect the excessive release of inflammatory cytokines,so as to achieve the anti-inflammatory effect.(4)Evaluation of immunoregulatory activity in vivo of sea cucumber visceral polysaccharides: SCVP-1 could significantly increase the immune organ index of immunosuppressive mice,promote the proliferation rate of spleen lymphocytes,enhance the phagocytic capacity of mice,effectively promote the production of hemolysin antibody,and increase the secretion level of cytokines IL-2,IL-4 and IFN-? in serum,effectively alleviate the delayed allergic reactions and improve the antioxidant capacity.However,SCVP-2 did not significantly improve the immune function and antioxidant capacity of immunosuppressed mice.(5)Application of sea cucumber visceral polysaccharide SCVP-1 in nutrition delivery system: The nanocomplex was constructed by self-assembly with negatively charged SCVP-1 and positively charged lysozyme by electrostatic force.The morphology of Ly/SCVP-1 nanocomplex was observed and characterized.The shape of the nanocomplex was spherical and the particle size was 197.1 nm.The curcumin(Cur)was loaded.Loaded with Cur Ly/SCVP-1 nanocomplex could significantly improve the stability of Cur in PBS and mouse serum,and improve the antitumor activity of Cur.The polysaccharide/protein nanocomposite system could improve the stability by loading unstable active ingredients,and had a good application prospect in the field of food and medicine.