Isolation, Purification, Structural Characterization And Immunostimulating Activities Of Jellyfish Polysaccharides

Jellyfish (Rhopilema esculetum) is an important marine creature for edible-medicinal use and widely cultured in traditional Chinese fishery. It has been reported that polysaccharide is one of the bioactive constituents of jellyfish. In our previous studies, the crude jellyfish polysaccharides were found to have the potential of immunostimulating activity. In this paper, isolation, purification, structural characterization and immunostimulating activities of jellyfish polysaccharide were studied. The obtained results were as follows:(1) The crude polysaccharide was extracted from jellyfish and a homogeneous fraction (JSP 11) was purified by DEAE Cellulose DE52 anion exchange chromatography and size exclusion charomatography of Sephacryl S-500. The average molecular weight of JSP 11 was estimated to be 1.25×106 Da by high performance liquid chromatography.(2) Monosaccharide composition analysis showed that JSP 11 was composed of mannose, galactose and glucuronic acid in a molar ratio of 2.18:1.00:1.94. Methylation analysis indicated that JSP 11 was composed of three glycosidic linkages of 1,3,6-Manp, 1,6-Galp and 1-GlcpA and there molar ratios were 1.98:1.00:2.03. Based on the structural features of JSP11, combined with NMR techniques, all 1H and 13C NMR signals were assigned to the sugar residues. Accordingly, the sequence of sugar residues in the backbone of JSP 11 was confirmed.(3) Methyl thiazolyl tetrazolium(MTT) assay showed JSP 11 can stimulate the proliferation of RAW264.7 cells when the concentration ranged from 10 to 200 μg/mL. Moreover, JSP11 could promote RAW264.7 cells to release NO (nitric oxide), TNF-a (tumor necrosis factor-a) and IL-1β (interleukin 1β) in a dose-dependent manner. When the cells were treated with 200 μg/mL JSP11, the production of NO, TNF-a and IL-1β were enhanced by 140%,77% and 53.8% than those of the normal group, indicating the good potential of immunostimulating activity of JSP11. These results were also ascertained by RT-PCR experiments for analysis of mRNA expression of iNOs, TNF-a and IL-1β. Western blot analysis displayed that JSP 11 promoted the nuclear translocation of NF-κB (Nuclear factor kappa-B) p65 in RAW264.7 cells. Compared with the control group, JSP 11 increased the phosphorylation of P38(Mitogen-activated protein kinase P38), ERK1/2 (Extracellular regulated protein kinase 1/2), JNK (Jun N-terminal kinase) and Akt (Serine/threonine protein kinase B) proteins. These results indicated that JSP 11 activates macrophages possibly through NF-κB, MAPK (Mitogen-activated protein kinases) and PI3K/Akt (phosphatidylinositol-3-kinase) signal transduction pathways.