Preparation And Biological Activities Of A Succinoglycan Riclin

Microbial polysaccharides are natural biological macromolecules and have been widely applied in food and medicine fields due to multiple biological activities such as anti-tumor,hypolipidemic,anti-oxidation,hypoglycemic,anti-coagulation,and anti-virus activities.Succinoglycan is a water-soluble exopolysaccharide that can be produced by Agrobacterium,Rhizobium genera etc.Succinoglycan is composed of one galactose residue per seven glucose residues along with some non-carbohydrate substituents,including pyruvate,succinate,and acetate.Succinoglycan mediates the interaction between bacteria and plants and plays an important role in symbiotic nitrogen fixation.However,there is little study about succinoglycan biological activities toward human and mammals in our knowledge.The present study aimed to prepare a stable-structure succinoglycan and explore the anti-tumor activity and cholesterol-lowering activity of succinoglycan.The main contents are as follows:1.A preparation method of structurally stable succinoglycan was developed.Compared with other eleven rhizobial strains from different origins,Agrobacterium sp.ZCC3656exhibited the highest exopolysaccharide(EPS)yield(18-20 g/L).EPS production was performed in modified M9 salts supplemented with sucrose as carbon source at 30�C for 48 h.The EPS was purified and the only one polysaccharide was designated riclin.The molecular weight of riclin is approximately 2.5�10~6 Da.The combined results of monosaccharide composition,methylation analysis,1D and 2D nuclear magnetic esonance analysis(NMR)indicated that riclin is comprised of glucose and galactose at a ratio of 7:1,along with pyruvate and succinate substituents.The structure of riclin is consistent with the typical succinoglycan.Riclin was suspended in water(1%w/v),and p H was adjusted to 9.0 with sodium hydroxide.After being autoclaved at 105�C for 20 min,the succinyl was removed completely,resulting in a more stable succinoglycan.2.The antitumor effect of de-succinyl riclin was investigated in sarcoma 180 tumor-bearing mice.The chemotherapy drug cyclophosphamide(CTX)was used as a positive control.The results showed that both intraperitoneally injection and orally gavage riclin inhibited the tumor growth significantly.The tumor inhibition rate of riclin(10 mg/kg)was 68%.Riclin could not inhibit the proliferation of S180 tumor cells directly.CTX decreased the body weight markedly,reduced white blood cells in the peripheral blood and caused serious damage to spleen structure in mice.Riclin increased the spleen index and decreased splenocytes apoptosis through Bcl-2/Bax/Caspase-3 apoptotic signaling pathway.~1H NMR-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes.Riclin increased the number of white blood cells and lymphocytes in the peripheral blood,and increased the level of tumor necrosis factor?(TNF?)in serum.Flow cytometer analysis showed that riclin promoted marcophages proliferation in tumor microenironment as well as CD4~+and CD8~+lymphocytes proliferation in tumor microenironment,lymph nodes,thymus and spleen.The fluorescent labeling of riclin with Alexa Fluor?488 Cadaverine showed that riclin can specifically recognize macrophages.Succinoglycan riclin performs strong immunogenicity and suppresses tumor growth effectively.3.The cholesterol-lowering effect of de-succinyl riclin was investigated in a hypercholesterolemia mouse model.We found that dietary riclin alleviated physiological changes effectively induced by high-cholesterol-diet(HCD).Compared with the HCD group,the high-riclin group significantly decreased levels of the serum total cholesterol,low density lipoprotein cholesterol,and hepatic cholesterol by 34,40,and 51%,respectively,consequently reducing proinflammatory cytokine expressions(TNFa,IL-6,IFN-?).Lipidomics and metabolomics analyses revealed that the riclin group partially reversed metabolic profile changes induced by the HCD,approaching that of the ND group.Riclin-treated mice exhibited significantly larger fecal outputs than those of ND and HCD mice.Riclin increased excretion of cholesterol and acid sterols.The results suggested that riclin inhibited the absorption of cholesterol.Riclin increased the bile acid-cholesterol solution viscosity and interfered in the emulsification process,thus decreasing cholesterol digestion and promoting cholesterol and bile acid excretion in the feces.Riclin improves lipid disorders induced by HCD.Succinoglycan riclin is a potential functional food additive in attenuating diet-induced hypercholesterolemia.In summary,Agrobacterium sp.ZCC3656 produces succinoglycan riclin with a fermentation yield of 18-20 g/L.The de-succinyl riclin inhibits sarcoma growth by enhancing immune function and reduces cholesterol absorption via interfering in the process of bile acid-cholesterol emulsification.The results contributes the basic understanding of succinoglycan and provides the new application of succinoglycan riclin in the fields of medicine and food.