Extraction And Purification Of Polysaccharides From Epimedium Koreanum Nakai And Studies On Its Tumor-immune Activity

Ginseng,Poria and many other polysaccharide-rich tonic herbs in the promotion of immune aspects have acquired extensive development.The utilization of polysaccharides assisted in the treatment of cancer has been widely recognized.As a traditional tonic herb,Epimedium has the effect on improving immunity and anti-tumor,but its polysaccharide composition reported in the literature is relatively less,in order to better explain the nutritional benefits of Epimedium,the extraction,isolation,purification and tumor immunological activity of the polysaccharides from Epimedium koreanum Nakai have been systematically studied based on the accumulated study of it in our labtory.We performed a series of experiments including physical and chemical properties,structure analysis and mechanism of tumor immunity on polysaccharide with high purity and the most desirable bioactivity.The main contents are as following:To establish the best technology for the Epimedium polysaccharide,the extraction process of polysaccharide was studied by Box-Benhnken center combination test and response surface analysis method.The optimal extraction scheme was 16 times the amount of water,extracted twice at 87 ?,and 3 hours per time.According to the above process,the average yield of polysaccharide was 1.023%.The theoretical prediction value of polysaccharide yield was 1.045%,and the relative error was 2.15%,which indicated that the model parameters were optimized and reliable by response surface method.In order to study the immunological activity of the crude polysaccharide from Epimedium koreanum Nakai in vivo,we have established a Lewis lung cancer model using C57BL6 mice.The results showed that the crude polysaccharides could effectively prevent the atrophy of the immune organs of the body with tumor,increase the levels of immune cytokines IL-2 and IFN-?in different degrees,and reverse the decrease of the proportion of CD4 + and CD8 + T cells caused by tumor development,inhibit the expression of IDO in tumor tissues,change the proportion of M1 and M2 macrophages in blood,promote spleen cell proliferation,resulting an indirect anti-tumor effect.The crude polysaccharide was dialyzed(MW:3000)and then purified by modern separation and purification technology.The results showed that the polysaccharides were primarily purified according to the different properties and surface charge of DEAE-52 and Sephadex-100.The LLC-cytotoxicity test,spleen cell proliferation experiment,peritoneal macrophage phagocytosis function,and levels of cytokines secretion in vitro were conducted to carry out activity screening experiment.Then we obtained F3 which possesses the best immune activity and named as EPS.Applied with high performance liquid chromatography,NMR spectrometry,infrared,ultraviolet and other means,we do the analysis of EPS in structure.The results indicated that the composition of monosaccharide was mannose,rhamnose,glucuronic acid,galactose,glucose,galactose,arabinose and fructose.Molar ratio is 1:10.97:0.43:7.41:2.77:10.34:11.33:1.28,high efficiency gel permeation chromatography suggested EPS has a single peak,with an average molecular weight(MV)180×104Da calculated by GPC software.Infrared displayed polysaccharide general properties and the results showed that EPS has absorption peaks in the wavenumber 1017,840,618,537cm-1,indicating that EPS contains pyran ring,strong absorption near 1640,1420,indicating the presence of acidic groups-COOH.UV spectrum displayed EPS has absorption between 270-290 nm,indicating the existence of C=O,and the physical and chemical characteristics indicated that the uronic acid content of EPS is high.The immune activity of mouse peritoneal macrophages and antigen-presenting DC cells and T cells was explored by using the purified polysaccharide EPS.The results showed that EPS could promote the proliferation of macrophages and DC cells,and elevate the antigen presenting function.Besides,it may increase the level of IL-6,TNF-a secreted by macrophage,enhance immune antitumor activity,and promote DC cells to secrete IL-12,inducing Th cell differentiation.Furthermore,in the presence of ovalbumin(OVA),EPS can promote the proliferation of CD4 + T cells,increase the level of INF-y,and enhance the function of T cells to kill tumor cells.