Study On Purification,Preliminary Structure And Immunological Activity Of Konjac Oligosaccharide

Konjac oligosaccharides(KOS)is the degradation product of Konjac Glucomannan(KGM),which has prebiotic activity.To study the immune activity of KOS,crude konjac oligosaccharides was purified by column separation technology,the structure information of KOS monosaccharide composition and glycosidic bond connection type were analyzed and researched via acid hydrolysis,periodate oxidation and Smith degradation,combining with the GC,LC-ESI-MS,IR and so on.In addition,in order to evaluate the immune activity of KOS,the immunosuppressed mice model induced by cyclophosphamide(CY)and KOS induced RAW264.7 cell immunomodulatory experiment in vitro was conducted.The main results were as follows:1.The separation,purification and structural analyses of KOSThree components of KOS-A,KOS-B,KOS-C were isolated from crude konjac oligosaccharides by DEAE 52 ion exchange column;the main collection components of KOS-A(accounts for 83.7% of the total amount of sugar)was further separated by Sephadex G-15,and one single component named KOS1 was obtained;KOS1 was purified and identified by Sephadex G-15 and ultraviolet band spectrum scanning,the results showed that KOS1 was the oligosaccharide with high homogeneous degree.Gas chromatographic analysis of KOS 1 proved that the monosaccharide composition of KOS1 contained D-mannose and D-glucose,and molecular ratio of Man:Glu were 1.6:1;the infrared spectrogram indicated KOS1 had the characteristic absorption peak of sugarmoieties,which also containing pyranoside and a-D-mannose;the results of periodate oxidation and Smith degradation indicated that six glycosidic bond types existed in KOS 1,contained 1?3,1?4,1?2,1?6 glycosidic bond,there is a nonreducing end group 1?or a 1?6 glycosidic bond per 4.3 residues,there is a 1?or a 1?6 glycosidic bond per 1.4 residues;KOSlcontained oligosaccharides with the degree of polymerization of 2-5 by the LC-ESI-MS/MS.2.The immunological activity of Konjac oligosaccharide in vivoKOS1 has antagonistic effect on immunity of the low immune mice induced by cyclophosphamide(CY).Compared with model control group,the thymus index and spleen index of each KOS1 dose group showed a rising trend,the thymus index of the mice at the dose of 1000 mg/(kg-bw)KOS1 had a significant difference(P<0.01);KOS1 could increase the secretion of IFN-?,IL-4 and TNF-? in a dose-dependent manner;and promote the proliferation of spleen lymphocytes in immunosuppressive mice cooperating with ConA or LPS,which also could enhance the tumor cell killing activity of NK cells in immunosuppression mice;1000 mg/(kg.bw)KOS1 could significant increase the phagocytic index of macrophage in immunosuppression mice induced by CY;the level of serum hemolysin was significant increased at the dose of 250-1000 mg/(kg.bw)KOS1 in immunosuppression mice(P<0.01),and which was near to the level of normal;the activity of Lactate dehydrogenase(LDH)at each KOS1 dose was significant increased in a dose-dependent manner(P<0.01).3.The immunoregulatory effects of Konjac oligosaccharide on peritoneal macrophagesRAW264.7 macrophages were used as the cell model to evaluate the immunological activity of KOS 1 in vitro.KOS1 can promote RAW264.7 cell proliferation,and there is no direct killing effect on the HepG-2 cells;however the culture medium collected from RAW264.7 induced by various doses of KOS1 and KOS 1-treated macrophages could significantly inhibit the proliferation of HepG-2.ELISA experimental results showed that the secretion of nitric oxide(NO),interleukin-1?(IL-1?),interleukin-6(IL-6)were increased in a dose-dependent manner with KOS1-treared RAW264.7 cells(P<0.05).RT-PCR results demonstrated that KOS1 can up-regulate the mRNA expression of iNOS,IL-1?,IL-6,which was consistent with the results of cytokines;the gene expression level of NF-?B P65 was also significantly increased,suggesting that the immunological mechanisms of KOS 1 may be related to the NF-?B P65 signaling pathway.