The Preliminary Exploration On The Degradation And The Active Oligosaccharides Fragments Of The Glycoconjugate LbGp4 From Fruit Of Lycium Barbarum L.

Lycium barbarum L, belongs to the family Solanaceae. The fruit has been used as a traditional herbal medicine, with the function of nourishing both the liver and kidney, producing nourishing essence and enhancing vision. The bioactive components of L.barbarum fruit have been mainly attributed to its polysaccharide LBP, which has been lucubrated in the last few decades, about the physicochemical properties, structural characterization and bioactivity. According the previous studies, LBP, especially LbGp4 can enhance the immune function.The bioactivities depend on the interactions between the oligosaccharide fragments and the receptors. The immunomodulatory activities were mediated by specific recepors. In this study, we will give a preliminary exploration on the degradation of the polysaccharide LbGp4 and the active oligosaccharides.1. Isolation, purification of glycoconjugate LbGp4 and its physicochemical propertiesCrude polysaccharide LBP was isolated from the fruit of Lycium barbarum L. by water extraction and ethanol precipitation, followed by removal of protein. Then the polysaccharide LbGp4 was purified by DEAE-Cellulose column chromatography followed by Sephadex G-100 column chromatography from the crude polysaccharide. HPGPC analysis showed that LbGp4 was homogeneous. The carbohydrate content of LbGp4 was 86.73% according to the phenol-sulfuric acid method. Monosaccharide composition analysis by GC revealed that it was composed of Ara, Gal, Rha and Xyl with a molar ratio of 1:1.3:0.11:0.082. The degradation of the glycoconjugate LbGp4By Pronase E andβ-elimination, the glycans LbGp4-OL and LbGp4-OL-βwere released. HPGPC showed they were both homogeneous and the monosaccharide composition was Rha, Ara and Gal (1:17.63:17.54 and 1:15.72:16.21). LbGp4-OL was treated with an endo-β-1,4-xylanase during 24 h,48 h and 72 h selectively, GC analysis revealed that the branched chain can't be cleaved efficiently. Then LbGp4-OL was partially hydrolyzed with 40 mmol/L H2SO4 at 80℃during 10 h. The dialyzable fraction LbGp4-OL-P mainly contained galactose. LbGp4-OL-P was further treated with 0.5 mol/L TFA. By ESI-MS analysis, oligosaccharides with a DP 2-8 were obtained, named OL-oligosaccharides.3. Effects of LbGp4 and its oligosaccharide fragments on splenocyte proliferationTo screen the immunomodulatory activities of LbGp4 and its oligosaccharide fragments, mouse splenocytes were stimulated with LbGp4 or its oligosaccharide fragments at serial concentrations. Lymphocytes proliferations were analyzed with 3H-TdR incorporation method using aβ-scintillation counter. We herein report that LbGp4-OL induced splenocyte proliferation from Balb/C mice in a dose-dependent manner, after derived by FITC, the immunomodulatory activities has no change. On the contrary, the activities of LbGp4-OL-P and OL-oligosaccharides reduce obviously, and OL-oligosaccharides can be an inhibiter in a high dose.Conclusion:A series of the oligosccharide fragments of glycoconjugate LbGp4 were obtained. By screening the immunomodulatory activities, the activities reduce obviously. But herein the methods for the degradation of the glycoconjugate LbGp4, the detection of the mass spectrum and the polysaccharide derived by fluorometric reagent, were proposed. The achievements lay a rationale for the further research. The bioactivities is closely bound up with the structural information, so in the subsequent research, it should focus on the controllable degradation of the polysaccharide, according to the structure-activity relationship.Thus, the active oligosaccharide fragments may be identified.