Structure Identification And Immune Activity Of Polysaccharides From Ampelopsis Japonica(Thunb.) Makino

Objective Ampelopsis jaPonica(Thunb.)Makino as traditional Chinese medicine raw material,were extracted and were separated by molecular weight using membrane separation technique.The structural characteristics were analyzed by chemical method and spectral method,to determine the immunoregulatory activity of the polysaccharide type,in order to further study its mechanism and formulation innovation to lay the foundation.Methods(1)The method of response surface analysis was used to optimize the extraction process of polysaccharide from water,and the protein was removed by TCA method.The molecular weight cut off of the membrane was 150 kDa,100 kDa,50 kDa,20 kDa,10 kDa and 6 kDa,by the membrane separation of the total polysaccharides(AJP).(2)The immunological activity of polysaccharides from seven different molecular weight segments were studied in vitro.The effects of polysaccharides on the proliferation of splenocytes in vitro were studied.Three in vitro immunoreactivity was selected for in vitro immunization.The effects of monocyte/macrophage function,the effects of T and B lymphocyte proliferative responses and the levels of TNF-? in serum of immunocompromised mice were studied by the effects of polysaccharide on the body weight and immune organ index of immunocompromised mice,The effect of IL-1 on immune function was studied.(3)The filtered polysaccharides were separated by DEAE Cellulose 52 cellulose column chromatography.The purity and molecular weight of polysaccharides were determined by high performance gel permeation chromatography.Infrared spectroscopy,high performance liquid chromatography,periodic acid oxidation,Smith degradation,methylation,Study on the Structural Characteristics of Homoptera Polysaccharide by Chromatography.(4)The immunological activity of the polysaccharide was confirmed by the immunological activity of mice in vitro..Results(1)On the basis of single factor test,the optimum process parameters were as follows: the ratio of material to liquid 1:20,extraction time 150 min,extraction temperature 100 ?,extraction rate of Ampelopsis japonica(Thunb.)Makino polysaccharide was 12.6 %.(>150 kDa?100~150 kDa?50~100 kDa?20~50 kDa?10~20 kDa?6~10 kDa ?<6 kDa)with different molecular weight ranges were obtained by membrane separation technique.AJP-1,AJP-3,AJP-4,AJP-4,AJP-5,AJP-6 and AJP-7,respectively.The proportion of total polysaccharides was 5.9%,42.3%,7.7%,6.4% 5.3%,4.5% and 27.9%,respectively.The contents of polysaccharides were 23.2%,39.3%,42.5%,49.7%,37.8%,40.7% and 29.5% respectively.Seven groups of AJP-4,AJP-6 and AJP-7 were screened by three groups of AJP with different molecular weight in vitro.(2)Immunoreactivity studies in immunocompromised mice determine that AJP-4 and AJP-7 are the most important immunoreactive substances.(3)AJP-4 and AJP-7a were isolated and purified by DEAE Cellulose 52 column chromatography.They were named AJP-4a,AJP-4b,AJP-4c and AJP-7a respectively.The molecular weight of four Ampelopsis japonica(Thunb.)Makino polysaccharides 4.76×104 Da?2.04×104 Da?3.80×104 Da ? 4.89×103 Da.AJP-4a is composed of Man,Rha,GlcA,Glc,Gal and Xyl,the molar ratio of 18.66: 16.81: 2.08: 1.00: 13.11: 8.23,the main chain may be(1?6)connected Glcp composition.AJP-4b is composed of Man,Rha,GlcA,GalA,Glc,Gal and Xyl,the molar ratio of which is 36.34: 39.04: 3.90: 4.15: 1.00: 27.47: 20.21,the main chain may be mainly composed of two Glycosidic linkages whose terminal residues are Rhap and Glcp.AJP-4c is composed of Man,Rha,GlcA,GalA,Glc,Gal and Xyl at a molar ratio of 18.06: 6.98: 2.00: 10.62: 1.00: 11.92: 5.81,and its main chain may be linked by(1?2)Galp.AJP-7a is composed of Man,Rha,GalA,Glc,Gal and Xyl with a molar ratio of 21.25: 8.57: 11.24: 1.00: 8.44: 5.91,consisting mainly of Glc and Gal.(4)AJP-4a,AJP-4b,AJP-4c,and AJP-7a significantly promoted the proliferation of spleen cells induced by ConA and LPS in vitro.Conclusions To determine the extraction process with water decoction for the extraction of Ampelopsis japonica(Thunb.)Makino polysaccharides.Different molecular weight segments AJP-4 and AJP-7 have obvious immune enhancement effect,and different molecular weight of white polysaccharide activity is different.Four homologous polysaccharides were obtained by separation and purification,and their structures were studied preliminarily.