Study On Purification,Characterization And Biological Activity Of Polysaccharides From Cynanchum Atratum Bunge

This thesis mainly includes three parts:the first part is the extraction and purification of Cynanchum atratum bunge's polysaccharide,the second part is the analysis of Cynanchum atratum bunge's homogeneous polysaccharide structure,and the third part is the study of the biological activity of Cynanchum atratum bunge's homogeneous polysaccharide.Cynanchum atratum bunge was degrease with 95%ethanol at 90?for 3h;repeated twice,then evaporated ethanol to obtain the dry residue and extracted with boiling water at 100?for 3h;repeated twice and concentrated the extracting solution,centrifuged and used 95%ethanol to precipitate until the final concentration reached75%,Finally,centrifuged and collected the precipitates,freeze-dried to obtain Cynanchum atratum bunge's crude polysaccharide,named CAP.Cynanchum atratum bunge's crude polysaccharide was divided into four polysaccharide parts with different charge by DEAE Sepharose Fast Flow Gel column chromatography that named CAP-W,CAP-1,CAP-2,CAP-5.Using HPLC method to determine their molecular weight distribution,then choose the appropriate gel filtration chromatography.Two homogeneous polysaccharides that named CAPW-1 and CAP-01-2 were isolated from the plant for the first time.Then the structure of homogeneous polysaccharides was analyzed.The primary structure of these two homogeneous polysaccharides was analyzed by various chemical methods?monosaccharides composition analysis,partial acid hydrolysis,methylation analysis,etc.?and spectroscopic methods?NMR,IR,HPGPC,GC-MS,etc.?,The molecular weight of CAPW-1 is 6.4×10⁴Dalton.The monosaccharide composition analysis shows that CAPW-1 is mainly composed of arabinose and galactose?Ara:Gal=4:1?.The main chain and fragments obtained from partial acid hydrolysis were analyzed by GC-MS.It was found that arabinose is mainly in branched chain,and the main chain is composed of galactose.Methylation results showed that galactose is mainly linked to 1,4-Galp,1,3-Galp,1,6-Galp and 1,3,6-galp,and arabinose is linked to 1,3-Araf,1,5-Araf,1,3,5-Araf.According to the experimental results,the structure of CAPW-1 is arabinose surrounded by the main chain formed by galactose.It is a kind of AG-II type polysaccharide.The molecular weight of CAP-01-2 is 1.9×10⁴ Da,and composes of galactose,connected by 1,4-galp,1,3,4-Galp,¹³C NMR map in the 108-150ppm field appeares some special signals:146.49ppm,108.5PPM,they are the glucuronic acid lost the carboxyl group in the4-bit and 5-bit unsaturated double bond after the display of the characteristic signal,that is the hexenuronic,in summary,CAP-01-2 is a 1,4-homogalacturonans that is linked together hexenuronic at C-3,is a class of pectin polysaccharide.The third part of the content is the activity research of two different types of homogeneous polysaccharides.First of all,the immune activity of two kinds of polysaccharides were studied,the experimental results showes that both the homogeneous polysaccharides have good immune enhancement activity,can promote the proliferation of RAW264.7 macrophages,while activating cells,they could promote NO's release and IL-6's secretion.They have significant proliferative effect on spleen lymphocytes,and have synergistic effect with Con A,and have the concentration dependence relation.On the other hand,the antioxidant activity of two kinds of polysaccharides was studied,and the experimental results showes that two homogeneous polysaccharides have a certain degree of antioxidant effect,and have concentration dependence,and the CAP-01-2 antioxidant effect of HG type is better,and the radical scavenging rate of CAP-01-2 reaches 90%when the concentration was2.5mg/m L,And CAPW-1 is only 70%,presumably associated with the presence of hexenuronic.