Structural Characterization,Anti-Tumor And Immune Activity Of CPP1c,A Pectic Polysaccharide From Wen Dangshen

Codonopsis pilosula polysaccharide（CPP）is the principle active constituents of Codonopsis pilosula（or Dangshen）.Nowadays,there are many reports about the study of CPP,however,most of them are about the basic phytochemical and bioactive studies.Little literatures reported the fine structure of CPP,and barely literatures elucidated the mechanisms of their bioactivities.Based on the previous study,this paper was aimed to analysis the fine structure,the antitumor and the immunoenhancing mechanisms of the homogeneous pectic polysaccharides from Wen Dangshen（CPP1a and CPP1c）.And the specific research contents are as follows:Part one:the structural characterization of CPP1a and CPP1cHigh performance gel permeation chromatography was used to determine the molecular weight.The results showed that the relative molecular weight and the absolute molecular weight of CPP1a and CPP1c were 1.01×10⁵ Da and 1.259×10⁵ Da,1.03×10⁵ Da and 1.491×10⁵ Da,respectively.Meanwhile,CPP1a and CPP1c were hyper-branched polymers in 0.9%NaCl and0.2%NaN₃ aqueous solution.The results of partial acidic hydrolysis,methylation analysis,GC,GC-MS,IR,NMR showed that CPP1a was composed of five types of linkages,1,4-linked Rha,1,5-linked Ara,1,4-linked GalA,1,6-linked Gal and 1,4-linked Glu at a molar ratio of 1:12:1:10:3.CPP1c was composed of four types of linkages,1,4-linked Rha,1,5-linked Ara,1,4-linked GalA and 1,6-linked Gal at a molar ratio of 3:1:33:2,and its side chain was just composed of 1,4-linked GalA.Additionally,TEM observations revealed that the ultrastructure of CPP1c was like a chain composed of ovoid-shape particles,with diameters of approximately 100 nm,while SEM showed CPP1c had a surface with a sheet-like appearance.Part two:the study on the antitumor activities of CPP1a and CPP1cHepG2 cell was employed for in vitro studies on changes of morphology by Wright’s stain method,cell migration with wound healing method,cell cycle distribution,and the expression of apoptosis-associated proteins（Bax,Bcl-2 and Caspase-3）to study the mechanism of CPP1a and CPP1c mediating HepG2 cell apoptotic.The results demonstrated that the mechanism of CPP1a and CPP1c induced apoptosis of HepG2 cell through a mitochondrial apoptotic pathway,and the content of GalA were correlated positively with the antitumor activity.Part three:the study on the immunoenhancing activities of CPP1cThe immunocompromised SAMP8 mice were used to study the immunoenhancing activities of CPP1c on levels of molecule,cell and body,which was unraveled by the studies about the influence of CPP1c on the splenocyte proliferation,the percentages of CD4⁺,CD8⁺,CD28⁺and CD152⁺T cells,the secretion of cytokines（IL-2,TNF-αand IFN-γ）,the levels of related CD28,PI3K,P38MAPK mRNA and protein,and the homing of lymphocytes.The results demonstrated that CPP1c had potential of enhancing splenocyte proliferation,modulating the percentages of CD4⁺,CD8⁺,CD28⁺and CD152⁺T cells,improving the secretion of IL-2,TNF-αand IFN-γ,up-regulating the levels of CD28,PI3K,p38MAPK mRNA and protein,and promoting the homing of lymphocytes.Therefore,we proposed that CPP1c may improve the immune function of SAMP8 mice by promoting T cell activation through TCR/CD28 signaling pathways.