The Separation, Purification And Bio-active Research On Polysaccharides From Clematis Chinensis Osbeck

In this paper, two homogeneous polysaccharides were separated and purified from Clematis chinensis Osbeck. The main contents of the papers are as follows:The optimization of extraction method of raw polysaccharides from Clematis chinensis Osbeck. First, monosaccharides, oligosaccharides and other micromolecule matters were removed by ethanol, and then orthogonal design method was used to optimize the extracting conditions. Considering the extraction efficiency and quality of polysaccharides, the final optimization extracting conditions were that the ratio of material to liquid ratio of 1:15,15h,80℃,and the ethyl alcohol concentration of 80%.The separation and purification of raw polysaccharides from Clematis chinensis Osbeck. First pigment and protein were removed from raw polysaccharides(CCP), then CCP was separated by DEAE-Sepharose Fast Flow column chromatography,We got three polysaccharides fractions: CCP-Ⅰ,CCP-Ⅱ,CCP-Ⅲ. CCP-Ⅲwas separated by Superdex-200 column chromatography, we got the fine purification polysaccharides: CCP-Ⅲa, CCP-Ⅲb.The study of the structure and the character of CCP-Ⅲa and CCP-Ⅲb preliminarily.CCP-Ⅲa and CCP-Ⅲb were analyzed and determined by phenol-sulphuricacid method, coomassie brilliant blue method,m-hydroxyldiphenyl, gel filtration, agarose gel electrophoresis, UV spectrum. Their chemical structures were characterized by periodate oxidation, GC and IR spectrum.The results of CCP-Ⅲa analysis were following: the total sugar content was 92.4%, uronic acid content was 27.6% and protein content was 6.9%. Its homogeneity was tested by agarose gel electrophoresis and HPLC. Its Mw was 3.1×10~5Da. The results of CCP-Ⅲb were following: the total sugar content was 89.9%, uronic acid content was 39.5% and protein content was 9.4%. Its homogeneity was tested by agarose gel electrophoresis and HPLC. Its Mw was 1.5×10~5Da. They had no obvious absorbance of protein and other substances. CCP-IIIa was an acidic polysaccharide consisting of Rha,Ara, Man, Glc, Gal, in the molar ratios 4.7:1.0:5.6:26.4:3.9 and CCP-Ⅲb was an also acidic polysaccharide consisting of Rha,Man,Glc,Gal,in the molar ratios 1.5:1.0:25.8:16.3. The configuration of them was mainlyβ-configuration. Their glucosidic bonds contained 1→3,1→6 linked-bonds.Pharmacological activity on anti-oxidation. CCP-Ⅲcould eliminate the hydroxyl free radical and the super oxygen anion free radical and it could raise the activities of SOD and GSH-PX in mice's serum and liver, reduce the content of MDA and liver index notably. CCP-Ⅲhad antioxidation effect in vitro and in vivo. Its antioxidation activities related to removing oxygen free radical.Pharmacological activity on anti-tumor. Effects of polysaccharides from Clematis chinensis Osbeck CCP,CCP-Ⅲ,CCP-Ⅲa,CCP-Ⅲb on the proliferation of two carcinoma cells were measured by MTT assay in vitro. The models of mice transplanted sarcoma S-180 were used for study the anti-tumor effects in vivo. Thymus indexes and spleen indexes were determined. The results showed they have no significant inhibition against tumor growth of HeLa cell and MCF-7 cell. All polysaccharides inhibited sarcoma S-180 at different degree in vivo. Among them, CCP-Ⅲshowed more strong anti-tumor activities. At the dose of 80 mg/kg, the average tumor inhibitory rate of CCP-Ⅲto S-180 was 41.8%.Furthermore, the effects of all polysaccharides on the immune organ indexes were different. The thymus indexes and spleen indexes in the groups treated with them were higher than control group.