Study On Preparation,Structure Characterization And Anti-tumor Activity Of Polysaccharide From Bupleurum Chinense DC.

Hepatocellular carcinoma(HCC)is a serious problem worldwide that can not be ignored.As a kind of traditional Chinese medicine,Bupleurum chinense DC.has a variety of activities,such as anti-inflammatory,analgesic,anti-virus and so on.Polysaccharide is one of the main biological activities of Bupleurum chinense DC.,but there are few reports on its anti-tumor activity.In this study,the root of Bupleurum chinense DC.was used as raw material by water extraction and alcohol precipitation.The basic structure,physicochemical properties and anti-tumor activity of an acidic homogeneous polysaccharide(BCP)were studied by optimizing the extraction process of crude polysaccharide from Bupleurum chinense DC.In this study,the water extraction and alcohol precipitation method was used.Through the single factor and response surface optimization experiment,the optimal extraction conditions of Bupleurum chinense DC.crude polysaccharide were determined as follows:the material liquid ratio was 1:20,the extraction temperature was 90℃,the extraction time was 3 hours,the extraction times were 3 times,and the alcohol precipitation concentration was 60%.Under these conditions,the extraction rate of crude Bupleurum chinense DC.polysaccharide was 5.35±0.16%.The crude polysaccharide of Bupleurum chinense DC.were deproteinized by Sevag method,purified by mobile water dialysis(100 k Da)and Sephadex G-150 gel column,pure polysaccharides from Bupleurum chinense DC.were obtained and named BCP.The total soluble sugar,protein and uronic acid contents were 93.58±1.34%,0.65±0.22%and 9.64±0.35%respectively.UV wavelength scanning results showed that BCP contained trace protein and nucleic acid.High performance liquid chromatography showed that BCP was a homogeneous polysaccharide with a molecular weight of 2.01×10~3k Da.By ion chromatography analysis,BCP was composed of rhamnose,arabinose,galactose,glucose and galacturonic acid,and the molar ratio was 0.063:0.788:0.841:1:0.196.Infrared and Nuclear Magnetic Resonance Spectroscopy showed that BCP was an acidic pyran polysaccharide,and the main chain was composed ofβ-D-Gal、β-L-Rha、α-L-Araf、α-D-Gal andα-D-Glu.The results of Congo Red and Circular Dichroism indicated that BCP had a triple helix structure.The results of Scanning Electron Microscope showed that BCP was flake or clastic,with rough surface and mainly layered structure.Thermogravimetric and Differential Scanning Calorimeter analysis showed that BCP had good thermal stability in the range of 0-200℃.X-ray diffraction experiment showed that BCP was a kind of amorphous polysaccharide.The antitumor activity of BCP in vivo was investigated by establishing H22tumor-bearing mouse model.Various physiological indexes of mice showed that BCP inhibited the growth of H22 solid tumor and protected immune organ from injury.In addition,the JC-1 FITC-Annexin V/PI staining and cell cycle analysis have collectively shown that BCP is sufficient to induce apoptosis and of H22 hepatocarcinoma cells in a dose-dependent manner.The inhibitory effect of BCP on tumor growth was to block tumor cells in the S phase of cell cycle through mitochondrial apoptosis pathway.In summary,this study is dedicated to discovering a new natural safe drug,Bupleurum chinense DC.polysaccharide,which provides effective information for the treatment of liver cancer and application in the field of food.