Study Of Extraction And Purification Of Polysaccharides From Anemone Raddeana Regel And Antitumor Activity Of HepG-2 Cells

Objective:Through the study of the Anemone raddean Regel polysaccharide,for further study ofitsextractionprocess,separation andpurification,chemicalstructure,pharmacology,antitumor activity in vitro and provide scientific basis for the study of the Anemone raddean Regel polysaccharide structure,biological activity and its application development.Method:Extraction of polysaccharides from both ends by water extraction and alcohol precipitation,the best extraction process of the Anemone raddean Regel polysaccharide polysaccharides was obtained by orthogonal experiment;three fractions designated（0.1mol/L、0.3mol/L、0.5mol/L）fromAnemone raddean Regel were isolated by the DEME-52 column separation and dialysis method,determination of polysaccharide content by phenolsulfuric acid method.The polysaccharide were hydrolyzed into monosaccharides with 2 mol·L^-1trifluoroaceticacid.The monosaccharide obtained with1-phenyl-3-methyl-5-pyrazolone（PMP）were separated by reverse-phase HPLC using gradient elution process,and monitored by ultraviolet detector at 250nm.The antineoplasmic activity of polysaccharide samples on Hela cell was by MTT bioassay in vitro.The effect of Anemone raddean Regel polysaccharide on HepG-2 cell apoptosis was observed by AO-EB double staining fluorescence method.Using flow cytometry instrument to detect Anemone raddean Regel HepG-2 on the liver cancer cells apoptosis effect.Result:According to the results of owater extraction conditionst,the material ratio is1:15,the time is 1.5,the duration is 3h,the concentration ratio 3,70%alcohol precipitating for 12h.The results showed that Anemone raddeana Regel compose of D-glucose,D-ribose,L-rhamnose,L-arabinose,L-fucose,D-mannose,D-galactose,D-xylosebyreverse-phase HPLC.The yields of them were 0.97:0.49:0.67:2.31:7.37:0.82.The antitumor activity of polysaccharide samples against HepG-2 cells was determined by MTT bioassay in vitro.The results showed that all samples had obvious antitumor effect.The results showed that all samples had obvious antitumor activity against HepG-2 cells,and the anti-tumor effect was enhanced with the increase of the concentration.AO-EB double staining can be used to observe the effects of different concentrations of two-tip polysaccharides on the apoptosis of hepatocellular carcinoma cells,With the increase of concentration,the cells gradually apoptosis,from the normal cell structure gradually transformed into orange-red round or solid and bulk-like apoptotic cells,and the number of cells also decreased.The apoptosis of hepatoma HepG-2 cells was detected by flow cytometry.The apoptosis of HepG-2cellswas increased and the number of apoptotic cells increased with the decrease of polysaccharide solution.Conclusion:The optimum extraction process of polysaccharides from both ends was obtained by single factor and orthogonal test.The purified polysaccharides were purified by DEAE-52columnseparationmethodanddialysismethod.Determinationof Monosaccharide Composition of Polysaccharides from Anemone raddean Regel by High Performance Liquid Chromatography.MTT assay,AO-EB double staining fluorescence method,flow cytometry results showed that the Anemone raddean Regel polysaccharides on liver cancer cells HepG-2 have a certain inhibitory effect,can promote cell apoptosis.The above results can lay a foundation for the further research and development of the two tip polysaccharides.