Optimization Of Decoloration And Deproteinization Of Cultured Cordyceps Sinensis Polysaccharides And The Detection Of Its Antitumor Activity To NCI-H446

Objective:The method for decoloration and deproteinization of Cordyceps sinensis polysaccharides was optimized; and the Cultured Cordyceps sinensis polysaccharide (CCSP-1) was abained by separation and purification.Then with the GC-MS analysis, the monosaccharide composition and molar ratio were figured out. And the impact of CCSP-1on non-small cell lung cancer NCI-H446cell were also studied. Through the preliminary exploration of the mechanism, the results can provide experimental data and theoretical foundation for the further study.Methods:1、The extraction of Cultured Cordyceps sinensis polysaccharides and its process optimization of decoloration and deproteinization:In the study, water circumfluence and ethanol precipitation was used to extracted the Cordyceps sinensis polysaccharides. According to the single-factor experiments and orthogonal test which evaluated by the decoloring rate and loss rate of polysaccharides, the best bleaching condition were confirmed. Three methods including sevag method, TCA method and HC1method were compared to finding the best deproteinization which evaluated by the protein loss rate and loss rate of polysaccharides.2、The research of monosaccharide composition of Cultured Cordyceps sinensis polysaccharides (CCSP-1):CCSP-1was obtained water extraction and ethanol precipitation, and then purified by DEAE-52cellulose column chromatography and SephadexG-100dextran column chromatography. TFA acidolysis and acetic anhydride derivation were used of the GC-MS analysis, and then the monosaccharide composition and its molar ratio of ccsp-1were confirmed.3、The influence of CCSP-1to NCI-H446lung cancer cell:The inhibition of CCSP-1to NCI-H446in vitro was observed by MTT assay; With the PI staining, we detected the cell cycle of the NCI-H446after treated with different concentrations of ccsp-1. And then the Annexin V-FITC/PI double staining were used to quantitativeiy determin the viable apoptotic cell and non-viable apoptotic cell/non-viable non-apoptotic cell.Results:1、The optimum decoloration conditions was50℃,10min,quantity of active carbon was1.5%and pH=3.5. Compared with TCA method and HC1method, sevag method was better for the deproteinization effect.2、Cultured Cordyceps sinensis polysaccharides (CCSP-1) were usually composed of mannose, glucose and galactose with a molar ratio of1.7:4.4:1.3、MTT assay showed that CCSP-1can inhibit the the growth of NCI-H446cells with dose-dependent, and the IC50value is347μg/ml; and CCSP-1was inhibited the NCI-H446cells in G2/M phase and increase the cell growth in vivo, while the Annexin V-FITC/PI double staining result indicated the CCSP-1has little effect to NCI-H446cells apoptosis.