Structural Analysis And Antitumor Activity Of Huanghuabai And Polysaccharides

Bletilla ochracea Schltr is a kind of perennial herbaceous medicine plants which belong to Bletilla of Orchidaceae,it has many uses and often used as substitute for Bletilla striata.B.ochracea as a kind of chinese traditional medicine,which is full of polysaccharide.So far,the medicinal component polysaccharide and medicinal function of B.ochracea are seldom reported.Base on above situation,this study researched the B.ochracea about its polysaccharide structure and medicinal functions,which could provide more information for exploitation of B.ochracea.The main results and conclusions:1.The optimization of polysaccharide extraction process:used the Response Surface Methodology(RSM)to analyse the extraction process,the results indicated that the optimal technological parameters as follows:extraction tempreture was 80?,extracted time was 90 min and liquid/solid ration was 30:1.Under this technology,the yield of polysaccharide was 26.45%.2.Preparation of the pure polysaccharide:use the optimal process to extract the BOP by water extract and alcohol precipitate.Removed out the pigment and protein to get the rough polysaccharide,then purified by DEAE-52 Cellulose and Sephadex G-150 column chromatography to get the pure polysaccharide.The Uv wavelength scanning was used to investigate the purity of polysaccharide,there was no wave in 260 nm and 280 nm indicated that the polysaccharide was pure,named it as B.ochracea polysaccharide(BOP).3.Analysed the structure of polysaccharide:the HPGPC measured out the molecular weight of BOP was 4.9×105 Da and the GC-MS indicated that BOP was mainly composed of glucose and mannose(molar ratio of 7.88:2.12).FT-IR,methylation,and NMR spectral analyses demonstrated the structure of BOP.4.The anticancer function of polysaccharide research in vitro:treated CT26 cells with 50 to 800 ?g/ml of BOP for 24 h or 48 h,the MTT assays showed that this polysaccharide had little impact on the proliferation or death of those cells in vitro,based on OD values that there were no significant difference among treatment groups(p>0.05).Therefore,BOP did not appear to have anti-tumor properties in vitro.Then the antitumor and immunity function of polysaccharide was researched in vivo:the CT26 tumor-bearing mice model was established and then the mice were assigned to five daily-treatment groups(eight mice per group),BOP doses of 50,100,or 200 mg/kg;positive control(20 mg/kg 5-Fu),or negative control(physiological saline solution).The tumor-bearing mice were treated with BOP for two weeks,then assessed the efficacy.The results showed that BOP significantly decreased tumor growth in a dose-dependent manner.After paraffin-sectioning and H&E-staining,tumors from the saline-control group showed compact cells with intact structures.In contrast,the cells were sparse and separated from each other in tumors from mouse groups that had received BOP,the tissue structure was more seriously damaged in tumors from the 5-FU and BOP(200 mg/kg)groups,exhibiting many nuclear fragments and obvious vacant sections.5.In order to study the mechanism of antitumor function in CT26 tumor-bearing mice model,the immune ability of tumor-bearing mouse was investigated and the results showed that the treated mice were more healthy.For example,in the treated mice,the spleen lymphocyte proliferation and the macrophage phagocytosis were all enhanced.Meanwhile,the TNF-?,IL-1? and NO began to release much more.Based on the above results,we can conclude that the BOP may have the function of immunoregulation and antitumor.