党参多糖硫酸酯抗人甲型流感病毒的评价及作用机制研究

Influenza A viruses mutate easily into new subtypes, which cause serious damage tohuman health. Now drug applications for ant Influenza A viruses in the clinical treatment exist adverse reactions and increasing drug resistance. Preliminary experiments found that the polysaccharide obtained from codonopsis pilosula has preventive effect on influenza virus H3N2, but after codonopsis pilosula polysaccharide sulfate was prepared by sulfuric acid modification, inhibition on the H3N2 virus is very significant. This project will develop in vivo and in vitro test against influenza a virus H1N1 and H3N2 role as a guide, using membrane separation and Sephadex chromatography column separation system to obtain the uniform codonopsis pilosula polysaccharide,chlorosulfonic acid-pyridine method prepared Influenza A viruses mutate easily into new subtypes, which cause serious damage to human health. Now drug applications for anti Influenza A viruses in the clinical treatment exist adverse reactions and increasing drug resistance. Preliminary experiments found that the polysaccharide obtained from codonopsis pilosula has preventive effect on influenza virus H3N2, but after codonopsis pilosula polysaccharide sulfate was prepared by sulfuric acid modification, inhibition on the H3N2 virus is very significant. This project will develop in vivo and in vitro test against influenza a virus H1N1 and H3N2 role as a guide, using membrane separation and Sephadex chromatography column separation system to obtain the uniform codonopsis pilosula polysaccharide,chlorosulfonic acid-pyridine method prepared corresponding codonopsis pilosula polysaccharide.sulfate, with using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry,ultraviolet visible spectrophotometry, infrared spectrophotometry, nuclear magnetic resonance spectroscopy and other spectroscopic methods for the weight of codonopsis pilosula polysaccharide sulfate structural characterization, and determination of the molecular structure.Research on molecular size of polysaccharide sulfate, sulfuric acid substitution degree, sulfuric acid substitution effect on the position of active anti influenza virus.and analyze its preliminary mechanism. The successful completion of this project, will select a series of codonopsis pilosula polysaccharide sulfate esters of influenza viruses are highly selective, as anti influenza drug material basis and the oretical basis for the development of highly selective on influenza A virus.corresponding codonopsis pilosula polysaccharide sulfate, with using liquid chromatography-mass spectrometry, gas chromatography -mass spectrometry,ultraviolet visible spectrophotometry, infrared spectrophotometry,nuclear magnetic resonance spectroscopy and other spectroscopic methods for the weight of codonopsis pilosula polysaccharide sulfate structural characterization, and determination of the molecular structure.Research on molecular size of polysaccharide sulfate, sulfuric acid substitution degree, sulfuric acid substitution effect on the position of active anti-influenza virus and analyze its preliminary mechanism. The successful completion of this project, will select a series of codonopsis pilosula polysaccharide sulfate esters of influenza viruses are highly selective, as anti influenza drug material basis and theoretical basis for the development of highly selective on influenza A virus.

甲型流感病毒易变异产生新的亚型。导致临床治疗中应用的药物耐药，本课题组预试验发现从中药党参中提取获得的党参多糖对甲型流感病毒H3N2具有预防效果，而经硫酸化修饰后制备得到的党参多糖硫酸酯，对H3N2病毒明显的抑制作用。该申请计划以体内、体外抗甲型流感病毒H1N1、H3N2作用为指导，用膜分离以及葡聚糖凝胶色谱柱系统分离获取分子量为6960Da的均一党参多糖，氯磺酸-吡啶法制备相应的党参多糖硫酸酯，采用液相色谱-质谱、气相色谱-质谱、紫外、红外、核磁共振等方法对党参多糖硫酸酯进行结构表征，测定其分子结构组成。研究党参多糖硫酸酯硫酸基取代度、硫酸基取代位置对甲型流感病毒的作用效果，分析其抗流感病毒的作用机制。筛选出对甲型流感病毒有高度选择性的党参多糖硫酸酯类化合物，为开发对甲型流感病毒具有高度选择性的新药提供基础。

本课题组从中药党参中提取分离获得党参多糖，用膜分离以及葡聚糖凝胶色谱柱系统分离获取10个不同分子量均一党参多糖，氯磺酸-吡啶法制备相应的党参多糖硫酸酯，采用液相色谱-质谱、气相色谱-质谱、紫外、红外、核磁共振等方法对党参多糖硫酸酯进行结构表征，测定其分子结构组成。（1）经硫酸化修饰后制备得到的党参多糖硫酸酯，对H1N1、H3N2病毒明显的抑制作用。研究党参多糖硫酸酯硫酸基取代度、硫酸基取代位置对甲型流感病毒的作用效果，分析其抗流感病毒的作用机制。（2）开展了党参均一多糖及其硫酸酯对人结肠癌细胞HT29、对胰腺癌细胞SW1990、对肝癌细胞（HepG2和SMCC7721）、人体结肠癌细胞HCT-116增殖毒性初步研究。（3）党参多糖的抗氧化活性初步研究。通过体外多糖清除DPPH自由基实验，表明党参多糖均具有一定的抗氧化活性，但分子量不同，结构相异，带来党参多糖的抗氧化能力不同。（4）构建体外免疫活性测试模型，在体外细胞上检测党参多糖对大鼠脾淋巴细胞和腹腔巨噬细胞增殖及相关细胞因子的影响，从而明确党参多糖的体外免疫调节活性；通过构建环磷酰胺诱导免疫功能低下大鼠模型，检测党参多糖对免疫功能低下大鼠免疫功能的调节作用，明确党参多糖的体内免疫调节活性及作用机制。（5）糖苷衍生物的设计合成及活性作用研究。对天麻素糖苷小分子结构进行改造以期设计合成活性高，毒性低的糖苷衍生物。利用细胞增殖抑制实验对目标化合物进行了抗肝癌测试，结果表明化合物对肝癌细胞株HepG2和SMCC7721都与一定的抑制作用。