维吾尔药源性多糖类物质的提取和动物抗病毒增强免疫活性及其作用机理的研究

The animal husbandry has developed rapidly in Xinjiang Uygur Autonomous Region and viral disease has been the important problem of the livestocks breeding. However, effective antiviral drugs and immunopotentiators have not been found at present. Xinjiang Uygur Autonomous Region is the place of origin of Uygur traditional medicine and the development of Uygur traditional medicine has been for thousands of years, its natural ingredient has many advantages that chemical drugs don't possess and unique curative effect. In order to utilize resources effectively, the effect of natural medicine can be improved through the molecular modification method which can enhance antiviral and antitumor activities of natural medicine, especially. In this study, uygur traditional medicine will be selected as the object.At first, total and fractional polysaccharides will be extracted respectively by water-decocting and one-step or stepwise ethanol precipitation method. Their antiviral and immune-enhancing activities in vitro will be compared and the active sites with better effects of polysaccharides will be selected and further sulfated. The antiviral and immune-enhancing activities of sulfated and natural polysaccharides will be determined in vitro and vivo, respectively. The best active polysaccharide will be eventually selected and clarify the mechanism.

新疆是我国畜牧业大省，病毒性传染病一直是我区养殖业的祸害，可至今仍尚未研发具有高效的抗病毒药和免疫增强制剂。新疆是维吾尔药的产地，又有着几千年的发展历史，其天然药物成分具有其它化学药物所不具备的优点和独特的疗效。为了有效的利用资源，通过分子修饰的方法可以提高天然药物功效，尤其是多糖结构修饰后,会使其加强抗病毒、增强免疫和抗肿瘤活性。本研究将8-10味维吾尔药作为研究对象，以NDV或IBDV作为指标、提取多糖类活性物质，活性追踪，筛选出效果较好的活性部位，对活性部位进行分子修饰，并体内外系统比较各多糖修饰前后抗病毒和增强免疫活性并筛选出活性最强的多糖同时阐明作用机理。本研究不仅对研究开发抗病毒和免疫增强制剂具有重要意义、而且为今后应用分子修饰研究其它新药提供研究思路和方法。

本课题选择葫芦、列当、阿魏、药桑、阿里红、一枝蒿、刺糖和芜菁为研究材料，以水提醇沉法提取各药物中的总多糖和分步醇沉多糖，用Sevag法脱蛋白，苯酚硫酸法测定糖含量，红外光谱法进行鉴定。通过体外实验筛选出各多糖中抗新城疫病毒（NDV）及淋巴细胞增殖作用较强的多糖，对筛选出的多糖进行纯化及硫酸化修饰，最后通过体内外试验筛选及验证纯化后的多糖或硫酸化多糖中抗NDV及增强免疫活性最强的活性成分。主要结果如下：.1. 采用Design Expert 8.0.6软件进行响应曲面试验优化葫芦多糖的最佳提取工艺。得到最佳提取条件为：提取温度82 ℃、水料比24:1 mL/g、提取时间2.3 h，提取次数2次，得到葫芦多糖提取率为5.810±0.240%。列当、药桑、阿魏、阿里红、一枝蒿、刺糖和芜菁多糖的提取率分别为：12.14%、1.37%、1.087%、1.27%、4.20%、10.2%及6.54%，提取到的各多糖在红外光谱下均具有多糖特征峰。.2. 体外抗NDV和免疫增殖作用的研究表明，各多糖中葫芦一步粗多糖（cLSPt）、阿魏50%分步醇沉多糖（cFSP50）、药桑70%分步醇沉多糖（cMP70）和阿里红一步粗多糖（cFAPt）有较强的抗NDV作用，病毒抑制率分别为61.85%、22.86%、20.07%及45.35%。各多糖中葫芦50%醇沉粗多糖（cLSP50）、阿魏50%分步醇沉多糖（cFSP50）和药桑30%分步醇沉多糖（cMP30）对淋巴细胞有较强的增殖作用。.3. 体内外抗新城疫病毒（NDV）和淋巴细胞增殖作用的研究表明，纯化后的多糖LSPt、FSP50、MP70、FAPt及硫酸化修饰物中，取代度为1.8的硫酸化葫芦多糖（sLSPt1.8）的体外抗NDV活性最好，病毒抑制率最高可达74.84%。sLSPt1.8注入新城疫模型雏鸡体内，能提高雏鸡抗体水平，降低死亡率，对鸡新城疫疾病有一定的疗效。LSP50、FSP50、MP30及硫酸化修饰物中，LSP50的淋巴细胞增殖作用最强，LSP50注入雏鸡体内，可显著提高新城疫疫苗接种的雏鸡一段时间内的抗体水平，提高淋巴细胞的增殖，增殖率为30.62%，显著提高IL-2、IL-6及IFN-γ等淋巴因子的分泌。