免疫活性多糖级分导向的黄精与酒黄精质量评价方法研究

As a kind of important effective components, polysaccharides were used as the quality evaluation index of many Chinese medicines. By acid hydrolysis, monosaccharides such as glucose were often used to calculate the content of polysaccharides, these methods lack specificity. In addition, the contents of polysaccharides calculated by these methods sometimes were inconsistent with the strength of activity, such as rhizoma polygonati and processed rhizoma polygonati. Our initial ultrafiltration separation-gel chromatography analysis showed, different molecular weight polysaccharide components in rhizoma polygonati and processed rhizoma polygonati had obvious differences. Because the polysaccharide activity was related with the structure, so the hypothesis was that, the immune activity differences between rhizoma polygonati and processed rhizoma polygonati, were caused by the composition differences of the activity molecular weight polysaccharide fraction.. This study will use ultrafiltration technology to obtain different molecular weight polysaccharide fractions; By peritoneal macrophages in vitro immunization and immunosuppression model in vivo, the activity fractions of polysaccharide were determined; Chemical analysis methods are used to contrast the differences of structure information in the activity fraction polysaccharide; Based on the immune activity polysaccharide, we establish the quality evaluation methods of rhizoma polygonati and processed rhizoma polygonati, and improve the quality standard of rhizoma polygonati and processed rhizoma polygonati, and also provide references for other Chinese medicines in which polysaccharide are used as quality control index.

多糖为重要的有效组分，很多中药以其作为质量评价指标。通过酸水解以单糖常用葡萄糖计算其含量，方法缺少专属性。更有研究显示，以此方法计算的多糖含量高低有时与活性强弱不符，如中药黄精与酒黄精，方法缺少科学性。我们初步超滤分离-凝胶色谱分析显示，不同分子量段的黄精与酒黄精多糖谱图有明显差异。由于多糖活性与结构有关。因此提出假说：黄精和酒黄精免疫活性的差异，是由于具有活性的分子量段多糖级分组成不同引起的。确定这些具有活性的多糖级分，并以其为指标，对于科学评价质量具有重要的实际应用价值。. 本研究运用超滤技术获取黄精和酒黄精不同分子量多糖级分；通过腹腔巨噬细胞体外免疫和免疫抑制体内模型，确定多糖活性级分；采用现代仪器分析技术对比具有活性的多糖级分结构信息差异；建立基于免疫活性多糖的黄精与酒黄精质量评价方法，完善黄精和酒黄精的质量标准，也为其他以多糖为质控指标的中药提供参考。

多糖是中药中一类重要的有效组分，很多中药以其作为质量评价指标。但常用的以单糖计算多糖含量，方法缺少专属性，且计算的多糖含量高低有时与活性强弱不符，如中药黄精与酒黄精，方法缺少科学性。. 本研究运用透析技术获取黄精和酒黄精不同分子量多糖级分；通过腹腔巨噬细胞体外免疫和免疫抑制体内模型，确定多糖活性级分；采用IR、NMR、单糖分析等方法对比具有活性的多糖级分结构信息差异；建立基于免疫活性多糖的黄精与酒黄精质量评价方法。. 结果显示，①从主产地购买到18批3个品种的黄精，多糖含量均符合药典规定，选取多糖含量较高的多花黄精为研究对象进行后续研究。②黄精在酒制后，多糖分子量分布存在明显变化，酒黄精产生新色谱峰，分子量为10386 kDa；③以截留分子量为5万的透析袋，可将黄精和酒黄精多糖的差异成分分开，获得分子量＞5万和分子量＜5万的多糖级分；④通过腹腔巨噬细胞体外免疫和免疫抑制体内模型，确定黄精酒制后免疫活性增强，且分子量＜5万的多糖级分是黄精和酒黄精的主要免疫活性成分；⑤采用FT-IR、NMR和HPLC-ELSD，分别对比黄精和酒黄精免疫活性多糖级分的糖苷键、功能团、构型、单糖组成等结构信息，初步研究显示分子量＜5万的多糖级分主要是以β（2→1）糖苷键相连的果聚糖，黄精和酒黄精果聚糖成分存在差异，但差异成分的信息还有待于深入研究；⑥建立黄精与酒黄精的特征图谱评价方法，完善黄精和酒黄精的质量标准，也为其他以多糖为质控指标的中药提供参考。. 项目证实黄精酒制后多糖分子量分布发生变量，免疫活性增强。其中以分子量＜5万的多糖级分的免疫调节作用最佳。因此，分子量＜5万的多糖级分是黄精酒制增强免疫调节作用的重要物质基础。本研究为阐明黄精酒制免疫活性增强提供了科学依据。