基于免疫“活性中心”寡糖片段筛选的黄芪多糖质量标志物研究

In light of polysaccharide as the main active ingredient of Radix Astragali, but it has been failed as an index of quality control to evaluate the germplasm resources of Radix Astragali due to its special properties and controllable problems which lead to the key scientific problem of the lack of quality marker of the polysaccharide. Based on the polysaccharide receptor theory and the research thought of quality marker (Q-marker), the immune functional polysaccharides were separated by the activity tracing method, and oligosaccharide fragments was prepared by enzymatic hydrolysis, step-by-step controlled acid hydrolysis of the active polysaccharides, and various chromatographic methods on foundation of the former youth fund. The characterization of these fragments was achieved via high resolution mass spectrometry and other structural analysis techniques. Afterwards, oligosaccharide fragments were used for construction of diverse structure oligosaccharide compound library. Different immune cells and animal models were employed to screen active oligosaccharides including immunoactive site of polysaccharide. At the same time, the structural specialization of the oligosaccharides was verified by comparison of oligosaccharide fragments acquired by degradation of polysaccharides from different Chinese materia medica and the Q-marker of Astragalus polysaccharide was ascertained. On the basis of the evaluation index of the active oligosaccharides, the special determination method of the contents of Astragalus polysaccharide was built for evaluating different standard and grade commodities of Radix Astragali. The research results will not only elucidate the material basis of the pharmacological function of Astragalus polysaccharide and enrich the polysaccharide receptor theory and provide new index of quality control for Radix Astragali, which will provide an effective solution to international problems of the quality control of polysaccharide drugs, but also lay a material foundation for the further development of carbohydrate compounds and the reservation of the strategic resources in the future and promote the research and development of polysaccharide drugs.

多糖虽为黄芪的主要活性成分，却因专属性和可控性难题，限制了在黄芪种质资源评控中的应用，缺乏多糖类成分质量标志物是关键科学问题。项目以多糖受体理论和中药质量标志物研究思路为指导，以黄芪中的免疫活性多糖为对象，在前一青年基金基础上采用可控式酸解及定向酶解等技术获得降解寡糖片段，以多种色谱分离纯化和高分辨质谱解析等技术，构建结构多样化的寡糖库，采用免疫细胞与动物模型筛选高活性寡糖，获得具有免疫“活性中心”的寡糖片段；同时对比其它药材多糖降解的寡糖结构验证其专属性，明确黄芪多糖质量标志物。建立以高活性寡糖为指标的黄芪多糖类成分专属性含量测定方法，评价不同规格等级商品黄芪质量。研究成果不仅为阐明黄芪多糖免疫功能的物质基础提供依据，丰富多糖受体理论，而且为黄芪提供新的质控标准，为解决多糖药品质控的国际性难题提供方案，同时还将为糖类化合物的深入开发及战略资源的储备奠定物质基础，促进药物研发。

多糖虽为黄芪的主要活性成分，却因专属性和可控性难题，限制了在黄芪种质资源评控中的应用，缺乏多糖成分质量标志物是关键科学问题。项目以多糖受体理论和中药质量标志物研究思路为指导，对黄芪多糖及其降解寡糖片段进行了系统研究。首先在多糖方面，表征了基于分子量分布的黄芪多糖糖谱，并对不同分子量黄芪多糖进行了分离制备和体内、外的特异性及非特异性免疫活性筛选，找出了主要的活性多糖组分APS-Ⅱ（10KD左右）, 以APS-Ⅱ为峰面积占比为指标，对比了仿野生黄芪与栽培黄芪差异，并采用ROC曲线方法确定了两种黄芪APS-Ⅱ峰面积占比的临界值89.75%，体现了仿野生黄芪的优质性。此外对建立的黄芪多糖糖谱进行了专属性验证和方法学考察，发现仿野生黄芪多糖糖谱相似度（≥99%）优于栽培黄芪（≥97%），可用于黄芪种质资源评价。. 根据以上结果，对活性多糖APS-Ⅱ进行了结构分析，据此确定了以α-1,4-葡萄糖苷水解酶和盐酸为主的降解方式，进行了单因素和正交试验确立了最佳降解条件，通过亲水色谱表征了近100余种寡糖片段，进行了不同寡糖片段组的制备，采用特异性及非特异性免疫细胞活性筛选，发现聚合度10~18低聚糖片段活性总体优于聚合度3~9的寡糖片段，且与多糖APS-Ⅱ活性相近。因此采用核磁、高分辨质谱进行了聚合度3~9的寡糖片段与10~18的低聚糖片段结构对比，研究发现低聚合度寡糖片段组主要是α-1,4-葡寡糖，高聚合度糖片段组主要以α-1,4连接、α-1,6连接的葡寡糖为主，且葡萄糖残基的2、6位上有分支位点，随着聚合度的逐渐增加，分支点占比越多，同时该类低聚糖片段还有半乳糖、鼠李糖等糖残基的出现，推测这些特征结构与低聚糖活性有密切关系。制备了聚合度3~9和10~18寡糖片段对照品，初步研究了对照品理化性质，建立了黄芪降解寡糖的糖谱分析方法，同时进行了专属性研究和方法学考察，通过两种栽培方式黄芪寡糖糖谱对比，发现仿野生黄芪寡糖聚合度≥10的色谱峰面积占比为11.835%~19.092%；栽培黄芪为2.778%~7.017%，表明仿野生黄芪多糖组分中活性寡糖片段多于栽培黄芪，方法可用于黄芪的品质评价。