薄层色谱质谱成像技术构建 “糖谱”与肠道微生物介导的免疫调节作用相关的党参多糖质量控制策略

Polysaccharides are the main bioactive components of traditional Chinese medicine, the effects of which are highly correlated with their structures. However, due to the complex structure of polysaccharides, qualitative and quantitative analysis based on its functional structure is still a worldwide problem, which makes it difficult to control the quality of traditional Chinese medicines containing polysaccharides. The current quality control methods of polysaccharides presented some bottlenecks such as non-specificity qualitative identification, inaccurate quantitative analysis, and non-correlation between quality control markers and clinical efficacy. In response to the above problems, the project uses polysaccharides from Codonopsis pilosula as the research carrier, which have been proven to have definite clinical efficacy by previous studies. Firstly, this project enriches different molecular weights of water-soluble non starch polysaccharides from C. pilosula by amylase enzymolysis and membrane separation rapidly. And the structure of different fractions will be analyzed using multi-component chromatography-mass spectrometry combining specific enzymatic hydrolysis and different derivatization methods. Meanwhile, a high-specificity, high-throughput, high-accuracy and high-structure information-based method using HPTLC-MALDI mass spectrometry imaging based on specific enzymatic hydrolysis and in situ derivation technology will be developed to construct the specific "saccharides mapping" of C. pilosula, which is achieved for qualitative identification. Secondly, a new method for quantitative analysis using HPSEC-MALLSD-RID will be established, which will be compared with complete acid hydrolysis method on the investigation on the accuracy and specificity. Finally, the active fraction of polysaccharides from C. pilosula will be determined by the evaluation of intestinal immune activity and intestinal microbial flora distribution combined with chemical structural characterization, and the effective substance and structure-function relationship are clarified. This project establishes a quality control method for polysaccharides from C. pilosula closely related to the clinical efficacy through one integrated evaluation method containing chemical characterization-activity detection, scientifically evaluates the quality of C. pilosula, and provides the reference for the quality control of traditional Chinese medicine containing polysaccharides.

多糖是中药的重要成分，其药效与结构特征相关。然而，由于多糖结构复杂，基于其功能结构的定性定量分析至今仍是世界性难题，导致含多糖的中药质量控制难以反映药材质量。现行多糖质量控制方法存在定性鉴别不能反映结构特征、定量分析不准确、检测指标与药效不关联等瓶颈问题。针对上述问题，本项目以药理活性确切的党参多糖为研究载体，开展如下研究：1）结合特异性酶解以及原位衍生技术开发HPTLC-MALDI质谱成像方法，解析多糖结构特征，构建党参多糖专属性“糖谱”，实现基于结构特征的定性鉴别。2）建立专属性的HPSEC-MALLSD-RID直接定量多糖的方法，与完全酸水解间接定量方法做比较。3）结合肠免疫活性评价及肠内菌群表征，锁定活性部位，明确药效物质基础，阐明谱效关系。本项目通过化学表征-活性检测整合评价方法建立与药效相关的党参多糖质量控制方法，科学评价党参的质量，也为含多糖类中药的质量控制提供借鉴意义。

多糖是中药的重要成分，其药效与结构特征相关。然而，由于多糖结构复杂，基于其功能结构的定性定量分析至今仍是世界性难题，导致含多糖的中药质量控制难以反映药材质量。现行多糖质量控制方法存在定性鉴别不能反映结构特征、定量分析不准确、检测指标与药效不关联等瓶颈问题。针对上述问题，本项目以党参多糖为研究对象，建立了党参多糖的富集纯化方法，获得了其中的特征性多糖，解析多糖结构特征，同时结合酶水解及HPTLC-MALDI构建了党参多糖专属性“糖谱”，实现了基于结构特征的定性鉴别。同时针对定量问题，优化了水解结合色谱的方法，建立了基于单糖信息的准确定量方法。针对活性组分结合肠免疫活性评价及肠内菌群表征，迅速锁定活性部位，明确药效物质基础，初步阐明了党参多糖的谱效关系。本项目的结果可以为多糖活性成分段的快速发现及基于结构的质控方法提供参考。