新型介孔分子筛固相萃取-液质联用中药五环三萜皂苷类成分鉴定新体系的构建

The aim of the project is to solve the key problem regarding purification of the trace components of Traditional Chinese medicine (TCM) in vitro and vivo and the conventional analytic strategy of mass spectrometry have the defects of time-consuming, low efficiency, blindness etc. It is imperative to develop new research models and technologies to solve the problems. Based on the previous studies, A miniaturized SPE approach was developed. The mesoporous molecular sieve SBA-15 as a solid sorbent was characterised by Fourier transform-infrared spectroscopy and scanning electron microscopy. Furthermore, all of the parameters, including the SBA-15 amount, solution pH, elution solvent, and the sorbent type, will be investigated in detail by UPLC-MS. To establish a universal strategy for finding and indentifying the chemome and metabolites of TCM in vitro and vivo by UPLC-TripleTOF-MS/MS. A technology platform combining mesoporous molecular sieves SPE with UPLC-TripleTOF-MS/MS was developed to discover and identify triterpenoid saponins and its metabolites of Pulsatilla by mass defect filter technique and background subtraction technique. The ultimate goal of this research project is to provide theoretical foundation and technical guidance for purifying and identifing triterpenoid saponins chemome and metabolites of TCM in vitro and vivo. This proposed provide a new thinking and method to tachy-identifying the trace components from TCM.

本研究拟针对中药体内外复杂体系中微（痕）量化学成分和代谢物分离纯化技术尚不完善及质谱分析策略普适性低的问题，在前期基础上，采用扫描电镜法、红外光谱法等技术评价介孔分子筛性能，用UPLC-MS法考察介孔分子筛SBA-15对五环三萜皂苷化合物吸附和色谱性能，建立以介孔分子筛为新型吸附剂的固相萃取技术；并与UPLC-TripleTOF-MS/MS技术相结合，利用采集后数据处理软件，构建普适性分析策略，建立中药化合物组和代谢物组快速鉴定分析平台。在此新型介孔分子筛SPE结合UPLC-MS/MS技术平台上选用白头翁属药材为研究对象，以五环三萜皂苷类成分为研究载体，拟引入数据采集模式和质谱库检索的集成法为思路，对中药五环三萜皂苷化合物组及代谢物组进行快速定性表征分析。本研究旨在为中药五环三萜皂苷类成分的分离纯化和识别鉴定提供有力技术支持，为快速鉴定中药体内外复杂体系中微（痕）量化合物提供新思路和方法。

本研究针对中药体内外复杂体系中微（痕）量化学成分和代谢物分离纯化技术尚不完善及质谱分析策略普适性低的问题，建立以介孔分子筛SBA-15为新型吸附剂的固相萃取技术；结合UPLC-QTOF-MS/MS技术建立中药化合物组和代谢物组快速鉴定分析平台。在此新型介孔分子筛SPE结合UPLC-MS/MS技术平台上选用白头翁属药材为研究对象，对其五环三萜皂苷化合物组及代谢物组进行快速定性表征分析。已正式发表论文8篇，SCI收录8篇，待发表2篇；编写论著1部；培养硕士研究生6名，已毕业3名。主要研究内容和成果如下：. 1. 建立以介孔分子筛材料SBA-15 为新型吸附剂的小型固相萃取方法，经萃取条件优化和方法学验证，并应用于五环三萜皂苷类化合物富集分离。. 2. 基于UHPLC-MS 技术分别总结五环三萜皂苷类化合物母核、糖链和同分异构体质谱裂解规律，建立快速识别和鉴定模式。. 3. 分别归纳羽扇豆烷型和齐墩果烷型五环三萜皂苷类代谢物裂解规律。. 4. 采用SBA-15固相萃取结合UPLC-Q-TOF-MS/MS法，对四种白头翁属药材中五环三萜皂苷类化合物进行定性和定量分析，明确区分四组药材，并鉴定了12种差异成分。. 5.基于介孔分子筛SPE结合UPLC-MS/MS 平台，共检测和鉴定羽扇豆烷型代谢产物64个，齐墩果烷型代谢物34个。. 6. 建立五环三萜皂苷化合物组和代谢物组数据库。. 7. 对白头翁汤治疗溃疡性结肠炎生物样本进行非靶向代谢组学研究发现了一系列差异性代谢物和潜在的诊断性生物标志物。. 本课题拓展了介孔分子材料的应用范围，并为固相萃取吸附剂提供新的选择，同时建立了一种普适性的筛选鉴定白头翁属药材中五环三萜皂苷类化合物组和代谢物组的快速分析方法，为中药五环三萜皂苷类成分的分离纯化和识别鉴定提供有力技术支持，为快速鉴定中药体内外复杂体系中微（痕）量化合物提供新思路和方法；同时为指导中药的临床合理应用提供参考。