基于理气功效和代谢组学技术的柑橘属药材效应物质基础及质量控制研究

Guided by the holistic view of traditional Chinese medicine theory, with the pharmacologically active herbal medicines from Citrus species as the research subjects, by utilizing multidisciplinary techniques, the purpose of the present project was to disclose the chemical profile and in vivo metabolism profile of Citrus herbs, to carry out multi-component integration pharmacokinetic studies on these Citrus herbs, and correlation network analysis of the in vitro and in vivo substance groups; We also aimed to observe and compare the efficacy of different Qi-activating herbs from the whole animal and isolated organ level; The non-targeted and targeted metabolomics approach was employed to identify the potential endogenous marker of the "Qi-activating herbs", the endogenous marker was also indirectly confirmed by the results of the pharmacological experiments. In the meantime, the multivariate analysis methods was used to quantitatively correlate the in vitro constituents, in vivo components and metabolomics potential markers and pharmacological indicators, to rapidly identify the potential therapeutic constituents from the view of "efficacy-time-quantity"; Based on the online chromatography coupled with mass spectrometry techniques, the knockout-fishing/spectral efficacy integration and characterization method was established to accurately capture the potential targeted active constituents in Citrus herbs and collect the remaining parts, their pharmacological activities and the contribution to the overall activity were individually evaluated to validate the potentially active constituents, and ultimately the quality control method based on the efficacy weighting was constructed for the Citrus herbs.

本课题拟在中医药整体观理论指导下，以药理活性明确的柑橘属药材为研究载体，综合利用多学科技术，解析柑橘属中药材体外化学成分谱和体内代谢谱，开展柑橘属药材多组分整合药代动力学研究，并对体内外物质组进行关联网络分析；在整体动物、离体器官层次观察比较柑橘属药材 的"理气"功效；运用非靶向和靶向代谢组学方法，寻找和确定"理气"相关的内源性潜在标志物，并根据直接的药理作用指标间接确证内源性标志物；同时运用多变量分析的方法将体外成分、体内成分与代谢组学潜在标志物或直接的药理作用指标量化关联，从其"效一时一量"的角度快速识别潜在的治疗成分；并基于在线色谱质谱技术建立成分群缺失-捕获/谱效集成表征方法，精准捕获柑橘属药材潜在目标成分（群）及同步收集缺失后剩余成分群，评价各成分（群）药理活性及对整体活性的贡献度，验证筛选出的潜在有效成分群，最终构建基于药效权重的柑橘属药材质量控制方法。

本课题拟在中医药整体观理论指导下，以药理活性明确的柑橘属药材为研究载体，综合利用多学科技术，解析柑橘属中药材体外化学成分谱和体内代谢谱，构建基于药效权重的柑橘属药材质量控制方法。我们采用植物代谢组学研究方法，将陈皮（CP）和广陈皮（GCP）挥发油样品进行GC-MS分析，基于主成分分析（PCA）和正交校正的偏最小二乘法（OPLS-DA）研究显示陈皮与广陈皮）样品可以较好的被区分。采用NIST标准库并结合保留指数对筛选得到的差异化合物进行结构鉴定，从陈皮和广陈皮样品中共鉴定出15个差异化合物；采用变波长液相色谱、强阳离子交换液相色谱、气质联用技术结合聚类分析、主成分分析、偏最小二乘法判别分析分别比较了枳实、枳壳药材中的黄酮、生物碱及挥发油，并寻找二者的差异性成分，所建立的色谱结合化学计量学方法相互补充，可用于枳实、枳壳药材的区分与质量评价；采用RRLC-ESI-QTOF/MS技术从化橘红中鉴定出22个化合物，2个黄酮-C-苷首次报道，果实中黄酮的含量随着果实的成熟而降低，抗氧化活性显示小果>花~中果>大果>叶~茎；从香圆和枸橼药材提取物中共鉴定了25个化学成分，香圆和枸橼的成分组成和含量有很大差别，香圆药材中含量最高的成分为柚皮苷，而枸橼药材中含量最高的成分为诺米林，香圆的总黄酮和总酚酸含量比枸橼的高，并且三种抗氧化活性实验也表明香圆的抗氧化活性优于枸橼；设计合成了43种绿色低共熔溶剂（DES），对中药三七、丹参等药材中的多类活性成分进行了提取比较，并采用响应面设计对提取条件进行了系统优化，结果显示DES对皂苷、酚酸、黄酮苷、生物碱提取效率显著高于甲醇，提示DES在中药活性成分提取方面具有巨大潜力。应用一测多评法，以活性成分群（橙皮苷、芸香柚皮苷、香蜂草苷、川陈皮素、橘皮素、3,5,6,7,8,3’,4’-七甲氧基黄酮）为指标，建立陈皮药材含量测定标准，已正式被美国药典收载。在本课题的资助下，共发表SCI研究论文11篇，获授权发明专利3项，起草美国药典标准1项，培养博士生4名，硕士生4名。