基于化学物质组学及代谢组学整合肠道菌群的蒲黄“生行熟止”效应物质基础及作用机制研究

Clarifing the effective material basis is the key to reveal the scientific connotation of the theory of processing of Chinese materia medica “Raw and Processed Chinese Herbal Pieces with Different Treatment”, and is also the precondition for formulation of quality standard for raw and processed pieces. Based on our preliminary work of chemical constituents and pharmacodynamics study of raw and carbonized products of Typhae Pollen, this study intends to take the multidimensional research model “Chemomics – Pharmacodynamics – Metabonomics - Intestinal Microflora” to reveal the effective material basis and mechanisms of Typhae Pollen with its raw product for activating blood circulation and its carbonized product for hemostasis. In this study, we intend to adopt metabonomics approach to construct metabolic profiling, build a mathematical model for quantitative characterization of metabolic profiling to reflect effect, clarify biomarkers related to syndrome of blood stasis, and then analyze metabolic pathway. Moreover, we intend to find out the important intestinal microflora high correlation to biomarkers by studying the correlation between intestinal microflora and biomarkers under the condition of disease, and then analyze the difference of action mechanisms between raw and carbonized products of Typhae Pollen from the intestinal microflora - the host metabolic angle. Additionally, we intend to use multivariate statistical analysis to study the correlation between in vitro and in vivo chemical group and biological effects including pharmacodynamics, metabonomics and intestinal microflora, and then find out effective material basis related to the difference of pharmacodynamics between raw and carbonized products of Typhae Pollen by multidimensional data mining for internal connecting link. This study can provide new study research model and thinking for studying on mechanisms of processing of Chinese materia medica “Raw and Processed Chinese Herbal Pieces with Different Treatment”.

药效物质基础的阐明是揭示中药炮制“生熟异治”理论科学内涵的关键，也是科学制定生熟饮片质量标准的前提。在前期研究蒲黄生品与炭品化学成分及药效学基础上，本项目采取“化学物质组学-药效学-代谢组学-肠道菌群”多维度研究模式，揭示蒲黄“生行熟止”效应物质基础及作用机制。本项目在明确蒲黄生品与炭品体内外化学物质组的基础上，采用代谢组学技术建立代谢轮廓谱，构建数学模型，定量表征代谢轮廓反映药效，阐明血瘀证相关生物标志物，分析代谢通路；并研究疾病状态下肠道菌群与生物标志物相关性，找出相关性强的重要肠道菌群，从肠道菌群-宿主代谢角度分析蒲黄生品与炭品的作用机制差异；并借助多元统计分析方法对体内外化学物质组与生物效应（药效学、代谢组学、肠道菌群三个层次）进行关联分析，多维度挖掘数据内在联系，找出蒲黄生品与炭品作用差异的效应物质基础。本项目可以为中药炮制“生熟异治”机理研究提供新的研究模式和思路。

药效物质基础的阐明是揭示中药炮制“生熟异治”理论科学内涵的关键。本项目在前期研究基础上，整合代谢组学与肠道菌群，来阐释蒲黄“生行熟止”效应物质基础及作用机制。本项目主要完成以下研究内容：（1）利用LC-MS技术对生蒲黄及蒲黄炭醇提物进行化学成分分析。结果，从生蒲黄及蒲黄炭醇提物中分别鉴定了32个和28个化学成分，主要有黄酮类、有机酸类、鞣质类成分。并且发现生蒲黄炒炭后黄酮类、有机酸类成分的含量降低，而鞣质类成分的含量升高。（2）研究了生蒲黄及蒲黄炭醇提物对急性血瘀大鼠血液流变性和凝血功能的影响。结果显示，在对血液流变性、血酶原时间（PT）、凝血酶时间（TT）及纤维蛋白原（FIB）的影响方面，生蒲黄干预效果优于蒲黄炭。但生蒲黄对活化部分凝血活酶时间（APTT）无明显影响，而蒲黄炭能显著缩短APTT，体现出了一定的止血作用。（3）利用LC-MS代谢组学技术测定大鼠血浆、尿液样品，找出与急性血瘀证相关的生物标志物，并进行代谢通路分析，同时观察了生蒲黄及蒲黄炭醇提物对急性血瘀大鼠血浆和尿液生物标志物水平的影响。结果发现了与急性血瘀证相关的血浆生物标志物18个，尿液生物标志物20个，涉及多条重要代谢通路，并且生蒲黄对生物标志物的干预效果优于蒲黄炭。（4）采用16S rDNA基因测序方法研究生蒲黄及蒲黄炭醇提物对急性血瘀大鼠肠道菌群的影响。结果发现了4个显著变化的菌属，且生蒲黄对差异菌属的干预效果优于蒲黄炭。（5）分析了差异菌属与血浆、尿液生物标志物之间的关系。结果发现了与差异菌属高度相关的5个生物标志物，分别是血浆生物标志物L-苯丙胺酸、亚油酸、花生四烯酸及尿液生物标志物尿刊酸和苯丙酮酸。（6）分析了生蒲黄化学成分与差异菌属之间的相关性，结果找出了与差异菌属高度相关的8个效应物质。（7）分析了蒲黄炭化学成分与APTT之间的相关性，结果找出了与APTT高度相关的3个鞣质类化学成分。本项目可以为中药炮制“生熟异治”机理研究提供新的研究模式和思路。