基于整体观的熟地黄“九蒸九晒”质-效相关性研究

Holistic quality-efficacy correlation study is a basic issue for the inheritence and innovation of traditional processing method of Chinese medicinal herb. “Nine cycles of steaming and sun-drying” is a traditional and classical way for processing of Rehmanniae Radix Praeparata, but previous quality-efficacy correlation studies were generally based on the determination of few chemical markers and pharmacological index, which are unable to reflect the well-accepted “multi-components against multi-targets” therapeutic feature of traditional Chinese medicines, thus are not objective to reveal the scientific connotation of the traditional “nine cycles of steaming and sun-drying” processing for Rehmanniae Radix Praeparata. In present study, based on our preliminary chemical profiling methods development, chemicalomics and glycomics by multi-dimensional hyphenated chromatographic techniques will be developed to analyze the whole non-sugar small molecules and glycome in herbs, respectively, to evaluate the variation of holistic quality of herbs; UPLC-QTOF-MS/MS-based metabolomics strategy would be established to determine the endogenous metabolome, 16S rRNA RT-PCR-based metagenomics to profile gut microbiota of model animals, and together with the common pharmacological index, to assess the change of holistic efficacy of herbs. With the aid of multivariate statistical analysis, the correlation between the holistic quality-efficacy would be then revealed in traditional “nine cycles of steaming and sun-drying” processing of Rehmanniae Radix Praeparata, so that the scientific connotation of the processing would be clarified, and innovative processing method would be objectively optimized. Moreover, the research outcomes can also offer new ideals and approaches for the inheritence and innovative research on traditional processing methods of other Chinese medicinal herbs.

体现整体观的质-效相关性研究是中药饮片传统炮制工艺继承创新的基础。“九蒸九晒”是熟地黄传统经典炮制方法，但既往基于少数活性成分和药效指标的质/效评价方法，不能体现“多成分多靶点”的中药整体作用特色，难以客观阐明“九蒸九晒”的科学内涵。本研究拟在前期分析方法创建的基础上，采用基于色-质联用技术的中药化学物质组学和中药糖组学方法分别表征地黄非糖小分子组和糖组，阐明“九蒸九晒”熟地黄整体“质”的变化规律；采用基于色-质联用技术的效应代谢组学方法表征内源性代谢产物组和基于基因序列分析的宏基因组学方法表征肠道菌群组成，并结合多种经典药效指标，阐明“九蒸九晒”熟地黄整体“效”的变化规律；再采用多变量统计方法进行“质”与“效”的关联分析，揭示熟地黄“九蒸九晒”炮制整体质-效相关性，为客观诠释熟地黄传统炮制工艺的科学内涵和创新工艺优化提供科学依据，也为其他中药传统炮制工艺的继承创新研究提供新思路和新方法。

体现整体观的质-效相关性研究是中药饮片传统炮制工艺继承创新的基础。“九蒸九晒”是熟地黄传统经典炮制方法，但既往基于少数活性成分和药效指标的质/效评价方法，不能体现“多成分多靶点”的中药整体作用特色，难以客观阐明“九蒸九晒”的科学内涵。为此，本课题开展了如下研究：①整合基于UPLC-QTOF-MS/MS技术的中药次生代谢物组学和基于HPGPC-ELSD、UPLC-PDA和HPLC-ELSD技术的中药糖组学方法，表征生、熟地黄次生代谢物组和糖组；并结合多变量统计方法，从次生代谢物组和糖组快速筛选区别生、熟地黄的化学标志物，通过对化学标志物模拟炮制后的产物进行化学物质组表征，探寻中药炮制化学转化机制，揭示地黄“九蒸九制”整体品质的变化规律；②基于血虚动物模型，建立基于UPLC-QTOF-MS/MS技术的效应代谢组学方法，结合基于16S rRNA高通量测序技术的肠道菌群多样性检测方法以及多种经典药效指标，构建地黄补血药效的生物学评价体系，阐明不同蒸晒次数所得熟地黄以及生地黄补血药效差异；③基于阴虚动物模型，通过比较效应代谢组、肠道菌群多样性以及多种经典药效指标，构建地黄滋阴药效的生物学评价体系，阐明生、熟地黄滋阴药效差异；④通过将化学成分-药效指标-肠道菌-代谢物加以关联，探讨生、熟地黄整体质-效差异的内在机制。研究结果表明：①“九蒸九制”过程中，地黄的次生代谢物组和糖组均发生较为明显的量变与质变，六次蒸制后熟地黄品质趋于稳定和均一，该炮制过程中化学转化机制主要涉及糖类成分和苷类成分通过脱水或脱糖基转化为呋喃醛衍生物；②效应代谢组学分析、肠道菌群分析及经典药效指标结果显示六次蒸晒熟地黄的补血作用明显强于生地黄及其他熟地黄；③经典药效指标结果显示六次蒸晒熟地黄的滋阴作用与生地黄相当，而效应代谢组学和肠道菌群分析结果表明二者可从不同角度改善代谢通路和肠道菌群紊乱；④化学成分-药效指标-肠道菌群-代谢物关联性分析表明，生、熟地黄的补血滋阴活性差异可能是其不同化学成分作用于特异性肠道菌及内源性代谢物，进而引发不同机制所致。该项研究为客观诠释熟地黄传统炮制工艺的科学内涵和创新工艺优化提供了科学依据，也为其他中药传统炮制工艺的继承创新研究提供了新思路和新方法。