基于“表里并重”动态关联“性效差异”的焦栀子饮片炒制过程质量传递动态变化规律及过程机理研究

This project focus on the dynamic association of internal chemical components、external appearance color、properties and pharmacodynamics of Gardeniae fructus Praeparatus (GFP) during heat processing in order to reveal the dynamic change of material basis and browning mechanism of GFP from internal and external multi-levels and multi-angles, this will be carried out in combination with multi-disciplinary research methods including Processing of Chinese medicine, Chemistry, Chemical analyses, Pharmacology, and Statistics, etc. Continuous dynamic variation of "processed products of GFP" will be taken as the breakthrough point. Processed products of GFP during heat processing will be identified by the correlation analysis on the appearance of color, the chemical components and their ratio. Label-free quantitative proteomics and network pharmacology are applied to construct the network including the effect composition (group) - protein target-pathway. Molecular docking is used to validate the effect composition (group) and the western blot is used to validate the protein targets. Then, the difference of pharmacodynamic indexes of pyrolysis and hemostasis and the molecular mechanism of GFP are revealed. Based on this, the dynamic variation of difference in property and efficacy of GFP and quality transfer process during heat processing is revealed by vivo experiments which involve anti-pyresis, hemostasis, label-free quantitative proteomics, network pharmacology and effect of energy metabolism in rat liver mitochondria, etc. . This project seeks to identify the quality marker by internal chemical components and external appearance color and to verify the biomarker by difference in properties and efficacy in dynamic association, which will be established to reveal the dynamic variation of quality transfer and the heat processing mechanism of GFP from different perspectives. This study will provide a demonstration for the other similar research. Meanwhile, this project will provide scientific basis to establish the technical standards and quality traceability system of processed products in the production process, and to lay down the foundation for the production process control and online detection of processed products of Chinese medicine.

本项目以焦栀子生产过程连续动态变化的“过程饮片”为研究对象，以其“表（外观性状）-里（内在物质）-性（药性）-效（药效）变化的动态关联性”为切入点，以“表里并重”多层次地表征栀子炒制过程化学物质动态变化规律，揭示饮片颜色褐变机理，辨识差异明显的“过程饮片”。利用定量蛋白质组学技术和网络药理学方法构建效应成分（群）-蛋白靶点-通路网络，采用分子对接技术确认药效物质群，通过蛋白水平验证药效物质群作用靶点，深入揭示焦栀子过程饮片在解热、止血方面差异性的药效指标群并揭示其分子机制；结合对肝线粒体功能影响评价其苦寒药性变化，阐明焦栀子炮制过程饮片“性效差异”动态变化规律。本项目探索以“表里并重”辨识质量标志物—“性效差异”确认生物标志物-动态关联分析阐释炮制过程机理研究模式，探明栀子饮片生产过程质量传递动态变化规律，为中药饮片生产过程控制技术标准的制定提供科学依据，为同类研究提供借鉴。

中药饮片生产过程控制粗糙、过程机理不清，制约着中药饮片的生产现代化进程。项目提出“过程饮片”概念，开展感官指标仿生数字化、物性特征客观化、多重物质分析、寒性药性变化和蛋白质组学药效评价等研究。.利用颜色-多成分关联分析、HPLC全谱比对，引入图像处理和机器学习算法等技术，多层面辨识出焦栀子炮制过程5个过程饮片。.建立电子眼、电子鼻、电子舌等对饮片外观的数字化表征和多特征成分的“表里关联”分析，构建颜色-气味-滋味-成分网络，4种颜色指标、9种气味传感器、6种味觉指标与10种内在物质成分有相关性；通过人工神经网络分析得到10个重要预测因子，即W6S、苦味、鸡屎藤次苷甲酯、甜味、G2、羟异栀子苷、a*、W5S、山栀苷、5-HMF等可用于焦栀子炮制过程潜在的质量标识物。.以总氨基酸含量、总还原糖含量、总多糖、A420nm（褐变程度）、pH值、5-HMF等多指标变化与颜色的关联分析，证实焦栀子炮制过程颜色褐变与美拉德反应相关，并从游离、结合糖/氨基酸变化基本明确了参与美拉德的反应物，为深入揭示焦栀子颜色褐变机理提供了可靠线索。.下丘脑/胃组织定量蛋白质组学结果表明，解热作用与调节产热、MAPK信号通路、TNF信号通路、加压素调节的水重吸收等途径中8种关键蛋白有关；氧化磷酸化、糖酵解/糖异生途径和与止血相关的血小板活化、血管平滑肌收缩通路中的19个蛋白是焦栀子炮制过程饮片凉血止血作用的潜在生物标志物。.5个能量代谢指标Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、cAMP和cGMP、UCP2的变化证明了栀子炒焦后寒性缓和的趋势，蛋白质组筛选得到Ndufs2、Lhpp、Thtpa和Tst等4个药性相关指标；性-效关联分析确定38个表征药性和药效的指标作为焦栀子炮制过程饮片的潜在生物标志物。.课题初步揭示了焦栀子饮片生产过程质量传递动态变化规律，构建了“表里并重”辨识质量标志物—“性效差异”确认生物标志物-动态关联分析阐释炮制过程机理研究模式，为系统诠释焦栀子炮制过程机理提供科了学依据。发现了可用于焦栀子过程控制的质量标志物和生物标志物，为中药饮片生产提供技术支撑。