基于代谢组学和谱效表征的栀子大黄汤保肝作用药效物质基础研究

Liver injury is the clinical common diseases which endangers human health. Because of the unique advantages of traditional Chinese medicine in the treatment of liver injury, it is significant for the treatment of liver diseases to research on liver protecting traditional Chinese medical prescriptions. The Zhi-Zi-Da-Huang (ZZDH) decoction is originally reported in the Synopsis of the Golden Chamber and has good curative effect against jaundice with damp-heat pathogen. In this project, the ZZDH decoction will be studied based on metabonomics research strategy and by taking the relationship between different herbal formulations of its components and its hepatoprotective effect as the cutting point. The effects of ZZDH decoction and decomposed prescriptions will be synthetically evaluated using different liver injury pharmacological models. Then, the in vitro and body fingerprints of ZZDH decoction and decomposed prescriptions will be established with analysis techniques such as LC-MS, and the correlation between the fingerprints and pharmacodynamics will be characterized. Meanwhile, the metabonomics studies of ZZDH decoction and the best efficacy prescription will be carried out. The biomarkers closely related with the action mechanism of ZZDH decoction will be found through analyzing the feature changes of the established 'metabolic profiles' of body fluids. In a word, embarking on concept of the wholism for traditional Chinese medicine theories, the hepatoprotective effect, therapeutic basis, compatibility and action mechanism of ZZDH decoction will be illustrated by means of the combinations of in vivo and in vitro study, chemical data and pharmacological data, metabolomics data and pathological physiology along with biochemistry indexes. This research will provide a theoretical support for the clinical application of ZZDH decoction, and will explore a new way with holism, efficiency and systematicness aiming at traditional Chinese medical prescriptions.

肝损伤是临床常见的危害人类健康的疾病。中医药治疗肝损伤具有独特优势，研究具有保肝作用的传统复方对肝病治疗具有重要意义。本课题以《金匮要略》治疗湿热黄疸之栀子大黄汤为研究对象，基于代谢组学研究策略，以该方药群配伍与保肝作用的关系为切入点，采用多种肝损伤模型综合评价栀子大黄汤全方及拆方的抗肝损伤作用；采用LC-MS等分析手段建立栀子大黄汤各组方的体外和体内指纹图谱，并进行谱效相关性表征；对栀子大黄汤全方和最佳药效组方进行代谢组学研究，建立并分析其体液"代谢谱"的变化，找寻与作用机理密切相关的生物标志物。本研究将从中医学整体观出发，通过体内与体外研究相结合、化学数据与药理数据相结合、代谢组学数据与病理生理及生化指标相结合的分析方法，评价栀子大黄汤的保肝药效，明确其药效物质基础，阐释其配伍及药理作用机制，从而为其临床应用提供理论支持，探索以整体性、高效性和系统性为特点的中药复方研究新思路。

本课题以传统经方栀子大黄汤为研究对象，通过抗肝损伤药效学研究表明了栀子大黄汤对ANIT诱导的胆汁淤积性肝损伤和酒精性肝损伤具有明显保护作用。建立了栀子大黄汤UFLC指纹图谱，考察了全方与各组成药味指纹图谱的相关性。建立了同时测定栀子大黄汤中20个化合物的LC-MS方法，并考察了不同配伍对这些成分溶出量的影响。对栀子大黄汤的15个不同拆方进行指纹图谱分析和药理作用评价，并进行了谱效相关研究。依据血清药物化学研究思路，建立了栀子大黄汤在大鼠体内的UFLC血清指纹图谱。进一步采用UHPLC-Q-TOF-MS技术对栀子大黄汤的体外、体内化学成分进行分离鉴定，并比较健康大鼠、胆汁淤积性肝损伤大鼠和酒精性肝损伤大鼠入血成分的差异。在栀子大黄汤中共检测到82个固有化学成分，在健康大鼠血浆中共检测到43种入血成分，包括21种原形和22种代谢物，在肝损伤大鼠血浆中共检测到39种入血成分，包括21种原形和18种代谢物。结果表明，环烯醚萜苷类、蒽醌类、黄酮类、香豆素类、西红花苷类、单萜苷类、单宁类、有机酸类和柠檬苦素类成分为栀子大黄汤的主要化学成分；葡萄糖醛酸化和硫酸化为环烯醚萜苷类和蒽醌类化合物的主要代谢途径，葡萄糖醛酸化为黄酮类化合物的主要代谢途径。建立了同时测定大鼠血浆中8种活性成分的UHPLC-MS/MS方法，药动学研究表明，8种活性成分在健康大鼠和肝损伤大鼠灌胃给予栀子大黄汤后的药动学行为存在明显差异。对栀子大黄汤在健康大鼠和肝损伤大鼠尿液中的代谢进行了分析，在健康大鼠尿液中共检测到59种成分，包括37种原形成分和22种代谢产物，在肝损伤大鼠尿液中均检测到这些原形成分及代谢物，从药物代谢的角度佐证栀子大黄汤发挥药效的体内化学物质基础。应用代谢组学方法探讨大鼠胆汁淤积性肝损伤和酒精性肝损伤的发病机制，并对栀子大黄汤的干预作用进行了研究。在大鼠血清、尿液和肝组织中鉴定出多个与肝损伤相关的生物标记物。结果表明，栀子大黄汤具有抗胆汁淤积性肝损伤作用，其发挥药效的机制可能与调节脂肪酸代谢、磷脂代谢、氨基酸代谢、胆固醇代谢和核苷酸代谢有关；栀子大黄汤具有抗酒精性肝损伤作用，其发挥药效的机制可能与调节脂肪酸代谢、磷脂代谢、氨基酸代谢和核苷酸代谢有关。本研究完成了课题的预期目标，为栀子大黄汤的临床应用和研究开发提供了理论依据。