胆黄连“寒者益寒”药性变化的物质基础及炮制机理研究

Rhizoma coptidis processed with pig's bile (RCPB) was a typical example of the processing method "cold to be more cold" in the pharmaceutics of traditional Chinese medicine (TCM) processing, which accorded with the processing theory of "Cong Zhi" . At present, research of RCPB was mostly about its processing technology and characterization of medicinal property, but its material foundation of medicinal property change and processing mechanism were rarely related to. So RCPB would be chosen as the research object, a series of studies would be developed around the medicinal properties, efficacy changes before and after processing. Based on the medicinal property enhancement of the cold, the biological effect of heat treatment was developed to characterize the medicinal property of RCPB. The differences of pharmacological effects before and after the processing of RCPB were carried out through the changes of body temperature, weight and biochemical indice (T3, T4,TSH, et al.). Rats' urine metabolic profile of RCPB treating heat syndromes before and after processing were analyzed based on metabolomics, and then synergistic efficacy of RCPB, Rhizoma coptidis and pig's bile were evaluated from the whole perspective of TCM. Because of pig's bile solubilization, total alkaloid, total bile acid and berberine, martin, hyodeoxycholic acid and bilirubin were determined, chemical fingerprints of RCPB before and after processing were studied, and then the relationship of spectrum and efficacy was built to confirm material foundation of drug property of RCPB. In addition, pharmacokinetic of active ingredients of RCPB which were absorbed in the body were studied before and after processing, in order to explain the effect of pig's bile on the absorption of RCPB in the body. Based on the comprehensive analysis of chemical composition -medicinal property, efficacy - pharmacokinetic studies, the scientific connotation of "Cong Zhi" processing of RCPB was further to be interpreted. This study would provide practicable methods for the modern research of TCM medicinal properties and the processing mechanism.

胆黄连的炮制是"寒者益寒"的典型代表，属"从制"范畴。其目前研究多以炮制工艺、药性表征为主，而很少涉及药性变化的物质基础及其炮制机理的相关研究。因此，本课题以胆黄连为研究对象，围绕其寒性增强的药性变化特征，采用实热证治疗为目的的生物效应实验方法，通过体温、体重、生化指标（T3、T4、TSH等）的变化和代谢组学研究，整体评价胆黄连炮制前后的药性、药效作用差异；同时基于猪胆汁的增溶作用，对总生物碱、总胆酸、小檗碱、巴马汀、猪去氧胆酸、胆红素等进行含量测定，并结合炮制前后化学指纹谱的研究，构建谱效关系，确定胆黄连"寒者益寒"药性变化的物质基础。另外对胆黄连炮制前后有效成分在体内吸收的药代动力学的差异进行研究，分析猪胆汁对胆黄连在体内吸收的影响。通过综合分析化学成分-药性、药效-药代动力学研究的结果，全面深入阐释胆黄连"从制"的炮制机理，为中药药性及炮制机理的现代化研究提供可行的方法。

胆黄连的炮制是“寒者益寒”、“从制”的经典实例，在中药炮制理论及临床应用上具有一定的科学性、合理性。本课题从化学成分、药代动力学和代谢组学等方面，对其“寒者益寒”的物质基础及炮制机理进行系统研究。. 通过HPLC法和紫外分光光度法，对黄连与胆黄连水煎液中的药根碱、巴马汀、小檗碱和总生物碱的溶出率进行了测定，研究胆黄连炮制前后对化学成分溶出变化的影响。结果表明辅料猪胆汁能够促进黄连中生物碱成分的溶出。. 采用附子、肉桂、干姜水溶液灌胃，并皮下注射干酵母混悬液造成大鼠热证模型，给药黄连、猪胆汁和胆黄连，进行解热药理作用研究。与模型组相比较，猪胆汁组、胆黄连组在3h时大鼠体温均显著性降低； 与模型组比较，黄连与胆黄连可使FT3水平显著性提高，猪胆汁组FT4显著性降低，rT3没有显著性变化。结果表明，猪胆汁对黄连的解热药效具有一定的协同作用。. 复制大鼠热证模型，比较黄连炮制前后中各生物碱成分的药代动力学参数的差异性。与黄连组相比较，胆黄连组小檗碱、药根碱、巴马汀的Tmax均提前2 h；小檗碱、巴马汀的Cmax增加一倍。 结果表明，辅料猪胆汁可明显促进黄连中生物碱类成分在大鼠体内的吸收。同时，对黄连、胆黄连进行整合药代动力学与解热药效作用的PK-PD相关性研究，结果表明，胆黄连中生物碱成分达峰时间1.11h，解热最佳时间3h；黄连、胆黄连中的生物碱成分在体内的动态变化过程与其解热药效作用消长之间的相关性良好。. 采用UPLC/LTQ-Orbitrap-MS方法对黄连、胆黄连尿液代谢组学的差异性进行研究，结果表明正常组与模型组在0-6 h和6-12 h达到分离，12-24 h出现重叠，分离趋势不明显；黄连组和胆黄连组0-6 h大鼠尿样与模型组分离，接近于正常组；黄连组和胆黄连组在0-6 h和6-12 h大鼠尿样有分离趋势。鉴定30个与热证相关的差异代谢物，结果表明，黄连经猪胆汁炮制后对热证大鼠的整体药效作用发生改变， 具有多靶点、起效快、作用强度较强的特点，主要通过对胆碱能神经递质、氨基酸代谢、嘌呤代谢的调节发挥解热作用。. 综上研究，胆黄连“寒者益寒”的药性变化是药效作用的物质基础发生了改变，主要是猪胆汁的增溶、协同作用，增强黄连中生物碱的溶出，促进生物碱成分在体内的吸收，疗效增强。