中药秦皮及其香豆素类活性成分的肝肠处置及调控机制

Cortex Fraxini is frequently used in clinical treatment of Traditional Chinese Medicine, which is attributed to its efficacy of clear heat and dry dampness, clear liver and improve eyesight, and relieve cough and asthma. Pharmacological researches had confirmed that Cortex Fraxini has a broad spectrum of bioactivity. Coumarin derivatives are the main bioactive ingredients of Cortex Fraxini. Many researches had demonstrated that intensive glucuronidation is the critical factor which resulted in poor bioavailability (<6%) of coumarin derivatives. However, studies on the disposition characteristics and its regulatory mechanism of bioactive coumarin derivatives are very limited. Esculetin is one of the important bioactive coumarin derivatives of Cortex Fraxini. Our preliminary studies had found that esculetin and 4-methylesculetin were intensively metabolized by UDP-glucuronosyltransferases (UGTs) in the liver and intestine. High concentrations of glucuronides of esculetin and 4-methylesculetin were detected in plasma, bile, and perfusate. Moreover, their glucuronides can be excreted by efflux transporter (ET) such as BCRP (Breast Cancer Resistance Protein). Further, structural selectivity of ET excretion action on glucuronides of esculetin and 4-methylesculetin was also observed. These results suggested that UGT/ET coupling is probably involved in the regulation of the enterohepatic disposition of Cortex Fraxini and its bioactive coumarin derivatives. Therefore, in the current study, advanced experimental models such as over-expressed UGT/ET cells (e.g. HeLa-UGT1A9 cells) and gene knock-out animals will be employed to elucidate the enterohepatic disposition characteristics of bioactive coumarin derivatives of Cortex Fraxini and its regulation mechanism. The effect of UGT/ET coupling on extract of Cortex Fraxini will also be evaluated in the current study. The outcomes of this study will not only clearly reveal the disposition characteristics of bioactive coumarin derivatives of Cortex Fraxini, and will also provide basic data for pharmacological and clinical researches of Cortex Fraxini in future.

秦皮能清热燥湿、清肝明目、平喘止咳，是中医临床常用中药；药理学也证实其有抗炎抗肿瘤等多种活性，香豆素类是其活性成分。研究表明，因易被葡萄糖醛酸转移酶（UGT）代谢，香豆素类生物利用度低（<6%）；但其体内处置及调控机制尚不清晰。我们研究发现，秦皮中香豆素类成分秦皮乙素及其衍生物4-甲基七叶亭主要在肝肠被UGT代谢，UGT代谢物是体内主要暴露形式，也易被外排转运蛋白（ET）如BCRP等外排，且外排存在结构选择性。这提示被UGT/ET共同调控的肝肠处置可能是香豆素类体内过程的关键调控机制。因此，本课题在此基础上，以秦皮和所含香豆素类为研究对象，采用过表达UGT/ET的细胞（如HeLa-UGT1A9）和基因敲除动物等前沿实验方法，系统深入研究秦皮香豆素类肝肠处置规律及其调控机制，并考察在秦皮总提取物中的差异。其结果不仅阐明香豆素类成分肝肠处置规律及调控机制，也为秦皮药理和临床研究提供基础依据。

秦皮具有“清热燥湿、清肝明目、平喘止咳”的功效，是中医临床使用频率非常高的中药。秦皮主要活性成分是香豆素类化合物。本研究采用Caco-2细胞和基因敲除动物，深入研究秦皮香豆素类肝肠处置规律及其调控机制。主要得到三方面结论。（1）秦皮香豆素类生物利用度低（0.27%-30%），较单体化合物，水提物中的秦皮香豆素类的生物利用度较高；秦皮香豆素类在体内主要经UGT代谢酶代谢，主要以UGT代谢产物形式存在，除肝脏外，其原型和UGT代谢物在胃、肾、小肠、肺和心脏中含量均较高；（2）秦皮香豆素类在体内主要生成7-O或8-O葡萄糖醛酸代谢产物；参与其UGT代谢的主要亚酶有UGT1A6，UGT1A7，UGT1A8和UGT1A9，其次是UGT1A1，UGT1A10，UGT2B7和UGT2B15；（3）外排转运蛋白BCRP和MRP2外排秦皮香豆素类UGT代谢产物，是调控秦皮香豆素类UGT代谢产物体内处置的重要分子因素。以上研究结果为秦皮及其香豆素类活性成分的药代动力学研究及临床用药提供依据。课题共发表SCI论文4篇，培养已毕业硕士研究生4名，培养广州市珠江科技新星1名。