中药钩藤抗帕金森病的体内药效物质及其多成分多途径调控机制

Uncariae Ramulus Cum Uncis is one of the famous traditional Chinese medicines (TCMs) for the clinical treatment of Parkinson's disease (PD). Preliminary results by our group and other groups showed that Uncariae Ramulus Cum Uncis effectively improved the behavior recovery of PD mice, and exhibited a significant protective effect on nerve cells and neurons. However, the pharmacologically active ingredients of Uncariae Ramulus Cum Uncis against PD and its regulatory mechanism are not clarified definitely. .In this project, we intends to clarify the in vivo bioactive constituents of Uncariae Ramulus Cum Uncis against PD from the perspectives of combinational analysis of the constituents and activity in vitro and those in vivo, and the single compound and multiple components, by using interdisciplinary research methods of Natural Medicinal Chemistry, Pharmacology, Drug Metabolism and Pharmacokinetics. Furthermore, systematical investigations on anti-Parkinson’s efficacy and molecular mechanism of the in vivo constituents will be carried out, for the purpose of establishing the "quantitative composition-activity relationship" and clarifying the regulatory mechanism of Ramulus Uncariae cum Unicisbased on multi-components and multi-pathways. .The implementation of this project will provide scientific evidences for the clinical precision use and innovative drug development based on Ramulus Uncariae cum Unicis, act as a useful strategy for the characterization of regulatory mechanism based on multi-components and multi-pathways, and also supply reference data for interpreting the scientific meaning of TCM theory of "smoothing the liver and suppressing the Yang, activating the meridians and extinguishing the wind" in the treatment of PD.

钩藤具有熄风定惊、清热平肝的功效，是临床治疗帕金森病（PD）的重要中药。本项目组和文献前期研究均表明，钩藤能有效改善PD小鼠的行为恢复，对神经细胞和神经元有显著保护作用。但钩藤抗PD活性的药效物质及作用机制尚不明确，因此本项目以中医药理论为指导，综合运用中药化学、药理学、药物代谢及药动学等多学科交叉的研究方法，从体外到体内、从单体成分到整体成分，阐明钩藤抗PD的体内药效物质；以主要体内活性成分（包括活性代谢产物的原型成分）在体内和钩藤中的含量为活性成分定量参数，以各活性成分在不同模型下的作用强度为活性参数，建立体内成分与活性之间的“定量组-效相关谱”，评价不同成分的活性贡献，系统表征钩藤抗PD的多成分、多途径调控机制。项目的实施将为钩藤临床精准用药和创新药物发现提供科学依据，为中药多成分、多途径作用表征的方法学提供有益探索，为诠释中医治疗PD的“平肝潜阳、活络熄风“理论提供重要参考。

钩藤具有熄风定惊、清热平肝的功效，是临床治疗帕金森病（PD）的重要中药，但其抗PD活性的药效物质及作用机制尚不明确。本项目在中医药理论指导下，综合运用中药化学、药理学、药物代谢及药动学等多学科交叉的研究方法，从体外到体内、从单体成分到整体成分对钩藤抗PD的体内药效物质和作用机制进行了系统研究，取得了一系列重要的、具有自主知识产权的、创新性科研成果：. ① 采用体内小鼠和大鼠两种动物模型明确了钩藤抗PD的药效作用及其有效部位为总生物碱（URA），并优化了URA制备工艺。.② 从钩藤中分离鉴定了97个化学成分，其中生物碱47个，三萜26个，明确了总生物碱活性部位的主要化学成分，阐明了钩藤生物碱的LC-MS质谱裂解规律，采用HPLC-UV方法系统表征了URA活性部位化学成分谱的主要特征。.③ 采用LC-MS从URA灌胃大鼠后血浆中检测到30个生物碱原型和41个可能的代谢产物，其中14个可通过血脑屏障在脑组织中检测到，描绘了16个生物碱的药代动力学特征，基本阐明了URA的体内药效物质。.④ 对URA中不同组份及32个代表性生物碱类化合物进行了神经保护作用、抗神经炎症、抗铁死亡、诱导自噬等体外活性评价，构建了“化合物-生物活性”图谱，初步阐释了URA主要活性成分群及活性组分。.⑤ 采用体外细胞实验证明了URA可以通过抑制JNK磷酸化抑制细胞的线粒体凋亡以及通过诱导线粒体自噬发挥神经保护作用；大鼠脑内黑质蛋白质组学和免疫组化分析结果表明给药URA可升高大鼠黑质UCHL1表达水平，并经体外细胞热转移分析和生物膜干涉分析验证。体外细胞实验表明去氢毛钩藤碱可能通过影响线粒体自噬与UCHL1发挥神经保护作用; 体外酶活性实验表明1个单萜吲哚生物碱和1个单萜吲哚生物碱苷具有AChE抑制活性；三萜类化合物则通过促进Nrf2表达发挥较好抗铁死亡活性。.上述研究结果为阐释钩藤抗PD的体内药效物质及其多成分、多靶点、多途径的调控作用机制提供了科学依据，为开发基于中药钩藤生物碱的成分明确、工艺稳定、质量可控的创新性抗PD药物奠定了扎实基础。