Tianjin University of Traditional Chinese Medicine, Tianjin, China.
Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.
J Nat Med. 2024 Sep;78(4):1044-1056. doi: 10.1007/s11418-024-01835-w. Epub 2024 Aug 5.
As a traditional Chinese medicine (TCM), Cortex Periplocae (CP) has a wide range of pharmacological effects, as well as toxic side effects. The main toxic components of it are cardiac glycosides, which tend to cause cardiotoxicity. Currently, it has also been reported in studies to cause hepatotoxicity, but it is not clear whether the hepatotoxicity is related to the toxicity caused by the reactive metabolites. This study aims to investigate the target components of CP that generate reactive metabolic toxicity. The fluorescent probe method was used to detect glutathione (GSH)-trapped reactive metabolites in a co-incubation system of CP extract with rat liver microsomes. Identification of GSH conjugates was performed by LC-MS/MS and that of the possible precursor components that produce reactive metabolites was conducted by UPLC-Q-TOF/MS. Cell viability assays were performed on HepG2 and L02 cells to determine the cytotoxicity of the target components. The findings of our study demonstrate that the extract derived from CP has the ability to generate metabolites that exhaust the intracellular GSH levels, resulting in the formation of GSH conjugates and subsequent cytotoxic effects. Through the utilization of the UPLC-Q-TOF/MS technique, we were able to accurately determine the molecular weight of the precursor compound in CP to be 355.1023. The primary evidence to determining the GSH conjugetes relies on the appearance of characteristic product ions resulting from central neutral loss (CNL) scanning of 129 Da and product scanning of m/z 660 in the positive MS/MS spectrum. Through analysis, it was ultimately ascertained that the presence of chlorogenic acid (CGA) and its isomers, namely neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA), could lead to the production of GSH conjugates, resulting in cytotoxicity at elevated levels. Taking these findings into consideration, the underlying cause for the potential hepatotoxicity of CP was initially determined.
作为一种传统中药（TCM），杠柳（CP）具有广泛的药理作用，也有一定的毒副作用。其主要毒性成分为强心苷，易引起心脏毒性。目前的研究还表明，它也会引起肝毒性，但尚不清楚肝毒性是否与活性代谢物引起的毒性有关。本研究旨在探讨 CP 中产生活性代谢毒性的靶成分。采用荧光探针法检测 CP 提取物与大鼠肝微粒体共孵育体系中谷胱甘肽（GSH）捕获的活性代谢物。通过 LC-MS/MS 鉴定 GSH 缀合物，通过 UPLC-Q-TOF/MS 鉴定可能产生活性代谢物的前体成分。采用 HepG2 和 L02 细胞活力测定法测定靶成分的细胞毒性。我们的研究结果表明，CP 提取物具有产生代谢物的能力，这些代谢物可耗尽细胞内 GSH 水平，形成 GSH 缀合物并随后产生细胞毒性。通过使用 UPLC-Q-TOF/MS 技术，我们能够准确确定 CP 中前体化合物的分子量为 355.1023。确定 GSH 缀合物的主要依据是在正 MS/MS 谱中通过中心中性丢失（CNL）扫描 129 Da 和产物扫描 m/z 660 出现特征产物离子。通过分析，最终确定绿原酸（CGA）及其异构体，即新绿原酸（NCGA）和隐绿原酸（CCGA）的存在可导致 GSH 缀合物的产生，从而导致细胞毒性升高。考虑到这些发现，CP 潜在肝毒性的潜在原因最初被确定。
