基于水液转运蛋白调控研究大戟属有毒中药醋制“毒-效”转化的共性机制

Traditional Chinese medicine Euphorbia pekinensis, Euphorbia kansui, Euphorbia ebracteolata (all belong to Euphorbia) are included in drastic water-expelling medicine in clinic. The crude drugs are poisonous and thus it is necessary to process before clinical application. The benefit of processing (commonly with vinegar) is to reduce toxicity and retain efficacy, to be specific, to alleviate the drastic toxicity, to achieve the efficacy of expelling water retention by purgation and to treat the disease like ascites. Previous studies have shown that the common substances of the drastic toxicity are the terpenoids of three kinds of Chinese medicines. Processing with vinegar caused the content of highly toxic terpenoids decreased, and the low toxic terpenoids increased, hence the toxicity decreased. In addition, the increase of low toxic terpenoids was associated with the structure transformation of highly toxic terpenoids. But how can vinegar processing retain efficacy? Is it related to the structure transformation of terpenoids? Some studies have shown that terpenoids have the effect of purgation, diuresis, and regulation of aquaporin. Ascites is associated with the disorder of body fluid transport. According to the above, we propose a hypothesis: “Terpenoids of the three Euphorbia toxic medicines develop quantitative and qualitative changes during the processing with vinegar, which is also the material basis of ‘toxicity - efficacy’ conversion. Terpenoids after processing with vinegar can promote the water discharge via regulating body fluid transport.” The project will use chromatographic separation, liquid chromatography-mass spectrometry, fluorescent protein labeling and other techniques, and apply ascites models to study the material basis of its water-expelling efficacy at the body, cell and molecular level; to clarify the common mechanism of ‘toxicity - efficacy’ conversion before and after processing with vinegar; to provide scientific basis for the quality control, clinical safety application, and reforming and innovating of the processing method of this three kinds of Chinese Herbal Pieces.

京大戟、甘遂、狼毒同属大戟属，均为峻下逐水药，有毒，需炮制后应用，均以醋制减毒存效，缓峻下毒性，达泻下逐水之效，疗胸腹积水等症。前期研究表明，其“峻下”毒性的共性物质是3种中药中萜类成分，醋制使高毒性萜类含量下降，低毒性萜类含量上升，毒性下降，且低毒萜类含量上升与高毒萜类结构转化有关。然醋制如何存效？是否与醋制时萜类成分转变有关？研究表明，萜类成分具泻下、利尿、调节水通道蛋白等作用；胸腹积水的发生与水液转运紊乱相关。据此提出“大戟属3种有毒中药，萜类成分在醋制过程中量和质的转变可能是其“毒-效”转化的物质基础；醋制后的萜类成分可通过调节机体水液转运，促使水液排出体外。”项目将采用色谱分离、液-质联用、荧光蛋白标记等技术，采用腹水动物模型，在整体、细胞、分子水平研究其泻下逐水效应物质，阐明醋制前后毒-效转化共性机制，为3种中药饮片质量控制、临床安全应用、炮制工艺改革与创新提供科学依据。

项目揭示了大戟科有毒中药狼毒、京大戟、甘遂醋制法炮制解毒-存效共性机制，发现炮制过程成分转化与毒性下降、效应相关的质量标志物（群），为3种有毒中药饮片质量控制及临床应用安全有效提供科学依据和实际指导。.1. 揭示3种有毒中药毒-效物质基础 狼毒、甘遂的毒性成分和药效成分均为二萜类；京大戟毒性成分为三萜、二萜、倍半萜类，药效成分主要为多酚类、有机酸类；3种有毒中药共性的毒性成分为萜类成分，可引起肠道炎症、肠道菌群紊乱；3种药物均具促进水液代谢、祛腹水效应，狼毒、京大戟主要通过利尿祛腹水，甘遂主要通过泻下祛腹水。.2. 阐释3种有毒中药醋制解毒-存效机制 狼毒、京大戟、甘遂中含有的多种萜类成分在醋制过程中结构发生转化，高毒性成分转化为低毒性成分，肠道致炎毒性显著下降；同时狼毒、甘遂萜类成分醋制后仍保留促进机体水液代谢的功效，醋制中辅料醋对萜类成分结构转化起关键作用，狼毒二萜类成分在醋制过程中可发生内酯环开环、羟基酯化、氧化、环氧环开环、醚键断裂等反应；京大戟三萜类以及倍半萜类成分醋制时发生氧化、消除反应，其中倍半萜类成分醋制后含量显著下降；甘遂醋制后12个萜类成分含量下降，4个萜类成分上升，其中2种主要成分3-O-(2'E,4'Z-decadienoyl)ingenol（EZDI）及-O-(2'E ,4'Z-decadienoyl)-20-O-acetylingenol（EZDA）具顺式结构，醋制后均转化为反式结构，毒性下降，效应增强。.3. 发现与效应相关，并可标识醋制炮制转化的质量标志物（群） 狼毒中二萜类成分在醋制过程中的转化可作为生品及炮制品的质量标志物（群），其中生狼毒中二萜二聚体Eupractenoid A（EA）在醋制时醚键断裂，形成转化产物Euphebracteolatin A （EHTA），醋制后含量显著上升，毒性下降，药效作用强于其转化前体EA，可作为醋狼毒的质量标志物；京大戟中倍半萜及三萜类成分可以作为京大戟毒性相关质量标志物（群），三萜FPZA在醋制后转化为产物FPZA-C1/C2，倍半萜Orobanone（OR）醋制后的转化产物OR-C2含量显著增加，可作为醋京大戟的质量标志物；甘遂中二萜类成分EZDI、EZDA及其转化产物EEDI、EEDA可以作为甘遂生品及醋制品的质量标志物。