Jiangsu Key Laboratory for High Technology Research of TCM Formulae, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine and Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, No. 138, Xianlin Road, Qixia District, Nanjing, 210023, PR China.
Key Laboratory of Shaanxi Administration of Traditional Chinese Medicine for TCM Compatibility, Shaanxi University of Chinese Medicine, Xi'an, 712046, Shaanxi Province, PR China.
J Pharm Biomed Anal. 2021 Feb 20;195:113828. doi: 10.1016/j.jpba.2020.113828. Epub 2020 Dec 7.
The dried roots of Euphorbia kansui (EK) are especially beneficial for the treatment of edema, but the severe toxicity limits their clinical applications. Euphorbia kansui stir-fried with vinegar (VEK) is traditionally employed to reduce the toxicity of EK. However, the material basis for the toxicity reduction with effectivity conservation is still unclear. Therefore, in this study, a rapid, sensitive, and reliable ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method was firstly established to simultaneously determine six ingenane-type diterpenoids, i.e. kansuiphorin C (1), 5-O-benzoyl-20-deoxyingenol (2), 20-deoxyingenol (3), 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (4), 20-O-(2'E,4'Z-decadienoyl)ingenol (5), and ingenol (6), in EK and VEK based on the processing conversion. Then, the toxicity evaluation on zebrafish embryos and modulation of the expression of aquaporin-3 (AQP3) proteins in HT-29 cells were employed to investigate the toxicity-activity of six compounds. Chromatographic separation was obtained on Waters BEH RP18 column (2.1 mm × 100 mm, 2.5 μm) with the mobile phase composed of 0.1 % formic acid in acetonitrile and water, respectively. The column temperature was 35 ℃ at a flow rate of 0.4 mL min. Multiple reaction monitoring was conducted in both positive and negative modes for quantitative analysis. The method was then successfully used for the determination of six compounds in EK and VEK. In addition, 1, 2, 4, and 5 had evident cardiotoxicity, intestinal irritation and nutrient absorption disorders on zebrafish larvae, while no in-vivo toxicity was seen for groups given 3 and 6 (LC > 200 μM). Meanwhile, 1, 2, 4, 5, and 6 significantly increased the expression of AQP3 protein (p < 0.05) to promote the excretion of water in the colon. This study demonstrated that toxic ingenane-type diterpenoids converted into the less toxic compounds with the same core structure through the breakage of multiple ester bonds in the side chain. At the same time, the laxative effect was retained, providing useful information for the optimization of the process of EK and quality evaluation of other similar toxic Chinese herbal medicines.
甘遂的干根尤其有益于治疗水肿，但严重的毒性限制了其临床应用。醋炙甘遂（VEK）传统上用于降低甘遂的毒性。然而，保持疗效降低毒性的物质基础仍不清楚。因此，本研究首次建立了一种快速、灵敏、可靠的超高效液相色谱串联质谱（UFLC-MS/MS）方法，用于同时测定甘遂中的六种异土木香二萜类化合物，即甘遂萜 C（1）、5-O-苯甲酰-20-去氧艾里新（2）、20-去氧艾里新（3）、3-O-（2'E，4'E-癸二烯酰基）-20-O-乙酰基艾里新醇（4）、20-O-（2'E，4'Z-癸二烯酰基）艾里新醇（5）和艾里新醇（6）。然后，通过对斑马鱼胚胎的毒性评价和 HT-29 细胞水通道蛋白 3（AQP3）蛋白表达的调节，研究了六种化合物的毒性-活性。色谱分离在 Waters BEH RP18 柱（2.1mm×100mm，2.5μm）上进行，流动相由乙腈和水中的 0.1%甲酸组成。柱温为 35℃，流速为 0.4mL/min。正、负离子模式下均进行多反应监测进行定量分析。该方法成功地用于测定甘遂和醋炙甘遂中的六种化合物。此外，1、2、4 和 5 对斑马鱼幼虫表现出明显的心脏毒性、肠道刺激和营养吸收障碍，而给予 3 和 6 的组未见体内毒性（LC>200μM）。同时，1、2、4、5 和 6 显著增加了 AQP3 蛋白的表达（p<0.05），促进了结肠中水的排泄。本研究表明，通过侧链中多个酯键的断裂，有毒的异土木香二萜类化合物转化为具有相同核心结构的毒性较小的化合物。同时保留了通便作用，为甘遂炮制工艺的优化和其他类似毒性中草药的质量评价提供了有用信息。
