Comparison of content-toxicity-activity of six ingenane-type diterpenoids between Euphorbia kansui before and after stir-fried with vinegar by using UFLC-MS/MS, zebrafish embryos and HT-29 cells.

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-1. 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 (LC50 > 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.

The water expelling effect evaluation of 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol and ingenol on H22 mouse hepatoma ascites model and their content differences analysis in Euphorbia kansui before and after stir-fried with vinegar by UPLC.

ETHNOPHARMACOLOGICAL RELEVANCE: Malignant ascites (MA) effusion is mainly caused by hepatocellular, ovarian, and breast cancer etc. It has been reported that Euphorbia kansui (EK), the root of Euphorbia kansui S.L.Liou ex S.B.Ho, possessing a therapeutic effect on MA. However, the clinical applications of EK are seriously restricted for its severe toxicity. Although studies demonstrated that vinegar-processing can reduce the toxicity and retain the water expelling effect of EK, its specific mechanism remains unknown. AIM OF THE STUDY: This study aims to explore the underlying mechanisms of toxicity reduction without compromising the pharmacological effects of EK stir-fried with vinegar (VEK). MATERIALS AND METHODS: 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3-O-EZ), a major diterpenoid of EK, could convert into ingenol after processing EK with vinegar. The H22 mouse hepatoma ascites model was replicated, and were given 3-O-EZ and ingenol seven days (110.14, 50.07 and 27.54 mg/kg). The histopathological observation, serum liver enzymes, serum Renin-Angiotensin-Aldosterone System (RAAS) levels, ascites volumes, pro-inflammatory cytokines levels and H22 cells apoptosis in ascites were examined. Then the intestine (Aquaporin 8, AQP8) and kidney (Aquaporin 2, AQP2; Vasopressin type 2 receptor, V2R) protein expression were detected, as well as the metabolomics of serum were analyzed. Finally, the content of 3-O-EZ and ingenol in EK and VEK were investigated. RESULTS: 3-O-EZ and ingenol can relieve hepatic and gastrointestinal injuries, reduce ascites volumes, enhance the H22 cells apoptosis, ameliorate abnormal pro-inflammatory cytokines and RAAS levels, and down-regulate the expression of AQP8, AQP2, V2R. The involved metabolic pathways mainly included glycerophospholipid metabolism and arachidonic acid metabolism. And the decreasing rate of 3-O-EZ in VEK was 19.14%, the increasing rate of ingenol in VEK was 92.31%. CONCLUSION: 3-O-EZ and ingenol possess significant effect in treating MA effusion, while ingenol has lower toxicity compared with 3-O-EZ. And provide evidence for the mechanism of attenuation in toxicity without compromising the pharmacological effects of VEK.

[Progress in application of bile acid metabolism in traditional Chinese medicine study].

Traditional Chinese medicine boasts aunique theoretical system and rich practical experience. However, traditional Chinese medicine has an unclear material basis, vague pharmacological mechanism, and potential toxicity, which is the key factor to hinder its modernization and wide application. Therefore, when the physico-chemical analysis of chemical components of traditional Chinese medicine is insufficient to reflect the characteristics and mechanisms, the multi-target biological system correlation analysis in conformity to the holistic view of the basic theory of traditional Chinese medicine has gradually attracted wide attention. Specifically, bile acids, as an important endogenous metabolite in the body, play an important role in regulating digestion, absorption and metabolism of nutrients, and greatly impact the health. In recent years, a number of studies have been made on the metabolism pathway of bile acids and their important regulatory effects in body metabolism, making bile acids as a significant target of traditional Chinese medicine on the body. In view of this, based on bile acid metabolism, the paper reviewed the biological functions of bile acids in regulating body metabolism and its interaction with intestinal microbiota, providing a basis for exploring the connotation of bile acid metabolism changes under physiological/pathological conditions of the body. The study progress of bile acid metabolism in traditional Chinese medicine efficacy/toxic mechanism is further reviewed, which provides a basis for exploring the efficacy and hepatotoxicity mechanism of traditional Chinese medicine with bile acid as a biomarker, thereby laying a foundation for the clinical safety of traditional Chinese medicine.