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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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (LC 50 > 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., Competing Interests: Declaration of Competing Interest All the authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Euphorbia kansui is a toxic Chinese herbal medicine and exhibits promising treatment to the malignant ascites (MA) in its traditional use. Ingenane-type and jastrophane-type diterpenes are demonstrated to be responsible for the toxicity and efficacy of kansui. Two representative compounds, kansuiphorin C (KPC) and kansuinin A (KA) in each type were proved to effectively reduce the ascites. The biological and toxicological effects are closely associated with the gastrointestinal tract, but the possible mechanism and related metabolic functions of KPC and KA treating MA through modulating the gut microbiota remain unclear., Aim of the Study: To investigate the possible mechanism and related metabolism of KPC and KA ameliorating malignant ascites through modulating gut microbiota., Materials and Methods: MA rats and normal rats were divided into different groups and administrated with KPC, KA, and positive drug, respectively. 16S rDNA gene sequencing and metagenomes analysis combined with the quantification of short-chain fatty acids of feces were performed to reflect the modulation of gut microbiota. Then, the metabolites of KPC and KA in rat feces under the normal and pathological circumstances were detected by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) to explore the in-vivo bacterial biotransformation., Results: KPC and KA were modulatory compounds for gut microbiota. The richness of Lactobacillus and the decreased abundance of Helicobacter involved in the carbohydrate metabolism and amino acid metabolism could be responsible for their prohibitory effects on malignant ascites. KPC exhibited stronger modulation of gut microbiota through making the abundance of Helicobacter about 3.5 times lower than KA. Besides, in-vivo microbial biotransformation of KPC and KA contained oxidation, hydrolysis, dehydration, and methylation to form metabolites of lower polarity. Besides, at the dosage of 10 mg kg -1 , the toxicity of both compounds had weaker influences on the gut microbiota of normal rats., Conclusion: KPC and KA could ameliorate malignant ascites by modulating gut microbiota mainly containing the increase of Lactobacillus and the decrease of Helicobacter and related carbohydrate and amino acid metabolism, providing a basis for their promising clinical usage., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The dried roots of Euphorbia kansui S.L.Liou ex S.B.Ho have long been used to treat edema in China. However, the severe toxicity caused by Euphorbia kansui (EK) has seriously restricted its clinical application. Although EK was processed with vinegar to reduce its toxicity, the detailed mechanisms of attenuation in toxicity of EK stir-fried with vinegar (VEK) have not been well delineated. Diterpenoids are the main toxic ingredients of EK, and changes in these after processing may be the underlying mechanism of toxicity attenuation of VEK. 3- O -(2' E ,4' Z -decadienoyl)-20- O -acetylingenol (3-O-EZ) is one of the diterpenoids derived from EK, and the content of 3-O-EZ was significantly reduced after processing. This study aims to explore the underlying mechanisms of toxicity reduction of VEK based on the change of 3-O-EZ after processing with vinegar. Based on the chemical structure of 3-O-EZ and the method of processing with vinegar, simulation experiments were carried out to confirm the presence of the product both in EK and VEK and to enrich the product. Then, the difference of peak area of 3-O-EZ and its hydrolysate in EK and VEK were detected by ultra-high-performance liquid chromatography (UPLC). Furthermore, the toxicity effect of 3-O-EZ and its hydrolysate, as well as the underlying mechanism, on zebrafish embryos were investigated. The findings showed that the diterpenoids (3-O-EZ) in EK can convert into less toxic ingenol in VEK after processing with vinegar; meanwhile, the content of ingenol in VEK was higher than that of EK. More interestingly, the ingenol exhibited less toxicity (acute toxicity, developmental toxicity and organic toxicity) than that of 3-O-EZ, and 3-O-EZ could increase malondialdehyde (MDA) content and reduce glutathione (GSH) content; cause embryo oxidative damage by inhibition of the succinate dehydrogenase (SDH) and superoxide dismutase (SOD) activity; and induce inflammation and apoptosis by elevation of IL-2 and IL-8 contents and activation of the caspase-3 and caspase-9 activity. Thus, this study contributes to our understanding of the mechanism of attenuation in toxicity of VEK, and provides the possibility of safe and rational use of EK in clinics.

Malignant ascites (MA) is one of the severe complications of gastrointestinal tumors, affecting the patients' survival time and quality of life. Euphorbia kansui is a commonly used toxic Chinese herbal medicine for malignant ascites. Our previous study showed that the biological and toxicological effects of kansui were closely related to the gastrointestinal tract. The ingenane-type and jastrophane-type diterpenoids are both toxic and active components of kansui. The contents of kansuiphorin C (KPC) and kansuinin A (KA) take highest accounts in each type of diterpene. Hence, in this study, the efficacy and toxicity of KPC and KA on normal rats and MA rats were firstly evaluated by serum liver enzymes (ALT and AST), oxidative damage indicators (GSH, SOD, MDA and LDH), inflammatory indexes (TNF-α, IFN-γ and IL-2) and the volume of ascites. Changes in the levels of these indices showed that although the toxicity of KPC on normal rats was stronger than KA, KPC exhibited better efficacy to the malignant ascites with no obvious side effects at the dose of 10 mg·kg -1 . Then, accurate and reliable methods for the determination of KPC and KA in the rat feces by ultra-fast liquid chromatography coupled with MS/MS detector (UFLC-MS/MS) were established, detected by the multiple reaction monitoring mode. The chromatographic separation was conducted on an XBbridge C18 column (50 mm × 2.1 mm, 2.5 μm) using gradient elution composed of 0.1% formic acid in water and acetonitrile. The flow rate was 0.5 mL·min -1 and column temperature was 30 °C. The method was finally applied to the comparative study on normal and malignant ascites rats given KPC and KA, respectively. Interestingly, the results showed that KPC's accumulative fecal excretion rate (normal, 19.22%±5.36%; model, 15.96%±3.47%) were much higher than that of KA (normal, 2.928%±0.741%; model, 2.835%±0.873%) at the same dose within 48 h. This suggested KPC had higher in-vivo transformations in comparison with KA, providing guidance for the further preclinical research of KPC and KA as promising compounds treating MA., (Copyright © 2019 Elsevier B.V. All rights reserved.)