| Kansui(short for the dried roots ofEuphorbia kansui T.N.Liou ex T.P.Wang),which was recorded in Shennong-Bencao,has multiple medical applications for edema,fullness,chest and abdomen ponding,retention of phlegm and fluid embolism.Kansui was widely used in clinical treatment of ascites due to cirrhosis,nephritis edema,infiltration of pleurisy and ascites due to advanced schistosomiasis in China for centuries.Many studies suggested that kansui exhibited promising effects in the treatment of leukemia,cancer,pancreatitis and intestinal obstruction.However,despite being effective in range of diseases,its use was always under scrutiny because of its irritation to the skin,oral and gastrointestinal,inflammation and tumor promotion.The processing method of stir-baking with vinegar was regularly used to reduce the toxicity of kansui,and our previous studies have also shown that the toxicity of kansui could be reduced after stir-baked with vinegar.However,the material basis and detoxication mechanism of kansui stir-baked with vinegar is still poorly defined and rarely reported.This research focuses on the mechanism of gastrointestinal irritation attenuation of kansui after vinegar-processing and lays the foundation for further demonstrating detoxication mechanism of vinegar treated kansui preparations.Based on the relevant literatures in home and abroad,the current research status of common traditional Chinese medicines with gastrointestinal toxicity,the mechanism of gastrointestinal toxicity and the methods to attenuate gastrointestinal toxicity of traditional Chinese medicine were summarize.With silica gel,preparative HPLC and TLC as main method,seventeen compounds were isolated from ethyl acetate extract of Euphorbia kansui and fifteen of them were elucidated on the basis of chemical and spectral methods.Six ingenane-type diterpenes were named as 3-O-(2,3-dimethylbutanoyl)-13-O-dodecanoyl-20-deoxylingenol(EK-01),3-0-(2,3-dimethylbutanoyl)-13-O-dodecanoyl-20-O-acetylingenol(EK-02),3-0-(2'E,4'Z-decadienoyl)-20-deoxyingnol(EK-03),3-0-benzoyl-20-deoxyingenol(EK-04),3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol(EK-05),3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol(EK-06);two jatrophane-type diterpene was named as kansuinin A(EK-11),kansuinin B(EK-12);four triterpenes such as 11-oxo-kansenonol(EK-07),euphol(EK-08),kansenone(EK-09),epi-kansenone(EK-10);other types such as ?-sitosterol(EK-13),palmitic acid(EK-14),isoscopletin(EK-15).Eight terpenoids and four triterpenes of kansui were selected for the gastrointestinal toxicity evaluation on the intestinal epithelial cell line IEC-6 by MTT assays.And the structure-activity relationship was discussed.The results show that ingenane-type diterpenoids like EK-03,EK-05,EK-06 exhibited stronge cytotoxicity on IEC-6 cells.Ingenane-type diterpenoids with 3-unsaturated aliphatic chain possessed stronger cytotoxicity,and there is a weak effect on the cytotoxicity with 20-0 group,such as EK-02.Triterpenoids with the structure of 8-ene-7-one such as EK-06,EK-07,EK-08 showed stronger cytotoxicity.All compounds of jatrophane diterpenoids showed very weak cytotoxicity on IEC-6 cells.The mechanism of toxicity on rat intestinal crypt epithelial cell line IEC-6 of kansenone was studied preliminary.First,the relative inhibitory rate of kansenone on IEC-6 cells was determined by MTT assay and cell morphology was observed under the inverted phase contrast microscope.The results showed that kansenone had strong toxicity of intestinal epithelial cells.The results of SOD and MDA kit showed that kansenone have oxidative damage to IEC-6 by reducing the activity of SOD and increasing the content of MDA.Then,cell cycle and apoptosis of IEC-6 cells of kansenone were determined by flow cytometry.Results showed that kansenone could arrest IEC-6 cells in G0/G1 phase and induce apoptosis of IEC-6 cells in a concentration-dependent manner.Further researches about apoptosis mechanism of kansenone on IEC-6 were taken.Typical characteristics of apoptosis were observed under a transmission electron microscopy,such as chromatin clumping under the nuclear membrane and mitochondria swelling.HCS results showed that kansenone significantly reduced the fluorescence intensity of JC-1 polymer and monomer ratio,suggesting that kansenone can significantly reduce the mitochondrial membrane potential of IEC-6 cells.Laser scaning confocal microscope results showed that kansenone can significantly increase the levels of reactive oxygen species(ROS)in IEC-6 cells.Activity of caspase-3,caspase-8 and caspase-9 was determined by Elisa kit and protein expression of Cytochrome C,Bax,Bcl-2,AIF,Apaf-1 by Western-blot.Results showed that kansenone could improve caspase-3,caspase-8,caspase-9 activity,promote the release of Cytochrome C,and significantly up-regulated Bax,AIF,Apaf-1 expression and down-regulated bcl-2 expression.Determining mRNA expression of Fas,FasL,TNFR1 and NF-? B using RT-PCR technology,the results showed that kansenone could enhance the mRNA expression of Fas,FasL,TNFR1,NF-?B.All above results suggest that kansenone induced IEC-6 cells apoptosis through mitochondria and death receptor apoptototic pathway.The content changes of euphol,kansenone and 11-oxo-kansenonol were evaluated by HPLC.The results showed that compared with unprocessed kansui,kansui stir-baked with vinegar could significantly reduce content of these three triterpenes and the content change rates were 13.43%,15.15%and 14.79%.The results indicated that triterpenes could be significantly reduced after stir-baked with vinegar and inferred that one of the possible mechanisms of detoxication was that reducing of toxic ingredients such as triterpene compounds. |
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