Study On The Mechanism Of Attenuation In Gastrointestinal Toxicity Of Euphorbia Kansui After Stir-baking With Vinegar

The roots of Euphorbia kansui T.N.Liou ex T.P.Wang are known as "Kan Sui" in Chinese medicine,recorded in Shenmung's Herbal as a low grade drug and has been used as a herbal remedy for edema,ascite,asthma in China for centuries.Kansui widely used in the clinical treatment of ascites due to cirrhosis,nephritis edema,permeability pleurisy,as well as advanced schistosomiasis due to ascites.However,the clinical use of kansui had some practical disadvantages because it might result in inflammation,irritation to the oral,skin,and gastrointestinal,and tumor-promotion.And recent studies have also reported that kansui have strong stimulating effect on mucous membranes of the gastrointestinal tract just like crotonic acid and cantharidin,the above toxic seriously restrict the clinical use of the drug.The vinegar stir-baking kansui was regularly used by Chinese in the past period,and our studies have also shown that vinegar stir-baking could reduce the toxicity of kansui.However,its detoxication mechanism remains unknown.This research is to carry out a systematic study on the mechanism of attenuated the gastrointestinal irritant action of Kansui Radix after vinegar-processing and lay the foundation for further demonstrating detoxication mechanism of vinegar treated kansui preparations.This dissertation is summarized as follows:Part one:Literature reviewBy means of reviewing relevant ancient and moderm documents,the current status of studied on the mechanism of gastrointestinal toxicity,common traditional Chinese medicines with gastrointestinal toxicity and the methods to attenuate gastrointestinal toxicity of traditional Chinese medicine were summarize.Part two:Confirm gastrointestinal toxic parts of Euphorbia kansuiThis section has compared the toxicity on normal gastric epithelial cell line GES-1 and rat intestinal crypt epithelial cell line IEC-6 of alcohol extract,ethyl acetate extract of Euphorbia kansui,ethyl acetate residue after extracting and ethyl acetate extract of Kansui Radix stir-baked with vinegar by MTT assay.The results showed that kansui significantly inhibited the proliferation and morphological changes of GES-1 cells and IEC-6 cells in concentration-dependent manner.However,kansui Radix stir-baked with vinegar significantly attenuated the toxicity on GES-1 and IEC-6 cells compared with the kansui Radix in concentration-dependent manner.The toxicity attenuation order as ethyl acetate extract of Euphorbia kansui>alcohol extract of Euphorbia kansui>ethyl acetate residue after extracting>ethyl acetate extract of Kansui Radix stir-baked with vinegar.Thus,the result show that gastrointestinal toxic parts of Euphorbia kansui was ethyl acetate extract and the vinegar stir-baking can significantly attenuate the toxicity on GES-1 and IEC-6 cells.Part three:chemical constituents of gastrointestinal toxic parts11 compounds were isolated from ethyl acetate extract of Euphorbia kansui with silica gel,HPLC,and TLC as the main method.Among them,10 compounds were elucidated on the basis of chemical and spectral methods.Four ingenane-type diterpenes were named as 20-O-(2 'E,4'Z-decadienoyl)ingenol(EK-01),3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol(EK-02),3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol(EK-03),5-O-benzoyl-20-deoxyingenol(EK-04);one jatrophane-type diterpene was named as kansuinin B(EK-05);four triterpenes such as 11-oxo-kansenonol(EK-06),Kansenonol(EK-07),Epi-kansenone(EK-08),euphol(EK-09);other types such as ?-sitosterol(EK-10).Part four:toxicity of chemical constituents on the IEC-6 cells9 terpenoids of kansui with different structures were selected to test for their inhibitions on the growth of IEC-6 cells by MTT assays.The result show that ingenane-type diterpenoids exhibited stronger potent cytotoxic activity against of IEC-6 cells.The compound of the ingenane-type diterpenoids with 3-unsaturated aliphatic chain possessed stronger cytotoxic activity,and there is a weak effect on the cytotoxic activity with 20-O group,such as EK-01.The compound of the triterpenoids showed stronger cytotoxic activity with the structure of 7-carbonyl group as EK-06,EK-07,EK-08.All compounds of jatrophane diterpenoids showed very weak cytotoxic activity against IEC-6 cells.Part five:Study the gastrointestinal toxicity attenuation and its mechanism of Kansui Radix stir-baked with vinegar in rats.After treated with Kansui and Kansui Radix stir-baked with vinegar,weight of ICR rats was reduced and diarrhea was happened.The results showed that Kansui can significantly alter the stomach,intestines pathological morphology(P<0.05,P<0.01);Kansui Radix stir-baked with vinegar can significantly improve the gastrointestinal pathological morphology(P<0.05,P<0.01).TEM showd Kansui groups rats gastric epithelial glands disorder.cytoplasmic electron density is uneven,irregular nuclei,Kansui Radix stir-baked with vinegar groups rats gastric epithelial cell morphology inherent disorder,individual cell apoptosis;Kansui groups rats intestinal microvilli reducing the number of height grow shorter,intercellular tight junctions near the surface of the shorter,Kansui Radix stir-baked with vinegar groups rats intestinal epithelial cells arranged rules,cell morphology consistent decrease in the number of microvilli surface height shorter,but more regular.In comparison with the control group,Kansui groups had lower SOD activities and GSH content,but higher MDA content and LDH activities(P<0.05,P<0.01);in comparison with Kansui groups,Kansui Radix stir-baked with vinegar groups had higher SOD activities and GSH content,but lower MDA content and LDH activities(P<0.05,P<0.01).HE staining of gastric and intestinal tissues showed less gastric mucosa and intestinal mucosa damage in Kansui Radix stir-baked with vinegar treated groups(P<0.05,P<0.01).Immunohistochemical staining of the expression of E-cad of Kansui treated groups were decreased,however,Kansui Radix stir-baked with vinegar treated groups were increased(P<0.05,P<0.01).Part six:the mechanism of toxicity attenuation on rat intestinal crypt epithelial cell line IEC-6 of EK-02This section has studied the toxicity on IEC-6 cells of EK-02 by MTT assay and the inverted phase contrast microscope.Results of Annexin-V-FITC/PI assay and flow cytometry showed that EK-02 significantly induced the IEC-6 cells apoptosis(P<0.05,P<0.01).HCS results showed that EK-02 can significantly reduce IEC-6 cells nuclear fluorescence intensity and the fluorescence intensity of the mitochondrial membrane potential(P<0.05,P<0.01).Elisa kit assay results show that EK-02 can significantly increased the activity of caspase-3,8,9.Intracellular reactive oxygen species(ROS)level detected with laser confocal microscopy results showed that EK-02 can increased ROS level(P<0.01).Western-blot analysis results showed that EK-02 can promoted the Cytochrome c release(P<0.01)and significantly up-regulated Bax,AIF,Apaf-1 expression(P<0.01)and down-regulated bcl-2 expression(P<0.01).The results indicated that EK-02 can significantly induce the IEC-6 cells apoptosis through mitochondrial apoptototic pathway.Part seven:structure and toxicity of EK-02 hydrolyzed productAfter hydrolysis,TLC results showed that a new substance generated with higher polarity than EK-02.The new substance clearly exist in Kansui Radix stir-baked with vinegar while low content in Kansui by HPLC.The structural analysis showed it was a ingenane-type diterpene alcohols and IC50to IEC-6 cells was 98.3?M.It was presumed that the toxicity attenuation and its mechanism might be ingenol-type diterpene esters hydrolysis converted into ingenane-type diterpene alcohols by the procedures of stir with baked and infiltration with vinegar.