Studies On Attenuated Mechanism Of Toxic Traditional Chinese Medicine Of Vinegar Processed Euphorbia

Toxic traditional Chinese medicine Euphorbia pekinensis, Euphorbia kansui, Euphorbia ebracteolata belong to the same genus——Euphorbiaceae Euphorbia genus. Three Euphorbia are included in drastic water-expelling medicinal in clinical and cure different kinds of edemas. The raw products of them are poisonous and stimulated. Using improperly can produce intense diarrhea, abdominal pain, bloody diarrhea and other gastrointestinal toxicity. Throughout the processing history, Euphorbia pekinensis, Euphorbia kansui, Euphorbia ebracteolata are used after vinegar processing to alleviate toxicity since the Song Dynasty and vinegar processed ways has been used till now. Three toxic traditional Chinese medicine have similar toxic behave, alexipharmic methods and clinical application, but toxic components and their transformation by vinegar processing have not been reported.Large numbers of research reveal that toxicity may be relevant to diterpene esters structure. Experimental research proved that diterpenes in Euphorbia pekinensis is Casbane type,Ingol type and Abietane type; diterpenes in Euphorbia kansui mainly include Ingenane type and Jatrophane type; diterpenes in Euphorbia ebracteolata include Abietane type and Ingenane type. These diterpenes all have esterfunction.The concept of "Plant Chemotaxonomy" and "Relatively Plants Chemistry" are already mooted by scholars at the beginning of19th century. More and more research on relation between develop of plant system and chemical components are studied by scholars from home and abroad since the20th Century. This thesis group consider that there is a certain existence of similar chemical components in Euphorbia. Preliminary study discovered that some chemical components have same polarity to terpenoid compounds.This paper is designed according to traditional theory of Chinese medicine——"vinegar processing can alleviate toxicity of Euphorbia pekinensis, Euphorbia kansui, Euphorbia ebracteolata". The research compare chemical contents and toxicity on raw and vinegar processed Euphorbia and find common toxicity components. The study show that Euphorbia pekinensis, Euphorbia kansui and Euphorbia ebracteolata have similar toxicity behave and vinegar processing can alleviate toxicity obviously. Also, the strongest toxic positions of three Euphorbia are affirmed and many diterpenes in strongest toxic positions are analyzed by LC-MS. Studies lay the groundwork for separating toxicity components and analysis on structure-activity relationship between the raw and vinegar processed Euphorbia. The main research contents and work are as follows:1、Screening evaluation model of gastrointestinal toxicity for toxic traditional Chinese medicine of Euphorbia The study adopt two whole animal model (Small intestinal accentuation of mice and pathological examination of mice’s digestive system after initial large-dose) and two cellular model (peritoneal macrophage NO release experiment and cytotoxic experiment on IEC-6) to evaluate toxicity of Euphorbia. Test results prove that Euphorbia pekinensis, Euphorbia kansui, Euphorbia ebracteolata all promote small intestinal accentuation and peritoneal macrophage NO release but can not cause organic destruction to mice’s digestive system and have not cytotoxicity to IEC-6. So, results locate small intestinal accentuation of mice and peritoneal macrophage NO release experiment as evaluation model of toxicity. Using toxic model compare toxicity for raw and vinegar processed Euphorbia. Results reveal that the toxicity of vinegar processd Euphorbia decreased obviously compared to crude one.2、Speculating on common toxic components of EuphorbiaUnder the same conditions, using TLC compare chemical composition for raw and vinegar processed Euphorbia. The result founded that Euphorbia pekinensis, Euphorbia kansui and Euphorbia ebracteolata have common toxic components by thin layer chromatography method (Silica gel as adsorbent, Cyclohexane:Ethyl acetate=8.5:1.5as developer). Common toxic components of raw and vinegar processed Euphorbia are separated into by preparative TLC chromatography and analyzed by HPLC. The chromatograph peak of common toxic components reduce and increase significantly or produce and degradate. In other words, vinegar processing have a material effect on common toxic components.3、Comparing chemical composition and toxicity of raw and vinegar processed Euphorbia in different polarity extractFor narrowing the existence range of common toxic components,70%ethanol extract of Euphorbia pekinensis, Euphorbia kansui and Euphorbia ebracteolata is further divided into four groups according to different polarity:petroleum ether extract, methylene chloride extract, ethyl acetate extract and water extract. Therefore, petroleum ether extract, methylene chloride extract, ethyl acetate extract and water extract of raw and vinegar processed Euphorbia are studied on toxicity by small intestinal accentuation of mice and peritoneal macrophage NO release experiment. The results show that two toxicity tests of Euphorbia pekinensis, Euphorbia kansui and Euphorbia ebracteolata are quite similar, the toxic manifestations are as follows:Euphorbia pekinensis——ethyl acetate extract> petroleum ether extract> methylene chloride extract> water extract; Euphorbia kansui——ethyl acetate extract> petroleum ether extract> methylene chloride extract> water extract; and Euphorbia ebracteolata——petroleum ether extract> methylene chloride extract> ethyl acetate extract> water extract. Also, toxicity reaction of ethyl acetate extract in Euphorbia pekinensis and Euphorbia kansui and petroleum ether extract in Euphorbia ebracteolata is lower significantly by vinegar processed.The results of TLC show that petroleum ether extract, methylene chloride extract and ethyl acetate extract of raw and vinegar processed Euphorbia all have common toxic components (Silica gel as adsorbent, Cyclohexane:Ethyl acetate=8.5:1.5as developer, Rf=0.5). And their chemical composition change obviously by vinegar processed.So ethyl acetate extract in Euphorbia pekinensis and Euphorbia kansui and petroleum ether extract in Euphorbia ebracteolata are defined as the most toxicity site.4-. Analyzing common diterpenoids and terpenoids of the EuphorbiaThis paper investigate whether there have Euphol in Euphorbia, develop an HPLC method for the determination of Euphol and study the changes of euphol in preparing process. The results show that determination of Euphol in Euphorbia pekinensis was obviously higher than Euphorbia kansui. Euphorbia ebracteolata have not Preparing process by vinegar had no significant effect on euphol. In addition, using small intestinal accentuation of mice model and cytotoxic experiment on IEC-6to evaluate toxicity of Euphol. Toxicity results founded that phlogogen of Euphol is very weak and have not the inhibition ratio on IEC-6cell. So Euphol is not the toxic components of Euphorbia.Being combined with data reported in literatures, diterpenoids of ethyl acetate extract in Euphorbia pekinensis and Euphorbia kansui and petroleum ether extract in Euphorbia ebracteolata are analyzed by LC-MS. Then possible chemical structures of diterpenoids in the most toxicity site of Euphorbia are contrasted and analyzed. Four possible diterpenoids including one Casbane type (Pekinenal) and three Abietane type(Helioscopinolide. Helioscopinolide B、 Helioscopinolide C) are identified in ethyl acetate extract of Euphorbia pekinensis. Twelve possible diterpenoids including nine Ingenane type (20-O-(2’E,4’E-decadienoyl)ingenol、20-O-(2’E,4’Z-decadienoyl) ingenol,3-O-(2’E,4’Z-decadienoyl)ingenol、3-O-(2’E,4’E-decadienoyl)ingenol、5-O-(2’E,4’E-decadienoyl)ingenol、3-O-(2’E,4’Z-decadienoyl)-5-O-acetylingenol、3-O-(2’E,4’Z-decadienoyl)-20-O-acetyl-ingenol、3-O-(2’E,4’E-decadienoyl)-20-O-acetylingenol、5-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol) and three Jatrophane type (Kansuinin A、Kansuinine H、Kansuinin F are identified in ethyl acetate extract of Euphorbia kansui. Seven possible diterpenoids including three Abietane type (Jolkinolide B、 ebraeteolatanolide B、ent-11β-hydroxyabiera-8(14),13(15)-dien-16,12β-olide)and four Ingenane type are identified in petroleum ether extract of Euphorbia ebracteolata. The results indicated that there are so many diterpene esters in the most toxicity site of Euphorbia.This paper worked at representative Chinese medicine of Euphorbia——Euphorbia pekinensis, Euphorbia kansui and Euphorbia ebracteolata under theory of Plant Chemotaxonomy. Making tracks for toxic component based on common angle and searching for common terpenoid compounds by model of toxicity will accelerate deeply discussion in mechanism of processing drugs.