Study On Acute Toxicity And Interaction Of The "Haizao,Daji,Gansui And Yuanhua Reverse Gancao" Based On Zebrafish

"Haizao,Daji,Gansui and Yuanhua all Reverse Gancao" is one verse of the eighteen incompatible medicaments of traditional Chinese medicine,and the eighteen incompatible medicaments is an important part of the incompatibility theory of traditional Chinese medicine,which is the core content in the ancient and modern.The safety of Chinese medicine has been received extensive attention in the society with the progress of human society and the continuous improvement of the healthful medical needs.This article is going to observe the acute toxicity and interaction of the Haizao-Gancao,Daji-Gancao,Gansui-Gancao and Yuanhua-Gancao based on the zebrafish,which will provide the experimental foundations for define the "Haizao,Daji,Gansui and Yuanhua all Reverse Gancao" is reverse or anti-reverse objectively and clarify the essence of science.This will have great significance for protect the safety of clinical drug effectively and for the enrichment and development of the traditional Chinese medicine theory.There are two segments in the first chapter.In the first segment,the model organisms is the essential material for the scientific research,this chapter introduce the common model organisms systematically and roundly,and which introduces theirs development,characteristic and so on primarily.In the second segment introduces the origin,development,characteristic and the application in the various disciplines of the zebrafish.And it clarified the application of zebrafish in the field of traditional Chinese medicine,in particular,the advantages in the field of acute toxicity,which will provide the evidence for choosing zebrafish as the experimental animal in this article.In the second chapter,Haizao-Gancao,Daji-Gancao,Gansui-Gancao and Yuanhua-Gancao with different proportions(1:150,1:67,1:39,1:25,1:17,1:11,1:6,1:4,1:2.8,1:1.8,1:1.2,1:0.84,1:0.6,1:0.36,1:0.2)were extracted by water based on the response surface analysis;Gansui-Gancao with different proportions(1:6,1:2.8,1:1.8,1:1.2,1:0.84,1:0.6,1:0.36,1:0.2)were extracted firstly by 95%alcohol then by water based on the response surface analysis,then obtained the 68 different samples.In the second segments,we got three huge halberd diterpene alcohol type compounds,one triterpenoids compound and two other ingredients by using silica gel column chromatography and preparative high performance liquid column chromatography,and so on.The iiterpenoid compounds was not only the toxic components of Gansui,but also the active ingredients of Gansui.There are sixth segments in the third chapter about the acute toxicity and interaction of the Gansui-Gancao on zebrafish.In the first segment,to evaluate the acute toxicity of different kansui extracts based on the model organism zebrafish.The kansui water extract,alcohol extract,water-alcohol extract were prepared by using the reflux extraction;The death of zebrafish was observed after exposed for 96h in the kansui extracts,in order to evaluate the toxicity of the different extracts.The half lethal concentrations(LC50)of different kansui extracts on zebrafish were calculated by using the SPSS 13.0 software.The LC50 values of the water extract,the alcohol extract and the water-alcohol extract were 31.00 ?g/mL,6.89 ?g/mL and 4.26 ?g/mL,respectively.The results demonstrated that the water-alcohol extract of kansui possessed the highest toxicity,whereas the water extract was lowest.The results provided evidence for the further understanding and evaluation on the toxicity of kansui.In the second segment,observed the acute toxicity and interaction of the Gansui-Gancao water sub-decoction liquid with 15 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.Counting the death rate of the different proportions extracts and drawing the Response Surface(RS),then select the ratio point which represented antagonistic,additive and synergistic accord to the RS,certificating these.The results showed that:it had synergistic when Gansui-Gancao was 1:20?2:1,the concentration range of Gansui was 31?62 ?g/mL and the concentration range of Gancao was 1414.5?9430 ?g/mL;it had antagonistic when Gansui-Gancao was 1:3?8:1,the concentration range of Gansui was 62?248 ?g/mL and the concentration range of Gancao was 471.5?1414.5 ?g/mL;it did not have very obvious synergistic or antagonistic in other scale and scope.Then choosed and verified the most representative point.At last,we found it had antagonistic when Gansui-Gancao was 1:30,and it had synergistic when Gansui-Gancao was 1:16,but it did not have additive when Gansui-Gancao was 1:8.We thought Gansui-Gancao water sub-decoction with different proportions had different function preliminarily.In the third segment,observed the acute toxicity and interaction of the Gansui-Gancao water-alcohol decoction liquid with 8 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.At last,we found the death rate of zebrafish went up with the increase in dose,but it did not show a good interaction.So we thought the interaction of the Gansui-Gancao water-alcohol decoction did not show very well on zebrafish.In the forth segment,observed the acute toxicity and interaction of the Gansui-Gancao water decoction liquid with 15 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.Counting the death rate of the different proportions extracts and drawing the Response Surface(RS).Then select the ratio point which represented antagonistic,additive and synergistic accord to the RS,certificating these on the zebrafish with bopsy.The results showed that:it had synergistic in the great majority of the pripotions,and it had the most synergistic when Gansui-Gancao was 1:30?2:1,the concentration range of Gansui was 31?93 pg/mL and the concentration range of Gancao was 471.5?943?g/mL;it had antagonistic when Gansui-Gancao was 1:12?2:3,the concentration range of Gansui was 434?651 ?g/mL and the concentration range of Gancao was 943?4715 ?g/mL;It did not have very obvious synergistic or antagonistic in other scale and scope.In the forth segment,choosed the three Different proportions which were represented of the synergistic,antagonistic,additive effection to study the toxicity to the organs of zebrafish.And the three groups all injuried the internal organs of the zebrafish,especially the liver and intestinal,and the synergistic group hurt most powerful.We thought Gansui-Gancao water sub-decoction with different proportions had different function preliminarily,and it was harmful to the zebrafish.In the sixth segment,choosed the two different proportions which were represented of the synergistic,antagonistic effection to study the differences in composition based on UPLC-TOF-MS,and analysis the indicator chemical composition which leaded to increase the toxicity by used the OPLS-DA to the established chemical fingerprint information.Founding that at least 14 potential chemical composition could significantly distinguish Gansui-Gancao water co-decocotion and sub-decoction of synergistic,antagonistic effection,and found four compositions appeared in the water co-decoction which did not appear in the water sub-decoction,a component in a fried mixture of water bill of lading does not appear in the water extraction and co-decoction,and one composition appeared in the water sub-decoction which did not appear in the water co-decoction.The experimental results showed that the different chemical composition might cause the toxicity of Gansui-Gancao water co-decoction increased.In the forth chapter,observed the acute toxicity and interaction of the Haizao-Gancao water decoction liquid with 15 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.Counting the death rate of the different proportions extracts and drawing the Response Surface(RS).The results showed that:it had synergistic when Haizao-Gancao was 1:1354?1:60,the concentration range of Haizao was 3.1341?6.268 mg/mL and the concentration range of Gancao was 9.43?94.3 mg/mL;it had the antagonistic in the great majority of the pripotions,and it had the most antagonistic when Gansui-Gancao was 1:16?1:3,the concentration range of Gansui was 6.268?31.34 mg/mL and the concentration range of Gancao was 0?94.3 mg/mL;it did not have very obvious synergistic or antagonistic in other scale and scope.Then choosed and verified the most representative point.In the fifth chapter,observed the acute toxicity and interaction of the Daji-Gancao water decoction liquid with 15 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.Counting the death rate of the different proportions extracts and drawing the Response Surface(RS).The results showed that:it had the most synergistic when Daji-Gancao was 1:123?1:9,the concentration range of Daji was 0?0.54 mg/mL and the concentration range of Gancao was 4.7?66.0 mg/mL;it had the most antagonistic when Daji-Gancao was 1:23?1:10,the concentration range of Daji was 0?188.0 mg/mL and the concentration range of Gancao was 0?1650.3 mg/mL;it did not have very obvious synergistic or antagonistic in other scale and scope.In the sixth chapter,observed the acute toxicity and interaction of the Yuanhua-Gancao water decoction liquid with 15 different proportions.The death of zebrafish was observed after exposed for 96h in the different samples,in order to evaluate the toxicity of the different proportions extracts.Counting the death rate of the different proportions extracts and drawing the Response Surface(RS).The results showed that:it had synergistic in the great majority of the pripotions,and it had the most synergistic when Yuanhua-Gancao was 1:380?1:38,the concentration range of Yuanhua was 0?98 ?g/mL and the concentration range of Gancao was 3.78?37.8 mg/mL;it had the moet antagonistic when Yuanhua-Gancao was 1:3?5:8,the concentration range of Yuanhua was 294?784 ?g/mL and the concentration range of Gancao was 471.5?2357.5 ?g/mL;it did not have very obvious synergistic or antagonistic in other scale and scope.Results:This paper established a rapid evaluation system to evaluate the toxicity and interaction of four groups of "Haizao,Daji,Gansui and Yuanhua all Reverse Gancao" based on the zebrafish.We found that not only the characteristic and extent of the toxicity and interaction of each group were different,but also had a significant synergistic increase in toxicity with the doseage and proportion in a certain range.In comparison,the group of Yuanhua-Gancao had the most significant synergistic increasing extent,followed by Daji,the last one was Gansui.And we also found the toxicity target organs of zebrafish was mainly the digestive system.These results showed that the four group of "Haizao,Daji,Gansui and Yuanhua all Reverse Gancao" all had the characteristics of "anti-",especially the Yuanhua-Gancao group.The results of this study not only provided a scientific basis for answering "Haizao,Daji,Gansui and Yuanhua" and"Gancao" was reverse or not reverse,but also provided evidences for evaluating the characteristics of the results of toxicity generated or enhanced.The main features and innovations in this dissertation are presented.1.Study on acute toxicity and interaction of the traditional Chinese medicine based on zebrafish,which provide a certain reference for zebrafish using in traditional Chinese medicine;2.Analyze the interaction of two herbs compatibility with using response surface methodology.It provides a certain reference for clinical medicine.