Study The Mechanism Of Reducing Toxicity And Preserving Effect Of Kansui Stir-fryed With Vinegar

Euphorbia kansui,the root of Euphorbia kansui S.L.Liou ex S.B.Ho,has the effect of draining water,dispersing swelling and dissipating binds.It can be used for the treatment of edema,hydrothorax,ascites,fecal and urinary stoppage and so on.However,it is toxic and has strong irritation to skin,mucous membrane and intestine,and can cause severe abdominal pain,diarrhea and other toxic and side effects.In clinic,vinegar is often used to reduce its toxicity and relieve its purgative effect.Previous studies of the research group found that the main toxicity of Kansui is intestinal toxicity,which can lead to diarrhea and intestinal inflammation,and the intestinal toxicity is weakened after stir-fryed with vinegar.The main effective substance of Kansui is its terpenoids.The composition of terpenoids will change significantly after stir-fryed with vinegar.The results show that terpenoids in kansui have dual physiological activities,which are toxic and effective components.So,the intestinal toxicity of Kansui after stir-fryed with vinegar decreased,does its effect of draining water and drinking still exist?Is the change of terpenoid composition related to the reducing toxicity and preserving effect of stir-fryed with vinegar?So far,there is no in-depth study on the moderating effect of stir-fryed with vinegar and the mechanism of reducing toxicity and preserving effect.In order to further explore the material basis of " Reduce toxicity and retain effect " of Kansui stir-fried with vinegar.Taking mice with cancerous ascites as the experimental model,and studies the changes of ascites eliminating and diarrhea effects before and after Kansui stir-fried with vinegar.Then terpenoids separated from kansui boiled with vinegar to explore the transformation rule of terpenoids by using HPLC and LC-MS/MS.Finally,the correlation between the transformation of terpenoids and the " Reduce toxicity and retain effect " of Kansui stir-fried with vinegar are evaluated by cell experiment.The main contents and results of this paper are as follows:1.The effects of different extract fractions of before and after Kansui stir-fryed with vinegar on mice with cancerous ascites.The dichloromethane fraction and non-dichloromethane fraction were obtained from kansui by dichloromethane and 95%ethanol.The results showed that the dichloromethane part of Kansui had the effect of purgation and elimination of ascites,while the non-dichloromethane part had little effect on mice.The dichloromethane part of Kansui stir-fryed with vinegar can also play the role of diarrhea and ascites elimination,but compared with the raw Kansui,the diarrhea effect is mild.The above results showed that dichloromethane was the effective fraction of Kansui,and the cathartic effect of Kansui stir-fryed with vinegar was mild,but the ascites eliminating effect still existed.In addition,the experiment also found that kansui had no significant inhibitory effect on cancer cells in ascites of mice with cancerous ascites,and had no significant promoting effect on urine volume.2.The effect of different extract fractions of before and after Kansui stir-fryed with vinegar on the expression of AQP1 and AQP3 protein in intestinal tract of mice with cancerous ascites.Since Kansui mainly expels ascites by purgative action,the intestinal aquaporins AQP1 and AQP3 were measured in order to find out whether the purgative action of Kansui is related to the regulation of aquaporins.The expression of AQP1 and AQP3 in duodenum,jejunum,ileum and colon were measured by Western blot.The results showed that kansui could reduce the protein expression of AQP1 and AQP3 in different segments of the intestine,resulting in the decrease of intestinal water absorption and diarrhea.Compared with the total extract group,there was no significant difference in the effect of dichloromethane fraction of Kansui,but the non-dichloromethane fraction had almost no effect,which further indicated that the dichloromethane fraction of Kansui was the effective fraction of Kansui.The effect of Kansui stir-fryed with vinegar on AQP1 and AQP3 in ileum was weakened,but not on AQP1 and AQP3 in colon.This may be the reason why Kansui stir-fryed with vinegar can alleviate diarrhea.3.Effects of different extract fractions of before and after Kansui stir-fryed with vinegar on the expression of IL-1β and TNF-α in intestinal tract of normal mice.Purgative effect is not only the effective way of removing ascites,but also the performance of intestinal irritation of Kansui.Literature studies have found that there is a certain relationship between the purgative effect of Kansui and inflammation.Therefore,in this part,the expression levels of IL-1 β and TNF-α were used as the toxicity indexes of Kansui,and the inflammatory factors in duodenum,jejunum,ileum and colon of mice were measured by ELISA,in order to further explore the toxic parts of Kansui and the toxic changes of Kansui after vinegar processing.The results showed that the total extract and dichloromethane fraction of Kansui could significantly increase the levels of IL-1 β and TNF-α in jejunum and ileum,and there was no significant difference between them.However,the non-dichloromethane fraction group had no significant effect on the expression of IL-1 βand TNF-α in intestinal inflammation.The results further showed that the dichloromethane fraction of Kansui was the toxic fraction of Kansui.In addition,the effect of Kansui vinegar on inflammation was significantly weakened,indicating that Kansui stir-fryed with vinegar can play a role of "detoxification".4.Component analysis of dichloromethane extract fraction and non-dichloromethane extract fraction Kansui.In order to further investigate whether the dichloromethane extract fraction of Kansui could be identified as the terpenoid fraction of Kansui,LC-MS/MS technology was used to identify and analyze the terpenoids in dichloromethane extract and non-dichloromethane extract fraction of Kansui.The results showed that 16 terpenoids were identified from the dichloromethane extract of Kansui,including 9 Euphorbiaceae diterpenoids,3 pseudoleucaena diterpenoids and 4 triterpenoids.Only one euphorbane diterpene and one pseudobruguiane diterpene were identified in the non-dichloromethane fraction,and they were also found in the dichloromethane fraction.The above results showed that the dichloromethane could enrich the terpenoids in kansui.5.Isolation,purification and identification of main components from dichloromethane extract fraction of Kansui.Kansui was extracted with dichloromethane and then separated by gradient elution of petroleum ether ethyl acetate and petroleum ether acetone with silica gel of different mesh numbers.The compounds were further purified by ODS column and C18 column.Finally,the compounds were identified by 1H-NMR and 13C-NMR.Finally,12 diterpenoids were identified as(5-O-benzoyl-20-deoxyingenol,3-O-benzoyl-20-deoxyingenol,3-O-(2E,4Z-Decadienoyl)-20-deoxyingenol,3-O-(2E,4E-Decadienoyl)-20-deoxyingenol,5-O-(2’E,4’E-decadienoyl)ingenol,3-O-(2’E,4’Z-decadienoyl)ingenol,3-0-(2’E,4’E-decadienoyl)ingenol,3-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol,kansuinine B,3-O-(2’E,4’E-decadienoyl)-20-O-acetylingenol,kansuinin A,4-O-acetyl-5-O-benzoyl-3β-hydroxy-20-deoxyingenol)and two fatty acids(α-linolenic acid,linoleic acid).6.Study on the correlation between the structure transformation of terpenoids and the change of toxicity and effect before and after Euphorbia kansui stir-fried with vinegar.The terpenoids were processed to explore the transformation of terpenoids in vinegar.Methods:the monomers were heated in a constant temperature heater(160℃)with 6%glacial acetic acid for 40 min,and then analyzed by HPLC and LC-MS/MS.The results showed that the content of most terpenoids decreased,and C6 and C8 compounds were transformed obviously.C6(3-O-(2’E,4’Z-decadienoyl)ingenol)can be converted to C7(3-O-(2’E,4’E-decadienoyl)ingenol)and C8(3-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol)can be converted to C10(3-O-(2’E,4’E-decadienoyl)-20-O-acetylingenol).It is found that the conversion of C6 and C10 compounds is that the spatial configuration of decadienyl group at position 3 changes from cis to trans.7.Study on the difference of toxicity and effect between C6 and C7,C8 and C10.The effects of C6,C7,C8 and C10 on AQP1 and AQP3 of IEC-6 cells and the expression of IL-1 β and TNF-α of macrophages were measured by Western blot.The results showed that there was no significant change in AQP1 and AQP3 of IEC-6 cells before and after transformation,but the inflammatory effect on macrophages was weakened after transformation.This result is consistent with the results of finishing animal experiment,which indicates that the transformation of this kind of components may be one of the material base of "reducing toxicity and preserving effect" of Kansui stir-fried with vinegar.Summary1.In this paper,the mice with cancerous ascites were used.The results showed that the dichloromethane part of Kansui had the function of purgation and elimination of ascites in mice with cancerous ascites.The dichloromethane part of Kansui stir-fried with vinegar also has the effect of purgation and ascites elimination,but compared with the raw Kansui,the purgation effect is mild and the effect on intestinal aquaporin is weakened.The non-dichloromethane fraction had no effect.In addition,the purgative effect of Kansui is related to the decrease of AQP1 and AQP3.In order to further explore the effect of Kansui stir-fried with vinegar on the intestinal toxicity of Kansui,the normal mouse model was also used.The results showed that dichloromethane could lead to the occurrence of intestinal inflammation in ileum and j ejunum.After stir-fried with vinegar,the pro-inflammatory effect was obviously.The non-dichloromethane fraction had no inflammatory effect.The above results showed that the dichloromethane fraction of Kansui was not only the effective fraction,but also the toxic fraction.2.The terpenoids in dichloromethane fraction and non-dichloromethane fraction of Kansui before and after stir-fried with vinegar were analyzed by LC-MS/MS.The results showed that almost all terpenoids were found in the dichloromethane extract fraction,but only a few terpenoids were found in the non-dichloromethane extract fraction.The above results showed that the extract of dichloromethane could represent the terpenoid fraction of Kansui.Twelve diterpenoids and two unsaturated fatty acids were isolated from dichloromethane extract fraction.3.The results showed that the content of most terpenoids decreased,and that C6(3-O-(2’E,4’Z-decadienoyl)ingenol)could be transformed into C7(3-O-(2’E,4’E-decadienoyl)ingenol)and C8(3-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol)could be transformed into C10(3-O-(2’E,4’E-decadienoyl)-20-O-acetyleneol).It is found that the conversion of C6 and C10 compounds is that the spatial configuration of decadienyl group at position 3 changes from cis to trans.IEC-6 cells and macrophages were used to evaluate the toxicity of the components before and after transformation.The results showed that the expression of AQP1 and AQP3 in IEC-6 cells was significantly decreased before and after the transformation,and there was no significant change before and after the transformation,but the inflammatory effect on macrophages was weakened after the transformation.These results indicate that the transformation of these components may be one of the material base of "reducing toxicity and preserving effect" of Kansui vinegar.