| Euphorbiae ebracteolatae(EER))and Euphorbia pekinensis(EPR)are toxic root herbs from Euphorbiaceae in Chinese Pharmacopoeia 2020.They have the effect of expeling water retention and removing swelling and resolving enlarged nodes.They are used for edema,hydrothorax,ascites and accumulation of phlegm and fluid and so on in clinical practice.Despite reporting good effects,there are serious gastrointestinal adverse reactions such as abdominal pain and diarrhea in the clinical use.In order to reduce the gastrointestinal adverse reactions,the method of processing with vinegar is adopted by the Chinese Pharmacopeia.Previous studies of our research group found that the toxicity of EER and EPR was mainly intestinal toxicity.The diterpenoids are the toxic components of EER and EPR,they could cause diarrhea,intestinal inflammation and the disorder of intestinal aquaporins expressions.After vinegar processing,the intestinal inflammation induced by the toxic fractions of EER and EPR was weakened,and the disorder of intestinal aquaporins were corrected.Further research has found that the compositions of terpenoids in toxic fractions from EER and EPR were significantly changed by the processing.But up to now,the mechanism of detoxification and efficacy retention effects is not clear,and there are no reports about the index components which can reflect the changes of EER and EPR before and after processing.In our research,the evaluation models of toxic and efficacy were established,and the efficacy and toxic fractions of EER and EPR were screened according to remove ascitic fluid and intestinal inflammation respectively.In order to further study the mechanism of detoxification and efficacy retention,the main components in the active fractions were isolated,and then the components were simulated processed with vinegar.The structure change rule and toxic and efficacy change of each components were analyzed after vinegar simulated processing.Finally,the content changes of main components and their vinegar processing products in raw and vinegar processing decoction pieces were investigated to clarify the mechanism of detoxification and efficacy retention by vinegar processing of EER and EPR based on the chemical structure transformation.The main research works are as follows:1.Screening of efficacy and toxic fractions of Euphorbiae ebracteolatae and Euphorbia pekinensisBased on the properties of diterpenoids and the previous studies about EER and EPR,the total extract,dichloromethane and non-dichloromethane fractions of EER and EPR were extracted.Screening of efficacy fraction:establishment of mice ascites model.The fecal water content,urine weight and the ascites weight were measured to screen the efficacy fraction.The results showed that both the dichloromethane fraction and the total extract of EER significantly reduced the ascites weight and increased the urine weight of ascites mice.High dose of EER significantly increased the fecal water content,but low dose had no effect on fecal water content.The non-dichloromethane fraction of EER had no diuretic and anti-ascitic effects,which indicated that the efficacy fraction of EER was dichloromethane fraction.EPR is not same as EER.The non-dichloromethane fraction of EPR decreased the ascites and increase the urine output,but it had no effect on the fecal water content.The dichloromethane fraction of EPR could significantly increase the fecal water content of ascites mice,but had no significant effect on the urine output and ascites weight.These results suggested that the anti-ascites fraction of EER was the dichloromethane fraction,and the efficacy fraction of EPR was the non-dichloromethane fraction.The tumor cells floated in ascites,the anti-tumor effects were evaluated by cell cycle and apoptosis assays using flow cytometry.The anti-tumor results showed that these efficacy fraction did not lead to cell cycle arrest and apoptosis.Therefore,EER and EPR were effective in anti-ascites through diuresis rather than anti-tumor and diarrhea.The kidney is the main organ for the formation of urine.The expression of AQPs in kidney is closely related to the formation of urine.In the research,Western blotting was used to detect the expression of aquaporin in the kidney of ascites mice.The results showed that EER could reduce ascites by inhibiting the expression of AQP1,AQP2,AQP3 and AQP4 proteins in kidney,while EPR only inhibited the expression of AQP2,AQP3 and AQP4 proteins.These results suggested that the effect of EER and EPR on anti-ascites and diuresis might be related to the intervention of AQPs expression in kidney.Screening of toxic fractions:the toxicity was evaluated by administration of EER and EPR in normal mice.The levels of TNF-? and IL-1? release from each intestinal segment were measured by ELISA kit,the results showed that the dichloromethane fraction of EER and EPR was toxic fraction,which could significantly increase TNF-? levels in ileum and IL-1? levels in jejunum and ileum.This section suggested that the dichloromethane fraction of EER was not only the efficacy fraction of anti-ascites,but also the toxic fraction of intestinal inflammation.The toxic fraction of EPR was same as EER,but the diuretic and anti-ascites fraction was different.Non-dichloromethane fraction of EPR was the efficacy fraction.2.Study on the changes of toxic and efficacy fractions of Euphorbiae ebracteolatae and Euphorbia pekinensis after vinegar processingAfter screened the toxic and efficacy fractions of EER and EPR,the efficacy and toxic fractions of EER and EPR before and after vinegar processing were prepared.The results showed that the dichloromethane fraction of EER before and after vinegar processing reduced ascites by urine output and inhibited the protein expression of AQP1,AQP2,AQP3 and AQP4 in kidney.There was no significant difference between raw and vinegar products.The non-dichloromethane fraction of EPR and vinegar processed EER could inhibit the AQP2,AQP3 and AQP4 expressions to reduce ascites by urine output,and there were no difference between raw and vinegar EPR in these indexes.The results of intestinal toxicity showed that the effects of the toxic fractions(dichloromethane fractions)of EER and EPR on fecal water content and intestinal inflammatory factors in mice were significantly reduced after vinegar processing compared to raw herbs.Therefore,the effect of diuresis and anti-ascites of EER and EPR processed with vinegar was not significantly reduced,but the toxicity of intestinal diarrhea and inflammation was significantly reduced.3.Isolation of chemical constituents from Euphorbiae ebracteolatae and EuphorbiapekinensisSilica gel,LH-20 gel column chromatography and high-pressure preparative liquid chromatography were used to isolate the main compounds from EER and EPR.12 compounds were isolated and identified from the dichloromethane fraction of EER.Diterpenoids were mainly contented in this fraction,including 5 abietane-type diterpenoids,2 rosane diterpenoids,1 norditerpene lactone diterpenoid,1 heterodimeric diterpenoid.In addition,2 acetophenones and docosyl ferulate were obtained.8 compounds were isolated and identified from EPR.Three phenolic acid derivatives were found in the efficacy fraction,and 2 triterpenoids and 2 sesquiterpenoids were the main toxic components of toxic fraction.4.Study on the chemical structure transformation role of the main components after vinegar simulated processing and the effect of excipient vinegar on chemical structure transformationThe isolated main component were added with acetic acid aqueous solution and heated to dry.Each component and its vinegar products were analyzed by HPLC and Q-TOF-MS/MS.The structure transformation rules of components from EER after vinegar simulated processing were as follows:the ether bond of heterodimeric diterpenoid were hydrolyzed to form rosane diterpenoid and abietane-type diterpenoid;the epoxy structure of abietane-type diterpenoid was easy to be opened by acetic acid heating;the lactone ring of abietane-type and norditerpene lactone diterpenoids were opened;when there are hydroxyl groups in the above components,they were eliminated to form a double bond,esterized to ester and oxidized to form a carbonyl group;When there are 1-methyl-1-vinyl cyclohexane units in the structure,the 1-methyl group was easy to be removed.The content and structure of phenolic acid derivatives in the efficacy fraction of EPR had no obvious change during the simulated processing,while the content of triterpenoids and sesquiterpenoids in the toxic fraction decreased significantly after vinegar processing.The change rules were as follows:the hydrogen on the alkane group in the molecule was oxidized to form hydroxyl,then the hydroxyl was further eliminated and dehydrated to form double bond.In order to study the effect of vinegar(processing excipients)on the structure transformation,the effects of acetic acid or water without acetic acid and single heating simulated processing were compared.The results showed that direct heating had no influence on the structure of abietane-type diterpenoid,and heating with acid significantly decreased the content of original abietane-type diterpenoid and promoted the structural transformation.The efficiency of composition transformation by heating with water is obviously lower than that by adding acid;the structure of norditerpene lactone diterpenoid was destroyed by direct heating and water heating,and no new transformed products was produced,but this diterpenoid were obviously transformed when they were processed with acetic acid.The results showed that the structure of triterpenoid was not transformed after direct heating and slightly transformed by heating with water.When heated with acetic acid,multiple new products were formed;the content of sesquiterpenoid decreased significantly by direct heating or heating with water,but the rate of transformation was low.When heated with acetic acid,new products were formed in large.Therefore,it is very important to use excipient vinegar in the processing of EER and EPR.Acetic acid in vinegar has a great influence on the chemical structure transformation of terpenoids.5.Study on the change of toxic and efficacy of the main components after vinegar simulated processingThe above results showed the structure transformation rule of components in EER and EPR after vinegar processing.In this part,we will further investigate the relationship between the structure transformation and the changes of their toxic and efficacy after vinegar processing.In this study,the expressions of AQPs in kidney cells(HK-2 cells and mIMCD3 cells)were detected by western blotting to evaluate the efficacy changes after vinegar processing.At the same time,the expressions of TNF-? and IL-1? in macrophages(RAW264.7 cells)were analyzed by western blotting to evaluate the toxicity.The results of efficacy of EER showed that the vinegar processing product of heterodimeric diterpenoid europactenoid A(EA)had stronger effects on AQP1,AQP2 and AQP4 than EA,and the effect of vinegar products on AQP3 protein expression was the same as EA.Eucebracteolatin A(EHTA),a transformation product of EA,had stronger effects on AQP1,AQP2 and AQP3 protein expressions than EA,and had the same inhibitory effect on AQP4 protein expression.Therefore,the effect of vinegar product of heterodimeric diterpenoid was enhanced;The effect of the vinegar product of abietane-type diterpenoid jolkinolide B(JNB)on AQP1 was weaker than JNB,but the inhibition of AQP2,AQP3 and AQP4 protein expressions were stronger than that of JNB.Therefore,vinegar process the abietane-type diterpenoid enhanced or retained the efficacy;When the fischeria A(FA)was vinegar processed,the regulatory effects on aquaporins were weakened but still existed.The results of the pro-inflammatory toxicity tests showed that above components significantly decreased pro-inflammatory effect after vinegar processing.In addition to the rosane diterpene EHTA,the other three types of diterpenoids had different pro-inflammatory effects in different concentrations and indicators.The efficacy fraction of EPR mainly contain phenolic acid derivatives,and the structure of these components is relatively stable.In three isolated components,only ethyl 3,4-dihydroxybenzoate could inhibit the expression of AQP2,AQP3 and AQP4 protein in vitro.The results of toxicity study of toxic components from EPR toxic fraction showed that the pro-inflammatory toxicity of triterpenoids and sesquiterpenoids was significantly reduced after simulated vinegar processing.Therefore,the toxicity of terpenoids from EER and EPR was weakened after processing by vinegar,but the diuretic effects still existed or remained.The structure transformation of terpenoids processed by vinegar is the mechanism of detoxification and efficacy retention.At the same time,euphorbacteolatin A from EER,fupenzic acid and orobanone from EPR could be used as quality control components of vinegar processing EER and EPR,respectively.6.Study on the chemical composition changes of toxic and efficacy fractions after vinegar simulated processingThe above transformation rules,toxic and efficacy evaluations were based on vinegar simulated processing,which is different from the decoction pieces vinegar processing.Therefore,the chemical composition changes of toxic and efficacy fractions from vinegar processing EER and EPR were determined by HPLC and Q-TOF-MS/MS.The dynamic changes of the mian active components from EER in different processing degree were assayed by HPLC,the content of euphracteolatin A,a transformed product from a heterodimeric diterpenoid,decreased firstly and then increased with the stir-frying time at 130?When the EER were fried to dry,the content of EHTA increased by 55.36%;the contents of other terpenoids first decreased,then increased and then decreased,but the final contents were equal to or slightly decreased compared with raw EER;The content of EHTA showed an obvious upward trend at 160?,and the content of other diterpenoids showed a downward trend after vinegar processing.The results showed that the content of EHTA in dichloromethane fraction from vinegar EER was 62.5%higher than that in raw herbs fraction,and the content reached 0.18%.Euphebractolatin A increased significantly after processing,and its content was the highest in raw and processed products.It can be used as an index component of EER quality control.It was found through the analysis of the products of chemical transformation,a variety of transformation products can be detected in EER and vinegar processed EER.Among them,the contents of transformation products EHTA and EA-C4 from heterodimeric diterpenoid EA,products HAO-C1 and HAO-C5 from HAO,products JNB-C2 from JNB and FA-C3 from FA were higher than that in the raw EER.8 components were identified from the efficacy fraction of EPR by Q-TOF-MS/MS,including phenolic acid derivatives and organic acids.The relative contents of 3,3',4'-tri-o-methyllactic acid,3,3'-di-o-methyllactic acid,ellagic acid,gallic acid and corilagin from the vinegar processed EPR were slightly increased or basically unchanged,while the relative contents of ethyl 3,4-dihydroxybenzoate,(-)-quinic acid and(-)-shikimic acid increased significantly.Nine compounds were identified from the toxic fraction of EPR,including diterpenes,triterpenes and sesquiterpenes.After vinegar processing,the relative content of terpenoids decreased by 10-75%,and sesquiterpenoids decreased the most,followed by diterpenes and triterpenoids.6 simulated processing products were identified in the toxic fraction of raw and processing EPR.Among them,the relative contents of FPZA transformation products and OR transformation product 2 increased.In conclusion,the aim of "detoxification and efficacy retention" can be achieved by reducing the content of terpenoids and promoting the structure transformation of terpenoids in the vinegar processing.In this research,pharmacological evaluation models were established.We screened the toxic and efficacy fractions of EER and EPR,further isolated and purified the active components,and researched the structural transformation rules of the main components in the vinegar processing.Meanwhile the relationship between the structures transformation and the changes of toxicity and efficacy of main components after vinegar processing was investigated,and the mechanism of detoxification and efficacy retention by vinegar processing was studied in depth.The mechanism of detoxification and efficacy retention was showed as follows:the toxic and effective components of EER are terpenoids.The toxic components of EPR are terpenoids,but the effective components are polyphenols.The structures of terpenoids from EER and EPR were transformed in the vinegar processing,these terpenoids were transformed to new structures by ether bond breaking,lactone ring opening,esterification,oxidation and hydroxyl dehydration.After vinegar processing,the toxicity of transformed products weaked than before,but the diuresis and anti-ascites activities still retained.The content of EHTA increased significantly after processing and was the highest in raw and processed products.It can be used as an index component of EER quality control.In the vinegar processing,vinegar plays a key role in the structural transformation of terpenoids.It is proved that vinegar processing of EER and EPR is scientific.The results of this research explained the scientific connotation of vinegar processing method of EER and EPR and provided scientific basis for processing technology optimization,quality improvement and clinical safe use of EER and EPR.This research provided reference for other toxic traditional Chinese medicine research. |
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