Study On The Mechanism Of Attenuated Hepatotoxicity Of Euphorbia Pekinensis After Vinegar-processing

Euphorbia pekinensis is the dry roots of Euphorbia pekinensis Rupr.It was a traditional Chinese medicine,first recorded in Shennong-Bencao and classified as a low-grade medicine.Euphorbia power and Ten Jujube Decoction are the classic recipes of Euphorbia pekinensis and has been widely used to treat cirrhosis and ascites and pleural effusion.Although Euphorbia pekinensis has been reported to have a wide array of therapeutic values,potential side effects of Euphorbia pekinensis have been revealed through experimental studies and clinical reports.But the decoction of Euphorbia pekinensis can cause hepatic injury.However,Euphorbia pekinensis after being processed by vinegar can release the toxicity.But the detoxication and mechanism of vinegar-processed on hepatic injury is still unknow.Thus,it makes sense to study on the hepatotoxic chemical constituent of Euphorbia pekinensis and the detoxication and mechanism of Euphorbia pekinensis after vinegar-processing on hepatotoxicity.This paper is summarized as follows:Part one:Literature reviewBy means of reviewing relevant ancient and modern literature,the pharmacognosy,chemical constituents,pharmacological action,toxicity,processing history,clinical application of Euphorbia pekinensis were summarized,and the current research status,existing problems and research direction were discussed,which provided reference for the subsequent research plan formulation and implementation.Part two:cytotoxicity test of before and after processed of Euphorbia pekinensisMTT assay and cell morphological observation were used to compare the inhibitory effect of different parts of before and after processed of Euphorbia pekinensis on L02 cell.The results showed that the inhibitory effect of alcohol extraction was stronger than the water extraction after alcohol extracting both in crude Euphorbia pekinensis and vinegar-processing Euphorbia pekinensis.And petroleum ether and ethyl acetate parts of alcohol extraction were the strongest,and the petroleum ether part of crude Euphorbia pekinensis was stronger than the petroleum ether part of vinegar-processing Euphorbia pekinensis,both have significant difference(P<0.05).Which infered that the hepatic cytotoxicity parts of Euphorbia pekinensis were the petroleum ether and ethyl acetate parts.Part three:The mechanism of detoxication on normal liver cells LO2 of Euphorbia pekinensis after vinegar-processingMTT assay、Hoechst 33258 staining、flow cytometry and so on were used to compare the toxicity on normal liver cells LO2 of before and after processed of Euphorbia pekinensis,and the possible mechanism of detoxication of Euphorbia pekinensis after vinegar-processing was discussed.The results showed that compared with control group,Euphorbia pekinensis can obviously inhibit the cell proliferation(P<0.05),can induce LO2 cell apoptosis,and can cause S-phase block;Euphorbia pekinensis can significantly increase the levels of ALT,AST,and LDH(P<0.05)in the supernatant of cell incubation,and can significantly decrease the level of SOD and the content of GSH(P<0.05),and can significantly increase the content of MDA(P<0.05).Compared with crude Euphorbia pekinensis group,Euphorbia pekinensis after vinegar-processing can significantly reduce these changes.Which infered that Euphorbia pekinensis after vinegar-processing can release the cytotoxicity of LO2 cell,the mechanism maybe was decreasing the oxidative damage of LO2 cell,accordingly,reducing the cell cycle arrest and apoptosis.Part four:The mechanism of detoxication on hepatotoxicity in mice of Euphorbia pekinensis after vinegar-processingTo verify the research results of part two and three and confirm the hepatotoxicity part of Euphorbia pekinensis,the ICR mice were given the hepatic cytotoxicity parts of crude Euphorbia pekinensis and vinegar-processing Euphorbia pekinensis to compare the hepatotoxicity in mice of before and after processed of Euphorbia pekinensis,and the possible mechanism of detoxication of Euphorbia pekinensis after vinegar-processing was discussed.The results showed that compared with control group,Euphorbia pekinensis can obviously increase the activity of ALT、AST and LDH and with the increase of dose,the difference was statistically significant(P<0.05);can increase the mice liver viscera coefficient;can significantly increase MDA content in liver(P<0.05);can significantly decrease SOD activity and GSH contents in liver(P<0.05).Compared with crude Euphorbia pekinensis group,Euphorbia pekinensis after vinegar-processing can significantly reduce these changes.Which infered that Euphorbia pekinensis can induce hepatotoxicity in mice and the mechanism perhapase is oxidative damage;Euphorbia pekinensis after vinegar-processing can release the hepatotoxicity in mice,the mechanism maybe is decreasing the oxidative damage.Part five:Study on chemical constituents of the hepatotoxicity parts of Euphorbia pekinensisThe silica gel column chromatography and preparative TLC were used to isolate the chemical constituents of the hepatotoxicity parts of Euphorbia pekinensis and the structures were identified on the basis of spectral data and their physical properties.MTT assay was used to evaluate the cytotoxicity of chemical constituents.Eleven compounds were isolated and identified as tetracosanol(1),octadecanol(2),myristic acid(3),euphol(4),octacosyl ferulate(5),β-sitosterol(6),(3β,12a,13a)-3,12-dihydroxypimara-7,15-dien-2-one(7),pekinenal(8),neomotiol(9),3,3’-di-O-methyl ellagicacid(10),isopekinenal(11).The compounds 1,3,7,9 and 11 were isolated from Euphorbia pekinensis for the first time,compound 7 exhibited hepatic cytotoxicity with IC50 values of 51.20 μg·mL-1;Compound 8 exhibited hepatic cytotoxicity with IC50 values of 15.72μg·mL-1;Compound 11 exhibited hepatic cytotoxicity with IC50 values of 25.49μg·mL-1.Part six:Toxicity of pekinenal isolated from Euphorbia pekinensis on normal liver cells L02MTT assay、Hoechst 33258 staining、flow cytometry and so on were used to evaluate the cytotoxicity of pekinenal and the cytotoxicity mechanism was disscussed.The results showed that compared with control group,pekinenal can obviously inhibit the L02 cell proliferation(P<0.05),can induce L02 cell apoptosis,and can cause G2/M-phase block;can significantly increase the levels of ALT,AST,and LDH(P<0.05)in the supernatant of cell incubation,and can significantly decrease the level of SOD and the content of GSH(P<0.05),and can significantly increase the content of MDA(P<0.05).Which infered that pekinenal showed direct toxic effect on L02 cell,the mechanism was associated with oxidative damage.Part seven:Simultaneous determination of three terpenoids in crude Euphorbia pekinensis and vinegar-processed Euphorbia pekinensis by UPLCChoosing three terpenoids which have pharmacological activities or hepartotoxicity as indexes,we first estabilished an UPLC method for simultaneous determination of isopekinenal、pekinenal and euphol in Euphorbia pekinensis.Then the method was used to compare contents of three terpenoids in Euphorbia pekinensis from defferent regions and compare contents of crude Euphorbia pekinensis and Euphorbia pekinensis after vinegar-processing.The results showed that there were great differences in the contents of three terpenoids in Euphorbia pekinensis of defferent regions;as Euphorbia pekinensis was processed with vinegar,the content of isopekinenal increased significantly(P<0.01),the content of pekinenal decreased significantly(P<0.01),the content of euphol decreased(P<0.05).These results suggested that exposure of isopekinenal、pekinenal and euphol to acid、high temperature cooking environment in vinegar-processing can resulted in reactions such as oxidation、hydrolysis、rearrangement,and pekinenal may translate into low hepatotoxicity isomers isopekinenal,thus release the hepatotoxicity.This experimental can further clarify the detoxication and mechanism of Euphorbia pekinens after vinegar-processing on hepatotoxicity.