Preliminary Study On The Toxicity Of Perennial Croton Cortex To Zebrafish Liver Based On Chemical Components And Zebrafish Mode

Objective:The aim of this study is to assess the liver toxicity,the hepatotoxicity mechanism and the main component of Periplocae Cortex decoction in zebrafish based on the traditional application approach.The character of high throughput of zebrafish was used to screen for liver toxicity of decoction and extracts of different polarity of Periplocae Cortex.Furthermore,the chemical components of the toxic extract of Periplocae Cortex were analyzed.Then the compositions of toxic extracts and two monomer compounds in toxic extracts that can get into zebrafish body were analysed after exposure treatment,Attemptting to analyze the source of toxicity from the chemical base.So that we hope to have a further understanding of the possible hepatic-toxicity mechanism and material basis of Periplocae Cortex.Method:（1）the effect of Periplocae Cortex decoction on 4dpf zebrafish larvae was evaluated firstly.Under the concentration of sub-lethal,the liver morphological phenotype（liver transparency,liver area,liver fluorescence intensity,hepatocyte apoptosis）,H&E staining and relevant enzymes activity（AST,ALT,SOD,GSH-Px,Caspase3）were used as the evaluation index to assess the hepatotoxicity of decoction and extracts of different polarity of Periplocae Cortex.Finally,the genes expression levels related to the hepatotoxicity were investigate to explore the the mechanism of toxicity.（2）Different polarity extracts were prepared by the extraction of different polar solvents（petroleum ether,dichloromethane,ethyl acetate,ethanol,and water）,then the hepatotoxicity and hepatotoxicity mechanisms of different samples were further examined and discussed with the same metods mentioned above.（3）UPLC-Q-TOF/MS^E technology,UNIFI1.8 data processing system and Periplocae Cortex compound database were used to qualitative analyze and character the chemical constituents of the toxic extracts.（4）Based on the UPLC-Q-TOF/MS^E technique,the in vivo toxic extracts and two monomer compounds from toxic extracts were analyzed.Result:（1）the concentration-mortality curve of Periplocae Cortex decoction for 4dpf zebrafish was y=-1.0843+0.0014x（R²=0.9524,r=0.9759,P<0.001）and LC₁₀ was 845.93 μg/mL.Compared with the control group,abnormal morphology of liver of wild zebrafish was observed in the treated group,including reduced transparency,dim intercellular substance,enlarged area of the liver with the exposure concentration-depandent manner;Also,the Periplocae Cortex decoction could result in the increased liver fluorescence area and decreased liver fluorescence density.The activity of SOD,GSH-Px decreased while the activity of AST,ALT increased with the significant difference with the control group（P<0.05）in the treated groups.The result of q-PCR showed that he expression of the apoptosis gene BAX,Caspase9,Caspase8and Caspase3 could be significantly up-regulated by the toxic extracts exposure treatment,and the expression of BCL-2,SOD1 and NOQ1 was downregulated,indicating that the hepatotoxicity of the water extract to the zebrafish may be achieved by destroying the oxidative stress balance in vivo and thereby inducing the apoptosis of the liver cells;（2）Through the toxicity screening with the similar assessment standards,it was found that the water extract was highest toxic to zebrafish and the LC₁₀ was 12.23ug/ml,far lower than LC₁₀ concentration of petroleum ether,dichloromethane,ethyl acetate and absolute ethanol extracts.The assessment of phenotype,physiological parameters and genes of zebrafish treated with the water extracts were similar to the result of zebrafish exposed to decoction.The toxic parts of the Periplocae Cortex were further identified as the water extract.（3）The chemical components of the toxic sites were detected by UPLC-QTOF/MS^E,combined with U.S.waters data processing software UNIFI and the network database（Chem Spider,Pub Chem,Sci Finder）for comparison and screening,and 24 components were estimated from the toxic site of the water extraction site.Including cardiac glycosides and their aglycons,C21 steroid glycosides and aldehydes,etc;that lay the foundation for further analysis of the toxic components of the body parts.（4）In vivo,that component of the monomer component of the Periplocin is found in the form of a prototype and metabolites,the detected metabolites were the Periplogenin and the Periplocymarin.The Periplogenin are present in the prototype.It reveals that the metabolic pathway in the zebra fish has a similar reaction to the existing model.（5）By comparing the enrichment of zebrafish body components in the blank group and the administration group,it is preliminarily concluded that the S-（8-（diethylphosphono）octyl）ethanethioate,Periplocymarin,Methyl-4-O（2-O-acetyl-β-D-digitalopyranosyl）-β-D-cymaropyranoside,PeriplocosideC and Periplogenin are entered into zebrafish,and the metabolic process in vivo remains to be further studied.