The Quality Control Of The Double-sided Needle And The Preliminary Toxicity Study Of The Two-sided Needle And Xanthium Sibiricus Based On The Zebrafish Model

Objective:1.The quality control of Zanthoxylum nitidum was studied by establishing the method of thin layer chromatographic identification,Ultra Performance Liquid Chromatography(UPLC)fingerprinting combined with quantitative determination of three active components,to provide a more comprehensive and effective evaluation method for the quality control of Zanthoxylum nitidum,and provide the basis for improving the quality standard of Zanthoxylum nitidum;2.The zebrafish was used to evaluate the developmental toxicity of Zanthoxylum nitidum,to provide developmental toxicity data of Zanthoxylum nitidum;3.The developmental toxicity and hepatotoxicity of Xanthium sibiricum were evaluated using zebrafish,to provide reference for the safety of clinical medications,and validate the applicability of zebrafish for rapid assessment of hepatotoxicity induced by traditional Chinese medicine.Methods:1.Ethyl acetate-methanol-concentrated ammonia(25:1:0.2)was used as developing solvent,the thin layer chromatography was used to distinguish the Zanthoxylum nitidum and its counterfeit species.2.The fingerprint of Zanthoxylum nitidum was established by using UPLC combined with similarity evaluation,cluster analysis and principal component analysis.The precision,repeatability and stability were investigated.3.The contents of nitidine chloride,ethoxychelerythrine and sesamin were determined by UPLC,and methodology was examined.4.To evaluate the developmental toxicity of water extract of Zanthoxylum nitidum in 6 hours post-fertilization(6 hpf)zebrafish embryos,morphological changes of embryos and the corresponding indicators were determined after drug exposure at the different time endpoints.5.The developmental toxicity and hepatotoxicity of water extract of Xanthium sibiricum were studied using 6 hours post-fertilization(6 hpf)zebrafish and 4 days post-fertilization(4 dpf)zebrafish model,respectively.In hepatotoxicity experiment,phenotype screening,evaluation of physiological and biochemical indexes,oil red O staining,acridine orange staining and histopathology were used to evaluate hepatotoxicity induceded by Xanthium sibiricus in zebrafish.Results:1.In the thin layer chromatography experiment,toddalolactone,nitidine chloride and ethoxychelerythrine could be separated by the developing solvent ethyl acetate-methanol-concentrated ammonia(25:1:0.2),indicating that the authenticity of Zanthoxylum nitidum can be identified by thin layer chromatography.2.The UPLC fingerprint of Zanthoxylum nitidum was established for the first time,and.25 characteristic peaks were selected as the common peaks,three characteristic peaks including nitidine chloride,ethoxychelerythrine and sesamin were identified.The similarity of 12 batchs of Zanthoxylum nitidum was 0.807?0.959;12 batches of Zanthoxylum nitidum were classified into two types based on the result of cluster analysis and principal component analysis,S1,S2,S4,S7 and S9 were one type,S3,S5,S6,S8,S10,S11 and S12 were the other type.There was a large difference between the chromatogram of its counterfeit species and the common mode of the Zanthoxylum nitidum fingerprint,indicating that the established fingerprint method could better distinguish the Zanthoxylum nitidum and its counterfeit species.3.The contents of nitidine chloride,ethoxychelerythrine and sesamin of Zanthoxylum nitidum were determined by UPLC method,and the methodology was examined.The contents of nitidine chloride,ethoxychelerythrine and sesamin were 1.2946?2.4975 mg·g-1,0.8972?4.4132 mg·g-1 and 0.2694?2.0540 mg·g-1.The method can be used as a valid content determination method.4.In the developmental toxicity assessment of Zanthoxylum nitidum,the zebrafish embryos exposed to low-concentration Zanthoxylum nitidu(?50?g·mL-1)showed normal embryonic development,and there was no significant difference compared with the control group(P>0.05).At the comcentration of the water extract of Zanthoxylum nitidum>100?g·mL-1,the embryo development appeared some abnormal phenotype including stunted the head and tail,lack of voluntary movements,reduced heart rate,decreased melanin deposition,pericardial enlargement,yolk cysts,and even cardiac arrest or death.Within a certain concentration range,heart rate,hatching rate and body length decreased significantly,deformity rate and mortality rate increased significantly in an exposure concentration depend-manner.5.In the developmental toxicity experiment of Xanthium sibiricum,the zebrafish embryos exposed to low-concentration Zanthoxylum nitidum(40 ?g·mL-1)showed normal embryonic development(P>0.05),while the zebrafish embryos were arrested at 6 hpf in the high concentration(280 ?g·mL-1)Xanthium sibiricum group(P<0.05).160 ?g·L-1 of water extract of Xanthium sibiricum exposure could result in the significant decrease of heart beats of zebrafish embryos at 48 hpf(P<0.05),and pericardial edema and delayed yolk sac absorption at 72 hpf.For the hepatotoxicity assessment of Xanthium sibiricum for 24 h,800?g·mL-1 of water extract of Xanthium sibiricum exposure could result in significant increase of liver area(P<0.05),decreased the transparency,and delayed the yolk sac absorption,unclear the outline of liver.Meanwhile,histopathological sections revealed vacuolation and loose arrangementof the liver cells.The oil red O staining showed accumulated fat and acridine orange staining showed apoptotic phenomenon in the liver area.Futhertime,superoxide dismutase(SOD)activity in the treated group was significantly reduced,while alanine transaminase(ALT)and aspartate transaminase(AST)activity and malondialdehyde(MDA)content were significantly increased(P<0.05).Compared with the control group,there was no significant difference in the 600 ?g mL-1 exposure group(P>0.05).In summary,Xanthium sibiricum may induce apoptosis through oxidative stress to induce hepatotoxicity in zebrafish.Conclusion:This project established a quality control method based on thin layer chromatographic identification,UPLC fingerprinting combined with multi-indicator components quantitative determination of Zanthoxylum nitidum,providing a more comprehensive and effective evaluation method for the quality control of Zanthoxylum nitidum,and providing the basis for improving the quality standard of Zanthoxylum nitidum;Toxicity experiment results showed that the water extracts of Zanthoxylum nitidum and Xanthium sibiricum had developmental toxicity in zebrafish embryos,providing reference for the safety of clinical medications.At the sub-lethal concentrations,Xanthium sibiricum induceded hepatotoxicity in zebrafish,which were consistent with the results reported in the literature using rat and mouse models,validating the applicability of zebrafish for rapid assessment of hepatotoxicity induced by traditional Chinese medicine.