The Vivo Metabolomics And Vitro Metabolism Study Of Ethanamiz Uril In Chicken

Ethanamizuril is a new type of three triazine anti coccidiosis drug,which is being studied by Chinese Academy of Agricultural Sciences Shanghai Veterinary Research Institute.The results show that the anticoccidial effects of the drug,low toxicity,and no cross resistance to diclazuril and other three triazine drugs,has good application prospects.In the research and development work with Ethanamizuril,related metabolic studies is essential in the process.Therefore,this experiment focuses on the vivo metabolomics and vitro vetabolism study of Ethanamizuril in chicken.on the one hand,in order to reveal the metabolic mechanism of Ethanamizuril,research the the change of endogenous metabolites of chicken body before and after administration,find out the main biomarkers,on the other hand,research the vitro metabolic mechanism of Ethanamizuril in chicken,establish a metabolic platform of chicken liver and kidney microsomal and cell,identification the metabolite of chicken in vitro.For the further study of Ethanamizuril,this provided many important data clues,such as lt?s mechanism of anticoccidiosis effect,pharmacodynamics and toxicology.At the same time,this laid the foundation for the further study of metabolism in chicken vitro?such as metabolic enzymes involved,Inhibition and induction of enzyme?,and make an important guiding function for the research of veterinary drug residues.The main contents include:1 Established a metabolomics platform of Ethanamizuril in chickens,Analyzed the difference of chicken plasma's metabolism spectrum after drug between experimental group and normal control group by GC-Q-TOF-MS.Using the method of unsupervised PCA,results showed that chicken's metabolic spectrum have a separation trend between experimental group and normal control group.Further use of supervised orthogonal partial least squares discriminant analysis?OPLS-DA?method,the metabolic differences was clearly observed with between the group and the normal control group.279 metabolites were identified and 28 differential metabolites were obtained after analysis.According to the analysis of metabolic pathways,the metabolic pathway has changed significantly,included:aminoacyl-tRNA synthesis,glycine,serine,threonine,methionine metabolism,cysteine metabolism,vitamin metabolism,cyano amino acid metabolism.Furthermore,the7 key metabolites of were identified,included:L-lysine,L-serine,L-methionine,D-mannose,stearic acid,xylose and glycerol.Through analyzing the changes of endogenous metabolites in chicken before and after chicken is gived medicine,the possible mechanism of anti coccidiosis was obtained.2 Established a method of the preparation of chicken liver kidney microsomal and it's activity detection,optimized the incubation condition in chicken liver kidney microsomal,and through the model,used the UPLC identified the metabolite of Ethanamizuril in chicken liver and kidney microsomal,use UPLC-Q-TOF-MS technology to carry on the preliminary qualitative analysis.This experiment successfully prepared chicken liver kidney microsomes by differential centrifugation,and through the Lowrry method to determine the content of microsomal protein.The results showed that protein content in liver and kidney microsomes were 17.37±0.55 mg/mL,5.37±0.24 mg/mL,use the method of carbon monoxide reduction differential spectroscopy determin the activity of microsomal,the results show that the maximum absorption peak at 450 nm,which has good activity.The final metabolites of Ethanamizuril in chicken liver and kidney microsomal are M,the relative molecular mass of M is 311.1,which is differ from AC₄ nearly 42,and it exists in[M+H]⁺and is deacetylated product of AC₄,the metabolism does not depend on coenzyme NADPH,and the same protein concentration of chicken kidney microsomes was significantly stronger than that of liver microsomes.3 respectively by in situ two step perfusion method and continuous trypsin digestion method to prepare chicken liver and kidney cells,after been cultured,do the in vitro incubation experiment of chicken kidney cells,use the UPLC-Q-TOF-MS technology to tentatively qualitative analyse of it's metabolites.The inverted microscope showed a good activity of cultured liver and kidney cells,through the model incubated,The results showed that AC₄ in the in vitro metabolism of liver and kidney cells are the same to cell metabolism in the liver and kidney,they all have the same metabolite M.