【作者】 吴光红；

【导师】 周光宏；

【作者基本信息】 南京农业大学 ， 食品科学， 2007， 博士

【摘要】 江苏的中华绒螯蟹已发展成为超过100亿元人民币的大产业,名牌大闸蟹畅销国内外。但是,与此同时,中华绒螯蟹的质量安全也经受着越来越严重的疾病暴发、兽药残留、种质退化的困扰。本工作侧重研究了中华绒螯蟹养殖过程中使用恩诺沙星的使用问题,旨在对该药品在中华绒螯蟹中的应用和推广提供科学的依据。研究主要分为如下几个部分:（1）中华绒螯蟹体内恩诺沙星等喹诺酮药物残留检测方法的研究;（2）恩诺沙星在中华绒螯蟹体內的药物代谢动力学研究;（3）水温对恩诺沙星在中华绒螯蟹体內药物代谢动力学的影响;（4）恩诺沙星与中华绒螯蟹血浆蛋白结合率的研究;（5）恩诺沙星在中华绒螯蟹体内使用修药期（withdrawal time）的研究。具体如下:1、建立了中华绒螯蟹体内恩诺沙星、环丙沙星和诺氟沙星残留量同时检测的反相高效液相色谱法（RP-HPLC）方法。考察了三种喹诺酮类药物残留的分离与流动相组成、pH值及乙腈含量的关系,优化了色谱条件。中华绒螯蟹肝脏成分复杂,脂肪类化合物含量高,色素等干扰物质多,极易干扰目标物的检测。通过改进前处理过程,采用振荡萃取-浓缩-高速离心萃取的前处理过程,有效地减少了干扰物质对检测结果的影响。研究表明,该方法线性关系和重复性良好,样品中三种喹诺酮类药物残留的回收率在73%～86%之间,相对标准偏差2.01%～4.29%（n=5）,最低检测限为10μg·kg^-1。用该方法对肌肉注射给药后72小时內中华绒螯蟹肝脏组织进行检测,恩诺沙星及其主要代谢物环丙沙星都可以检测到,为弄清楚恩诺沙星在中华绒螯蟹体内的分布、代谢的研究提供了检测方法。2、在17±2℃条件下,研究了中华绒螯蟹肌肉注射每只蟹0.1 mL恩诺沙星(恩诺沙星原粉用双蒸水配成6.25 mg·mL^-1)的药代动力学。中华绒螯蟹肌肉注射恩诺沙星后,血液中药物浓度很快达到峰值,并迅速向组织中分布,血药经时过程符合一级吸收二室开放代谢模型,主要药动学参数为:T_1/2α5.29h,T_1/2β92.42h,K₂₁0.078h^-1,K₁₂0.048 h^-1,K₁₀0.012h^-1,AUC304.80μg·ml^-1h^-1,CLs0.016 L·kg^-1h^-1。药物在血液中的药时曲线方程为C_blood=6.68e^-0.49t+3.38e^-0.013t。在中华绒螯蟹的血液及肌肉、肝胰腺、鳃组织中均能检测到恩诺沙星的代谢物环丙沙星残留,最低检测限为10μg·kg^-1。3、研究在17、23和29℃水温下,恩诺沙星（ENR）在中华绒螯蟹血淋巴、肌肉和肝脏组织中的代谢规律和组织分布的影响。以3.0 mg·kg^-1剂量单次肌肉注射给药,于给药后1、10 min及0.5、1、2、8、16、24、48、72和168 h取各组织样品,药物含量由高效液相色谱法同时测定。研究结果表明,在3个温度下,血淋巴和肌肉组织中ENR含量瞬时达到峰值,而肝脏中ENR含量则随给药时间,先上升后下降,各组织含量从大到小依次为:血淋巴、肝脏、肌肉。水温23和29℃下,CIP含量及CIP与两种药物含量之比从大到小依次为:肝脏、血淋巴、肌肉;各组织中CIP含量及CIP与两种药物含量之比与水温呈正相关。应用药代动力学计算软件3P97分析结果表明:肝脏组织中ENR的代谢规律符合二室开放模型,在给定3个温度下,吸收半衰期（分别为:1.81、0.97和0.14 h）、消除半衰期（分别为:240.27、60.57和37.59 h）、药时曲线下总面积(分别为:2148.7、324.2和264.5μg·mL^-1·h^-1)与温度呈负相关,而总消除率与溫度呈正相关,分别是0.0014、0.0093和0.011 L·kg^-1·h^-1。4、研究了恩诺沙星与中华绒螯蟹血浆的蛋白结合率。实验结果显示:恩诺沙星与牛血清白蛋白(1.0mg·mL^-1)的蛋白结合率在13.0%～29.0%之间。实验测定的中华绒螯蟹血浆蛋白浓度为64.0±0.3mg·mL^-1,蛋白分子量主要集中在66.2KDa～97.1KDa之间,恩诺沙星与中华绒螯蟹血浆蛋白的结合率在50.0～78.8%之间。实验结果认为,恩诺沙星与中华绒螯蟹血浆50%左右的蛋白结合率能充分保证其药效的发挥,可以在中华绒螯蟹养殖过程中考虑使用。5、研究了单次肌肉注射恩诺沙星在中华绒螯蟹体內的休药期。实验数据通过药代动力学分析软件3P97在电脑上进行分析。中华绒螯蟹在肌肉注射恩诺沙星后经时过程符合二室开放模型,并计算出其主要药动学参数。以Michaelis-Menten动力学理论作为基础,以中国药理学会数学药理专业委员会编写的3P97实用药动学计算程序计算出的参数作为条件,建立了利用迭代法推算渔药休药期的方案。并确定,中华绒螯蟹肌肉注射恩诺沙星的休药期定在600度日（天*℃）。综上所述,本文较系统研究了恩诺沙星应用于中华绒螯蟹的药物残留检测方法、代谢动力学、蛋白结合率,并确定了休药期,对中华绒螯蟹质量安全生产具有明确的指导作用,实际效益显著。更多还原

【Abstract】 It was more than 10 billion yuan/year of Chinese mitten-handed crab,Eriocheir sinens can be sold from the lakes and rivers in Jiangsu.At the same time,Quality safety of Eriocheir sinens about sickness,drug residues and specie deterioration becomes a more and more serious problem.Usage of Enrofloxaxin in Eriocheir sinens has been studied in this paper,including（1） RP-HPLC detecting method of Enrofloxacin,ciprofloxacin and norfloxacin in Eriocheir sinens;（2） Pharmacoknetics of enroflxacin in Eriocheir sinensis after intramuscular injection at 17±2℃;（3） Pharmacoknetics of enroflxacin in Eriocheir sinensis after intramuscular injection at 17,23 and 29℃water temperatures;（4） Study on protein binding data of enrofloxacin with plasma protein in Eriocheir sinens;（5）Study on withdrawal time of enrofloxacin in Eriocheir sinen.1.A RP-HPLC method was developed for the determination of residues of norfloxacin, ciprofloxacin and enrofloxacin in the liver of Eriocheir sinensis.The effect of composition, pH value of the mobile phase on the separation of the above said quinolone mixtures was studied and the chromatographic conditions were thus optimized.The pre-treatment steps as vibration extraction,concentration and centrifugation were carefully improved to minimize the interferences caused by the complicated compositions,high content of lipid and pigments etc.in the liver of Eriocheir sinensis.The developed method is with required accuracy and precision as well as a good linearity in the interested concentration range of 0.01 to 1.0μg·mL^-1.The recovery at spiking levels of 0.01,0.02 and 0.05μg·mL^-1 is.73 -86%,respectively,with a relative standard deviation（n=5） of 2.01%～4.29%from the liver sample.The limit of detection of the method as estimated by 3 times of signal to noise ratio is 10μg·kg^-1.The low residue levels of enrofloxacin and its major metabolites ciprofloxacin could be identified in the liver of Chinese mitten-handed crab 72h after intramuscular injection by the method developed in this work.This should be helpful for the future study on the distribution and metabolism of quinolones in Eriocheir sinensis.2 This is first report about pharmacoknetics of enroflxacin and its activated metabolite ciprofloxacin in Eriocheir sinensis after intramuscular injection at 17±2℃was reported. The concentration of medicament was determined by HPLC.The data were analyzed with the pharmacokinetics computer program 3P97.The pharmacyoknetic results showed that the concentration-time curve of enrofloxacin in plasma after intramuscular injection could be described by a two-compartment open model.The main pharmacokinetics parameters are as follows:T_1/2α 5.29h,T_1/2β 92.42h,K₂₁0.078h^-1,K₁₂0.048 h^-1,K₁₀0.012h^-1, AUC304.80μg·ml^-1h^-1,CLs0.016 L·kg^-1hour^-1.The theoretical equation was C_blood= 6.68e^0.49t+3.38e^-0.013t.The concentration-time course of enrofloxacin in muscle and liver can be described by a two-compartment open model,too.The main pharmacokinetics parameters are as follows:Ciprofloxacin,the metabolite of enrofloxacin at concentration level as low as 10μg·kg^-1 can be indentified in all three tissues.3 Study on pharmacokinetic of enrofloxacin and its metabolite ciprofloxacin following a single intramuscular administration(3.0 mg.kg^-1) was carried out in the Chinese mitten-handed crab at 17,23 and 29℃water temperatures.Hemolymph,muscular and liver samples were collected at 1,10 min and 0.5,1,2,8,16,24,48,72 and 168 h post-injection.The concentration of ENR and CIP were simultaneously determined by HPLC method.The ENR level in hemolymph and muscular reached maximum momentarily,while those in liver appeared to be a maximum then declined.The liver data was analyzed with Practical Pharmacokinetics Program（3P97）.The kinetic profile of ENR in liver was conformed to a two-compartment model.The absorption half-life（1.81,0.97 and 0.14 h,respectively）,elimination half-life（240.27,60.57 and 37.59 h,respectively） and the area under the concentration-time curve from zero to infinity(2148.65,324.16 and 264.47μg·mL^-1·h^-1,respectively) of ENR in liver was found to have a negative relationship with temperature.Whereas total body clearance had a positive relationship with temperature,and were 0.0014,0.0093 and 0.011 L·kg^-1·h^-1,respectively.4 Protein binding data of enrofloxacin with plasma protein in Chinese Mitten-Handed Crab,Eriocheir sinensis were determined in this paper.Results showed that:percent of enrofloxacin binding to bovine serum albumin（BSA） was 13%～28.95%.In the test,it was found that the concentration of plasma proteins in Chinese Mitten-Handed Crab,Eriocheir sinensis was 63.95±0.29mg/mL.And molecular weight of these proteins were mainly between 66.2KDa～97.1KDa.Protein binding level of enrofloxacin and crab plasma proteins was 49.97～78.81%,which showed more than 50%of the drug could be bound to the proteins.Above all,it could come into the conclusion that enrofloxacin bound with plasma proteins tightly,which assured the efficient utilizing of drug in the crab.It seemed that enrofloxacin could be used in Chinese Mitten-Handed Crab,Eriocheir sinensis.5 The tissue concentration of enrofloxacin and ciprofloxacin were determined simultaneously by a HPLC method.The data were analyzed with Practical Pharmacokinetic Program 3P97（Mathpharmacology Committee,Chinese Academy of Pharmacology, Beijing,China） on a personal computer to automatically determine the type of compartment model and pharmacokinetic parameters.The model was selected on the basis of the residual sum of squares and the minimum Akaike’s information criterion（AIC） value.Finally,it can be concluded that the WDT in Eriocheir sinens is 600 day*℃.Generally speaking,this paper totally studied enrofloxacin residual detecting method, pharmaeokinetics,protein bingd data in Chinese mitten-handed crab,Eriocheir sinens,including,And at the end,withdrawal time of enrofloxaein was assured,which is very useful in safe crab producing.更多还原