【作者】 陶长戈；

【导师】 彭成；

【作者基本信息】 成都中医药大学 ， 方剂学， 2008， 硕士

【摘要】 目的:研究乌头类有毒中药中双脂型总生物碱、乌头碱、新乌头碱和次乌头碱在体内吸收（A）、分布（D）、代谢（M）和消除（E）的过程,探讨乌头类有毒中药的毒性机制。方法与结果:1方法学本实验采用的HPLC-MS-MS分析方法在用于检测包括大鼠血浆、胆汁、尿液和不同组织脏器等生物样品时发现方法其灵敏度高、专属性强、稳定性好,能很好的符合生物样品分析的要求。所做标准曲线的相关系数（r）均符合要求。故在实验中用该方法研究了,口服给予乌头类有毒中药生物碱后乌头碱、新乌头碱、次乌头碱的毒代动力学特征,口服连续给予乌头类有毒中药生物碱后乌头碱、新乌头碱、次乌头碱的体内组织脏器分布及静脉分别给予毒性剂量乌头碱、新乌头碱、次乌头碱后其在大鼠体内排泄规律。2乌头类有毒中药生物碱口服给药后毒代动力学研究口服给予附子总生物碱后,将药时数据用中国药理学会3p97药代动力学程序进行自动拟合处理。以理论血药浓度值与实验测定值的相关系数最大和AIC最小作为判断标准,结果:乌头碱的药动学曲线经拟合均符合口服给药的二室模型,主要药动参数为:T_1/2alpha=3.326±1.564min,T_1/2beta=886.609±242.136min,t_1/2Ka=2.5192±0.846min,AUC=86557.852±9462.485（ng/ml）×min,T_（peak）=6.989±10546min,C（max）=83.5489±10.4591ng/ml,CL（s）=0.000004±0.000001mg/kg/min/（ng/ml）。新乌头碱的药动学曲线经拟合均符合口服给药的二室模型,主要药动学参数为:T_1/2alpha=15.4989±4.8712min,T_1/2beta=1255.8081±684.891min,t_1/2Ka=3.618±1.254min,AUC=297212.38±74641.91（ng/ml）×min,T_（peak）=15.782±7.541min,CL（s）=0.000013±0.000009mg/kg/min/（ng/ml）,C（max）=202.983±30.781ng/ml。次乌头碱的药动学曲线经拟合均符合口服给药的二室模型,主要药动学参数为:T_1/2alpha=125.482±51.654min,T_1/2beta=1007.7575±349.4852min,t_1/2Ka=1.8541±1.4648min,AUC=241205.797±9146.841（ng/ml）×min,CL（s）=0.000005±0.000002mg/kg/min/（ng/ml）,T_（peak）=16.7646±5.4783min,C（max）=164.3022±20.8914 ng/ml。3乌头类有毒中药生物碱的脏器组织分布研究口服连续给予附子总生物碱后,乌头碱、新乌头碱和次乌头碱在大鼠体内组织脏器的分布较为广泛。乌头碱:心、肝、脾、肺和肾的样品中均检测到乌头碱,其中肺的含量最高,其次分别为肝,心,肾,脾。脑内有乌头碱原型药的存在,但其含量及其低微。新乌头碱:在心、肝、脾、肺和肾样品中均可检测到新乌头碱,其中肝内含量最高,其次分别为肺、肾、脾和心。脑的6个样品我们均发现了有新乌头碱存在但其含量很低,仅能定性,无法定量。次乌头碱:在心、肝、脾、肺和肾样品中均可检测到次乌头碱,其中肝内含量最高,其次分别为肺、肾、脾和心。在脑的6个样品中检测到了次乌头碱,但其含量限低,无法定量。另外实验中发现,动物的个体差异较大,不同动物的同一器官中同一种生物碱的含量存在一定差异。4乌头类有毒中药生物碱的排泄研究4.1乌头类有毒中药生物碱的排泄定量研究大鼠尾静脉注射乌头碱、新乌头碱、次乌头碱后,我们分别收集了6h胆汁和24h尿液,在对胆汁及尿液样品定量分析后发现:乌头碱:尿液中24h平均排除量为2.756±00262μg,24h平均排除率为32.83±2.39%。提示尿液是乌头碱原型药排泄的一个重要途径。在各胆汁样品中均未检测出乌头碱。说明胆汁中乌头碱的含量极低,理论上不高于13.5ng,即大鼠静注乌头碱后6h内由胆汁排泄的乌头碱原型药含量理论上不高于13.5ng。提示乌头碱原药排泄的主要途径可能不是胆汁。新乌头碱:尿液中24h平均排除量为2.515±0.337μg,24h平均排除率为29.94±3.45%。提示尿液是新乌头碱原型药排泄的一个重要途径。在各胆汁样品中均未检测出新乌头碱。说明胆汁中新乌头碱的含量极低,理论上不高于10.4ng,即大鼠静注新乌头碱后6h内由胆汁排泄的新乌头碱原型药含量理论上不高于10.4ng。提示新乌头碱原药排泄的主要途径可能不是胆汁。次乌头碱:尿液中24h平均排除量为2.507±0.203μg,24h平均排除率为15.36±1.29%。提示尿液是新乌头碱原型药排泄的一个重要途径。4.2乌头类有毒中药生物碱注射给药后胆汁及尿液内可能代谢产物的研究本实验研究采用注射给药方式,对乌头类有毒中药生物碱给药后在胆汁及尿液内生物转化进行研究。乌头碱可能的生物转化途径尿液中发现了M0:m/z 646.4,M1:m/z 703.6,M2:m/z 597.5,M3:m/z555.1,M4:m/z 522.5等五个准分子离子[M+H]⁺有存在离子流峰。胆汁中发现了M0:m/z 646.4,M1:m/z 597.5,M2:m/z 586.2,M3:m/z 521.3,M4:m/z 502.5等五个准分子离子[M+H]⁺有存在离子流峰。新乌头碱可能的生物转化途径尿液中发现了M0:m/z 632.5,M1:m/z 694.5,M2:m/z 687.5,M3:m/z 496.6等四个准分子离子[M+H]⁺存在离子流峰。胆汁中发现了M0:m/z 632.5,M1:m/z 687.5,M2:m/z 496.6等三个准分子离子[M+H]⁺存在离子流峰。次乌头碱可能的生物转化途径尿液中发现了M0:m/z 616.7,M1:m/z 678.3,M2:m/z 602.5,M3.m/z 574.5等四个准分子离子[M+H]⁺存在离子流峰。胆汁中发现了M0:m/z 616.7,M1:678.3m/z,M2:m/z 574.5等三个准分子离子[M+H]⁺存在离子流峰。结论:大鼠口服附子总生物碱后,发现三种生物碱吸收快,并在短时间内血药浓度快速下降,提示吸收后分布快,在30-360分钟内血药浓度保持相对平稳,同时具有多峰现象。脏器分布研究表明,乌头碱、新乌头碱和次乌头碱在大鼠体内组织脏器的分布较为广泛,肝脏、肺脏中的含量较高。排泄研究提示尿液是乌头碱、新乌头碱及次乌头碱原型药的重要排除途径,其中乌头碱、新乌头碱的24h平均排除率均为30%左右,次乌头碱的24h平均排除率相对较低,仅为乌头碱、新乌头碱的一半即15.36±1.29%,提示次乌头碱在体内相对较难于排出。在各胆汁样品中均未检测出乌头碱、新乌头碱及次乌头碱。提示乌头碱、新乌头碱及次乌头碱原药排泄的主要途径可能不是胆汁。更多还原

【Abstract】 Object:To study the absorption（A）,distribution（D）,and elimination（E） after iv. aconitine,mesaconitine and hypaconitine,respectively,and po.Aconitum alkaloid; discuss the toxic mechanism of the herbs of genus Aconitum in family of Ranunculaceae.Methods and Results1 MethodsWe successfully established the method of HPLC-MS-MS to detect aconitine mesaconitine and hypaconitine in biological samples including plasma,bile,urine and organs.The method（HPLC-MS-MS） is simple and efficient with excellent accuracy, precision,reproducibility and low detection limit.It can offer biological analysis in all calibration curves.We carried organ distribution after po aconitum alkaloid; metabolites after iv aconitine,mesaconitine and hypaconitine;elimination after iv. aconitine,mesaconitine,hypaconitine by the method.2 The study of toxicokineties after po aconitum alkaloidPo aconitum alkaloid in a single dose,the toxicokinetic parameters were estimated by the 3p97 program（designed by Chinese Pharmacological Society）.According to the compartment model judgment principle,we chose the most suitable compartment model.Aconitine was a two compartment Model after po.The major toxicokinetic parameters are as follow.T_1/2alpha=3.326±1.564min,T_1/2beta=886.609±242.136min, t_1/2Ka=2.5192±0.846min,AUC=86557.852±9462.485（ng/ml）×min,T_（peak）=6.989 ±1.546min,CL（s）=0.000004±0.000001mg/kg/min/（ng/ml）,C（max）=83.5489±10.4591ng/ml.Mesaconitine was a two compartment Model after po.The major toxicokinetic parameters are as follow.T_1/2alpha=15.4989±4.8712min,t_1/2Ka=3.618±1.254min,T_1/2beta =1255.8081±684.891min,T_peak=15.782±7.541min,C_max=202.983±30.781ng/ml,AUC =297212.38±74641.91（ng/ml）×min,CL（s）=0.000013±0.000009mg/kg/min/（ng/ml）. Hypaconitine was a two compartment Model after po.The major toxicokinetic parameters are as follow.T_1/2alpha=125.482±51.654min,T_1/2beta=1007.7575±349.4852 min,t_1/2Ka=1.8541±1.4648min,AUC=241205.797±9146.841（ng/ml）×min,T_peak= 16.7646±5.4783min,CL（s）=0.000005±0.000002mg/kg/min/（ng/ml）,C_max=164.3022±20.8914ng/ml.3 the study of organ distribution after po aconitum alkaloidWe found that aconitine,mesaconitine and hypaconitine were distributed in SD rats organs widely after multiple po aconitum alkaloid.Aconitine:samples of heart,liver,spleen,lung and kidney were all detected aconitine. The distribution of aconitine was in a descending order of lung,liver,heart,kidney, spleen.Samples of brain were also detected aconitine.Mesaconitine:samples of heart,liver,spleen,lung and kidney were all detected mesaco:aitine.The distribution of mesaconitine was a descending order of liver,lung, kidney,spleen and heart.In all brain and samples,we detected mesaconitine,but it was not enough to quantitate.Hypaconitine:samples of heart,liver,spleen,lung and kidney were all detected mesaconitine.The distribution of mesaconitine was a descending order of liver,lung, kidney,spleen and heart.We detected hypaconitine in all brain samples.But it was not enough to quantitate.In addition,because of individual variation,we found the content of same aconitum alkaloid had some difference in the same organ with different rat.4 the study of elimination after iv aconitine,mesaconitine and hypaconitine.We collected bile and urine to prepare samples after iv aconitine,mesaconitine and hypaconitine.In urine,the average elimination quantity of aconitine, mesaconitine,and hypaconitine after 24h iv the drugs were2.756±0.262/μg, 2.515±0.337μg,2.507±0.203μg,respectively.The ratio of elimination in the first 24h were 32.83±2.39%,29.94±3.45%,15.36±1.29%,respectively.In bile,we did not find aconitine,mesaconitine,and hypaconitine in all samples.It hinted us the drugs density were very insignificance.Conclusions:The research of organ distribution indicated that the aconitum alkaloid kept a high level in lung and liver.According to the study of aconitum alkaloid biotransformation and elimination,we basically understood aconitum alkaloid metabolic process and the rule of elimination in vivo.更多还原