附子质量评价及乌头碱类成分药动学研究

【作者】 王瑞；

【导师】 乔延江；

【作者基本信息】 北京中医药大学 ， 中药学， 2007， 博士

【摘要】 本论文分附子质量评价研究和附子中乌头碱类成分药动学研究两部分。一、第一部分应用HPLC以及HPLC／MS技术对附子生药指纹图谱的建立与附子炮制品的含量测定进行了研究。附子（Radix Aconiti Lateralis Praeparata）为毛茛科乌头属植物乌头Aconitum carmichaeli Debx.的子根的加工品，从附子中分离鉴定的化学成分主要有生物碱和脂类两大类化合物。国内外研究表明，附子生物碱类化合物具有多方面的药理活性和临床功效。历版《中华人民共和国药典》对于附子的质量控制都是对其酯型生物碱的限量检查。为了更科学、客观地反映附子的内在质量，建立基于中药成分系统研究基础上的指纹图谱是一种有效的手段。将LC／MS技术应用于中药指纹图谱的研究中，是对常规色谱强有力的支持，通过对多种流出成分的质谱检测，可增加指纹图谱的“可知性”。HPLC技术对于中药指纹图谱研究中药材提取方法的研究，实验条件的选择与优化以及MS技术对于中药成分的归属等均起着重要的作用。本论文对附子生药指纹图谱建立中的分析方法进行了研究，采用Hepersil BDS C₁₈色谱柱进行附子生物碱成分液相分离的色谱条件；运用HPLC／MS技术对附子生药中的主要色谱峰进行了初步归属；利用中药色谱指纹图谱相似度评价软件评价了各批样品指纹图谱间的相似程度。研究结果表明，以HPLC法建立的附子生药指纹图谱较全面地反映了附子的内在质量，可作为附子生药质量控制及其炮制品指纹图谱建立的依据之一。在指纹图谱研究的基础上，本论文还对附子生药及其炮制品中的主要生物碱成分-双酯型生物碱乌头碱、中乌头碱和次乌头碱以及单酯型生物碱苯甲酰乌头原碱、苯甲酰中乌头原碱和苯甲酰次乌头原碱的含量进行了测定，为附子生药及其炮制品质量控制的建立提供科学支撑。二、第二部分建立了体外血浆温孵法测定乌头碱、中乌头碱和次乌头碱水解规律的方法。空白兔血浆加入不同浓度的3种生物碱混合对照品溶液，通过对温孵不同时间血浆样品的预处理（沉淀蛋白、除杂）、分离和检测等过程实现复杂生物样品中待测成分的分离分析。所建立的体外水解方法简单、稳定、可控，对上述3种成分的体内药动学研究具有一定的参考意义。在体外血浆温孵试验和急性毒性试验的基础上，建立了附子总生物碱中主要成分（乌头碱、中乌头碱和次乌头碱）在大鼠血浆样品中UPLC／MS／MS测定法。大鼠血浆中加入一定体积磷酸，以固相萃取法提取待测成分，采用UPLC／ESI／MS／MS联用技术，多反应监测（MRM）扫描方式（乌头碱监测离子对646.3→586.0和646.3→367.9；中乌头碱监测离子对632.3→572.0和632.3→354.0；次乌头碱监测离子对616.3→556.0和616.3→337.9）同时测定大鼠血浆中附子总碱各主要成分的浓度；优化的色谱条件为Acquity UPLC^TM BEH C₁₈色谱柱，乙腈-0.05％氨水溶液为流动相，梯度洗脱，3种生物碱类成分在3 min内即可完全分离，内源性物质不干扰样品的测定；血浆样品提取回收率均高于85％；定量限乌头碱、中乌头碱和次乌头碱分别为0.0516 ng·mL^-1，0.0744 ng·mL^-1和0.0427 ng·mL^-1；日内和日间精密度的RSD均小于6％。该测定方法具有灵敏度高、专属性强、快速的优点，符合生物样品分析要求。选取3只大鼠灌胃（ig）附子总碱，灌胃给药剂量为0.2 g附子生药·kg^-1。从附子总碱大鼠灌胃的血药浓度-时间曲线可以看出，乌头碱、中乌头碱和次乌头碱的药时曲线均为双峰，无法用房室模型拟合，推测双峰是由于3种成分的肠肝循环造成的。乌头碱、中乌头碱和次乌头碱按非线性统计矩法计算的AUC0-t分别为4.277±0.754，7.950±2.909和24.75±4.05 ng·mL^-1·h；t_1／2分别为1.40±0.26，1.49±0.08和1.73±0.03 h；MRT分别为3.758±0.524，3.645±0.477和4.012±0.381 h。通过上述研究，初步得出了乌头碱、中乌头碱和次乌头碱药动学的变化规律。更多还原

【Abstract】 In this thesis, there are two sections: 1. Study on the quality assessment of Radix AconitiLateralis Praeparata; 2. Study on the pharmacokinetics of aconitum alkaloids in RadixAconiti Lateralis in rats.1. In section one, HPLC and LC/MS were used for establishing the fingerprints of RadixAconiti Lateralis in this study. Aconitic alkaloids and aconitic lipoids are main componentsextracted from Radix Aconiti Lateralis. Aconitic alkaloids are effective components. They have alot of pharmacological and clinical function. For quality control of Radix Aconiti LateralisPraeparata, Limit test of ester-alkaloids content is required in Pharmacopoeia of People’sRepublic of China. In order to control the quality of Radix Aconiti Lateralis roundly, fingerprintsbased on the chemical components investigation are one of the methods to control the quality oftraditional Chinese medicine.Application of LC/MS techniques on the fingerprints of traditional Chinese medicine is astrong support to routine chromatography. Combined the HPLC as a high efficiency separationmeans with MS as a high sensitive and special detector, the LC/MS techniques show unique powerto separate and identify the compounds in a complex matrix. LC/MS techniques have an importantrole in study of extraction methods, choice and optimum of experimental conditions, identificationof components for traditional Chinese medicine. In this thesis, we studied the methodology of thefingerprints of Radix Aconiti Lateralis by using HPLC and LC/MS, focused on the choice ofliquid chromatographic parameters, such as the type of column, the composition of mobile phaseand UV detector wavelength, etal. In this thesis, we used Hypersil BDS C₁₈ column and basicmobile phase to separate aconitic alkaloids. Main alkaloids of Radix Aconiti Lateralis wer eelucidated by using HPLC/MS, it provided experimental basis for establishment of the fingerprintsof Radix Aconiti Lateralis. The similarity of the chromatographic resulted from different batchesof samples have also been evaluated by chromatographic fingerprint evaluation software. The aboveexperimental results proved that the fingerprints obtained with HPLC can be used for the qualityevaluation of Radix Aconiti Lateralis and the establishment of its prepared materials.Based on the fingerprints of Radix Aconiti Lateralis, we determined the content of aconitine,mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in RadixAconiti Lateralis and its prepared materials. This also can be used for the quality control of RadixAconiti Lateralis and its prepared materials.2. In section two, we developed a platform of incubation of plasma in vitro for analysis ofconponents of traditional Chinese medicine. The aconitic alkaloids were as one of the examples.Aconitine, mesaconitine ans hypaconitine were added to blank rabbit plasma and incubated forvarious time. The plasma samples were processed with acetonitrile to denature and deposit the protein and extracted the three alkaloids, and the acetonitrile solution was analyzed by a RPchromatographic system. A simple, stable and controllable method has been established, which hassome reference value for pharmacokinetic study of aconitic alkaloids.Based on the incubation of plasma in vitro and the acute toxicity research, a sensitive, specificand rapid UPLC/MS/MS method for determination of the main components including aconitine,mesaconitine and hypaconitine in Radix Aconiti Lateralis in rat plasma was developed. The maincomponents in aconitic alkaloids were extracted from rats plasma using solid phase extraction, thenseparated on an Acquity UPLC^TM BEH C₁₈ column. The mobile phase consisted of acetonitrile-0.05% ammonia water （gradient elution）. Detection was performed on a tandem mass spectrometerequipped with an ESI interface and operated in positive-ionization mode. Multi-reactionsmonitoring （MRM） scan mode was employed for determination of the main components in aconiticalkaloids in rats plasma （the parent-daughter ion pairs of m/z 646.3→586.0 and 646.3→367.9 foraconitine; 632.34→572.0 and 632.3→354.0 for mesaconitine; 616.3→556.0 and 616.3→337.9 for hypaconitine was selected as MRM ions pair）. The linear calibration curves wereobtained. The extraction recovery was more than 85%. The minimum quantitative limitation foraconitine, mesaconitine and hypaconitine were 0.0516 ng·mL^-1, 0.0744 ng·mL^-1 and 0.0427 ng·mL^-1,respectively. The RSD of intra-day and inter-day assays were all less than 6%. The method wassuccessfully applied to determinate the concentration of the main components in aconitic alkaloidsin rats plasma after ig (0.2 g·kg^-1) Radix Aconiti Lateralis to rats （n=3）. The plasma drugconcentration-time curves for aconitine, mesaconitine and hypaconitine can not conform tocompartment models because they were double-peaks which may be due to enterohepaticcirculation. Their pharmacokinetic parameters were computated by Nonlinear Statistical MomentMethod, the AUC0-t was 4.277±0.754 ng·mL^-1·h （aconitine）, 7.950±2.909 ng·mL^-1·h（mesaconitine） and 24.75±4.05 ng·mL^-1·h （hypaconitine）; the mean value of t_1/2 was 1.40±0.26h, 1.49±0.08 h and 1.73±0.03 h, respectively; the MRT was 3.758±0.524 h, 3.645±0.477 hand 4.012±0.381 h, respectively.更多还原