The Aconite Quality Evaluation And Aconitum Composition Pharmacokinetic Study

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.