| Traditional Chinese Medicine (TCM) and its prescriptions are Chinese national treasures; they are also the important fountainhead of developing independent innovative new drugs in our country. Studies on pharmacokinetic of TCM prescriptions can help reveal the pharmacodynamic material basis and mechanism and have a great significance to the modernization and internationalization of TCM.Xiao-Xu-Ming-Dectotion (XXMD) prescription was a traditional Chinese prescription and firstly recorded in'Bei Ji Qian Jin Yao Fang' which was written by Chinese ancient Si-Miao Sun of Tang Dynasty. XXMD is effective in treating theoplegia and the sequela of theoplegia and has been widely used in clinical treament. In this paper, modern analytical techniques were applied to identify active compounds and their metabolites after oral administration of XXMD to rats and to study the pharmacokinetics of three constitutes in plasma. The research results can provide the basis for the interpretation of the metabolic mechanism of XXMD in vivo.In the present work a liquid chromatography connected with electrospray ionization mass spectrometry (HPLC-FTICRMS and HPLC/LTQ-MS") method has been developed for the identification of the chemical constitutes in the active fraction from XXMD and XXMD prescription and the metabolites in the plasma, urine, feces and tissues of rats. The samples were separated on a HYPERSIL C18 column (250×4.6 mm,5μm). The mobile phase consisted of acetonitrile (A) and 0.4%(v/v) acetic acid (B) was delivered at a flow rate of 0.8 mL/min with the gradient program. The column temperature was maintained at 30℃. Detection was performed via electrospray ionization (ESI) source. Mass spectra were recorded in a mass range of m/z 100 to 1500. The operating parameters in the positive ion mode were as follows:collision gas, ultrahigh-purity helium (He); nebulizing gas, high-purity nitrogen (N2); ion spray voltage,3.5 kV; capillary temperature,300℃; capillary voltage,40 V; sheath gas flow rate,35 (arbitrary units); auxiliary gas flow rate,10 (arbitrary units); sweep gas flow rate,5 (arbitrary units); and tube lens,120 V. The column effluent was split in a ratio of 3:1, so that 200μL/min entered the source of the mass spectrometer.Fourteen constitutes were identified both in the active fraction from XXMD and in XXMD prescription including eight flavonoids (baicalin, liquiritigenin, oroxylin A-7-O-glucuronide, wogonoside, baicalein, wogonin, chrysin, oroxylin A), three chromones (prim-o-glucosylcimifugin, cimifugin,4'-O-(3-D-glucosyl-5-O-methylvisamminol), two triterpenes (glycyrrhizic acid, glycyrrhetinic acid) and one monoterpene (paeoniflorin). Eleven constitutes were identified in the plasma of rats treated with the active fraction from XXMD, including 5-O-methylvisammiol, which was the metabolite of 4'-O-β-D-glucosyl-5-O-methylvisamminol. Fourteen and nine constitutes were detected in the urine and feces of rats, separately. In the plasma, urine and feces of rats treated with XXMD prescription, ten, fourteen and eight compounds were identified, separately. The compounds detected in rats treated with both active fraction from XXMD and XXMD prescription were almost the same, which indicated that the major chemical basis of the pharmacologic actions of the two prescriptions had little differences.The same fifteen constituents were identified in the tissues of rats dosed active fraction and XXMD prescription. The chemical constituent distribution showed the similar characterization in rat tissues dosed active fraction or XXMD prescription. However, the number of compounds detected in some of the tissues of rats treated with XXMD prescription was a little more than that of rats given with the active fraction. The constituents apeared in tissues on more time point after rats dosed XXMD prescription than dosed active fraction. In addition, the paper made an exploratory analysis of the tissue distribution using Principle Component Analysis (PCA).In order to study the pharmacokinetics of XXMD, an HPLC-MS/MS method had been developed and validated for the identification and quantification of liquiritigenin, oroxylin A-7-O-glucuronide and wogonoside in rat plasma after oral administration of active fraction from XXMD. Biological samples were prepared by solid phase extraction. Icariin was used as internal standard (IS). The chromatographic separation was accomplished on a Waters Symmetry C18 (100×2.1mm,3.5μm). The mobile phase consisted of acetonitrile and 0.2% aqueous acetic acid (30:70, V/V) was delivered at a flow rate of 0.3 mL/min, the column oven was maintained at 30℃. Detection was performed with a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The linearity of liquiritigenin, oroxylin A-7-O-glucuronide and wogonoside was established for the range of concentrations 2.55-2.04×103 ng/mL, 1.88-1.50×103 ng/mL and 2.50-2.00×103 ng/mL separately, with the coefficient of determination (r) of 0.99. The RSD of intra-and inter-day precision was lower than 9.4% and the accuracy ranged from 92.6 to 113.4%. The lower limit of quantification was lower than 2.55 ng/mL for all the three analytes. The concentration-time curves of the three analytes showed double-peak phenomenon at 5 min and 960 min after oral administration of active fraction from XXMD to rats. There might be enterohepatic circulation and metabolic transformation in vivo. The concentration of wogonoside was the highest in rat plasma among the three compounds. In contrast, the concentration of liquiritigenin was the lowest. However, in the active fraction from XXMD, the contents of the three compounds have only a little difference relatively, demonstrating that there was obvious selectivity for the absorption of the three compounds. The results provide scientific data for elucidation of pharmaceutical mechanism after oral administration of the active fraction to rats. |
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