| Wuzhuyu Tang (WZYT) is a widely used traditional Chinese Medicine (TCM) formula composed of Fructus of Evodiae Rutaecarpa, Rhizome of Zingiberi officinale, Radix of Panax Ginseng, and Fructus of Ziziphus jujuba. It has a broad spectrum of applications in treatment of nausea, migraine headache, beriberi, heart failure, etc. In this paper, chemical basis, quality control method, pharmacokinetics and metabonomics of Wuzhuyu Tang were studied systematically.A high performance liquid chromatographic tandem mass spectrometric (HPLC-MS/MS) method combined with liquid-liquid extraction sample preparation was developed for screening and identification of the main constituents of Wuzhuyu Tang. Chromatographic peaks of 44 were detected,18 of which were identified including dehydroevodiamine, N13-methyl-2-methoxyrutaecarpine, evodianinine,12a-hydroxylimonin, limonin, evodol, evodiamide, rutaevine acetate, evodiamine, rutaecarpine and chlorogenic acid from Evodiae fructus,6-ginerol form Zingiber officinale, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro and ginsenoside Rd from Panax Ginseng. The HPLC-MS/MS method was also applied to screen the constituents in rat plasma after oral administration of Wuzhuyu Tang.5 compounds were detected in rat plasma,4 of which were identified including dehydroevodiamine, N13-methyl-2-methoxyrutaecarpine, evodianinine and limonin.A high performance liquid chromatographic (HPLC) method with diode array detection was developed and validated to simultaneously determine five compounds in Evodiae fructus including chlorogenic acid, dehydroevodiamine, limonin, evodiamine and rutaecarpine. The established method was applied to determine the amounts of the five compouds in thirteen samples from different regions of China and processed Euodia Rutaecarpa after several different ways. An HPLC method with diode array detection was developed and validated to simultaneously determine six compounds in Wuzhuyu Tang including chlorogenic acid, dehydroevodiamine, limonin, evodiamine, rutaecarpine and 6-ginerol. The established method was applied to determine the amounts of the six compounds in three batches Wuzhuyu Tang, and the extraction of the six compounds in different herb combination was investigated by the HPLC method. The interaction of herbs was observed when the herbs were boiled in different compatibilities.A high performance liquid chromatographic-mass spectrometric (HPLC-MS) method was developed and validated for the simultaneous determination of dehydroevodiamine and limonin from Evodiae fructus in rat plasma for the first time. After addition of the internal standard domperidone, plasma samples were extracted by liquid-liquid extraction (LLE) method with ethyl acetate and separated on an Apollo C18 column (250 x 4.6 mm,5μm) using a mobile phase composed of methanol-0.01% formic acid water (60:40, v/v) within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0-500 ng/ml for dehydroevodiamine and 2.0-1,000 ng/ml for limonin with the lower limit of quantitation of 1.0 and 2.0 ng/ml, respectively. The intra-day and inter-day precisions (RSD%) were both within 6.0% and 10.9% for the two analytes, while the accuracy (relative error, RE%) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to the comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, aqueous extract of Evodiae fructus and Wuzhuyu Tang, respectively. After dehydroevodiamine, aqueous extract of Evodiae fructus or Wuzhuyu Tang were orally treated, the Cmax of dehydroevodiamine was 122.5±78.6,53.36±7.67 and 36.79±9.11 ng/ml, AUCo-∞was 798.2±354.4,677.1±297.0 and 149.2±42.9 ngxh/ml,T1/2 was 4.4±1.4,10.1±2.9 and 3.8±1.3 h, respectively. After limonin, aqueous extract of Evodiae fructus or Wuzhuyu Tang were orally treated, the Cmax of limonin was 67.78±46.25,695.6±265.8 and 765.9±256.1 ng/ml, AUC0-∞was 205.9±56.2,3992±1444 and 1691±400 ngxh/ml,T1/2 was 4.9±1.9,5.5±2.8 and 3.9±2.4 h, respectively. The results indicated that there were obvious differences in the pharmacokinetic behaviors after oral administration of the mono substance, aqueous extract of Evodiae fructus or Wuzhuyu Tang.The metabonomics was introduced to study the influence of Wuzhuyu Tang on migraine model rats. The migraine rat model was copied by glyceryltrinitrate infusion. An UPLC-MS method was developed and applied to analyze the plasma and urine from control group, model group, Wuzhuyu Tang group and flunarizine group. The analytical data were processed via principle component analysis (PCA). PCA score piots of analytical data indicated the general differences among different groups and the influence of Wuzhuyu Tang on migraine. The dominant metabolites obtained from loading plot of Masslynx were the differential metabolites among different groups, ten of which were conformed. They are 16:0 LPC,18:0 LPC,18:2 LPC, 20:4 LPC, and cholic acid form plasma, xanthurenic acid, kynurenic acid, hippuric acid, phenylacetylglycine and hexadecas-phinganine form urine. The mechanism of pathological changes may be elucidated with the up- or down-regulated metabolic pathways. |
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