RESUMO
This study aimed to develop a rapid, sensitive, and specific LC-tandem mass spectrometry method for the determination of nootkatone in rat plasma. α-Cyperone was chosen as the internal standard (IS). The plasma was processed using a one-step acetonitrile protein precipitation method. Chromatographic separation of nootkatone was achieved on a Phenomenex Kinetex XB-C18 column (2.10 × 50 mm, 2.6 µm) at 35°C with a mobile phase consisting of acetonitrile and water under a gradient elution at a flow rate of 0.35 mL/min. An electrospray ionization source was applied and operated in positive ion and multiple reaction monitoring modes. Nootkatone and IS were quantified using the transitions of m/z 219.200 â 163.110 and m/z 219.200 â 111.000, respectively. The calibration curves were linear over the range of 10-2000 ng/mL (r = 0.9943). The lower limit of quantification was 10 ng/mL. The intra- and inter-day precision (relative standard deviation) ranged from 2.56% to 8.41%, with the accuracy values ranging from 98.9% to 99.17% for four different concentration levels. The matrix effect and extraction recovery were within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of nootkatone in rats after oral and intravenous administration at three dosages. The main pharmacokinetic parameters were calculated, showing low bioavailability of nootkatone.
Assuntos
Cromatografia Líquida/métodos , Sesquiterpenos Policíclicos , Espectrometria de Massas em Tandem/métodos , Animais , Feminino , Limite de Detecção , Modelos Lineares , Sesquiterpenos Policíclicos/sangue , Sesquiterpenos Policíclicos/química , Sesquiterpenos Policíclicos/farmacocinética , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Plant secondary metabolites are known to not only play a key role in the adaptation of plants to their environment, but also represent an important source of active pharmaceuticals. Alpinia oxyphylla capsular fruits, made up of seeds and pericarps, are commonly used in traditional East Asian medicines. In clinical utilization of these capsular fruits, inconsistent processing approaches (i.e., hulling pericarps or not) are employed, with the potential of leading to differential pharmacological effects. Therefore, an important question arises whether the content levels of pharmacologically active chemicals between the seeds and pericarps of A. oxyphylla are comparable. Nine secondary metabolites present in A. oxyphylla capsular fruits, including flavonoids (e.g., tectochrysin, izalpinin, chrysin, apigenin-4',7-dimethylether and kaempferide), diarylheptanoids (e.g., yakuchinone A and B and oxyphyllacinol) and sesquiterpenes (e.g., nootkatone), were regarded as representative constituents with putative pharmacological activities. This work aimed to investigate the abundance of the nine constituents in the seeds and pericarps of A. oxyphylla. Thirteen batches of A. oxyphylla capsular fruits were gathered from different production regions. Accordingly, an ultra-fast high performance liquid chromatography/quadrupole tandem mass spectrometry (UFLC-MS/MS) method was developed and validated. We found that: (1) the nine secondary metabolites were differentially concentrated in seeds and fruit capsules; (2) nootkatone is predominantly distributed in the seeds; in contrast, the flavonoids and diarylheptanoids are mainly deposited in the capsules; and (3) the content levels of the nine secondary metabolites occurring in the capsules varied greatly among different production regions, although the nootkatone levels in the seeds were comparable among production regions. These results are helpful to evaluating and elucidating pharmacological activities of A. oxyphylla capsular fruits. Additionally, it may be of interest to elucidate the mechanisms involved in the distinct accumulation profiles of these secondary metabolites between seeds and pericarps.
Assuntos
Alpinia/química , Flavonoides/classificação , Extratos Vegetais/análise , Sementes/química , Sesquiterpenos/classificação , China , Cromatografia Líquida de Alta Pressão/métodos , Clima , Flavonoides/isolamento & purificação , Geografia , Especificidade de Órgãos , Sesquiterpenos/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Galangin (3,5,7-trihydroxyflavone) is present in high concentrations in herbal medicine such as Alpinia officinarum Hance. Galangin shows multifaceted in vitro and in vivo biological activities. The number and position of hydroxyl groups in this molecule play an important role in these biological activities. However, these hydroxyl groups undergo glucuronidation and sulfation in in vitro assay system. However, the systemic exposure to galangin after dosing in animals and/or humans remains largely unknown. Thus it is not clear whether the galangin exists in the body at concentrations high enough for the biological effects. Furthermore, the metabolite identification and the corresponding plasma pharmacokinetics need to be characterized. RESULTS: Two LC-MS/MS methods were developed and validated and successfully applied to analyze the parent drug molecules and aglycones liberated from plasma samples via ß-glucuronidase hydrolysis. Our major findings were as follows: (1) The routes of administration showed significant influences on the systemic exposure of galangin and its metabolites. (2) Galangin was preferentially glucuronidated after p.o. dosing but sulfated after i.v. medication. (3) Kaempferol conjugates were detected demonstrating that oxidation reaction occurred; however, both glucuronidation and sulfation were more efficient. (4) Oral bioavailability of free parent galangin was very low. CONCLUSIONS: Systemic exposure to galangin and its metabolites was different in rat plasma between oral and intravenous administration. Further research is needed to characterize the structures of galangin conjugates and to evaluate the biological activities of these metabolites. Graphical abstractGalangin was preferentially glucuronidated after p.o. dosing but sulfated after i.v. medication.
RESUMO
Alpinia oxyphylla (Yizhi) capsularfruits are commonly used in traditional medicine. Pharmacological studies have demonstrated that A. oxyphylla capsularfruits have some beneficial roles. Besides volatile oil, sesquiterpenes, diarylheptanoids and flavonoids are main bioactive constituents occurring in the Yizhi capsularfruits. The representative constituents include tectochrysin, izalpinin, chrysin, apigenin-4',7-dimethylether, kaempferide, yakuchinone A, yakuchinone B, oxyphyllacinol and nootkatone. Their content levels in the fruit and its pharmaceutical preparations have been reported by our group. The nine phytochemicals are also the major components present in the Yizhi alcoholic extracts, which have anti-diarrheal activities. However, the fates of these constituents in the body after oral or intravenous administration remain largely unknown. In the present study, we focus on these phytochemicals albeit other concomitant compounds. The chemicals and their metabolites in rat plasma were identified using liquid chromatography/tandem mass spectrometry with selected reaction monitoring mode after orally administered Yizhi extract to rats. Rat plasma samples were treated by methanol precipitation, acidic or enzymatic hydrolysis. This target analysis study revealed that: (1) low or trace plasma levels of parent chemicals were measured after p.o. administration of Yizhi extract, Suoquan capsules and pills to rats; (2) flavonoids and diarylheptanoids formed mainly monoglucuronide metabolites; however, diglucuronide metabolites for chrysin, izalpinin and kaempferide were also detected; (3) metabolic reduction of Yizhi diarylheptanoids occurred in rats. Yakuchinone B was reduced to yakuchinone A and then to oxyphyllacinol in a stepwise manner and subsequently glucuronidated by UDP-glucuronosyl transferase. Further research is needed to characterize the UDP-glucuronosyl transferase and reductase involved in the biotransformation of Yizhi chemicals.