RESUMEN
Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Geraniltranstransferasa/genética , Matricaria/enzimología , Matricaria/genética , Oxilipinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Aminoácidos , Biocatálisis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Genes de Plantas , Geraniltranstransferasa/química , Geraniltranstransferasa/aislamiento & purificación , Matricaria/efectos de los fármacos , Matricaria/crecimiento & desarrollo , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/química , Proteínas de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
A rapid liquid chromatography-electrospray mass spectrometry (LC-ES-MS) method with in vivo microdialysis for the determination of free-form of cocaine (COC) in rat brain has been developed. A C18 column and a gradient elution were employed for the separation. The [M+H]+ (m/z=304) and a fragmented ion (m/z=182) were detected using positive ion mode detection. Selective ion monitoring was utilized for quantitative measurement. The linearity of this assay was good ranging from 0.01 to 1.0 microM (r2=0.999). The inter- and intra-day precisions showed relative standard deviations ranging from 1.0% to 3.3% and 1.0% to 3.6%, respectively. In addition, the detection of one COC metabolite, benzoylecgonine (BE), by this assay was also investigated. The linearity, precision, and detection limit associated with this method for BE were determined. The application of this newly developed method was demonstrated by examining the pharmacokinetics of COC in rat brain.
Asunto(s)
Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Cocaína/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Cocaína/química , Cocaína/farmacocinética , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
This paper describes a newly developed high performance liquid chromatography/electrospray/mass spectrometry (HPLC/ES/MS) method for the determination of flunarizine (FZ) in artificial cerebrospinal fluid. The optimization for the detection of FZ in biological fluid by LC/ES/MS was investigated. The effects of solvent composition, the addition of modifier and flow rate on the detection of FZ by ES/MS were examined. The detection limit of this method ( approximately 0.8 nM) proved to be much better than previously reported methods. Satisfactory accuracy (98.2-106.0%) of this newly developed method was obtained. The application of this method was demonstrated by analyzing FZ in rat microdialysis samples.