RESUMEN
RATIONALE: Cucumber, as a popular fruit and vegetable, has tremendously contributed to providing a sufficient and high-quality food supply. However, the cucumber plant metabolites, which may possess potential benefits for human health, were rarely reported. In addition, rapid detection of these metabolites from the complex biological matrix of cucumber samples is a tremendous challenge. METHODS: A rapid detection method was established to systematically screen cucurbitacins and cucurbitacin glycosides in cucumber plants by combining high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) with in-source fragmentation (ISF). Moreover, the alkali cations, including acetic acid, 0.1% LiCl, 0.1% NH4 Cl, 0.1% NaCl, and 0.1% KCl, were added to the mobile phase for improving the ion response. RESULTS: The fragmentation pathways of seven cucurbitacins and cucurbitacin glycosides were primarily investigated. The characteristic ISF ions at m/z 501.3211 and 503.3367 were identified and employed to screen 40 cucurbitacins and cucurbitacin glycosides from the complex biological matrix. Their structures were identified by their tandem mass spectrometry (MS/MS) spectra and fragmentation pathways of references. Finally, the metabolic distribution and network of cucurbitacins and cucurbitacin glycosides in cucumber plants were also proposed. CONCLUSIONS: This work marks the first systematic and comprehensive study of the metabolites in cucumber plants using HPLC-Q-TOF-MS technology, providing a template for screening and identifying the triterpenoids from other plant-derived medicines or food.
Asunto(s)
Cucumis sativus , Cucurbitacinas , Álcalis , Cromatografía Líquida de Alta Presión/métodos , Cucurbitacinas/análisis , Glicósidos/química , Humanos , Iones , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Agarwood is derived from Aquilaria trees, the trade of which has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. RESULTS: In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. DNA and RNA data are utilized to annotate many genes and protein functions in the draft genome. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. agallocha to biotic stress and a set of homologous genes in Arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qRT-PCR. CONCLUSIONS: This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria. The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants.
Asunto(s)
Cucurbitacinas/análisis , Genoma de Planta , Thymelaeaceae/genética , Cromatografía Líquida de Alta Presión , Cucurbitacinas/química , Cucurbitacinas/metabolismo , Enzimas/genética , Enzimas/metabolismo , Biblioteca de Genes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ARN , Espectrometría de Masa por Ionización de Electrospray , Thymelaeaceae/química , Thymelaeaceae/metabolismoRESUMEN
Bitterness in cucumber fruit and foliage is due to the presence of cucurbitacins. Several genes have been described that control the trait, with bi (bi-1) making fruit and foliage bitter free and Bt (Bt-1) making the fruit highly bitter. Previous studies have reported the inheritance and molecular markers linked to bi-1 or Bt-1, but we were interested in studying the inheritance of fruit bitterness in the progeny of 2 nonbitter fruit inbred lines. The objective was to determine the inheritance of cucumber fruit and foliage bitterness and to locate them on a current linkage map using a recombinant inbred lines (RILs) population derived by crossing 9110Gt and 9930. It was concluded from the inheritance analysis that there were 2 loci controlling fruit bitterness in the population. One locus was in the same position as the location previously identified for bi-1, and another locus was for bi-3. Using a simple sequence repeat (SSR) linkage map, 2 loci for fruit bitterness in this RILs population were mapped. The locus of bi-1 was located at the region between SSR0004 and SSR02309 within the genetic distance of 5.2 cM on chromosome 6. The locus of bi-3 was placed in the region of SSR00116-SSR05321 within the genetic distance of 6.3 cM on chromosome 5. The physical distances for the regions of bi-1 and bi-3 were 11,430.94 Kb with 160 predicted genes and 1528.23 Kb with 198 predicted genes, respectively. Among 160 predicted genes for bi-1, there is a terpene synthase gene named Csa008595, which was speculated as the candidate gene of bi-1.
Asunto(s)
Cucumis sativus/genética , Cucurbitacinas/genética , Frutas/genética , Hojas de la Planta/genética , Transferasas Alquil y Aril/genética , Mapeo Cromosómico , Biología Computacional , Cruzamientos Genéticos , Cucumis sativus/química , Cucurbitacinas/análisis , Cartilla de ADN/genética , Genes de Plantas/genética , Marcadores Genéticos/genética , Escala de Lod , Repeticiones de Microsatélite/genética , Anotación de Secuencia Molecular , Sitios de Carácter Cuantitativo/genéticaRESUMEN
OBJECTIVE: To establish an HPLC method for the simultaneous determination of four bioactive cucurbitacins in Cucubita pepo cv Dayangua. METHODS: The HPLC chromatography was carried out with a lineal gradient programming and detection wavelength at 215 nm. Kromasil C8 column (150 mm x 4.6 mm ID, 5 microm)was used. The mobile phase was acetonitrile-water (containing 2.0% HAc) and the flow rate was 1.0 ml/min. RESULTS: The linear range of 23, 24-dihydrocucurbitacin F was 0.28-5.6 microg/ml (r = 0.9978), 23, 24-dihydrocucurbitacin D 0.39-7.8 microg/ml (r = 0.9986), cucurbitacin B 0.304-6.08 microg/ml (r = 0.9983), cucurbitacin E 2. 52 -50. 4 microg/ml (r = 0.9998). The method was accurate with variation less than 5% and recovery more than 95%. CONCLUSION: The method is successfully applied to determination of the four cucurbitacins from Cucubita pepo cv dayangua.