Introduction: The genus Clusia L. is mostly recognised for the production of prenylated benzophenones and tocotrienol derivatives.Objectives: The objective of this study was to map metabolome variation within Clusia minor organs at different developmental stages.Material and Methods: In total 15 organs/stages (leaf, flower, fruit, and seed) were analysed by UPLC‐MS and 1H‐ and heteronuclear multiple‐bond correlation (HMBC)‐NMR‐based metabolomics.Results: This work led to the assignment of 46 metabolites, belonging to organic acids(1), sugars(2) phenolic acids(1), flavonoids(3) prenylated xanthones(1) benzophenones(4) and tocotrienols(2). Multivariate data analyses explained the variability and classification of samples, highlighting chemical markers that discriminate each organ/stage. Leaves were found to be rich in 5‐hydroxy‐8‐methyltocotrienol (8.5 μg/mg f.w.), while flowers were abundant in the polyprenylated benzophenone nemorosone with maximum level detected in the fully mature flower bud (43 μg/mg f.w.). Nemorosone and 5‐hydroxy tocotrienoloic acid were isolated from FL6 for full structural characterisation. This is the first report of the NMR assignments of 5‐hydroxy tocotrienoloic acid, and its maximum level was detected in the mature fruit at 50 μg/mg f.w. Seeds as typical storage organ were rich in sugars and omega‐6 fatty acids.Conclusion: To the best of our knowledge, this is the first report on a comparative 1D‐/2D‐NMR approach to assess compositional differences in ontogeny studies compared with LC‐MS exemplified by Clusia organs. Results derived from this study provide better understanding of the stages at which maximal production of natural compounds occur and elucidate in which developmental stages the enzymes responsible for the production of such metabolites are preferentially expressed.

Background/Aim: Tubugi-1 is a more stable and accessible synthetic counterpart of natural tubulysins. This study aimed to evaluate its cytotoxic potential against anaplastic human melanoma cells. Materials and Methods: The viability of A-375 cells was determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assay. The type of cell death and proliferative rate were investigated using flow cytometry and fluorescent microscopy, while the molecular background was evaluated by western blot. Results: Tubugi-1 reduced the viability of A-375 cells, inducing massive micronucleation, followed by augmented expression of inhibitor of nuclear factor-κB and caspase-2, typical of a mitotic catastrophe. Disturbed proliferation and G2M block with prominent caspase activity, weakened the expression of B-cell lymphoma 2 and B-cell lymphoma 2-associated X transient up-regulation, coexisted with intensive autophagy. Specific inhibition of autophagy by chloroquine resulted in conversion from mitotic catastrophe to rapid apoptosis. Conclusion: Multilevel anticancer action of tubugi-1 is extended by co-application of an autophagy inhibitor, giving a new dimension in further preclinical advancement of this potential agent.

The β ‐carboline alkaloids harmane (1 ) and norharmane (2 ) were isolated from fruiting bodies of Hygrophorus eburneus (Bull.) Fr. as well as brunnein A (3 ) from Hygrophorus hyacinthinus Quél. (Tricholomataceae, Agaricales) for the first time. Their occurrence within the genus was investigated using liquid chromatography/electrospray ionisation tandem mass spectrometric methods, especially by selected reaction monitoring. Based on these results their chemotaxonomical relevance is discussed.

A simple method involving polyamide column chromatography in combination with HPLC‐PAD and HPLC‐ESI[sol ]MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4°C from which two main compounds were purified by semi‐preparative HPLC. In comparison with the UV and ESI‐MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction[sol ]separation technique was validated using an internal standard method. Copyright © 2005 John Wiley & Sons, Ltd.

Natural isothiocyanates, produced during plant tissue damage from methionine‐derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell‐free leaf extracts as inducers of murine QR, which reduces sample preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine‐derived glucosinolates, as shown by the analysis of wild‐type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate‐based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate‐producing species.

After application of [17‐D2]‐GA34 to seedlings of Phaseolus coccineus L. cv. Prizewinner, both metabolically formed conjugates [17‐D2]‐GA34‐2‐O‐glucoside and [17‐D2]‐GA34‐glucosyl ester could be established structurally. The identification was based on data from GC‐MS as well as LC‐ESI‐tandem MS (negative ions) techniques (daughter ion scan, parent ion scan, selected ion monitoring, selected reaction monitoring), and NMR studies.

Conjugates of 3,7‐trans cucurbic acid isomers with either (S )‐ or (R )‐isoleucine were synthesised from the diastereomeric conjugate of (3R , 7R )‐jasmonic acid and (3S , 7S )‐jasmonic acid by sodium borohydride reduction. The resulting diastereomers were characterised by nuclear magnetic resonance spectra. The authentic substances were subjected to liquid chromatography using a reversed‐phase C18 matrix and the chiral stationary phase Chiralpak AS. For all (6RS )‐hydroxy epimeric pairs, a baseline separation could be observed. The elution sequences obtained indicate that the 3,6‐trans oriented epimers with (S )‐isoleucine elute prior to the 3,6‐cis configured individuals independent of the mode of chromatography. In contrast, the elution pattern of the conjugates containing (R )‐isoleucine was reversed on the chiral stationary phase. The epimers with a 3,6‐cis configured hydroxy group and the acid side chain eluted faster than those with the 3,6‐trans configuration. The chromatographic conditions described are suitable for resolving mixtures of isomeric N‐(cucurbinoyl)‐isoleucines in order to assign their stereochemistry and to obtain chiral reference materials on a preparative scale.

The brassinosteroids brassinolide, castasterone and 24‐epi‐castasterone could be isolated and identified from seeds of Daucus carota . Furthermore, a new pregnanolone glucoside was identified as β‐D ‐glucopyranosyl‐(1‐2)‐β‐D ‐glucopyranosyl‐3β‐hydroxy‐5α‐pregnane‐20‐one (sophorosylpregnanolone) by nuclear magnetic resonance spectroscopy, liquid chromatography‐mass spectrometry and gas chromatography‐mass spectrometry.

Coenzyme A thioesters of anthranilic acid and N‐methylanthranilic acid were synthesized. The corresponding N‐hydroxysuccinimidyl esters proved to be useful as activated intermediates to prepare anthraniloyl‐CoA and N‐methylanthraniloyl‐CoA. These compounds were characterized by positive and negative ion liquid secondary ion mass spectrometry. Acridone synthase from suspension cultures of Ruta graveolens catalyses the condensation of N‐methylanthraniloyl‐CoA and malonyl‐CoA. The reaction product 1,3‐dihydroxy‐N‐methylacridone was directly identified after the extraction of the assay mixture by electron impact mass spectrometry and capillary gas chromatography.