Paul A. Steenkamp

Analyses of chlorogenic acids and related cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method.

BackgroundChlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures.ResultsUsing an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof.ConclusionUsing this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.

Mycotoxic nephropathy in Bulgarian pigs and chickens: complex aetiology and similarity to Balkan Endemic Nephropathy

Spontaneous nephropathy in Bulgaria, which is observed frequently during meat inspection and which differs morphologically from the classical description of mycotoxic porcine/chicken nephropathy as made in Denmark, was found to have a multi-mycotoxic aetiology being mainly provoked by a combined effect of ochratoxin A, penicillic acid and fumonisin B1 in addition to a not-yet-known metabolite. Mean contamination levels of ochratoxin A were consecutively low (188.8 and 376.4 µg kg−1) in contrast to high contamination levels of fumonisin B1 (5564.1 and 3254.5 µg kg−1) and penicillic acid (838.6 and 904.9 µg kg−1) for 2006 and 2007, respectively. Some other mycotoxins with lower importance such as citrinin, penitrem A, etc., may also influence clinicopathological picture of this nephropathy. A heavy contamination with Gibberella fujikuroi var. moniliformis (Fusarium verticillioides) and Penicillium aurantiogriseum complex (mainly Penicillium polonicum) was observed in almost all examined feed samples coming from pig and chick farms with nephropathy problems from Bulgaria. In contrast, low contamination with Aspergillus ochraceus, Penicillium verrucosum and Penicillium citrinum was observed in the same feed samples and these species were isolated as very rare components of the mycobiota.

Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells.

Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0–1000 nM) of ergosterol and incubated for different time periods (0–24 h). Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC×GC-TOF-MS, UHPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The ‘defensome’ involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin) and other metabolites (abscisic acid and phytosterols) putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.

Metabolomic Analysis of Methyl Jasmonate-Induced Triterpenoid Production in the Medicinal Herb Centella asiatica (L.) Urban

Centella asiatica is an important source of biologically active pentacyclic triterpenoids. The enhancement of the biosynthesis of the centellosides by manipulation of associated metabolic pathways is receiving much attention. Jasmonates play critical roles in plant metabolism by up-regulating the expression of genes related to secondary metabolites. Here, we investigated the effect of methyl jasmonate (MeJa) in C. asiatica through targeted metabolomic profiling of asiaticoside and madecassoside as well as their aglycones, asiatic acid and madecassic acid. Cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days. Liquid chromatography coupled to mass spectrometry (LC-MS) was used to explore induced changes in metabolite profiles, both qualitatively and quantitatively. Principal component analysis (PCA)-derived scores plots revealed clusters of sample replicates for control and treated samples at 2, 4 and 6 days while loading plots aided in identifying signatory biomarkers (asiatic acid and madecassic acid, as well as asiaticoside and madecassoside) that clearly demonstrate the variability between samples. In addition to increased biosynthesis of the targeted centelloids, other differential changes in the intracellular metabolite profiles reflected the response of the C. asiatica cells to the MeJa-treatment as a reprogramming of the metabolome.

A one-pot synthesis of 1,4-naphthoquinone-2,3-bis-sulfides catalysed by a commercial laccase

Oxidative C–S bond formation with aryl and alkyl thiols was catalysed under mild conditions in a reaction vessel open to air at pH 4.5 and 7.15 in the presence of a commercial laccase (Novozym 51003) and a co-solvent (DMF) to afford 1,4-naphthoquinone-2,3-bis-sulfides. The synthesis of 1,4-naphthoquinone-2,3-bis-sulfides from two different 1,4-naphthohydroquinone substrates was investigated with regard to pH and number of equivalents of the thiol.

Preferential alkali metal adduct formation by cis geometrical isomers of dicaffeoylquinic acids allows for efficient discrimination from their trans isomers during ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE Caffeoylquinic acid (CQA) derivatives are a group of structurally diverse phytochemicals that have attracted attention due to their many health benefits. The structural diversity of these molecules is due in part to the presence of regio- and geometrical isomerism. This structural diversity hampers the accurate annotation of these molecules in plant extracts. Mass spectrometry (MS) is successfully used to differentiate between the different regioisomers of the CQA derivatives; however, the accurate discrimination of the geometrical isomers of these molecules has proven to be an elusive task. METHODS UV-irradiated methanolic solutions of diCQA were analyzed using an ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) method in negative ionisation mode. An in-source collision-induced dissociation (ISCID) method was optimized by varying both the capillary and cone voltages to achieve differential fragmentation patterns between UV-generated geometrical isomers of the diCQAs during MS analyses. RESULTS Changes in the capillary voltage did not cause a significant difference to the fragmentation patterns of the four geometrical isomers, while changes in the cone voltage resulted in significant differences in the fragmentation patterns. The results also show, for the first time, the preferential formation of alkali metal (Li(+), Na(+) and K(+)) adducts by the cis geometrical isomers of diCQAs, compared to their trans counterparts. CONCLUSIONS Optimized QTOFMS-based methods may be used to differentiate the geometrical isomers of diCQAs. Finally, additives such as metal salts to induce adduct formation can be applied as an alternative method to differentiate closely related isomers which could have been difficult to differentiate under normal MS settings.

Multivariate statistical models of metabolomic data reveals different metabolite distribution patterns in isonitrosoacetophenone-elicited Nicotiana tabacum and Sorghum bicolor cells

Isonitrosoacetophenone (INAP, 2-keto-2-phenyl-acetaldoxime) is a novel inducer of plant defense. Oxime functional groups are rare in natural products, but can serve as substrates depending on existing secondary pathways. Changes in the metabolomes of sorghum and tobacco cells treated with INAP were investigated and chemometric tools and multivariate statistical analysis were used to investigate the changes in metabolite distribution patterns resulting from INAP elicitation. Liquid chromatography combined with mass spectrometry (UHPLC-MS) supplied unique chemical fingerprints that were generated in response to specific metabolomic events. Principal component analysis (PCA) together with hierarchical cluster analysis (HCA) and Metabolic Trees were used for data visualization. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and shared and unique structure (SUS) plots were exploited in parallel to reveal the changes in the metabolomes. PCA indicated that the cells responded differentially to INAP through changes in the metabolite profiles. Furthermore, HCA and Metabolic Trees showed that INAP induced metabolic perturbations in both cell lines and that homeostasis was re-established over time. OPLS-DA-based shared and unique structure (SUS) plots confirmed the results and revealed differences in the metabolites distribution patterns between tobacco and sorghum cells. Chemometric analyses of metabolomic data offers insight into changes in metabolism in response to chemical elicitation. Although similar, the response in sorghum cells was found to be more consistent and well-coordinated when compared to tobacco cells, indicative of the differences in secondary metabolism between cyanogenic and non-cyanogenic plants for oxime metabolism.

Expeditious synthesis and biological evaluation of novel 2,N6-disubstituted 1,2-dihydro-1,3,5-triazine-4,6-diamines as potential antimalarials.

A small set of novel 2,N6-disubstituted 1,2-dihydro-1,3,5-triazine-4,6-diamines was prepared possessing a flexible tether between the exocyclic nitrogen bonded to C-6 of the 1,2-dihydro-1,3,5-triazine-4,6-diamine heterocycle and the distal aryl ring. Three zones were varied in this series of compounds, namely the nature of the substituent(s) on C-2; the nature of the substituent(s) on the distal aryl ring; as well as the nature and length of the flexible tether between the rings. The compound showing the best antimalarial activity (cycloguanil-resistant FCR-3 Plasmodium falciparum IC50=0.99 μM) was N6-(3-(4-chlorophenoxy)propyl)-2-(furan-2-yl)-1,2-dihydro-1,3,5-triazine-4,6-diamine hydrochloride.

Development and Optimization of an UPLC-QTOF-MS/MS Method Based on an In-Source Collision Induced Dissociation Approach for Comprehensive Discrimination of Chlorogenic Acids Isomers from Momordica Plant Species

Chlorogenic acids (CGA) have been profiled in the leaves of Momordica balsamina, Momordica charantia, and Momordica foetida. All three species were found to contain the trans and cis isomers of 4-acyl para-coumaroylquinic acid (pCoQA), caffeoylquinic acid (CQA), and feruloylquinic acid (FQA). To the best of our knowledge, this is the first report of pCoQA and FQA and their cis isomers in these Momordica species. These profiles were obtained by a newly developed UPLC-qTOF-MS method based on the in-source collision induced dissociation (ISCID) method optimized to mimic the MS2 and MS3 fragmentation of an ion trap-based MS. The presence of the cis isomers is believed to be due to high UV exposure of these plants. Furthermore, the absence of the 3-acyl and 5-acyl CGA molecules points to a metabolic mark that is unusual and represents a very interesting biochemical phenotype of these species. Our optimized ISCID method was also shown to be able to distinguish between the geometrical isomers of all three forms of CGA, a phenomenon previously deemed impossible with other common mass spectrometry systems used for CGA analyses.

Ergosterol-Induced Sesquiterpenoid Synthesis in Tobacco Cells

Plants have the ability to continuously respond to microbial signals in their environment. One of these stimuli is a steroid from fungal membranes, ergosterol, which does not occur in plants, but acts as a pathogen-associated molecular pattern molecule to trigger defence mechanisms. Here we investigated the effect of ergosterol on the secondary metabolites in tobacco (Nicotiana tabacum) cells by profiling the induced sesquiterpenoids. Suspensions of tobacco cells were treated with different concentrations (0–1,000 nM) of ergosterol and incubated for different time periods (0–24 h). Metabolites were extracted with a selective dispersive liquid-liquid micro-extraction method. Thin layer chromatography was used as a screening method for identification of sesquiterpenoids in tobacco extracts. Liquid chromatography coupled to mass spectrometry was used for quantitative and qualitative analyses. The results showed that ergosterol triggered differential changes in the metabolome of tobacco cells, leading to variation in the biosynthesis of secondary metabolites. Metabolomic analysis through principal component analysis-scores plots revealed clusters of sample replicates for ergosterol treatments of 0, 50, 150, 300 and 1,000 nM and time-dependent variation at 0, 6, 12, 18 and 24 h. Five bicyclic sesquiterpenoid phytoalexins, capsidiol, lubimin, rishitin, solavetivone and phytuberin, were identified as being ergosterol-induced, contributing to the altered metabolome.