The occurrence and human health risk assessment of microcystins in diverse food matrixes during production.

Globally, the presence of cyanotoxins in water supplies and food has been widely investigated for over a decade. Cyanotoxins are harmful metabolites produced by toxic cyanobacterial genera. These metabolites belong to diverse chemical classes, with a variety of physicochemical properties, chemical structures, and toxic activities. The present study seeks to investigate the occurrence of cyanotoxins in water supplies destined for food processing and assess the human health risk from exposure to cyanotoxins. To achieve this, a simple, sensitive, and reliable analytical method was developed for the determination of microcystins (MC-RR, MC-LR, MC-YR) in process water, raw maize meal, and cooked maize (porridge) at ppb (parts per billion) levels. These compounds were extracted using Solid Phase Extraction (SPE) with optimized parameters; thereafter, Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) was used for the rapid determination of the analytes selected for the present study. The method developed was applied to samples collected from the meal grinding station located in Mawoni village in South Africa; and was able to detect and quantify all the target cyanotoxins. MC-LR, MC-YR and MC-RR were detected at concentrations ranging from 10 to 11.2 µg/L, 9.1-9.4 µg/L, and 2.3-3.5 µg/L, in water samples, respectively. However, MC-YR was not detected in ground water sample. Moreover, MC-LR, MC-YR, and MC-RR concentrations in maize and porridge samples ranged between 9.2 and 11.2, 5.5-8.6, and 6.3-9.3 µg/kg dry weight, respectively. The hazard quotient index (HQi) levels found in the present study ranged between 2.2 - 8.4 and 0.11-8.9 for adults and children, respectively, representing potential risks to human health. Findings from LC-MS/MS reveal that cyanotoxins can be transferred from water to food during food processing using cyanotoxins contaminated water. Furthermore, the methods developed can be used by environmental and health agencies to strengthen the monitoring of cyanotoxins in water and food.

Defence-related metabolic changes in wheat (Triticum aestivum L.) seedlings in response to infection by Puccinia graminis f. sp. tritici.

Stem rust caused by the pathogen Puccinia graminis f. sp. tritici is a destructive fungal disease-causing major grain yield losses in wheat. Therefore, understanding the plant defence regulation and function in response to the pathogen attack is required. As such, an untargeted LC-MS-based metabolomics approach was employed as a tool to dissect and understand the biochemical responses of Koonap (resistant) and Morocco (susceptible) wheat varieties infected with two different races of P. graminis (2SA88 [TTKSF] and 2SA107 [PTKST]). Data was generated from the infected and non-infected control plants harvested at 14- and 21- days post-inoculation (dpi), with 3 biological replicates per sample under a controlled environment. Chemo-metric tools such as principal component analysis (PCA), orthogonal projection to latent structures-discriminant analysis (OPLS-DA) were used to highlight the metabolic changes using LC-MS data of the methanolic extracts generated from the two wheat varieties. Molecular networking in Global Natural Product Social (GNPS) was further used to analyse biological networks between the perturbed metabolites. PCA and OPLS-DA analysis showed cluster separations between the varieties, infection races and the time-points. Distinct biochemical changes were also observed between the races and time-points. Metabolites were identified and classified using base peak intensities (BPI) and single ion extracted chromatograms from samples, and the most affected metabolites included flavonoids, carboxylic acids and alkaloids. Network analysis also showed high expression of metabolites from thiamine and glyoxylate, such as flavonoid glycosides, suggesting multi-faceted defence response strategy by understudied wheat varieties towards P. graminis pathogen infection. Overall, the study provided the insights of the biochemical changes in the expression of wheat metabolites in response to stem rust infection.

Metabolome profile variations in common bean (Phaseolus vulgaris L.) resistant and susceptible genotypes incited by rust (Uromyces appendiculatus).

The causal agent of rust, Uromyces appendiculatus is a major constraint for common bean (Phaseolus vulgaris) production. This pathogen causes substantial yield losses in many common bean production areas worldwide. U. appendiculatus is widely distributed and although there have been numerous breakthroughs in breeding for resistance, its ability to mutate and evolve still poses a major threat to common bean production. An understanding of plant phytochemical properties can aid in accelerating breeding for rust resistance. In this study, metabolome profiles of two common bean genotypes Teebus-RR-1 (resistant) and Golden Gate Wax (susceptible) were investigated for their response to U. appendiculatus races (1 and 3) at 14- and 21-days post-infection (dpi) using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-qTOF-MS). Non-targeted data analysis revealed 71 known metabolites that were putatively annotated, and a total of 33 were statistically significant. Key metabolites including flavonoids, terpenoids, alkaloids and lipids were found to be incited by rust infections in both genotypes. Resistant genotype as compared to the susceptible genotype differentially enriched metabolites including aconifine, D-sucrose, galangin, rutarin and others as a defence mechanism against the rust pathogen. The results suggest that timely response to pathogen attack by signalling the production of specific metabolites can be used as a strategy to understand plant defence. This is the first study to illustrate the utilization of metabolomics to understand the interaction of common bean with rust.

Mass spectrometry DDA parameters and global coverage of the metabolome: Spectral molecular networks of momordica cardiospermoides plants.

INTRODUCTION: Molecular networking (MN) has emerged as a key strategy to organize and annotate untargeted tandem mass spectrometry (MS/MS) data generated using either data independent- or dependent acquisition (DIA or DDA). The latter presents a time-efficient approach where full scan (MS1) and MS2 spectra are obtained with shorter cycle times. However, there are limitations related to DDA parameters, some of which are (i) intensity threshold and (ii) collision energy. The former determines ion prioritization for fragmentation, and the latter defines the fragmentation of selected ions. These DDA parameters inevitably determine the coverage and quality of spectral data, which would affect the outputs of MN methods. OBJECTIVES: This study assessed the extent to which the quality of the tandem spectral data relates to MN topology and subsequent implications in the annotation of metabolites and chemical classification relative to the different DDA parameters employed. METHODS: Herein, characterising the metabolome of Momordica cardiospermoides plants, we employ classical MN performance indicators to investigate the effects of collision energies and intensity thresholds on the topology of generated MN and propagated annotations. RESULTS: We demonstrated that the lowest predefined intensity thresholds and collision energies result in comprehensive molecular networks. Comparatively, higher intensity thresholds and collision energies resulted in fewer MS2 spectra acquisition, subsequently fewer nodes, and a limited exploration of the metabolome through MN. CONCLUSION: Contributing to ongoing efforts and conversations on improving DDA strategies, this study proposes a framework in which multiple DDA parameters are utilized to increase the coverage of ions acquired and improve the global coverage of MN, propagated annotations, and the chemical classification performed.

Metabolomics and Molecular Networking to Characterize the Chemical Space of Four Momordica Plant Species.

Momordica plant species (Cucurbitaceae), have been used for centuries in traditional medicine and for nutritional purposes. Plants from this family are thus claimed to be phytochemically rich, representing an inexhaustible source of natural products. However, the chemical space of these Momordica species has not yet been fully decoded, and due to the inherent complexity of plant metabolomes, the characterization of the Momordica phytochemistry remains challenging. Thus, in this study we propose the use of molecular networking to unravel the molecular families within the metabolomes of four Momordica species (M. cardiospermoides, M. balsamina, M. charantia and M. foetida) and highlight the relevance of molecular networking in exploring the chemotaxonomy of these plants. In silico annotation tools (Network Annotation Propagation and DEREPLICATOR) and an unsupervised substructure identification tool (MS2LDA) were also explored to complement the classical molecular networking output and integration using MolNetEnhancer within GNPS. This allowed for the visualisation of chemical classes and the variety of substructures within the molecular families. The use of computational tools in this study highlighted various classes of metabolites, such as a wide range of flavonoids, terpenoids and lipids. Herein, these species are revealed to be phytochemically rich plants consisting of many biologically active metabolites differentially distributed within the different species, with the metabolome of M. cardiospermoides dereplicated in this paper for the first time.

Application of Plant Growth Regulators Modulates the Profile of Chlorogenic Acids in Cultured Bidens pilosa Cells.

Plant cell culture offers an alternative to whole plants for the production of biologically important specialised metabolites. In cultured plant cells, manipulation by auxin and cytokinin plant growth regulators (PGRs) may lead to in vitro organogenesis and metabolome changes. In this study, six different combination ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (BAP) were investigated with the aim to induce indirect organogenesis from Bidens pilosa callus and to investigate the associated induced changes in the metabolomes of these calli. Phenotypic appearance of the calli and total phenolic contents of hydromethanolic extracts indicated underlying biochemical differences that were investigated using untargeted metabolomics, based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS), combined with multivariate data analysis. The concentration and combination ratios of PGRs were shown to induce differential metabolic responses and, thus, distinct metabolomic profiles, dominated by chlorogenic acids consisting of caffeoyl- and feruloyl-derivatives of quinic acid. Although organogenesis was not achieved, the results demonstrate that exogenous application PGRs can be used to manipulate the metabolome of B. pilosa for in vitro production of specialised metabolites with purported pharmacological properties.

Proportional coexistence of okanin chalcone glycoside and okanin flavanone glycoside in Bidens pilosa leaves and theoretical investigation on the antioxidant properties of their aglycones.

Bidens pilosa plant has been shown to produce okanin flavanone glycoside and its chalcone derivative. In most other plants, due to chalcone isomerase enzyme, the flavanone tends to exist in higher proportions than their chalcone precursors. Herein we have utilized liquid chromatography-mass spectrometry approach and shown that within the leaves of Bidens pilosa plant the two okanin glycosides exist in unusual equal proportional distribution, which indicates that Bidens pilosa plant is an alternative rich source of these highly sought-after antioxidant molecules. The aglycone okanin chalcone (ONC) and okanin flavanone (ONF) have experimentally been shown to exhibit antioxidant activity. However, experimental findings have not conclusively determined which of the two compounds is a more potent antiradical than the other. Herein, the density functional theory (DFT) method is utilized to establish, from structural and thermodynamic energetic considerations, the preferred antioxidant molecule between the two aglycone okanins. A theoretical study on the antioxidant properties of ONC and ONF has been performed by considering their radical scavenging and metal cation (Mn+, where M = Cu(II) or Fe (III)) chelation ability. The study has been performed using B3LYP/6-31 + G(d,p) method. In the case of the metal chelation mechanism, the LANL2DZ pseudo-potential was selected to describe the selected Mn+ cations. The results of the study suggest that ONC is a better radical scavenger than ONF because of the extended electron delocalization on its neutral radical, which is due to the presence of conjugation within the ONC neutral radical after hydrogen atom abstraction. In the metal chelation mechanism, it is noted that the binding energies depend on the media, the nature of the ligand and the cation and the cation coordination site on the ligand. The charge and the spin density on Mn+ decrease on coordination to the ligand. The ability of the ligands to reduce Mn+ cations, coupled with the strong Mn+ binding properties, has significant implication on the antioxidant ability of both okanins. However, since ONC⋅⋅⋅M+n interaction results in higher binding energy than ONF⋅⋅⋅M+n interaction, the implication is that ONC is a preferred free metal ion chelator than ONF.

Profiling of Altered Metabolomic States in Bidens pilosa Leaves in Response to Treatment by Methyl Jasmonate and Methyl Salicylate.

Bidens pilosa (Asteraceae) is an edible medicinal plant with many bioactivities reported to have a health-beneficial role in controling various diseases. Though B. pilosa contain a diverse array of natural products, these are produced in relatively low concentrations. A possible way to enhance secondary metabolite production can be through the use of elicitors. Here, the effects of exogenous treatments with two signal molecules-methyl jasmonate (MeJA) and methyl salicylate (MeSA)-on the metabolomic profiles of B. pilosa leaves were investigated. Plants were treated with 0.5 mM of MeJA or MeSA and harvested at 12 h and 24 h. Metabolites were extracted with methanol and separated on an ultra-high performance liquid chromatography system hyphenated to quadrupole time-of-flight mass spectrometry detection. Data was subjected to multivariate statistical analysis and modeling for annotation of metabolites. Hydroxycinnamic acid (HCA) derivatives, such as caffeoylquinic acids (CQAs), tartaric acid esters (chicoric acid and caftaric acid), chalcones, and flavonoids were identified as differentially regulated. The altered metabolomes in response to MeSA and MeJA overlapped to a certain extent, suggestive of a cross-talk between signaling and metabolic pathway activation. Moreover, the perturbation of isomeric molecules, especially the cis geometrical isomers of HCA derivatives by both treatments, further point to the biological significance of these molecules during physiological responses to stress. The results highlight the possibility of using phytohormones to enhance the accumulation of bioactive secondary metabolites in this plant.

Application of an agitation-assisted dispersed solvent microextraction for analysis of naphthalene and its derivatives from aqueous matrices.

Agitation-assisted dispersive liquid-liquid extraction without a dispersing solvent is lately receiving considerable attention owing to the low to no solvent loss relative to its predecessor, which suffers severe extracting solvent loss. Herein, we report the application of a simple agitation-assisted dispersive liquid-liquid microextraction method, without a disperser solvent, for the extraction of naphthalene and its derivatives from aqueous solutions. Under the optimised conditions, namely, 25 µL 3:1 mixture of dichloroethane and ethylacetate with 20 s agitation, in 2-mL aqueous solutions containing 10% NaCl, the method demonstrated acceptable figures of merit: linearity-R2 ≥ 0.9914 in the concentration range 0.5-50 ng/mL, repeatability (%RSD ≤ 12.9 for n = 15) and limits of detection (0.034-0.081 ng/mL). The recoveries obtained from the spiked dam water sample were also satisfactory (94-103%). These parameters are comparable with those reported in literature, especially for dispersive liquid-liquid microextraction techniques albeit for different analytes. Despite only naphthol being detected in one of the three sampled sites, the method shows considerable promise for routine monitoring of river and dam water quality subject to accuracy validation using certified reference materials.

Mass Spectrometric Approaches to Study the Metabolism of Jasmonates: Biotransformation of Exogenously Supplemented Methyl Jasmonate by Cell Suspension Cultures of Moringa oleifera.

Jasmonic acid (JA) and derivatives play a crucial role in plant adaptation to environmental stress. The exogenous application of methyl jasmonate (MeJA) results in activation of stress-related genes and subsequent production of secondary metabolites. This implies the biotransformation of the hormone into a more active form. In this study, Moringa oleifera cell suspension cultures were treated with 100 µM MeJA. Methanolic cell extracts were analyzed with reverse phase ultrahigh performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight high definition mass spectrometer (QTOF-HDMS, with MSE data acquisition). Using a targeted approach for jasmonate profiling, extracted ion chromatograms were generated. MS fragmentation patterns were subsequently derived and molecular formulae calculated from the MS data of each ion. Furthermore, triple quadrupole (QqQ) MS, operated in selected reaction monitoring (SRM) mode, was employed to target masses of interest. In addition to MeJA and JA, three jasmonoyl-amino acids were annotated: jasmonoyl-valine (JA-Val), jasmonoyl-isoleucine/leucine (JA-Ile/Leu), and jasmonoyl-phenylalanine (JA-Phe), based on characteristic precursor and product ions. Furthermore, JA conjugated to a hexose was observed, as well as hydroxylated and carboxylated derivatives of JA-amino acid conjugates. The data point to active metabolism of the externally added MeJA by the M. oleifera cells through biotransformation and bioconversion reactions that can be investigated in depth using advanced mass spectrometric analyses.

Deciphering the Resistance Mechanism of Tomato Plants Against Whitefly-Mediated Tomato Curly Stunt Virus Infection through Ultra-High-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS)-Based Metabolomics Approaches.

Begomoviruses, such as the Tomato curly stunt virus (ToCSV), pose serious economic consequences due to severe crop losses. Therefore, the development and screening of possible resistance markers is imperative. While some tomato cultivars exhibit differential resistance to different begomovirus species, in most cases, the mechanism of resistance is not fully understood. In this study, the response of two near-isogenic lines of tomato (Solanum lycopersicum), differing in resistance against whitefly-mediated ToCSV infection were investigated using untargeted ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS)-based metabolomics. The responses of the two lines were deciphered using multivariate statistics models. Principal component analysis (PCA) scores plots from various time intervals revealed that the resistant line responded more rapidly with changes to the metabolome than the susceptible counterpart. Moreover, the metabolic reprogramming of chemically diverse metabolites that span a range of metabolic pathways was associated with the defence response. Biomarkers primarily included hydroxycinnamic acids conjugated to quinic acid, galactaric acid, and glucose. Minor constituents included benzenoids, flavonoids, and steroidal glycoalkaloids. Interestingly, when reduced to the level of metabolites, the phytochemistry of the infected plants' responses was very similar. However, the resistant phenotype was strongly associated with the hydroxycinnamic acid derivatives deployed in response to infection. In addition, the resistant line was able to mount a stronger and quicker response.

Comparison of Soxhlet and reflux techniques for extraction and characterisation of potential endocrine-disrupting compounds from solid waste dumpsite soil.

Landfill leachate contains a myriad of hazardous chemicals; as such, they should always be planned and constructed following approved guidelines. A sample of soil collected from the old quarry designated as the official solid waste disposal site in Maseru, the capital city of Lesotho, was exposed to two extraction techniques, namely Soxhlet and reflux extractions, for characterisation of the potential endocrine-disrupting chemicals in the leachate. Principal component analysis was used to compare the extractability of these chemicals between the two methods, and it revealed that phthalates extract better in Soxhlet than in reflux extraction. Other compounds do not show as much difference. Qualitative analysis of the extracts revealed several compounds of environmental health interest, namely anthracene, bis-di-ethylhexyl-phthalates and di-tert-butylphenol. A review of the literature on some of the annotated compounds was explored for the likely sources thereof. It was discovered that most of the compounds that were identified have plastic origins and are listed as potential endocrine disruptors. The identified compounds were similar to those reported elsewhere in the literature.

Untargeted Metabolomics Reveal Defensome-Related Metabolic Reprogramming in Sorghum bicolor against Infection by Burkholderia andropogonis.

Burkholderia andropogonis is the causal agent of bacterial leaf stripe, one of the three major bacterial diseases affecting Sorghum bicolor. However, the biochemical aspects of the pathophysiological host responses are not well understood. An untargeted metabolomics approach was designed to understand molecular mechanisms underlying S. bicolor⁻B. andropogonis interactions. At the 4-leaf stage, two sorghum cultivars (NS 5511 and NS 5655) differing in disease tolerance, were infected with B. andropogonis and the metabolic changes monitored over time. The NS 5511 cultivar displayed delayed signs of wilting and lesion progression compared to the NS 5655 cultivar, indicative of enhanced resistance. The metabolomics results identified statistically significant metabolites as biomarkers associated with the sorghum defence. These include the phytohormones salicylic acid, jasmonic acid, and zeatin. Moreover, metabolic reprogramming in an array of chemically diverse metabolites that span a wide range of metabolic pathways was associated with the defence response. Signatory biomarkers included aromatic amino acids, shikimic acid, metabolites from the phenylpropanoid and flavonoid pathways, as well as fatty acids. Enhanced synthesis and accumulation of apigenin and derivatives thereof was a prominent feature of the altered metabolomes. The analyses revealed an intricate and dynamic network of the sorghum defence arsenal towards B. andropogonis in establishing an enhanced defensive capacity in support of resistance and disease suppression. The results pave the way for future analysis of the biosynthesis of signatory biomarkers and regulation of relevant metabolic pathways in sorghum.

Rapid Screening of Volatile Organic Compounds from Aframomum danielli Seeds Using Headspace Solid Phase Microextraction Coupled to Gas Chromatography Mass Spectrometry.

Volatile organic compounds (VOCs) derived from plants have been used in the fragrance industry since time immemorial. Herein we report on the rapid screening of VOCs from seeds of ripe Aframomum danielli (family, Zingiberaceae) using a polydimethylsiloxane fibre headspace solid phase microextraction coupled to a gas chromatography mass spectrometry (SPME-GC/MS) instrument. Portions of 0.25, 0.35, and 0.50 g of ground sample were weighed and extraction of volatile organic compounds (VOCs) was achieved using a 100 µm polydimethylsiloxane solid phase microextraction (PDMS SPME) fibre, with the equilibrium time of 40 minutes and extraction temperature of 50°C; the following compounds with their respective relative abundances were obtained as the top ten most abundant and annotated ones using NIST, Wiley, and Fragrances Libraries: eucalyptol (58%); ß-pinene (22%); α-pinene (7.5%); α-terpineol (4%), α-terpinyl acetate (2%); α-bergamotene (1%); pinocarveol (0.39%); α-copaene (0.35%); caryophyllene (0.34); and ß-bisabolene (0.31%). These compounds have been reported elsewhere in the literature and listed in the Fragrances Library, incorporated into the Saturn QP2020 GCMS Solution® software used for their analysis.

The Chemistry of Plant-Microbe Interactions in the Rhizosphere and the Potential for Metabolomics to Reveal Signaling Related to Defense Priming and Induced Systemic Resistance.

Plant roots communicate with microbes in a sophisticated manner through chemical communication within the rhizosphere, thereby leading to biofilm formation of beneficial microbes and, in the case of plant growth-promoting rhizomicrobes/-bacteria (PGPR), resulting in priming of defense, or induced resistance in the plant host. The knowledge of plant-plant and plant-microbe interactions have been greatly extended over recent years; however, the chemical communication leading to priming is far from being well understood. Furthermore, linkage between below- and above-ground plant physiological processes adds to the complexity. In metabolomics studies, the main aim is to profile and annotate all exo- and endo-metabolites in a biological system that drive and participate in physiological processes. Recent advances in this field has enabled researchers to analyze 100s of compounds in one sample over a short time period. Here, from a metabolomics viewpoint, we review the interactions within the rhizosphere and subsequent above-ground 'signalomics', and emphasize the contributions that mass spectrometric-based metabolomic approaches can bring to the study of plant-beneficial - and priming events.

Leishmanicidal activity of the root bark of Erythrophleum Ivorense (Fabaceae) and identification of some of its compounds by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS).

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis is one of the neglected tropical disease caused by a protozoan of the genus Leishmania transmitted by sandflies. High cost and lack of oral formulation of existing drugs, rapid developments of resistance by the parasite coupled with serious side effects require new treatments to augment or replace currently available therapies. The major merits of herbal medicine seem to demonstrate perceived efficacy, low incidence of serious adverse effects and low cost. Erythrophleum plants possess beneficial biological properties and, as such, characterization of the bioactive components of these plants is imperative. Previous work has shown an overwhelming presence of cassaine alkaloids in these plants. However, amongst these plants, the African based specie (Erythrophleum ivorense) is the least studied. OBJECTIVE: In the current study, the in vitro anti-leishmanial activity of the crude extract, its fractions and isolated compounds were evaluated using direct counting assay of promastigotes of Leishmania donovani using amphotericin B as positive control. MATERIALS AND METHODS: The anti-leishmanial activity of E. ivorense extract was evaluated in vitro against the promastigote forms of Leishmania Donovani using a direct counting assay based on growth inhibition. Different crude extracts from ethyl acetate, pet-ether, and methanol as well as pure isolated compounds of E. ivorense: Erythroivorensin, Eriodictyol and Betulinic acid were screened. To know the possible components of the active methanolic extract, attempt was made to elucidate the extract using ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS/MS). RESULTS: This afforded a weak pet-ether fraction, a moderately active ethyl acetate fraction and a significantly active methanol fraction (IC50 = 2.97µg/mL) compared to Amphotericin B (IC50 = 2.40±0.67µg/mL). The novel diterpene erythroivorensin, betulinic acid and the flavanone Eriodictyol, from the ethyl acetate fraction, showed weak activity. UPLC-QTOF-MS/MS was used to identify the cassaine diterpenoids from the active methanol fraction. Here, 10 compounds of this type were putatively identified from the ethanol crude extract. CONCLUSION: The fragmentation mechanism of these metabolites is also proposed and are expected to serve as reference template for identification of these and related compounds in future. The presence of these compounds is an indication that they are an inherited and evolutionary component of plants belonging to the Erythrophleum genus. Our results further present another dimension where these compounds and their relative abundances can be used as chemo-taxonomical bio-markers of the genus. The present study also successfully demonstrated/re-affirmed the use of UPLC-QTOF-MS/MS as a robust technique for the characterization of natural products.

Highlighting mass spectrometric fragmentation differences and similarities between hydroxycinnamoyl-quinic acids and hydroxycinnamoyl-isocitric acids.

BACKGROUND: Plants contain a myriad of metabolites which exhibit diverse biological activities. However, in-depth analyses of these natural products with current analytical platforms remains an undisputed challenge due to the multidimensional chemo-diversity of these molecules, amplified by both isomerization and conjugation. In this study, we looked at molecules such as hydroxyl-cinnamic acids (HCAs), which are known to exist as positional and geometrical isomers conjugated to different organic acids namely quinic- and isocitric acid. OBJECTIVE: The study aimed at providing a more defined distinction between HCA conjugates from Amaranthus viridis and Moringa oleifera, using mass spectrometry (MS) approaches. METHODS: Here, we used a UHPLC-MS/MS targeted approach to analyze isobaric HCA conjugates extracted from the aforementioned plants. RESULTS: Mass spectrometry results showed similar precursor ions and fragmentation pattern; however, distinct differences were seen with ions at m/z 155 and m/z 111 which are associated with isocitric acid conjugates. CONCLUSION: Our results highlight subtle differences between these two classes of compounds based on the MS fingerprints, enabling confidence differentiation of the compounds. Thus, these findings provide a template reference for accurate and confident annotation of such compounds in other plants.

Gamma radiation treatment activates glucomoringin synthesis in Moringa oleifera

Abstract Plants are a very rich source of pharmacologically relevant metabolites. However, the relative concentrations of these compounds are subject to the genetic make-up, the physiological state of the plant as well as environmental effects. Recently, metabolic perturbations through the use of abiotic stressors have proven to be a valuable strategy for increasing the levels of these compounds. Oxidative stress-associated stressors, including ionizing radiation, have also been reported to induce metabolites with various biological activities in plants. Hence, the aim of the current study was to investigate the effect of gamma radiation on the induction of purported anti-cancerous metabolites, glucomoringin and its derivatives, in Moringa oleifera Lam., Moringaceae. Here, an UHPLC-qTOF-MS-based targeted metabolic fingerprinting approach was used to evaluate the effect of gamma radiation treatment on the afore-mentioned health-beneficial secondary metabolites of M. oleifera. Following radiation, an increase in glucomoringin and three acylated derivatives was noted. As such, these molecules can be regarded as components of the inducible defense mechanism of M. oleifera as opposed to being constitutive components as it has previously been assumed. This might be an indication of a possible, yet unexplored role of moringin against the effects of oxidative stress in M. oleifera plants. The results also suggest that plants undergoing photo-oxidative stress could accumulate higher amounts of glucomoringin and related molecules.

A Metabolomics-Guided Exploration of the Phytochemical Constituents of Vernonia fastigiata with the Aid of Pressurized Hot Water Extraction and Liquid Chromatography-Mass Spectrometry.

Vernonia fastigiata is a multi-purpose nutraceutical plant with interesting biological properties. However, very little is known about its phytochemical composition and, thus the need for its phytochemical characterization. In the current study, an environmentally friendly method, pressurized hot water extraction (PHWE), was used to extract metabolites from the leaves of V. fastigiata at various temperatures (50 °C, 100 °C, 150 °C and 200 °C). Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) analysis in combination with chemometric methods, particularly principal component analysis (PCA) and liquid/gas chromatography mass spectrometry (XCMS) cloud plots, were used to descriptively visualize the data and identify significant metabolites extracted at various temperatures. A total of 25 different metabolites, including hydroxycinnamic acid derivatives, clovamide, deoxy-clovamide and flavonoids, were noted for the first time in this plant. Overall, an increase in extraction temperature resulted in an increase in metabolite extraction during PHWE. This study is the first scientific report on the phytochemical composition of V. fastigiata, providing insight into the components of the chemo-diversity of this important plant.

Structural Elucidation of cis/trans Dicaffeoylquinic Acid Photoisomerization Using Ion Mobility Spectrometry-Mass Spectrometry.

Due to the recently uncovered health benefits and anti-HIV activities of dicaffeoylquinic acids (diCQAs), understanding their structures and functions is of great interest for drug discovery efforts. DiCQAs are analytically challenging to identify and quantify since they commonly exist as a diverse mixture of positional and geometric (cis/trans) isomers. In this work, we utilized ion mobility spectrometry coupled with mass spectrometry to separate the various isomers before and after UV irradiation. The experimental collision cross sections were then compared with theoretical structures to differentiate and identify the diCQA isomers. Our analyses found that naturally the diCQAs existed predominantly as trans/trans isomers, but after 3 h of UV irradiation, cis/cis, cis/trans, trans/cis, and trans/trans isomers were all present in the mixture. This is the first report of successful differentiation of cis/trans diCQA isomers individually, which shows the great promise of IMS coupled with theoretical calculations for determining the structure and activity relationships of different isomers in drug discovery studies.