Charting the Cannabis plant chemical space with computational metabolomics.

INTRODUCTION: The chemical classification of Cannabis is typically confined to the cannabinoid content, whilst Cannabis encompasses diverse chemical classes that vary in abundance among all its varieties. Hence, neglecting other chemical classes within Cannabis strains results in a restricted and biased comprehension of elements that may contribute to chemical intricacy and the resultant medicinal qualities of the plant. OBJECTIVES: Thus, herein, we report a computational metabolomics study to elucidate the Cannabis metabolic map beyond the cannabinoids. METHODS: Mass spectrometry-based computational tools were used to mine and evaluate the methanolic leaf and flower extracts of two Cannabis cultivars: Amnesia haze (AMNH) and Royal dutch cheese (RDC). RESULTS: The results revealed the presence of different chemical compound classes including cannabinoids, but extending it to flavonoids and phospholipids at varying distributions across the cultivar plant tissues, where the phenylpropnoid superclass was more abundant in the leaves than in the flowers. Therefore, the two cultivars were differentiated based on the overall chemical content of their plant tissues where AMNH was observed to be more dominant in the flavonoid content while RDC was more dominant in the lipid-like molecules. Additionally, in silico molecular docking studies in combination with biological assay studies indicated the potentially differing anti-cancer properties of the two cultivars resulting from the elucidated chemical profiles. CONCLUSION: These findings highlight distinctive chemical profiles beyond cannabinoids in Cannabis strains. This novel mapping of the metabolomic landscape of Cannabis provides actionable insights into plant biochemistry and justifies selecting certain varieties for medicinal use.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

An Integrated Molecular Networking and Docking Approach to Characterize the Metabolome of Helichrysum splendidum and Its Pharmaceutical Potentials.

South Africa is rich in diverse medicinal plants, and it is reported to have over 35% of the global Helichrysum species, many of which are utilized in traditional medicine. Various phytochemical studies have offered valuable insights into the chemistry of Helichrysum plants, hinting at bioactive components that define the medicinal properties of the plant. However, there are still knowledge gaps regarding the size and diversity of the Helichrysum chemical space. As such, continuous efforts are needed to comprehensively characterize the phytochemistry of Helichrysum, which will subsequently contribute to the discovery and exploration of Helichrysum-derived natural products for drug discovery. Thus, reported herein is a computational metabolomics work to comprehensively characterize the metabolic landscape of the medicinal herb Helichrysum splendidum, which is less studied. Metabolites were methanol-extracted and analyzed on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Spectral data were mined using molecular networking (MN) strategies. The results revealed that the metabolic map of H. splendidum is chemically diverse, with chemical superclasses that include organic polymers, benzenoids, lipid and lipid-like molecules, alkaloids, and derivatives, phenylpropanoids and polyketides. These results point to a vastly rich chemistry with potential bioactivities, and the latter was demonstrated through computationally assessing the binding of selected metabolites with CDK-2 and CCNB1 anti-cancer targets. Molecular docking results showed that flavonoids (luteolin, dihydroquercetin, and isorhamnetin) and terpenoids (tiliroside and silybin) interact strongly with the CDK-2 and CCNB1 targets. Thus, this work suggests that these flavonoid and terpenoid compounds from H. splendidum are potentially anti-cancer agents through their ability to interact with these proteins involved in cancer pathways and progression. As such, these actionable insights are a necessary step for further exploration and translational studies for H. splendidum-derived compounds for drug discovery.

A Relook into the Flavonoid Chemical Space of Moringa oleifera Lam. Leaves through a Combination of LC-MS and Molecular Networking.

Moringa oleifera Lam. is a functional tree that is known to produce a variety of metabolites with purported pharmacological activities. It is frequently called the "miracle tree" due to its utilization in numerous nutraceutical and pharmacological contexts. This study was aimed at studying the chemical space of M. oleifera leaf extracts through molecular networking (MN), a tool that identifies metabolites by classifying them based on their MS-based fragmentation pattern similarities and signals. In this case, a special emphasis was placed on the flavonoid composition. The MN unraveled different molecular families such as flavonoids, carboxylic acids and derivatives, lignin glycosides, fatty acyls, and macrolactams that are found within the plant. In silico annotation tools such as network annotation propagation (NAP) and DEREPLICATOR, an unsupervised substructure identification tool (MS2LDA), and MolNet enhancer were also explored to further compliment the classic molecular networking output within the Global Natural Product Social (GNPS) site. In this study, common flavonoids found within Moringa oleifera were further annotated using MS2LDA. Utilizing computational tools allowed for the discovery of a wide range of structurally diverse flavonoid molecules within M. oleifera leaf extracts. The expansion of the flavonoid chemical repertoire in this plant arises from intricate glycosylation modifications, leading to the creation of structural isomers that manifest as isobaric ions during mass spectrometry (MS) analyses.

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data.

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms' role in ecology and human health.

Rationalising the retro-Diels-Alder fragmentation pattern of viscutins using electrospray interface-tandem mass spectrometry coupled to theoretical modelling.

RATIONALE: Although mass spectrometry (MS) is a powerful tool in structural elucidation of unknown flavonoids based on their unique fragmentation patterns, proposing the correct fragmentation mechanism is still a challenge from tandem mass spectrometry data only. In recent years, computational tools such as molecular networking and MS2LDA have played a major role in the identification of structurally related compounds through an in-depth survey of their fragmentation patterns. METHODS: Therefore, in this study, three viscutin molecules in Viscum combreticola Engl. crude extracts were characterised using ultra-high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry and MS2LDA, a computational tool. Ion-trap mass spectrometry and density functional theoretical modelling were used as confirmatory tools to rationalise the unique fragmentation patterns observed for these molecules. RESULTS: Here, MS2LDA revealed the presence of a unique Mass2Motif in all the three viscutin molecules at m/z 137, which was confirmed to be a 1,3 A- RDA (retro-Diels-Alder) fragmentation product using liquid chromatography-ion-trap mass spectrometry and density functional theoretical modelling. Moreover, MS2LDA proved to be useful in differentiating this spectral feature that was specific to viscutin molecules in the presence of other isobaric ions at m/z 137 occurring in compounds in other molecular families. CONCLUSION: Therefore, the results of the current study showed that computational tools such as MS2LDA are essential in uncovering some gas-phase fragmentation reactions of molecules in MS and that theoretical modelling is a powerful tool in rationalising these reactions in metabolite identification.

Evaluation of a Chaotrope and Kosmotrope in the Multivariate Optimization of PHW-ATPE of Solasodine from Leaves of Solanum mauritianum.

A hyphenated pressurized hot water­aqueous two-phase extraction (PHW-ATPE) method was applied to extract solasodine from Solanum mauritianum (S. mauritianum). A central composite design (CCD) was applied to determine the optimal conditions for the extraction of solasodine. The parameters evaluated included the percentage concentration of salt (NaCl or Na2CO3) and temperature. The fit of the central composite design response surface model for PHW-ATPE to the data generated a model with a good quadratic fit (R2 = 0.901). The statistically significant (p < 0.05) parameters, such as the linear and quadratic effects of the concentration of salt (%) powder, had a significant impact on the extraction of solasodine. The application of multiply charged salts such as Na2CO3 (kosmotrope) was shown to be a comparably better extractant of solasodine than NaCl (chaotrope) due to the salting-out effect. The optimized conditions for extraction of solasodine with NaCl or Na2CO3 were a temperature of 80 °C at a salt concentration of 20%. The maximum extraction of solasodine was 300.79 mg kg−1 and 162.34 mg kg−1 for Na2CO3 and NaCl, respectively.

Diverse chemical modifications of the chlorogenic acid composition of Viscum combreticola Engl.: A premise for the state of readiness against excessive sunlight exposure.

Mistletoe plants that are positioned on the canopy of their hosts are more susceptible to UV radiation exposure. These aerial plants are resistant to damage by UV radiation due the presence of epidermal constituents such as the cuticle, cork layer, trichomes and antioxidant secondary metabolites. In response to the photo-oxidative stress associated with UV exposure, plants generally deploy photo-protective responsive mechanisms that involve the biosynthesis of UV absorbing phenolic compounds such as chlorogenic acids (CGAs). The hydroxycinnamic acid moieties of these CGAs are predominantly in the trans configuration, naturally. However, excessive sunlight exposure of plants containing these compounds can result in geometrical isomerisation, characterized by the formation of cis isomers. Therefore, in this study, the effect of UV light radiation on the CGA composition of Viscum combreticola Engl. (Santalacaeae) plants using an in vitro model was unravelled through UHPLC-q-TOF-MS-based metabolic profiling. Interestingly, the findings of this study revealed that this plant has a diverse chemical composition of CGAs that is characterized by epimerization, monoacylation, homodiacylation and heterodiacylation of the quinic acid (QA), thereby, contributing to the state of readiness in these plants against sunlight or UV exposure. In addition to the commonly reported cinnamoyl containing heterodiacylated CGAs, hydroxybenzoyl containing heterodiacylated CGAs were also reported in this study. Moreover, cis isomers (24 in total) of some CGAs were identified in the non-irradiated samples and the formation of these compounds has been reported to help plants in the mitigation of photo-oxidative stress. An additional 28 cis isomers of CGAs and HCA derivatives were observed in the UV-irradiated samples, hence, further increasing the complexity of the metabolome of this plant, with a total of 108 compounds identified in this study. The presence of epimers, positional and geometrical isomers of these compounds could be a biochemical strategy to maximize the chemical arsenal of this plant to withstand the photo-oxidative stress posed by UV radiation from the sunlight. Due to purported pharmacological properties associated with the identified compounds these parasitic plants can be a rich source of prospective therapeutic compounds that can be employed as drug discovery leads. Moreover, UV radiation might be essential in future to produce potent drugs since plants naturally produce these compounds in low quantities.

Parallel validation of a green-solvent extraction method and quantitative estimation of multi-mycotoxins in staple cereals using LC-MS/MS.

In this study, 15 different mycotoxins were estimated in three staple cereals from selected agro-ecological regions in Nigeria using a 'novel' green extraction method, pressurized hot water extraction (PHWE) in comparison to a conventional solvent extraction method. Discrimination of the results of PHWE and solvent extraction using principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) did not yield any differential clustering patterns. All maize samples (n = 16), 32% (n = 38) of sorghum and 35% (n = 37) of millet samples were positive for at least one of the 15 tested mycotoxins. Contamination levels for the cereals were higher in the warm humid rain forest region and gradually decreased towards the hot and arid region in the north of the country. The results demonstrate the applicability of PHWE as a possible alternative extraction method to conventional methods of extraction, which are solvent based.

Development, chemometric-assisted optimization and in-house validation of a modified pressurized hot water extraction methodology for multi-mycotoxins in maize.

Effective management of mycotoxins rely on stringent regulation and routine surveillance of food/feed commodities via efficient analysis, hence the continuous need for improved methods. The present study developed, optimized and validated a modified pressurized hot water extraction (PHWE) method for the simultaneous extraction of multi-mycotoxins from maize and subsequent quantification on LC-MS/MS. The PHWE system was modified using ethanol (EtOH) as a cosolvent, while a numerical modelling approach, the central composite design (CCD), was adopted for the optimization of the extraction conditions. Using the optimized method, it was possible to effectively extract and quantify 15 different mycotoxins from maize in a single step with satisfactory linearities (0.986-0.999), recoveries (14-124%) and other associated method validation parameters. Further efficacious application of the method to real samples re-affirmed the prospects of PHWE as a suitable, cost-effective and greener alternative to traditional methods of mycotoxin extraction.

Scribbling the Cat: A Case of the "Miracle" Plant, Moringa oleifera.

This paper reviews the properties of the most cultivated species of the Moringaceae family, Moringa oleifera Lam. The paper takes a critical look at the positive and the associated negative properties of the plant, with particular emphasis on its chemistry, selected medicinal and nutritional properties, as well as some ecological implications of the plant. The review highlights the importance of glucosinolates (GS) compounds which are relatively unique to the Moringa species family, with glucomoriginin and its acylated derivative being the most abundant. We highlight some new research findings revealing that not all M. oleifera cultivars contain an important flavonoid, rutin. The review also focuses on phenolic acids, tannin, minerals and vitamins, which are in high amounts when compared to most vegetables and fruits. Although there are numerous benefits of using M. oleifera for medicinal purposes, there are reports of contraindications. Nonetheless, we note that there are no major harmful effects of M. oleifera that have been reported by the scientific community. M. oleifera is suspected to be potentially invasive and moderately invasive in some regions of the world because of its ability to grow in a wide range of environmental conditions. However, the plant is currently classified as a low potential invasive species and thus there is a need to constantly monitor the species. Despite the numerous benefits associated with the plant, there is still a paucity of data on clinical trials proving both the positive and negative effects of the plant. We recommend further clinical trials to ascertain the properties associated with the plant, especially regarding long term use.

Numerical optimization of temperature-time degradation of multiple mycotoxins.

Mycotoxins are potent food contaminants that exert significant deleterious effects on human and animal health, yet, there is limited and often conflicting data on their thermal stability. The present study systematically investigated the thermal degradation patterns of multiple mycotoxins as a function of temperature and time, in pure form and spiked into a food matrix (maize flour), using a numerical modelling approach. Mycotoxins under investigation included aflatoxins (AFs), fumonisins (FBs), zearalenone and its analogue α and ß epimers (ZEAs), ochratoxins (OTs), T-2 toxin (T-2), alternariol monomethyl ether (AME) and sterigmatocystin (STEG). A set of statistically-designed experiments were conducted, and a second-order optimization function fitted to the experimental data. The resultant models were well fit with R2 values ranging from 0.87 to 0.99 and 0.89 to 0.99, for pure mycotoxin standards and spiked maize flour, respectively. It was also possible to statistically determine the optimum degradation conditions which were 216.57 °C/63.28 min and 210.85 °C/54.71 min for pure mycotoxins and spiked into maize flour, respectively. Our observations herein could be critical for food safety applications targeted at reducing or at best eliminating completely multi-mycotoxins in food using heat processing while limiting the destruction of food quality factors.

Efficient discrimination of natural stereoisomers of chicoric acid, an HIV-1 integrase inhibitor.

Plants from the Asteraceae family are known to contain a wide spectrum of phytochemicals with various nutraceutical properties. One important phytochemical, chicoric acid (CA), is reported to exist in plants, such as Sonchus oleraceus and Bidens pilosa, as stereoisomers. These CA molecules occur either as the naturally abundant RR-chicoric acid (RR-CA), or the less abundant RS-chicoric acid (RS-CA), also known as meso-chicoric acid. To date, little is known about the biological activity of RS-CA, but there is evidence of its anti-human immunodeficiency virus (HIV) properties. In this study, a reliable analytical method was developed to distinguish between the two stereoisomers detected in S. oleraceus and B. pilosa. For structure identification and characterization of CA molecules, liquid chromatography-mass spectrometry (LC-MS) was used in combination with ultraviolet radiation (UV)-induced geometrical isomerization, molecular dynamics (MD) simulations, and density functional theory (DFT) models. Optimized structures from DFT calculations were used for docking studies against the HIV-1 integrase enzyme. Different retention times on the reverse phase chromatograms revealed that the plants produce two different CA stereoisomers: S. oleraceus produced the RR-CA isomer, while B. pilosa produced the RS-CA isomer. DFT results demonstrated the RR-CA molecule was more stable than RS-CA due to the stabilizing force of intra-molecular hydrogen bonding. Differences in the HIV-1 integrase enzyme binding modes were observed, with the RR-CA being a more potent inhibitor than the RS-CA molecule. The results highlight the significance of plant metabolite structural complexity from both chemical and biological perspectives. Furthermore, the study demonstrates that induced-formation of geometrical isomers, in combination with the predictive ability of DFT models and the resolving power of the LC-MS, can be exploited to distinguish structurally closely related compounds, such as stereoisomers.

Distribution patterns of flavonoids from three Momordica species by ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry: a metabolomic profiling approach

ABSTRACT Plants from the Momordica genus, Curcubitaceae, are used for several purposes, especially for their nutritional and medicinal properties. Commonly known as bitter gourds, melon and cucumber, these plants are characterized by a bitter taste owing to the large content of cucurbitacin compounds. However, several reports have shown an undisputed correlation between the therapeutic activities and polyphenolic flavonoid content. Using ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry in combination with multivariate data models such as principal component analysis and hierarchical cluster analysis, three Momordica species (M. foetida Schumach., M. charantia L. and M. balsamina L.) were chemo-taxonomically grouped based on their flavonoid content. Using a conventional mass spectrometric-based approach, thirteen flavonoids were tentatively identified and the three species were found to contain different isomers of the quercetin-, kaempferol- and isorhamnetin-O-glycosides. Our results indicate that Momordica species are overall very rich sources of flavonoids but do contain different forms thereof. Furthermore, to the best of our knowledge, this is a first report on the flavonoid content of M. balsamina L.

Activation of camalexin biosynthesis in Arabidopsis thaliana in response to perception of bacterial lipopolysaccharides: a gene-to-metabolite study.

Lipopolysaccharides (LPS), as lipoglycan microbe-associated molecular pattern molecules, trigger activation of signal transduction pathways involved in defence that generate an enhanced defensive capacity in plants. The transcriptional regulation of the genes for tryptophan synthase B, TSB1, and the cytochrome P450 monooxygenases CYP79B2 and CYP71B15, involved in the camalexin biosynthetic pathway, were investigated in response to LPS treatment. GUS-reporter assays for CYP71B15 and CYP79B2 gene promoter activation were performed on transgenic plants and showed positive histochemical staining in response to LPS treatment, indicating activation of the promoters. Quantitative PCR revealed that transcripts of TSB1, CYP79B2 and CYP71B15 exhibited differential, transient up-regulation. TSB1 transcript levels were up-regulated between 6 and 9 h after LPS-induction, while CYP71B15 and CYP79B2 both exhibited maxima at 12 h. To obtain information on the gene-to-metabolite network, the effect of the transcriptome changes on the metabolome was correlated to camalexin production. Increases in camalexin concentration were quantified by ultra pressure liquid chromatography-mass spectrometry and both absorbance spectra and elemental composition confirmed its identity. The concentrations increased from 0.03 to 3.7 µg g(-1) fresh weight over a 24-h time period, thus indicating that the up-regulation of the biosynthetic pathway in response to LPS was accompanied by a time-dependent increase in camalexin concentration. Metabolomic analysis through principal component analysis-derived scores plots revealed clusters of sample replicates for 0, 6, 12, 18 and 24 h while loadings plots for LPS data identified camalexin as a biomarker that clearly demonstrated the variability between samples.