A comprehensive analytical framework integrating liquid chromatography-tandem mass spectrometry metabolomics with chemometrics for metabolite profiling of lettuce varieties and discovery of antibacterial agents.

This study comprehensively characterized the metabolite profiles of six lettuce varieties and established the correlation between the elucidated profiles and their antivirulence effects. A total of 195 metabolites were annotated using LC-QTOF-MS/MS metabolomics assisted by molecular networking and integrated with chemometrics. Red varieties (red longifolia and lolla rosa) demonstrated higher chlorogenic and chicoric acids suggesting their antioxidant properties. In parallel, amino acids and disaccharides were enriched in romaine longifolia rationalizing its palatable taste and nutritional potential, while crispa, capitata, and lolla bionda presented a high ß-carboline alkaloid content. The antibacterial and antihemolytic potential of all varieties against methicillin-sensitive and methicillin-resistant Staphylococcus aureus was assessed and validated by prominent downregulation of α-hemolysin transcriptional levels in both strains. Moreover, correlation analysis revealed sesquiterpenes, ß-carboline alkaloids, amino acids, and oxy-fatty acids as the main bioactives. Results emphasize lettuce significance as a functional food and nutraceutical source, and highlight varieties naturally rich in antibacterial agents to adapt breeding programs.

Unveiling the functional components and antivirulence activity of mustard leaves using an LC-MS/MS, molecular networking, and multivariate data analysis integrated approach.

Plant extracts have recently received increased attention as alternative sources of antimicrobial agents in the fight against multidrug-resistant bacteria. Non-targeted metabolomics liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, molecular networking, and chemometrics were used to evaluate the metabolic profiles of red and green leaves of two Brassica juncea (L.) varieties, var. integrifolia (IR and IG) and var. rugosa (RR and RG), as well as to establish a relationship between the elucidated chemical profiles and antivirulence activity. In total, 171 metabolites from different classes were annotated and principal component analysis revealed higher levels of phenolics and glucosinolates in var. integrifolia leaves and color discrimination, whereas fatty acids were enriched in var. rugosa, particularly trihydroxy octadecadienoic acid. All extracts demonstrated significant antibacterial activity against Staphylococcus aureus and Enterococcus faecalis, presenting the IR leaves the highest antihemolytic activity against S. aureus (99 % inhibition), followed by RR (84 %), IG (82 %), and RG (37 %) leaves. Antivirulence of IR leaves was further validated by reduction in alpha-hemolysin gene transcription (∼4-fold). Using various multivariate data analyses, compounds positively correlated to bioactivity, primarily phenolic compounds, glucosinolates, and isothiocyanates, were also identified.

Bioherbicidal Activity and Metabolic Profiling of Allelopathic Metabolites of Three Cassia species using UPLC-qTOF-MS/MS and Molecular Networking.

INTRODUCTION: Compared to synthetic herbicides, natural products with allelochemical properties can inhibit weed germination, aiding agricultural output with less phytotoxic residue in water and soil. OBJECTIVES: To identify natural product extracts of three Cassia species; C. javanica, C. roxburghii, and C. fistula and to investigate the possible phytotoxic and allelopathic potential. METHODS: Allelopathic activity of three Cassia species extracts was evaluated. To further investigate the active constituents, untergated metabolomics using UPLC-qTOF-MS/MS and ion-identity molecular networking (IIMN) approach was performed to identify and determine the distribution of metabolites in different Cassia species and plant parts. RESULTS: We observed in our study that the plant extracts showed consistent allelopathic activity against seed germination (P < 0.05) and the inhibition of shoot and root development of Chenopodium murale in a dose-dependent manner. Our comprehensive study identified at least 127 compounds comprising flavonoids, coumarins, anthraquinones, phenolic acids, lipids, and fatty acid derivatives. We also report the inhibition of seed germination, shoot growth, and root growth when treated with enriched leaf and flower extracts of C. fistula, and C. javanica, and the leaf extract of C. roxburghii. CONCLUSION: The present study recommends further evaluation of Cassia extracts as a potential source of allelopathic compounds in agricultural systems.

Unveiling metabolome heterogeneity and new chemicals in 7 tomato varieties via multiplex approach of UHPLC-MS/MS, GC-MS, and UV-Vis in relation to antioxidant effects as analyzed using molecular networking and chemometrics.

Tomatoes show diverse phytochemical attributes that contribute to their nutritive and health values. This study comprehensively dissects primary and secondary metabolite profiles of seven tomato varieties. UHPLC-qTOF-MS assisted molecular networking was used to monitor 206 metabolites, 30 of which were first-time to be reported. Flavonoids, as valuable antioxidants, were enriched in light-colored tomatoes (golden sweet, sun gold, and yellow plum) versus high tomatoside A, an antihyperglycemic saponin, in cherry bomb and red plum varieties. UV-Vis analysis revealed similar results with a strong absorbance corresponding to rich phenolic content in light varieties. GC-MS unveiled monosaccharides as the main contributors to samples' segregation, found abundant in San Marzano tomato accounting for its sweet flavor. Fruits also demonstrated potential antioxidant activities in correlation to their flavonoids and phospholipids. This work provides a complete map of tomatoes' metabolome heterogeneity for future breeding programs and a comparative approach utilizing different metabolomic platforms for tomato analysis.

Iphiona mucronata (Forssk.) Asch. & Schweinf. A Comprehensive Phytochemical Study via UPLC-Q-TOF-MS in the Context of the Embryo- and Cytotoxicity Profiles.

Iphiona mucronata (Family Asteraceae) is widely distributed in the Eastern desert of Egypt. It is a promising plant material for phytochemical analysis and pharmacologic studies, and so far, its specific metabolites and biological activity have not yet been thoroughly investigated. Herein, we report on the detailed phytochemical study using UPLC-Q-TOF-MS approach. This analysis allowed the putative annotation of 48 metabolites belonging to various phytochemical classes, including mostly sesquiterpenes, flavonoids, and phenolic acids. Further, zebrafish embryotoxicity has been carried out, where 100 µg/mL extract incubated for 72 h resulted in a slow touch response of the 10 examined larvae, which might be taken as a sign of a disturbed peripheral nervous system. Results of in vitro testing indicate moderate cytotoxicity towards VERO, FaDu, and HeLa cells with CC50 values between 91.6 and 101.7 µg/mL. However, selective antineoplastic activity in RKO cells with CC50 of 54.5 µg/mL was observed. To the best of our knowledge, this is the first comprehensive profile of I. mucronata secondary metabolites that provides chemical-based evidence for its biological effects. A further investigation should be carried out to precisely define the underlying mechanisms of toxicity.

Phoenix dactylifera L. date tree pollen fertility effects on female rats in relation to its UPLC-MS profile via a biochemometric approach.

Date palm (Phoenix dactylifera L.) is among the most ancient cultivated crops, of special value owing to its fruits high nutritive and economic benefits. Asides, date palm pollen is a high energy material that has been used traditionally used for fertility enhancement. In this study, effects of date palm pollen crude extract and its fractions viz., petroleum ether, methylene chloride, ethyl acetate and n-butanol on the female reproductive system were evaluated for the first time in relation to its metabolite fingerprint. Fertility activity was evaluated in immature female rats by assessing their FSH-, LH- and estrogen- activities. To pinpoint active hormonal agents in crude pollen extract and fractions, UPLC- MS analysis was employed for metabolites profiling, and in correlation to extract/fraction bioassays using multivariate OPLS analysis. Results revealed that both polar n-butanol and non-polar petroleum ether fractions exhibited the strongest activities; with a significant increase in FSH (25.7 mIU/ml in n-butanol group), estradiol (414.7 pg/ml in petroleum ether group) and progesterone levels (122.4 pg/ml in n-butanol group). Correlation between UPLC-MS and fraction bioassays was attempted using multivariate OPLS analysis to reveal for bioactive hits in these fractions. This study provides the first report on the fertility effect of date palm pollen in female rats and in relation to its metabolite fingerprint.

Nuclear Magnetic Resonance Metabolomics Approach for the Analysis of Major Legume Sprouts Coupled to Chemometrics.

Legume sprouts are a fresh nutritive source of phytochemicals of increasing attention worldwide owing to their many health benefits. Nuclear magnetic resonance (NMR) was utilized for the metabolite fingerprinting of 4 major legume sprouts, belonging to family Fabaceae, to be exploited for quality control purposes. Thirty-two metabolites were identified belonging to different classes, i.e., fatty acids, sugars, amino acids, nucleobases, organic acids, sterols, alkaloids, and isoflavonoids. Quantitative NMR was employed for assessing the major identified metabolite levels and multivariate data analysis was utilized to assess metabolome heterogeneity among sprout samples. Isoflavones were detected exclusively in Cicer sprouts, whereas Trigonella was characterized by 4-hydroxyisoleucine. Vicia sprouts were distinguished from other legume sprouts by the presence of L-Dopa versus acetate abundance in Lens. A common alkaloid in all sprouts was trigonelline, detected at 8-25 µg/mg, suggesting its potential role in legume seeds' germination. Trigonelline was found at highest levels in Trigonella sprouts. The aromatic NMR region data (δ 11.0-5.0 ppm) provided a better classification power than the full range (δ 11.0-0.0 ppm) as sprout variations mostly originated from secondary metabolites, which can serve as chemotaxonomic markers.

Sensory Metabolite Profiling in a Date Pit Based Coffee Substitute and in Response to Roasting as Analyzed via Mass Spectrometry Based Metabolomics.

Interest in developing coffee substitutes is on the rise, to minimizing its health side effects. In the Middle East, date palm (Phoenix dactylifera L.) pits are often used as a coffee substitute post roasting. In this study, commercially-roasted date pit products, along with unroasted and home-prepared roasted date pits, were subjected to analyses for their metabolite composition, and neuropharmacological evaluation in mice. Headspace SPME-GCMS and GCMS post silylation were employed for characterizing its volatile and non-volatile metabolite profile. For comparison to roasted coffee, coffee product was also included. There is evidence that some commercial date pit products appear to contain undeclared additives. SPME headspace analysis revealed the abundance of furans, pyrans, terpenoids and sulfur compounds in roasted date pits, whereas pyrroles and caffeine were absent. GCMS-post silylation employed for primary metabolite profiling revealed fatty acids' enrichment in roasted pits versus sugars' abundance in coffee. Biological investigations affirmed that date pit showed safer margin than coffee from its LD50, albeit it exhibits no CNS stimulant properties. This study provides the first insight into the roasting impact on the date pit through its metabolome and its neuropharmacological aspects to rationalize its use as a coffee substitute.

Metabolites profiling of date palm (Phoenix dactylifera L.) commercial by-products (pits and pollen) in relation to its antioxidant effect: a multiplex approach of MS and NMR metabolomics.

INTRODUCTION: Phoenix dactylifera L. (date palm) is one of the most valued crops worldwide for its economical and nutraceutical applications of its date fruit (pericarp). Currently date pits, considered as a waste product, is employed as coffee substitute post roasting. Whereas, pollen represents another valuable by-product used as a dietary supplement. OBJECTIVES: In this study, a large-scale comparative metabolomics approach was performed for the first characterization and standardization of date palm by-products viz., date pits and pollen. Moreover, roasting impact on date pit metabolite composition was also assessed. METHODS: Metabolites profiling of pits and pollen was determined via a multiplex approach of UPLC-MS and NMR, coupled to multivariate analysis, in relation to its antioxidant activities. RESULTS: Chemical analyses led to the identification of 67 metabolites viz., phenolic acids, flavonols, fatty acids, sphingolipids, steroids and saponins of which 10 are first time to be reported. The enrichment of steroids in date pollen accounts for its fertility promoting properties, whereas date pit was found a rich source for antioxidant polyphenols using metabolomics.

Comparative metabolite profiling and fingerprinting of genus Passiflora leaves using a multiplex approach of UPLC-MS and NMR analyzed by chemometric tools.

Passiflora incarnata as well as some other Passiflora species are reported to possess anxiolytic and sedative activity and to treat various CNS disorders. The medicinal use of only a few Passiflora species has been scientifically verified. There are over 400 species in the Passiflora genus worldwide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Herein, large-scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Passiflora species leaves' chemical composition. Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) spectra of extracted components derived from 17 Passiflora accessions and from different geographical origins were analyzed using multivariate data analyses. A total of 78 metabolites were tentatively identified, that is, 20 C-flavonoids, 8 O-flavonoids, 21 C, O-flavonoids, 2 cyanogenic glycosides, and 23 fatty acid conjugates, of which several flavonoid conjugates are for the first time to be reported in Passiflora spp. To the best of our knowledge, this study provides the most complete map for secondary metabolite distribution within that genus. Major signals in (1)H-NMR and MS spectra contributing to species discrimination were assigned to those of C-flavonoids including isovitexin-2″-O-xyloside, luteolin-C-deoxyhexoside-O-hexoside, schaftoside, isovitexin, and isoorientin. P. incarnata was found most enriched in C-flavonoids, justifying its use as an official drug within that genus. Compared to NMR, LC-MS was found more effective in sample classification based on genetic and/ or geographical origin as revealed from derived multivariate data analyses. Novel insight on metabolite candidates to mediate for Passiflora CNS sedative effects is also presented.

Mechanistic evidence of Passiflora edulis (Passifloraceae) anxiolytic activity in relation to its metabolite fingerprint as revealed via LC-MS and chemometrics.

Passiflora edulis Sims F. flavicarpa along with several other plants belonging to the genus Passiflora have been reported as sedatives and for treatment or prevention of central disorders. This study evaluated the anxiolytic effect of P. edulis ethanol extract and its fractions (viz. chloroform, ethyl acetate and butanol) using the elevated plus-maze model of anxiety and assessment of γ-aminobutyric acid levels. The results revealed that butanol and chloroform extracts exhibit the strongest effect followed by ethyl acetate suggesting that a combination of different classes of metabolites is likely to mediate for P. edulis anxiolytic effect in these fractions. To further pinpoint bioactive agents in fractions, ultra-performance liquid chromatography (UPLC) coupled to high resolution qTOF-MS was used for secondary metabolite profiling. A total of 65 metabolites were characterized including O-flavonoids, C-flavonoids, cyanogenic glycosides and fatty acids. Harman type alkaloids found in P. incarnata were not detected in P. edulis ethanol extract or any of its fractions suggesting that they do not mediate for its CNS modulating effects. Multivariate data analysis (PCA) was further applied to identify metabolite markers for fractions and revealed that enrichment of C-glycoside type flavonoids in chloroform/ethyl acetate fractions versus the exclusive presence of cyanogenic glycosides in its butanol fraction.