Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Selective Chlorophyll Removal Method to "Degreen" Botanical Extracts.

Chlorophylls are present in all extracts from the aerial parts of green plant materials. Chlorophylls may act as in vitro bioassay nuisance compounds, possibly preventing the reproducibility and accurate measurement of readouts due to their UV/vis absorbance, fluorescence properties, and tendency to precipitate in aqueous media. Despite the diversity of methods used traditionally to remove chlorophylls, details about their mode of operation, specificity, and reproducibility are scarce. Herein, we report a selective and efficient 45 min liquid-liquid/countercurrent chlorophyll cleanup method using Centrifugal Partition Chromatography (CPC) with a solvent system composed of hexanes-EtOAc-MeOH-water (5:5:5:5, v/v) in elution-extrusion mode. The broader utility of the method was assessed with four different extracts prepared from three well-characterized plant materials: Epimedium sagittatum (leaves), Senna alexandrina (leaves), and Trifolium pratense (aerial parts). The reproducibility of the method, the selectivity of the chlorophyll removal, as well as the preservation of the phytochemical integrity of the resulting chlorophyll-free ("degreened") extracts were evaluated using HPTLC, UHPLC-UV, 1H NMR spectroscopy, and LC-MS as orthogonal phytochemical methods. The cleanup process adequately preserves the metabolomic diversity as well as the integrity of the original extracts. This method was found to be sufficiently rapid for the "degreening" of botanical extracts in higher-throughput sample preparation for further biological screening.

The DESIGNER Approach Helps Decipher the Hypoglycemic Bioactive Principles of Artemisia dracunculus (Russian Tarragon).

Complementing classical drug discovery, phytochemicals act on multiple pharmacological targets, especially in botanical extracts, where they form complex bioactive mixtures. The reductionist approach used in bioactivity-guided fractionation to identify single bioactive phytochemicals is inadequate for capturing the full therapeutic potential of the (bio)chemical interactions present in such complex mixtures. This study used a DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources) approach to selectively remove the known bioactives, 4'-O-methyldavidigenin (1; 4,2'-dihydroxy-4'-methoxydihydrochalcone, syn. DMC-1) and its isomer 4-O-methyldavidigenin (2; syn. DMC-2), from the mixture of phytochemicals in an ethanol extract from Artemisia dracunculus to determine to what degree the more abundant 2 accounts for the established antidiabetic effect of the A. dracunculus extract. Using an otherwise chemically intact "knock-out extract" depleted in 2 and its regioisomer, 1, in vitro and in vivo outcomes confirmed that 2 (and likely 1) acts as major bioactive(s) that enhance(s) insulin signaling in skeletal muscle, but also revealed that 2 does not account for the breadth of detectable biological activity of the extract. This is the first report of generating, at a sufficiently large preparative scale, a "knock-out extract" used as a pharmacological tool for in vitro and in vivo studies to dissect the biological impact of a designated bioactive in a complex phytochemical mixture.

Estrogen Receptor (ER) Subtype Selectivity Identifies 8-Prenylapigenin as an ERß Agonist from Glycyrrhiza inflata and Highlights the Importance of Chemical and Biological Authentication.

Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERß activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/ß41 breast cancer cells (ERß). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERß agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.

Evolution of Quantitative Measures in NMR: Quantum Mechanical qHNMR Advances Chemical Standardization of a Red Clover (Trifolium pratense) Extract.

Chemical standardization, along with morphological and DNA analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. Achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. Employing quantitative 1H NMR (qHNMR), this study determined the total isoflavone content (TIfCo; 34.5-36.5% w/w) via multimarker standardization and assessed the stability of a 10-year-old isoflavone-enriched red clover extract (RCE). Eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with LC-based standardization. Two advanced quantitative measures in qHNMR were applied to derive quantities from complex and/or overlapping resonances: a quantum mechanical (QM) method (QM-qHNMR) that employs 1H iterative full spin analysis, and a non-QM method that uses linear peak fitting algorithms (PF-qHNMR). A 10 min UHPLC-UV method provided auxiliary orthogonal quantitation. This is the first systematic evaluation of QM and non-QM deconvolution as qHNMR quantitation measures. It demonstrates that QM-qHNMR can account successfully for the complexity of 1H NMR spectra of individual analytes and how QM-qHNMR can be built for mixtures such as botanical extracts. The contents of the main bioactive markers were in good agreement with earlier HPLC-UV results, demonstrating the chemical stability of the RCE. QM-qHNMR advances chemical standardization by its inherent QM accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.

Structural Sequencing of Oligopeptides Aided by 1H Iterative Full-Spin Analysis.

This report describes an approach using 1H NMR iterative full-spin analysis (HiFSA) to extract definitive structural information on unknown peptides from 1D 1H NMR data. By comparing the experimental data and HiFSA fingerprint of a known analogue, it is possible to isolate the characteristic 1H subspectrum of the different amino acids and, thus, elucidate the structure of the peptide. To illustrate this methodology, a comprehensive analysis of five new anti-Mycobacterium tuberculosis peptides (2-6), all analogues of ecumicin (1), was carried out. The method was validated by demonstrating congruence of the HiFSA-based structures with all available data, including MS and 2D NMR. The highly reproducible HiFSA fingerprints of the new ∼1600 amu peptides were generated in this process. Besides oligo-peptides, the HiFSA sequencing approach could be extended to all oligomeric compounds consisting of chains of monomers lacking H-H spin-spin coupling across the moieties. HiFSA sequencing is capable of differentiating complex oligomers that exhibit minor structural differences such as shifted hydoxyl or methyl groups. Because it employs the basic and most sensitive 1D 1H NMR experiment, HiFSA sequencing enables the exploration of peptide analogues up to at least 2000 amu, even with basic contemporary spectrometers and when only sub-milligram amounts of isolates are available.

Dissemination of original NMR data enhances reproducibility and integrity in chemical research.

The notion of data transparency is gaining a strong awareness among the scientific community. The availability of raw data is actually regarded as a fundamental way to advance science by promoting both integrity and reproducibility of research outcomes. Particularly, in the field of natural product and chemical research, NMR spectroscopy is a fundamental tool for structural elucidation and quantification (qNMR). As such, the accessibility of original NMR data, i.e., Free Induction Decays (FIDs), fosters transparency in chemical research and optimizes both peer review and reproducibility of reports by offering the fundamental tools to perform efficient structural verification. Although original NMR data are known to contain a wealth of information, they are rarely accessible along with published data. This viewpoint discusses the relevance of the availability of original NMR data as part of good research practices not only to promote structural correctness, but also to enhance traceability and reproducibility of both chemical and biological results.

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 µg/mL), and LigC (1 µM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 µM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 µM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 µg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

Induction of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) by Glycyrrhiza Species Used for Women's Health: Differential Effects of the Michael Acceptors Isoliquiritigenin and Licochalcone A.

For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme, NAD(P)H: quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human hepatoma (HepG2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in nontumorigenic breast epithelial MCF-10A cells. In female Sprague-Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 in vivo most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability in vivo. These data show the chemopreventive potential of licorice species in vitro could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the in vivo effects in the rat model after four-day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible.

Digital NMR profiles as building blocks: assembling ¹H fingerprints of steviol glycosides.

This report describes a fragment-based approach to the examination of congeneric organic compounds by NMR spectroscopy. The method combines the classic interpretation of 1D- and 2D-NMR data sets with contemporary computer-assisted NMR analysis. Characteristic NMR profiles of key structural motifs were generated by (1)H iterative full spin analysis and then joined together as building blocks to recreate the (1)H NMR spectra of increasingly complex molecules. To illustrate the methodology described, a comprehensive analysis of steviol (1), seven steviol glycosides (2-8) and two structurally related isosteviol compounds (9, 10) was carried out. The study also assessed the potential impact of this method on relevant aspects of natural product research including structural verification, chemical dereplication, and mixture analysis.

Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals.

Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on (1)H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements.

K-targeted metabolomic analysis extends chemical subtraction to DESIGNER extracts: selective depletion of extracts of hops (Humulus lupulus).

This study introduces a flexible and compound targeted approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources, generating DESIGNER extracts, by means of chemical subtraction or augmentation of metabolites. Targeting metabolites based on their liquid-liquid partition coefficients (K values), K targeting uses countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture, according to the following equation: DESIGNER extract = total extract ± target compound(s). Expanding the scope of the recently reported depletion of extracts by immunoaffinity or solid phase liquid chromatography, the present approach allows a more flexible, single- or multi-targeted removal of constituents from complex extracts such as botanicals. Chemical subtraction enables both chemical and biological characterization, including detection of synergism/antagonism by both the subtracted targets and the remaining metabolite mixture, as well as definition of the residual complexity of all fractions. The feasibility of the DESIGNER concept is shown by K-targeted subtraction of four bioactive prenylated phenols, isoxanthohumol (1), 8-prenylnaringenin (2), 6-prenylnaringenin (3), and xanthohumol (4), from a standardized hops (Humulus lupulus L.) extract using specific solvent systems. Conversely, adding K-targeted isolates allows enrichment of the original extract and hence provides an augmented DESIGNER material. Multiple countercurrent separation steps were used to purify each of the four compounds, and four DESIGNER extracts with varying depletions were prepared. The DESIGNER approach innovates the characterization of chemically complex extracts through integration of enabling technologies such as countercurrent separation, K-by-bioactivity, the residual complexity concepts, as well as quantitative analysis by (1)H NMR, LC-MS, and HiFSA-based NMR fingerprinting.

Orthogonal Analysis Underscores the Relevance of Primary and Secondary Metabolites in Licorice.

Licorice botanicals are produced from the roots of Glycyrrhiza species (Fabaceae), encompassing metabolites of both plant and rhizobial origin. The composition in both primary and secondary metabolites (1°/2°Ms) reflects the physiologic state of the plant at harvest. Interestingly, the relative abundance of 1°Ms vs 2°Ms in licorice extracts remains undetermined. A centrifugal partition chromatography (CPC) method was developed to purify liquiritin derivatives that represent major bioactive 2°Ms and to concentrate the polar 1°Ms from the crude extract of Glycyrrhiza uralensis. One objective was to determine the purity of the generated reference materials by orthogonal UHPLC-UV/LC-MS and qHNMR analyses. The other objectives were to evaluate the presence of 1°Ms in purified 2°Ms and define their mass balance in a crude botanical extract. Whereas most impurities could be assigned to well-known 1°Ms, p-hydroxybenzylmalonic acid, a new natural tyrosine analogue, was also identified. Additionally, in the most polar fraction, sucrose and proline represented 93% (w/w) of all qHNMR-quantified 1°Ms. Compared to the 2°Ms, accounting for 11.9% by UHPLC-UV, 1°Ms quantified by qHNMR defined an additional 74.8% of G. uralensis extract. The combined orthogonal methods enable the mass balance characterization of licorice extracts and highlight the relevance of 1°Ms, and accompanying metabolites, for botanical quality control.

Species-specific Standardisation of Licorice by Metabolomic Profiling of Flavanones and Chalcones.

INTRODUCTION: Major phenolics from licorice roots (Glycyrrhiza sp.) are glycosides of the flavanone liquiritigenin (F) and its 2'-hydroxychalcone isomer, isoliquiritigenin (C). As the F and C contents fluctuate between batches of licorice, both quality control and standardisation of its preparations become complex tasks. OBJECTIVE: To characterise the F and C metabolome in extracts from Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. ex DC. by addressing their composition in major F­C pairs and defining the total F:C proportion. MATERIAL AND METHODS: Three types of extracts from DNA-authenticated samples were analysed by a validated UHPLC/UV method to quantify major F and C glycosides. Each extract was characterised by the identity of major F­C pairs and the proportion of Fs among all quantified Fs:Cs. RESULTS: The F and C compositions and proportions were found to be constant for all extracts from a Glycyrrhiza species. All G. uralensis extracts contained up to 2.5 more Fs than G. glabra extracts. Major F­C pairs were B-ring glycosidated in G. uralensis, and A-/B-ring apiosyl-glucosidated in the G. glabra extracts. The F:C proportion was found to be linked to the glycosidation site: the more B-ring F-C glycosides were present, the higher was the final F:C proportion in the extract. These results enable the chemical differentiation of extracts from G. uralensis and G. glabra, which are characterised by total F:C proportions of 8.37:1.63 and 7.18:2.82, respectively. CONCLUSION: Extracts from G. glabra and G. uralensis can be differentiated by their respective F and C compositions and proportions, which are both useful for further standardisation of licorice botanicals.

Correction to "Diving into the Molecular Diversity of Aplysina cavernicola's Exometabolites: Contribution of Bromo-Spiroisoxazoline Alkaloids".

[This corrects the article DOI: 10.1021/acsomega.2c05415.].

Absolute configuration of naturally occurring glabridin.

The title compound {systematic name: 4-[(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano[2,3-f]chromen-3-yl]benzene-1,3-diol, commonly named glabridin}, C20H20O4, is a species-specific biomarker from the roots Glycyrrhiza glabra L. (European licorice, Fabaceae). In the present study, this prenylated isoflavan has been purified from an enriched CHCl3 fraction of the extract of the root, using three steps of medium-pressure liquid chromatography (MPLC) by employing HW-40F, Sephadex LH-20 and LiChroCN as adsorbents. Pure glabridin was crystallized from an MeOH-H2O mixture (95:5 v/v) to yield colorless crystals containing one molecule per asymmetric unit (Z' = 1) in the space group P212121. Although the crystal structure has been reported before, the determination of the absolute configuration remained uncertain. Stereochemical analysis, including circular dichroism, NMR data and an X-ray diffraction data set with Bijvoet differences, confirms that glabridin, purified from its natural source, is found only in a C3 R configuration. These results can therefore be used as a reference for the assignment of the configuration and enantiopurity of any isolated or synthetic glabridin sample.

In Situ Capture and Real-Time Enrichment of Marine Chemical Diversity.

Analyzing the chemical composition of seawater to understand its influence on ecosystem functions is a long-lasting challenge due to the inherent complexity and dynamic nature of marine environments. Describing the intricate chemistry of seawater requires optimal in situ sampling. Here is presented a novel underwater hand-held solid-phase extraction device, I-SMEL (In Situ Marine moleculELogger), which aims to concentrate diluted molecules from large volumes of seawater in a delimited zone targeting keystone benthic species. Marine benthic holobionts, such as sponges, can impact the chemical composition of their surroundings possibly through the production and release of their specialized metabolites, hence termed exometabolites (EMs). I-SMEL was deployed in a sponge-dominated Mediterranean ecosystem at a 15 m depth. Untargeted MS-based metabolomics was performed on enriched EM extracts and showed (1) the chemical diversity of enriched seawater metabolites and (2) reproducible recovery and enrichment of specialized sponge EMs such as aerothionin, demethylfurospongin-4, and longamide B methyl ester. These EMs constitute the chemical identity of each targeted species: Aplysina cavernicola, Spongia officinalis, and Agelas oroides, respectively. I-SMEL concentrated sponge EMs from 10 L of water in a 10 min sampling time. The present proof of concept with I-SMEL opens new research perspectives in marine chemical ecology and sets the stage for further sustainable efforts in natural product chemistry.

Diarylheptanoids from Dioscorea villosa (Wild Yam).

A fractionation methodology aimed at the metabolomic mining of new phytoconstituents for the widely used botanical, wild yam (Dioscorea villosa), makes use of 1D qHNMR and 2D NMR profiles along the preparative fractionation pathway. This quantifiable and structural guidance led to the isolation of 14 diarylheptanoids (1-14), including five new compounds (1-5) with a tetrahydropyrano core skeleton. The structures, including the absolute configurations of both new and previously known diarylheptanoids, were assigned by a combination of HRESIMS, 1D and 2D NMR, (1)H iterative full spin analysis (HiFSA), and Mosher's ester method. The isolation yields were consistent with yields predicted by qHNMR, which confirms the (semi)quantifiable capabilities of NMR-based preparative metabolomic mining. The qHNMR-aided approach enabled the identification of new and potentially significant chemical entities from a small fraction of the plant extract and, thereby, facilitated the characterization of the residual complexity of the D. villosa secondary metabolome. LC-MS profiling of different D. villosa accessions further confirmed that the diarylheptanoids represent genuine secondary metabolites, which can serve as a new class of markers for botanical integrity analysis of D. villosa.

LC-MS Based Phytochemical Profiling towards the Identification of Antioxidant Markers in Some Endemic Aloe Species from Mascarene Islands.

Aloe plant species have been used for centuries in traditional medicine and are reported to be an important source of natural products. However, despite the large number of species within the Aloe genus, only a few have been investigated chemotaxonomically. A Molecular Network approach was used to highlight the different chemical classes characterizing the leaves of five Aloe species: Aloe macra, Aloe vera, Aloe tormentorii, Aloe ferox, and Aloe purpurea. Aloe macra, A. tormentorii, and A. purpurea are endemic from the Mascarene Islands comprising Reunion, Mauritius, and Rodrigues. UHPLC-MS/MS analysis followed by a dereplication process allowed the characterization of 93 metabolites. The newly developed MolNotator algorithm was usedfor molecular networking and allowed a better exploration of the Aloe metabolome chemodiversity. The five species appeared rich in polyphenols (anthracene derivatives, flavonoids, phenolic acids). Therefore, the total phenolic content and antioxidant activity of the five species were evaluated, and a DPPH-On-Line-HPLC assay was used to determine the metabolites responsible for the radical scavenging activity. The use of computational tools allowed a better description of the comparative phytochemical profiling of five Aloe species, which showed differences in their metabolite composition, both qualitative and quantitative. Moreover, the molecular network approach combined with the On-Line-HPLC assay allowed the identification of 9 metabolites responsible for the antioxidant activity. Two of them, aloeresin A and coumaroylaloesin, could be the principal metabolites responsible for the activity. From 374 metabolites calculated by MolNator, 93 could be characterized. Therefore, the Aloe species can be a rich source of new chemical structures that need to be discovered.