Molecular networking-based mass spectral identification of Brucea javanica (L.) Merr. metabolites and their selective binding affinities for dengue virus enzymes.

Brucea javanica, a valued traditional medicinal plant in Malaysia, known for its fever-treating properties yet remains underexplored for its potential antiviral properties against dengue. This study aims to simultaneously identify chemical classes and metabolites within B. javanica using molecular networking (MN), by Global Natural Product Social (GNPS), and SIRIUS in silico annotation. Liquid chromatography-mass spectrometry (LC-MS2)-based MN explores chemical diversity across four plant parts (leaves, roots, fruits, and stem bark), revealing diverse metabolites such as tryptophan-derived alkaloids, terpenoids, and octadecadenoids. Simultaneous LC-MS2 and MN analyses reveal a discriminative capacity for individual plant components, with roots accumulating tryptophan alkaloids, fruits concentrating quassinoids, leaves containing fusidanes, and stem bark primarily characterised by simple indoles. Subsequently, extracts were evaluated for dengue antiviral activity using adenosine triphosphate (ATP) and plaque assays, indicates potent efficacy in the dichloromethane (DCM) extract from roots (EC50 = 0.3 µg/mL, SI = 10). Molecular docking analysis of two major compounds; canthin-6-one (264) and 1-hydroxy-11-methoxycanthin-6-one (275) showed potential binding interactions with active sites of NS5 RNA-dependent RNA polymerase (RdRp) of dengue virus (DENV) protein. Subsequent in vitro evaluation revealed compounds 264 and 275 had a promising dengue antiviral activity with SI value of 63 and 1.85. These identified metabolites emerge as potential candidates for further evaluation in dengue antiviral activities.

Mirror, mirror on the wall, which phytochemicals in Clinacanthus nutans inhibits advanced glycation end products of them all?

In our quest to discover advanced glycation end products (AGEs) inhibitors from Clinacanthus nutans (Burm.f.) Lindau leaves, we conducted a bioactivity-based molecular networking. This approach integrates LC-MS2 profiling and in vitro antiglycation data to predict bioactive compounds. We began by screening three extracts: 100% ethanol, 70% ethanol and 100% water alongside the in vitro antioxidant activity, total phenolics content (TPC) and schaftoside content. Among these extracts, 100% ethanol extract exhibited the highest total AGEs inhibition effects (IC50 = 80.18 ± 11.6 µg/mL), DPPH scavenging activity (IC50 = 747.40 ± 10.30 µg/mL) and TPC (26.54 ± 2.09 µg GAE /mg extract). Intriguingly, 100% ethanol extract contained the lowest amount of schaftoside, suggesting the involvement of other phytochemicals in the antiglycation effects. The molecular networking and in silico structural annotations of 401 LC-MS features detected in the fractions from 100% ethanol extract predicted 21 bioactive compounds (p < 0.05, r > 0.90), including several C40 carotenoids, alkaloids containing tetrapyrrole structures and fatty acids. On the contrary, all phenolics showed weak correlations with antiglycation effects. These predictions were further validated in vitro, where carotenoid lutein showed half maximal inhibitory concentration, IC50 = 96 ± 8 µM and selected flavonoid-C-glycosides exhibited weaker inhibitions (IC50 between 568 and 1922 µM). Notably, lutein content was higher in freeze-dried leaves (12.42 ± 0.82 mg/100 g) than oven-dried, although the former was associated with elevated mercury levels. In summary, C. nutans exhibited potential antiglycation and antioxidant activity, and lutein was identified as the main bioactive principle.

Rolling forms the diversities of small molecular nonvolatile metabolite profile and consequently shapes the bacterial community structure for Keemun black tea.

The detailed dynamics of small molecular nonvolatile chemical and bacterial diversities, as well as their relationship are still unclear in the manufacturing process of Keemun black tea (KMBT). Herein, mass spectrometry-based untargeted metabolomics, Feature-based Molecular Networking (FBMN) and bacterial DNA amplicon sequencing were used to investigate the dense temporal samples of the manufacturing process. For the first time, we reveal that the pyrogallol-type catechins are oxidized asynchronously before catechol-type catechins during the black tea processing. Rolling is the key procedure for forming the small molecular nonvolatile metabolite profile (SMNMetProf), increasing the metabolite richness, and then shaping the bacterial community structure in the KMBT manufacturing process, which decreases both molecular weight and molecular polarity of the small molecular nonvolatile metabolites. The SMNMetProf of black tea is formed by the endogenous enzymatic oxidation of tea leaves, rather than bacterial fermentation.

Targeted isolation, identification, and antioxidant evaluation of aromatic polyketides from a plant-derived fungus Ophiobolus cirsii LZU-1509.

There are >350 species of the Ophiobolus genus, which is not yet very well-known and lacks research reports on secondary metabolites. Three new 3,4-benzofuran polyketides 1-3, a new 3,4-benzofuran polyketide racemate 4, two new pairs of polyketide enantiomers (±)-5 and (±)-7, two new acetophenone derivatives 6 and 8, and three novel 1,4-dioxane aromatic polyketides 9-11, were isolated from a fungus Ophiobolus cirsii LZU-1509 derived from an important medicinal and economic crop Anaphalis lactea. The isolation was guided by LC-MS/MS-based GNPS molecular networking analysis. The planar structures and relative configurations were mainly elucidated by NMR and HR-ESI-MS data. Their absolute configurations were determined by using X-ray diffraction analysis and via comparing computational and experimental ECD, NMR, and specific optical rotation data. 9 possesses an unreported 5/6/6/6/5 five-ring framework with a 1,4-dioxane, and 10 and 11 feature unprecedented 6/6/6/5 and 6/6/5/6 four-ring frames containing a 1,4-dioxane. The biosynthetic pathways of 9-11 were proposed. 1-11 were nontoxic in HT-1080 and HepG2 tumor cells at a concentration of 20 µM, whereas 3 and 5 exerted higher antioxidant properties in the hydrogen peroxide-stimulated model in the neuron-like PC12 cells. They could be potential antioxidant agents for neuroprotection.

Chemical and biological investigation of Indigofera ammoxylum (DC.) Polhill. red and white phenotypes through feature-based molecular networking.

Chemical investigation of ethyl acetate bark extracts of Indigofera ammoxylum red and white phenotypes led to the bio-guided isolation of four previously undescribed flavonoids, named (2S,3R)-3',7-dihydroxy-4',6-dimethoxyflavanol (1), (2S,3R)-6-methoxy-7-hydroxyflavanol (2), 2',3',7-trihydroxy-4',6-dimethoxyisoflavone (7) and 2',5' -dimethoxy-4',5,7-trihydroxyisoflavanone (8), along with 14 known compounds (3-6 and 9-18). The previously undescribed structures were characterized based on NMR, HRESIMS, UV and IR data. Published spectroscopic data were used to deduce the structure of the known compounds. Eleven of the 18 isolated metabolites were evaluated for anti-inflammatory activity and cytotoxic activity against human liver carcinoma cells and human colon and colorectal adenocarcinoma cells. All tested compounds showed an anti-inflammatory activity (IC50 NO < 25 µg/mL), and compounds 2 and 3 were more selective than the positive control dexamethasone. Afromorsin (6) showed promising cytotoxic properties against both cancer cell lines (IC50 18.9 and 11.4 µg/mL). Feature-based molecular networking approach applied to bark and leaves extracts of the two phenotypes allowed to detect bioactive analogues, belonging to the families of flavones, isoflavones, flavanones, flavanols and flavonols, and to explore the chemodiversity of the species. The red and white phenotypes have a similar composition, whereas bark and leaves contain specific chemical entities. Finally, this approach highlighted a cluster of potentially bioactive and undescribed metabolites.

Identification of tyrosinase inhibitors in defatted seeds of evening primrose (Oenothera biennis L.) by affinity-labeled molecular networking.

The defatted seeds of evening primrose (DE), a by-product of evening primrose oil extraction, are currently underutilized. This study aimed to valorize DE by examining its effects on melanogenesis and tyrosinase activity in zebrafish embryos and in vitro, and an innovative affinity-labeled molecular networking workflow was proposed for the rapid identification of tyrosinase inhibitors in DE. Our results indicated DE significantly reduced melanin content (53.3 % at 100 µg/mL) and tyrosinse activity (80.05 % for monophenolase and 70.40 % for diphenolase at 100 µg/mL). Furthermore, through the affinity-labeled molecular networking approach, 20 compounds were identified as potential tyrosinase inhibitors within DE, predominantly flavonoids and tannins characterized by catechin and galloyl substructures. Seven of these compounds were isolated and their inhibitory effects on tyrosinase were validated using functional assays. This study not only underscores the potential of DE as a rich source of natural tyrosinase inhibitors but also establishes the effectiveness of affinity-labeled molecular networking in pinpointing bioactive compounds in complex biological matrices.

Rapid identification of the compounds of Bushen Huoxue Prescription based on offline two-dimensional liquid chromatography with high-resolution mass spectrometry and molecular network technology.

The comprehensive and efficient characterization of components in traditional Chinese medicine is crucial for elucidating its active constituents and uncovering its mechanism. Identifying the compounds of the Bushen Huoxue Prescription (BHP) is difficult because of its complex composition and the large difference in concentration among its compounds. In this study, a hydrophilic interaction liquid chromatography coupled with reversed-phase LC (HILIC × RPLC) offline 2D-LC tandem high-resolution mass spectrometry method was established to analyze the total compounds of the BHP. Database screening and molecular networking were performed to identify the compounds. In contrast to conventional 1D chromatography, 2D chromatography increased peak capacity, enriched trace ingredients, and prevented the masking of high-abundance compounds. A total of 165 compounds were identified, and 14 potential compounds needed to be further identified. This study provided an effective method for comprehensively analyzing the complex system of traditional Chinese medicine compounds.

Tandem mass spectrometry (MS/MS) molecular networking guided profiling of small molecules from Aquilaria sinensis (Lour.) Gilg leaves and their bioactivity evaluation.

INTRODUCTION: Agarwood, a fragrant resinous wood mainly formed by Aquilaria spp., is used worldwide as a natural fragrance and traditional medicine. A large amount of Aquilaria sinensis (Lour.) Gilg leaves are underutilised during the process of the agarwood industry, and the development of A. sinensis leaves as tea has recently attracted more and more attention. However, the small molecule profile of A. sinensis leaves and their bioactivities has been rarely reported. OBJECTIVE: To conduct a rapid untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of A. sinensis leaves with a molecular networking (MN) strategy and evaluate its antioxidant and antidiabetic value. METHOD: A MN-assisted tandem mass spectrometry (MS/MS) analysis strategy was used to investigate the small molecule profile of A. sinensis leaves. Additionally, the integration of antioxidant and α-glucosidase inhibitory assays with MN analysis was executed to expeditiously characterise the bioactive compounds for potential prospective application. RESULTS: Five main chemical groups including phenol C-glycosides, organic acids, 2-(2-phenylethyl) chromones, benzophenone O-glycosides and flavonoids were rapidly revealed from the A. sinensis leaves. Eighty-one compounds were provisionally identified by comparing their MS/MS fragments with canonical pathways. The featured xanthone C-glycosides and benzophenone C-glycosides were recognised as the primary components of A. sinensis leaves. Several dimers and a trimer of mangiferin were reported firstly in A. sinensis leaves. Furthermore, 17 and 14 potential bioactive molecules were rapidly annotated from antioxidant and α-glucosidase inhibitory fraction, respectively. CONCLUSION: Our findings will help expand the utilisation of A. sinensis leaves and thus promote the high-quality development of agarwood industry.

Untargeted metabolomics-based molecular networking for chemical characterization of selected Apiaceae fruit extracts in relation to their antioxidant and anti-cellulite potentials.

Stress, obesity, hormonal changes, and aging have been connected to cellulite aggravation resulting in skin dimpled appearance, a very common painless skin disorder with a female preponderance. Several Apiaceae plants have been traditionally used for cosmetic applications. However, their screening for anti-cellulite potential has not been deeply investigated. In this work, UPLC-HRMS/MS coupled with molecular networking was employed to glean a holistic overview of the chemodiversity of the metabolome of nine Apiaceae fruits. Additionally, the extracts were screened for in vitro antioxidant and anti-cellulite activities. Apium graveolens and Petroselinum crispum revealed excellent free radical scavenging activity, remarkably increased lipolysis, and decreased adipogenesis. Furthermore, apigenin and its glycosides were identified to be the major components in both extracts, which might be responsible for the antioxidant activity and anti-cellulite potential. Conclusively, these results signify the potent antioxidant and anti-cellulite properties of A. graveolens and P. crispum fruit extracts, holding potential for the development of plant derived products for cellulite management.

Bioprospecting for Rhizobacteria with the Ability to Enhance Drought Tolerance in Lessertia frutescens.

Lessertia frutescens is a multipurpose medicinal plant indigenous to South Africa that is used for the management of cancer, stomach ulcers, wounds, etc. The use and demand for the raw materials from this plant have been increasing steadily over the years, putting strain on the dwindling wild populations. Although cultivation may provide relief to the strained supply, the persistent drought climate poses a threat to the plant's growth and productivity. This study explored three plant-growth-promoting rhizobacteria isolates, TUTLFNC33, TUTLFNC37 and TUTLFWC74, obtained from the root nodules of Lessertia frutescens as potential bioinoculants that can improve yield, biological activities and the production of secondary metabolites in the host plant. Isolate TUTLFNC37 was identified as the most promising isolate for inoculation of Lessertia frutescens under drought conditions as it induced drought tolerance through enhanced root proliferation, osmolyte proline accumulation and stomatal closure. Superior biomass yield, phenolics, triterpenes and antioxidant activity were evident in the extracts of Lessertia frutescens inoculated with TUTLFNC37 and under different levels of drought. Furthermore, the metabolomics of the plant extracts demonstrated the ability of the isolate to withstand drastic changes in the composition of unique metabolites, sutherlandiosides A-D and sutherlandins A-D. Molecular families which were never reported in the plant (peptides and glycerolipids) were detected and annotated in the molecular networks. Although drought had deleterious effects on Lessertia frutescens, isolate TUTLFNC37 alleviated the impact of the stress. Isolate TUTLFNC37 is therefore the most promising, environmentally friendly alternative to harmful chemicals such as nitrate-based fertilizers. The isolate should be studied to establish its field performance, cross infectivity with other medicinal plants and competition with inherent soil microbes.

Characterization of Brazilian green propolis as a photosensitizer for LED light-induced antimicrobial photodynamic therapy (aPDT) against methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-intermediate Staphylococcus aureus (VISA).

Staphylococcus aureus is the primary cause of skin and soft tissue infections. Its significant adaptability and the development of resistance are the main factors linked to its spread and the challenges in its treatment. Antimicrobial photodynamic therapy emerges as a promising alternative. This work aimed to characterize the antimicrobial photodynamic activity of Brazilian green propolis, along with the key bioactive compounds associated with this activity. Initially, a scanning spectrometry was conducted to assess the wavelengths with the potential to activate green propolis. Subsequently, reference strains of methicillin-resistant Staphylococcus aureus (MRSA ATCC 43300) and vancomycin-intermediate Staphylococcus aureus (VISA ATCC 700699) were exposed to varying concentrations of green propolis: 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg /mL and 100 µg/mL and were stimulated by blue, green or red LED light. Finally, high-performance liquid chromatography coupled with a diode array detector and tandem mass spectrometry techniques, along with classic molecular networking analysis, was performed to identify potential bioactive molecules with photodynamic activity. Brazilian green propolis exhibits a pronounced absorption peak and heightened photo-responsiveness when exposed to blue light within the range of 400 nm and 450 nm. This characteristic reveals noteworthy significant photodynamic activity against MRSA and VISA at concentrations from 5 µg/mL. Furthermore, the propolis comprises compounds like curcumin and other flavonoids sourced from flavone, which possess the potential for photodynamic activity and other antimicrobial functions. Consequently, Brazilian green propolis holds promise as an excellent bactericidal agent, displaying a synergistic antibacterial property enhanced by light-induced photodynamic effects.

Simultaneous extraction of crocin and geniposide from gardenia fruits (Gardenia jasminoides Ellis) by probe-type ultrasound-assisted natural deep eutectic solvents and their inhibition effects on low density lipoprotein oxidation.

The simultaneous extraction of crocin and geniposide from gardenia fruits (Gardenia jasminoides Ellis) was performed by integrating natural deep eutectic solvents (NADES) and ultrasound-assisted extraction (UAE). Among the eight kinds of NADES screened, choline chloride-1,2-propylene glycol was the most suitable extractant. The probe-type ultrasound-assisted NADES extraction system (pr-UAE-NADES) demonstrated higher extraction efficiency compared with plate-type ultrasound-assisted NADES extraction system (pl-UAE-NADES). Orthogonal experimental design and a modified multi-index synthetic weighted scoring method were adopted to optimize pr-UAE-NADES extraction process. The optimal extraction conditions that had a maximum synthetic weighted score of 29.46 were determined to be 25 °C for extraction temperature, 600 W for ultrasonic power, 20 min for extraction time, and 25% (w/w) for water content in NADES, leading to the maximum yields (7.39 ± 0.20 mg/g and 57.99 ± 0.91 mg/g, respectively) of crocin and geniposide. Thirty-three compounds including iridoids, carotenoids, phenolic acids, flavonoids, and triterpenes in the NADES extract were identified by LC-Q-TOF-MS2 coupled with a feature-based molecular networking workflow. The kinetics evaluation of the conjugated dienes generation on Cu2+-induced low density lipoprotein (LDL) oxidation via the four-parameter logistic regression model showed that crocin increased the lag time of LDL oxidation in a concentration-dependent manner (15 µg/mL, 30 µg/mL, 45 µg/mL) by 12.66%, 35.44%, and 73.42%, respectively. The quantitative determination for fluorescence properties alteration of the apolipoprotein B-100 exhibited that crocin effectively inhibited the fluorescence quenching of tryptophan residues and the modification of lysine residues caused by reactive aldehydes and malondialdehydes. The pr-UAE-NADES showed significant efficiency toward the simultaneous extraction of crocin and geniposide from gardenia fruits. And this study demonstrates the potential utility of gardenia fruits in developing anti-atherogenic functional food.

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.

Mining Xanthine Oxidase Inhibitors from an Edible Seaweed Pterocladiella capillacea by Using In Vitro Bioassays, Affinity Ultrafiltration LC-MS/MS, Metabolomics Tools, and In Silico Prediction.

The prevalence of gout and the adverse effects of current synthetic anti-gout drugs call for new natural and effective xanthine oxidase (XOD) inhibitors to target this disease. Based on our previous finding that an edible seaweed Pterocladiella capillacea extract inhibits XOD, XOD-inhibitory and anti-inflammatory activities were used to evaluate the anti-gout potential of different P. capillacea extract fractions. Through affinity ultrafiltration coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS), feature-based molecular networking (FBMN), and database mining of multiple natural products, the extract's bioactive components were traced and annotated. Through molecular docking and ADMET analysis, the possibility and drug-likeness of the annotated XOD inhibitors were predicted. The results showed that fractions F4, F6, F4-2, and F4-3 exhibited strong XOD inhibition activity, among which F4-3 reached an inhibition ratio of 77.96% ± 4.91% to XOD at a concentration of 0.14 mg/mL. In addition, the P. capillacea extract and fractions also displayed anti-inflammatory activity. Affinity ultrafiltration LC-MS/MS analysis and molecular networking showed that out of the 20 annotated compounds, 8 compounds have been previously directly or indirectly reported from seaweeds, and 4 compounds have been reported to exhibit anti-gout activity. Molecular docking and ADMET showed that six seaweed-derived compounds can dock with the XOD activity pocket and follow the Lipinski drug-like rule. These results support the value of further investigating P. capillacea as part of the development of anti-gout drugs or related functional foods.

Guided isolation of secondary metabolites from Nectria sp. MHHJ-3 by molecular network strategy.

The fungus Nectria sp. MHHJ-3 was isolated from Illigera rhodantha. A molecular networking-guided the secondary metabolites investigation of Nectria sp. MHHJ-3 led to the isolation of ten metabolites (1-10), including two new naphthalenone derivatives, nectrianaphthalenones A (1) and B (2), and two new steroids, nectriasteroids A (3) and B (4). Their structures were elucidated by extensive spectroscopic analysis including the HRESIMS, 1D/2D NMR and electronic circular dichroism (ECD) spectra. A plausible biosynthetic pathway for 1-2 was proposed. Compounds 1 and 2 exhibited moderate acetylcholinesterase (AChE) inhibitory activities. Compounds 3 and 4 showed significant cytotoxic activity against selected tumor cells. Particularly, compound 3 exhibited the strongest activity against A549 cells with an IC50 value of 13.73 ± 0.03 µM, which was at the same grade with that of positive control cisplatin.

Comparative LC-MS/MS-based molecular networking, DNA fingerprinting, and in vitro anti-Helicobacter pylori activity of three Egyptian Ficus cultivars.

Ficus species (Moraceae) have been used for nutrition and traditional medicine, and plants from this family are phytochemically abundant and serve as a potential source of natural products. As a result of the inherent complexity of the plant metabolomes and the fact that these Ficus species chemical space has not yet been fully decoded, it is still difficult to characterize their phytochemistry. Therefore, this study, we suggest the use of the molecular networking to elucidate the chemical classes existing in leaves of three Ficus species (F. deltoidei Jack, F. drupacea Thunb and F. sycomorus L.) and highlight the importance of molecular networking in examining their chemotaxonomy . By using computational tools, 90 metabolites were annotated , including phenolic acids, flavonoids, furanocoumarins, fatty acids and terpenoids. Phenolic acids were detected as the main class present in the three studied species. Flavonoids-C-glycosides, flavonoids-O-glycosides and isoflavonoids were mainly present in F. drupacea and F. sycomorus, while furanocoumarins were proposed in F. sycomorus. Vomifoliol-based sesquiterpenes were proposed in F. deltoidei. The chemotaxonomic differentiation agreed with the DNA fingerprinting using SCOT and ISSR markers. F. deltoidei, in particular, had a divergent chemical fingerprint as well as a different genotype. Chemotype differentiation using chemical fingerprints, in conjunction with the proposed genetic markers, creates an effective identification tool for the quality control of the raw materials and products derived from those three Ficus species. As well, F. drupacea exploited the most potent inhibition of H. pylori with MIC of 7.81 µg/ mL compared with clarithromycin. Overall, molecular networking provides a promising approach for the exploration of the chemical space of plant metabolomes and the elucidation of chemotaxonomy.

Japonins A-D, cyathane diterpenoids with neurite outgrowth-promoting activity isolated from Onychium japonicum using NMR and MS/MS-based molecular networking.

Guided by MS/MS-based molecular networking strategy, four new cyathane diterpenoids japonin A-D (1-4), together with the known analogues (5 and 6), have been isolated from aerial parts of Onychium japonicum. The structures of the new compounds were elucidated through a combination of NMR and MS experiments. Through single-crystal X-ray diffraction analysis, and comparison of experimental and calculated computational electronic circular dichroism (ECD) spectra, the absolute configurations of compounds 1-4 were determined. The new compound 1 showed promoting effects on the differentiation of PC12 at a concentration of 40 µM.

Genomic and Metabolomic Analysis of the Endophytic Fungus Fusarium sp. VM-40 Isolated from the Medicinal Plant Vinca minor.

The genus Fusarium is well-known to comprise many pathogenic fungi that affect cereal crops worldwide, causing severe damage to agriculture and the economy. In this study, an endophytic fungus designated Fusarium sp. VM-40 was isolated from a healthy specimen of the traditional European medicinal plant Vinca minor. Our morphological characterization and phylogenetic analysis reveal that Fusarium sp. VM-40 is closely related to Fusarium paeoniae, belonging to the F. tricinctum species complex (FTSC), the genomic architecture and secondary metabolite profile of which have not been investigated. Thus, we sequenced the whole genome of Fusarium sp. VM-40 with the new Oxford Nanopore R10.4 flowcells. The assembled genome is 40 Mb in size with a GC content of 47.72%, 15 contigs (≥50,000 bp; N 50~4.3 Mb), and 13,546 protein-coding genes, 691 of which are carbohydrate-active enzyme (CAZyme)-encoding genes. We furthermore predicted a total of 56 biosynthetic gene clusters (BGCs) with antiSMASH, 25 of which showed similarity with known BGCs. In addition, we explored the potential of this fungus to produce secondary metabolites through untargeted metabolomics. Our analyses reveal that this fungus produces structurally diverse secondary metabolites of potential pharmacological relevance (alkaloids, peptides, amides, terpenoids, and quinones). We also employed an epigenetic manipulation method to activate cryptic BGCs, which led to an increased abundance of several known compounds and the identification of several putative new compounds. Taken together, this study provides systematic research on the whole genome sequence, biosynthetic potential, and metabolome of the endophytic fungus Fusarium sp. VM-40.

6-Oxofurostane and (iso)Spirostane Types of Saponins in Smilax sieboldii: UHPLC-QToF-MS/MS and GNPS-Molecular Networking Approach for the Rapid Dereplication and Biodistribution of Specialized Metabolites.

Identifying novel phytochemical secondary metabolites following classical pharmacognostic investigations is tedious and often involves repetitive chromatographic efforts. During the past decade, Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight-Tandem Mass Spectrometry (UHPLC-QToF-MS/MS), in combination with molecular networking, has been successfully demonstrated for the rapid dereplication of novel natural products in complex mixtures. As a logical application of such innovative tools in botanical research, more than 40 unique 3-oxy-, 3, 6-dioxy-, and 3, 6, 27-trioxy-steroidal saponins were identified in aerial parts and rhizomes of botanically verified Smilax sieboldii. Tandem mass diagnostic fragmentation patterns of aglycones, diosgenin, sarsasapogenin/tigogenin, or laxogenin were critical to establishing the unique nodes belonging to six groups of nineteen unknown steroidal saponins identified in S. sieboldii. Mass fragmentation analysis resulted in the identification of 6-hydroxy sapogenins, believed to be key precursors in the biogenesis of characteristic smilaxins and sieboldins, along with other saponins identified within S. sieboldii. These analytes' relative biodistribution and characteristic molecular networking profiles were established by analyzing the leaf, stem, and root/rhizome of S. sieboldii. Deducing such profiles is anticipated to aid the overall product integrity of botanical dietary supplements while avoiding tedious pharmacognostic investigations and helping identify exogenous components within the finished products.

Dereplication of polar extracts from Croton antisyphiliticus Mart. roots and its anti-inflammatory and antinociceptive potential.

Croton antisyphiliticus Mart. is a plant popularly used in folk medicine by traditional communities from Brazilian savannah to treat general inflammation. According to ethnopharmacological data, this specie can be considered a source of biologically active molecules for the development of new drugs. Thereby, this study reports the results of the dereplication approach of C. antisyphiliticus roots extracts and the in vivo evaluation of its potential antinociceptive and anti-inflammatory in albino Swiss mice. Based on HPLC coupled to Q-Exactive Orbitrap Mass Spectrometer and using GNPS, a total of thirteen polyphenolic compounds were noticed, including four compounds that have been reported for the first time in the genus Croton. Ethanolic and aqueous roots extracts demonstrated a dose-dependent inhibition for the number of writes, reduced pain induced by formalin and hyperalgesia induced by carrageenan. These extracts also reduced paw edema, cell migration, and myeloperoxidase activity, with effects similar to indomethacin and dexamethasone drugs.