Antiproliferative Effects of Methanolic Fruit Extract of Solanum xenthocarpum (L.) on Human Breast Cancer Cells.

Solanum xanthocarpum, a perennial herb native to India, contains steroidal glycoalkaloids with notable anticancer properties. This study investigated the antioxidant and antiproliferative effects of methanolic fruit extract of S. xanthocarpum on human breast cancer cells (MDA-MB-231). Phytochemical screening and LC-HRMS analysis confirmed presence of various primary and secondary metabolites. Antioxidant activity was assessed through DPPH, ABTS radical scavenging, reducing power, and phosphomolybdate assays. The extract demonstrated significant antioxidant potential with EC50 values of 60.10 ± 0.88 µg/mL (DPPH) and 392.29 ± 3.93 µg/mL (ABTS). Cytotoxicity against MDA-MB-231 cells was evaluated via morphological analysis, MTT assays, and IC50 determination (24.19 ± 0.56 µg/L). Apoptosis was confirmed using dual staining techniques (AO/EB, Hoechst 33342/PI, DAPI), revealing condensed nuclei, apoptotic bodies, and reduced mitochondrial membrane potential, as indicated by Rhodamine staining. Additionally, increased reactive oxygen species (ROS) levels were observed using H2-DCF-DA staining. The total phenolic and flavonoid contents of the extract were 127.78 ± 3.547 mg GAE/g and 98.06 ± 4.289 mg QE/g, respectively. These findings suggest that the methanolic fruit extract of S. xanthocarpum possesses strong antioxidant and anticancer activities, indicating its potential role in cancer treatment. Further studies are warranted to explore its bioactive compounds for developing novel anticancer therapies.

LC-HRMS Profiling of Phytochemicals with Assessment of Antioxidant, Anticholinesterase, and Antimicrobial Potentials of Astragalus brachystachys DC.

This study provides the first comprehensive report on the chemical constituents and biological activities of an important medicinal plant, Astragalus brachystachys DC. The aerial part samples were collected from Adana, Türkiye, and an ethanol extract was prepared with these parts. The secondary metabolites of the extract were determined by an LC-HRMS analysis. The LC-HRMS analysis showed the presence of 39 different constituents, hyperoside (303.419±10.50 µg/g extract), p-coumaric acid (256.975±8.51 µg/g extract), and rutin (72.684±2.23 µg/g extract) were determined as major compounds in the aerial parts ethanol extract. Attributed to its high total phenolic (58.53±1.30 µg PEs/mg extract) and total flavonoid content (29.98±0.83 µg QEs/mg extract), the extract demonstrated strong antioxidant activity according to three different assays namely DPPH free (IC50: 33.08±0.61 mg/mL), and ABTS cation radical scavenging (IC50: 15.39±0.72 mg/mL) and CUPRAC activity (A0.5: 36.25±0.28 mg/mL) methods. In vitro assays showed that cholinesterase inhibitory activity results were found to be exceptional with 85.95±0.52 % inhibition on acetylcholinesterase and 66.32±1.33 % inhibition on butyrylcholinesterase at 200 mg/mL. Regarding antimicrobial properties, Astragalus brachystachys DC extract was found to be effective against Enterococcus faecalis with a MIC value of 39.06 µg/mL.

Integrated targeted and untargeted metabolomics profiling of Vanilla species from the Atlantic Forest: Unveiling the bioeconomic potential of Vanilla cribbiana.

In this study, we employ both targeted and untargeted approaches to explore the metabolomic profiles of Vanilla spp., with a particular focus on V. cribbiana (VCR) and its comparison with V. planifolia (VP). We also examine V. bahiana and V. chamissonis using targeted approaches. Through advanced analytical techniques, our untargeted LC-HRMS approach led to the annotation of 60 metabolites, revealing a complex chemical composition with 34 novel compounds in the Vanilla genus in VCR and VP. These findings highlight significant flavoring compounds and lay the foundation for a subsequent quantitative estimation approach. Our targeted analysis, which measured key molecules, underscores VCR's potential in producing vanillin and acetovanillone at levels comparable to the commercially valuable VP and even higher levels of vanillic acid. This research enriches our understanding of flavor composition in vanilla species and emphasizes the importance of exploring wild relatives of vanilla crop for sustainable production and biodiversity conservation.

Lentil Waste Extracts for Inflammatory Bowel Disease (IBD) Symptoms Control: Anti-Inflammatory and Spasmolytic Effects.

BACKGROUND/OBJECTIVES: In the contest of agro-industrial waste valorization, we focused our attention on lentil seed coats as a source of health-promoting phytochemicals possibly useful in managing inflammatory bowel diseases (IBDs), usually characterized by inflammation and altered intestinal motility. METHODS: Both traditional (maceration) and innovative microwave-assisted extractions were performed using green solvents, and the anti-inflammatory and spasmolytic activities of the so-obtained extracts were determined through in vitro and ex vivo assays, respectively. RESULTS: The extract obtained through the microwave-assisted procedure using ethyl acetate as the extraction solvent (BEVa) proved to be the most useful in inflammation and intestinal motility management. In LPS-activated Caco-2 cells, BEVa down-regulated TLR4 expression, reduced iNOS expression and the pro-inflammatory cytokine IL-1 production, and upregulated the anti-inflammatory cytokine IL-10 production, thus positively affecting cell inflammatory responses. Moreover, a significant decrease in the longitudinal and circular tones of the guinea pig ileum, with a reduction of transit speed and pain at the ileum level, together with reduced transit speed, pain, and muscular tone at the colon level, was observed with BEVa. HPLC separation combined with an Orbitrap-based high-resolution mass spectrometry (HRMS) technique indicated that 7% of all the identified metabolites were endowed with proven anti-inflammatory and antispasmodic activities, among which niacinamide, apocynin, and p-coumaric acid were the most abundant. CONCLUSIONS: Our results suggest that lentil hull extract consumption could contribute to overall intestinal health maintenance, with BEVa possibly representing a dietary supplementation and a promising approach to treating intestinal barrier dysfunction.

LC-HRMS-based global metabolomics profiling unravels the distinct metabolic signature of lapatinib-resistant and trastuzumab-resistant HER2+ breast cancer cells.

The effectiveness of lapatinib (LAP) and trastuzumab (TRZ), the first-line therapies for HER2+ breast cancer, has been limited owing to the development of acquired resistance in patients with HER2+. This study aimed to investigate the alterations in metabolic signatures in LAP-resistant HCC1954 and TRZ-resistant HCC1954 and pathways in human HER2+ breast cancer cells using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and enrichment analysis. The HCC1954 parental cells were sequentially treated 13 rounds with LAP or TRZ to develop resistant cells and then tested for their cytotoxicity using the MTT assay. Metabolites were prepared from HCC1954 parental (MBXWT), HCC1954-LAP (MBXLAP), and HCC1954-TRZ (MBXTRZ) cells prior to LC-HRMS, chemometric, enrichment, and joint pathway analyses. LAP- and TRZ-resistant cells were successfully developed from HCC1954, and 29 and 17 differentially expressed metabolites (DEMs) were identified between MBXWT-MBXLAP and MBXWT-MBXTRZ, respectively. The analysis of DEMs between MBXWT and MBXLAP revealed significant enrichment in D-amino acid metabolism, while MBXWT and MBXTRZ identified valine, leucine, isoleucine biosynthesis, ascorbate, and aldarate metabolism. Joint pathway enrichment analysis of LAP-resistant DEMs and differentially expressed genes (DEGs) showed enrichment in glutathione metabolism, while that of TRZ-resistance and DEGs showed enrichment in carbohydrate metabolism, namely pentose and glucuronate interconversions, starch and sucrose metabolism, and galactose metabolism. The findings from this study indicate considerable metabolic changes in LAP- and TRZ-resistant HCC1954 cells, which are crucial for understanding the resistance mechanisms and developing strategies to overcome these problems.

Identification of organic contaminants in water and related matrices using untargeted liquid chromatography high-resolution mass spectrometry screening with MS/MS libraries.

Nontargeted and suspect screening with liquid chromatography-high resolution mass spectrometry (LC-HRMS) has become an indispensable tool for quality assessment in the aquatic environment - complementary to targeted analysis of organic (micro)contaminants. An LC-HRMS method is presented, suitable for the analysis of a wide variety of water related matrices: surface water, groundwater, wastewater, sediment and sludge, including extracts from passive samplers and on-site solid phase enrichment, while focusing on the data processing aspect of the method. A field study is included to demonstrate the practical application and versatility of the whole process. HRMS/MS data were recorded following LC separation in both (ESI) positive and negative ionization modes using data dependent as well as data independent acquisition. Two vendor (Agilent's Personal Compound Database and Library and from National Institute of Standards and Technology) and one open (MassBank/EU) tandem mass spectral libraries were utilized for the identification of compounds via mass spectral match. The development of a novel software tool for parsing, grouping and reduction of MS/MS features in data files converted to mascot generic format (MGF) helped to substantially decrease the amount of time and effort needed for MS library search. While applying the method, in the course of the entire field study, 18771 detections (from 870 individual compounds) in total were recorded in 275 samples, resulting in 68.3 identified compounds per sample, on average. Among the top ten most frequently detected contaminants across all samples and sample types were pharmaceutical compounds carbamazepine, 4-acetamidoantipyrine, 4-formylaminoantipyrine, tramadol, lamotrigine and phenazone and industrial contaminants toluene-2-sulfonamide, tolytriazole, tris(2-butoxyethyl) phosphate and benzotriazole. Exploratory data analysis methods and tools enabled us to discover organic pollutant occurrence patterns within the comprehensive sets of qualitative data collected from various projects between the years 2018-2023. The results may be used as valuable inputs for future water quality monitoring programs.

Per- and polyfluoroalkyl substances in food and beverages: determination by LC-HRMS and occurrence in products from the Belgian market.

Per- and polyfluoroalkyl substances (PFAS) are industrial chemicals encompassing thousands of compounds. Due to their persistent, bioaccumulative and toxic character, PFAS have become environmental contaminants, and exposure to these chemicals may lead to adverse health effects. This study aimed to provide a sensitive analytical method for the quantification of 25 PFAS in food including food for the young population and beverages, and to gather the missing occurrence data for the dietary exposure evaluation for the Belgian population. More than a decade ago, such assessment was performed only for PFOS and PFOA and is currently outdated. For the determination of PFAS in foodstuffs, an extraction based on a "quick, easy, cheap, effective, rugged, and safe" (QuEChERS) protocol and combined with a two-step purification using solid-phase extraction (SPE) was optimised. The quantitative analysis was performed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). The method was validated, and the achieved limits of quantification (LOQs) ranged from 0.002 to 0.3 µg/kg, with the exception of HFPO-DA (1 µg/kg). The LC-HRMS analysis of 268 food products from the Belgian market demonstrated that 43% of samples contained at least one PFAS with a maximum of eleven PFAS measured in a stew of wild pork. PFOS was the most detected compound found in 19% of samples, followed by PFBA (18%) and PFOA (15%), while PFTeDA, PFPeS, PFHpS, PFDS, PFUnDS, PFDoDS, PFTrDS, Minor F53B and HFPO-DA were not detected. The concentrations of the different PFAS in commercial food varied from

[Analysis of the relationship between neonatal birth weight and meconium metabolites based on birth cohort metabolomics].

Neonatal birth weight is a crucial indicator of intrauterine growth and development with important implications for child development and adult health. The birth weight of a newborn is closely linked to the nutrition and health of the mother during pregnancy as well as genetic factors. Therefore, assessing the metabolic status of the fetus in utero is greatly significant for understanding the mechanisms responsible for abnormal birth weight. While previous studies often analyzed the impact of maternal metabolism on fetal development using umbilical cord blood from pregnant women, such blood may not accurately reflect the actual intrauterine environment owing to the barrier function of the placenta; moreover, obtaining biological samples during the fetal period is challenging. Meconium, the first feces excreted by a newborn, provides ideal biological material for studying maternal and infant health. Metabolomics can reveal metabolic changes in living organisms by analyzing small molecules in biological samples; hence studying meconium samples using metabolomics technology is expected to reveal fetal metabolic changes during pregnancy, thereby providing new insights into fetal nutritional intake, growth, and development, as well as metabolic pathways related to birth weight. To gain a deeper understanding of the metabolic changes associated with birth weight, this study collected metabolomic data from the meconium of 484 newborns in the established Xiaogan birth cohort using an untargeted metabolomics technique based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) and analyzed the association between meconium metabolites and birth weight. This cohort exhibited incidence rates of low birth weight (<2500 g) and macrosomia (>4000 g) of 3.3% and 7.2%, respectively, which were roughly equivalent to the national average. Orthogonal partial least squares discriminant analysis revealed significant differences between the meconium metabolomes of the low birth weight and macrosomic groups when compared to the normal weight group. We discovered significant distinctions between the differential metabolites of newborns of low birth weight and those of normal weight, as well as between macrosomic and normal weight newborns that point to disparate biological pathways. Newborns with low birth weight exhibited significantly lower levels of critical amino acids, such as glutamate and proline, compared to the normal weight group, which may be associated with placental dysfunction and maternal nutritional deficiencies. Conversely, the meconium of macrosomic newborns contained significantly elevated levels of hormone metabolites such as estrone that reflected the pathophysiological state associated with maternal metabolic diseases or excessive placental hormone levels. Our study suggests that the metabolomic profile of the meconium reflects the metabolic pathways and regulatory mechanisms at play during fetal growth and development, and offers potential metabolic biomarkers and directions for future in-depth research into diseases related to fetal development. However, this study was based solely on the Xiaogan birth cohort, which was limited to specific regions and populations. A multicenter, multiethnic, and multiregional study is expected to help validate the universality of our research findings.

Metabolic Characterization of Vamorolone in Human Liver Microsomes: Implications for Anti-Doping.

Vamorolone, a potential alternative to conventional glucocorticoids, shows significant promise in sports medicine due to its reduced side effects and superior pharmacodynamic properties. This study aims to investigate the metabolic characteristics of this novel synthetic cyclodextrin-steroid anti-inflammatory drug and elucidate the metabolic pathways in human liver microsomes (HLMs) in vitro, thereby providing a scientific basis for assessing its potential risks for athletes. All compounds are detected by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and metabolite identification was performed using Compound Discoverer 3.3 software. In the HLMs model, 12 metabolites of vamorolone are successfully identified, including 10 phase I metabolites and 2 phase II metabolites. Among these, the reduction metabolite M1 exhibited the highest peak area, indicating it as one of the primary metabolic pathways. The dehydrogenated compound M2 had the second highest peak area, further elucidating the metabolic characteristics of vamorolone. This study systematically identifies the metabolite structures of vamorolone in HLMs and provide crucial data for the pharmacokinetics and biomarker research of this drug. The findings not only enhance the understanding of its metabolic mechanisms but also offer a scientific basis for evaluating its safety and efficacy in sports medicine. Meanwhile, these discoveries can contribute to better regulation and control of Vamorolone's use in competitive sports, ensuring fairness in competitions.

Liquid chromatography-high-resolution mass spectrometry-based metabolomics revealing the effects of zearalenone and alpha-zearalenol on human endometrial cancer cells.

Human exposure to mycotoxins through food involve a mixture of compounds, which can be harmful to human health. The Fusarium fungal species are known to produce zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, and its metabolite alpha-zearalenol (α-ZEL), both of which possess endocrine-disruptive properties. Given their potential harm to human health through food exposure, investigating the combined effects of ZEN and α-ZEL becomes crucial. Hence, the combined impact of ZEN and α-ZEL study hold significant importance. This in vitro study delves into the critical area, examining their combined impact on the proliferation and metabolic profile of endometrial cancer Ishikawa cells via sulforhodamine, clonogenic, proliferating cell nuclear antigen (PCNA) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) based untargeted metabolomics. Low concentrations of ZEN (25 nm), α-ZEL (10 nm), or a combination of both were observed to significantly enhance cell proliferation of Ishikawa cells, as evidenced by PCNA immunostaining, immunoblotting as well and clonogenic assays. The metabolomics revealed the perturbations in glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and phenylalanine, tyrosine, tryptophan biosynthesis provides valuable insights into potential mechanism by which these mycotoxins may facilitate cell proliferation. However, further investigations are warranted to comprehensively understand the implications of these findings and their possible implications for human health.

New metabolites of methyltrienolone by in vitro human microsome and characterized using Liquid Chromatography/High Resolution Mass Spectrometry for doping control purposes.

Methyltrienolone (17ß-hydroxy-17α-methylestra-4,9,11-trien-3-one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti-Doping Agency (WADA). The biotransformation of methyltrienolone is performed in vitro by human hepatocytes microsomes. Both phase I and phase II experiments are investigated. The incubation samples were extracted and injected to Liquid Chromatography/High Resolution Mass Spectrometry (LC-HRMS), or reacted with methoxylamine (29 Da added) or hydroxylamine (15 Da added), to increase the detection sensitivity. The extracted ion chromatograms of the negative control and the positive samples are compared, and 7 groups of phase I metabolites were found. The metabolic pathways including 17-epimerization, mono-hydroxylation, dihydroxylation, reduced combining mono- or dihydroxylation, as well as 18-nor which are observed and tentatively identified according to the mass spectra by high resolution mass spectrometry. The 18-nor metabolites will potentially be the long-term metabolite in doping control analysis. Its main phase II metabolites, glucuronides of metabolites are all obtained and analyzed.

Implementation of a community-based LC-UV drug checking service: promising preliminary findings on feasibility and validity.

BACKGROUND: The increasing diversity of psychoactive substances on the unregulated drug market poses significant health, psychological, and social risks to people who use drugs (PWUD). To address these risks, various harm reduction (HR) policies have been implemented, including drug checking services (DCS). Many analytical methods are used for DCS. While qualitative methods (e.g., thin layer chromatography, spectroscopy) are easier to implement, they are not as accurate as quantitative methods (e.g., LC-UV, LC-MS). Some HR programmes have implemented high-performance liquid chromatography coupled with UV detection (LC-UV). This article presents the cross-validation of this quantitative method with a reference liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) method. METHODS: Drug samples were provided by PWUD to a DCS called DrugLab in Marseille, France. The samples were weighed and prepared through dissolution in methanol, followed by ultrasonic bathing. Samples were analysed onsite using LC-UV analysis. They were then subsequently analysed with the reference LC-HRMS method. The LC-UV instrument in DrugLab was calibrated after being purchased; analysis of standard solutions was routinely performed once a month and after maintenance operations. For the LC-HRMS instrument, calibration and quality control procedures followed European Medicines Agency (EMA) guidelines. Statistical analyses were conducted including Spearman correlation tests using IBM® SPSS® Statistics version 20. RESULTS: A total of 102 samples representing different product classes and cutting agents were cross-validated. Differences between both analyses methods for each molecule analysed were ≤ 20%, with significant correlations between both methods' results for most substances. Notably, LC-HRMS provided lower concentration values for cocaine and acetaminophen, whereas it provided higher values for other substances. Correlations were significant for cocaine, ketamine, MDMA, heroin, amphetamine, caffeine, acetaminophen, and levamisole. CONCLUSIONS: This study demonstrates that the results provided by DrugLab were accurate and reliable, making LC-UV an adaptable, stable, and suitable analytical method for simple matrices like drugs in a DCS context. However, this cross validation does not guarantee accuracy over time. A proficiency test project in HR laboratories across France is currently under development in order to address potential drifts in LC-UV accuracy.

Characterization of disulfide bridges containing cyclic peptide Linaclotide and its degradation products by using LC-HRMS/MS.

Linaclotide (LINA) is a first-in-class guanylate cyclase agonist used for treating irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation. Stress degradation studies were performed to examine LINA's intrinsic stability, adhering to International Council for Harmonisation of Technical ICH) guidelines Q1A (R2). The current study endeavours to elucidate the stability behavior of LINA by exposing various stress conditions. A simple LC method was developed for effective separation of all LINA degradation products using a Waters Symmetry C18 column (150 ×4.6 mm, 3.5 µm) as the stationary phase. The generated degradation products were identified and characterized by using high-resolution mass spectrometry (LC-HRMS), MS/MS studies. The mechanistic fragmentation pathway for the seven degradation products was established and the chemical structure for the identified degradation products was elucidated. LINA was susceptible to degrade under acidic, basic, neutral, photolytic, and oxidative conditions. A total of three Pseudo DPs, DP-1, DP-2, and DP-3, were formed under acidic conditions while using methanol as the co-solvent. Additionally, degradation products (DPs) were identified: DP-4 formed under basic stress condition and DP-5 under neutral, thermal, and photolytic conditions. Furthermore, DP-6 and DP-7 were formed under oxidative stress condition. This study established the mechanistic fragmentation pathways and elucidated the chemical structures of the degradation products, offering valuable insights for generics and novel formulation drug development.

Histopathological and biochemical evaluation of the protective efficacy of Prunus spinosa L. extract in a rat model of indomethacin-induced gastric ulcer.

Objectives: Some species of Prunus L. are popularly used to treat gastric ulcers. However, the possible healing mechanisms of the anti-ulcer activity of P. spinosa, which has proven antioxidant, anti-inflammatory, and wound-healing properties, are unclear. Materials and Methods: Ethanol extracts of P. spinosa fruits were administered orally at 100 mg/kg and 200 mg/kg to Wistar albino rats, with an indomethacin-induced gastric ulcer model. The ulcerous areas on the stomach surface were examined macroscopically. Tissues were examined histopathologically and biochemically. LC-HRMS revealed the phytochemical content. Results: TNF-α, IL-6, IL-1ß, IL-8, and NF-kB levels were higher in the gastric ulcer group than in the extract groups. The VEGF values did not differ in each group. A significant difference was found between the lansoprazole group and the high-dose P. spinosa group regarding PGE2 levels. A histopathologically significant difference was observed between the healthy group and the indomethacin-applied groups in terms of neutrophilic infiltration of the gastric mucosa. Ascorbic acid (1547.521 µg/g), homoprotocatechuic acid (1268.217 µg/g), and genistein (1014.462 µg/g) were found as the main compounds in the P. spinosa extract by LC-HRMS. Conclusion: Our results demonstrated that P. spinosa protected the gastric mucosa from inflammation and also modulated the PGE2 pathway. When considered in terms of TNF-α, IL-1ß, IL-8, IL-6, PGE2, and NF-kB values, it can be concluded that it has a similar or even more positive effect than the reference substance. P. spinosa showed its effects in a dose-dependent manner.

Investigating drug-gut microbiota interactions: reductive and hydrolytic metabolism of oral glucocorticoids by in vitro artificial gut microbiota.

Elucidation of the role of gut microbiota in the metabolism of orally administered drugs may improve therapeutic effectiveness and contribute to the development of personalized medicine. In this study, ten different artificial gut microbiota (AGM), obtained by culturing fecal samples in a continuous fermentation system, were challenged for their metabolizing capacity on a panel of six glucocorticoids selected from either prodrugs or drugs. Data from metabolic stability assays highlighted that, while the hydrolysis-mediated conversion of prodrugs to drugs represented only a minor metabolic pathway, significant differences in the stability of parent compounds and in their conversion rates to multiple reductive metabolites were obtained for the selected drugs. In the latter case, a taxonomic composition-dependent ability to convert parent drugs to metabolites was observed. Indeed, the artificial microbial communities dominated by the genus Bacteroides showed the maximal conversion of parent glucocorticoids to several metabolites. Furthermore, the effect of drugs on AGM was also evaluated through shallow shotgun sequencing and flow cytometry-based total bacterial cell count highlighting that these drugs can affect both the taxonomic composition and growth performances of the human gut microbiota.

Investigating the effects of thermal processing on bitter substances in atemoya (Annona cherimola × Annona squamosa) through sensory-guided separation.

Atemoya (Annona cherimola × Annona squamosa) is a specialty crop in Taiwan. Thermal treatment induces bitterness, complicating seasonal production adjustments and surplus reduction. In this research, sensory-guided separation, metabolomics, and orthogonal partial least squares discrimination analysis (OPLS-DA) are used for identifying the bitterness in atemoya which originates from catechins, epicatechin trimers, and proanthocyanidins. Different thermal treatments (65 °C, 75 °C, and 85 °C) revealed that the glucose and fructose contents in atemoya significantly decreased, while total phenols, flavonoids, and tannins significantly increased. The concentration of 5-hydroxymethylfurfural (5-HMF) increased from 23.16 ng/g in untreated samples to 400.71 ng/g (AP-65), 1208.59 ng/g (AP-75), and 2838.51 ng/g (AP-85). However, these levels are below the 5-HMF bitterness threshold of 3780 ng/g. Combining mass spectrometry analysis with sensory evaluation, OPLS-DA revealed that atemoya treated at 65 °C, 75 °C, and 85 °C exhibited significant bitterness, with the main bitter components being proanthocyanidin dimers and trimers.

Untargeted Liquid Chromatography-High-Resolution Mass Spectrometry Metabolomic Investigation Reveals Altered Lipid Content in Leishmania infantum Lacking Lipid Droplet Protein Kinase.

Leishmaniasis is a complex disease caused by different species of Leishmania. To date, no vaccine for humans or ideal therapy has been developed owing to the limited efficacy and toxicity of available drugs, as well as the emergence of resistant strains. Therefore, it is necessary to identify novel therapeutic targets and discover therapeutic options for leishmaniasis. In this study, we evaluated the impact of deleting the lipid droplet protein kinase (LDK) enzyme in Leishmania infantum using an untargeted metabolomics approach performed using liquid chromatography and high-resolution mass spectrometry. LDK is involved in lipid droplet biogenesis in trypanosomatids. Thirty-nine lipid metabolites altered in the stationary and logarithmic growth phases were noted and classified into five classes: (1) sterols, (2) fatty and conjugated acids, (3) ceramides, (4) glycerophosphocholine and its derivatives, and (5) glycerophosphoethanolamine and its derivatives. Our data demonstrated that glycerophosphocholine and its derivatives were the most affected after LDK deletion, suggesting that the absence of this enzyme promotes the remodeling of lipid composition in L. infantum, thus contributing to a better understanding of the function of LDK in this parasite.

Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS.

Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of "Aspergillus unguis isolate SP51-EGY" on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1ß, and IL-6 through the NF-κB signaling pathway. In conclusion, "Aspergillus unguis isolate SP51-EGY", isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.

Evaluation of Nontargeted Mass Spectral Data Acquisition Strategies for Water Analysis and Toxicity-Based Feature Prioritization by MS2Tox.

The machine-learning tool MS2Tox can prioritize hazardous nontargeted molecular features in environmental waters, by predicting acute fish lethality of unknown molecules based on their MS2 spectra, prior to structural annotation. It has yet to be investigated how the extent of molecular coverage, MS2 spectra quality, and toxicity prediction confidence depend on sample complexity and MS2 data acquisition strategies. We compared two common nontargeted MS2 acquisition strategies with liquid chromatography high-resolution mass spectrometry for structural annotation accuracy by SIRIUS+CSI:FingerID and MS2Tox toxicity prediction of 191 reference chemicals spiked to LC-MS water, groundwater, surface water, and wastewater. Data-dependent acquisition (DDA) resulted in higher rates (19-62%) of correct structural annotations among reference chemicals in all matrices except wastewaters, compared to data-independent acquisition (DIA, 19-50%). However, DIA resulted in higher MS2 detection rates (59-84% DIA, 37-82% DDA), leading to higher true positive rates for spectral library matching, 40-73% compared to 34-72%. DDA resulted in higher MS2Tox toxicity prediction accuracy than DIA, with root-mean-square errors of 0.62 and 0.71 log-mM, respectively. Given the importance of MS2 spectral quality, we introduce a "CombinedConfidence" score to convey relative confidence in MS2Tox predictions and apply this approach to prioritize potentially ecotoxic nontargeted features in environmental waters.

Prioritization of the Secondary Metabolites for the Rapid Annotation Based on Liquid Chromatography-High Resolution Mass Spectrometry Assessment: Varanasine and Schroffanone from Murraya paniculata and Cytotoxic Evaluation.

The liquid chromatography-high resolution mass spectrometry (LC-HRMS) technique enables the detection of phytochemicals present in the extracts. LC-HRMS-generated mass list showed abundant compounds of interest, artifacts, and primary metabolites. The identification of a secondary metabolite of interest within the extract is very challenging. We hypothesized that identifying the "new metabolite" in the whole metabolome is more challenging than identifying it within the class of metabolites. The proposed prioritization strategy focused on the elimination of unknown and prioritizing the known class of secondary metabolites to identify new metabolites. The prioritization strategy demonstrated on Murraya paniculata for the identification of new metabolites. LC-HRMS-generated information is used as a filter to target the secondary metabolite and the new metabolites. This strategy successfully annotated the new coumarin and coumarin alkaloids from the mass list of 1448 metabolites. Varanasine (3), schroffanone (4), schroffanene (5), and O-methylmurraol (9) are new compounds, and coumarin (1, 2, and 6-8) are known. Varanasine (3) is the first naturally occurring 7-aminocoumarin with additional N-formyl functionality. The isolates were screened for cytotoxicity against the panel of cancer cell lines. Varanasine (3) and minumicrollin (6) showed significant cytotoxicity and apoptosis-inducing potential. The immunoblot analysis confirmed inhibition of apoptotic protein PARP-1 and caspase-3 expression by 3 and 6.