Liquid Chromatography Electrospray Ionization High-resolution Mass Spectrometry Research Articles

Quantification Approaches in Non-Target LC/ESI/HRMS Analysis: An Interlaboratory Comparison.

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15× and over 83% of compounds quantified within 10× error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

Quantitative Determination of the Cytotoxic Compounds in Different Organs of Arctium minus (Hill) Bernh. by LC-HRESIMS Using Respond Survey Methodology.

Arctium minus (Hill) Bernh., commonly known as "Burdock", is a species within the Arctium genus of the Asteraceae family. Determining the optimum extraction conditions to obtain a concentrated extract with targeted active ingredients guides the most efficient use of natural products. Herein, ultrasound-assisted extraction (UAE) was optimized by using response surface methodology (RSM) to extract bioactive compounds from different organs of A. minus. Furthermore, phytochemical composition of extracts of the A. minus was investigated by using liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS), antioxidant potential by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), diazanium;3-ethyl-2-[(3-ethyl-6-sulfonato-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonate (ABTS), CUPRAC, and metal chelation assays, and cytotoxic activities by using human breast cancer cell line (MDA-MB-231) and hepatocellular carcinoma cancer cell line (HepG2), and compared against conventional methods; Soxhlet and maceration. The RSM was employed to investigate the influence of ultrasound power, extraction time, and extraction temperature on the antioxidant potential assessed by the DPPH free radical scavenging assay. In UAE of A. minus leaves, flowers, and branches, the conditions resulting in the minimum IC50 values: 20 °C for 6 min at 50 W for leaves, 20 °C for 3 min at 100 W for flowers, and 20 °C for 3 min at 100 W for branches. Chlorogenic acid was identified as the major phenolic compound in the extracts obtained by UAE, with concentrations of 24,666.96 μg/g in leaves, 1054.92 μg/g in flowers, and 3,501.24 μg/g in branches. Flowers of A. minus had significantly higher levels of arctiin and arctigenin than those of leaves and branches. Extracts from leaves and flowers were more effective against MDA-MB-231 and HepG2 cancer cell lines than arctiin and arctigenin.

Pharmacokinetic study of osilodrostat and identification of mono-hydroxylated metabolite in equine plasma for the purpose of doping control.

Osilodrostat is an inhibitor of 11-beta-hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma. Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre- and post-administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS). [Correction added on 27 January 2023, after first online publication: In the preceding sentence, "C-HRMS" was changed to "LC/ESI-HRMS" in this version.] For quantification of osilodrostat, a strong cation exchange solid-phase extraction was employed, and the extracts were analyzed using LC/ESI-triple quadrupole tandem mass spectrometry (LC/ESI-QqQ-MS/MS)to establish its elimination profile. Such extracts were further analyzed using LC/ESI-HRMS to investigate the detectability of osilodrostat and its identified mono-hydroxylated metabolite over a 2-week sampling period. Mono-hydroxylated osilodrostat was identified based on the differential analysis and mass spectrometric interpretations, and it was found to be the most abundant metabolite in plasma. Elimination profile of osilodrostat in plasma was successfully established over the 24-h post-administration period. Both osilodrostat and its mono-hydroxylated metabolite were detected up to the last sampling point at 2weeks using HRMS, and osilodrostat could be confirmed up to 8-day post-administration with its reference material using HRMS as well. For doping control, screening of both the parent drug osilodrostat and its mono-hydroxylated metabolite in equine plasma would be recommended due to their extended detection windows of up to 2weeks. Given the availability of reference material for potential confirmation in forensic samples, osilodrostat is considered the most appropriate monitoring target.

Apple extract protects against indomethacin-induced gastric ulcers in rats by suppressing oxidative stress – The implication of Nrf-2/HO-1 signaling pathway: In silico and in vivo studies

Gastric ulcers represent a worldwide health problem, characterized by erosions with different degrees in the gastrointestinal mucosa. In the current study, we explore the protective effects of the extract of apple seeds (Malus domestica, family: Rosaceae), regarded as environmental wastes, against indomethacin-induced gastric ulcers in rats. The chemical composition of the seeds extract was investigated through liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). The metabolomics profiling resulted in identification of 17 compounds (1–17) of different chemical classes. The gene ontology analysis demonstrated that the total extract of M. domestica seeds possess an antiulcer activity through PI3K-Akt signaling pathway as the top identified signaling pathway. The present research work evidenced the reuse of apple seeds waste as an effective therapy to treat NSAIDs-induced gastric ulcer, attributed to its anti-inflammatory and antioxidant properties. It is important to note that the extract does not cause the undesirable effects of traditional antisecretory drugs.

Moutai Distiller's grains Polyphenol extracts and rutin alleviate DSS-induced colitis in mice: Modulation of gut microbiota and intestinal barrier function （R2）

Distiller's grains, byproducts of the brewing process, represent a valuable resource for extracting natural phenolic compounds due to their significant global production. This study presents the first evidence of the protective effects of Moutai distiller's grain polyphenol extract (MDGP) on dextran sulfate sodium (DSS)-induced colitis in mice. These protective effects manifest predominantly through the amelioration of general colitis indices and histopathological improvements. Utilizing liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS), the main components of MDGP were identified as rutin, quercetin, naringenin, and dihydroquercetin. Moreover, a novel mechanism was elucidated by which rutin, the primary active component of MDGP, alleviates DSS-induced colitis. Assessment of intestinal barrier function, microbial sequencing, fecal transplantation, and antibiotic depletion experiments revealed that rutin suppresses the abundance of pathogenic bacteria (Helicobacter, Klebsiella, and Veillonella) while promoting the proliferation of beneficial bacteria (Ruminococcus_torques_group, Lachnoclostridium, and norank_f__Muribaculaceae). This modulation culminates in elevated butyric acid concentrations within short-chain fatty acids (SCFAs), amplified integrity of tight (ZO-1, occludin) and adherent (E-cadherin, β-catenin) junctional complexes, fortified intestinal barrier function, and diminished intestinal inflammation.This investigation accentuates the innovative therapeutic potential of MDGP and its main active component, rutin, in assuaging DSS-induced intestinal inflammation and fortifying the intestinal barrier through a mechanism predominantly mediated by the intestinal microbiota. Such insights potentially elevate the prominence of distiller's grains in the realm of functional food development.

Dose-dependent Action of Zingiber officinale on Colonic Dysmotility and Ex Vivo Spontaneous Intestinal Contraction Modulation.

Ginger (Zingiber officinale) rhizomes are commonly used in foods and employed for many ailments including gastrointestinal disorders. Our main objective was to evaluate the effect of Zingiber officinale aqueous extract (ZOAE) on gastrointestinal (GI) physiological motility and colonic dysmotility. Thereby, Wistar rats were given loperamide (LP, 3 mg/kg, b.w.) and ZOAE (75, 150, and 300 mg/kg, b.w.) or yohimbine (YOH, 2 mg/kg, b.w.). ZOAE-action on intestinal secretion was assessed using Ussing chamber technique and intestinal motility with isometric transducer. GI-transit (GIT) and gastric emptying (GE) were evaluated with the charcoal meal test and the red phenol methods. ZOAE-bioactive components were analyzed by liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS). Constipation was induced with LP and the different indicators such as stool composition, GIT, oxidative stress biological parameters, and colonic mucosa histological alteration were performed. Anti-constipation effect of ZOAE was confirmed on stool composition, GIT (53.42% to 85.57%), GE (55.47% to 98.88%), and re-established oxidative balance. ZOAE induces an amplitude increase of spontaneous intestinal contraction with EC50 of 10.52 μg/mL. No effect of ZOAE was observed on electrogenic transport of intestinal fluid. These findings suggest that ZOAE-bioactive candidates might exert an anti-constipation action and spontaneous intestinal contraction modulation.

HPLC-ESI-HRMS2 Determination of N-(2-Hydroxyethyl)-l-valyl-l-leucine in Human Urine: Method Validation.

Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N-terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25-10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98-100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10-150 weeks differed in the range of 2.2-9.7%. The stability study on urine samples (-20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring.

Guide to Semi-Quantitative Non-Targeted Screening Using LC/ESI/HRMS.

Non-targeted screening (NTS) with reversed phase liquid chromatography electrospray ionization high resolution mass spectrometry (LC/ESI/HRMS) is increasingly employed as an alternative to targeted analysis; however, it is not possible to quantify all compounds found in a sample with analytical standards. As an alternative, semi-quantification strategies are, or at least should be, used to estimate the concentrations of the unknown compounds before final decision making. All steps in the analytical chain, from sample preparation to ionization conditions and data processing can influence the signals obtained, and thus the estimated concentrations. Therefore, each step needs to be considered carefully. Generally, less is more when it comes to choosing sample preparation as well as chromatographic and ionization conditions in NTS. By combining the positive and negative ionization mode, the performance of NTS can be improved, since different compounds ionize better in one or the other mode. Furthermore, NTS gives opportunities for retrospective analysis. In this tutorial, strategies for semi-quantification are described, sources potentially decreasing the signals are identified and possibilities to improve NTS are discussed. Additionally, examples of retrospective analysis are presented. Finally, we present a checklist for carrying out semi-quantitative NTS.

Exploring clinically isolated Staphylococcus sp. bacteriocins revealed the production of amonabactin, micrococcin, and α-circulocin.

Background and Objectives:Bacteriocins are considered alternative non-conventional antimicrobials produced by certain bacteria with activity against closely related species. The present study focuses on screening, characterization, and partial purification of bacteriocins produced by Staphylococcus sp. isolated from different clinical sources such as pus and blood.Materials and Methods:A total of 100 Staphylococcus isolates were screened for bacteriocin production using spot on lawn assay and agar diffusion method against five indicator bacteria. Bacteriocins from five selected highly active isolates were subjected to proteinase-K enzyme, different pH, and heating at different temperatures, and investigated the stabilities of their antimicrobials. Two selected isolates, MK65 and MK88, were molecularly identified by 16S rRNA gene sequencing, explored for the presence of 18 bacteriocin genes, and liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) was used to identify their different metabolites.Results:Twenty isolates exhibited inhibitory effect against at least one indicator bacteria. Micrococcus luteus ATCC 4698 showed the highest sensitivity to such bacteriocins. Proteinase K, acidic pH, and heating at 100°C triggered marked activity inhibition. However, amylase enzyme, alkaline pH, and heating at 80°C caused trivial effects. Four out of eighteen bacteriocin genes were detected using PCR. Fermentation, partial purification, and LC-HRESIMS of total protein extracts of two selected isolates, MK65 and MK88, revealed the production of different antimicrobial peptides.Conclusion:To the best of our knowledge, this is the first study to report the production of micrococcin and α-circulocin from Staphylococcus aureus MK65 and the production of amonabactin from Staphylococcus epidermidis MK88.

Momordicine-I, a Bitter Melon Bioactive Metabolite, Displays Anti-Tumor Activity in Head and Neck Cancer Involving c-Met and Downstream Signaling.

Simple SummaryThe incidence of head and neck cancer (HNC), one of the most aggressive cancers, is increasing rapidly globally. Conventional and targeted therapies show limited success with several undesirable side effects. Thus, there is a critical clinical need to identify additional alternative therapeutic strategies for successfully managing the disease. Preclinical and clinical studies indicate the crucial roles of dietary phytochemicals to manage different cancers. We and others previously showed the potential anticancer effect of bitter melon extract (BME) to prevent various cancers, including HNC. In this study, we identified momordicine-I (M-I) as a bioactive component in the BME. Subsequent mechanistic study showed that M-I inhibited HNC cell (JHU022, JHU029, Cal27) proliferation involving c-Met and downstream signaling. In pre-clinical mouse models, M-I showed similar effectiveness to prevent HNC tumor growth in mice with no apparent toxic side effect, suggesting an additional option for HNC therapy.Head and neck cancer (HNC) is one of the most aggressive cancers, and treatments are quite challenging due to the difficulty in early diagnosis, lack of effective chemotherapeutic drugs, adverse side effects and therapy resistance. We identified momordicine-I (M-I), a bioactive secondary metabolite in bitter melon (Momordica charantia), by performing liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. M-I inhibited human HNC cell (JHU022, JHU029, Cal27) viability in a dose-dependent manner without an apparent toxic effect on normal oral keratinocytes. Mechanistic studies showed that M-I inhibited c-Met and its downstream signaling molecules c-Myc, survivin, and cyclin D1 through the inactivation of STAT3 in HNC cells. We further observed that M-I was non-toxic and stable in mouse (male C57Bl/6) blood, and a favorable pharmacokinetics profile was observed after IP administration. M-I treatment reduced HNC xenograft tumor growth in nude mice and inhibited c-Met and downstream signaling. Thus, M-I has potential therapeutic implications against HNC.

Detection and longitudinal distribution of GW1516 and its metabolites in equine hair for doping control using liquid chromatography/high-resolution mass spectrometry.

GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using a Q-Exactive instrument. This method was successfully validated and applied to post-administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516. After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), and 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post-administration and GW1516 could be detected and confirmed up to 6 months post-administration. The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q‐Exactive high‐resolution mass spectrometry for doping control

The use of GW1516, a peroxisome proliferator-activated receptor δ (PPAR δ) agonist, is strictly prohibited in both horseracing and equestrian competitions. However, little is known about its metabolic fate in horses. To the best of our knowledge, this is the first reported metabolic study of GW1516 in equine urine. Urine samples obtained from a thoroughbred after nasoesophageal administration with GW1516 were protein-precipitated and the supernatants were subsequently analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) with a Q-Exactive mass spectrometer. Monoisotopic ions of GW1516 and its metabolites were monitored from the full-scan mass spectral data of pre- and post-administration samples. A quantification method was developed and validated to establish the excretion profiles of GW1516, its sulfoxide, and its sulfone in equine urine. GW1516 and its nine metabolites [including GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), methyl 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylate (MMTC), 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), and M1 to M4] were detected in post-administration urine samples. GW1516 sulfoxide and GW1516 sulfone showed the longest detection times in post-administration urine samples and were therefore recommended as potential screening targets for doping control purposes. Quantitative analysis was also conducted to establish the excretion profiles of GW1516 sulfoxide and GW1516 sulfone in urine. For the purposes of doping control of GW1516, the GW1516 sulfoxide and GW1516 sulfone metabolites are recommended as the target analytes to be monitored in equine urine due to their high specificities, long detection times (1 and 4 weeks, respectively), and the ready availability of their reference materials.

Phenolic Compounds Characterization and Antioxidant Properties of Monocultivar Olive Oils from Northeast Algeria

In Algeria, the olive tree is one of the main fruit species and plays a very important socioeconomic role. The objective of this study was firstly, to identify and quantify the phenolics of some Algerian olive oils, and secondly, to assess the antioxidant activity of the samples. The olive oils used in this study were derived from Algerian cultivars, including Tefahi, Gelb Elfarroudj, Chemlal, and imported cultivar Manzanilla and Zebboudj. For this purpose, gas chromatography—mass spectrometry (GC-MS) was used to identify olive oil fatty acids profile, while the individual phenolic compounds were assessed by ultra-high-performance liquid chromatography–electrospray ionization–high-resolution mass spectrometry (UHPLC-HESI-MS). To verify the antioxidant capacity, five in vitro free radical assays were used. Questionable values of particular physico-chemical parameters, such as the high value of free acidity and the low concentration of monounsaturated fatty acids in oil from the Zebboudj cultivar, indicate that improvements in olive cultivation and oil production practices are needed. Gelb Elfarroudj, Tefahi, and Manzanilla oils contain quantities of monounsaturated fatty acids in accordance with EU regulations. The oil obtained from the Zebboudj cultivar is not usable for food purposes due to the high value in free acidity and the low concentration of monounsaturated fatty acids. Tefahi and Manzanilla cultivars have given oils with the best antioxidant activity as compared to other studied cultivars; this is attributable to their composition in bioactive phenolic compounds, such as secoiridoids, which play an important role in human health as scavengers of free radicals. The results are interesting for producers and consumers to promote the culture of olive oils derived in particular from the Tefahi cultivar. However, in order to improve the health qualities of this oil, the agronomic techniques essentially linked to the time of harvesting of the olives destined for oil production must be improved.

The biosurfactants iturin, lichenysin and surfactin, from vaginally isolated lactobacilli, prevent biofilm formation by pathogenic Candida.

Lactic acid bacteria (LAB), particularly lactobacilli, are major components of the vaginal microbiota. Lactobacilli are facultative anaerobes forming a critical line of defense against pathogenic microorganisms, including those forming biofilms, such as Candida spp. This study aimed to investigate the anti-adhesion capabilities of vaginal Lactobacillus isolates against biofilms formed by pathogenic Candida species. When the extracellular biosurfactant activities of culture supernatants from 120 Lactobacillus isolates were evaluated by the oil-spreading method, clear spreading zones were recognized. Biofilm formation was quantified by the crystal violet plate assay, and different isolates exhibited anti-adhesion activity that ranged from 65.6to 74.4% inhibition against Candida spp. biofilms. Liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HRESIMS) identified biosurfactants, extracted from three representative Lactobacillus isolates, as surfactin, iturin and lichenysin. Finally, the distribution of representative genes from six different biosynthetic clusters, related to the production of different biosurfactants, was investigated by the polymerase chain reaction. In conclusion, surfactin, iturin and lichenysin were identified for the first time in vaginal Lactobacillus spp. These biosurfactants, which showed strong anti-adherence activity may be used as promising antibiofilm agents in equipment care to prevent vaginal infections by pathogenic Candida spp. with the prospect of reducing nosocomial infections.

Liquid chromatography high-resolution mass spectrometry analysis, phytochemical and biological study of two aizoaceae plants: A new kaempferol derivative from Trianthema portulacastrum L.

Background: Natural remedies used for the treatment of liver diseases have a major concernworldwide. Objectives: Evaluation of the cytotoxic potential of successive fractions of two Aizoaceae plants; Trianthema portulacastrum L. and Aizoon canariense L. against human hepatocellular carcinoma (HepG2) cell lines. Moreover, metabolomic profiling of the successive fractions of both plants was carried out. Materials and Methods: Cytotoxic activity of successive fractions of the two plants against hepatocellular carcinoma (HCC) HepG2 cell lines were evaluated using viability assay, whereas metabolomic profiling was carried out using Liquid Chromatography High-Resolution ElectroSpray Ionization Mass Spectrometry (LC-HR-ESI-MS). Results: Significant cytotoxic activity of the n-hexane and n-butanol extracts of A. canariense (24.7 ± 3.5 and 55.3 ± 4.9 μg/mL, respectively) is recorded. On the other hand, metabolomic profiling of both plants resulted in dereplication of 27 metabolites belonging to different chemical classes; for example, sterols, flavonoids, diterpenes, triterpenes, tetraterpenes, alkaloids, lignans, hydrocarbons, and nucleosides. Phytochemical study of the biologically active fractions resulted in the isolation of one new compound; kaempferol-3-O-(2''-O-β-D-glucopyranosyl)-6''-O-E-feruloyl-β-D-glucopyranosid (T1). Biological testing of the isolated compounds indicated significant activity of T1 against HCC (IC50 =7.19 ± 0.27 μg/mL). Conclusion: Phytochemicals isolated from T. portulacastrum L and A. canariense L. may be responsible for their cytotoxic activity against HCC HepG2 cell lines.

In vitro metabolism of olaparib in liver microsomes by liquid chromatography/electrospray ionization high-resolution mass spectrometry.

Olaparib is a Poly (ADP-ribose) Polymerase (PARP) inhibitor which has been developed as an anti-cancer agent. The purpose of this study was to characterize the metabolites of olaparib from liver microsomes and to reveal the interspecies differences between animals and humans. Olaparib (20 μM) was incubated with different species of liver microsomes at 37°C for 1 h in the presence of NADPH. The incubation samples were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) operated in positive ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. A total of 12 metabolites were detected and the structures of the metabolites were characterized based on their accurate masses, fragment ions and retention times. Four metabolites, i.e., M1, M10, M11 and M12, were unambiguously identified by using reference standards. The metabolic pathways of olaparib included hydroxylation, bis-hydroxylation, hydrolysis, dealkylation, dehydrogenation, and alcohol oxidation. Compared with animal species, no human-specific metabolite was found in HLM. Dog also had a closer metabolic profile to humans. This study will be helpful for a better understanding of the species difference in pharmacokinetics/pharmacodynamics.

Exploring the Phytochemical Landscape of the Early-Diverging Flowering Plant Amborella trichopoda Baill.

Although the evolutionary significance of the early-diverging flowering plant Amborella (Amborella trichopoda Baill.) is widely recognized, its metabolic landscape, particularly specialized metabolites, is currently underexplored. In this work, we analyzed the metabolomes of Amborella tissues using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). By matching the mass spectra of Amborella metabolites with those of authentic phytochemical standards in the publicly accessible libraries, 63, 39, and 21 compounds were tentatively identified in leaves, stems, and roots, respectively. Free amino acids, organic acids, simple sugars, cofactors, as well as abundant glycosylated and/or methylated phenolic specialized metabolites were observed in Amborella leaves. Diverse metabolites were also detected in stems and roots, including those that were not identified in leaves. To understand the biosynthesis of specialized metabolites with glycosyl and methyl modifications, families of small molecule UDP-dependent glycosyltransferases (UGTs) and O-methyltransferases (OMTs) were identified in the Amborella genome and the InterPro database based on conserved functional domains. Of the 17 phylogenetic groups of plant UGTs (A–Q) defined to date, Amborella UGTs are absent from groups B, N, and P, but they are highly abundant in group L. Among the 25 Amborella OMTs, 7 cluster with caffeoyl-coenzyme A (CCoA) OMTs involved in lignin and phenolic metabolism, whereas 18 form a clade with plant OMTs that methylate hydroxycinnamic acids, flavonoids, or alkaloids. Overall, this first report of metabolomes and candidate metabolic genes in Amborella provides a starting point to a better understanding of specialized metabolites and biosynthetic enzymes in this basal lineage of flowering plants.

Effect of Phoenix dactylifera L. Sap Against Bleomycin-Induced Pulmonary Fibrosis and Oxidative Stress in Rats: Phytochemical and Therapeutic Assessment

Lung fibrosis is a lethal interstitial disease characterized by massive proliferation of fibroblast inducing excessive collagen deposition. We aimed to investigate whether Date palm sap (DPS) can play a protective effect on bleomycin (BLM)-induced lung fibrosis in rats. MaleWistar rats were given single dose of BLM (4 mg/kg, intra-tracheal), while DPS (45 mg/kg, intraperitoneal) was administered three days later and continued for three weeks (BLM/DPS group). Characterization of phenolic compounds in DPS was evaluated by LC-HRESIMS analysis. Hematoxylin-eosin and Masson’s Trichome staining were used for the revelation of lung architecture, collagen deposition, and fibrosis score. Antioxidant effects of DPS and hydroxyproline content in lung tissues were studied using standard spectrophotometric methods. We reveled by liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HRESIMS) an important amount of vitamins and phenolic compounds in DPS. BLM increased lipid peroxidation (MDA) and superoxide dismutase (SOD) levels and decreased catalase (CAT) activity. BLM also induced inflammation and accumulation of bundles of collagen in lung. DPS treatment normalized MDA, SOD, and CAT levels, decreased hydroxyproline level and morphological lesions induced by BLM. In conclusion, DPS has a protective effect against BLM-induced murine lung fibrosis due to its richness in phenolic compounds and vitamins.

UHPLC-HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities.

Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R.fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography-high resolution-electrospray ionization-mass spectrometry (UHPLC-HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C.glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularlyactive against human pathogenic fungi with MIC80 values ranging from 8 to 62.5μg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.

A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore.

The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes.IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.