Nucleos'ID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides.

As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.

Selenoneine-inspired selenohydantoins with glutathione peroxidase-like activity.

Chronic diseases such as cystic fibrosis, inflammatory bowel diseases, rheumatoid arthritis, and cardiovascular illness have been linked to a decrease in selenium levels and an increase in oxidative stress. Selenium is an essential trace element that exhibits antioxidant properties, with selenocysteine enzymes like glutathione peroxidase being particularly effective at reducing peroxides. In this study, a series of synthetic organoselenium compounds were synthesized and evaluated for their potential antioxidant activities. The new selenohydantoin molecules were inspired by selenoneine and synthesized using straightforward methods. Their antioxidant potential was evaluated and proven using classical radical scavenging and metal-reducing methods. The selenohydantoin derivatives exhibited glutathione peroxidase-like activity, reducing hydroperoxides. Theoretical calculations using Density Functional Theory (DFT) revealed the selenone isomer to be the only one occurring in solution, with selenolate as a possible tautomeric form in the presence of a basic species. Cytocompatibility assays indicated that the selenohydantoin derivatives were non-toxic to primary human aortic smooth muscle cells, paving the way for further biological evaluations of their antioxidant activity. The results suggest that selenohydantoin derivatives with trifluoro-methyl (-CF3) and chlorine (-Cl) substituents have significant activities and could be potential candidates for further biological trials. These compounds may contribute to the development of effective therapies for chronic diseases such cardiovascular diseases.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, ßAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77 mg L-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L-1 in casein hydrolysate. Graphical abstract.

New approach in the characterization of bioactive compounds isolated from Calycotome spinosa (L.) Link leaves by the use of negative electrospray ionization LITMSn, LC-ESI-MS/MS, as well as NMR analysis.

Two novel compounds were isolated for the first time from Calycotome spinosa (L.) Link, an alkaloid 5-Hydroxy-1H-indole (4) and a cyclitol D-pinitol (5), together with the three well-known flavonoids; Chrysin-7-O-(ß-D-glucopyranoside) (1), Chrysin-7-O-ß-D-(6″-acetyl)glycopyranoside (2) and Apigenin-7-O-ß-D-glycopyranoside (3). The chemical structures of the isolated compounds were elucidated by spectroscopic data and mass spectrometric analyses; including a fresh approach 1D-NMR, 2D-NMR with LC-ESI-MS/MS. In this study, the new compound (4) that has been obtained from the leaves MeOH extract presented the best radical scavenging activity (DPPH) (IC50 < 10 µg/mL) compared to the standard butylated hydroxytoluene (BHT, IC50 = 34.73 ± 0.23 µg/mL) and showed the highest total antioxidant capacity (TAC = 985.54 ± 0.13 mg AAE/g extract) in contrast to ascorbic acid (TAC = 905.95 ± 0.07 mg AAE/g extract). Furthermore, the strongest reducing power (EC50 = 344.82 ± 0.02 µg/mL), as well as the remarkable scavenging potential by ABTS assay (IC50 = 7.8 ± 0.43 µg/mL), were exhibited by the same composite (4). Followed by the methanol crude extract and the compound (3) that also showed a potent antioxidant (DPPH; IC50 = 41.04 ± 0.15 and 47.36 ± 0.21 µg/mL, TAC; 671.02 ± 0.21 and 608.67 ± 0.34 mg AAE/g extract, FRAP; EC50 = 763.73 ± 0.32 and 814.61 ± 0.31 µg/mL, ABTS; IC50 = 19.18 ± 0.06 and 63.72 ± 0.64 µg/mL, respectively), but less than the previous samples. On the opposite side, compound (5) had the lowest activity, in which its values were less interesting to determine. Moreover, compound (4) has equally exerted an attractive antibacterial activity against Staphylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATTC- 27853) and Salmonella abony (NCTC 6017), as measured by the disc diffusion assay, with inhibition zones of 16 ± 0.5, 9.83 ± 0.29 and 8 ± 0.28 mm, in that order. To the best of our knowledge, 5-Hydroxy-1H-indole was isolated from plants for the second time in our current work. Thus, the obtained results from this investigation propose that the leaves of C. spinosa are a rich natural source for value molecules as potential antioxidants and antimicrobial agents for best human health.

Higher-energy collision-induced dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitroso-glutathione in an in vitro model of the intestinal barrier.

RATIONALE: The potency of S-nitrosoglutathione (GSNO) as a nitric oxide (NO) donor to treat cardiovascular diseases (CVDs) has been highlighted in numerous studies. In order to study its bioavailability after oral administration, which represents the most convenient route for the chronic treatment of CVDs, it is essential to develop an analytical method permitting (i) the simultaneous measurement of GSNO metabolites, i.e. nitrite, S-nitrosothiols (RSNOs) and nitrate and (ii) to distinguish them from other sources (endogenous synthesis and diet). METHODS: Exogenous GSNO was labeled with 15 N, and the GS15 NO metabolites after conversion into the nitrite ion were derivatized with 2,3-diaminonaphthalene. The resulting 2,3-naphthotriazole was quantified by liquid chromatography/tandem ion trap mass spectrometry (LC/ITMS/MS) in multiple reaction monitoring mode after Higher-energy Collision-induced Dissociation (HCD). Finally, the validated method was applied to an in vitro model of the intestinal barrier (monolayer of Caco-2 cells) to study GS15 NO intestinal permeability. RESULTS: A LC/ITMS/MS method based on an original transition (m/z 171 to 156) for sodium 15 N-nitrite, GS15 NO and sodium 15 N-nitrate measurements was validated, with recoveries of 100.8 ± 3.8, 98.0 ± 2.7 and 104.1 ± 3.3%, respectively. Intra- and inter-day variabilities were below 13.4 and 12.6%, and the limit of quantification reached 5 nM (signal over blank = 4). The permeability of labeled GS15 NO (10-100 µM) was evaluated by calculating its apparent permeability coefficient (Papp ). CONCLUSIONS: A quantitative LC/ITMS/MS method using HCD was developed for the first time to selectively monitor GS15 NO metabolites. The assay allowed evaluation of GS15 NO intestinal permeability and situated this drug candidate within the middle permeability class according to FDA guidelines. In addition, the present method has opened the perspective of a more fundamental work aiming at studying the fragmentation mechanism leading to the ion at m/z 156 in HCD tandem mass spectrometry in the presence of acetonitrile.

Correlative Analysis of Fluorescent Phytoalexins by Mass Spectrometry Imaging and Fluorescence Microscopy in Grapevine Leaves.

Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.

2-(Thienothiazolylimino)-1,3-thiazolidin-4-ones inhibit cell division cycle 25 A phosphatase.

Cell division cycle dual phosphatases (CDC25) are essential enzymes that regulate cell progression in cell cycle. Three isoforms exist as CDC25A, B and C. Over-expression of each CDC25 enzyme is found in cancers of diverse origins. Thiazolidinone derivatives have been reported to display anti-proliferative activities, bactericidal activities and to reduce inflammation process. New 2-(thienothiazolylimino)-1,3-thiazolidin-4-ones were synthesized and evaluated as inhibitors of CDC25 phosphatase. Among the molecules tested, compound 6 inhibited CDC25A with an IC50 estimated at 6.2±1.0µM. The binding of thiazolidinone derivative 6 onto CDC25A protein was reversible. In cellulo, compound 6 treatment led to MCF7 and MDA-MB-231 cell growth arrest. To our knowledge, it is the first time that such 4-thiazolidinone derivatives are characterized as CDC25 potential inhibitor.

Screening of indeno[1,2-b]indoloquinones by MALDI-MS: a new set of potential CDC25 phosphatase inhibitors brought to light.

Quinones and quinones-like compounds are potential candidates for the inhibition of CDC25 phosphatases. The combination of MALDI-MS analyses and biological studies was used to develop a rapid screening of a targeted library of indeno[1,2-b]indoloquinone derivatives. The screening protocol using MALDI-TOFMS and MALDI-FTICRMS highlighted four new promising candidates. Biological investigations showed that only compounds 5c-f inhibited CDC25A and -C phosphatases, with IC50 values around the micromolar range. The direct use of a screening method based on MALDI-MS technology allowed achieving fast scaffold identification of a new class of potent inhibitors of CDC25 phosphatases. These four molecules appeared as novel molecules of a new class of CDC25 inhibitors. Assessment of 5c-e in an MRC5 proliferation assay provided an early indicator of toxicity to mammalian cells. Compound 5d seems the most promising hit for developing new CDC25 inhibitors.

Natural and Synthetic Coumarins with Effects on Inflammation.

In this review, we will present the different aspects of coumarins and derivatives, from natural origins or synthetically prepared, and their action on inflammation. Coumarins and also furo- and pyranocoumarins are found in many different plants. These compounds are very often investigated for antioxidant properties. Other biological properties are also possible and anti-inflammation activity is one of these. As coumarins are also available quite easily via synthesis, natural ones can be prepared this way but derivatives with special substituents are also feasible. A review on the same topic appeared in 2004 and our contribution will take into account everything published since then.

A novel coumarin-quinone derivative SV37 inhibits CDC25 phosphatases, impairs proliferation, and induces cell death.

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation by regulating CDK/cyclin complexes. Overexpression of these enzymes is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, targeting CDC25 by compounds, able to inhibit their activity, appears a good therapeutic approach. Here, we describe the synthesis of a new inhibitor (SV37) whose structure is based on both coumarin and quinone moieties. An analytical in vitro approach shows that this compound efficiently inhibits all three purified human CDC25 isoforms (IC50 1-9 µM) in a mixed-type mode. Moreover, SV37 inhibits growth of breast cancer cell lines. In MDA-MB-231 cells, reactive oxygen species generation is followed by pCDK accumulation, a mark of CDC25 dysfunction. Eventually, SV37 treatment leads to activation of apoptosis and DNA cleavage, underlining the potential of this new type of coumarin-quinone structure.

New Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1 for Anti-Vascular Targeted Photodynamic Therapy.

Photodynamic therapy (PDT) is a cancer treatment modality that requires three components, namely light, dioxygen and a photosensitizing agent. After light excitation, the photosensitizer (PS) in its excited state transfers its energy to oxygen, which leads to photooxidation reactions. In order to improve the selectivity of the treatment, research has focused on the design of PS covalently attached to a tumor-targeting moiety. In this paper, we describe the synthesis and the physico-chemical and photophysical properties of six new peptide-conjugated photosensitizers designed for targeting the neuropilin-1 (NRP-1) receptor. We chose a TPC (5-(4-carboxyphenyl)-10,15, 20-triphenyl chlorine as photosensitizer, coupled via three different spacers (aminohexanoic acid, 1-amino-3,6-dioxaoctanoic acid, and 1-amino-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid) to two different peptides (DKPPR and TKPRR). The affinity towards the NRP-1 receptor of the conjugated chlorins was evaluated along with in vitro and in vivo stability levels. The tissue concentration of the TPC-conjugates in animal model shows good distribution, especially for the DKPPR conjugates. The novel peptide-PS conjugates proposed in this study were proven to have potential to be further developed as future NRP-1 targeting photodynamic therapy agent.

MALDI mass spectrometry imaging for the simultaneous location of resveratrol, pterostilbene and viniferins on grapevine leaves.

To investigate the in-situ response to a stress, grapevine leaves have been subjected to mass spectrometry imaging (MSI) experiments. The Matrix Assisted Laser Desorption/Ionisation (MALDI) approach using different matrices has been evaluated. Among all the tested matrices, the 2,5-dihydroxybenzoic acid (DHB) was found to be the most efficient matrix allowing a broader range of detected stilbene phytoalexins. Resveratrol, but also more toxic compounds against fungi such as pterostilbene and viniferins, were identified and mapped. Their spatial distributions on grapevine leaves irradiated by UV show their specific colocation around the veins. Moreover, MALDI MSI reveals that resveratrol (and piceids) and viniferins are not specifically located on the same area when leaves are infected by Plasmopara viticola. Results obtained by MALDI mass spectrometry imaging demonstrate that this technique would be essential to improve the level of knowledge concerning the role of the stilbene phytoalexins involved in a stress event.

Hyphenation of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with separation methods: The art of compromises and the possible - A review.

This review provides an overview of the online hyphenation of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) with separation methods to date. The online coupling between separation techniques (gas and liquid chromatography, capillary electrophoresis) and FT-ICR MS essentially raises questions of compromise and is not look as straightforward as hyphenation with other analyzers (QTOF-MS for instance). FT-ICR MS requires time to reach its highest resolving power and accuracy in mass measurement capabilities whereas chromatographic and electrophoretic peaks are transient. In many applications, the strengths and the weaknesses of each technique are balanced by their hyphenation. Untargeted "Omics" (e.g. proteomics, metabolomics, petroleomics, …) is one of the main areas of application for FT-ICR MS hyphenated to online separation techniques because of the complexity of the sample. FT-ICR MS achieves the required high mass measurement accuracy to determine accurate molecular formulae and resolution for isobar distinction. Meanwhile separation techniques highlight isomers and reduce the ion suppression effects extending the dynamic range. Even if the implementation of FT-ICR MS hyphenated with online separation methods is a little trickier (the art of compromise), this review shows that it provides unparalleled results to the scientific community (the art of the possible), along with raising the issue of its future in the field with the relentless technological progress.

Daniellia oliveri (Rolfe) Hutch and Dalziel: Antimicrobial Activities, Cytotoxicity Evaluation, and Phytochemical Identification by GC-MS.

During a previous study that identified plants used in traditional medicine in Togo to treat infectious diseases, Daniellia oliveri was specifically reported to treat intertrigo and candidiasis. Consequently, to explore the anti-infective potential of this plant, we investigated the antibacterial and the antifungal activity of the plant's parts, as well as the cytotoxic activities of raw extracts and subsequent fractions, and the chemical composition of the most active fractions. In order to evaluate the antimicrobial activity, MICs were determined using the broth dilution method. Then, the most active fractions were evaluated for cytotoxicity by using normal human cells (MRC-5 cells) via the MTT assay. Finally, the most active and not toxic fractions were phytochemically investigated by GC-MS. Interestingly, all the raw extracts and fractions were active against the bacteria tested, with MICs ranging from 16 µg/mL to 256 µg/mL, while no antifungal activity was observed at 256 µg/mL, the highest tested concentration. Moreover, no toxicity was observed with most of the active fractions. The subsequent chemical investigation of the most interesting fractions led to identifying terpenes, phytosterols, phenolic compounds, and fatty acids as the main compounds. In conclusion, this study demonstrated that D. oliveri possesses valuable antibacterial activities in accordance with traditional use.

Protocol for Etching Bare-Fused Silica Capillaries for Sheathless Capillary Electrophoresis-Mass Spectrometry Coupling.

Homemade capillaries are a very common practice for the users of capillary electrophoresis (CE), notably in CE-UV. With the advent of the capillary electrophoresis-mass spectrometry coupling since the end of the 1980s, several interfaces have been developed. Among those interfaces, the porous tip sprayer allows great sensitivity at nano flow rates and has been used in numerous applications over the past few years. However, the homemade implementation of a suitable capillary for the porous tip sprayer is more challenging. The porous tip is created by etching the bare-fused silica capillary with hydrofluoric acid. Here we describe the complete process of etching bare-fused silica capillaries, from length cutting to quality control of the newly etched capillary.

Optimization of nucleotides dephosphorylation for RNA structural characterization by tandem mass spectrometry hyphenated with separation methods.

As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA)1 from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP)2 consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA.

Evaluation of combined matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry experiments for peptide mass fingerprinting analysis.

Peptide Mass Fingerprinting (PMF) is still of significant interest in proteomics because it allows a large number of complex samples to be rapidly screened and characterized. The main part of post-translational modifications is generally preserved. In some specific cases, PMF suffers from ambiguous or unsuccessful identification. In order to improve its reliability, a combined approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) was evaluated. The study was carried out on bovine serum albumin (BSA) digest. The influence of several important parameters (the matrix, the sample preparation method, the amount of the analyte) on the MOWSE score and the protein sequence coverage were evaluated to allow the identification of specific effects. A careful investigation of the sequence coverage obtained by each kind of experiment ensured the detection of specific peptides for each experimental condition. Results highlighted that DHB-FTICRMS and DHB- or CHCA-TOFMS are the most suited combinations of experimental conditions to achieve PMF analysis. The association (convolution) of the data obtained by each of these techniques ensured a significant increase in the MOWSE score and the protein sequence coverage.

Elicitation of Antimicrobial Active Compounds by Streptomyces-Fungus Co-Cultures.

The bacteria of the genus Streptomyces and Basidiomycete fungi harbor many biosynthetic gene clusters (BGCs) that are at the origin of many bioactive molecules with medical or industrial interests. Nevertheless, most BGCs do not express in standard lab growth conditions, preventing the full metabolic potential of these organisms from being exploited. Because it generates biotic cues encountered during natural growth conditions, co-culture is a means to elicit such cryptic compounds. In this study, we explored 72 different Streptomyces-fungus interaction zones (SFIZs) generated during the co-culture of eight Streptomyces and nine fungi. Two SFIZs were selected because they showed an elicitation of anti-bacterial activity compared to mono-cultures. The study of these SFIZs showed that co-culture had a strong impact on the metabolic expression of each partner and enabled the expression of specific compounds. These results show that mimicking the biotic interactions present in this ecological niche is a promising avenue of research to explore the metabolic capacities of Streptomyces and fungi.

New Pregnane Glycosides Isolated from Caralluma hexagona Lavranos as Inhibitors of α-Glucosidase, Pancreatic Lipase, and Advanced Glycation End Products Formation.

Caralluma hexagona Lavranos (Family Asclepiadaceae) is an endemic herb in Yemen and Saudi Arabia, traditionally used to treat diabetes, abdominal pain, and stomach ulcers. Different extracts, fractions, and main constituents of C. hexagona were evaluated for their inhibitory activity against key enzymes in diabetes and hyperlipidemia, i.e., α-glucosidase and pancreatic lipase. In addition, the antioxidative effect and inhibition of advanced glycation end products (AGEs) were also assayed. Using a bioguided approach, the crude aqueous, methanolic extracts, methylene chloride (CH2Cl2), Diaion HP20 50% MeOH (DCF-1), and 100% MeOH (DCF-2) fractions of C. hexagona were evaluated for their possible α-glucosidase and pancreatic lipase inhibition and antioxidant activity. In addition, inhibition of AGE generation using bovine serum albumin (BSA)-fructose, BSA-methylglyoxal, and arginine-methylglyoxal models was carried out. Moreover, the main constituents of the most active fraction were isolated and identified using different chromatographic and sprectroscopic methods. From the most active CH2Cl2 fraction, four new pregnane glycosides were isolated and identified as 12ß-O-benzoyl 3ß,8ß,12ß,14ß,20-pentahydroxy-(20S)-pregn-5-ene-3-O-ß-d-glucopyranosyl-(1 → 4)-O-ß-d-digitaloside (1), 3ß,8ß,14ß,20-tetrahydroxy-(20S)-pregn-5-ene-3-O-ß-d-glucopyranosyl-(1 → 4)-O-ß-d-digitaloside-20-O-3-isoval-ß-d-glucopyranoside (2), 3ß,8ß,14ß,20-tetrahydroxy-(20R)-pregn-5-ene-3-O-ß-d-glucopyranosyl-(1 → 4)-O-ß-d-digitaloside-20-O-3-isoval-4-benzoyl-ß-d-glucopyranoside (3A), and 3ß,8ß,14ß,20-tetrahydroxy-(20R)-pregn-5-ene-3-O-ß-d-glucopyranosyl-(1 → 4)-O-ß-d-digitaloside-20-O-3,4 di-benzoyl-ß-d-glucopyranoside (3B). Among the tested samples, the highest trolox equivalent (TE) antioxidant capacity (TEAC) was observed for DCF-1 with values of 128.53 ± 5.07, 378.58 ± 5.19, and 106.71 ± 5.66 µM TE/mg using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant potential (FRAP) assays, respectively. The isolated apigenin-8-C-neohesperoside showed the highest antioxidant capacity (168.80 ± 1.80 and 278.21 ± 13.26 µM TE/mM) in DPPH and FRAP, respectively, while luteolin 4'-O-ß-d-neohesperidoside had the highest TEAC (599.19 ± 9.57 µM TE/mM) in ABTS assay. Compounds 1, 2, and the mixture 3A and 3B inhibited α-glucosidase with IC50 values of 0.92 ± 0.02, 0.67 ± 0.01, and 0.74 ± 0.02 mM, respectively. In the AGE assays, DCF-1 showed the highest inhibitory effect in BSA-fructose and arginine-methylglyoxal models with IC50 values of 0.39 ± 0.02 and 0.77 ± 0.10 mg/mL, respectively. Among the isolated compounds, flavonoid compounds showed the highest antiglycation effect, while pregnanes revealed higher α-glucosidase inhibition. In conclusion, the current study revealed that C. hexagona is a promising Yemeni natural remedy, of which the major content of pregnane glycosides and flavonoids could be considered as a new therapeutic candidate targeting the metabolic syndrome.

Labeling nitrogen species with the stable isotope 15N for their measurement by separative methods coupled with mass spectrometry: A review.

Nitrogen and its numerous hydrogenated and oxygenated derivatives are of main importance in our environment and in living cells as well in both qualitative and quantitative aspects. Their monitoring is needed to evaluate all disturbances occurring in the nitrogen cycle and in pathophysiological events related to variations of nitric oxide (NO) bioavailability. Many analytical methods are devoted to the measurement of nitrogen species, especially those related to NO, in the environmental, biological and pharmacological fields, and they have already been compiled and discussed in numerous reviews. Nitrogen isotope (15N) is stable and has a low level of natural abundance. Labeling nitrogen species with 15N associated with mass spectrometry (MS) gives rise to more mechanistic information and improved analytical performances compared to conventional methods. The present review is dedicated to the 15N labeling of related nitrogen species to monitor their interconversion and metabolism, the different chemical probes used for their derivatization and the corresponding separative methods coupled with MS for analyzing resulting adducts. The fragmentation mode of the different adducts and the resulting selectivity and sensitivity are discussed.