Electrospray Research Articles

Abstract P209: The novel angiotensin Alamandine-(1-5) is increased in plasma of pediatric patients with primary nephrotic syndrome

Background and Aim: The Renin Angiotensin System (RAS) has an important role in the pathophysiology of primary nephrotic syndrome (NS). It was previously found that urinary levels of angiotensin converting enzyme 2 (ACE2) are reduced in patients with NS in comparison to health controls. The aim of this study was to measure the novel angiotensin peptide, Alamandine-(1-5), in plasma samples of patients with primary NS and to compare with sex and age-matched controls. Methods: Blood samples of pediatric patients with primary NS (n=11) and of sex and age-matched health controls (n=11) were collected in sterile tubes with 7.5% (w/v) EDTA. After plasma separation and purification by solid phase extraction, the circulating levels of Alamandine and of Alamandine-(1-5) were measured by Mass Spectrometry using multiple reaction monitoring on an ACQUITY I-Class UPLC system coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS/MS Xevo TQ-S). Data were compared between patients and controls and associated with laboratory findings. Continuous variables were presented as mean ± standard error of the mean. Results: A total of 11 patients (7 males) with NS with mean age of 10.65±3.66 were compared to 11 controls (6 males) and with 10.86±4.35 years. In terms of disease response, 5 patients were in partial remission, 5 patients in total remission and only 1 patient was in disease relapse. All patients have normal renal function. Plasma levels of Alamandine-(1-5) were significantly higher in patients when compared to controls (28.03±7.54 vs. 10.68±3.02 pg.mL-1 in controls). Despite a mild decrease in patients, plasma levels of Alamandine did not differ between groups (19.54±6.33 vs 26.09±6.12 pg.mL-1 in controls). No correlations were found between RAS molecules and proteinuria and with the medications in use. However, Alamandine-(1-5) negatively correlated with plasma levels of albumin (r= -0.695; p=0.038). In addition, Alamandine negatively correlated with total cholesterol (r=-0.700; p = 0.036), LDL (r=-0.800; p=0.004), and HDL (r = -0.762; p = 0.038). Conclusion: The novel RAS peptide Alamandine-(1-5) is significantly increased in primary NS and inversely correlated with plasma albumin levels. These findings suggest a potential role of Alamandine-(1-5) in NS

Discovery of antitumor diterpenoids from Casearia graveolens targeting VEGFR-2 to inhibit angiogenesis

Eight novel clerodane diterpenoids (1−8) were isolated from the twigs of Casearia graveolens. Their structures were elucidated through comprehensive nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and electronic circular dichroism (ECD) analyses. In addition to structural determination, surface plasmon resonance (SPR) assays were conducted to investigate molecular interactions, revealing that compound 8 exhibited high affinity for vascular endothelial growth factor receptor 2 (VEGFR2), a key regulator of tumor angiogenesis. Subsequent in vivo experiments demonstrated that compound 8 effectively inhibited angiogenesis and displayed significant antitumor activity by suppressing tumor proliferation and metastasis in zebrafish xenograft models. These findings suggest that compound 8 holds promise as an anticancer lead compound targeting VEGFR-2 to obstruct tumor angiogenesis.

Bioactive secondary metabolites from an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus

This study focused on the bioactive secondary metabolites of an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus. The secondary metabolites from Aspergillus sp. CCH-1E were isolated by using various chromatographic methods [such as normal-phase and reversed-phase chromatography and high-performance liquid chromatography(HPLC)], and their structures were identified by various spectroscopic methods [e.g., ultraviolet(UV) spectroscopy, infrared(IR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy, and high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)]. Twelve compounds were yielded and identified from Aspergillus sp. CCH-1E, which are chermesinone H(1), chermesinone I(2), chermesinone B(3), 8,11-didehydrochermesinone B(4), chermesinone C(5), chermesinone A(6), chevalone B(7), barbacenic acid(8), 3,6,8-trihydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin(9), 5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone(10), 1-hydroxy-6,8-dimethoxy-3-methylanthracene-9,10-dione(11), and 7-drimen-9α,11,12-triol(12). Among them, compounds 1 and 2 are new compounds. The growth inhibition effects of all compounds were evaluated against non-small cell lung cancer cell lines A549 and NCI-H1650, as well as human cervical cancer cell line HeLa by using methylthiazolyldiphenyl-tetrazolium bromide(MTT). Compound 7 significantly inhibited the growth of three tumor cells with the IC_(50) values of 1.22-2.43 μmol·L~(-1), respectively. Compounds 1-6 showed moderate cell growth inhibition with the IC_(50) values of 16.24-35.28 μmol·L~(-1).

Rapid determination of chlorate and perchlorate in tea by ion exchange chromatography-tandem mass spectrometry

Ion exchange chromatography-tandem mass spectrometry (IEC-MS/MS) has recently become the preferred method for detecting ionic substances in tea. In this study, an IEC-MS/MS method was developed for the rapid determination of chlorate and perchlorate residues in tea samples. The optimal sample extraction process, pretreatment column, and chromatographic and mass spectrometric conditions were systematically investigated. In the optimal process, the tea samples were ultrasonically extracted with methanol-water (13∶7, v/v), and a PRiME HLB SPE column was used to purify the sample extract. An AceChrom Hybri-A IEC column (150 mm×2.1 mm, 5.0 μm) was used for separation, and 100 mmol/L ammonium acetate-acetonitrile (40∶60, v/v) was used as the mobile phase for isocratic elution. The flow rate was 0.3 mL/min, the column temperature was 40 ℃, and the injection volume was 5.0 μL. The mass spectrometric data were collected in negative electrospray ionization mode combined with multiple reaction monitoring (MRM) mode to achieve the rapid and accurate separation and qualitative analysis of the desired chemical components. Quantification was performed using the internal standard (IS) method. The measurement results showed a good linear relationship when the mass concentrations of chlorate and perchlorate were between 2.00-200 and 1.00-100 μg/L, respectively, with correlation coefficients (r2) greater than 0.9990. The average recoveries of chlorate and perchlorate at three spiked levels of low, medium, and high ranged from 88.54% to 97.25% with relative standard deviations (RSDs, n=7) of 3.2%-5.2%. The limits of detection for chlorate and perchlorate were 12.0 and 8.0 μg/kg, respectively, while the limits of quantification were 40.0 and 26.6 μg/kg, respectively. The results of tests conducted to assess the linearity, specificity, accuracy, precision, and applicability of the method to the analysis of chlorate and perchlorate in 15 tea samples collected from a local market demonstrated its validity for the routine analysis of tea samples. The proposed method is simple, rapid, sensitive, and accurate, and can meet requirements for the rapid screening and quantitative analysis of residual trace chlorate and perchlorate in large quantities of tea samples.

A Flavonoid Concentrate from Moringa Oleifera Lam. Leaves Extends Exhaustive Swimming Time by Improving Energy Metabolism and Antioxidant Capacity in Mice.

Moringa oleifera Lam. leaves contain various nutrients and bioactive compounds. The present study aimed to assess the anti-fatigue capacity of a flavonoids concentrate purified from M. oleifera Lam. leaves. The total flavonoids in the purified extract were analyzed by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS/MS). The mice were supplemented with purified M. oleifera Lam. leaf flavonoid-rich extract (MLFE) for 14 days. The weight-loaded forced swimming test was used for evaluating exercise endurance. The 90-min non-weight-bearing swimming test was carried out to assess biochemical biomarkers correlated to fatigue and energy metabolism. UPLC-MS/MS analysis identified 83 flavonoids from MLFE. MLFE significantly increased the swimming time by 60%. Serum lactate (9.9 ± 0.9 vs. 8.9 ± 0.7), blood urea nitrogen (BUN) (8.8 ± 0.8 vs. 7.2 ± 0.5), and nonesterified fatty acid (NEFA) (2.4 ± 0.2 vs. 1.7 ± 0.3) were significantly elevated; phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GCK), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression were significantly downregulated; and heme oxygenase 1 mRNA expression was significantly upregulated in muscle after swimming. MLFE supplement significantly decreased serum lactate (8.0 ± 1.0 vs. 9.9 ± 0.9), BUN (8.6 ± 0.4 vs. 8.9 ± 0.8), and NEFA (2.3 ± 0.4 vs. 2.4 ± 0.2) and increased the protein and mRNA expression of GCK, PEPCK, and Nrf2. The enhancement of glucose metabolism and antioxidant function by MLFE contributes partly to its anti-fatigue action.

Longitudinal characterization of the muscle metabolome in dairy cows during the transition from lactation cessation to lactation resumption

Skeletal muscle is vital in maintaining metabolic homeostasis and adapting to the physiological needs of pregnancy and lactation. Despite advancements in understanding metabolic changes in dairy cows around calving and early lactation, there are still gaps in our knowledge, especially concerning muscle metabolism and the changes associated with drying off. This study aimed to characterize the skeletal muscle metabolome in the context of the dietary and metabolic changes occurring during the transition from the cessation of lactation to the resumption of lactation in dairy cows. Twelve Holstein dairy cows housed in tie stalls were dried off 6 weeks (wk) before the expected calving date. Cows were individually fed ad libitum total mixed rations composed of grass silage, corn silage, and concentrate during lactation and of corn silage, barley straw, and concentrate during the dry period. The metabolome was characterized in skeletal muscle samples (M. longissimus dorsi) collected on wk -7 (9 d before dry-off), -5 (6 d after dry-off), and wk -1, and 1 relative to calving. The targeted metabolomics approach was conducted using the MxP Quant 500 kit (Biocrates Life Sciences AG) with liquid chromatography, flow injection, and electrospray ionization triple quadrupole mass spectrometry. Statistical analysis on the muscle metabolite data was performed using MetaboAnalyst 5.0, which allowed us to conduct various multivariate analyses such as principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), informative heat map generation, and hierarchical clustering. The statistical analysis revealed a clear separation between pregnancy (wk -7, -5, and -1) and post-calving (wk 1). Starting 5 wk before calving and continuing through the first wk thereafter, the concentration of 3-methylhistidine (3-MH) in the muscle increased. This coincided with an increase in the concentrations of 11 AA (Phe, His, Tyr, Trp, Arg, Asn, Leu, Ile, Gly, Ser, and Thr) in the first wk after calving, whereas Gln decreased. l-arginine pathway metabolites (homoarginine, ornithine, citrulline, and asymmetric dimethylarginine), betaine, and sarcosine followed a similar pattern, increasing from wk -7 to -5, but decreasing from wk -1 to 1. The transition from pregnancy to lactation was associated with an increase in concentrations of the long-chain acylcarnitine species C16, C16:1, C18, and C18:1 in the muscle, whereas the concentrations of phosphatidylcholine and sphingomyelin in the muscle remained stable. The significant changes observed in the metabolome mainly concerned the AA and AA-related metabolites, indicating muscle protein breakdown in the first wk after calving. The metabolites produced by the L-Arg pathway might contribute to regulating skeletal muscle mass and function in periparturient dairy cows. The elevated concentrations of long-chain acylcarnitine species in the muscle in the first wk after calving suggest incomplete fatty acid oxidation, likely due to insufficient metabolic adaptation in response to the fatty acid load around the time of calving.

Phytochemicals, Two New Sulphur Glycosides and Two New Natural Products, from Shepherd's Purse Seed and Their Activities.

Two new sulfur glycosides, bursapastoris A-B (3-4), were extracted and isolated from shepherd's purse seed, along with two new natural products, 11-(methylsulfinyl)undecanoic acid (2) and 10-(methylsulfinyl)decanoic acid (1). Their structures were determined though infrared spectroscopy, one-dimensional nuclear magnetic resonance (1H and 13C), and electrospray ionization mass spectrometry. Additionally, the structures of 3-4 were further identified by two-dimensional nuclear magnetic resonance (HMBC, HSQC, 1H-1H COSY, and NOESY). Compounds 1-4 showed relatively favorable docking to NF-κB. Unfortunately, we only discovered that compound 1-4 had weak anti-radiation activity at present. Therefore, further research regarding the biological activity of these organosulfur compounds is required at a later stage.

A 1H-imidazo[4,5-f][1,10]phenanthroline based ligand and its binuclear Cu(I) complexes: Syntheses, structures and luminescence sensing for Cr2O72− and MnO4−

Dichromate (Cr2O72−) and permanganate (MnO4−) ions have been important targets for sensing and detection in the field of luminescent complexes and their corresponding ligands due to their significant impact on the ecological environment and human health. However, many Cu(I) complexes with good room-temperature phosphorescence are rarely used as luminescent sensors for ions in water due to the insolubility and the disproportionation of the Cu(I) ion. Herein, 2,6-di-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl-4-tertbutylphenol (diptpH3) and its two luminescent binuclear Cu(I) complexes, [Cu(diptpH3)(POP)]ClO4 (1) and [Cu(diptpH3)(xantphos)]ClO4 (2), were designed and synthesized (POP = bis[(2-diphenylphosphino)phenyl] ether, and xantphos = 9,9-dimethyl-4,5-bis(diphenylphosphino)-9H-xanthene). All compounds in DMSO/H2O (1: 9, v: v) show high stability to water, which was confirmed by the results of electrospray ionization mass spectrometry (ESI-MS). Cr2O72− and MnO4− ions showed high luminescence quenching coefficients (Cr2O72−: 3.042 × 104 − 2.946 × 105 M−1; MnO4−: 1.476 × 104 − 2.254 × 105 M−1) for all the three compounds. DiptpH3, complexes 1 and 2 exhibited good luminescent sensing properties toward Cr2O72− and MnO4− ions with short response times, good selectivity and low detection limits (Cr2O72−: 1.845 × 10−7 − 2.359 × 10−6 mol⋅L−1; MnO4−: 1.850 × 10−7 − 6.221 × 10−6 mol⋅L−1) in DMSO/H2O. The plausible sensing mechanisms were fully discussed.

Pharmacokinetics of Oleandrin Following Administration of a Nerium oleander Extract in Mice

Background. Nerium oleander Linn is native of Mediterranean regions of Africa and Europe, where it is used in folk medical practices. The whole plant is known to be toxic. Various studies have shown its antimicrobial and anticancer properties. Oleandrin, the main toxic component in Nerium oleander is an inhibitor of P-glycoprotein. Cardiac glycosides, including oleandrin, are substrates of P-glycoprotein. The presence of other inhibitors in the alcoholic extract may potentially modify the pharmacokinetics of oleandrin. Given the therapeutic potential and the associated toxicity of the alcoholic extract, it is important to investigate the kinetics of oleandrin within this extract. Purpose. The objective of this study was to determine the pharmacokinetic parameters in mice following oral and I.V. administrations of a hydroalcoholic extract of Nerium oleander. Methods. Pharmacokinetic investigations of oleandrin, a cardiotonic glycoside and main active compound in Nerium oleander, were conducted in mice following intravenous administration (30 mg/kg) and oral administration (150 mg/kg) of the hydrolcoholic extract of Nerium oleander. For the oral route, seven times were chosen for the mice sampling. Four times were chosen for the intravenous route. Three mice per group were used at each time point. In the excretion study, seven mice were housed in a mouse urine collection device and a dose of 150 mg/g was administered. A urine sample was collected after 48 hours. Oleandrin was measured by LC-MS/MS validated method. MS data were acquired in the positive ion electrospray ionization (ESI) mode. Two deuterated internal standards, cocaine-d3 and digoxin-d3, were used. The retention times were 8.47 min for oleandrin, 4.78 min for deuterated internal standard cocaine d3 and 5,37 min for deuterated internal standard digoxin d3. Results. The equivalent doses of oleandrin administred to mice were 1710 ug/kg for the oral route and 342 ug/kg for the intravenous route. Oleandrin was rapidly absorbed after oral administration (Cmax at 10 min). The AUC0-inf (ug/L*min) values obtained after intravenous and oral dosing were 34797.7 and 107222 respectively, resulting in an oral bioavailability of 61.6 %. The apparent volume of distribution (Vss) was 0.55 L/kg and Clearance ClT was 0.01 kg* L/min.

Quantitative analysis of phenolics in Trifolium pratense L. flowers and evaluation of antioxidant activity by sensory

Trifolium pratense L. flowers (TPF) were collected and dried in shade in this study. After extraction in methanol, a diluted solution was applied to the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) device to determine the bioactive compounds quantitatively. Isoquercitrin (38.64 mg/g extract), coumarin (13.66 mg/g extract), and catechin (12.52 mg/g extract) were verified as major products. Antioxidant activity of TPF was performed using a potentiometric PVC membrane sensor to evaluate 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content of TPF. TPF inhibited the DPPH radical as 31.4 ± 0.02% at the 500-ppm concentration. However, the DPPH activity of gallic acid was determined as 81.43 ± 0.07% and 92.44 ± 0.1% at the TPF concentrations of 12.5 ppm and 25 ppm, respectively. In addition, the total phenolic content was calculated to be 82.4 ± 0.15 mg gallic acid eq/g extract. It was observed that TPF has the potential to be an antioxidant and a valuable source of isoquercitrin, coumarin, and catechin.

The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HRMS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys34*Pro and the single amino acid residue His* were produced. The use of proteinaseK yielded Cys34*ProPhe and theuse of pepsin generated ValThrGlu48*Phe, AlaGlu230*ValSerLysLeu, and LeuGlyMet329*Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu230 and Met239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys34(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu86, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.

Assessment of Flavor Compounds in Blended Malt Whisky Aged with Acacia, Cedar and Oak Wood Using Liquid Chromatography Hyphenated to Electrospray Ionization and Tandem Mass Spectrometry (LC-ESI-MS/MS)

Selected flavor compounds from wood were quantified, with liquid chromatography hyphenated to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS), in four samples of base spirit matured with acacia, cedar, virgin oak, and used port wine oak wood cubes and the results were used to describe and differentiate their different wood congener profiles. ESI-MS/MS parameters were optimized in terms of capillary voltage, cone- and desolvation gas flow, source- and desolvation temperature, cone voltage, and collision energy. The method was validated in terms of linearity (R2 > 0.995), limit of detection, limit of quantification, recovery, and repeatability. One-way analysis of variance (ANOVA) showed that for most compounds there were no significant differences (α = 0.05) between seven to 30 days of aging plus a heat treatment at 40 °C, but wood type did significantly influence the wood congener profile. Principal component analysis discriminated the spirits based on relative amounts of wood congeners extracted, and the chemical profile. Aging with cedar wood led to a less pronounced chemical profile, not very distinct from the base spirit. Aging with acacia and virgin oak wood produced a second chemical profile with relatively higher levels of synapaldehyde, syringaldehyde, scopoletin, and coniferyl aldehyde. Aging with port oak wood produced a third chemical profile with relatively higher levels of 5-HMF, gallic acid, furfural, vanillic acid, and syringic acid.

Advantages of online supercritical fluid extraction and chromatography hyphenated to mass spectrometry to analyse plastic additives in laboratory gloves

Plastic additives are introduced in plastic material formulations, along with organic polymers, to offer different properties such as stability, plasticity or color. However, plastic additives may migrate from the plastic material to the content (in case of plastic containers) or to the material in contact with the plastic, like human skin. In the case of plastic medical devices, this migration is of particular interest, as plastic additives may be deleterious to health. In the present paper, we examined the interest of combining supercritical fluid extraction (SFE) to supercritical fluid chromatography (SFC) hyphenated to mass spectrometry (MS) in an online system to characterize plastic additives in laboratory gloves, taken as samples of medical devices. A set of target compounds comprising 18 plasticizers, 4 antioxidants and 2 lubricants was defined and their detectability with MS was examined, where it appeared that electrospray ionization (ESI) provided better detectability than atmospheric pressure chemical ionization (APCI). After examining possible stationary phases with the help of Derringer desirability function, an isocratic chromatographic method (CO2:methanol 95:5) was developed on Shim-pack UC Phenyl column. The extraction method was examined with a 3-level full factorial design of experiments to optimize the extraction temperature (40 °C) and pressure (200 bar). The online SFE-SFC-MS method was compared to offline methods where the samples were extracted with liquid solvents at atmospheric pressure or high pressure then analysed with SFC-MS. In all cases, offline methods showed significant contaminants (like the oleamide lubricant) issuing from laboratory plastic materials as nitrogen drying station, syringes and filters, while the online method allowed a complete elimination of laboratory contaminations. Furthermore, the online method saved time, solvents and laboratory consumables. It will also show that transferring a compressible fluid from a loading loop is favourable to high efficiency, as the resulting chromatographic peaks are much thinner than when transferring a liquid. Compared to injecting liquid heptane, the efficiency increase was 3.4-fold, while compared to injecting liquid methanol (a common practice in SFC), the efficiency increase was 13-fold. Finally, the additive composition of different laboratory gloves was compared.

Untargeted metabolic analysis of Epaltes mexicana by LC-QTOF-MS: Terpenes with activity against human cancer cell lines

Epaltes mexicana is a plant widely used in traditional medicine and as a food in Mexico; however, its phytochemical and pharmacological studies are limited. This study aimed to identify the active secondary metabolites of Epaltes mexicana and determine its cytotoxic activity on cancer cell lines. Three organic extracts were obtained by maceration using n-hexane, dichloromethane, and methanol. The n-hexane extract was fractioned by simple column chromatography. Eight terpenes were annotated in collection 6 (C6) by LC-QTOF-MS using a gradient elution and Electrospray Ionization (ESI) in positive ion mode: 1) Gibberellin A15, 2) farfugin A, 3) dehydromyodesmone, 4) eremopetasitenin A1, 5) hydroxyisonobilin, 6) anhydrocinnzeylanine, 7) nigakilactone H and 8) taxodione. On the other hand, C6 showed a concentration-dependent cytotoxic effect on cancer cell lines MCF-7 (Emax = 74.69 ± 6.19 % and IC50 = 6.31 μg/mL), MDA-MB-231 (Emax = 79.28 ± 12.12 % and IC50 = 124.21 μg/mL), and SiHa (Emax = 82.96 ± 6.02 % and IC50 = 124.31 μg/mL). The C6 did not show a cytotoxic effect against DU-145 and non-cancerous cells from the mammary glands MCF-10A. These results indicate cytotoxic specificity on cancer cell lines and support the hypothesis that terpenes identified in E. mexicana must be investigated and developed for non-clinical and clinical trials as potential anti-cancer drugs.

New Meroterpenes from South China Sea Soft Coral Litophyton brassicum.

A chemical investigation of the extracts from the soft coral Litophyton brassicum led to the isolation and identification of four new meroterpenes, brassihydroxybenzoquinone A and B (1 and 2) and brassinaphthoquinone A and B (3 and 4), along with two known related meroterpenes (5 and 6). Their structures were elucidated using high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR) spectroscopy, and a comparison with the literature data. All compounds were evaluated for antibacterial activity against six pathogenic bacterial strains and for cytotoxic activity against three cancer cell lines. In the cytotoxic assay, all compounds were inactive at 10 μM against the A549, HeLa, and MDA-MB-231 cell lines. In the antibacterial assay, compounds 1 and 2 exhibited moderate inhibitory activity with minimum inhibitory concentrations (MIC) ranging from 8 to 64 μg/mL.

Exploring Triazole-Connected Steroid-Pyrimidine Hybrids: Synthesis, Spectroscopic Characterization, and Biological Assessment.

Molecules originating from natural sources are physicochemically and biologically diverse. The conjugation of two active biomolecules has become the foundation for medical and pharmaceutical sciences. An effective synthesis of 11 new steroid-pyrimidine conjugates containing 1,2,3-triazole rings was carried out. The group of 3α-OH bile acids (lithocholic, deoxycholic, cholic) and 3β-OH sterols (cholesterol, cholestanol) were respectively modified to azidoacetates. 2-thiouracil was converted into N(1)S and N(3)S dipropargyl derivatives. Azide-alkyne cycloaddition in the presence of copper(I) of the obtained compounds led to the preparation of 1,2,3-triazole derivatives. Based on a series of spectroscopic (1H NMR, 13C NMR, Fourier-transform infrared (FT-IR)), spectrometric analyses (Electrospray ionization-mass spectrometry (ESI-MS), electron impact-mass spectrometry (EI-MS)), and semiempirical calculations, the structures of all compounds were confirmed. In silico biological tests and molecular docking (for domain 1KZN, 2H94, 5V5Z, 1EZF, 2Q85) were performed for selected compounds. The tests performed indicate the theoretical antimicrobial potential of the obtained ligands.

Chrysomycin A Reshapes Metabolism and Increases Oxidative Stress to Hinder Glioblastoma Progression.

Glioblastoma represents the predominant and a highly aggressive primary neoplasm of the central nervous system that has an abnormal metabolism. Our previous study showed that chrysomycin A (Chr-A) curbed glioblastoma progression in vitro and in vivo. However, whether Chr-A could inhibit orthotopic glioblastoma and how it reshapes metabolism are still unclear. In this study, Chr-A markedly suppressed the development of intracranial U87 gliomas. The results from airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) indicated that Chr-A improved the abnormal metabolism of mice with glioblastoma. Key enzymes including glutaminase (GLS), glutamate dehydrogenases 1 (GDH1), hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD) were regulated by Chr-A. Chr-A further altered the level of nicotinamide adenine dinucleotide phosphate (NADPH), thus causing oxidative stress with the downregulation of Nrf-2 to inhibit glioblastoma. Our study offers a novel perspective for comprehending the anti-glioma mechanism of Chr-A, highlighting its potential as a promising chemotherapeutic agent for glioblastoma.

Clustering of biphenyl oxamide ions by chiral recognition.

Gels created by self-assembly of small organic molecules are dynamic soft materials that have unique properties and demanding characterization. Four chiral gelators, with two valinol- or leucinoloxamido arms attached to the 2,2'-positions of the proatropisomeric biphenyl group were chosen to show that the electrospray ionization mass spectrometry (ESI-MS) could be used to differentiate the gelation feature of the chiral compounds 1-4 and also to shed light on the gelation processes. By inspecting the gelation of several solvents, we showed that 1 (R, R) proved to be the most efficient gelator, forming the largest observable assemblies in the gas phase. The strong intermolecular H-bonds hold single-charged assemblies consisting of up to five monomer units detectable by ESI MS. Enantiomer 1 (R, R) is a good gelator due to favorable intramolecular interactions that remain preserved in the gas phase. Compound 3 (meso) does not have gelator properties and detected signals of larger assemblies in the gas phase. So, the detected signals correlate with the conformations of the studied compounds. MS could be used to elucidate the preferential type of noncovalent interaction due to the chiral recognition. The study paves a novel way to investigate the influence of chirality on the molecular assembly and consequently macroscopic properties and functions of materials.

Ensiling conditions and changes of cannabinoid concentration in industrial hemp.

Hemp (Cannabis sativa L.) is an important source of fibre and seed oil and protein. By-products of industrial hemp fibre production, like hemp seeds and cakes, can be used as feed for all animal species as fat and protein source and the whole hemp plant (including stalk and leaves) might be a suitable fibre source for ruminants. However, a previous feeding experiment with leaf-flower-seed hemp silage, made from an industrial hemp variety, demonstrated detrimental effects on cow health parameters and a significant transfer of several cannabinoids, including the psychoactive tetrahydrocannabinol (∆9-THC), into cow's milk, posing a potential risk to the safety of consumers. Based on those observations, the present study tested the hypothesis that anaerobic fermentation, as during ensiling, increases the content of ∆9-THC in hemp. Therefore, silages of whole plants from the industrial hemp Cannabis sativa L. var. Ivory were prepared in a multifactorial design, with the four treatments 1) untreated control (CON), 2) addition of 10 mL per kg fresh weight homofermentative lactobacilli at 105 cfu/mL (LBAC), 3) addition of 10 mL per kg fresh weight homofermentative lactobacilli at 105 cfu/mL plus 30 g molasses (LBACmol) and 4) addition of propionic acid (10 mL/kg fresh weight) (PRO). Ultra high performance liquid chromatography coupled with tandem mass spectrometry with electrospray ionisation (UHPLC-MS/MS) was performed for analysis of cannabinoids in fresh hemp material and after 10 and 90 days of ensiling. The study revealed that ensiling decreased all acid forms of analysed cannabinoids in hemp at about 40-65% of the initial values after 90 days of storage, with the exception of cannabinolic acid (CBNA), and CBGA, the acidic form of cannabigerol (CBG). This decrease in most acidic forms was accompanied by an increase of the corresponding non-acidic forms of all cannabinoids, including the psychoactive ∆9-THC. Thus, although ensiling decreases the total cannabinoid content, psychoactive compounds like ∆9-THC can increase, enhancing the risk for animal health and a transfer of these substances into animal derived products.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.