Effect of ultra-high pressure pretreatment on the phenolic profiles, antioxidative activity and cytoprotective capacity of different phenolic fractions from Que Zui tea.

This study aims to explore whether ultra-high pressure (UHP) pre-treatment strengthened the bioaccessibility and bioactivities of the free (QF), esterified (QE) and insoluble-bound phenolics (QIB) from Que Zui tea (QT). The results revealed that the extraction yields, the total phenolic (TPC) and total flavonoid contents (TFC) of three phenolic fractions from QT were markedly increased after ultra-high pressure (UHP) processing (p < 0.05). A total of 19 and 20 compounds were characterized and quantified in non- and UHP-treated QT, respectively, including the content of 6'-O-caffeoylarbutin (11775.68 and 13248.87 µg/g of dry extract) was highest in QF, the content of caffeic acid was highest in QE (2131.58 and 7362.99 µg/g of dry extract) and QIB (9151.89 and 10930.82 µg/g of dry extract). QF, QE and QIB from QT after UHP processing had better antioxidant, ROS scavenging, and anti-apoptosis effects. The possible mechanism of cytoprotective effect was related to Keap1-Nrf2 pathway.

Mutations in Rht-B1 Locus May Negatively Affect Frost Tolerance in Bread Wheat.

The wheat semi-dwarfing genes Rht (Reduced height) are widely distributed among the contemporary wheat varieties. These genes also exert pleiotropic effects on plant tolerance towards various abiotic stressors. In this work, frost tolerance was studied in three near-isogenic lines of the facultative variety 'April Bearded' (AB), carrying the wild type allele Rht-B1a (tall phenotype), and the mutant alleles Rht-B1b (semi-dwarf) and Rht-B1c (dwarf), and was further compared with the tolerance of a typical winter type variety, 'Mv Beres'. The level of freezing tolerance was decreasing in the order 'Mv Beres' > AB Rht-B1a > AB Rht-B1b > AB Rht-B1c. To explain the observed differences, cold acclimation-related processes were studied: the expression of six cold-related genes, the phenylpropanoid pathway, carbohydrates, amino acids, polyamines and compounds in the tricarboxylic acid cycle. To achieve this, a comprehensive approach was applied, involving targeted analyses and untargeted metabolomics screening with the help of gas chromatography/liquid chromatography­mass spectrometry setups. Several cold-related processes exhibited similar changes in these genotypes; indeed, the accumulation of eight putrescine and agmatine derivatives, 17 flavones and numerous oligosaccharides (max. degree of polymerization 18) was associated with the level of freezing tolerance in the 'April Bearded' lines. In summary, the mutant Rht alleles may further decrease the generally low frost tolerance of the Rht-B1a, and, based on the metabolomics study, the mechanisms of frost tolerance may differ for a typical winter variety and a facultative variety. Present results point to the complex nature of frost resistance.

Identification of Erythromycin and Clarithromycin Metabolites Formed in Chicken Liver Microsomes Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Nontargeted analysis can be used for the rapid screening and confirmatory analysis of veterinary drugs and their metabolites, which are important for the comprehensive safety evaluation of animal-derived foods. Here, a novel nontargeted screening approach based on liquid chromatography coupled with electrospray ionization-high-resolution mass spectrometry (LC/ESI-HR-MS) was developed to determine erythromycin, clarithromycin, and their metabolites in chicken liver microsomes. Erythromycin and clarithromycin were incubated in vitro in the presence of NADPH for 60 min to generate metabolites in chicken liver microsomes. After the incubation, the supernatant was extracted using ultrasonic shaking, orbital shaking, and centrifugation before analysis using LC/ESI-HR-MS in positive ion mode on an Agilent Eclipse Plus C18 column (100 mm × 2.1 mm; i.d. 3.5 µm) with 0.1 percent formic acid-water and acetonitrile as the mobile phases for gradient elution at 0.4 mL/min. The results show that erythromycin can produce N-desmethyl-erythromycin A in chicken liver microsomes, but clarithromycin cannot produce N-desmethyl-clarithromycin in chicken liver microsomes. The N-desmethyl-erythromycin A and N-desmethyl-clarithromycin were tentatively identified in chicken liver microsomes using the established quick analytic method, which will provide a theoretical foundation for future research on pharmacokinetics and drug elimination in poultry.

Design, synthesis, and biological evaluation of quinazolin-4(3H)-one derivatives co-targeting poly(ADP-ribose) polymerase-1 and bromodomain containing protein 4 for breast cancer therapy.

This study was aimed to design the first dual-target small-molecule inhibitor co-targeting poly (ADP-ribose) polymerase-1 (PARP1) and bromodomain containing protein 4 (BRD4), which had important cross relation in the global network of breast cancer, reflecting the synthetic lethal effect. A series of new BRD4 and PARP1 dual-target inhibitors were discovered and synthesized by fragment-based combinatorial screening and activity assays that together led to the chemical optimization. Among these compounds, 19d was selected and exhibited micromole enzymatic potencies against BRD4 and PARP1, respectively. Compound 19d was further shown to efficiently modulate the expression of BRD4 and PARP1. Subsequently, compound 19d was found to induce breast cancer cell apoptosis and stimulate cell cycle arrest at G1 phase. Following pharmacokinetic studies, compound 19d showed its antitumor activity in breast cancer susceptibility gene 1/2 (BRCA1/2) wild-type MDA-MB-468 and MCF-7 xenograft models without apparent toxicity and loss of body weight. These results together demonstrated that a highly potent dual-targeted inhibitor was successfully synthesized and indicated that co-targeting of BRD4 and PARP1 based on the concept of synthetic lethality would be a promising therapeutic strategy for breast cancer.

Metabolite profiling and antioxidant activity of the polar fraction of Italian almonds (Toritto and Avola): Analysis of seeds, skins, and blanching water.

The health benefits of almonds along with their ability to prevent atheroschlerosis as well as cardiovascular and diabetes risks are well known. Previous works were focused mainly on the lipid fraction, thus a lack of information about the polar constituents still persists. In order to provide deeper insight into the chemical composition of almond, the polar fraction of Italian almond cultivars (Toritto and Avola) was investigated. The MeOH extract of the Toritto cultivar was submitted to LC-ESI-(HR)MS/MS experiments, highlighting the occurrence of several compounds, mainly cyanogenic glycosides, glycosylated flavonoids, proanthocyanidins, and a diterpene glycoside. EtOH and EtOH:H2O solution were also employed for extractions, resulting selective for flavonoids and proanthocyanidins, while cyanogenic glycosides were mainly detected in EtOH:H2O extract. Almonds were also blanched to afford skins and blanching water which were analyzed by LC-ESI-(HR)MS/MS experiments in order to establish the source of the identified compounds. Cyanogenic glycosides were detected in the peeled almonds, while flavonoids and proanthocyanidins were detected in almond skins and blanching water. The LC-ESI-(HR)MS/MS analyses of the almonds of the Avola cvs (Fascionello, Pizzuta and Romana) were compared with those of the Toritto cv., showing clear differences in their metabolome, probably due to the different growing conditions. Finally, total phenolic content and radical scavenging activity of the extracts and blanching waters were evaluated by Folin-Ciocalteu assay and by DPPH and ABTS+ assays, highlighting interesting antioxidant properties possessed by almond skins and blanching water, suggesting their potential employment in pharmaceutical, cosmetic and nutraceutical formulations.

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry.

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.

Investigation of Fragmentation Pathways of Fentanyl Analogues and Novel Synthetic Opioids by Electron Ionization High-Resolution Mass Spectrometry and Electrospray Ionization High-Resolution Tandem Mass Spectrometry.

The global drug market is characterized by the fast development of new psychoactive substances such as fentanyl analogues and novel synthetic opioids, the detection of which is complicated by the lack of appropriate quality control procedures and references. Herein, we analyze the fragmentation pathways and characteristic ions of 25 novel fentanyl analogues and 5 novel synthetic opioids by electron ionization (EI) and electrospray ionization (ESI) high-resolution mass spectrometry to provide a reference for the identification of these species. In the ESI mode, fentanyl analogues mainly undergo piperidine ring degradation, phenethyl and piperidine ring dissociation, and piperidine ring and amide moiety cleavage, while piperidine ring degradation and phenethyl and piperidine ring dissociation are the major pathways in the EI mode. The five novel synthetic opioids largely undergo amide group dissociation and N-cyclohexyl bond cleavage in the ESI mode. Thus, this work facilitates the detection and quantitation of fentanyl analogues and novel synthetic opioids or other substances with similar structures in forensic laboratories.

Identification and quantification of limonoid aglycones content of Citrus seeds

ABSTRACT Citrus fruits are recognized as an important source of bioactive molecules such as limonin and nomilin. However, these molecules exhibit low bioavailability, therefore, obtaining these molecules using biotechnological techniques may be an alternative to harvesting them directly from fruits. The aim of this study was to quantify and identify limonoids in the dichloromethane extracts of Citrus seeds of Criolla orange, Oneco tangerine, Tangerine-lemon, Sour orange and Valencia orange from department of Antioquia-Colombia by high performance liquid chromatography with diode array detection, and high-resolution mass spectrometry. Although in all the samples total glycosidic free limonoids were present, Oneco tangerine seeds had the highest concentration, followed by Tangerine-lemon seeds, equivalent to 0.75% and 0.53% per total dry weight, respectively. These results suggest Oneco tangerine seeds may be used as an elite material for biotechnological processes looking for increased production of limonoids to support research and drug development.

Quantitative analysis of 4ß- and 4α­hydroxycholesterol in human plasma and serum by UHPLC/ESI-HR-MS.

Cholesterol oxidation product 4ß­hydroxycholesterol (4ß­OHC) may possibly be used as an endogenous biomarker of CYP3A enzyme activity and as CYP3A4 is involved in the metabolism of approximately 50% of the drugs in clinical use, the monitoring of CYP3A activity by 4ß­OHC plasma or serum levels, may be of clinical significance. The plasma and serum concentrations of 4α­hydroxycholesterol (4α­OHC), an isomer of 4ß­OHC, increase during uncontrolled storage conditions and therefore serve as an indicator of proper handling of samples. A sensitive and simple high-throughput method for the simultaneous quantification of both 4α­OHC and 4ß­OHC in human plasma and serum was developed utilizing ultrahigh performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC/ESI-HR-MS). The chromatographic analysis was carried out on a Waters HSS T3 C18 reversed phase column with a mobile phase composed of 0,1% formic acid with 200 mg/l sodium acetate, and methanol. 4ß­OHC and 4α­OHC and also internal standard d7­4ß­OHC were monitored using HR-MS as sodium adducts, which could not be used as a precursor ions in conventional tandem mass spectrometry methods due to their extensive stability in collision for MS/MS. The use of HR-MS detection enabled avoiding laborious sample derivatization, which is required with triple quadrupole mass spectrometer-based methods to achieve adequate analytical sensitivity for 4ß­OHC, as the underivatized molecule is otherwise poorly ionized to other molecular ions than sodium adduct. Chromatographic separation of 4α­OHC and 4ß­OHC was obtained and confirmed with standard samples prepared in blank surrogate matrix. The lower limits of quantitation in the assay were 0.5 ng/ml for 4ß­OHC, and 2 ng/ml for 4α­OHC. Endogenous levels of 4ß­OHC can vary between 10 and 100 ng/ml depending on the possible induction or inhibition of CYP3A4, whereas the levels of 4α­OHC can vary between 5 and 100 ng/ml, depending on the storage conditions of the samples. Thus, the sensitivity of the assay developed allows for the simultaneous measurement of endogenous levels of 4α­OHC and 4ß­OHC cost-effectively and with high throughput. The method was successfully used for the determination of 4ß­OHC and 4α­OHC concentrations in clinical plasma and serum samples collected before and after treatment with a known CYP3A4 inducer rifampicin. The endogenous levels in clinical human samples before treatment varied between 13.4 and 31.9 ng/ml for 4ß­OHC, and between 3.53 and 5.65 ng/ml for 4α­OHC, and a three-fold increase in 4ß­OHC plasma levels was observed after the rifampicin treatment, while 4α­OHC levels remained unaffected.

Mechanistic Study of Copper-Catalyzed C-H Hydroxylation/C-S Coupling by ESI-HR MS and DFT Calculations.

The reaction mechanism of Cu-catalyzed C-H hydroxylation/C-S coupling was studied using electrospray ionization high resolution mass spectrometry (ESI-HR MS) and density functional theory calculations (DFT). Notably, a series of CuI and CuIII complexes were observed as key intermediates and identified using ESI-HR MS. Furthermore, a catalyst cycle involving proton abstraction/oxidative addition/reductive elimination was proposed. This study is important and valuable with respect to C-H functionalization.

Antioxidant activity, cytotoxic activity and metabolic profiling of juices obtained from saffron (Crocus sativus L.) floral by-products.

Juices obtained from cold-pressed saffron (Crocus sativus L.) floral by-products were evaluated as a potential source of compounds with antioxidant and cytotoxic activities. Floral by-products were split in two batches for extraction 24 and 48h after flower harvesting, respectively. The in vitro anti-oxidant activity of these extracts was tested using the FRAP and DPPH assays, and two biological models of lipid oxidation (activity in preventing cholesterol degradation and protection against Cu(2+)-mediated degradation of the liposomal unsaturated fatty acids). The cytotoxic activity was evaluated using the MTT assay. The results show that extracts obtained 48h post-harvest contained higher levels of total polar phenols and had the highest antioxidant activity in all of the performed assays. The LC-DAD and LC-ESI-(HR)MS(n) metabolic profiles showed high levels of kaempferol derivatives and anthocyanins. This study suggests that juices from saffron floral by-products could potentially be used to develop new products for the food and health industry.

Phenolic profiling in the pulp and peel of nine plantain cultivars (Musa sp.).

The present study investigated the phenolic profiles of the pulp and peel of nine plantain cultivars and compared them to those of two dessert bananas of commercial interest (Grand Nain and Gros Michel), alongside a newly created hybrid, resistant to black sigatoka disease (F568). Identification and quantification of phenolic compounds were performed by means of HPLC-ESI-HR-MS and HPLC-DAD. Hydroxycinnamic acids, particularly ferulic acid-hexoside with 4.4-85.1 µg/g of dry weight, dominated in the plantain pulp and showed a large diversity among cultivars. Flavonol glycosides were predominant in plantain peels, rutin (242.2-618.7 µg/g of dry weight) being the most abundant. A principal component analysis on the whole data revealed that the phenolic profiles of the hybrid, the dessert bananas and the pure plantains differed from each other. Plantain pulps and peels appeared as good sources of phenolics, which could be involved in the health benefits associated with their current applications.