On the Identification and Quantification of Ergothioneine and Lovastatin in Various Mushroom Species: Assets and Challenges of Different Analytical Approaches.

In recent years, mushrooms have drawn the attention of agro-industries and food-industries as they were considered to be valuable natural sources of health promoting compounds such as ß-glucans, ergothioneine, and lovastatin. The detection and quantification of such compounds by implementing reliable analytical approaches is of the utmost importance in order to adjust mushrooms' cultivation conditions and maximize the production in different species. Toward this direction, the current study focuses on the comparison of ultraviolet-visible (UV-Vis) spectrometry and liquid chromatography-mass spectrometry (LC-MS) methods (a) by evaluating the content of ergothioneine and lovastatin in mushrooms and (b) by highlighting any possible substrate-based interferences that hinder the accurate determination of these two compounds in order to propose the technique-of-choice for a standardized bioactive compounds monitoring. For this purpose, mushrooms produced by three species (i.e., Agaricus bisporus, Pleurotus ostreatus, and P. citrinopileatus) on various cultivation substrates, namely wheat straw (WS), winery (grape marc (GM)), and olive oil (OL) by-products, were examined. Among the two applied techniques, the developed and validated LC-MS methods, exhibiting relatively short analysis time and higher resolution, emerge as the methods-of-choice for detecting ergothioneine and lovastatin in mushrooms. On the contrary, UV-Vis methods were hindered due to co-absorbance of different constituents, resulting in invalid results. Among the studied mushrooms, P. citrinopileatus contained the highest amount of ergothioneine (822.1 ± 20.6 mg kg-1 dry sample), whereas A. bisporus contained the highest amounts of lovastatin (1.39 ± 0.014 mg kg-1 dry sample). Regarding the effect of different cultivation substrates, mushrooms produced on OL and WS contained the highest amount of ergothioneine, while mushrooms deriving from GM-based substrates contained the highest amount of lovastatin.

Expanding the Role of Sub-Exploited DOE-High Energy Extraction and Metabolomic Profiling towards Agro-Byproduct Valorization: The Case of Carotenoid-Rich Apricot Pulp.

Traditional extraction remains the method-of-choice for phytochemical analyses. However, the absence of an integrated analytical platform, focusing on customized, validated extraction steps, generates tendentious and non-reproducible data regarding the phytochemical profile. Such a platform would also support the exploration and exploitation of plant byproducts, which are a valuable source of bioactive metabolites. This study deals with the incorporation of (a) the currently sub-exploited high energy extraction methods (ultrasound (UAE)- and microwave-assisted extraction (MAE)), (b) experimental design (DOE), and (c) metabolomics, in an integrated analytical platform for the extensive study of plant metabolomics and phytochemical profiling. The recovery of carotenoids from apricot by-products (pulp) is examined as a case study. MAE, using ethanol as solvent, achieved higher carotenoid yields compared to UAE, where 1:1 chloroform-methanol was employed, and classic extraction. Nuclear magnetic resonance (NMR)-based metabolomic profiling classified extracts according to the variations in co-extractives in relation to the extraction conditions. Extracts with a lower carotenoid content contained branched-chain amino acids as co-extractives. Medium carotenoid content extracts contained choline, unsaturated fatty acids, and sugar alcohols, while the highest carotenoid extracts were also rich in sugars. Overall, the proposed pipeline can provide different the phytochemical fractions of bioactive compounds according to the needs of different industrial sectors (cosmetics, nutraceuticals, etc.).

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery.

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood-brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs.

Bioenergetic Profiling in Glioblastoma Multiforme Patients with Different Clinical Outcomes.

The accumulation of cell biomass is associated with dramatically increased bioenergetic and biosynthetic demand. Metabolic reprogramming, once thought as an epiphenomenon, currently relates to disease progression, also in response to extracellular fate-decisive signals. Glioblastoma multiforme patients often suffer misdiagnosis, short survival time, low quality of life, and poor disease management options. Today, tumor genetic testing and histological analysis guide diagnosis and treatment. We and others appreciate that metabolites complement translational biomarkers and molecular signatures in disease profiling and phenotyping. Herein, we coupled a mixed-methods content analysis to a mass spectrometry-based untargeted metabolomic analysis on plasma samples from glioblastoma multiforme patients to delineate the role of metabolic remodeling in biological plasticity and, hence, disease severity. Following data processing and analysis, we established a bioenergetic profile coordinated by the mitochondrial function and redox state, lipids, and energy substrates. Our findings show that epigenetic modulators are key players in glioblastoma multiforme cell metabolism, in particular when microRNAs are considered. We propose that biological plasticity in glioblastoma multiforme is a mechanism of adaptation and resistance to treatment which is eloquently revealed by bioenergetics.

Thermal, dynamic and structural properties of drug AT1 antagonist olmesartan in lipid bilayers.

It is proposed that AT1 antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT1 receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC), 13C magic-angle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles. 13C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as 13C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche:trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile.

ENT-A010, a Novel Steroid Derivative, Displays Neuroprotective Functions and Modulates Microglial Responses.

Tackling neurodegeneration and neuroinflammation is particularly challenging due to the complexity of central nervous system (CNS) disorders, as well as the limited drug accessibility to the brain. The activation of tropomyosin-related kinase A (TRKA) receptor signaling by the nerve growth factor (NGF) or the neurosteroid dehydroepiandrosterone (DHEA) may combat neurodegeneration and regulate microglial function. In the present study, we synthesized a C-17-spiro-cyclopropyl DHEA derivative (ENT-A010), which was capable of activating TRKA. ENT-A010 protected PC12 cells against serum starvation-induced cell death, dorsal root ganglia (DRG) neurons against NGF deprivation-induced apoptosis and hippocampal neurons against Aß-induced apoptosis. In addition, ENT-A010 pretreatment partially restored homeostatic features of microglia in the hippocampus of lipopolysaccharide (LPS)-treated mice, enhanced Aß phagocytosis, and increased Ngf expression in microglia in vitro. In conclusion, the small molecule ENT-A010 elicited neuroprotective effects and modulated microglial function, thereby emerging as an interesting compound, which merits further study in the treatment of CNS disorders.

Comparison of thermal effects of stilbenoid analogs in lipid bilayers using differential scanning calorimetry and molecular dynamics: correlation of thermal effects and topographical position with antioxidant activity.

In previous studies it was shown that cannabinoids (CBs) bearing a phenolic hydroxyl group modify the thermal properties of lipid bilayers more significantly than methylated congeners. These distinct differential properties were attributed to the fact that phenolic hydroxyl groups constitute an anchoring group in the vicinity of the head-group, while the methylated analogs are embedded deeper towards the hydrophobic region of the lipid bilayers. In this work the thermal effects of synthetic polyphenolic stilbenoid analogs and their methylated congeners have been studied using differential scanning calorimetry (DSC). Molecular dynamics (MD) simulations have been performed to explain the DSC results. Thus, two of their phenolic hydroxyl groups orient in the lipid bilayers in such a way that they anchor in the region of the head-group. In contrast, their methoxy congeners cannot anchor effectively and are embedded deeper in the hydrophobic segment of the lipid bilayers. The MD results explain the fact that hydroxystilbenoid analogs exert more significant effects on the pretransition than their methoxy congeners, especially at low concentrations. To maximize the polar interactions, the two phenolic hydroxyl groups are localized in the vicinity of the head-group region, directing the remaining hydroxy group in the hydrophobic region. This topographical position of stilbenoid analogs forms a mismatch that explains the significant broadening of the width of the phase transition and lowering of the main phase-transition temperature in the lipid bilayers. At high concentrations, hydroxy and nonhydroxy analogs appear to form different domains. The correlation of thermal effects with antioxidant activity is discussed.

Three-Dimensional Cell Metabolomics Deciphers the Anti-Angiogenic Properties of the Radioprotectant Amifostine.

Aberrant angiogenesis is a hallmark for cancer and inflammation, a key notion in drug repurposing efforts. To delineate the anti-angiogenic properties of amifostine in a human adult angiogenesis model via 3D cell metabolomics and upon a stimulant-specific manner, a 3D cellular angiogenesis assay that recapitulates cell physiology and drug action was coupled to untargeted metabolomics by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The early events of angiogenesis upon its most prominent stimulants (vascular endothelial growth factor-A or deferoxamine) were addressed by cell sprouting measurements. Data analyses consisted of a series of supervised and unsupervised methods as well as univariate and multivariate approaches to shed light on mechanism-specific inhibitory profiles. The 3D untargeted cell metabolomes were found to grasp the early events of angiogenesis. Evident of an initial and sharp response, the metabolites identified primarily span amino acids, sphingolipids, and nucleotides. Profiles were pathway or stimulant specific. The amifostine inhibition profile was rather similar to that of sunitinib, yet distinct, considering that the latter is a kinase inhibitor. Amifostine inhibited both. The 3D cell metabolomics shed light on the anti-angiogenic effects of amifostine against VEGF-A- and deferoxamine-induced angiogenesis. Amifostine may serve as a dual radioprotective and anti-angiogenic agent in radiotherapy patients.

Development and Validation of a UPLC-ESI(-)-MS/MS Methodology for the Simultaneous Quantification of Hesperidin, Naringin, and their Aglycones in Chicken Tissue Samples.

BACKGROUND: The dietary supplementation of livestock with antioxidants to improve the meat quality represents an active research area of high commercial impact. In order to investigate the optimal dosing, analytical methodologies need to be developed in various tissues to evaluate which concentration does remain in the tissue. OBJECTIVE: We aimed to develop and validate a sensitive and specific methodology for the simultaneous quantitative determination of hesperidin, naringin, hesperetin, and naringenin in chicken tissue samples employing ultra-performance LC-tandem MS. METHODS: Lipid extraction using cold chloroform was performed followed by protein precipitation by cold acetone. Chromatography was performed on a C18 column using a ternary gradient of water, acetonitrile, and isopropanol-acetonitrile-acetone (58+40+2, v/v) as the mobile phase. Detection was performed by electrospray ionization in negative ion mode with the selected reaction monitoring technique. RESULTS: Calibration plots exhibited good linearity (r2 > 0.99) over the concentration range from 0.125 to 25 µg/g tissue for the four analytes, and the lower LOQ for the four analytes was 0.125 µg/g tissue. The repeatability as percent relative SD and precision as percent accuracy were <20 and >80%, respectively. CONCLUSIONS: The developed methodology was applied for the quantitative determination of hesperidin, naringin, hesperetin, and naringenin in tissue samples after dietary supplementation with 1.5 g/kg hesperidin and 1.5 g/kg naringin in Ross 308 broiler chickens. HIGHLIGHTS: This is the first methodology to access naringin, naringenin, hesperidin, and hesperetin in chicken tissue. It involved simple sample preparation, and the mass spectrometry based detection ensures high specificity and sensitivity.

Macular carotenoids in lipid food matrices: DOE-based high energy extraction of egg yolk xanthophylls and quantification through a validated APCI(+) LC-MS/MS method.

Lutein and zeaxanthin exhibit significant biological activities therefore their dietary intake through carotenoid-rich foods and supplements is strongly recommended as preventive approach. Hence their extraction from natural substrates targets to their commercial exploitation as nutraceuticals and ocular pharmaceuticals. Since carotenoids' bioavailability is higher in fat-containing substrates, egg yolk is considered an ideal food matrix. DOE-based optimization of novel high energy extraction practices achieves efficient recovery of xanthophylls from lipid sources. In this research, 23 full factorial and Box-Behnken designs (BBD) were applied for optimizing ultrasound- (UAE) and microwave-assisted extraction (MAE) variables (i.e. extraction solvent, temperature, time, US or MW power and solvent/material ratio). LC-MS/MS results pointed out the precedence of UAE in lutein and zeaxanthin extraction, where higher yields were obtained with 1:1 n-hexane-acetone as solvent mixture at 19 min, 600 W and 35 mL g-1. UAE carotenoid content was higher than MAE due to the different mechanisms laying behind the two processes and due to more complete granule rupture caused by higher US power. Evaluating the current results, DOE-based UAE analytical methodology stands out as an auspicious and sustainable alternative for commercial-based extraction of lipidic bioactive compounds for food and drug industrial applications.

UHPLC-HRMS-based tissue untargeted metabolomics study of naringin and hesperidin after dietary supplementation in chickens.

To date numerous metabolomic studies have been performed in order to characterize nutritional intervention studies. The aim of the current study was to present a comprehensive pipeline for characterizing the metabolic changes that occur in chickens tissues in response to naringin and hesperidin dietary supplementation. Forty-nine chickens were randomly divided into 3 groups: the first one fed with diet supplemented with naringin, the second with hesperidin whereas the control group was fed by commercial basal diet. After 30 days of administration chicken muscle samples were analyzed by UHPLC-HRMS whereas data were analyzed by the proposed pipeline. Three significant variables were detected to discriminate the control from the group after naringin administration and thirteen variables after hesperidin supplementation. Furthermore, a more detailed pipeline (encompassing multiple internal standards, internal validation of the clustering, extended statistical significance scores and multiple identification procedures) has been proposed aiming towards a more accurate untargeted analysis.

Evaluating Modern Techniques for the Extraction and Characterisation of Sunflower (Hellianthus annus L.) Seeds Phenolics.

Recently there is a great interest in using high energy techniques (HET) which involve microwave or ultrasound-assisted extraction (MAE and UAE) for isolation of natural bioactive compounds from plant foods. Such bioactive compounds are phenolics which were determined from sunflower (Helianthus annuus L.) kernels and hulls (defatted) utilising two different high energy extraction techniques, ultrasound and microwave assisted solvent extraction. All samples were characterised by ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS). The effect of parameters such as the nature of the solvent, volume of solvent, temperature and time is discussed. It is proved that the techniques applied had reduced solvent consumption and shorter extraction times, and extraction yields of the analytes were equal to or to some extent higher than those obtained with conventional techniques. Total Phenolic Composition (TPC) of samples examined was studied by the Folin-Ciocalteu method and results were presented in µg gallic acid equivalents (GAE)/g dry extract. Kernels proved to have the higher amount of TPC while the press residues had shown comparable TPC results. The antioxidant activity of samples was spectrophotometrically determined by 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay using Butylated hydroxyl toluene (BHT) as reference compound to compare with samples. Sunflower seeds (kernels) showed again the highest antiradical efficiency (AE) compared to hulls and press-residue extract. Afterwards, ferric reducing ability of plasma (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays were used for measuring the antioxidant capacity of samples. Press residue, a by-product of sunflower oil extraction, contained phenolics as shown by UHPLC-ESI-MS analysis. Hence, later on these compounds can be possibly utilised by food or neutraceutical industries. Phenolic substances characterised in hulls, kernels, and press residue were phenolic acids, mainly chlorogenic, caffeic, cinnamic, 4-hydroxybenzoic and p-coumaric.

Post-acquisition spectral stitching. An alternative approach for data processing in untargeted metabolomics by UHPLC-ESI(-)-HRMS.

INTRODUCTION: In the case of the MS-based metabolomics, the large number of false positives remains a fundamental issue. OBJECTIVE: The aim of this study was to develop a new strategy, which highlights the number of the reliable features i.e. the detected features that correspond to a consistent peak according to chromatographic and mass spectrometric criteria. METHOD: For the analysis blood samples from 20 chickens, which were administrated with naringin and 9 samples from control, were analyzed by UHPLC-HRMS (Orbitrap Velos). Two methodologies have been compared for data processing. In the first one (classical approach), all data in the 100-900 m/z mass-to charge range were included for the data processing procedure whereas for the newly developed methodology, the data were shred in 100Da slices generating 8 datasets, which have been then subjected to the downstream MS data processing. Each dataset was treated separately and the m/z_tR features obtained by either VIP's or t-test values were merged and used as the input for the construction of the general model. RESULTS: The new methodology resulted to a 4-fold increase of the peaks that could be considered chromatographically and mass spectrometrically valid. CONCLUSION: A new strategy was reported on the detection of chromatographically reliable features during a metabolomic approach. The shredding of the LC-MS chromatograms into multiple m/z ranges increased the number of the identified chromatographically reliable features.

Antileishmanial ring-substituted ether phospholipids.

Three series of ring-substituted ether phospholipids were synthesized carrying N,N,N-trimethylammonium, N-methylpiperidino, or N-methylmorpholino headgroups. The first series is substituted by 2-cyclohexyloxyethyl or 2-(4-alkylidenecyclohexyloxy)ethyl groups, the second series by cyclohexylidenealkyl or adamantylidenealkyl moieties, and the third series by 2-aryloxyethyl or 6-aryloxyhexyl groups in the alkyl portion of the molecule. The antileishmanial activity of the new compounds was evaluated in vitro against the promastigote forms of L. donovani and L. infantum using an MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide)-based microassay as a marker of cell viability. Analogues 12, 15, 24, 30, 32, 41, 43, and 45 were more potent than the control compound miltefosine (hexadecylphosphocholine) against both L. donovani and L. infantum while, derivatives 13 and 42 were equipotent to miltefosine. Analogues 16, 17, 19, 20 were more potent than miltefosine against L. infantumand compounds 27, 31, 44 were more active than miltefosine against L. donovani. Differential scanning calorimetry (DSC) was used to probe the role of individual ether phospholipids on the physicochemical properties of model membranes. The DSC scans showed that the active compounds have a more profound effect on the thermotropic properties of model membrane bilayers than the less active ones.

An integrated approach using UHPLC-PDA-HRMS and 2D HSQC NMR for the metabolic profiling of the red alga Laurencia: dereplication and tracing of natural products.

The global metabolic profile of Laurencia crude red algal extracts was addressed by applying high-throughput analytical techniques, namely UHPLC­PDA­HRMS and 2D HSQC NMR. An integrated platform including software tools and databases, such as Xcalibur, ToxID, ACD/Labs and MarinLit, has been developed to mine the complex analytical data towards the accelerated identification of known metabolites and the detection of new natural products at the early stages of phytochemical analysis. In parallel, a searchable 'in-house' Laurencia-focused NMR database incorporating chemical structures, NMR spectroscopic data and reported biological activities has been generated. The screening strategy has been developed as a tool to prioritize the crude extracts to be further subjected to phytochemical analysis by tracing the presence of new natural products among the pool of known compounds. The successful application of this integrated methodology in the crude extract of Laurencia chondrioides led to the rapid detection of two new C15 bromoallene acetogenins (1 and 2), which were subsequently isolated and characterized.

Growth and membrane fluidity of food-borne pathogen Listeria monocytogenes in the presence of weak acid preservatives and hydrochloric acid.

This study addresses a major issue in microbial food safety, the elucidation of correlations between acid stress and changes in membrane fluidity of the pathogen Listeria monocytogenes. In order to assess the possible role that membrane fluidity changes play in L. monocytogenes tolerance to antimicrobial acids (acetic, lactic, hydrochloric acid at low pH or benzoic acid at neutral pH), the growth of the bacterium and the gel-to-liquid crystalline transition temperature point (T m) of cellular lipids of each adapted culture was measured and compared with unexposed cells. The T m of extracted lipids was measured by differential scanning calorimetry. A trend of increasing T m values but not of equal extent was observed upon acid tolerance for all samples and this increase is not directly proportional to each acid antibacterial action. The smallest increase in T m value was observed in the presence of lactic acid, which presented the highest antibacterial action. In the presence of acids with high antibacterial action such as acetic, hydrochloric acid or low antibacterial action such as benzoic acid, increased T m values were measured. The T m changes of lipids were also correlated with our previous data about fatty acid changes to acid adaptation. The results imply that the fatty acid changes are not the sole adaptation mechanism for decreased membrane fluidity (increased T m). Therefore, this study indicates the importance of conducting an in-depth structural study on how acids commonly used in food systems affect the composition of individual cellular membrane lipid molecules.

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.