Preparative three-dimensional GC and nuclear magnetic resonance for the isolation and identification of two sesquiterpene ethers from Dictyota Dichotoma.

Separation science plays a crucial role in the isolation of novel compounds contained in complex matrices. Yet their rationale employment needs preliminary structure elucidation, which usually requires sufficient aliquots of grade substances to characterize the molecule by nuclear magnetic resonance experiments. In this study, two peculiar oxa-tricycloundecane ethers were isolated by means of preparative multidimensional gas chromatography from the brown alga species Dictyota dichotoma (Huds.) Lam., aiming to assign their 3D structures. Density functional theory simulations were carried out to select the correct configurational species matching the experimental NMR data (in terms of enantiomeric couples). In this case, the theoretical approach was crucial as the protonic signal overlap and spectral overcrowding were preventing any other unambiguous structural information. Just after the identification through the density functional theory data matching of the correct relative configuration it was possible to verify an enhanced self-consistency with the experimental data, confirming the stereochemistry. The results obtained further pave the way toward structure elucidation of highly asymmetric molecules, whose configuration cannot be inferred by other means or strategies.

Cytoprotective and Antioxidant Effects of Hydrolysates from Black Soldier Fly (Hermetia illucens).

The black soldier fly (BSF), Hermetia illucens, has been recognized as one of the most promising insect species for its ability to valorize organic waste while producing a valuable larval biomass with a great potential as a sustainable source of nutrients, including proteins and bioactive molecules. In the present study, BSF larvae were used to produce and characterize the protein hydrolysates (BPHs) that were then evaluated for their potential biological activity in vitro. The BPHs obtained from the BSF larvae proteins by enzymatic digestion were characterized by Nuclear Magnetic Resonance (NMR) and polyacrylamide gel electrophoresis and assessed for their antioxidant activity (BPHs in the range of 0.1 to 1.5 mg/mL) in L-929 cells. Our findings show that BPHs can exert a dose-dependent cytoprotective role against H2O2-iduced oxidative stress in cells. This antioxidant activity relies on the reduction of ROS levels in challenged cells as measured by flow cytometry and fluorescence microscopy, together with the induction and nuclear translocation of Nrf2, as evaluated by qPCR and indirect immunofluorescence analysis, respectively. Overall, our findings on the remarkable biological activity of the BPHs obtained in a large-scale process strongly suggest the application of BPHs as ingredients promoting animal health in feed formulations.

A Fast and Efficient Ultrasound-Assisted Extraction of Tocopherols in Cow Milk Followed by HPLC Determination.

A fast HPLC method with fluorescence detector (FD) was developed for the determination of three tocopherols (TOCs) in milk samples from Modicana cattle breed. The ultrasound-assisted procedure was optimized for the extraction of TOCs prior to HPLC/FD analysis, reducing sample preparation time and allowing a fast quantification of α-tocopherol, δ-tocopherol and γ tocopherol. The optimized ultrasonic extraction combines an efficient and simple saponification at room temperature and a rapid HPLC quantification of TOCs in milk. The precision of the full analytical procedure was satisfactory and the recoveries at three spiked levels were between 95.3% and 87.8%. The linear correlations were evaluated (R2 > 0.99) and the relative standard deviation (RSD) values for intra-day and inter-day tests at three spiked levels were below 1% for the retention time and below 5.20% for the area at low level spiking. The proposed procedure, reducing the experimental complexity, allowed accurate extraction and detection of three TOCs in milk samples from Modicana cattle breed.

Protein hydrolysates from anchovy waste: purification and chemical characterization.

The aim of this study is to prompt the recovery of industrial by-products through the production of new functional foods; it takes advantage from new throughput technologies with low environmental impact and high economic sustainability. In the field of fish processing, in order to recover the worthy protein-rich fish waste, residues from the production of Anchovies (Engraulis encrasicolus) have been converted into hydrolysate through enzymatic treatment. The obtained hydrolysate product showed a promising biological and nutritional content made by differently sized peptides and free amino acids endowed with assessed benefic effects. The study showed the possibility to produce a dry powder with an activity water (aw) of 0.3-0.5 and an essential amino acids (EAA) fraction of 42.0% over the total amino acids (TAAs). These results pave the way to the smart recovery of commercial products featured by high nutritional value, either as stand-alone items or as components of functional foods.

Properties of a New Food Supplement Containing Actinia equina Extract.

Marine species represent a great source of biologically active substances; Actinia equina (AE), an Anthozoa Cnidaria belonging to the Actinidiae family, have been proposed as original food and have already been included in several cooking recipes in local Mediterranean shores, and endowed with excellent nutraceutical potential. The aim of this study was to investigate some unexplored features of AE, through analytical screening and an in-vitro and in-vivo model. An in-vitro study, made on RAW 264.7 stimulated with H2O2, showed that the pre-treatment with AE exerted an antioxidant action, reducing lipid peroxidation and up-regulating antioxidant enzymes. On the other hand, the in-vivo study over murine model demonstrated that the administration of AE extracts is able to reduce the carrageenan (CAR)-induced paw edema. Furthermore, the histological damage due to the neutrophil infiltration is prevented, and this highlights precious anti-inflammatory features of the interesting food-stuff. Moreover, it was assessed that AE extract modulated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and The nuclear factor erythroid 2-related factor 2 (Nrf-2) pathways. In conclusion, our data demonstrated that thanks to the antioxidant and anti-inflammatory properties, AE extract could be used as a new food supplement for inflammatory pathology prevention.

Theoretical prediction of 13C NMR spectrum of mixed triglycerides by mean of GIAO calculations to improve vegetable oils analysis.

This pioneering study based on GIAO-DFT methods is aimed to the best prediction of 13C Nuclear Magnetic Resonances (NMR) arising from triglycerides (and also glycerols), known to be the main component of vegetable oils. Provided that fatty esters bound to the glycerol moiety are not affected by the other esterification chains, and slightly affected by their own esterification position (2- internal, or 1/3- external), eight natural molecules are first optimized despite the challenging presence of many non-hydrogen atoms and the large conformational freedom. This preliminary study sheds light on the total chemical shift prediction concerning five fatty esters (Oleic, Palmitic, Linoleic, Stearic and Linolenic) either present in internal or external positions (ten fragments in total); these results display a very good matching to the experimental profile recorded for several vegetable oils chosen as natural mixtures of glycerides. In order to further improve the theoretical to experimental matching, ten simplified triglycerides with the mentioned fatty esters in the two different esterification positions, and flanked by acetyl esters, were studied and optimized. Beyond the best matching reached so far, we notice that the theoretical rationalisation of the overcrowding in the 28.7-29 ppm spectral region in unable to decode the necessary resolution, nonetheless the same theoretical prediction can still drive the appropriate assignments (as for the fifth and sixth carbon attribution of every chain) even against actual misleading reports.

Oleic Acid Is not the Only Relevant Mono-Unsaturated Fatty Ester in Olive Oil.

(1) Background: Extra-virgin olive oil (EVOO) is a precious and universally studied food matrix. Recently, the quantitative chemical composition was investigated by an innovative processing method for the nuclear magnetic resonance (NMR) experiments called Multi-Assignment Recovered Analysis (MARA)-NMR. (2) Methods: Any EVOO 13-carbon NMR (13C-NMR) profile displayed inconsistent signals. This mismatch was resolved by comparing NMR data to the official gas-chromatographic flame ionization detection (GC-FID) experiments: the analyses concerned many EVOOs but also the "exotic" Capparis spinosa oil (CSO). (3) Results: NMR and GC-FID evidenced the overwhelming presence of cis-vaccenic esters in the CSO and, more importantly, cis-vaccenic 13C-NMR resonances unequivocally matched the misunderstood 13C-NMR signals of EVOOs. The updated assignment revealed the unexpected relevant presence of cis-vaccenic ester (around 3%) in EVOOs; it was neglected, so far, because routine and official GC-FID profiles did not resolve oleic and cis-vaccenic signals leading to the total quantification of both monounsaturated fatty esters. (4) Conclusions: The rebuilt MARA-NMR and GC-FID interpretations consistently show a meaningful presence of cis-vaccenic esters in EVOOs, whose content could be a discrimination factor featuring specific cultivar or geographical origin. The study paves the way toward new quantification panels and scientific research concerning vegetable oils.

Accumulation of PCBs, PAHs, plasticizers and inorganic elements in Hexanchus griseus from the strait of Messina (Central Mediterranean sea).

This study investigates the inorganic elements and the persistent organic pollutants (POPs) accumulated in liver of a sexually mature Hexanchus griseus living in the Mediterranean Sea. The casual finding of a specimen in the Strait of Messina (April 2018) allowed us to carefully analyse its liver which can be considered a very important biological indicator. The determination of inorganic elements was carried out by ICP-MS technique. Quantitative determination of polycyclic aromatic hydrocarbons (PAH) and poly-chloro-biphenyl compounds (PCB), distinguished in dioxine-like (DL) and not-dioxine-like (NDL) derivates, has been carried out by HRGC/MS and HRGC-MS/MS respectively. Worrying levels of PCB-DL (TEQ = Σ(PCB-DL)*TEF = 5.96 ng g-1), PCB-NDL (Σ(PCB-NDL) = 1390.4 ng g-1) together with the presence of pesticides and plasticizers were found in the shark liver oil. The levels of heavy metals were below the legal limits.

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) as quick and direct insight of almonds.

Almonds are the tasty seeds of Prunus dulcis plants globally appreciated for the pleasant palatability and remarkable nutritional value, therefore it is very spread as snack and as basic ingredient of the confectionery products. The HR-MAS-NMR is a simple spectroscopy able to directly and quickly explore the chemical composition of powdered seed samples dispersed in D2O. 1H spectra witness the remarkable presence of triglyceride fatty esters together with sucrose; other minor water soluble metabolites are also detectable. This very rough approach is effectively providing chemical profiles featuring almond samples. In this analysis we were able to statistically distinguish the "Avola" almonds from other marketed products submitted to the same analysis. This is just a first investigation based on the main compounds but it might pave the way toward the quantitative evaluation of many other compounds in the almond therefore implementing the HR-MAS-NMR knowledge of these precious seeds.

Collection and identification of an unknown component from Eugenia uniflora essential oil exploiting a multidimensional preparative three-GC system employing apolar, mid-polar and ionic liquid stationary phases.

The present research deals with the collection and structural elucidation of an unknown component, accounting for about 35% of the essential oil obtained upon distillation of the leaves of Eugenia uniflora L., harvested during summer (January, 2017) in Paraná State (Southern Brazil). A multidimensional gas chromatographic preparative system, based on the coupling of three GC systems equipped with apolar, PEG and ionic liquid-based stationary phases, was successfully applied for the isolation of the chromatographic band relative to the unknown molecule. The use of wide-bore columns allowed for an increased sample capacity compared to conventional micro-bore columns, thus the injection of a neat sample was feasible, greatly reducing the total collection time. A higher chromatographic efficiency was afforded by the use of a multidimensional approach in the heart-cut mode, exploiting the different selectivity of three stationary phases, which ensured the attainment of a highly pure fraction. In only five runs, more than 3 milligrams were collected, with an average purity greater then 95%. Finally, the unknown component was subjected to nuclear magnetic resonance spectroscopy, mass spectrometry and condensed phase Fourier-transform infrared spectroscopy, leading to the identification of 6-ethenyl-6-methyl-3,5-di(prop-1-en-2-yl)cyclohex-2-en-1-one. The presented approach has been demonstrated to be effective for the isolation and structural elucidation of unknown molecules in complex samples, which will allow for further in-depth studies, like biological evaluation or pharmacological tests.

Multiple analytical approaches for the organic and inorganic characterization of Origanum vulgare L. samples.

Origanum vulgare L. samples, marketed in different geographic locations, were characterized by their organic and inorganic chemical composition. A total of 35 commercial samples were collected from various sites and analyzed to determine the qualitative and quantitative profile of essential oils, phenolic compounds and some inorganic elements. The variation in the content and composition of the essential oil was assessed by GC and GC-MS analyses, the phenolic fraction was investigated by UPLC®/PDA, and the inorganic elements were determined by ICP-MS. The Principal Component Analysis (PCA) was applied with the aim to sort out the Origanum vulgare L. samples with different composition according to the different belonging origins. The results showed appreciable qualitative and quantitative differences among samples from different geographic origin.

Anti-cancer activity of di- and tri-organotin(IV) compounds with D-(+)-Galacturonic acid on human tumor cells.

We have compared the anti-proliferative activity in vitro, of R2SnGala (1-3) [R = Me, n-Bu, Ph] and novel R3SnGala (4, 5) [R = Me, n-Bu] with D-(+)-Galacturonic acid [HGala; Galaq-, q = (2) and (1) for R2SnGala and R3SnGala, respectively] compounds, towards human tumor cell lines of intestinal carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). The new synthesized 4 and 5 compounds were characterized, in solution, by 1H, 13C and 119Sn NMR, that showed that HGala acts as monoanionic moiety and evidenced the dynamic behavior of the compounds, due to inter-conversions involving the anomeric carbon atom of the ligand. Cell viability, apoptosis induction and cell cycle distribution were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry, respectively. The cytotoxicity of the compounds, in the micro-submicromolar range, changed in the order of the organotin(IV) moieties, according to 5 > 3 > 2, while 1 and 4, containing MenSn(IV) (n = 2,3) moieties, were ineffective. Compound 5 showed peculiar cytotoxic effects. It did not cause time dependent inhibition of cell growth nor accumulated into the cells. Cell death induced by the active 2, 3, and 5, was shown to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and the loss of mitochondrial potential. All the cytotoxic compounds induced an accumulation of cells in the subG0/G1phase, while only 2 and 3 perturbed the cell cycle confining viable cells in G0/G1phase. Finally, none of the compounds investigated affected the viability of normal intestinal or liver cells, indicating selectivity towards tumor cells.

Chemical characterization of a variety of cold-pressed gourmet oils available on the Brazilian market.

Different specialty extra virgin oils, produced by cold-pressing fruits/nuts (olive, pequi, palm, avocado, coconut, macadamia and Brazil nut) and seeds (grapeseed and canola), and retailed in the Brazilian region of Minas Gerais, were chemically characterized. Specifically, for each type of oil, the fatty acid composition was elucidated by GC-FID, the contents of selected polyphenols and squalene were determined respectively by UHPLC-MS and UHPLC-PDA, whereas minerals were explored by means of ICP-MS. Olive oil was confirmed to have the highest MUFA content due to a valuable level of oleic acid, while oils from grapeseed, Brazil nut and canola were marked by nutritionally important PUFA levels. The highest SFA content found in coconut oil was mainly due to the high levels of lauric acid, known for its advantageous HDL-raising effects. As for polyphenols, gourmet oils from palm, coconut and canola showed higher levels of phenolic acids (e.g. p-hydroxybenzoic, ferulic, syringic, acids) than olive oil, which was though characterized by peculiar antioxidants, such as tyrosol and hydroxytyrosol. Also, olive oil had the highest amount of squalene, followed by the oil from Brazil nut. Finally, all the investigated oils had very low levels (order of µg/kg) of pro-oxidant elements, such as Cu, Fe and Mn. Overall, these findings may fill the gaps still present in literature on certain compositional aspects of commercially available gourmet oils.

Combination of separation and spectroscopic analytical techniques: application to compositional analysis of a minor citrus species.

The composition of juice and essential oil of Citrus limetta Risso was investigated. Multidimensional and enantio-gas chromatography were used for the elucidation of the volatile profile and the assessment of enantiomeric distribution. Predominant compounds were linalyl acetate (13.06 g/100 g), ß-pinene (6.79 g/100 g), myrcene (1.40 g/100 g) and sabinene (1.05 g/100 g). Through a ß-cyclodextrin column eight chiral pairs were separated, equally distributed between laevorotatory and dextrorotatory enantiomers. Liquid chromatography with triple quadrupole ESI-MS and PDA detection showed the presence of flavonoids and phenolic compounds (791.34 ± 36.83 and 32.97 ± 1.92 mg L-1, respectively) in the juice. Sugars (i.e. glucose 202.23 ± 26.81 and fructose 146.73 ± 3.17 mM, respectively), aminoacids (i.e. proline 29.40 ± 3.78 and GABA 8.90 ± 0.95 mM) and organic acids (i.e. citrate 4.68 ± 0.81 mM) were determined in juice by means of 1H NMR spectroscopy.

Quali-quantitative characterization of the volatile constituents in Cordia verbenacea D.C. essential oil exploiting advanced chromatographic approaches and nuclear magnetic resonance analysis.

Cordia verbenacea D.C. (Boraginaceae, Varronia curassavica Jacq. synonym) is a medicinal plant, native from Brazil, especially the leaves are used in folk medicine. The aim of this study was to extend the characterization of the volatile fraction of the essential oil obtained from this plant, by using GC-FID, GC-MS, and chiral GC. Moreover, to further clarify the composition of the volatile fraction, preparative multidimensional-GC (prep-MDGC) was used to collect unknown compounds, followed by NMR characterization. Specifically, the chemical characterization, both qualitative and quantitative, of the volatile fraction of the essential oil obtained from Cordia verbenacea cultivated in the Minas Gerais area (central area of Brazil) was investigated for the first time. The principal components from a quantitative point of view were α-pinene (25.32%; 24.48g/100g) and α-santalene (17.90%; 17.30g/100g), belonging to the terpenes family. Chiral-GC data are reported for the enantiomeric distribution of 7 different components. Last, to obtain the complete characterization of the essential oil constituents, prep-MDGC analysis was used to attain the isolation of two compounds, not present in the principal MS databases, which were unambiguously identified by NMR investigation as (E)-α-santalal and (E)-α-bergamotenal, reported for the first time in Cordia verbenacea essential oil.

Determination and quantification of carotenoids in sea sponges Raspaciona aculeata and Dictyonella marsilii present in the Ganzirri Lake (Messina), Italy.

Considering the crucial role of carotenoids exploitable both as nutraceuticals and also as dyes in food industry, there are many efforts in seeking for new sources of these pigments, especially in the marine world. In this study, for the first time, we extracted carotenoids from sea sponges Raspaciona aculeata and Dictyonella marsilii taken from Ganzirri Lake Messina (Italy). The determination and quantification of carotenoids was made by UPLC-PDA-MS. Remarkable results concern renieratene content in R. aculeate found to be over 2570 ppm.

Evaluation of Chronic Nanosilver Toxicity to Adult Zebrafish.

Nanotechnology is rapidly growing with nanoparticles produced and utilized in a wide range of commercial products worldwide. Among the different types of nanomaterials produced, silver nanoparticles (AgNPs) occupy a predominant position and they are used in electronics, clothing, food industry, cosmetics and medical devices. Nanosilver has also showed excellent performance in antibacterial application. Nowadays, the increasing use of AgNPs has put the evidence on their possible toxicity to the human health and the impact on the environment. This paper focus on adverse effects of AgNPs in adult of Danio rerio. Fishes exposed to increasing concentrations (8, 45, and 70 µg/l) silver nanoparticles (AgNPs, 25 nm in average diameter) and after treatment for 30 days, was quickly euthanized in MS-222. We have evaluated bioaccumulation of AgNPs using ICP-MS and analyzed histological changes, biomarkers of oxidative damage and gene expression in the gut, liver and gills tissues of AgNPs-treated zebrafish. The histological analysis showed lesions of secondary lamellae of the gills with different degrees of toxicity such as hyperplasia, lamellar fusion, subepithelial edema, and even in some cases telangiectasia. Huge necrosis of the intestinal villi was found in the gut. No lesion was detected in the liver. The analysis revealed a high expression of metallothioneins 1 (MTs 1) in animals exposed to AgNPs compared to the control group. The ICP-MS analysis shows that the amount of particles absorbed in all treated samples is almost the same. We can affirm that AgNPs toxicity linked more to their size and state of aggregation than to their concentrations. Silver nanoparticles can damage gills and gut because they are able to pass through the mucosal barrier thanks to their small size. The damage is still reversible because it is not documented injury to the basal membrane.

Metabolite and mineral profiling of "Violetto di Niscemi" and "Spinoso di Menfi" globe artichokes by 1H-NMR and ICP-MS.

Globe artichoke has been long considered a nutraceutical food for its valuable content of bioactive compounds. However, beside a well-known polyphenol profile, poor information is available about its metabolite and mineral composition. The aim of this study was to investigate edible parts of Sicilian artichokes, 'Spinoso di Menfi' and 'Violetto di Niscemi', by 1H NMR and ICP-MS for elucidating these compositional aspects. Although bracts and hearts of both artichokes shared a very similar metabolite pattern, 'Spinoso di Menfi' showed a higher number of metabolites, such as amino acids and polyphenols, than 'Violetto di Niscemi'. 'Spinoso di Menfi' was also marked by higher levels of macro- and microelements when compared to 'Violetto di Niscemi'. Also, artichoke heart demonstrated to accumulate higher mineral levels than bracts. 1H NMR and ICP-MS successfully profiled metabolites and metals in such plant food, partially covering the lack of literature data about 'Spinoso di Menfi' and 'Violetto di Niscemi' artichokes.

Determination of 1,2/1,3-diglycerides in Sicilian extra-virgin olive oils by 1H-NMR over a one-year storage period.

This study is aimed to monitor by 1H NMR spectroscopy the effect of a 12-month storage period on the 1,2-diglycerides over 1,3-diglycerides ratio for five mono-cultivar 'extra virgin olive oils' (EVOO) (Arbequina, Arbosana, Cerasuola, Nocellara and FS17) and one blend of two different cultivars (Nocellara + Biancolilla) preserved in the dark and at room temperature. These quantifications, at 500 MHz, are readily extracted through a specific and original integration difference method. Albeit it was known that the isomerisation rate is affected by the free acidity, we here demonstrate that it also depends on the presence of specific macromolecules (lipases), indeed, different EVOO cultivars with similar free acidity, show different isomerisation rate. Our results are consistent with similar diglyceride monitoring performed on Greek and Spanish EVOOs by 31P NMR.

Discovery of 4-[(5-arylidene-4-oxothiazolidin-3-yl)methyl]benzoic acid derivatives active as novel potent allosteric inhibitors of protein tyrosine phosphatase 1B: In silico studies and in vitro evaluation as insulinomimetic and anti-inflammatory agents.

New 4-{[5-arylidene-2-(4-fluorophenylimino)-4-oxothiazolidin-3-yl]methyl}benzoic acids (5) and 2-thioxo-4-thiazolidinone analogues (6) were synthesised as a part of a continuing search for new inhibitors of protein tyrosine phosphatase 1B (PTP1B), an enzyme which is implicated in metabolic disorders and inflammatory signaling. Most of the tested compounds were shown to be potent PTP1B inhibitors. Moreover, their inhibition mechanism was markedly influenced by the substituents in the positions 2 and 5, as kinetic studies indicated. Docking experiments suggested that certain derivatives 5 and 6 may efficiently fit into an allosteric site positioned between the ß-sheet including Leu71 and Lys73 and a lipophilic pocket closed by the loop consisting of Pro210 to Leu 204. In cellular assays, several of these new 4-thiazolidinone derivatives showed insulinomimetic and anti-inflammatory properties. Out of them, compound 5b exhibited the most promising profile, being able to promote the activation of both insulin receptor and downstream Akt protein as well as to increase 2-deoxyglucose cellular uptake. Interestingly, compound 5b was also able to interrupt critical events in inflammatory signaling.