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.

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.

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.

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.

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.

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.

Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 µM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the ß5/ß6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (Ki = 0.42 µM). The solution structure of 9 and 38 was solved by (1)H NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 µM) is slightly more potent than 9 (IC50 = 34.3 µM) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors.

Analysis of the unsaponifiable fraction of lipids belonging to various milk-types by using comprehensive two-dimensional gas chromatography with dual mass spectrometry/flame ionization detection and with the support of high resolution time-of-flight mass spectrometry for structural elucidation.

The present investigation is focused on the use of a comprehensive two-dimensional GC (GC×GC) method, with dual mass spectrometry/flame ionization detection (MS/FID), for the qualitative and quantitative analysis of the unsaponifiable fraction of milk lipids (cow butter, buffalo, ewe, and goat milks). The structure of many constituents (particularly sterols) was confirmed by using GC-high resolution time-of-flight MS. The GC×GC column set used consisted of a low-polarity first dimension, and a medium-polarity secondary one, both characterized by a high thermal stability. The use of dual detection enabled the attainment of both mass spectral information and relative % FID data. The complexity of the fingerprint, generated by the unsaponifiable fraction, justified the employment of the two-dimensional GC technology. However, it was two other GC×GC characteristics that contributed most to the attainment of promising results, namely sensitivity enhancement and the formation of group-type patterns. Because many milk lipid constituents were not contained in the MS databases employed, exact mass information proved to be valuable for identification purposes.

Rapid collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric analysis.

The present research reports the use of a three-dimensional preparative gas chromatography (prep GC) system, equipped with three Deans-switch devices and 5%diphenyl/wax/mid-polarity ionic liquid stationary phases, for the isolation of volatile components from a complex natural source, namely wampee essential oil (derived from Clausena lansium Skeels leaves). Collection was performed by using a simple and effective lab-constructed trapping device. Initially, an unknown (and abundant) wampee oil constituent was erroneously identified as α-sinensal, through an MS database search (a low similarity match was attained), performed after a GC-quadMS experiment., The unknown compound was then the isolated by using the novel prep GC system, in a highly pure form (at the mg level), and was correctly identified by using nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR) and mass spectrometry (MS). Both FTIR and MS data were used to confirm the NMR information. The name given to the molecule was (2E,6E)-2-methyl-6-(4-methylcyclohex-3-enylidene)hept-2-enal. The results herein described will demonstrate the need for a high-resolution GC step, prior to analyte collection, in the prep GC analysis of complex samples.

catena-Poly[[[µ-aqua-penta-aqua-dizinc(II)]-µ(4)-benzene-1,2,4,5-tetra-carboxyl-ato] dihydrate].

The asymmetric unit of the title compound, {[Zn(2)(C(10)H(2)O(8))(H(2)O)(6)]·2H(2)O}(n), contains two distinct Zn atoms joined by a bridging water molecule and two bridging carboxyl-ate groups belonging to distinct halves of benzene-1,2,4,5-tetra-carboxyl-ate (tbec) tetra-anionic ligands, both lying on crystallographic inversion centres. The structure of this new isopolymorphic one-dimensional coordination polymer features asymmetric bimetallic octa-hedral knots. O-H⋯O hydrogen bonds between water molecules and carboxylate O atoms help to consolidate the crystal packing.

catena-Poly[bis-(4-amino-pyridinium) [[tetra-aqua-nickel(II)]-µ-benzene-1,2,4,5-tetra-carboxyl-ato] dihydrate].

The asymmetric unit of the title compound, {(C(5)H(7)N(2))(2)[Ni(C(10)H(2)O(8))(H(2)O)(4)]·2H(2)O}(n), contains an Ni(II) atom, two water mol-ecules of coordination, one half of a benzene-1,2,4,5-tetra-carboxyl-ate (btec) anionic ligand, one 4-amino-pyridinium cation (papy) and an uncoordinated water mol-ecule. The metal center lies on an inversion center and adopts an octa-hedral geometry with the carboxyl-ate groups tilted out of the mean plane formed by the btec. In the crystal, mol-ecules are linked into one-dimensional coordination polymers running along the ac diagonal. The crystal structure is consolidated by N-H⋯O and O-H⋯O hydrogen bonds.

5-Arylidene-2-imino-4-thiazolidinones: design and synthesis of novel anti-inflammatory agents.

The synthesis and pharmacological activity of 5-arylidene-2-imino-4-thiazolidinones (3a-8a) are described. All derivatives exhibited significant activity levels in models of acute inflammation such as carrageenan-induced paw and pleurisy edema in rats. In particular, 5-(3-methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (3a) displayed high levels of carrageenan-induced paw edema inhibition comparable to those of indomethacin. In addition the ability of such a new class of anti-inflammatory agents to inhibit COX isoforms was assessed in murine monocyte/macrophage J774 cell line assay. 5-(4-Methoxyphenylidene)-2-phenylimino-3-propyl-4-thiazolidinone (6a), the most interesting compound in such an experiment, was docked in the known active site of COX-2 protein and showed that its 4-methoxyarylidene moiety can easily occupy the COX-2 secondary pocket considered as the critical interaction for COX-2 selectivity.