Rui Gonçalves

Comparative study of phytochemicals and antioxidant potential of wild edible mushroom caps and stipes

A comparative study of the organic acids and phenolics composition and of the total alkaloids content of entire wild edible mushrooms (Russula cyanoxantha, Amanita rubescens, Suillus granulatus and Boletus edulis) and correspondent caps and stipes was performed. All species presented oxalic, citric, malic and fumaric acids, with A. rubescens exhibiting the highest total organic acids content. Organic acids were preferably fixed in the cap. Among phenolics, only p-hydroxybenzoic acid was found in A. rubescens and S. granulatus, in very low amounts. B. edulis was the species that presented the highest total alkaloid amounts. Except for this species, alkaloids mainly accumulated in the cap. All of the species exhibited a concentration-dependent scavenging ability against DPPH(·). B. edulis revealed the highest antioxidant capacity. The cap seemed to be the part with highest antioxidant potential. Some relationships between chemical composition and antioxidant capacity were considered.

Mechanisms of tannin-induced trypsin inhibition: a molecular approach.

Association of procyanidins with enzymes has drawn attention over the past few years. This work aimed to bring insights on interaction of the protease trypsin with the procyanidin dimer (B3). This interaction was characterized by fluorescence quenching, saturation transfer difference (STD) NMR, molecular modeling, and through an enzymatic inhibition assay. Further studies were conducted regarding the influence of pectin on the binding process. A general overview of the binding process may be outlined as follows: a) at low procyanidin concentrations (below the critical micellar concentration-(CMC)) a specific interaction probably driven by hydrogen bonds between the protein backbone and the procyanidin occurs and is associated with the reduction of both enzyme activity and fluorescence; b) at high procyanidin concentration (above the CMC) the interaction becomes nonspecific. This variation in both nature and extent of the interaction with the variation of procyanidin concentration shows how tannin self-association may affect the interaction between tannins and proteins. It was also shown that the mechanism through which pectin affects the interaction between procyanidin B3 and trypsin is of a competitive type.

Volatile composition of Catharanthus roseus (L.) G. Don using solid-phase microextraction and gas chromatography/mass spectrometry

A total of 88 volatile and semi-volatile components were formally or tentatively identified in flowers, leaves and stems of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye), by headspace solid-phase microextraction (HS-SPME) and by dichloromethane extraction, combined with gas chromatography-mass spectrometry (GC-MS). These include some diterpenic compounds (manool and manoyl oxides), a sesquiterpen (alpha-bisabolol), and some pyridine, pyrazine, indol and carotenoid derivatives. Applying multivariate analysis (principal component analysis and agglomerative hierarchic cluster analysis) to the HS-SPME-GC-MS data, it was possible to characterize each part of the vegetal material using a relative small number of compounds. Hence, flowers were richer in terpenic molecules (including limonene), alpha-bisabolol, methyljasmonate, cis-jasmone, 2-phenylethanol, phenylacetaldehyde, trans-2-octenal, benzylic alcohol and 2-isobutyl-3-methoxypyrazine. Leaves can be characterized by the methyl and propyl esters of fatty acids, mono- and disaturated, trans-phytol, carotenoid derivative compounds, hydrofarnesylacetone, methylanthranilate, manool and epi-manool oxide, while stems have high levels of volatile aldehydes, such as hexanal, octanal, cis-2-nonenal, cis-2-decenal, cis, trans-2,6-nonadienal, trans, trans-2,4-decadienal and cis, trans-2,4-decadienal. Dichloromethane extraction allowed also the identification of some alkaloid-like compounds that were not detected by HS-SPME.

Interaction of phenolic compounds with bovine serum albumin (BSA) and α-amylase and their relationship to astringency perception.

The ability of grape seed extracts to bind to bovine serum albumin (BSA) and α-amylase was studied by fluorescence quenching of protein intrinsic fluorescence and nephelometry. The influence of grape seed ripeness on astringency was also evaluated. From the spectra obtained, the modified Sterm-Volmer (K(app)) and the bimolecular quenching constants were calculated. Results showed that grape seed extracts had good affinity for proteins. The association strength of tannin-protein interactions varied with changes in tannin structure associated with the degree of ripeness affecting the binding/quenching process. In all cases studied, higher values of K(app) were obtained in samples at harvest which have greater ability to bind to proteins than have samples at post-veraison time. Nephelometric assays show the same trend as do fluorescence quenching studies. A possible explanation for this is that, as seeds ripen, their tannins increase in molecular mass, which relates to an increase in hydrophobicity of the molecules, and this increases protein affinity. However, that is contrary to the reported decrease in astringency of grape seeds during maturity. This indicates that tannin-protein interactions are not the only explanation for the complex sensations of astringency of grape seeds.

Free Water-Soluble Phenolics Profiling in Barley (Hordeum vulgare L.)

The phenolic profile of barley ( Hordeum vulgare L.) leaves, seeds, awns, and stems, collected in two different locations from Portugal, was determined by a high-performance liquid chromatography/diode array detector (HPLC/DAD). A total of 28 compounds were identified and quantified, which included 4 phenolic acids, 6 C-glycosylflavones, and 18 O-glycosyl-C-glycosyl flavones, with some of them acylated. Distinct profiles were noticed among the analyzed materials. The greatest diversity of compounds was found in barley leaves (26 flavonoids and 2 phenolic acid derivatives), which also exhibited the highest concentration of phenolics. Isoorientin-7-O-glucoside (lutonarin) was the major compound in leaves, while, in general, the pair isovitexin-7-O-rutinoside plus isoscoparin-7-O-glucoside were the main phenolics in the other materials. Thus, barley leaves may constitute an important dietary source of protective compounds, which could be used, for example, to take profit from the wastes resulting from alcoholic drink obtainment.

On the bioavailability of flavanols and anthocyanins: flavanol-anthocyanin dimers.

The bioavailability of flavanols, anthocyanins and anthocyanin-derived pigments like flavanol-anthocyanin dimers already reported to occur in food products is a major unsolved issue. The absorption of the flavanol-anthocyanin dimer (+)-catechin-(4,8)-malvidin-3-O-glucoside (Cat-Mv3glc) through Caco-2 cells was assessed by performing transepithelial transport assays. The ability of Cat-Mv3glc to cross Caco-2 cells was compared with that of malvidin-3-glucoside (Mv3glc), (+)-catechin (Cat) and procyanidin B3 (Cat-Cat), in order to evaluate the influence of some structural features on the transport efficiency. The flavanol-anthocyanin dimer was absorbed in this intestinal model although with a lower efficiency than the monomers Cat and Mv3glc. On the other hand, Cat-Mv3glc was found to cross the intestinal barrier model more significantly than Cat-Cat. This feature may be related to the presence of the glucose moiety in its structure. Overall, this study brings more insights into the bioavailability of anthocyanins and flavanols and represents the first report on the bioavailability of flavanol-anthocyanins.

Biological Relevance of the Interaction between Procyanidins and Trypsin: A Multitechnique Approach

The interactions between the digestive protease trypsin type IX-S from porcine pancreas and grape seed procyanidins were monitorized by fluorescence quenching, dynamic light scattering, nephelometry, circular dichroism, and enzymatic inhibition assay. This work reports that the inhibition of trypsin activity by grape seed procyanidins and the respective quenching of intrinsic protein fluorescence are closely related. These two phenomena increase with the molecular weight of the tested procyanidins. The interaction between procyanidins and enzyme was shown to involve a specific interaction as inferred from the fluorescence assays. It was also shown by fluorescence spectroscopy that the binding of procyanidin molecules to the enzyme does not induce significant structural modifications. A relationship between aggregate formation, using dynamic light scattering and nephelometry, and fluorescence quenching was observed with maxima achieved for similar stoichiometric ratios. The binding of procyanidins to trypsin affects only slightly protein structure as seen by circular dichroism.

AUV Control and Communication using Underwater Acoustic Networks

Underwater acoustic networks can be quite effective to establish communication links between autonomous underwater vehicles (AUVs) and other vehicles or control units, enabling complex vehicle applications and control scenarios. A communications and control framework to support the use of underwater acoustic networks and sample application scenarios are described for single and multi-AUV operation.

Study of the Interaction of Pancreatic Lipase with Procyanidins by Optical and Enzymatic Methods

The interactions between porcine pancreatic lipase (PL) and grape seed procyanidins were studied by an enzymatic assay, fluorescence quenching, nephelometry, and dynamic light scattering (DLS). An inhibitory effect of grape seed procyanidins on lipase hydrolytic activity was found. Both the inhibition of lipase activity by procyanidins and the respective quenching of intrinsic protein fluorescence increased with the average degree of polymerization of the tested procyanidins. The association between procyanidins and enzyme involves a specific interaction as inferred from the fluorescence assays despite not changing significantly the tertiary structure of the protein. For all tested procyanidins it was shown, both by DLS and by nephelometry, that an increase in aggregation occurs up to a stoichiometric maximum after which further procyanidin addition causes a decrease in aggregation of aggregates. The maximum size of aggregates was shown to be closely related to the maximum overall aggregation. It was also shown that the inhibition of enzyme activity is to a large extent independent of the formation of aggregates.

Understanding the binding of procyanidins to pancreatic elastase by experimental and computational methods.

Human diets are rich in secondary metabolites such as polyphenols. These compounds perform a wide range of crucial functions in biological systems and are of great interest for the pharmaceutical and food industries. In this work, the binding mode of the natural polyphenolic compounds from grape seed on the porcine pancreatic elastase surface was studied by experimental and computational methods. Fluorescence quenching, circular dichroism, nephelometry, dynamic light scattering (DLS), molecular docking, and molecular dynamics simulation studies were performed. A decrease in fluorescence intensities was observed with addition of increasing polyphenol concentrations. The order of binding ability obtained was oligomeric fraction of procyanidins (OFP) > tetramer > trimer > dimer B3 procyanidins. Thus a relationship between higher molecular weight and binding ability was observed. The interaction between these molecules and the enzyme occurs by a static mechanism, as inferred from the high apparent fluorescence and bimolecular quenching constants. A blue shift in the maximal emission wavelength could be seen, which indicates that the tryptophan residues acquire a more hydrophobic character upon procyanidin binding. Molecular docking and dynamics simulations also demonstrate that the SASA (solvent-accessible surface area) values of tryptophans decrease with the binding of these compounds, preventing the accessibility of water molecules, which agrees with the referred blue shift. Circular dichroism studies indicate a decrease in alpha-helix content, followed by an increase in the beta-sheet component of secondary structures of this enzyme. DLS and nephelometry techniques also indicate a relationship between large procyanidins and aggregate formation ability.