Exploring the Potential of Vine Shoots as a Source of Valuable Extracts and Stable Lignin Nanoparticles for Multiple Applications.

Large amounts of vine shoots are generated every year during vine pruning. This residue still presents many of the compounds found in the original plant, including low molecular weight phenolic compounds and structural compounds such as cellulose, hemicellulose, and lignin. For wine-producing regions, the challenge is to develop alternatives that will increase the value of this residue. This work proposes the full valorization of vine shoots, focusing on the extraction of lignin by mild acidolysis for the preparation of nanoparticles. The effect of the pretreatment solvents (ethanol/toluene, E/T, and water/ethanol, W/E), on the chemical and structural features of lignin, was evaluated. The chemical analysis suggests similar composition and structure regardless of the pretreatment solvent, although lignin isolated after pretreatment of biomass with E/T showed a higher content of proanthocyanidins (11%) compared with W/E (5%). Lignin nanoparticles (LNPs) presented an average size ranging from 130-200 nm and showed good stability for 30 days. Lignin and LNPs showed excellent antioxidant properties (half maximal inhibitory concentration, IC50 0.016-0.031 mg/mL) when compared to commercial antioxidants. In addition, extracts resulting from biomass pretreatment showed antioxidant activity, with W/E presenting a lower IC50 (0.170 mg/mL) than E/T (0.270 mg/mL), correlated with the higher polyphenol content of W/E, with (+)-catechin and (-)-epicatechin being the main compounds detected. Overall, this work shows that the pre-treatment of vine shoots with green solvents can yield (i) the production of high-purity lignin samples with antioxidant properties and (ii) phenolic-rich extracts, promoting the integral reuse of this byproduct and contributing to sustainability.

Study of Serial Exposures of an Astringent Green Tea Flavonoid Extract with Oral Cell-Based Models.

It is well known that repeated exposure to phenolic compounds (PCs) raises astringency perception. However, the link between this increase and the oral cavity's interactions with salivary proteins (SPs) and other oral constituents is unknown. To delve deeper into this connection, a flavonoid-rich green tea extract was tested in a series of exposures to two oral cell-based models using a tongue cell line (HSC3) and a buccal mucosa cell line (TR146). Serial exposures show cumulative PC binding to all oral models at all concentrations of the green tea extract; however, the contribution for the first and second exposures varies. The tongue mucosal pellicle (HSC3-Mu-SP) may contribute more to first-stage astringency (retaining 0.15 ± 0.01 mg mL-1 PCs at the first exposure), whereas the buccal mucosal pellicle (TR146-Mu-SP) retained significantly less (0.08 ± 0.02 mg mL-1). Additionally, increased salivary volume (SV+), which simulates the stimulation of salivary flow brought by a food stimulus, significantly enhances PC binding, particularly for TR146 cells: TR46-Mu-SP_SV+ bound significantly higher total PC concentration (0.17 ± 0.02 mg mL-1) than the model without increased salivary volume TR146-Mu-SP_SV- (0.09 ± 0.03 mg mL-1). This could be associated with a higher contribution of these oral cells for astringency perception during repeated exposures. Furthermore, PCs adsorbed in the first exposure to cell monolayer models (+TR146 and +HSC3) change the profile of PCs bound to these models in the second exposure. Regarding the structure binding activity, PCs with a total higher number of hydroxyl groups were more bound by the models containing SP. Regarding the SP, basic proline-rich proteins (bPRPs) may be involved in the increased perception of astringency upon repeated exposures. The extent of bPRP precipitation by PCs in mucosal pellicle models for both cell lines (HSC3 and TR146) in the second exposure (76 ± 13 and 83 ± 6%, respectively) was significantly higher than in the first one (25 ± 14 and 5 ± 6%, respectively).

Unravelling the immunomodulatory role of apple phenolic rich extracts on human THP-1- derived macrophages using multiplatform metabolomics.

Apples represent a significant source of dietary phenolic compounds with evidenced anti-inflammatory and immunomodulatory activities. Nevertheless, the effect of the whole apple matrix on human macrophages is unknown. In this context, our study attempts to evaluate the effect of apple-derived phenolic compounds-rich extracts (pulp, peel and leaf) on IL-1ß production in THP-1-differentiated macrophages and derived metabolic alterations through untargeted metabolomics. Our results have showed that apple pulp treatment inhibited the release of the pro-inflammatory cytokine IL-1ß induced by LPS in THP-1 macrophages by ELISA analysis. Metabolomics demonstrate that different proportions of phenolic compounds led to differential alterations in the metabolism of THP-1 macrophages. Indeed, apple extracts promoted alterations in lipid, carbohydrate, amino acid and vitamins as well as cofactors metabolism. Specifically, leaf extracts were characterized by alteration of galactose metabolism while the extracts derived from the fruit showed predominant alterations in lipids metabolism. All extracts mimicked the response observed under normal conditions in LPS-stimulated macrophages, inhibiting LPS response. Thus, the phenolic enriched extracts from apples will be a good source of natural compounds with a beneficial effect against inflammation, and they may be applied as a food supplement and/or functional ingredient for the treatment of inflammatory diseases.

The Antidiabetic Effect of Grape Pomace Polysaccharide-Polyphenol Complexes.

Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic metabolic diseases of the 21st century. Nevertheless, its prevalence might be attenuated by taking advantage of bioactive compounds commonly found in fruits and vegetables. This work is focused on the recovery of polyphenols and polysaccharide-polyphenol conjugates from grape pomace for T2DM management and prevention. Bioactives were extracted by solid-liquid extraction and by pressurized hot water extraction (PHWE). Polyphenolic fraction recovered by PHWE showed the highest value for total phenolic content (427 µg GAE.mg-1), mainly anthocyanins and proanthocyanidins, and higher antioxidant activity compared to the fraction recovered by solid-liquid extraction. Polysaccharide-polyphenol conjugates comprehended pectic polysaccharides to which approximately 108 µg GAE of phenolic compounds (per mg fraction) were estimated to be bound. Polyphenols and polysaccharide-polyphenol conjugates exhibited distinct antidiabetic effects, depending on the extraction methodologies employed. Extracts were particularly relevant in the inhibition of a-glucosidase activity, with free polyphenols showing an IC50 of 0.47 µg.mL-1 while conjugates showed an IC50 of 2.7, 4.0 and 5.2 µg.mL-1 (solid-liquid extraction, PHWE at 95 and 120 °C, respectively). Antiglycation effect was more pronounced for free polyphenols recovered by PHWE, while the attenuation of glucose uptake by Caco-2 monolayers was more efficient for conjugates obtained by PHWE. The antidiabetic effect of grape pomace bioactives opens new opportunities for the exploitation of these agri-food wastes in food nutrition, the next step towards reaching a circular economy in grape products.

Bioactive Peptides and Dietary Polyphenols: Two Sides of the Same Coin.

The call for health-promoting nutraceuticals and functional foods containing bioactive compounds is growing. Among the great diversity of functional phytochemicals, polyphenols and, more recently, bioactive peptides have stood out as functional compounds. The amount of an ingested nutrient able to reach the bloodstream and exert the biological activity is a critical factor, and is affected by several factors, such as food components and food processing. This can lead to unclaimed interactions and/or reactions between bioactive compounds, which is particularly important for these bioactive compounds, since some polyphenols are widely known for their ability to interact and/or precipitate proteins/peptides. This review focuses on this important topic, addressing how these interactions could affect molecules digestion, absorption, metabolism and (biological)function. At the end, it is evidenced that further research is needed to understand the true effect of polyphenol-bioactive peptide interactions on overall health outcomes.

Oral interactions between a green tea flavanol extract and red wine anthocyanin extract using a new cell-based model: insights on the effect of different oral epithelia.

Phenolic compounds (PC) are linked to astringency sensation. Astringency studies typically use simple models, with pure PC and/or proteins, far from what is likely to occur in the oral cavity. Different oral models have been developed here, comprising different oral epithelia (buccal mucosa (TR146) and tongue (HSC-3)) and other main oral constituents (human saliva and mucosal pellicle). These models, were used to study the interaction with two PC extracts, one rich in flavanols (a green tea extract) and one rich in anthocyanins (a red wine extract). It was observed that within a family of PC, the PC seem to have a similar binding to both TR146 and HSC-3 cell lines. When the oral constituents occur altogether, flavanols showed a higher interaction, driven by the salivary proteins. Conversely, anthocyanins showed a lower interaction when the oral constituents occur altogether, having a higher interaction only with oral cells. Epigallocatechin gallate, epicatechin gallate, epigallocatechin-3-O(3-O-methyl) gallate were the flavanols with the highest interaction. For the studied anthocyanins (delphinidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and malvidin-3-glucoside), there was not a marked difference on their interaction ability. Overall, the results support that the different oral constituents can have a different function at different phases of food (PC) intake. These differences can be related to the perception of different astringency sub-qualities.

Impact of grape pectic polysaccharides on anthocyanins thermostability.

The impact of grape pectic polysaccharides on malvidin-3-O-ß-d-glucoside thermostability was evaluated in model solutions. Pectic polysaccharides richer in homogalacturonan domains, with less neutral sidechains (chelator fraction) showed higher binding with malvidin-3-O-ß-d-glucoside, by 1H NMR (Ka=505 M-1). Binding affinity with water soluble extract was estimated to be 10-fold lower, possibly due to the presence of neutral branched regions and more compacted structure, hampering the binding. Hydrophobic domains, such as rhamnogalacturonans-I domains in acid soluble polysaccharides, may participate in the formation of complexes with malvidin-3-O-ß-d-glucoside. The thermostability of anthocyanin-polysaccharides complexes was evaluated at different temperatures, assessing anthocyanins degradation by HPLC-DAD. Polysaccharides showed to improve anthocyanin thermostability, with chelator and acid extract having the highest impact at lowest temperatures. Electrostatic interactions, additionally stabilized by hydrophobic effect contribute to the anthocyanin-polysaccharides binding and to the consequent thermostabilization. The protection provided by grape pectic polysaccharides foresees innovative anthocyanin food products with improved thermostability and colour features.

The effect of pectic polysaccharides from grape skins on salivary protein - procyanidin interactions.

In this study, water and chelator-soluble pectic polysaccharide fractions were obtained from white grape skins, aiming to study their impact on the interaction between low polymerized grape seed procyanidins and salivary proteins. Water and chelator-soluble polysaccharide fractions were composed by uronic acids and neutral sugars, mainly arabinose and galactose, with water polysaccharide fraction showing a higher amount of branched pectic polysaccharides. Both polysaccharide fractions were able to mitigate salivary protein-procyanidin interactions, by a competition mechanism, resulting in a decrease of the amount of precipitated protein. Water polysaccharide fraction was the most effective in inhibiting salivary protein precipitation, especially for acidic proline-rich proteins, due to the higher affinity to interact with procyanidins (KA = 22222 M-1 and KA = 365 M-1 for water and chelator polysaccharides, respectively). The interaction between polysaccharides and procyanidins showed to be mainly governed by hydrophobic effect.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation.

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

Purple-fleshed sweet potato acylated anthocyanins: Equilibrium network and photophysical properties.

Two anthocyanins from purple-fleshed sweet potato were isolated and characterized by LC-MS and NMR analysis. They were identified as peonidin-3-(6'-hydroxybenzoyl)-sophoroside-5-glucoside and peonidin-3-(6'-hydroxybenzoyl-6″-caffeoyl)-sophoroside-5-glucoside. The acid-base dynamics of these acylated anthocyanins was evaluated by means of pH jump techniques. Equilibrium and kinetic constants were determined and, in general, these anthocyanins demonstrated a higher capacity in retaining the red and blue colors at acidic and basic pH values, suggesting a higher resistance to pH variations compared to the parent anthocyanin, peonidin-3-O-glucoside. The presence of acyl groups and additional glucoside moieties seems to determine this particular characteristic. The fluorescence properties of these anthocyanins were evaluated. Overall, the species present at higher pH values (7-9) showed higher fluorescence intensity for both anthocyanins, with an optimum λex/λem pair at λex 610 nm/λem 640 nm. The fluorescence characteristics of these anthocyanins were used to evaluate their location in gastric and intestinal cells by fluorescence microscopy.

Effect of in vitro digestion on the functional properties of Psidium cattleianum Sabine (araçá), Butia odorata (Barb. Rodr.) Noblick (butiá) and Eugenia uniflora L. (pitanga) fruit extracts.

Brazilian native fruits are reported to be promising sources of bioactive compounds; however their bioactivity depends on their stability along the digestive process. This study evaluated the α-glucosidase inhibition, antioxidant activity and total phenolic content (TPC) stability of araçá, butiá and pitanga fruit extracts using an in vitro digestion model. Additionally, the individual phenolic compound recovery of the most stable and active extract was evaluated by HPLC-DAD-ESI-MS/MS. Overall, the antioxidant activity of all extracts decreased along the process. Araçá fruit extracts, at the end of digestion, showed α-glucosidase inhibition values similar to their non-digested extracts and the highest TPC recovery (28%). Recovery of individual phenolic compounds of red araçá fruit extract revealed a negative impact on the stability of ellagitannins. Araçá fruit extract seems to provide phenolic compounds with α-glucosidase inhibitory properties after the gastrointestinal digestion, indicating their potential to be used in the control of type II diabetes.

Wine industry by-product: Full polyphenolic characterization of grape stalks.

In this research work grape stalk samples from the Douro region were analyzed concerning polyphenolic composition. Different solid-liquid extraction conditions were tested for reaching the optimal parameters to obtain higher amounts of polyphenols. In general, the best conditions comprise the use of a mixture of acetone/ethanol/water (1:1:1) at room temperature during 20 min, using a pre-wash treatment with deionized water. The polyphenolic extractions with acetone are generally more effective than without acetone. Total phenolic determination (Folin-Ciocalteu), antioxidant capacity (DPPH/FRAP) and more accurate methods such as HPLC-DAD and HPLC-ESI-MS were performed. An intensive polyphenolic characterization (including a possible identification of a new polyphenol) and compound quantification was achieved. A comparative study between these two analysis approaches was accomplished, which allowed to conclude that the Folin-Ciocalteu and DPPH/FRAP determinations do not always allow a direct correspondence between high phenolic content or antioxidant/reducing capacity and real high polyphenolic content on crude polyphenolic extracts.

The effect of anthocyanins from red wine and blackberry on the integrity of a keratinocyte model using ECIS.

There is a growing market demand for the incorporation of plant-derived ingredients into new products for the cosmetic industry. Anthocyanins are polyphenols arising from plant secondary metabolism that have been shown to possess many bioactive properties such as free radical scavenging, antimicrobial, and chemopreventive activities. In this work, the biological activities of red wine and blackberry anthocyanins were assessed by developing a new keratinocyte barrier model using the HaCat cell line and a microelectrode-based biosensor device, referred to as Electric Cell-Substrate Impedance Sensing (ECIS). Cells were seeded at the optimal cellular density of 1.6 × 106 cells per mL and the half-time was calculated to be 3.55 ± 0.67 hours. The compounds' cytotoxicity was assessed and anthocyanin pigments showed no cytotoxicity towards keratinocyte cells. Wound healing assays were also performed using ECIS and it was observed that the tested pigments enhanced the healing rate of keratinocyte cells by reducing the healing time more than 50%. Cyanidin-3-glucoside presented the best results recovering 50% of the injured area in 1.48(±0.15) hours, followed by the blackberry anthocyanins (2.01 ± 0.18 hours), malvidin-3-glucoside (2.03 ± 0.09 hours) and red wine anthocyanins (2.36 ± 0.76 hours). All presented significant differences from the control 4.91(±1.11) hours.

Gastrointestinal absorption, antiproliferative and anti-inflammatory effect of the major carotenoids of Gardenia jasminoides Ellis on cancer cells.

The gastrointestinal absorption of the main carotenoids present in Gardenia jasminoides Ellis, crocetin, crocin-1 and crocin-2, was assayed through transport studies on MKN-28 and Caco-2 cell lines. Overall, crocetin was the compound that presented the highest gastrointestinal transport efficiency. Additionally, and since after absorption crocins are metabolized into crocetin, the antiproliferative capacity of crocetin was assayed in MKN-28 (stomach), MCF-7 (breast) and Caco-2 (colon) cancer cell lines. The results point to an antiproliferative effect of crocetin on the three cell lines tested. Anti-inflammatory properties were also assayed. Overall, crocetin showed a potential involvement in the downregulation of IL-1ß and TNF-α but not IL-6. Altogether, these results suggest that these compounds can have an important role against cancer proliferation, highlighting the importance of Gardenia jasminoides Ellis as a nutraceutical food source.

Impact of a pectic polysaccharide on oenin copigmentation mechanism.

Copigmentation plays an important role in the colors provided by anthocyanins. However, little attention has been paid to the interaction between anthocyanins and cell wall compounds (e.g. polysaccharides) and the impact of this interaction on anthocyanins color, a fundamental issue to be considered in industrial applications of these pigments as food colorants. The copigmentation binding constants (KCP) for the interaction between malvidin-3-O-glucoside and (+)-catechin in the presence of low methoxylated pectic polysaccharide were determined. The values obtained showed that in the presence of pectic polysaccharide the copigmentation binding constants decreased. These results probably suggest the occurrence of competition equilibrium in which the presence of pectin limited the association between catechin and oenin. (1)H NMR studies revealed that the dissociation constant determined for these complexes was very similar in absence and presence of 1.5g/L pectin with this polysaccharide apparently not affecting the strength of anthocyanin-catechin binding.

Antioxidant and antiproliferative properties of 3-deoxyanthocyanidins.

The study of the antioxidant properties of six deoxyanthocyanidins (deoxypeonidin, deoxymalvidin, luteolinidin, apigeninidin, guaiacylcatechinpyrylium and syringylcatechinpyrylium) and an anthocyanin (cyanidin-3-glucoside) was carried out. The aim was to evaluate the relationship between the structure and the antioxidant properties of individual deoxyanthocyanidins, compared to a common anthocyanin derivative, cyanidin-3-glucoside. The ability of these compounds to inhibit lipid peroxidation in a liposome membrane system was examined by monitoring oxygen consumption and the antiradical and reducing capacities were determined using the DPPH and FRAP assay, respectively. The results showed that all the compounds tested presented antioxidant properties. Cyanidin-3-glucoside presented higher antiradical and reducing activities in the DPPH and FRAP assay, although in the liposome model, the guaiacylcatechinpyrylium was more effective inhibiting lipid peroxyl radicals. Additionally, the anti-proliferative effects of deoxyanthocyanidins, have been evaluated against two cancer cell lines from stomach (AGS, MKN-28) and one colon cancer cell (Caco-2), and compared with the effect of the respective anthocyanins. Considering the antiproliferative activity, all compounds were active against Caco-2 cell line, being the ones with glucose moiety and oaklin Scp the most active. Deoxyanthocyanidins, and in particular, guaiacylcatechinpyrylium may be regarded as potential food colorants.

Direct identification and characterization of phenolic compounds from crude extracts of buds and internodes of grapevine (Vitis vinifera cv Merlot).

The crude methanol extracts of latent buds and internodes Vitis vinifera L. cv. Merlot were used for the determination of phenolic compounds by a combination of reverse phase HPLC with diode array detection (HPLC-DAD) and mass spectrometry (LC-MS). This method allowed the identification of 9 phenolic compounds without purification or fractionation. These 9 compounds were divided into three groups: procyanidins, flavonols and stilbenes. Detection by HPLC-DAD at different wave lengths of 280 nm to 320 nm, allowed the estimation of concentrations of those compounds. This method permitted, for the first time, both characterization and quantification of polyphenolic compounds in buds of grapevine. Comparison with the results obtained in internodes showed that quercetin, resveratrol tetramer and ε-viniferin had similar levels in buds and internodes while six other compounds identified had higher levels in buds.

Understanding the molecular mechanism of anthocyanin binding to pectin.

Association between anthocyanins and carbohydrates has drawn attention over the past few years and this interaction is of particularly importance in food chemistry since these compounds are often found together in plants and foodstuffs. This work intended to bring insights on the interaction between ionic carbohydrates (pectin) and two anthocyanins (cyanidin-3-O-glucoside, cy3glc and delphinidin-3-O-glucoside, dp3glc). The interaction between the flavylium cation and hemiketal anthocyanin forms was characterized by saturation transfer difference (STD) NMR spectroscopy and the respective dissociation constant (Kd) was obtained. This binding was also studied by Molecular Dynamics simulation. In the presence of the anthocyanin hemiketal form a weak interaction between anthocyanins and pectin seems to occur. A variation in the extent of this interaction was also noticed for the two anthocyanins with dp3glc bearing three hydroxyl groups, revealing to be a stronger binder to pectin (Kd ≈ 180 µM for dp3glc and Kd ≈ 250 µM for cy3glc). Experiments performed at acidic pH (flavylium cation) revealed a much stronger interaction (Kd ≈ 2 µM). These experimental results were also supported by theoretical studies which also revealed a stronger interaction in the presence of the anthocyanin flavylium cation and also a stronger interaction between pectin and dp3glc than with cy3glc (for the hemiketal form).

Antioxidant and antiproliferative properties of methylated metabolites of anthocyanins.

Anthocyanins are major flavonoids in many plant foods and have been related to health promotion. In the human organism anthocyanins are metabolised to different metabolites. One of the most important phase II reactions of flavonoids is the methylation of the catechol group. This feature is expected to have an effect on the antioxidant and antiproliferative properties of flavonoids including anthocyanins. In this work, delphinidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside methylated metabolites were obtained by enzymatic hemi-synthesis. The compounds were identified as monomethylated products by HPLC-MS and NMR. The methylated metabolites were found to still retain significant radical scavenging activity and reducing activity, suggesting that they could act as potential antioxidants in vivo. The antiproliferative activity of the metabolites in comparison with the parental anthocyanins was also evaluated in three cancer cell lines by sulforhodamine B assay. The conjugation with methyl groups decreased or did not alter the antiproliferative effect of the original anthocyanin.

Different phenolic compounds activate distinct human bitter taste receptors.

Bitterness is a major sensory attribute of several common foods and beverages rich in polyphenol compounds. These compounds are reported as very important for health as chemopreventive compounds, but they are also known to taste bitter. In this work, the activation of the human bitter taste receptors, TAS2Rs, by six polyphenol compounds was analyzed. The compounds chosen are present in a wide range of plant-derived foods and beverages, namely, red wine, beer, tea, and chocolate. Pentagalloylglucose (PGG) is a hydrolyzable tannin, (-)-epicatechin is a precursor of condensed tannins, procyanidin dimer B3 and trimer C2 belong to the condensed tannins, and malvidin-3-glucoside and cyanidin-3-glucoside are anthocyanins. The results show that the different compounds activate different combinations of the ~25 TAS2Rs. (-)-Epicatechin activated three receptors, TAS2R4, TAS2R5, and TAS2R39, whereas only two receptors, TAS2R5 and TAS2R39, responded to PGG. In contrast, malvidin-3-glucoside and procyanidin trimer stimulated only one receptor, TAS2R7 and TAS2R5, respectively. Notably, tannins are the first natural agonists found for TAS2R5 that display high potency only toward this receptor. The catechol and/or galloyl groups appear to be important structural determinants that mediate the interaction of these polyphenolic compounds with TAS2R5. Overall, the EC(50) values obtained for the different compounds vary 100-fold, with the lowest values for PGG and malvidin-3-glucoside compounds, suggesting that they could be significant polyphenols responsible for the bitterness of fruits, vegetables, and derived products even if they are present in very low concentrations.