Antioxidative effects and percutaneous absorption of five polyphenols.

A new strategy was designed to evaluate the antioxidant effectiveness of five topically applied polyphenols following skin penetration profiles. The antioxidants were the following polyphenol derivatives: epicatechin, resveratrol, rutin, quercetin, and trolox, which was used as the reference antioxidant. The hydrophilic/lipophilic character of these compounds was evaluated, and their antioxidant activity was measured by the DPPH method. The percutaneous absorption of these polyphenols was obtained by an in vitro methodology using porcine skin biopsies. This methodology involves the quantification of the antioxidants present in each specific skin layer to evaluate antioxidant effectiveness. The antioxidant activity in each skin layer was also determined by the DPPH method. The results indicated that lipophilic antioxidants (epicatechin, resveratrol, quercetin, and trolox) penetrated deeper into the skin layers, whereas a more hydrophilic compound, rutin, remained on the skin surface. The antioxidant evaluation of each skin compartment suggested that resveratrol and rutin were the most effective topically applied compounds in view of their antioxidant activity and their skin penetration profile.

Laccase-assisted formation of bioactive chitosan/gelatin hydrogel stabilized with plant polyphenols.

Laccase-assisted simultaneous cross-linking and functionalization of chitosan/gelatin blends with phenolic compounds from Hamamelis virginiana was investigated for the development of bioactive hydrogel dressings. The potential of these hydrogels for chronic wound treatment was evaluated in vitro, assessing their antibacterial and inhibitory effect on myeloperoxidase and collagenase. Rheological studies revealed that the mechanical properties of the hydrogels were a function of the enzymatic reaction time. Stable hydrogels and resistant to lysozyme degradation were achieved after 2 h laccase reaction. The inhibitory capacity of the hydrogel for myeloperoxidase and collagenase was 32% and 79% respectively after 24 h incubation. Collagenase activity was additionally suppressed by adsorption (20%) of the enzyme onto the hydrogel. Therefore, the bioactive properties of the hydrogels were due to the effect of both released phenolic compounds and the permanently functionalized platform itself. The hydrogels showed antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

A new approach to produce plant antioxidant-loaded chitosan for modulating proteolytic environment and bacterial growth.

This work reports a straightforward approach for incorporation of plant-derived flavonoid-based antioxidants into an intrinsically antimicrobial chitosan platform in order to generate chitosan-based materials suitable for biomedical applications. The novelty of this approach consists in the use of thioacidolysis - a common analytical method for the characterization of proanthocyanidins - to covalently functionalize natural macromolecules such as chitosan with bioactive phenolic moieties. It is known that proanthocyanidins undergo depolymerization in acid medium, where in the presence of nucleophiles they form stable flavan-3-ol derivatives. Based on this reaction, flavan-3-ol moieties from the polyphenol extract of Hamamelis virginiana are incorporated covalently into a previously thiolated chitosan matrix. The phenolics-functionalized chitosan possesses improved antioxidant and antimicrobial properties, and inhibitory efficiency over deleterious wound enzymes in vitro.

Metabolites in contact with the rat digestive tract after ingestion of a phenolic-rich dietary fiber matrix.

Grape antioxidant dietary fiber (GADF) is a phenolic-rich dietary fiber matrix. The aim of this work was to determine which phenolic compounds come into contact with colonic epithelial tissue after the ingestion of GADF. By use of HPLC-ESI-MS/MS techniques phenolic metabolites were detected in feces, cecal content, and colonic tissue from rats. Free (epi)catechin (EC) was detected in all three sources, and more than 20 conjugated metabolites of EC were also detected in feces. Fourteen microbially derived phenolic metabolites were also identified in feces, cecal content, and/or colonic tissue. These results show that during transit along the digestive tract, proanthocyanidin oligomers and polymers are depolymerized into EC units. After ingestion of GADF, free EC and its conjugates, as well as free and conjugated microbially derived phenolic metabolites, come into contact with the intestine epithelium for more than 24 h and may be partly responsible for the positive influence of GADF on gut health.

Role of galloylation and polymerization in cytoprotective effects of polyphenolic fractions against hydrogen peroxide insult.

Byproducts and wastes generated by agricultural, food, and forestry industries contain large amounts of polyphenols, which can be potentially used as sources of natural or semisynthetic antioxidants. This study examined and compared the protection against peroxidative damage induced in erythrocytes and 3T3 cell line of polyphenolic fractions from white grape pomace, pine bark, and witch hazel bark. The work pays special attention to the different degrees of polymerization and galloylation of the extracts to contribute to the understanding of their mechanisms of action. Fractions demonstrated different protections against erythrocyte lipid peroxidation, hemolysis, and 3T3 cytotoxicity caused by H(2)O(2). Galloylation is claimed to be related to antioxidant protective capacity, and it is also responsible for the pro-oxidant effect observed at high doses. The results show that not only the percentage of galloylation but also the degree of polymerization are important modulators of their antioxidant capacity. In this sense, it is crucial that novel polyphenolic fractions were prepared attending a value of 3 for the mean degree of polymerization and did not exceed a 30% of galloylation to reach the highest antioxidant capacity with the lowest cytotoxic effects. For this reason, the grape extracts appear to be the best strategy to fight against hydrogen peroxide cell damage.

Antioxidant activities of hydroxytyrosol main metabolites do not contribute to beneficial health effects after olive oil ingestion.

Hydroxytyrosol (HOTYR) and tyrosol (TYR), main phenolic compounds of olive oil, have been reported to contribute to the prevention of cardiovascular diseases due to their antioxidant activities, e.g., protection of low-density lipoprotein (LDL) oxidation. Their bioavailability in humans is poor, and they are found in biological fluids mainly as conjugated metabolites. Low concentrations of free phenols are unlikely to explain the biological activities seen in humans after olive oil intake. In this context, antioxidant activities of conjugated metabolites, in a range of concentrations compatible with their dietary consumption, were evaluated. Concentrations of metabolites and their core compounds were estimated in an intervention study of 11 healthy volunteers whose diet was supplemented with 50 ml of virgin olive oil, using high-performance liquid chromatography coupled to mass spectrometry for the simultaneous analysis of 3'-O- and 4'-O-HOTYR-glucuronides and 4'-O-glucuronides of TYR and homovanillyl alcohol in human urine. Glucuronides and core compounds were tested for their chemical (hydrogen donation by 1,1-diphenyl-2-picrylhydrazyl free radical test) and in vitro biological (inhibition of Cu-mediated LDL oxidation) antioxidant activities at the concentration ranges observed in human biological fluids (range, 0.01-10 muM) after dietary olive oil consumption. None of the glucuronides displayed significant antioxidant activities at the concentrations tested.

Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic fermentation and proanthocyanidin metabolites in human plasma.

Proanthocyanidins (PAs) or condensed tannins, a major group of dietary polyphenols, are oligomers and polymers of flavan-3-ol and flavan-3, 4-diols widely distributed in plant foods. Most literature data on PAs' metabolic fate deal with PAs that can be extracted from the food matrix by aqueous-organic solvents ( extractable proanthocyanidins). However, there are no data on colonic fermentation of non-extractable proanthocyanidins (NEPAs), which arrive almost intact to the colon, mostly associated to dietary fibre (DF). The aim of the present work was to examine colonic fermentation of NEPAs associated with DF, using a model of in vitro small intestine digestion and colonic fermentation. Two NEPA-rich materials obtained from carob pod (Ceratonia siliqua L. proanthocyanidin) and red grapes (grape antioxidant dietary fibre) were used as test samples. The colonic fermentation of these two products released hydroxyphenylacetic acid, hydroxyphenylvaleric acid and two isomers of hydroxyphenylpropionic acid, detected by HPLC-ESI-MS/MS. Differences between the two products indicate that DF may enhance the yield of metabolites. In addition, the main NEPA metabolite in human plasma was 3,4-dihydroxyphenyl acetic acid. The presence in human plasma of the same metabolites as were detected after in vitro colonic fermentation of NEPAs suggests that dietary NEPAs would undergo colonic fermentation releasing absorbable metabolites with potential healthy effects.

Phenolic metabolites of grape antioxidant dietary fiber in rat urine.

Grape antioxidant dietary fiber (GADF) combines the putative health benefits of fiber and polyphenols. Polyphenolic metabolites may play a key role in the overall biological effects of this supplement. We identified phenolic GADF metabolites in rat urine at different times after oral administration, using HPLC-ESI-MS/MS techniques. The phenolic metabolic outcome of GADF is essentially an array of mono- and polyconjugated epicatechins and free or conjugated smaller phenolic acids, some of them never reported before. We have detected 18 mono-, di-, and triconjugates of epicatechin with glucuronide, methyl and sulfate moieties and small phenolic acids both free and conjugated. The results suggest that the procyanidin oligomers are both depolymerized in the digestive tract into epicatechin conjugates and degraded by the colonic microbiota into phenolic acids and their conjugates. For several hours after ingestion of GADF, a great variety of phenolic species, including some with an intact catechol group, are in contact with the digestive tract tissues before, during and after metabolization, and many of them are systemically bioavailable before being excreted.

The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana.

The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway.

Witch hazel (Hamamelis virginiana) fractions and the importance of gallate moieties--electron transfer capacities in their antitumoral properties.

Witch hazel (Hamamelis virginia) extracts are used in traditional medicine. They are particularly rich in gallate esters included in proanthocyanidins, hydrolyzable tannins (galloylated sugars), and methyl gallate. This study examines the response of human colon cancer cells to treatment with fractions obtained from a witch hazel polyphenolic extract. The results are compared with those obtained previously with homologous fractions from grape (less galloylated) and pine (nongalloylated). Witch hazel fractions were the most efficient in inhibiting cell proliferation in HT29 and HCT116 human colon cancer cell lines, which clearly shows that the more galloylated the fractions, the more effective they were at inhibiting proliferation of colon cancer cells. Witch hazel fractions were, in addition, more potent in arresting the cell cycle at the S phase and inducing apoptosis; they also induced a significant percentage of necrosis. Interestingly, the apoptosis and cell cycle arrest effects induced were proportional to their galloylation. Moreover, witch hazel fractions with a high degree of galloylation were also the most effective as scavengers of both hydroxyl and superoxide radicals and in protecting against DNA damage triggered by the hydroxyl radical system. These findings provide a better understanding of the structure-bioactivity relationships of polyphenolics, which should be of assistance in choosing an appropriate source and preparing a rational design for formulations of plant polyphenols in nutritional supplements.

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber.

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.

Highly galloylated tannin fractions from witch hazel (Hamamelis virginiana) bark: electron transfer capacity, in vitro antioxidant activity, and effects on skin-related cells.

Witch hazel ( Hammamelis virginiana) bark is a rich source of both condensed and hydrolizable oligomeric tannins. From a polyphenolic extract soluble in both ethyl acetate and water, we have generated fractions rich in pyrogallol-containing polyphenols (proanthocyanidins, gallotannins, and gallates). The mixtures were highly active as free radical scavengers against ABTS, DPPH (hydrogen donation and electron transfer), and HNTTM (electron transfer). They were also able to reduce the newly introduced TNPTM radical, meaning that they included some highly reactive components. Witch hazel phenolics protected red blood cells from free radical-induced hemolysis and were mildly cytotoxic to 3T3 fibroblasts and HaCat keratinocytes. They also inhibited the proliferation of tumoral SK-Mel 28 melanoma cells at lower concentrations than grape and pine procyanidins. The high content in pyrogallol moieties may be behind the effect of witch hazel phenolics on skin cells. Because the most cytotoxic and antiproliferative mixtures were also the most efficient as electron transfer agents, we hypothesize that the final putative antioxidant effect of polyphenols may be in part attributed to the stimulation of defense systems by mild prooxidant challenges provided by reactive oxygen species generated through redox cycling.

Comparative antioxidant and cytotoxic effect of procyanidin fractions from grape and pine.

There is a great interest in characterizing the biological properties of natural compounds obtained from plants, especially polyphenols. We studied the structure-activity-cytotoxicity relationships of polyphenolic fractions obtained from grape pomace and pine bark. These fractions contained similar polymerised flavonoids but different percentages of pyrogallol groups that confer on them different biological properties. The human keratinocyte cell line HaCaT and the mouse fibroblast cell line 3T3 were used to study the cytotoxicity of the different fractions after 24, 48, and 72 h of exposure. Antioxidant activity of the fractions was evaluated by measuring the inhibition of hemolysis mediated by AAPH. Our results demonstrate that the polyphenolic fractions studied show high antioxidant capacity in a concentration range that is not harmful to normal human cells. Pine fractions presented slightly lower antioxidant activity than grape fractions but are less cytotoxic. This data provides useful information to help design safe antioxidant products that act without altering critical cell functions.

Electron-transfer capacity of catechin derivatives and influence on the cell cycle and apoptosis in HT29 cells.

Galloylated and nongalloylated catechin conjugates with cysteine derivatives have been synthesized and evaluated for their capacity to scavenge free radicals and to influence crucial functions (cell cycle, apoptosis) in HT29 colon carcinoma cells. We show that the nonphenolic part of the molecule modified the capacity of catechins to donate hydrogen atoms and to transfer electrons to free radicals. Nongalloylated derivatives did not significantly influence either the cell cycle or apoptosis. Among the galloylated species, 4beta-[S-(O-ethyl-cysteinyl)]epicatechin 3-O-gallate, which showed a high electron-transfer capacity (5 e- per molecule), arrested the cell cycle and induced apoptosis as expected for galloylated catechins such as tea (-)-epigallocatechin 3-O-gallate. 4beta-[S-(N-Acetyl-O-methyl-cysteinyl)]epicatechin 3-O-gallate, which showed the highest hydrogen-donating capacity (10 H per molecule) while keeping the electron-transfer capacity low (2.9 e- per molecule), did not trigger any significant apoptosis. The gallate moiety did not appear to be sufficient for the pro-apoptotic effect of the catechin derivatives in HT29 cells. Instead, a high electron-transfer capacity is more likely to be behind this effect. The use of stable radicals sensitive exclusively to electron transfer may help to design molecules with either preventive scavenging action (high hydrogen donation, low electron transfer) or therapeutic pro-apoptotic activity (high electron transfer).

Procyanidin fractions from pine (Pinus pinaster) bark: radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells.

Pine (Pinus pinaster) bark is a rich source of procyanidin oligomers. From a total polyphenolic extract, we have generated fractions of different procyanidin composition. The mixtures, devoid of gallate esters, were active as free radical scavengers against ABTS(*+), DPPH, and HNTTM. Pine bark fractions were tested for antioxidant activity in solution (hydrogen donation and electron transfer) and emulsion (inhibition of lipid peroxidation) and compared with their galloylated counterparts from grape origin. While galloylation clearly influenced the free radical scavenging efficiency in solution, it did not seem to play a determinant role in protection against lipid peroxidation in emulsion. The fractions were very mild inhibitors of cell proliferation. Because gallate esters appear to interfere with crucial cell functions, gallate free pine procyanidins may be the innocuous chemopreventative agents of choice for many applications in food and skin protection.