Grape polyphenols and propolis mixture inhibits inflammatory mediator release from human leukocytes and reduces clinical scores in experimental arthritis.

Polyphenols from red fruits and bee-derived propolis (PR) are bioactive natural products in various in vitro and in vivo models. The present study shows that hematotoxicity-free doses of grape polyphenols (GPE) and PR differentially decreased the secretion of pro-inflammatory cytokines from activated human peripheral blood leucocytes. While GPE inhibited the monocytes/macrophage response, propolis decreased both monokines and interferon γ (IFNγ) production. When used together, their distinct effects lead to the attenuation of all inflammatory mediators, as supported by a significant modulation of the transcriptomic profile of pro-inflammatory genes in human leukocytes. To enforce in vitro data, GPE+PR were tested for their ability to improve clinical scores and cachexia in chronic rat adjuvant-induced arthritis (AA). Extracts significantly reduced arthritis scores and cachexia, and this effect was more significant in animals receiving continuous low doses compared to those receiving five different high doses. Animals treated daily had significantly better clinical scores than corticoid-treated rats. Together, these findings indicate that the GPE+PR combination induces potent anti-inflammatory activity due to their complementary immune cell modulation.

Preparative purification of antiamyloidogenic stilbenoids from Vitis vinifera (Chardonnay) stems by centrifugal partition chromatography.

Five stilbenoids, E-resveratrol, E-piceatannol, (+) E-(epsilon)-viniferin, (+)-ampelopsin A and vitisin C were isolated from methyl tert-butyl ether (MtBE) stem extract of Vitis vinifera (Chardonnay cv). Their purification on a preparative scale was obtained by centrifugal partition chromatography (CPC) using quaternary Arizona solvent systems composed of n-heptane/ethyl acetate/methanol/water. We tested 23 Arizona solvent systems to partition the extract and found that systems K and M (Hept/EtOAc/MeOH/water, 1:2:1:2 and 5:6:5:6, respectively; v/v) were the best to separate the stilbenes mentioned above. This support-free liquid-liquid chromatographic procedure made it possible to isolate ampelopsin A from V. vinifera for the first time. The antiamyloidogenic activity of the isolated stilbenes was evaluated versus beta-amyloid fibrils. E-resveratrol and (+)-ampelopsin A were found to be the most active compounds with 63 and 46% inhibition at 10microM, respectively. These findings suggest that E-resveratrol and (+)-ampelopsin A may function as attractive new candidates for protecting against brain cell dysfunction in vivo in AD by inhibiting the aggregation of Abeta.

Role of peptide primary sequence in polyphenol-protein recognition: an example with neurotensin.

Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol-protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-D-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.

Is the C-terminal region of bradykinin the binding site of polyphenols?

Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts beta-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-terminus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.

Polyphenols newly extracted in red wine from southwestern France by centrifugal partition chromatography.

Polyphenols from the ethyl acetate extracts of red wine were successfully fractionated using a four-step process (solvent extraction, ion-exchange column chromatography, centrifugal partition chromatography, and semipreparative HPLC), which resulted in the isolation of 22 compounds belonging to different classes of polyphenols (stilbenes, cinnamic acids, flavonoids). Five of them are red wine constituents reported for the first time. The newly isolated compounds include resveratrol dimers, dihydroflavonols, and a cinnamic derivative.

Regioselective and stereospecific glucuronidation of trans- and cis-resveratrol in human.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol present in wine, which has been reported to have anti-inflammatory, anti-platelet, and anti-carcinogenic effects. The glucuronidation of this compound and that of the cis-isomer also naturally present, has been investigated in human liver microsomes. Both isomers were actively glucuronidated. The reaction led to the formation of two glucuronides (3-O- and 4'-O-glucuronides), whose structure was characterized by LC-MS and proton NMR. Glucuronidation was regio- and stereoselective. It occurred at a faster rate with the cis-isomer and preferred the 3-position on both isomers. In addition, the glucuronidation of resveratrol was tested using several recombinant UDP-glucuronosyltransferase (UGT) isoforms. The reaction was catalyzed by UGT of the family 1A (UGT1A1, 1A6, 1A7, 1A9, 1A10). The bilirubin conjugating UGT1A1 was mainly involved in the 3-O-glucuronidation of trans-resveratrol, whereas the phenol conjugating UGT1A6 activity was restricted to cis-resveratrol. The UGT1A9 and 1A10 were active toward both isomers. The activity supported by UGT2B7 and UGT2B15 was very low and restricted to cis-resveratrol. UGT1A3, 1A4, 2B4, and 2B11 were unable to form resveratrol glucuronides.

Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures.

Moderate consumption of wine is associated with a reduced risk of cancer. Grape plant cell cultures were used to purify 12 phenols: the stilbenoids trans-astringin, trans-piceid (2), trans-resveratroloside, trans-resveratrol, trans-piceatannol, cis-resveratroloside, cis-piceid, and cis-resveratrol; the flavans (+)-catechin, (-)-epicatechin, and epicatechin 3-O-gallate; and the flavan dimer procyanidin B2 3'-O-gallate. These compounds were evaluated for potential to inhibit cyclooxygenases and preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture. At 10 micrograms/ml, trans-astringin and trans-piceatannol inhibited development of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesions in mouse mammary glands with 68.8% and 76.9% inhibition, respectively, compared with untreated glands. The latter compound was the most potent of the 12 compounds tested in this assay, with the exception of trans-resveratrol (87.5% inhibition). In the cyclooxygenase (COX)-1 assay, trans isomers of the stilbenoids appear to be more active than cis isomers: trans-resveratrol [50% inhibitory concentration (IC50) = 14.9 microM, 96%] vs. cis-resveratrol (IC50 = 55.4 microM). In the COX-2 assay, among the compounds tested, only trans- and cis-resveratrol exhibited significant inhibitory activity (IC50 = 32.2 and 50.2 microM, respectively). This is the first report showing the potential cancer-chemopreventive activity of trans-astringin, a plant stilbenoid recently found in wine. trans-Astringin and its aglycone trans-piceatannol were active in the mouse mammary gland organ culture assay but did not exhibit activity in COX-1 and COX-2 assays. trans-Resveratrol was active in all three of the bioassays used in this investigation. These findings suggest that trans-astringin and trans-piceatannol may function as potential cancer-chemopreventive agents by a mechanism different from that of trans-resveratrol.

Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera cells producing anthocyanins.

We have previously reported that sucrose modulates anthocyanin biosynthesis in cell suspension cultures of Vitis vinifera L. The main role of sugar in this response does not seem to be that of general carbohydrate source for the supply of energy. In the present work, a number of pharmacological agents were used to further investigate the components of the signal transduction pathway involved in the induction of anthocyanin biosynthesis by sugar. We found that the phosphorylation of hexose by hexokinase, but not its transport, has to be taken into account for the sucrose signal transduction leading to anthocyanin accumulation. Indeed, 3-O-methylglucose, a glucose analog transported into cells but not phosphorylated by hexokinase, has no effect on anthocyanin production. Mannose mimics the effect of sucrose in grape cells, and mannoheptulose, a specific inhibitor of hexokinase, reduces the accumulation of anthocyanins in response to sucrose. The results with the two latter analogs are discussed. Ca2+ channel blockers, verapamil and LaCl3, which were used to investigate the role of extracellular Ca2+, all inhibited the sugar response. Ca2+ depletion by pretreatment with ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) also blocked the sugar response, which was partially recovered when Ca2+ was added exogenously after Ca2+ depletion. The use of two potent calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphtalenesulphonamide (W7) and chlorpromazine, showed that calmodulin is involved in the sugar signal transduction. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), and the protein phosphatase inhibitors, endothall and cantharidin, also inhibited the sugar response. The results of the present study suggest the involvement of several components of general signal transduction pathways such as Ca2+, calmodulin, and protein kinases phosphatases in the induction of anthocyanin biosynthesis by sugar.

Deactivation of cultured human liver myofibroblasts by trans-resveratrol, a grapevine-derived polyphenol.

Liver myofibroblasts are major actors in the development of liver fibrosis and cancer progression. There is a large interest in drugs that might deactivate these cells. Many studies have shown that the grapevine-derived polyphenol, trans-resveratrol, and other stilbenes have therapeutic potential in some diseases. In this work, we have studied the effect of grapevine polyphenols on cultured human liver myofibroblasts. We have shown that trans-resveratrol profoundly affects myofibroblast phenotype. Trans-resveratrol induced morphological modifications. It markedly reduced proliferation of myofibroblasts in a dose-dependent manner. Trans-resveratrol also decreased the expression of alpha smooth muscle actin (alpha-SMA) without affecting vimentin or beta-cytoplasmic actin expression. It decreased myofibroblast migration in a monolayer wounding assay. We also showed that trans-resveratrol inhibited the messenger RNA (mRNA) expression of type I collagen. Finally, it decreased the secretion of matrix metalloproteinase 2 (MMP-2). We conclude that trans-resveratrol can deactivate human liver myofibroblasts. In the second part of this study, we have shown that neither trans-piceid (a glycosylated analog) nor trans-piceatannol (a hydroxylated analog) reproduces trans-resveratrol effects on liver myofibroblasts. We finally show that, although trans-resveratrol decreases the proliferation of skin fibroblast and vascular smooth muscle cells, it does not affect their expression of alpha-SMA, which indicates some cell specificity.

Determination of stilbenes (trans-astringin, cis- and trans-piceid, and cis- and trans-resveratrol) in Portuguese wines.

Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method is described for their direct determination in different types of wine using high-performance liquid chromatography with ultraviolet detection. In a survey of 120 commercial wines from Portugal and France, the highest concentrations of stilbenes were found in red wines. The glucosides of resveratrol were present in higher concentrations than the free isomers. Isolation from wine and characterization of trans-astringin in a large quantity are described for the first time.

High-performance liquid chromatography coupled with fluorescence detection for the determination of trans-astringin in wine.

In this study a high-performance liquid chromatography (HPLC) method was developed for the determination of trans-astringin in wine using fluorescence detection. This is the first time the occurrence of trans-astringin has been reported in wine. The method allows analysis of both red and white wine samples with no prior treatment. The quantification threshold is 0.03 mg/l. Levels of trans-astringin in the French wines analyzed ranged from 0.09 mg/l to 0.29 mg/l. The reproducibility of the method was measured and the CV was less than 4.8% for both red and white wines.

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures.

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars.

Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 M sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells.

Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests.

Vitis vinifera cell suspensions were used to isolate and characterize the flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes) found in red wine. Furthermore, we showed that astringin is produced although this stilbene has not previously been reported to be a constituent of V. vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical. Antioxidant activities were assessed by their capacity to prevent Fe2+-induced lipid peroxidation in microsomes and their action on Cu2+-induced lipid peroxidation in low-density lipoproteins. The results showed that astringin has an important antioxidant effect similar to that of trans-resveratrol, and a higher radical scavenger activity than the latter. Astringinin appeared to be more active. These data indicate that phenolic compounds (stilbenes, catechins, anthocyanins) exhibit interesting properties which may account in part for the so-called "French paradox," i.e. that moderate drinking of red wine over a long period of time can protect against coronary heart disease.

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures.

Suspension cultures of Vitis vinifera were cultured in different media in order to establish a model system for promoting high levels of phenolic substances identical with those found in wine. These media were: a low sucrose maintenance medium (MM) and four high sucrose media (differing mainly in sucrose and mineral contents) which were shown to induce secondary metabolism. In MM medium, polyphenol accumulation in the cells was low, and concentrations of 0.1 mg/gfw for condensed tannins and 0.3 mg/gfw for anthocyanins were reached within two weeks of cultivation. Values of 1.4 and 6.4 mg/gfw, respectively, were obtained with a low nitrate and high sucrose medium (HM1), but cell proliferation was reduced. To obtain a maximal production of polyphenols, we investigated the most effective conditions for cell growth and polyphenol production (a high mineral and high sucrose medium, IM1; inoculum dilution of 1.25:10). Under these conditions, the cells produced mainly anthocyanins (1100 mg/l), proanthocyanidins (300 mg/l) and catechins (25 mg/l).

Putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions.

The effects of cytokinins on the different branches of the indole alkaloid pathway were investigated in Catharanthus roseus cell cultures. Addition of zeatin to a 2,4-dichlorophenoxyacetic acid-containing medium decreased tryptamine levels and increased the bioconversion of secologanin to ajmalicine. Zeatin also enhanced the geraniol-10 hydroxylase activities and modified the indole alkaloid pattern. The results are discussed in the light of previous works showing that cytokinins have a positive effect on indole alkaloid accumulation in some lines of C. roseus.

Cytokinin-enhanced accumulation of indole alkaloids in Catharanthus roseus cell cultures - The factors affecting the cytokinin response.

Cytokinins were found to stimulate the alkaloid synthesis induced by removing auxin from the medium of a cell line of Catharanthus roseus. Diluting the mineral salts of the culture medium decreased the alkaloid production but increased the "sensitivity" of the cells. Addition of high levels of Ca(2+), Mg(2+) or Sr(2+) to B5 media in which the mineral salts were diluted to 5-40%, increased the alkaloid production. The latter effect is related only partially to enhanced osmotic potential.

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media.

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.

Influence of Sucrose on Levels of Ajmalicine, Serpentine, and Tryptamine in Catharanthus roseus Cells in vitro.

The fates of major nutritional elements and the synthesis of tryptophan, tryptamine and 2 alkaloid markers in C. ROSEUS cells, cultivated in media containing 20 and 60 g/l of sucrose, were studied. Sucrose effects were greatest, with these parameters, during the stationary growth phase. Intracellular ajmalicine and serpentine levels increased with sucrose concentration, whereas intracellular tryptamine (nitrogen precursor) levels were unaffected. The role of sucrose in the alkaloid pathway is discussed.