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.

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.

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.

Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from vitis vinifera cell cultures

Suspension cultures of Vitis vinifera L. (Vitaceae) produce many hydroxylated stilbene glucosides found in red wine. From these cells, we isolated and characterized glycosylated stilbenes, (Z)-piceatannol (3,5,3',4'-tetrahydroxystilbene) -3-O-beta-d-glucopyranoside (6) and (E)- and (Z)-resveratrol (3,5, 4'-trihydroxystilbene)-4'-O-beta-d-glucopyranoside (2 and 7, respectively), which have not previously reported to be constituents of Vitis vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1 diphenyl-2-picryl-hydrazyl, a stable free radical. Antioxidant activities were assessed by their capacity to prevent Cu2+-induced lipid peroxidation in human low-density lipoprotein.

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.

Effects of bolesatine on a cell line from the SP2/O thymic lymphosarcoma.

Bolesatine, a toxic protein isolated from Boletus satanas Lenz inhibits in vitro protein synthesis in a concentration-dependent manner in a cell line from a radiation-induced thymic lymphosarcoma (SP2/O) with a 50% inhibitory concentration (IC50) of 9.5 nM (0.6 microgram/ml). In vivo, an i.p. single injection of bolesatine, corresponding to 1/6 and 1/10 of 24-h 50% lethal dose, in Balb/c mice having ascitic tumour induced by the i.p. preinjection of SP2/O cells allows a remission of 50% and 30%, respectively. Treated mice survived 120 days after the treatment, i.e. 90 days after the death of control animals.

Effect of bolesatine, a glycoprotein from Boletus satanas, on rat thymus in vivo.

Bolesatine is a glycoprotein purified to homogeneity from Boletus satanas Lenz, a toxic mushroom which causes serious gastroenteritis. This lectin possesses a mitogenic activity on human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro in several systems. The mitogenic activity on peripheral blood T lymphocytes in vitro has been shown to be at least 200-fold higher than the activity of the well studied phytohemagglutinin (PHA). In order to verify this property in vivo, the effect of bolesatine has been studied in thymus of rats given orally bolesatine. Two groups of bolesatine-treated animals were used in addition to the control group. One group was given every 48 h, 28 micrograms of bolesatine/kg body weight seven times and 150 micrograms/kg body weight 48 h before the sacrifice. The other group was given 55 micrograms of bolesatine/kg body weight according to the same protocol and 150 micrograms/kg body weight 48 h before the sacrifice. In these conditions, the ratio thymus weight/body weight is increased by 10% and 28%, respectively, in groups 1 and 2. Similarly, the DNA synthesis is increased by more than 50%, indicating that (i) bolesatine probably possesses a mitogenic effect on thymocytes in vivo (ii) that the increase of the ratio thymus weight/body weight is not due to swelling by water retention, but rather to a multiplication of thymocytes. These results are confirmed in a second run of experiments in which bolesatine given orally to rats in lower doses of 3-12 micrograms/kg induces an increase of both thymus weight by 47% to 54% and an increase of total proteins by 52% to 56%, respectively, whereas the ratio total protein/g of thymus does not change. Thus bolesatine, known to be mitogenic to human lymphocytes in vitro is also mitogenic to rat thymocytes in vivo.

New insecticidal cyclodepsipeptides from the fungus Isaria felina. I. Production, isolation and insecticidal properties of isariins B, C and D.

Three new cyclodepsipeptides related to the previously described isariin were isolated from a strain of Isaria felina. They were named isariins B, C and D. Isariin D and, to a lesser extent, isariin C, exhibited insecticidal activity against Galleria mellonella larvae, whereas isariin B and isariin itself proved inactive.

New insecticidal cyclodepsipeptides from the fungus Isaria felina. II. Structure elucidation of isariins B, C and D.

Isariins B, C and D, isolated from a strain of Isaria felina, were shown to be cyclodepsipeptides constituted by a pentapeptide cyclized through a beta-hydroxyacid. The nature of the latter and the sequence of the peptide were determined for each compound. Relations between insecticidal activity and structure of the depsipeptides were pointed out.

New antibiotics from the fungus Epicoccum nigrum. I. Fermentation, isolation and antibacterial properties.

An atmosphere isolate of the fungus Epicoccum nigrum was found to exhibit an activity against Staphylococcus aureus. A more active, non-sporulating variant of this strain was selected. From its fermentation broth, two novel compounds, epicorazines A and B, were isolated by preparative TLC and tested against a series of bacteria.

New antibiotics from the fungus Epicoccum nigrum. II. Epicorazine A: structure elucidation and absolute configuration.

An antibiotic, epicorazine A, isolated from a particular strain of the fungus Epicoccum nigrum, was shown to be a new epidithiodiketopiperazine from its UV, IR, mass, NMR and CD spectra. X-Ray diffraction measures confirmed this structure and its absolute configuration.

New antibiotics from the fungus Epicoccum nigrum. III. Epicorazine B: structure elucidation and absolute configuration.

Comparison of UV, IR, PMR and CD spectra of epicorazine B with those of epicorazine A, a previously isolated metabolite of Epicoccum nigrum, showed that they were isomers with the same epidithiodiketopiperazine skeleton. X-Ray determination of epicorazine B indicated that the difference is related to a cis-trans configuration.