George J. Soleas

The red wine phenolics trans-resveratrol and quercetin block human platelet aggregation and eicosanoid synthesis: Implications for protection against coronary heart disease

A number of lines of evidence suggest that red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease (CHD) mortality. This protection over and above that due to ethanol itself may be explained by phenolic components with which red wines are richly endowed. We have studied the effects of the trihydroxy stilbene trans-resveratrol on human platelet aggregation and on the synthesis of three eicosanoids from arachidonate by platelets, i.e. thromboxane B2 (TxB2), hydroxyheptadecatrienoate (HHT) and 12-hydroxyeicosatetraenoate (12-HETE). These effects were compared with the actions of other wine phenolics (quercetin, catechin and epicatechin) and antioxidants (alpha-tocopherol, hydroquinone and butylated hydroxytoluene). trans-Resveratrol and quercetin demonstrated a dose-dependent inhibition of both thrombin-induced and ADP-induced platelet aggregation, whereas ethanol inhibited only thrombin-induced aggregation. The other compounds tested were inactive. trans-Resveratrol also inhibited the synthesis of TxB2, HHT, and to a lesser extent 12-HETE, from arachidonate in a dose-dependent manner. Quercetin inhibited only 12-HETE synthesis, and hydroquinone caused slight inhibition of TxB2 synthesis, the remaining compounds being ineffective. De-alcoholized red wines inhibited platelet aggregation; their ability to inhibit the synthesis of TxB2 but not that of 12-HETE from labelled arachidonate by washed human platelets was proportional to their trans-resveratrol concentration. These results are consistent with the notion that trans-resveratrol may contribute to the presumed protective role of red wine against atherosclerosis and CHD.

Wine as a biological fluid: History, production, and role in disease prevention

Wine has been part of human culture for 6,000 years, serving dietary and socioreligious functions. Its production takes place on every continent, and its chemical composition is profoundly influenced by enological techniques, the grape cultivar from which it originates, and climatic factors. In addition to ethanol, which in moderate consumption can reduce mortality from coronary heart disease by increasing high‐density lipoprotein cholesterol and inhibiting platelet aggregation, wine (especially red wine) contains a range of polyphenols that have desirable biological properties. These include the phenolic acids (p‐coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids), trihydroxy stilbenes (resveratrol and polydatin), and flavonoids (catechin, epicatechin, and quercetin). They are synthesized by a common pathway from phenylalanine involving polyketide condensation reactions. Metabolic regulation is provided by competition between resveratrol synthase and chalcone synthase for a common precursor pool of acyl‐CoA derivatives. Polymeric aggregation gives rise, in turn, to the viniferins (potent antifungal agents) and procyanidins (strong antioxidants that also inhibit platelet aggregation). The antioxidant effects of red wine and of its major polyphenols have been demonstrated in many experimental systems spanning the range from in vitro studies (human low‐density lipoprotein, liposomes, macrophages, cultured cells) to investigations in healthy human subjects. Several of these compounds (notably catechin, quercetin, and resveratrol) promote nitric oxide production by vascular endothelium; inhibit the synthesis of thromboxane in platelets and leukotriene in neutrophils, modulate the synthesis and secretion of lipoproteins in whole animals and human cell lines, and arrest tumour growth as well as inhibit carcinogenesis in different experimental models. Target mechanisms to account for these effects include inhibition of phospholipase A2 and cyclo‐oxygenase, inhibition of phosphodiesterase with increase in cyclic nucleotide concentrations, and inhibition of several protein kinases involved in cell signaling. Although their bioavailability remains to be fully established, red wine provides a more favourable milieu than fruits and vegetables, their other dietary source in humans. J. Clin. Lab. Anal. 11:287–313, 1997.

Absorption of three wine-related polyphenols in three different matrices by healthy subjects.

BACKGROUND Despite their powerful biologic activities conducive to protection against atherosclerosis, cancer and inflammatory diseases demonstrated in vitro, there is considerable doubt whether the polyphenolic constituents present in red wine and other dietary components are effective in vivo. OBJECTIVE We have tested the absorptive efficiency of three of these constituents (trans-resveratrol, [+]-catechin and quercetin) when given orally to healthy human subjects in three different media. DESIGN Twelve healthy males aged 25 to 45 were randomly assigned to three different groups consuming orally one of the following polyphenols: trans-resveratrol, 25 mg/70 kg; [+]-catechin 25 mg/70 kg; quercetin 10 mg/70 kg. Each polyphenol was randomly administered at 4-week intervals in three different matrices: white wine (11.5% ethanol), grape juice, and vegetable juice/homogenate. Blood was collected at zero time and at four intervals over the first four hours after consumption; urine was collected at zero time and for the following 24-h. The sums of free and conjugated polyphenols were measured in blood serum and urine by a gas-chromatographic method. RESULTS All three polyphenols were present in serum and urine predominantly as glucuronide and sulfate conjugates, reaching peak concentrations in the former around 30-min after consumption. The free polyphenols accounted for 1.7 to 1.9% (trans-resveratrol), 1.1 to 6.5% ([+]-catechin) and 17.2 to 26.9% (quercetin) of the peak serum concentrations. The absorption of trans-resveratrol was the most efficient as judged by peak serum concentration, area-under-the curve (4 h) and urinary 24-h excretion (16-17% of dose consumed). [+]-Catechin was the poorest by these criteria (urine 24-h excretion 1.2%-3.0% of dose consumed), with quercetin being intermediate (urine 24-h excretion 2.9%-7.0% of dose consumed). Some significant matrix effects were observed for the serum polyphenol concentrations, but in the case of urine no matrix promoted significantly higher excretion than the other two. CONCLUSIONS The absorption of these three polyphenols is broadly equivalent in aqueous and alcoholic matrices but, at peak concentrations of 10 to 40 nmol/L, is inadequate to permit circulating concentrations of 5 to 100 micromol/L consistent with in vitro biologic activity. The voluminous literature reporting powerful in vitro anticancer and antiinflammatory effects of the free polyphenols is irrelevant, given that they are absorbed as conjugates.

A comparison of the anticarcinogenic properties of four red wine polyphenols.

BACKGROUND There has been growing interest in the analysis of certain polyphenols in wine, especially flavonoids, trihydroxystilbenes and phenolic acids, stimulated by intense research into their potential benefits to human health. One of their main properties in this regard is their antioxidant activity, which enables them to attenuate the development of atherosclerosis, inflammatory diseases, and cancer. METHODS A two stage CD-1 mouse skin cancer model using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and phorbol 12-myristate 13-acetate (TPA) as promoter was employed to compare the antitumorigenic activities of one polyphenol from each of four different classes: flavanols [(+)-catechin], stilbenes (trans-resveratrol), flavonols (quercetin) and hydroxybenzoic acids (gallic acid). Animals were treated with specific polyphenols at doses ranging from 0 to 25 micromoles (dissolved in 200 microL acetone), twice a week for eighteen weeks. The solution was applied topically to the shaved dorsal region of each animal. The relative potencies of the polyphenols were compared by evaluating the percentage inhibition of tumor formation in individual mice and the number of mice developing one or more tumors with the different dose schedules. RESULTS Probit analysis revealed that quercetin was the most (ED(50)<1 micromole) and gallic acid the least effective (ED(50) 5-10 micromoles). (+)-Catechin and trans-resveratrol were intermediate, with ED(50) values of 5 and 6 micromoles, respectively. CONCLUSION We have shown recently that trans-resveratrol is absorbed much more efficiently than (+)-catechin and quercetin in humans after oral consumption. Taking this and the relative concentrations in red wine into account, together with the present results, we conclude that trans-resveratrol may be the most effective anticancer polyphenol present in red wine as consumed po by healthy human subjects.

Ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 microl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 microg/l and 0.1 microg/l, respectively, for all compounds. Recovery at 1 microg/l and 10 microg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 microg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5-1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.

Assay of resveratrol glucosides and isomers in wine by direct-injection high-performance liquid chromatography

Abstract We have developed a normal-phase high-performance liquid chromatographic method to measure the glucosides of cis - and trans -resveratrol as well as the free isomers in commercial wines. The procedure utilises a novel technique for calibration requiring only the trans -resveratrol in pure form and the conversion of the glucosides to the free isomers by hydrolysis with β-glucosidase. Only 20 μl of sample is needed for direct injection without prior preparation, and isocratic elution separates the 4 compounds of interest in approximately 50 min. Calibration curves are linear, and stable for several months. Recovery was around 100% for all compounds, and replicate analyses gave coefficients of variation of 0.4–6.7%. The limits of sensitivity were 0.2 and 0.1 μmoles l −1 for the glucosides and free isomers of cis - and trans -resveratrol, respectively. A preliminary survey of sixteen wines from eight countries or regions revealed the presence of all four compounds in fourteen; the glucosides of resveratrol were present in higher concentrations than the free isomers in five of the wines.

The World of Resveratrol

Since the discovery of trans-resveratrol (3,5,4’-trihydroxystilbene) as a constituent of wine by Siemann and Creasy, first reported in 1992 (1), the possibility that this compound, almost unique to red wine among constituents of the human diet, may in large measure account for the putative health benefits of this beverage beyond its mere content of vulgar ethanol, excited the imagination of the scientific and medical communities, initiating a ferment of research and enquiry that continues to this day. Indeed, ripples of these activities from time to time flow into the pages of the lay press, so that resveratrol has become a molecule impacting the consciousness of many well-informed members of the lay public. In March, 1997 we published a major review incorporating 183 references forming the bulk of the world literature on resveratrol up to that time (2). Our bottom line was that the future of resveratrol did not look particularly promising given the reality that, despite its miraculous performances in the culture dish and the test tube, the intact bodies of mice and men proved to be an inhospitable millieu robbing it of its presumed powers. To us, it seemed to be a compound for whom the bell tolled , but others did not share this gloomy prognosis. In fact, so much new work on this topic has been published in a mere two years-and-a-bit that a reappraisal of the situation deserves a welcome and is mandated by the present Symposium. Resveratrol exists as trans and cis isomers. Very little is known about the latter. When the nature of resveratrol is not specified, the reader should assume that the text refers to the trans isomer.

A Multiresidue Derivatization Gas Chromatographic Assay for Fifteen Phenolic Constituents with Mass Selective Detection

We have developed a GC/MS method to simultaneously measure the concentrations of 15 biologically active phenolic components of wine:  vanillic acid, gentisic acid, m- and p-coumaric acid, gallic acid, ferulic acid, caffeic acid, cis- and trans-resveratrol, epicatechin, catechin, morin, quercetin, and cis- and trans-polydatin. Wine (1 mL) was diluted 1:1 with water to reduce the alcohol content and extracted on a preconditioned C-8 solid-phase extraction cartridge. The phenolic compounds were eluted with ethyl acetate, evaporated to dryness, and derivatized with bis(trimethylsilyl)trifluoroacetamide/pyridine. The TMS derivative of each phenolic compound was analyzed on a GC/MSD coupled to a DB-5HT capillary column using one target and two qualifying ions for each compound in a total run time of 26 min. Resolution and quantitation of all compounds were excellent, with linear calibration curves over a wide range. The lowest detection limit was for gentisic acid (24 μg/L) and highest for quercetin (843 μg/L). The average percent recovery and coefficient of variation (mean precision) ranged from 90.7 to 104.6 (except morin, 72.2%) and 4.0 to 10.2 (except morin, 16.1%, and quercetin, 16.0%) respectively. This method has been applied to solid vitaceous plant materials as well as wine and should be suitable to measure polyphenols in fruit, vegetables, and other foods provided that efficient extraction techniques are employed.

Multiresidue analysis of seventeen pesticides in wine by gas chromatography with mass-selective detection.

We have developed a multiresidue method permitting the simultaneous quantitation of 17 pesticides in wine: dicloran, dimethoate, diazinon, chlorpyrifos-methyl, vinclozolin, carbaryl, methiocarb, dichlofluanid, parathion-ethyl, triadimefon, procymidone, myclobutanil, iprodione, imidan, dicofol, phosalone and azinphos-methyl. Solid-phase extraction of 0.5 ml of wine sample is followed by direct injection of 1 microl of the eluent onto a DB-5 MS gas chromatographic column followed by mass-selective detection using one target and two qualifier ions for each pesticide. The extraction and injection steps are carried out with automatic instrumentation. Good resolution of all compounds was achieved with a run-time approximating 23 min. Detection and quantitation limits were around 2 microg/l and 10 microg/l, respectively, with linear calibration curves up to 3 mg/l for most constituents. Recovery in half the compounds was >90%, and >80% in most of the remainder. Imprecision (relative standard deviation) was <10% for most pesticides and <18% in all. Further analytes can be added to the repertoire without difficulty. The method merits consideration together with four other multiresidue methods now available that offer similar analytical characteristics, slower run-times, and a different selection of analytes.

Method development and cultivar-related differences of nine biogenic amines in Ontario wines

We have developed a new high-performance liquid chromatographic method using pre-column derivatization with orthophthalaldehyde, gradient elution and fluorescence detection to assay the concentrations of nine biogenic amines in wines: histamine, 1-methylhistamine, methylamine, ethylamine, tyramine, tryptamine, 2-phenylethylamine, putrescine and cadaverine. The method shows excellent analytical characteristics. It has been used to measure the concentration of these biogenic amines in 73 monovarietal wines from five red and six white cultivars. All wines were from the Niagara viticultural region of southern Ontario and were certified as to origin. Pinot noir wines had the highest content of total amines, and also of histamine, putrescine, cadaverine, ethylamine and 1-methylhistamine. Among the white wines, those from Chardonnay had the highest content of total amines and also of histamine, tyramine, methylamine and 1-methylhistamine. It is suggested that longer ageing as well as Sur-lie fermentation (Chardonnay) can account, at least in part, for these findings.