Winter 2001 Issue — Provinols™: Red Wine Polyphenols

Redacted by R. Andriansitohain

CNS Strasbourg, France

D. Ageron

SFD, France

M-R Desrumaux

Seppic, France

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Introduction

Plant polyphenols contain a multitude of compounds and are one of the most important and widely distributed groups of compounds in plants. They are present in many foods including fruits, vegetables, dried fruits and drinks. Epidemiological studies have suggested that dietary factors including consumption of plant foods rich in polyphenols decreased the incidence of cardiovascular insults especially coronary heart diseases (Hertog et al., 1995). Cardiovascular diseases are responsible for approximately 50% of deaths in Western countries. One of the major processes giving rise to these cardiovascular diseases is atherosclerosis, as a consequence of poorly balanced nutrition. These observations have attracted the attention of many researchers into the quality of food and its importance in human health.

Interest of red wine polyphenols

Some polyphenols - and particular those contained in red wine - mostly flavonoids are believed to be the active compounds responsible for protective effects against cardiovascular diseases. The antioxidant and free radical scavenging properties of polyphenols may explain their capacity in reducing oxidation of serum low density lipoprotein and platelet aggregation (Frankel et al., 1993, Renaud and Logeril, 1992; Osman et al., 1998). Furthermore, polyphenols have been reported to suppress smooth muscle cell proliferation and to stabilise immune cell for review (Middleton et al., 2000). Hence, because of the above properties, polyphenols may interfere with the atherogenesis process and/or the thrombotic phenomena associated with atherosclerosis, which could explain at least partially the beneficial effects of these substances. Finally, wine derived from polyphenol extracts are capable of acting directly on blood vessel function, including an endothelium-dependant relaxation that involves the nitric oxide (NO) pathway (Fitzpatrick et al., 1993).

Provinols™: red wine polyphenols

Further to these potential health benefits of polyphenols from red wine, INRA (Institut National de Recherche Agronomique) in Montpellier-France had the idea of extracting polyphenols from red wine. Provinols™ was then developed by INRA in partnership with the Société Française de Distilleries, a French wine producing company specialised in the production of wine based products.

Provinols™ is an alcohol-free red wine extract polyphenol concentrate that contains at least 95% of polyphenols in its composition. It comes from red wine produced in the Languedoc-Roussillon area in the South-east of France, which has been specifically selected for its quality and antioxidant property. After removal of alcohol contained in red wine, Provinols™ is obtained after a concentration process without any solvents or spray drying without additives. It is water soluble dark red to purple fine powder with a tannic taste.

The different polyphenol families of Provinols™ are exactly the same as that of the wine they are extracted from and consequently allows the preservation of beneficial activities of moderate wine consumption for human health. It is perfectly suited for use in Drinks (alcoholic and alcohol-free), dairy products and desserts, dietary supplement bars, tablets, capsules or sachets. 100mg of Provinols™ corresponds to the polyphenol content of 1 glass of red wine.

Studies on Provinols™

Since 1996, the group of R. Andriatsithohaina, a researcher working at UMR CNRS (Centre National de la Recherche Scientifique) 7034 of Strasbourg-France carried out studies for a better understanding of the potential beneficial effects of Provinols™ on cardiovascular diseases, which are still a major health concern nowadays.

The evidence that Provinols™ elicited directly NO generation and endothelium-dependant relaxation within blood vessels is reported. Cellular and molecular mechanisms by which Provinols™ mediate their effects within endothelial cells in situ or in culture were determined. The effects of acute and short-term oral administration of Provinols™ on haemodynamic parameters and on vascular reactivity have also been analysed in vivo in the rat. Finally, the mechanisms by which chronic administration of Provinols™ might protect against the increase in blood pressure and the associated deleterious effects of cardio-vascular structure and function was also assessed in rat model of hypertension.

Vasodilator properties of the RWPC and, in particular, Provinols™

The blood vessel consists of 3 layers:

1. The endothelium, which lies in the internal surface of the vessel wall and is in contact with circulating blood;
2. The medial layer composed of smooth muscle cells that possess contractile properties;
3. The adventitial layer, at the outside part of the vessel composed of fibroblasts, macrophages and nerve endings.

The vascular endothelium plays an important role in the regulation of blood flow and vascular tone. It secretes various vasodilator factors including NO, prostanoid derivatives and the hyperpolarizing factor and vasoconstrictor factors such as endothelin, angiotensin II and reactive oxygen species (Furchgott and Vanhoutte, 1989). Endothelial dysfunction leads not only to a decrease in the release of vasodilator facture such as NO, but also to the increase of platelet aggregation and the increase of the flux of the atherogenic plasma protein into the artery walls leading to the acceleration of atherosclerosis process.

The effects of RWPC and Provinols™, in particular, on vasomotricity have been studied in detail using different pharmacological approaches in the blood vessels and in the cells which make up the blood vessels, particularly endothelial cells Andriantsithaina et al., 1999). RWPC and Provinols™ have been shown to produce vascular relaxation by eliciting the release of vasodilator compound from the endothelium (Andriambeloson et al., 1997, 1999).

The use of electronic paramagnetic resonance which allows direct measurements of NO in vascular tissue demonstrate that wine extract directly stimulates the production of NO in the blood vessels (Andriambeloson et al., 1997). This is due to the direct action of polyphenols on the endothelial cells, stimulating the release of NO and other endothelial relaxing factors, such as the hyperpolarizing factor. The positive active principles that support the endothelial NO-dependent relaxation produced by RWPC1 (a standard polyphenol extract (Cabernet-Sauvignon) supplied by the INRA in Montpellier) has been investigated in rat aorta (Andriambeloson et al., 1998). The results show that the chromatographically resolved fractions enriched into anthocyanins and those enriched into oligomeric condensed tannins inhibit the interaction between calmodulin and phosphodiesterase I and between calmodulin and a fluorescent probe. The latter results suggest that the active compounds contained in RWPC1 interact with a site presenting structural analogies with the lipophilic domain exposed on calmodulin upon activation by Ca²+ (Klaiber et al., 2001).

NO may produce both beneficial and deleterious cardiovascular effects, depending on its level of concentration and its interactions with various targets, including superoxide anions (Stoclet et al., 1998). Among the beneficial effects of NO include vascular relaxation protection against platelet aggregation, adhesion and leukocyte migration and inhibition of vascular cells proliferation following endothelial damage. In addition, NO is able to induce genes protective of the cardiovascular system (Kim et al., 1997; Marschall et al., 2000). Finally, NO is capable of interacting with process leading to apoptosis or "programmed cell death" in vascular cells. These above properties of NO might explain the protective role of wine extract - and, in particular, Provinols™ - in against vascular diseases such as coronary artery diseases and atherosclerosis.

Intracellular mechanisms governing the production of NO

The mechanisms by which Provinols™ induced endothelial-dependent relaxation have been investigated in rat thoracic aorta rings (Andriambeloson et al., 1999). The results suggest that Provinols™ produces endothelial-NO derived vasorelaxation through an extracellular Ca²+-dependant mechanism. The associated mechanisms do not involve the activation of known secondary messengers, such as those produced by PLC or phospholipase A2, nor G proteins.

The mechanisms of action of RWPC from different sources (RWPC1 and Provinols™), and the anthocyanin, Delphinndin in bovine aortic endothelial cells have been assessed with respect to calcium handling using Ca²+ imaging techniques with CCD camera and confocal microscopy (Andriambeloson et al., 1997; Martin et al., 2000). The three have similar pharmacological profiles in blood vessels and all increase cytosolic free calcium, by releasing Ca²+ entry; a cascade bio-assay showed the latter is essential for NO production. RWPC1, Provinols™ and the defined polyphenol, Delphinidin, display differences in the processes leading to the increase in cytosolic Ca²+ in endothelial cells. In any view of the results obtained, several conclusions can be drawn. First of all, Provinols™ stimulates an elevation in Ca²+ levels and the production of endothelial factors by acting on multiple molecular targets. Secondly, the mechanisms underlying the elevation in cytosolic Ca²+ in response to Provinols™ and to INRA wine extract are different, although both are capable of producing NO.

Effect of in vivo administration of Provinols™

Acute i.v. infusion of Provinols™ produces a dose-dependent decrease of the mean arterial blood pressure (the maximal effect reached at 1 mg/kg) without any effect on the heart rate in anaesthetised rat. The dose producing maximal hypertensive effect is compatible to that required to induce maximal endothelium-dependent relaxation of rat aortic rings in vitro. The short term oral administration (20 mg/kg, once daily) of Provinols™ for one week reduced blood pressure in normotensive rats without affecting heart rate. The haemodynamic effects are associated with an enhanced endothelium-dependent relaxation and a subtle induction of gene expression (or inducible NO-synthase and cyclo-oxygenase-2) within the arterial wall which together maintain unchanged agonist-induced contractility. (Diebolt et al., 2001).

All these results suggest that the polyphenols in Provinols™ are absorbed in vivo and that, under these experimental conditions, polyphenols are present in the blood stream at concentrations that can influence blood pressure, cardiac and vascular functions. Release of vaso-active and induction of gene expression in the vascular wall play a significant role in the above mentioned effect of Provinols™.

Protective effect of Provinols™ on hypertension

The effect of Provinols™ on blood pressure, left ventricular hypertrophy and NO-synthase activity during regression of hypertension in a model of hypertensive rats (i.e. NO-deficient rats using the NO-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) has been assessed (Bernatova et al., 2000). Provinols™ accelerates the regression of blood pressure and prevents the development cardiovascular remodelling (myocardial fibrosis and aortic stiffness) and vascular dysfunction (endothelial and smooth muscle abnormalities) in a model of chronic inhibition of NO synthesis. Increased NO synthase activity contributes to the beneficial effects of Provinols™. Finally in the same model of hypertension, Provinols™ partially prevents the increase in blood pressure and the decrease of NO-synthase activity but not LV hypertrophy produced by L-NAME (Pechanova et al., 2000).

Conclusions

The above results suggest that NO formation and release caused by Provinols™ trigger various cardioprotective mechanisms both ex vivo and in vivo. Cardiovascular effect's of Provinols™, at least partially through NO, may contribute to the anti-hypertensive and anti-arteriosclerotic properties of plant derived polyphenols. Thus, polyphenols present in red wine may therefore be of therapeutic benefit in the future and may represent a new class of medicinal products against cardiovascular diseases which remain, at present, a major public health problem.

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