WO2014131884A1 - Novel combinations comprising a polyphenolic extract of grape marc with an antihypertensive and uses thereof - Google Patents

Abstract

The present invention relates to a combination comprising a polyphenolic extract of grape marc with an antihypertensive, and to the use thereof in the treatment of hypertension. The present invention also relates to a polyphenolic extract of grape marc and to the use thereof as a food supplement or dietetic food intended for special medical purposes, in the treatment of hypertension.

Description

 Novel combinations comprising a polyphenol extract of grape marc with an antihypertensive and their uses

The present invention relates to a combination comprising a polyphenol extract of grape marc with an antihypertensive agent, and its use in the treatment of hypertension.

 The present invention also relates to a polyphenol extract of grape marc and its use as a dietary supplement and / or as a food for special medical purposes, in the treatment of hypertension.

 High blood pressure (HTA) is one of the major public health problems in developed and emerging countries. This is the most common cardiovascular disease. In the 20-year-olds, the percentage of hypertensives is very low but then increases steadily to reach 40% at age 65 and 90% at age 85. According to the World Health Organization, the number of hypertensive individuals is in perpetual growth and is estimated at 1.56 billion people worldwide by 2025.

 There are two types of hypertension: essential hypertension and secondary hypertension. In 95% of cases, hypertension is said to be essential or primary, that is to say that it is of unknown cause, but strongly related to heredity or the environment. Age, sex and race are important risk factors in the development of essential hypertension. There are also lifestyle risk factors including obesity, alcohol consumption, salt intake, smoking, stress and lack of exercise (Carretero, OA, & Oparil, S. (2000) Circulation, 101 (3), 329-335). Other cases of hypertension are called secondary and are caused by another identifiable pathology such as kidney damage, adrenal tumors and hormonal imbalances (Sukor, N. (201 1).) Postgraduate Medical Journal, 57 (1032) , 706-713).

Hypertension is also considered a risk factor in the development of cardiovascular diseases. Elevated blood pressure can cause changes in the heart and vessels, including stroke, ischemic heart disease (angina, myocardial infarction), heart failure (left ventricular hypertrophy), and renal failure (hyperactivity). of the Renin-Angiotensin-Aldosterone system) as well as aneurysms (Leifert, WR, & Abeywardena, MY (2008), Nutrition Research, 28 (11), 729-737). The altering effect of hypertension on artery walls promotes fat deposition and plaque accumulation associated with atherosclerosis, especially at the coronary level.

 Currently, the management of hypertension includes the administration of hypotensive active ingredients - or antihypertensives - of synthesis, as well as hygienic and dietary measures (reduction of salt consumption for example).

 Several classes of synthetic antihypertensives are known such as angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, renin inhibitors, calcium channel blockers (also known as calcium channel blockers), diuretics and betablockers.

 However, the effectiveness of these antihypertensives is not completely satisfactory.

Indeed, most hypertensive patients require polytherapy, that is to say the combination of several active ingredients from different classes, more or less long term to obtain a satisfactory level of tension: a dual therapy is often necessary, even a triple therapy. Resistance to some antihypertensives may also appear, making them ineffective. This is particularly the case of calcium channel blockers.

 There is therefore a need to find new treatments for hypertension. There is also a need to improve existing hypertension treatments. Finally, there is a need to diversify the management of hypertension.

The purpose of the present invention is to provide a new treatment for hypertension.

 A second object of the present invention is to provide an improved treatment of hypertension.

 A third object of the present invention is to avoid and / or delay the use of polytherapy in the treatment of hypertension.

 A fourth object of the present invention is to reduce the effective doses required of active ingredients currently used in the treatment of hypertension.

 A fifth object of the present invention is to overcome the resistances acquired against the antihypertensive active principles of synthesis.

 The present invention also aims to diversify the management of hypertension.

 An object of the present invention is therefore to provide a food for special medical purposes, in particular for the treatment of hypertension.

An object of the present invention is therefore to provide a dietary supplement to an antihypertensive treatment. Another object of the present invention is to provide a food supplement and / or a food intended for special medical purposes, in order to improve the antihypertensive effect of a synthetic antihypertensive active principle.

 Another object of the present invention is to provide a food supplement and / or a food intended for special medical purposes in the prevention of arterial hypertension.

 According to the present invention, these different goals are achieved by means of polyphenolic extracts from grape marc. Surprisingly, the present inventors have in fact discovered that polyphenolic extracts from grape marc potentiate the antihypertensive activity of synthetic antihypertensive active ingredients, and more particularly antihypertensives of the family of calcium channel blockers.

 Said polyphenolic extracts, in combination with a calcium channel blocker, make it possible in particular to obtain a satisfactory blood pressure level in the hypertensive patient.

 Said polyphenolic extracts, in combination with a calcium channel blocker, have a synergistic effect: the antihypertensive activity of the combination is greater than the sum of the antihypertensive effects obtained separately with a polyphenol extract alone and with a calcium channel blocker alone.

 This synergy has the advantage of avoiding and / or delaying the taking of several synthetic antihypertensive active ingredients to obtain a satisfactory blood pressure level. This synergy makes it possible in particular to overcome the resistance to certain synthetic antihypertensive active ingredients.

 This combination also has the advantage of being able to maintain and / or reduce the doses of the calcium channel blocker.

 Another advantage lies in the fact that the side effects are reduced by taking said combination: only one active principle of synthesis is prescribed, possibly at a reduced dose.

Finally, the polyphenolic extract makes it possible to diversify the management of hypertension, when it is, for example, presented as a dietary supplement, and / or as a food intended for special medical purposes in the treatment. hypertension. The present invention thus relates to a combination comprising a polyphenol extract of grape marc and an antihypertensive agent. The present invention relates to more particularly a combination comprising a polyphenol extract of grape marc and an antihypertensive of the family of calcium channel blockers.

 The term "polyphenol extract of grape marc", hereinafter called "polyphenol extract", an extract obtained by aqueous extraction or alcoholic extraction of one or more grape marc (s).

 "Grape marc" means the dandruff and / or seeds obtained after pressing the grapes during the preparation of the wine, after separation of the must.

 A marc of reason according to the present invention can thus be fermented or non-fermented, depending on whether the pressing takes place after the fermentation step (case of the preparation of a red wine) or before the fermentation step (case of the preparation of a white wine).

 According to a particular embodiment, the films and / or seeds used are obtained from fresh grapes, that is to say unfermented grapes. According to another particular embodiment, the films and / or seeds are obtained from fermented grapes. More particularly, the dandruff and / or pips are obtained from fermented grapes used in the preparation of the wine.

 According to one particular embodiment, the aqueous extractions are carried out by placing seeds and / or grape pomace films (for example 3 kg) in distilled water (for example 10 kg), generally for a period of time. 30 minutes to 1 h 30, at a temperature generally between 80 ° C and 120 ° C, preferably for 1 hour at 100 ° C.

 According to one particular embodiment, the alcoholic extractions are carried out by placing pips of pomace (for example 3 kg) in ethanol at 70% vol (for example 10 kg) generally for a period of 30 minutes to 1 hour 30 minutes. a temperature generally between 80 ° C and 120 ° C, preferably for 1 hour at 100 ° C.

 According to a particular embodiment, the alcoholic extractions are carried out by placing dough films (for example 3 kg) in ethanol at 70% vol. (For example 10 kg) for a period of 30 minutes to 1 h 30, at a temperature between 30 ° C and 70 ° C, preferably for 1 hour at 50 ° C.

 According to a particular embodiment, following the extraction, the aqueous or alcoholic solvents are then evaporated in order to obtain a dry residue of polyphenol extract of grape marc. The aforementioned evaporation techniques are techniques generally known to those skilled in the art.

According to a particular embodiment, the concentration of the extracts, that is to say the evaporation of the aqueous or alcoholic solvent, is carried out in a rotavapor (ie under vacuum) with a maximum temperature of 35 ° C. Said polyphenolic extract is characterized in particular by the presence of polyphenols.

 The term "polyphenols", also called "phenolic compounds", a family of organic molecules present in the plant kingdom. Natural polyphenols include a vast group of chemical substances comprising at least one aromatic nucleus bearing one or more hydroxyl groups. They can include simple molecules, such as phenolic acids (gallic acid for example), and highly polymerized compounds of more than 30000 Dalton, such as tannins. Phenolic compounds have antioxidant properties that protect the body against the excessive production of free radicals. In addition, phenolic compounds appear to play a role in the prevention of arterial hypertension and angiotensin II-induced endothelial dysfunction in part by preventing vascular oxidative stress-dependent NADPH oxidase and the formation of vasoconstrictor metabolites.

 The polyphenols contained in grapes, wine or plants are generally classified as flavonoids and non-flavonoids. Stilbenes and phenolic acids (non-flavonoids) are thus found on the one hand, flavones, flavonols, flavan-3-ols and anthocyanins (flavonoids) on the other hand:

 - simple phenols, phenolic acids (derived from benzoic or cinnamic acid),

 - coumarines,

 - naphthoquinones,

 stilbenoids or stilbenes,

 - flavonoids,

 - isoflavonoids,

 anthocyanins;

 - lignans,

 - lignins,

 - condensed tannins and proanthocyanidins.

The extracts according to the invention comprise in particular procyanidines and / or anthocyanins.

Among the procyanidins mention may be made of (Epi) catechin monomers, (Epi) catechin gallate, (Epi) catechin dimers, gallate dimers, (Epi) catechin trimer, Gallo ( epi) Catechine trimers, Trimeres gallate, Tetramers Catechin tetramers, Gallate tetramers, Pentamers of (Epi) catechin, Gallo (epi) Catechine pentamers, and Hexamers, Heptamers, Octamers, Nonamers and Decamers of (Catechine tetramers). epi) catechin.

 Among the anthocyanins that may be mentioned: Delphinidin-3-O-glucoside, Cyanidin-3-O-glucoside, Petunidin-3-O-glucoside, Peonidin-3-O-glucoside, Malvidin-3-O glucoside, Malvidine-3-O-glucoside-acetaldehyde (Vitisin B), Delphinidin-3-O- (6 "-O-acetyl) glucoside, Mv-Cat Dimer, Malvidin-3-O-glucoside pyruvate (Vitisin A), Mv-Cat Dimer, Mv-Cat Dimer, Peonidine-3-0- (6 "-O-acetyl) glucoside, Malvidine-3-0- (6" -O-acetyl) glucoside Delphinidine-3-0- (6 "-O-coumaroyl) glucoside, Malvidine-3-0- (6" -O-caffeoyl) glucoside, Cyanidin-3-0- (6 "-0-coumaroyl) glucoside, Petunidine-3-0- (6 "-O-coumaroyl) glucoside and Malvidine-3-0- (6" -O-coumaroyl) glucoside.

 Among the procyanidines, there may be mentioned monomers, dimers, trimers, tetramers and pentamers of (Epi) catechin.

 According to one particular embodiment, the aqueous and / or alcoholic extracts of pips have a concentration of monomers between 25 and 80 mg / g of dry matter equivalent of (-) - epicatechin. According to another embodiment, the aqueous and / or alcoholic film extracts have a monomer concentration of 1 to 30 mg / g dry matter equivalent of (-) - epicatechin. More particularly, the aqueous and / or alcoholic extracts of seeds have a dimeric concentration of between 5 and 33 mg / g dry matter equivalent of (-) - epicatechin. More particularly, the aqueous and / or alcoholic film extracts have a dimer concentration of 1 to 10 mg / g dry matter equivalent of (-) - epicatechin.

 Among the anthocyanins, particular mention may be made of glycosylated anthocyanins, acetylated anthocyanins and coumaroylated anthocyanins, and more particularly Delphinidin-3-O-glucoside, Malvidine-3-O-glucoside, Petunidine-3-O-glucoside and Malvidine-3-0- (6 "-O-coumaroyl) glucoside.

 According to one embodiment, the aqueous and / or alcoholic extracts of pips have a concentration of total glycosylated anthocyanins between 0.15 and 29 mg / g of dry matter in the equivalent of malvidin-3-O-glucoside. According to another embodiment, the alcoholic extracts of marc film comprise between 9 and 29 mg / g of dry matter in equivalent of malvidine-3-O-glucoside.

The term "antihypertensives" or "hypotensives" means active ingredients administered to reduce arterial hypertension. High blood pressure is defined as systolic blood pressure above 140 mmHg and / or blood pressure diastolic greater than 90 mmHg. Preferably, the measurements must be carried out at the medical office and confirmed at least by two measures per consultation, during three successive consultations.

 The term "satisfactory blood pressure level" means blood pressure values below 140 mmHg for systolic blood pressure and below 90 mmHg for diastolic blood pressure. More particularly, the systolic blood pressure is greater than 90 mmHg, preferably between 90 mmHg and 140 mmHg (a systolic blood pressure below 90 mmHg is generally considered to be hypotension). The satisfactory blood pressure level is determined by the physician, depending on the patient (for example, for a diabetic patient or a patient with renal insufficiency, the satisfactory blood pressure level is a systolic blood pressure of less than 130 mmHg and / or a diastolic blood pressure less than 80 mmHg).

 Calcium inhibitors are a class of synthetic active ingredients used in the treatment of hypertension. This class includes:

dihydropyridines such as nifedipine, amlodipine, felodipine, isradipine, lacidipine, nicardipine and nitrendipine;

 phenylalkylamines such as verapamil;

 benzothiazepines such as diltiazem.

 In one embodiment of the invention, the calcium channel blocker is selected from one of the specific calcium channel block inhibitors listed above. In a preferred embodiment, the calcium channel blocker is verapamil.

According to one embodiment of the invention, the polyphenolic extract is an aqueous extract or a hydroalcoholic extract, preferably hydroalcoholic. According to a particular embodiment, the hydroalcoholic extract is produced with ethyl alcohol at 70% vol.

 According to one embodiment, the combination as defined above comprises a polyphenol extract as defined below.

 According to one embodiment, in the combination as defined above, the antihypertensive agent is verapamil.

The invention also relates to a polyphenol extract of grape marc that can be obtained by aqueous extraction and / or alcohol extraction by contacting with the grape marc, in particular by aqueous or alcoholic extractions as defined above. More particularly, the grape pomace polyphenol extract is a dry extract, the solvent of which (for example distilled water or 70% vol ethanol alcohol) has been evaporated. According to a particular embodiment, the solvent used is suitable for the food consumption of the extract. According to a particular embodiment, the solvent is not sulphited water. The dry extract may then be treated according to methods generally known to those skilled in the art: it may especially be ground, pulverized and / or sieved. It is in the form of a tablet, granules, or powder, preferably in the form of a powder. According to one embodiment, the polyphenolic extract is derived from one or more pomace grape marc (s) and / or grape skins. According to a particular embodiment, the polyphenolic extract is derived from grape seeds. According to another particular embodiment, the polyphenol extract is derived from grape skins. According to one embodiment, the polyphenol extract of grape marc is derived from the grapes belonging to the variety Vitis viniferas. According to a particular embodiment, said polyphenol extract of grape marc is derived from red grapes.

 According to one embodiment, the polyphenol extract of grape marc is derived from one or more grapes belonging to one of the grape varieties selected from Grenache Long du Coudoulet, Grenache Face aux Pins, Syrah du Plantier. , the Syrah of Haut de Julien, Carignan, Mourvèdre, Counoise and Alicante.

 In a particular embodiment, the grape marc is chosen from:

 the marc of pips of Grenache Long of the Coudoulet, the marc of seeds of Syrah of Plantier,

 - the marc of Syrah dandruff from the Haut de Julien,

 the pomace marc of Carignan,

 the film of Mouvèdre film,

 the marc of Alicante dandruff, and their mixtures.

 Preferably, the polyphenol extract of grape pomace is selected from the grape pomace of Grenache Long Coudoulet, the marc of dandruff Alicante and mixtures thereof.

The present invention also relates to the combination as defined above, for its use as a medicament, and more particularly for its use in the treatment of hypertension. The present invention relates to the use of the combination as defined above for the preparation of a medicament for treating hypertension.

 The present invention more particularly relates to the combination as defined above, for its use in hypertensive patients, preferably treated with a calcium channel blocker.

 The combination according to the invention can therefore be a pharmaceutical specialty within the meaning of the European Directive 2001/83 / EC. The present invention relates to a pharmaceutical composition comprising the combination as defined above with at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include derivatives of cellulose or microcrystalline cellulose, alkaline earth carbonates, magnesium phosphate, starches, modified starches, lactose for solid forms, cocoa butter or polyethylene glycol stearates, water, aqueous solutes, saline solution, isotonic solutes, etc.

According to an alternative embodiment, the combination according to the invention is in the form of a pharmaceutical kit for the treatment of hypertension, in which the polyphenol extract of grape marc and the antihypertensor of the family of inhibitors calcium are formulated separately for simultaneous, separate or staggered use.

The invention also relates to the polyphenol extract of grape marc as defined above, for its use in the treatment of hypertension, preferably in the treatment of hypertensive patients treated with calcium channel blocker.

The present invention also relates to a dietary supplement comprising the polyphenol extract of grape marc as defined above. The food supplement is understood in the sense of the European Directive 2002/46 / EC.

 The present invention also relates to the use of a dietary supplement as defined above, in the treatment of hypertension.

 The present invention also relates to the use of a dietary supplement as defined above, in the treatment of hypertension, in addition to a calcium channel blocker and more particularly in patients treated with a calcium channel blocker.

By user is meant any person suffering from hypertension, especially who is following an antihypertensive treatment, such as a calcium channel blocker. The polyphenolic extract according to the invention may especially be a food or a functional food (or food).

 Functional foods, or nutraceuticals, are generally defined as a conventional food, or one that appears to be part of the normal diet, and has the characteristic of providing beneficial physiological effects beyond its normal nutritional functions or reducing the risk of chronic diseases.

 The polyphenolic extract according to the invention may preferably be a "dietary food for special medical purposes" as defined by Directive 1999/21 / EC. This type of food is defined as belonging to a category of foods for a particular diet, which are specially treated or formulated and intended to meet the nutritional needs of patients and which can only be used under medical supervision. They are intended to constitute the exclusive or partial feeding of patients whose capacities of absorption, digestion, assimilation, metabolization or excretion of ordinary foods or of some of their ingredients or metabolites are diminished, limited or disturbed. or whose state of health determines other special nutritional needs that can not be met by a change in the normal diet or by a diet consisting of foods for a particular diet or a combination of both.

 Among these foods, there are several categories depending on whether they may or may not be the sole source of food for the people for whom they are intended.

 The polyphenolic extracts according to the invention are particularly considered to belong to the category of nutritionally incomplete foods which, with a composition that is normal or adapted to meet the specific needs of a pathology, a disorder or a disease, can not constitute the only source of power. They can also be used to replace part of the patient's diet or serve as a supplement.

 In the context of the present invention, the polyphenolic extract as defined above may be a dietary food for special medical purposes, more particularly for hypertensive patients, even more particularly for hypertensive patients whose treatment comprises a calcium channel blocker. . Preferably, the calcium channel blocker is verapamil.

The present invention also relates to a kit comprising the combination as defined above, for simultaneous, separate or staggered use for the treatment of hypertension. The present invention also comprises a kit comprising:

 the polyphenolic extract as defined above, and

 a calcium channel blocker,

for simultaneous, separate or staggered use for the treatment of hypertension.

 According to one embodiment of the kit defined above, the calcium channel blocker is verapamil.

For the purposes of the invention, "simultaneous" means that the polyphenolic extract and the calcium channel blocker are administered by the same route, preferably orally and at the same time (they may for example be mixed), "separated" means that they are administered by different routes and / or at different times, and "spread over time" means that they are administered separately, at different times.

The present invention also provides a method of treating hypertension, comprising administering to a hypertensive patient a combination as defined above. The present invention also provides a method of treating hypertension, comprising administering to a patient treated with a calcium channel blocker the polyphenolic extract as defined above.

Said combinations, extract and kit according to the invention are useful as preventive and / or curative, therapeutic and / or nutritional, for human and / or animal use. The excipients necessary for their industrial shaping can be chosen from all the acceptable substances according to the regulation (food, dietary supplement, functional food, pharmaceutical compound) which governs this product.

 The combination and / or the polyphenolic extract according to the invention may be presented in forms intended for oral, sublingual, topical, local, intratracheal, intranasal or rectal administration, in particular oral administration.

 They can therefore be present especially in the form of solutes or multi-dose vials, or in the form of naked or coated tablets, coated tablets, capsules, soft or hard capsules, granules, pills, cachets, powders, suppositories or rectal capsules, solutions or suspensions, or creams, gels, ointments, pastes, patches, etc.

Said dietary supplement is advantageously formulated in the form of a dose, namely in a form of presentation defined by the European Directive 2002/46 / EC, such as "capsules, lozenges, tablets, pills and other similar forms, as well as sachets of powder, ampoules of liquid, vials with an drop and other similar forms of liquid or powder preparations intended to be taken in units of a small quantity '.

 The polyphenolic extracts according to the invention may especially be used in freeze-dried form in the pharmaceutical or food compositions according to the invention.

 The present invention is particularly aimed at gastroresistant formulations such as pasta or capsules, in particular gastroresistant capsules comprising the combination and / or the polyphenolic extract according to the invention. The dose and / or dosage of the combination and / or polyphenol extract may vary within the important limits and may depend on the route of administration, as well as the age and weight of the subject.

 There may be special cases where higher or lower dosages are appropriate; such dosages are not outside the scope of the invention. According to the usual practice, the appropriate dosage for each subject is determined according to the mode of administration, the weight and the response of said subject.

 According to one embodiment, the polyphenolic extract is formulated in a unitary form comprising for example between 0.5 gram and 5 grams of polyphenols, especially between 0.5 gram and 2 grams.

Description of the figures:

 Figure 1 shows the effect of polyphenolic extracts according to the invention on the arterial pressure of SHR rats.

 Figure 2 shows the effects of verapamil on systolic blood pressure in rats.

 Figure 3 shows the effect of the combination of polyphenol extracts with verapamil on arterial blood pressure of SHR rats.

EXAMPLES

EXAMPLE 1 Preparation of Extracts According to the Invention 1. Materials and Methods:

 1. A. Solvents:

Ethyl alcohol 70% vol (70% volume),

Deionized water purified with Milli-Q system (Millipore, Bedford, MA), Solvents provided by Prolabo VWR ®. 1. B. Vegetable raw materials:

 The extracts were made on grapes mares (V.vinifera) of the 2010 vintage from the Château de Beaucastel located in the Rhône Valley under the name Châteauneuf-du-Pape. The grape varieties selected are Grenache (from two different locations: Grenache Long from Coudoulet-GLC and Grenache from Pins-GFP), Syrah (from two different locations: Syrah Plantier-SyR and Syrah Haut from Julien-SyH), Carignan (CAR), Mourvèdre (MOU), Counoise (COU) and Alicante (ALI).

1. C. Sample Preparation and Extraction:

 Prior to extraction, a seed-dandruff separation was carried out at Beaucastel Castle using a plucking machine.

 The extraction of pips and skins of pomace from each variety was carried out according to the following extraction protocol:

 1) Aqueous extraction:

The seed pips (3 kg) are placed in distilled water (10 kg) for 1 hour at 100 ° C.

 The dough sheets (3 kg) are placed in water (10 kg) for 1 hour at 100 ° C.

 2) Alcoholic extraction:

 The seed pips (3 kg) are placed in water (10 kg) for 1 hour at 100 ° C. The dough films (3 kg) are placed in ethanol at 70% vol. (10 kg) for 1 hour at 50 ° C.

 The concentration of the extracts is rotavapor (vacuum) with a maximum temperature of 35 ° C.

The seeds and dandruff of each variety have undergone two different types of extraction. For the same sample, an aqueous extract (EAQ) and a 70% hydroalcoholic extract (EA70) (ethyl alcohol at 70% vol) were synthesized, as shown in the table below:

Pellets film

 EAQ EA70 EAQ EA70

GLC Grenache long Coudoulet X X

 GFP Grenache facing the pines X X

SyP Syrah plantier XXXX SyH Syrah Top of Julien XX

 Carignan Carignan Joseph X X X X

 MOUVEDRE MOU X X

 COU Counoise

 ALI Alicante X X

EXAMPLE 2 Antioxidant Activity and Composition of Polyphenolic Extracts According to the Invention

1 Materials and methods:

 1. A. Solvents and reagents:

 Deionized water purified with a Milli-Q system (Millipore, Bedford, MA). The solvents are of HPLC quality and come from Prolabo-VWR (Fontenay-sous-Bois, France): acetonitrile, ethyl acetate, chloroform, methanol, ethanol and acetone.

 Reagents come from Sigma-Aldrich (St Quentin Fallavier, France) and Polyphenols Biotech (Villenave d'Ornon, France):

 (+) - Catechin, (-) - epicatechin, B1 [(-) - epicatechin- (4b-8) - (+) - catechin], B2 [(-) - epicatechin- (4b-8) - (-) -epicatechin], B3 [(+) - catechin- (4a-8) - (+) - catechin] and B4 [(+) - catechin- (4a-8) - (-) - epicatechin], cyanidin-3 O-glucoside chloride, delphinidin-3-O-glucoside chloride, malvidin-3-O-glucoside chloride, peonidin-3-O-glucoside chloride, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), acid 6 2-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammoniumimide (ABTS), potassium persulfate, fluorescein, 2, 2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH), sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, 2,4,6-tri (2-pyridyl) -s-triazine (TPTZ), iron chloride hexahydrate, iron sulfate heptahydrate, Folin Ciocalteu reagent (2N), sodium carbonate, sodium bisulfite, phloroglucinol, L (+) - tartaric acid, L-ascorbic acid, hydroxide Sodium, hydrochloric acid (37%), formic acid and acetic acid.

1. B. Chemical analyzes:

 at. Sample preparation:

The extracts analyzed are those of Example 1. The extracts were solubilized and suitably diluted in a model solution composed of water / ethanol (90:10, v / v, pH 3, adjusted with tartaric acid). b. Determination of total phenolic compounds:

The total polyphenol concentration of the extracts was determined by the Folin-Ciocalteu reagent which is a mixture of phosphotungstic (H3PW12O14) and phosphomolybdic acid (Η ₃ ΡΜθι ₂ 0 ₄ ο). The hydroxyl groups of the phenols are oxidized and the acids reduced to tungsten and molybdenum. Depending on the amount of phenols present and in an alkaline medium, a blue color appears (VL Singleton and Joseph A. Rossi, J. (1965) 1965. In American Journal of Enology and Viticulture, 16 (pp. 158)).

 The extract solutions (500 μί) were placed in the presence of Folin-Ciocalteu reagent (2.5 ml) and sodium carbonate solution (10 ml). The reaction medium was made up to the mark (50 mL) with distilled water. After 30 minutes of incubation at room temperature, the absorbance of the samples is measured at 760 nm under 10 mm optical path relative to the distilled water (UV-vis spectrophotometer, Genway-6305). A blank is analyzed under the same conditions. The absorbance of the samples is related to a gallic acid standard range. The concentration of total polyphenols of the extracts is expressed in gallic acid equivalent (mg / g of extract). vs. Assays of total tannins:

 The total tannin content of the extracts was determined using the Bate-

Smith (Ribéreau-Gayon, PS, E. (1966), Analytical Chemistry (48), 188-196) which is based on the property of proanthocyanidines to transform into anthocyanins stained in acidic medium, heated to 100 ° C and quantifiable by Electrophotometry at 550 nm. The test extract solution was put in two tubes (2 ml in each) and are brought into contact with 1 ml of distilled water and 3 ml of hydrochloric acid (12N). One of the two tubes, hermetically closed, was placed in a water bath at 100 ° C. for 30 minutes and then cooled in ice for 10 minutes. In each tube, 500 μl of ethanol was added to each of the two tubes. The difference in absorbance was measured at 550 nm with respect to water under 1 cm of optical path. The concentration was calculated as mg of dry matter by the formula:

[Total tannins] = 19.33 / 50 ^χ (OD ₅₅ onm control tube - OD ₅₅ onm hydrolyzed tube) d. Total anthocyanin assays:

Total anthocyanins were determined by the bisulfite decolorization method proposed by Ribéreau Gayon, P. a. S., E. (1965). Bulletin of the Chemical Society of France (9), (2649-2652). Total anthocyanins are found under several forms: anthocyanins in the free state and anthocyanins combined with tannins, one fraction of which is decolorizable by S0 ₂ and the other insensitive. A first solution was prepared by adding 1 ml of extract to be assayed, 1 ml of ethanol acidified with 0.1% hydrochloric acid (12N) and 20 ml with 2% hydrochloric acid (12N).

 In a first test tube, 10 mL of the above solution was added to 4 mL of distilled water (control tube) and then in a second test tube, 10 mL of the first solution was added to 4 mL of bisulfite. 15% sodium (bisulphite tube). Absorbance was measured at 520 nm under an optical path of 1 cm with respect to water after 20 minutes of incubation at room temperature. The concentration was calculated in mg / g dry matter by the following formula:

[Total anthocyanins] = 875 ^χ (OD ₅₅ nm control tube - OD ₅₅ μm bisulfite tube) e. Identification and quantification of proanthocyanidins by HPLC-fluo

The extracts to be assayed were solubilized in a water / methanol solution (50/50 v / v), filtered (0, 45 μΜ) and analyzed according to the method adapted by Silva, MA, Ky, I., Jourdes, M. , & Teissedre, PL (201 1). European Food Research and Technology, 1-5.). The analyzes were performed on a Thermo-Finnigan Surveyor (Thermo, electron Corporation) consisting of a pump module (Surveyor LC pump Plus), a smother (Thermo-Finnigan autosampler) and a UV-Vis detector with diode array (250-700nm) (Surveyor PDA Plus). This system is coupled to a Fluorometer (FL plus Detector). The UV detector is controlled by the XCalibur software and the fluorescence detector by the ChromQuest 4.2 software (Thermo, Electron corporation). The separation was carried out using a Lichrospher C18 reverse phase column (250 mm × 4 mm, 5 μηι) eluted with a flow rate of 1 mL / min and whose injection volume was set at 20 μL · Both solvents are water / formic acid (99/1, v / v) (solvent A) and acetonitrile / formic acid (99/1, v / v) (solvent B). The gradient of the mobile phase is as follows: 8% B at 0 min, 18% B at 21 min and 100% B at 22 min. The column is washed with 100% B for 3 min and rebalanced with 8% B for 5 min before the next injection. Fluorescence detection is set for an excitation value at 280 nm and emission at 320 nm. The identification of the molecules was carried out by comparing their retention times with those of the commercial standards (C, EC, B1, B2, B3, B4 and C1 with C being catechin, EC being Epicatechin, B1 being Procyanidin dimer B1, B2 being the B2 dimer Procyanidin, B3 being the d3 Procyanidin B3, B4 being the D4 procyanidin dimer and C1 the trimer Procyanidin C1). The results are expressed in mg / g of dry matter. f. Identification and quantification of anthocyanins by HPLC-UV:

 Before injection, the extracts to be assayed were solubilized in a water / methanol solution (50/50, v / v) and filtered (0.45 μl). The separation device consists of a Thermo-Accela UPLC system consisting of a UV-Vis detector (Accela PDA detector), a sample changer (Thermo-Accela autosampler) and a pump module (Accela 600 pump). ). The set is driven by Xcalibur software. The analysis was performed using a Kinetex C18 column (100 mm x 2.1 mm, 1. 7 μηι) with an injection volume set at 2 μί and an elution of 200 μΙ_Ληίη. The two solvents are water / formic acid (95/5, v / v) (solvent A) and acetonitrile / formic acid (95/5, v / v) (solvent B). The gradient of the mobile phase is as follows: 7% of B at 0 min, 26% of B at 18 min and 100% of B at 19 min. The column is washed with 100% B for 3 min and rebalanced with 7% B for 5 min before the next injection. UV-visible detection is carried out at 520 nm.

 The identification of the molecules was performed by comparing their retention times with those of commercial standards (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O glucoside). Acetylated and coumaroyl anthocyanins are expressed as malvidin-3-O-glucoside equivalent. The results are expressed in mg / g of dry matter. boy Wut. Evaluation of antioxidant properties

ORAC test:

The ORAC test (Oxygen Radical Absorbance Capacity) was applied according to the method described in Ou, B., Hampsch-Woodill, M., & Prior, RL ((2001) Journal of Agricultural and Food Chemistry, 49 (10), 4619-4626) modified by Devvalos, A., Gomez-Cordovés, C., & Bartolomé, B. ((2004) Journal of Agricultural and Food Chemistry, 52 (1), 48-54). The analysis was performed using a BMG FLUOstar Omega plate reader equipped with a fluorescence detector. The dilution of the extracts and the reaction were carried out with phosphate buffer (75 mM, pH 7.4). The solutions of extracts to be tested (30 μl) are brought into contact with fluorescein (180 μl, 17 nM) and AAPH (90 μl, 40 mM). Fluorescence is recorded for 1 hour at 37 ° C at excitation wavelengths of 485 nm and 530 nm emission. A blank and a standard range of Trolox standard were analyzed under the same conditions. The area under the curve was calculated for each sample and a regression line was established between the area under the curve and the Trolox concentration. The antioxidant power of the samples is expressed in Trolox μΜ equivalent per gram of dry matter. FRAP test:

The Free Radical Scavenging Potential (FRAP) test was adapted according to the procedure of Benzie, IFF, & Strain, JJ ((1996) Analytical Biochemistry, 239 (1), 70-76 (1996)). The procedure is based on the reduction, at low pH, of the colorless ferric complex (Fe ³⁺ - tripyridyltriazine) to ferrous blue complex (Fe ²⁺ -tripyridyltriazine) by the action of electron donor antioxidant. The reduction is measured by a change in absorbance at 593 nm. The FRAP reagent is freshly prepared for each use by mixing 10 volumes of sodium acetate buffer (300mM, pH 3.6), 1 volume of TPTZ (2,4,6-tri (2-pyridyl) -s-triazine) (10mM in 40mM hydrochloric acid) and 1 volume of aqueous solution of iron chloride hexahydrate (20mM). 300 μί of FRAP reagent are added to 40 μl of test extract previously placed on a 96-well plate. After 4 minutes of incubation at 37 ° C, the sample absoibance is measured using a BMG FLUOstar plate reader. A blank is analyzed under the same conditions. The antioxidant power is obtained by the difference between the absorbance of the samples and the white. The results are related to a standard range of iron sulphate (Fe ²⁺ ) and are expressed in equivalent mmol Fe ² 7g of dry matter.

ABTS test:

The antioxidant potency of the extracts was also evaluated using the ABTS assay (Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C. ( 1999) Free Radical Biology and Medicine, 26 (9-10), 1231-1237.1999). By reacting with potassium persulfate (K ₂ S ₂ 0 ₈ ), ABTS (2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid)) forms the green ABTS ^{* +} radical after 12 hours. at 16 incubation in the dark. The addition of antioxidants will reduce this radical and cause discoloration of the mixture which is proportional to the concentration of antioxidants contained in the extract. For assays, the ABTS ^{* +} solution is diluted with distilled water until an absorbance of C = 0.7 ± 0.02 at 734 nm is obtained. 200 μl of ABTS ^{* +} are added to 10 μΐ of sample previously placed on a 96-well plate. After 10 minutes of incubation at room temperature, the absorbance of the samples is measured using the BMG FLUOstar plate reader at 734 nm. A blank is analyzed under the same conditions. The antioxidant power is obtained by the difference between the absorbance of the samples and the white. The results are related to a standard range of Trolox and are expressed in Trolox μηιοΙ equivalent / g of dry matter. DPPH test:

 The DPPH test was applied according to the method of Brand-Williams, W., Cuvelier, ME, & Berset, C. ((1995) LWT - Food Science and Technology, 28 (1), 25-30) modified by Miliauskas , G., Venskutonis, PR, & Van Beek, (TA (2004) Food Chemistry 85 (2), 231-27 & Van Beek, (2004)). In its free and stable form, the DPPH radical has an intense purple color that is discolored when reduced by an antioxidant. 200 μl of DPPH are added to 10 μl of sample previously placed on a 96-well plate. After 20 minutes incubation at room temperature, the absorbance of the samples is measured using the BMG FLUOstar 515 nm plate reader. A blank is analyzed under the same conditions. The antioxidant power is obtained by the difference between the absorbance of the samples and the white. The results are related to a standard range of Trolox and are expressed in Trolox μηιοΙ equivalent / g of dry matter. 2. Results of in vitro tests:

 For all samples, overall analyzes such as total polyphenols, total tannins and total anthocyanins were performed. Extensive HPLC-UV-Fluo analysis yielded the composition of individual tannins and anthocyanins. The antioxidant potency of the extracts was assessed using four different antioxidant tests: ORAC, FRAP, ABTS and DPPH.

2. A. Overall compositions of extracts:

 The aqueous (EAQ) and aqueous-alcoholic extracts 70% (EA70) were characterized overall by analysis of total polyphenols, total tannins and total anthocyanins after being solubilized and diluted to the correct concentration.

 The results show that, overall, extraction with ethanol 70% vol allows in particular a very satisfactory extraction of the phenolic compounds that it is at the level of the seeds or films.

 Among the pips of pomace, the Syrah Plantier and Carignan pips are particularly rich in polyphenols, tannins and anthocyanins, whether in the EAQ or EA70. In the latter, Syrah Plantier seeds are richer in total tannins (up to 455.42 mg / g DM) whereas those in Carignan contain a greater amount of total anthocyanins (57.34 mg / g DM) .

For the films, the statistical analyzes showed that in ASR, Alicante films had the highest levels of phenolic compounds for all three analyzes (total polyphenols: 196.71 mg GAE / g MS, tannins total: 221, 40 mg / g MS and total anthocyanins: 21, 40 mg / g DM) whereas in EA70, Syrah Plantier pellets contain the most (total polyphenols: 224.92 mg GAE / g MS, total tannins: 312.46 mg / g MS and total anthocyanins: 86.68 mg / g MS).

 As previously noted, in EA70, the amounts of total polyphenols, total tannins and total anthocyanins are greater.

 This result illustrates that ethanol at 70% compared to water, allows a better extraction. Ethanol could facilitate the solubilization of tissues and thus the release of a larger amount of polyphenols. Indeed, between the EAQ and EA70 extracts, the total polyphenols and total tannins are respectively 1, 5 to 2.5 and 1 to 3 times higher, depending on the grape varieties in the EA70s. The anthocyanins are the compounds whose extraction varies the most according to the grape varieties: from 2.5 to 25.8 times in Alicante skins (21, 40 mg / g DM in EAQ and 54.41 mg / g DM in EA70) and top Syrah from Julien (1.76 mg / g DM in EAQ and 45.38 mg / g DM in EA70) respectively.

2. B. Composition in individual tannins of the extracts (HPLC-Fluo):

 Monomers of flavan-3-ol (catechin and epicatechin) as well as dimers (B1, B2, B3 and B4) and trimer C1 were identified and quantified in aqueous and hydroalcoholic extracts 70% of seeds and dandruff of grapes by HPLC-Fluo.

 For both types of extracts, Syrah Plantier seeds are the most rich in tannins at the monomer level (8.88 mg / g DM in EAQ and 13.84 mg / g DM in EA70), dimers (6.87 mg / g DM in EAQ and 7.10 mg / g DM in EA70) and trimer C1 (2.00 mg / g DM in EAQ and 1.253 mg / g DM in EA70). In films, the results varied in QARs from 0.89 mg / g DM to 3.4 mg / g DM for the sum of the monomers, from 0.91 mg / g DM to 2.34 mg / g DM for the sum of dimers and in EA70s from 1.88 mg / g MS to 7.71 mg / g DM for sums of monomers and from 1.61 mg / g DM to 4.83 mg / g DM for sum of dimers .

 The varietals containing the most individual tannins are Alicante and Syrah (Syrah Plantier) for both types of extracts.

2. C. Composition in individual anthocyanins extracts (HPLC-UV):

 The anthocyanin profile was established by HPLC-UV. In all varieties and both types of extracts, malvidin-3-O-glucoside appears to be the major anthocyanin.

Among the aqueous extracts, it is the Syrah (Syrah Plantier) seeds that contain the most anthocyanins (3.24 mg / g MS of glycosylated anthocyanins, 2.20 mg / g MS acetylated anthocyanins and 1.86 mg / g MS coumaroylated anthocyanins). Carignan seeds are particularly rich in glycosylated anthocyanins (3.16 mg / g MS in total) and consist mainly of malvidin-3-O-glucosides (1.35 mg / g DM) and delphinidin-3-O- glucoside (0.63 mg / g MS). Their levels of acetylated and coumaroyl anthocyanins are lower than those of Grenache seeds (Grenache Long of the Coudoulet) which consists of 2.03 mg / g MS of acetylated anthocyanins and of 1.37 mg / g MS of coumaroylated anthocyanins .

 For 70% hydroalcoholic extracts, Carignan seeds contain a large amount of glycosylated anthocyanins (14.66 mg / g DM) and coumaroylées (4.15 mg / g DM). The Syrah pips (Syrah Plantier) are particularly rich in acetylated anthocyanins (up to 2.98 mg / g DM).

 For dandruff, in aqueous extracts and among glycosylated anthocyanins, petunidin-3-O-glucoside and paeonidine-3-O-glucoside are the two major anthocyanins after malvidin-3-O-glucoside, whereas in extracts hydroalcoholic 70% are petunidine-3-O-glucoside and delphinidin-3-O-glucoside. The richest extracts are the Syrah (Syrah Plantier), Carignan and Alicante dandruff in the EAQ and Grenache (Grenache Face aux Pins) and Carignan (EA70) films. 2. D. Evaluation of the antioxidant activity:

 The antioxidant potency of the extracts was assessed through four different tests. Three spectrophotometric tests: FRAP, ABTS and DPPH and a spectrofluorometric test: ORAC.

 For glitches, whether at the level of the QARs and EA70s, the results of the various antioxidant tests are correlated. On the one hand, the strongest antioxidant powers were found in Syrah seed extracts (Syrah Plantier). The latter has, in fact, the greatest amount of tannins and anthocyanins according to the global analyzes and the characterization by HPLC-UV-Fluo.

The results obtained for dandruff are more heterogeneous than those of pips, especially at the level of EA70 extracts. In the aqueous extracts, the highest grape varieties with antioxidant capacity are Syrah (Syrah Plantier) and Alicante. This finding was confirmed by the four antioxidant tests. In addition, there is a correlation between the results obtained previously which showed that the Syrah (Syrah Plantier) and Alicante films contain the largest quantities of tannins and anthocyanins. Concerning the EA70 extracts, the Syrah (Syrah Plantier) films can be retained as having an important antioxidant potential among the others. Syrah (Syrah Plantier) is classified as being either the first or the second grape variety with the most activity according to the tests (ORAC: 1912.56 μηιοΙ TE / g MS, FRAP: 1, 52 mmol Fe ² 7g MS, ABTS: 2614.50 μπιοΙ TE / g MS, DPPH: 1391, 69 μπιοΙ TE / g MS).

 In aqueous extracts, the ORAC, FRAP, ABTS and DPPH tests give more comparable results than in the 70% hydroalcoholic extracts where the heterogeneity is greater, especially in the films. This could be due to an extraction of different compounds and richer by 70% ethanol.

 As described above, the extracts contain different phenolic compounds present at different concentrations according to the grape varieties, the parts (seeds or films) and the extraction processes (water or ethanol 70%). With different operating principles and different sensitivities of the tests used, the heterogeneity of the results seems normal.

3. Conclusions of in vitro experiments:

 In this in vitro study, the aqueous and hydroalcoholic extracts 70% of pips and dandruff of six typical grape varieties characteristic of the Rhône Valley were characterized.

 Concentrations of total polyphenols, total anthocyanins and total tannins were evaluated as well as identification and quantification in individual tannins and anthocyanins. The antioxidant powers of the extracts were subsequently appreciated.

 The results showed that 70% ethanol generally allows better extraction of phenolic compounds. Among the aqueous extracts, the Syrah (Syrah Plantier) and Carignan seeds and the Alicante films have high levels of polyphenols. These results are correlated with the evaluation of their antioxidant power.

 Among hydroalcoholic extracts, seed and seed extracts of Syrah Plantier and Carignan have important antioxidant properties.

EXAMPLE 3 Complementary Analysis of the Procyanidin Compositions of the Extracts of Grape Marks

 1 Methods:

The extracts of dandruff and pips of marcs according to Example 1 were solubilized in a water / methanol solution (50/50, v / v) acidified with 1% of formic acid. The Analyzes were carried out on a Thermo-Finnigan Surveyor (Thermo, Electron Corporation) consisting of a pump module (Surveyor LC pump Plus), a 4 ° C regulated flow regulator (Thermo-Finnigan autosampler), a UV-Vis diode array detector (200-600nm) (Surveyor PDA Plus) and a fluorescence detector (FP-920m Jasco (UK) Ltd.). The fluorescence detection was set at an excitation and emission wavelength of 230 and 320 nm respectively. After passing through the measuring cell of the diode array detector, the eluate is separated and 0.2 mL is sent to a Finnigan LCQ Duo mass ion trap mass spectrometer equipped with an electrospray ionization interface. The analyzes are carried out in negative ionization with a mode of scanning of all the masses between m / z 200 and 1000. The set is controlled by the software Xcalibur. The separation is performed on a column Develosil diol 100A (250 x 4.6 mm id 5 μηι) (Phenomenex, Cheshire, UK) maintained at 35 ^Q C with an injection volume set at 10 \ xL for extracts of seeds and 20 \ xL for film extracts and elution of 1 mL / min. The two solvents are acidified acetonitrile ((A), CH ₃ CN: HOAc, 98: 2, v / v) and methanol / water / acetic acid ((B), CH ₃ OH: H ₂ O: HOAc, 95 : 3: 2; v / v / v). The mobile phase consists of a gradient of 7% B for 3 min and then 7% to 37.6% in 57 min. The column is washed with 100% B for 7 min before being reconditioned at 7% B for 6 min. The column is rebalanced with 7% B for 10 min. The detection of the molecules is carried out thanks to the fluorescence and confirmed by mass spectrometer. The desired ions are described in Table 1. Two different gains in fluorescence were used: on the one hand for the quantification of the monomers and dimers and on the other hand for the quantification of the molecules at Dpm 2 (Dpm greater than 2). The concentrations are expressed in (-) - epicatechin equivalent and expressed in mg / g dry matter (DM).

2. Results

 Table 1: Characteristics of Procyanidins Detected in Seed Extracts and Pellets

 Peaks Rt (min) Procyanidins [M] (m / z)

1 4.92 Monomers (epi) C 289

 2 6.98 (Epi) Catechin gallate (epi) Cg 441

 3 8.67 Dimers (epi) C- (epi) C 577

 13.26 Gallate (epi) dimers C- (epi) Cg 729

15.5 Trimers (epi) C- (epi) C- (epi) C 865 6 17.25 Gallo (epi) Catechin (epi) C- (epi) - (epi) GC 881 trimers

 7 20.69 Gallate epi (epi) C- (epi) C- (epi) Cg 1017

22.59 Tetramers (epi) C- (epi) C- (epi) C-1 153

 (epic

 9 23.52 Gallo (epi) Catechin (epi) C- (epi) C- (epi) - 1,169 tetramers (epi) GC

 10 27.08 Tetramers gallate (epi) C- (epi) C- (epi) C-1305

 (Epi) -C

 11 29.28 Pentamers (epi) C- (epi) C- (epi) C-1441

 (Epi) C- (epi) C

 12 30.23 Gallo (epi) Catechin (epi) C- (epi) C- (epi) C- (epi) - 1457 pentamers (epi) GC

 13 34.79 Hexamers (epi) C- (epi) C- (epi) C-1729

 (Epi) C- (epi) C- (epi) C

 14 39.74 Heptamers (epi) C- (epi) C- (epi) C- 2017

 (Epi) C- (epi) C- (epi) - (epi) C

 43.78 Octamers (epi) C- (epi) C- (epi) C-2305

 (epi) C- (epi) C- (epi) C- (epi) C- (epi) C

 47.16 Nonamers (epi) C- (epi) C- (epi) C-2593

 (epi) C- (epi) C- (epi) C- (epi) C- (epi) C- (epi) C

 17 50.21 Decamerons (epi) C- (epi) C- (epi) C-2881

 (epi) C- (epi) C- (epi) C- (epi) C- (epi) C- (epi) - (epi) C

Table 2: Procyanidin composition in aqueous extracts (QAR) of pips of marc

Procyanidin Pics GLC SyP CAR

 1 Monomers 37.74 ± 0.06 76.30 ± 0.39 45.42 ± 0.93

 2 (Epi) Catechin gallate 0.70 ± 0.00 0.77 ± 0.01 0.66 ± 0.01

 3 Dimer 1 1 .65 ± 0.13 21 .52 ± 0.16 15.82 ± 0.22

4 Gallate dimers 0.22 ± 0.00 1 .20 ± 0.01 0.40 ± 0.02 5 Trimers 3.81 ± 0.04 17.76 ± 0.21 4.94 ± 0.07

 6 Gallo (epi) Catechin 0.25 ± 0.01 1.27 ± 0.03 0.21 ± 0.04

 trimers

 7 gallate trimer 0.27 ± 0.02 0.68 ± 0.02 0.18 ± 0.01

 8 Tetramers 1.56 ± 0.06 7.05 ± 0.05 1.89 ± 0.00

 9 Gallo (epi) Catechine 0.91 ± 0.04 2.66 ± 0.07 0.92 ± 0.01

 tetramers

 10 Tetramers gallate 0.01 ± 0.00 0.15 ± 0.00 0.04 ± 0.00

 11 Pentamers 0.79 ± 0.01 3.95 ± 0.06 1.29 ± 0.02

 12 Gallo (epi) Catechine 0.12 ± 0.01 0.61 ± 0.01 0.26 ± 0.00

 pentamers

 13 Hexamers 0.55 ± 0.02 1.64 ± 0.06 0.67 ± 0.02

 14 Heptamers 0.22 ± 0.01 1.01 ± 0.01 0.39 ± 0.02

 15 Octamers 0.10 ± 0.00 0.43 ± 0.01 0.16 ± 0.01

 16 Nonamers 0.02 ± 0.00 0.17 ± 0.01 0.07 ± 0.01

 17 Decamers n / a 0.07 ± 0.00 0.02 ± 0.00

Table 3: Composition of procyanidins in the hydroalcoholic extracts 70% (EA70) of pips of marc

Procyanidin Pics GLC SyP CAR

 1 Monomers 32.59 ± 0.6 72.11 ± 0.93 29.72 ± 0.35

 2 (Epi) Catechine 1.01 ± 0.02 1.34 ± 0.05 0.79 ± 0.02

 gallate

 3 Dimer 10.63 ± 0.17 28.15 ± 0.11 9.22 ± 0.15

 4 Gallate dimers 0.33 ± 0.01 1.94 ± 0.00 0.34 ± 0.00

 5 Trimers 47ΐΊΓ ± 023 20.74 ± 0.03 3.49 ± 0.04

 6 Gallo (epi) Catechine 0.63 ± 0.01 2.18 ± 0.02 0.58 ± 0.01

 trimers

7 gallate trimeres ^" 0.30 ± 0.00 1.42 ± 0.01 0.28 ± 0.00

 8 Tetramers 1.98 ± 0.06 9.46 ± 0.19 1.69 ± 0.04

 9 Gallo (epi) Catechine 0.97 ± 0.13 4.5 ± 0.04 0.8 ± 0.01

 tetramers

 10 tetramers gallate 0.04 ± 0.01 0.82 ± 0.05 0.06 ± 0.00

11 Pentamers 1.30 ± 0.01 6.27 ± 0.15 1.25 ± 0.00 12 Gallo (epi) Catechine 0.32 ± 0.01 1.37 ± 0.05 0.2 ± 0.00

 pentamers

 13 Hexamers 0.92 ± 0.03 3.43 ± 0.05 0.83 ± 0.02

 14 Heptamers 0.51 ± 0.00 1.82 ± 0.01 0.51 ± 0.00

 15 Octamers 0.28 ± 0.00 0.90 ± 0.00 0.24 ± 0.01

 16 Nonamers 0.13 ± 0.01 0.43 ± 0.00 0.15 ± 0.01

 17 Decamers 0.09 ± 0.00 0.24 ± 0.01 0.07 ± 0.00

Table 4: Procyanidin Composition in Aqueous Extracts (EAQ) of Dandruff

Procyanidin Picks GFP SyP SyH CAR MOU ALI

1 Monomers 13.01 ± 26.3 ± 17.44 ± 14.2 ± 10.19 ± 12.2 ±

 0.34 0.05 0.00 0.03 0.15 0.00

2 (Epi) Catechin 0.20 ± 0.37 ± 0.28 ± 0.31 ± 0.17 ± 0.24 ± gallate 0.01 0.01 0.02 0.02 0.00 0.01

3 Dimer 4.17 ± 7.96 ± 5.32 ± 5.29 ± 3.92 ± 3.73 ±

 0.1 0.06 0.05 0.02 0.06 0.07

4 Gallate dimers 0.37 ± 0.83 ± 0.35 ± 0.45 ± 0.37 ± 2.25 ±

 0.01 0.03 0.02 0.01 0.01 0.01

5 Trimers 5.48 ± 11.24 ± 6.13 ± 6.68 ± 5.64 ± 19.44 ±

 0.00 0.29 0.15 0.11 0.09 0.11

6 Gallo 0.82 ± 1.45 ± 0.90 ± 1.25 ± 0.48 ± 2.15 ±

(epi) Catechine 0.01 0.01 0.02 0.04 0.01 0.01 trimers

 7 gallate trimer 0.23 ± 0.35 ± 0.17 ± 0.20 ± 0.16 ± 0.94 ±

 0.01 0.07 0.00 0.03 0.01 0.00

8 Tetramers 1.94 ± 4.19 ± 2.14 ± 2.50 ± 2.33 ± 7.13 ±

 0.06 0.00 0.01 0.04 0.02 0.01

9 Gallo 0.89 ± 1.17 ± 0.70 ± 1.06 ± 0.73 ± 2.64 ±

(epi) Catechine 0.02 0.00 0.02 0.04 0.00 0.09 tetramers

10 Tetramers ¹ n / a N / A N / A N / A N / A N / A Gallate

 11 Pentamers 1.03 ± 2.41 ± 1.05 ± 1.45 ± 1.32 ± 3.42 ±

0.00 0.08 0.02 0.02 0.01 0.01 12 Gallo 0.69 ± 0.66 ± 0.66 ± 1 .04 ± 0.66 ± 1 .26 ±

(epi) Catechine 0.00 0.01 0.03 0.00 0.02 0.01 pentamers

 13 Hexamers 0.80 ± 1 .68 ± 0.58 ± 0.70 ± 0.92 ± 1 .90 ±

 0.00 0.00 0.02 0.00 0.01 0.01

14 Heptamers 0.36 ± 0.86 ± 0.27 ± 0.34 ± 0.39 ± 0.92 ±

 0.01 0.01 0.01 0.01 0.03 0.01

15 Octamers 0.08 ± 0.30 ± nd 0.15 ± 0.07 ± 0.29 ±

 0.00 0.02 0.01 0.01 0.03

16 Notamers nd 0.1 ± na na na 0.10 ±

 0.01 0.00

17 Decameres ¹ n / a N / A N / A N / A N / A 0.04 ±

 0.00

Table 5: Composition in procyanidines in hydroalcoholic extracts 70%

(EA70) of dandruff

Procyanidin Picks GFP SyP SyH CAR MOU ALI

1 Monomers 9.89 ± 19.28 ± 9.62 ± 12.58 ± 3.80 ± 12.71 ±

 0.08 0.28 0.05 0.05 0.07 0.03

2 (Epi) Catechin 0.28 ± 0.47 ± 0.64 ± 0.49 ± 0.02 ± 0.44 ± gallate 0.00 0.01 0.02 0.03 0.00 0.01

3 Dimers 4.05 ± 6.76 ± 2.70 ± 4.65 ± 1 .9 ± 4.51 ±

 0.03 0.09 0.02 0.02 0.05 0.08

4 Gallate dimers 0.40 ± 0.89 ± 0.49 ± 0.61 ± 0.17 ± 0.44 ±

 0.01 0.00 0.00 0.00 0.00 0.01

5 Trimers 6.31 ± 10.51 ± 4.35 ± 7.91 ± 2.34 ± 4.52 ±

 0.08 0.01 0.06 0.14 0.01 0.06

6 Gallo 0.62 ± 0.78 ± 0.49 ± 0.35 ± 0.07 ± 0.08 ±

(epi) Catechine 0.00 0.01 0.04 0.01 0.00 0.00 trimers

 7 gallate trimer 0.27 ± 0.47 ± 0.23 ± 0.38 ± 0.06 ± 0.31 ±

 0.00 0.01 0.01 0.01 0.00 0.00

8 Tetramers 2.79 ± 4.79 ± 1 .94 ± 3.58 ± 1 .14 ± 1 .97 ±

0.01 0.01 0.01 0.04 0.00 0.01 9 Gallo 1 .03 ± 1 .16 ± 0.45 ± 1 .30 ± 0.33 ± 0.67 ±

(epi) Catechine 0.00 0.01 0.01 0.03 0.02 0.03 tetramers

 10 Tetramers n / a 0.1 1 ± 0.09 ± 0.40 ± nd 0.05 ± gallate 0.01 0.01 0.01 0.00

11 Pentamers 1 .87 ± 2.78 ± 1 .10 ± 2.42 ± 0.84 ± 1 .22 ±

 0.00 0.00 0.02 0.01 0.01 0.00

12 Gallo 0.56 ± 0.36 ± 0.37 ± 0.80 ± 0.18 ± 0.16 ±

(epi) Catechine 0.00 0.00 0.01 0.04 0.00 0.00 pentamers

 13 Hexamers 1 .16 ± 1 .61 ± 1 .02 ± 1 .74 ± 0.47 ± 0.69 ±

 0.01 0.00 0.02 0.01 0.00 0.01

14 Heptamers 0.87 ± 0.93 ± 0.32 ± 1 .00 ± 0.30 ± 0.28 ±

 0.00 0.01 0.01 0.07 0.01 0.00

15 Octamers 0.18 ± 0.44 ± 0.19 ± 0.59 ± 0.16 ± 0.17 ±

 0.00 0.01 0.00 0.00 0.00 0.00

16 Nonamers 0.16 ± 0.20 ± 0.26 ± 0.07 ± 0.08 ±

 0.00 0.01 0.01 0.00 0.00

17 Decamers 0.03 ± 0.1 1 ± nd 0.03 ± 0.04 ± 0.03 ±

 0.00 0.00 0.00 0.00

EXAMPLE 4 Complementary Analysis of the Anthocyanin Compositions of the Marks Extracts

1. Methods :

The extracts of dandruff and pips of marcs according to Example 1 were solubilized in a water / methanol solution (50/50, v / v) acidified with 1% of formic acid. The analyzes were carried out on a Thermo-Finnigan Surveyor (Thermo, Electron Corporation) consisting of a pump module (Surveyor LC pump Plus), a 4 ° C controlled flow regulator (Thermo-Finnigan autosampler) and a UV-Vis detector with diode array (200-600nm) (Surveyor PDA Plus). The set is driven by Xcalibur software. The separation is carried out using a RP-column column Max (4 μηι, 250 ^χ 4.6 mm) maintained at 40 ^Q C with an injection volume set at 10 μ \ - and elution of 1 ml / min. The two solvents are water / formic acid (99/1, v / v) (solvent A) and methanol / formic acid (99/1, v / v) (solvent B). The mobile phase consists of a gradient ranging from 10% to 45% of B in 65 minutes. After passing through the measuring cell of the diode array detector, the eluate is separated and 0.2 ml are sent to an LCQ Deca XP ion trap mass spectrometer equipped with an electrospray ionization interface. The analyzes are carried out in positive ionization with a mode of scanning of all the masses between m / z 100 and 1000. The desired ions are described in table 6. The detection of the molecules is carried out at 520 nm and by comparison of their retention time with those of commercial standards. The concentrations are expressed as malvidine-3-O-glucoside equivalent and expressed as mg / g dry matter (DM).

Table 6: Characteristics of the compounds identified in the seed extracts and dandruff

Rt MS2

(min) Compounds λ max MW [m / z] + (m / z)

20.46 Delphinidin-3-O-glucoside 520 464 465 303

24.33 Cyanidin-3-O-glucoside 520 448 449 287

27.58 Petunidine-3-O-glucoside 520 478 479 317

31 .09 Peonidine-3-O-glucoside 520 462 463 301

33.32 Malvidine-3-O-glucoside 520 492 493 331

 Malvidin-3-O-glucoside-

38.75 520 516 517 355, 331 acetaldehyde (vitisin B)

 Delphinidin-3-0- (6 "-0-

39.97 520 506 507 303 acetyl) glucoside

 44.47 Dimer Mv-Cat 520 808 809

 Malvidin-3-O-glucoside-

46.01 520 560 561 339, 331 pyruvate (vitisin A)

 46.63 Dimer Mv-Cat 520 808 809

 49.71 Dimer Mv-Cat 520 808 809

 Peonidin-3-0- (6 "-0-acetyl)

 50.3 520 504 505 301 glucoside

 Malvidin-3-0- (6 "-0-acetyl)

 51.01 520 534 535 331 glucoside

 Delphinidin-3-0- (6 "-0-

54.31 520 610 61 1 303 coumaroyl) glucoside Malvidin-3-0- (6 "-0-caffeoyl)

 55.75 520 654 655 331 glucoside

 Cyanidin-3-0- (6 "-0-

57.89 520 594 595 287 coumaroyl) glucoside

 Petunidin-3-0- (6 "-0-

59.34 520 624 625 317 coumaroyl) glucoside

 Malvidine-3-0- (6 "-0-

62.77 520 638 639 331 coumaroyl) glucoside

Table 7: Composition in anthocyanins in aqueous extracts (EAQ) of marc seeds

Compounds (mg eq malvidin / g MS) GLC SyP CAR

Delphinidin-3-O-glucoside 0.03 ± 0 0.08 ± 0 0.4 ± 0

Cyanidin-3-O-glucoside 0.02 ± 0 0.02 ± 0 0.05 ± 0

Petunidine-3-O-glucoside 0.05 ± 0 0.14 ± 0.43 ± 0

Péonidine-3-O-glucoside 0.11 ± 0 0.12 ± 0 0.15 ± 0.01

Malvidin-3-O-glucoside 0.39 ± 0 0.93 ± 0 1.48 ± 0.02

Malvidin-3-O-glucoside-acetaldehyde (vitisin B) nd 0.02 ± 0 0.02 ± 0

Delphinidin-3-0- (6 "-0-acetyl)

 glucoside nd 0.02 ± 0 nd

Dimer Mv-Cat 0.01 ± 0 0.03 ± 0 nd

Malvidin-3-O-glucoside-pyruvate

 (vitisin A) 0.02 ± 0 0.04 ± 0 0.04 ± 0

Dimer Mv-Cat 0 ± 0 0.07 ± 0 0.05 ± 0

Mv-Cat Dimer 0.01 ± 0 0.03 ± 0 0.02 ± 0

Peonidine-3-0- (6 "-O-acetyl) glucoside 0.01 ± 0 0.07 ± 0 nd

Malvidine-3-0- (6 "-0-acetyl) glucoside 0.02 ± 0 0.32 ± 0 0.09 ± 0

Delphinidin-3-0- (6 "-0-coumaroyl)

 glucoside 0 ± 0 0.06 ± 0 0.1 ± 0

Malvidin-3-0- (6 "-0-caffeoyl)

 glucoside 0.01 ± 0 0.04 ± 0 0.02 ± 0

Cyanidin-3-0- (6 "-0-coumaroyl)

glucoside 0.01 ± 0 0.02 ± 0 0.02 ± 0 Petunidin-3-0- (6 "-0-coumaroyl)

glucoside 0.01 ± 0 0.11 ± 0 0.11 ± 0

Malvidin-3-0- (6 "-0-coumaroyl)

glucoside 0.06 ± 0 1.14 ± 0.01 0.65 ± 0

Total glycosylated anthocyanins 0.6 ± 0 1.3 ± 0 2.52 ± 0.03

Total acetylated anthocyanins 0.03 ± 0 0.41 ± 0 0.09 ± 0

Total coumaroyl anthocyanins 0.08 ± 0 1.32 ± 0.01 0.88 ± 0

Total Anthocyanin 0.76 ± 0 3.26 ± 0.01 3.63 ± 0.03

Table 8: Composition in anthocyanins in hydroalcoholic extracts 70% (EA70) of pips of marc

Compounds (mg eq malvidin / g MS) GLC SyP CAR

Delphinidine-3-O-glucoside 0.19 ± 0 0.31 ± 0 3.11 ± 0.02

Cyanidin-3-O-glucoside 0.09 ± 0 0.05 ± 0.23 ± 0

Petunidine-3-O-glucoside 0.33 ± 0.01 0.58 ± 0.02 3.18 ± 0.01

Péonidine-3-O-glucoside 0.56 ± 0.03 0.47 ± 0.02 1.11 ± 0

Malvidin-3-O-glucoside 2.36 ± 0.12 3.57 ± 0.08 10.52 ± 0.11

Malvidin-3-O-glucoside-acetaldehyde

(vitisin B) 0.01 ± 0 0.05 ± 0 0.03 ± 0

Delphinidin-3-0- (6 "-0-acetyl)

glucoside nd 0.09 ± 0 0.06 ± 0

Mv-Cat Dimer 0.04 ± 0 0.05 ± 0 0.06 ± 0

Malvidin-3-O-glucoside-pyruvate

(vitisin A) 0.12 ± 0 0.07 ± 0 0.14 ± 0

Dimer Mv-Cat nd 0.13 ± 0 0.17 ± 0

Dimer Mv-Cat 0.1 ± 0 0.13 ± 0 0.16 ± 0

Peonidine-3-0- (6 "-O-acetyl) glucoside 0.07 ± 0.32 ± 0.01 0.12 ± 0

Malvidine-3-0- (6 "-0-acetyl) glucoside 0.06 ± 0 0.9 ± 0.03 0.31 ± 0

Delphinidin-3-0- (6 "-0-coumaroyl)

glucoside 0.05 ± 0 0.18 ± 0 0.58 ± 0.01

Malvidine-3-0- (6 "-0-caffeoyl) glucoside 0.08 ± 0 0.11 ± 0.33 ± 0

Cyanidin-3-0- (6 "-0-coumaroyl)

glucoside 0.07 ± 0 0.07 ± 0 0.11 ± 0 Petunidin-3-0- (6 "-0-coumaroyl)

glucoside 0.08 ± 0 0.35 ± 0.01 0.66 ± 0

Malvidin-3-0- (6 "-0-coumaroyl)

glucoside 0.8 ± 0 2.85 ± 0 4.51 ± 0.04

Total glycosylated anthocyanins 3.53 ± 0.16 4.99 ± 0.12 18.15 ± 0.09

Total acetylated anthocyanins 0.13 ± 0 1.3 ± 0.02 0.49 ± 0

Total coumaroyl anthocyanins 1 ± 0 3.44 ± 0.01 5.86 ± 0.02

Total Anthocyanin 5 ± 0.17 10.28 ± 0.14 25.38 ± 0.11

Table 9: Composition in Anthocyanins in Aqueous Extracts (EAQ) of Dandruff

Compounds (mg eq

 malvidin / g MS) GFP SyR SyH CAR MOU ALI

Delphinidin-3-O-CL24 + 0.01 ± 0.53 ±

 glucoside 0.01 0.15 ± 0 0 0 0.2 ± 0 0.31 ± 0

 0.05 ±

 Cyanidin-3-O-glucoside 0.09 ± 0 0.01 ± 0 0 ± 0 0 0.07 ± 0 0.11 ± 0

 0.33 ± 0.25 ± 0.03 ± 0.54 ± 0.47 ±

Petunidine-3-O-glucoside 0.01 0.01 0 0 0.3 ± 0 0.02

 0.35 ± 02 + 0.02 ± 0.19 ± 1.49 ±

Peonidin-3-O-glucoside 0.01 0.01 0 0.22 ± 0.03

1.78 ± 1.54 ± 0.11 ± 1.74 ± ^'Γδί ± 3.14 ±

Malvidine-3-O-glucoside 0.05 0.05 0 0.02 0.01 0.03

Malvidin-3-O-glucoside-0.02 ±

 acetaldehyde (vitisin B) 0.01 ± 0 0.02 ± 0 nd 0 0.01 ± 0 nd

Delphinidine-3-0- (6 "-0- 0.02 ±

 acetyl) glucoside na 0.05 ± 0 na 0 na na

Dimer Mv-Cat 0.01 ± 0 0.03 ± 0 n / a n / a 0.01 ± 0 0.03 ± 0

Malvidin-3-O-glucoside-0.02 ± 0.04 ±

 pyruvate (vitisin A) 0.05 ± 0 0.04 ± 0 0 0 0.06 ± 0 0.1 ± 0

 0.05 ±

 Mv-Cat dimer nd 0.08 ± 0 0 ± 0 0 0.01 ± 0 0.05 ± 0

 0.01 ± 0.01 ±

Mv-Cat Dimer 0.01 ± 0 0.03 ± 0 0 0 0.01 ± 0 0.03 ± 0 Péonidine-3-0- (6 "-0- 0.01 ± 0.02 ±

 acetyl) glucoside 0.02 ± 0 0.1 ± 0.0 0 0.01 ± 0 0.09 ± 0

Malvidine-3-0- (6 "-0- 0.02 ± 0.07 ±

 acetyl) glucoside nd 0.52 ± 0 0 0 0.04 ± 0 0.19 ± 0

Delphinidine-3-0- (6 "-0- 0.13 ±

 coumaroyl) glucoside nd 0.09 ± 0 n / a 0 0.02 ± 0 0.06 ± 0

Malvidine-3-0- (6 "-0- 0.03 ±

 caffeoyl) glucoside 0.02 ± 0 0.05 ± 0 nd 0 0.02 ± 0 0.05 ± 0

Cyanidine-3-0- (6 "-0- 0.03 ±

 coumaroyl) glucoside 0.01 ± 0 0.02 ± 0 nd 0 0.03 ± 0 0.03 ± 0

Petunidine-3-0- (6 "-0- 0.12 ±

 coumaroyl) glucoside 0.04 ± 0 0.16 ± 0 nd 0 0.03 ± 0 0.07 ± 0

Malvidin-3-0- (6 "-0- 0.01 ± 0.68 ± 1.11 ± coumaroyl) glucoside 0.22 ± 0 1.59 ± 0 0 0.01 0.15 ± 0 0.01

Anthocyanins 2.8 ± 2.15 ± 0.17 ± 3.05 ± 1.8 ± 5.52 ± total glycosylated 0.07 0.06 0 0.02 0.01 0.08

Anthocyanins 0.03 ± 0.11 ±

 total acetylated 0.02 ± 0 0.67 ± 0 0 0 0.05 ± 0 0.27 ± 0

Anthocyanins 0.01 ± 0.96 ± 1.27 ± total coumaroyl 0.27 ± 0 1.86 ± 0 0 0.01 0.24 ± 0 0.01

 3.19 ± 4.92 ± 0.24 ± 4.25 ± 2.2 ± 7.32 ±

Total Anthocyanin 0.08 0.06 0.03 0.01 0.08

Table 10: Anthocyanin Compositions in 70% Alcoholic Acid Extracts (EA70) from Dandruff

Compounds (mg eq

 malvidin / g MS) GFP SyR SyH CAR MOU ALI

Delphinidin-3-O- 1.43 ± 0.78 ± 0.97 ± 5.35 ± 2.35 ± 1.06 ± glucoside 0 0 0 0.21 0 0.01

 0.34 ± 0.05 ± 0.12 ± 0.39 ± 0.52 ± 0.24 ±

Cyanidin-3-O-glucoside 0.02 0 0 0.01 0 0

 2.05 ± 1.29 ± 1.53 ± 5.04 ± 3.38 ± 1.65 ±

Petunidine-3-O-glucoside 0.08 0 0.21 0.02 0.03 1 .91 ± 0.87 ± 0.94 ± 1 .71 ± 2 ± 5.32 ±

Péonidine-3-O-glucoside 0.02 0.01 0.02 0.03 0.01 0.01

 10.96 ± 7.59 ± 6.76 ± 14.82 ± 10.55 ± 1 1 .18 +

Malvidine-3-O-glucoside 0.22 0.04 0.09 0.42 0.03 0.05

Malvidin-3-O-glucoside-0.03 ± 0.06 ± 0.07 ± 0.05 ± 0.04 ± 0.03 ± acetaldehyde (vitisin B) 0 0 0 0 0 0

Delphinidin-3-0- (6 "-0- 0.04 ± 0.16 ± 0.13 ± 0.09 ± 0.05 ± 0.05 ± acetyl) glucoside 0 0 0 0 0 0

 0.05 ± 0.07 ± 0.1 1 ± 0.06 ±

Dimer Mv-Cat 0 0 0 nd 0.1 ± 0 0

Malvidin-3-O-glucoside-0.22 ± 0.17 ± 0.39 ± 0.18 ± 0.32 ± pyruvate (vitisin A) 0.01 0.01 0 0.01 0 0.2 ± 0

 0.12 ± 0.27 ± 0.21 ± 0.16 ± 0.07 ± 0.14 ±

Dimer Mv-Cat 0 0.01 0 0 0 0

 0.1 ± 0.21 ± 0.08 ± 0.23 ±

Dimer Mv-Cat 0.1 ± 0 0 0.01 0.1 ± 0 0 0

Peonidine-3-0- (6 "-0- 0.17 ± 0.52 ± 0.39 ± 0.19 ± 0.52 ± acetyl) glucoside 0 0.01 0.01 0.1 ± 0 0 0.14

Malvidine-3-0- (6 "-0- 0.46 ± 2.1 1 ± 1 .06 ± 0.41 ± 0.39 ± 0.61 ± acetyl) glucoside 0.01 0.05 0 0.02 0 0

Delphinidin-3-0- (6 "-0- 0.25 ± 0.12 ± 0.35 ± 0.75 ± 0.27 ± coumaroyl) glucoside 0.01 0 0.01 0 0.3 ± 0 0

Malvidine-3-0- (6 "-0- 0.34 ± 0.15 ± 0.56 ± 0.24 ± 0.32 ± 0.25 ± caffeoyl) glucoside 0.01 0 0 0 0 0

Cyanidin-3-0- (6 "-0- 0.13 ± 0.05 ± 0.17 ± 0.13 ± 0.52 ± 0.14 ± coumaroyl) glucoside 0 0 0.01 0 0 0

Petunidine-3-0- (6 "-0- 0.36 ± 0.51 ± 0.82 ± 0.65 ± 0.55 ± coumaroyl) glucoside 0 0.2 ± 0 0.01 0.01 0 0

Malvidine-3-0- (6 "-0- 3.09 ± 1 .53 ± 4.12 ± 3.8 ± 3.24 ± 6.25 ± coumaroyl) glucoside 0.02 0 0.03 0 0.04

Anthocyanins 16.68 ± 10.59 ± 10.33 ± 27.3 ± 18.79 ± 19.45 ± total glycosylated 0.34 0.05 0.1 0.88 0.02 0.08

Acetylated anthocyanins 0.67 ± 2.79 ± 1.59 ± 0.6 ± 0.62 ± 1.18 ± total 0.01 0.04 0.01 0.02 0 0.14 Anthocyanins 3.82 ± 1.89 ± 5.14 ± 5.494 ± 4.71 ± 7.21 ± total coumaroylated 0.01 0 0.01 0.035 0 0.04

 22.03 ± 16.1 ± 18.6 ± 34.11 ± 25.06 ± 28.74 ±

Total Anthocyanin 0.35 0.1 0.11 0.95 0.01 0.01

These data show that epicatechin monomers and total glycosylated anthocyanins, more particularly malvidine-3-O-glucoside, are predominant in the extracts according to the invention. They also confirm that alcoholic extracts allow a greater extraction of polyphenols compared to aqueous extracts.

EXAMPLE 5 Effect of the combination according to the invention on rats

 The following in vivo experiments were performed on a genetically hypertensive rat animal model commonly referred to as SHR for Spontaneaously Hypertensive Rats. Among the models of hypertensive rats, SHR is the model of choice for the screening of antihypertensive agents (Wagner, A. et al., 201 1) Journal of Hypertension, 29 (1), 43-50 and Yang, N.C. et al (2012), Food Chemistry, 132 (4), 1796-1801) and is currently the most widely used animal for studying hypertension. Their normotensive controls are Wistar-Kyoto rats (WKY).

The polyphenol extracts of marc chosen to carry out this in vivo study are hydro-alcoholic extracts (EA70) as defined in Example 1.

1. Materials and methods:

 1. A. Animals and treatments:

 The SHR rats and their normotensive WKY control come from the January laboratory (Genest St. Isie, France). The animals were kept at a temperature of 23 ° C, with a cycle of 12 hours of light / 12 hours of darkness. Water and food were administered ad libitum.

 The polyphenol extracts were administered at a dose of 21 mg / kg of polyphenols per day which is equivalent to a consumption of polyphenols contained in 500ml of red wine by a 70kg adult. The extracts are solubilized in 3% ethanol and then administered by gavage.

Three experimental groups have been established Study 1: Screening for antihypertensive effects of 6 polyphenolic extracts in SHR rats

 Male SHR strain rats and their 9-week normotensive WKY controls were distributed as follows:

a group of six control rats (WKY);

 a group of five SHR rats treated with 3% ethanol (SHR control);

 -Six groups of four SHRs treated with hydroalcoholic polyphenolic EA extracts

70% following:

^* n ° 1: Grenache seeds Long Coudoulet (SHR 1);

 * n ° 2: Syrah Plantier seeds HR 2);

^* n ° 3: dandruff of Syrah Haut-Julien HR 3);

^* No. 4: Carignan seeds (SHR 4);

^* n ° 5: Mourvèdre films 3HR 5);

^* n ° 6: films of Alicante HR 6).

 Study 1 was conducted over six weeks with three weeks of treatment, one week off, and two weeks of treatment.

Study 2: Evolution of blood pressure in SHR rats treated with verapamil

Three male Wistar Kyoto (WKY) rats and three SHRs, nine weeks old, were used. Arterial blood pressure monitoring was performed for two weeks and then SHR rats were treated successively with three doses of verapamil: 30 mg / kg / day, 20 mg / kg / day and 40 mg / kg / day for ten weeks.

Study 3: Effect of polyphenolic extracts in combination with verapamil

 This study itself took place in three stages:

 • Development of the model with a polyphenolic extract: three male rats of SHR strain previously treated with verapamil of study 1 were treated with polyphenol extract # 1: Grenache Long Coudoulet seeds.

 • Comparison of the different polyphenol extracts: under the same conditions as above, the effects of polyphenolic extracts # 2 to # 6 were compared.

• Confirmation of the results obtained with a second polyphenolic extract: seven male rats of SHR strain were treated with the extract n ° 6 (marc of Alicante).

 1. B. Measurement of blood pressure:

Blood pressure was measured on a LETICA LE 5002 device by the sphingomanometry technique (tail-cuff). The cuff is placed on the tail of the rat 2 cm from the end, the temperature of the room must be between 29 ° C and 32 ° C. The animal is placed in a quiet and dark room to minimize stress as well as handled with care and covered with a cloth during the measurement. The animal must be trained to measure. For each rat, the value used is the average of three successive measurements if the maximum difference between two measurements is less than 20 mmHg.

3. Results of in vivo tests:

2. A. Study 1: Determination of the anti-hepertensive effects of 6 polphenol extracts in SHR rats

 The systolic blood pressure of SHR control rats is between 150 mmHg at the beginning of the experiment and 1 90 mmHg after 5 weeks (see Figure 1). The polyphenolic extracts administered to SHR rats have little effect on the animals' systolic blood pressure, which increases gradually (except for lot 1 after 2 weeks of treatment) (see Figure 1). However, after 3 weeks of treatment, an intolerance to gavage made the administrations very difficult and forced the interruption of treatment for 1 week (see week 4 in Figure 1). This discontinuation of treatment was associated in batches of SHR 1, 2 and 6 rats at an increase in systolic blood pressure higher than the control SHR rats (see Figure 1). This rise can be interpreted as a rebound effect, often observed with antihypertensive products and could mean an antihypertensive potential of the extracts concerned. However, the resumption of administration of polyphenols at the fifth week did not allow to find a significant drop in blood pressure (see Figure 1), nor a rebound effect to a new stoppage of treatment (not shown). 2. B. Study 2: Evolution of blood pressure in SHR treated with verapamil

The blood pressure of the WKY rats remains stable between 100 and 110 mmHg. Before the start of treatment, there was a gradual increase in blood pressure in SHR rats from 1 58 mmHg to 1 94 mmHg. Verapamil treatment at 30 mg / kg / day causes a sudden drop in blood pressure to 1 20 mmHg followed by a progressive rise to 21 7 mmHg at 20 mg / kg / day. The increase in the dose administered at 40 mg / kg / day causes a decrease in blood pressure to 180 mmHg followed by a rise to 200 mmHg in 10 days (see Figure 2). This result illustrates the resistance of SHR rats to standard antihypertensive treatments, including calcium antagonists. 2. C. Study 3: Determination of the pressure effects of phenol extracts in the presence of verapamil

 Study 2 showed that verapamil, a major antihypertensive calcium inhibitor, gradually lost its antihypertensive activity in the SHR rat model.

 This third study was conducted in three stages:

 A) Firstly, treatment with extract no. 1 at 21 mg / kg (Grenache Long Coudoulet seeds) combined with verapamil at 40 mg / kg of the three animals previously treated with verapamil was put in place. (Lot 1 ). Following this treatment, a progressive decrease in arterial blood pressure (maximum after three weeks of treatment) compared to control SHR rats (see Figure 3) or treated only with verapamil was observed (see Figure 2, in comparison with Figure 3).

 B) The effects of extracts No. 2 to No. 6 (21 mg / kg) assayed with verapamil (40 mg / kg) were determined in a comparative manner under the same experimental conditions (see Table 1 below).

 C) Extract No. 6 (Alicante film) was used to reinforce the preliminary result obtained previously. The number of animals treated with extract No. 6 (21 mg / kg) combined with verapamil (40 mg / kg) was increased to a total of seven. This experiment confirmed the efficacy of extract No. 6 in combination with verapamil (see Figure 3, Lot 6).

 An animal has in fact almost normalized its blood pressure. This phenomenon is quite exceptional for this model.

Table 1: Effect of the effects of the various polyphenolic extracts on treated SHR rats treated with verapamil (Study 3B)

 Lots 6 days 13 days 20-23 days

 108.5 ± 1, 2 108.5 ± 1, 2 108.5 ± 1, 2

WKY

 (102-121) (102-121) (102-121)

 200.3 ± 1, 8

 SHR 200.3 ± 1.8 (1 90-214) 200.3 ± 1.8 (1 90-214)

 (190-214)

 198.0 ± 1, 5 198.0 ± 1, 5 198.0 ± 1, 5

SHR + V ^a

 (195-202) (195-202) (195-202)

SHR + V + Γ 193, 1 ± 3.2 161, 4 ± 1 0.5 146.3 ± 5.9

SHR + V + 2 ^at 191, 7 ± 3.7 192.5 ± 0.8

SHR + V + 3 ^was 188.7 ± 2.7 192 ± 1

SHR + V + 4 ^at 187.2 ± 5.8 189.5 ± 5.5 SHR + V + 5 ^at 186.3 ± 0.0 185.3 ± 5.7

SHR + V + 6 ^a 168.8 ± 10.6 173.0 ± 24.7 171, 8 ± 18.8

SHR + 6 ^at 200.9 ± 2.8 195-204 aV, Verapamil; V + 1 to V + 6, Verapamil + extract # 1 to # 6; SHR + 6, SHR treated with extract # 6.

 The values in parentheses correspond to the minimum and maximum values recorded over the entire measurement period considered.

3. Conclusion of in vivo experiments:

The SHR rat used in this study is a model of essential hypertension that is difficult to correct, including first-line antihypertensive drugs. This notion was found in this study using a calcium antagonist, verapamil. Resistance to verapamil even developed in SHR rats treated with verapamil alone (see Figure 2).

Used chronically at a dose equivalent to that ingested by a 70 kg man consuming 500 ml of red wine, the 70% hydro-alcoholic polyphenolic extracts studied did not induce a significant drop in blood pressure. Neither polyphenolics nor verapamil showed sufficient antihypertensive activity alone for a sufficient period of time.

Surprisingly, the combination of polyphenol extracts with verapamil allowed a decrease in the SHR rats' blood pressure, compared with SHR control rats, over a period of up to 23 days. Even more surprisingly, the combination of polyphenolic extracts with verapamil overcame the resistance developed against verapamil in SHR rats (see Study 3A).

The polyphenolic extracts associated with verapamil demonstrated a synergistic action, the antihypertensive effect of the combination being greater than the sum of the effects of the two agents, the antihypertensive effect of verapamil alone, the polyphenolic extracts did not produce significant drop in tension alone.

Claims

A combination comprising a polyphenol extract of grape marc and an antihypertensive agent of the family of calcium channel blockers.

The combination of claim 1 wherein the antihypertensive agent is verapamil.

Combination according to any one of claims 1 or 2, wherein the polyphenol extract is obtainable by an alcoholic or aqueous extraction of one or more grape pomace (s).

Combination according to any one of the preceding claims, wherein the polyphenolic extract is obtainable by extraction of grape pomace composed of pips and / or grape skins.

Combination according to any one of the preceding claims, wherein the polyphenol extract is likely to be obtained by an extraction of grape pomace whose grape belongs to one of the grape varieties selected from Grenache Long du Coudoulet, Grenache Face in Pines, Syrah from Plantier, Syrah from Haut de Julien, Carignan, Mourvedre, Counoise, Alicante and their mixtures.

Combination according to any one of the preceding claims, in the form of a pharmaceutical composition for the treatment of hypertension.

Combination according to any one of claims 1 to 6, in the form of a pharmaceutical kit for the treatment of hypertension, in which the polyphenol extract of grape marc and the antihypertensor of the family of calcium channel blockers are formulated separately for simultaneous, separate or staggered use