LU92908B1 - Extract from the vine as an active ingredient able to stabilize the oil / water interfaces - Google Patents

Abstract

Extract from the vine capable of stabilizing the emulsions at the interface and inhibiting two key enzymes of the arachidonic cascade at the same time, sPLA2-V and lipoxygenase, but also capable of inhibiting collagenase type I, stimulate Tyrosinase, and having the ability to reduce wrinkles in topical application.

Description

Extract from the vine as an active ingredient able to stabilize the oil / water interfaces.

The present invention relates, as a new and useful industrial product, to an extract derived from grapevine as an inhibitor of the enzyme Phospholipase A2 (PLA2), and in particular PLA2 of group V. The invention also relates to the use of this extract as a dermo-cosmetic active principle and its use in topical pharmaceutical compositions for use in human or veterinary medicine or for cosmetic use. The invention also relates to the use of this extract as a stabilizer of O / W and W / O emulsions and its use as a food, cosmetic, veterinary or pharmaceutical composition.

PLA2 is an enzyme produced by cells at the membrane level. It is the first enzyme of the inflammatory cascade, also called "arachidonic cascade". Indeed, by releasing arachidonic acid from membrane phospholipids, PLA2 is the key factor triggering the release of pro-inflammatory mediators in mammalian tissues, namely ecosanoids.

PLA2 is a super-family of enzymes consisting of 4 major families, calcium-dependent secreted PLA2 (sPLA2), calcium-dependent cytosolic PLA2 (cPLA2), calcium independent cytosolic PLA2 (ÎPLA2), and PLA2 activation factor platelet acetyl hydrolase. PLA2 are then classified into 15 groups according to the composition of their primary molecular sequence. In the vast majority of inflammation-related cells such as macrophages and mast cells, only PLA2 belonging to the first two families mentioned, secreted calcium dependent PLA2 (sPLA2) and calcium dependent cytosolic PLA2 (cPLA2), are involved in release of eicosanoid mediators, and more particularly groups IV (cPLA2) and V (sPLA2).

Phospholipase A2 type V (sPLA2-V) plays a decisive role in the pro-inflammatory process because it is also capable of inducing the expression of cycloxygenase -2 (COX-2) [1]. It is also demonstrated that sPLA2-V is able to bind to membrane phosphatidylcholines and hydrolyze substrates thereof in a much more efficient manner than any other PLA2. In light of these results, SPLA2-V is an enzyme whose activity is major in the inflammatory process, and the inhibitors of this enzyme can more effectively stop the inflammatory process and thus relieve or soothe the affected tissues more effectively.

It appears that a keen interest exists for extracts or natural substances capable of inhibiting group V phospholipase A2, especially for topical cosmetic, pharmaceutical or veterinary applications which aim to soothe and / or reduce the inflammation of the tissue. The anti-inflammatory activity is as evoked a process under the control of "the arachidonic cascade", the PLA2 is the first link, then another enzyme 5'-Lipoxygenase can take over to produce new mediators of the Inflammation, 5'-Lipoxygenase, hereinafter referred to as "lipoxygenase", is, like PLA2, a membrane enzyme. It intervenes downstream of the release of arachidonic acid by PLA2, in the conversion of this acid into leukotrienes, specific mediators of inflammation. The inhibition of PLA2 is not correlated with the inhibition of lipoxygenase, which is why it is very rare to find substances or molecular complexes capable of modulating by inhibition these two key enzymes of the inflammatory cascade.

Recent studies demonstrate the involvement of leukotrienes in skin swelling and skin fibrosis, especially via leukotriene cysteinyl [2]. These swellings of the skin tissue and skin growths are particularly unsightly, and From a cosmetic point of view, the inhibition of leukotrienes by skin treatment is a cosmetic care technique that makes it possible to improve the appearance of the skin, and more particularly when it is in an inflammatory state. In the case of topical applications, the substances having, in addition to the group V phospholipase A 2 inhibition properties, properties which are capable of improving the skin or of treating it advantageously by giving it protection, are particularly sought after. active against the production of leukotrienes, the degradation of cutaneous collagen, allowing it to increase its ability to produce melanin without getting in the sun, allowing it to limit wrinkles. These substances can therefore be advantageously used in the manufacture of active ingredients for the treatment of the skin, cosmetic topical preparations, dermopharmaceutical or veterinary. The object of the present invention has therefore been to provide substances having the ability to inhibit SPLA2-V, to inhibit lipoxygenase, to inhibit collagenase type I, to stimulate tyrosinase, and to the ability to reduce wrinkles.

The systematic tests carried out by the Applicant have made it possible to select an extract derived from the vine which, surprisingly, was capable of responding to the complex requirement profile depicted.

The extracts from the vine are known in the prior art and among the active ingredients for cosmetics extracted from the vine, we can find extracts of vine shoots, vine leaves, pork and other by-products from vinification (FR2804864) most rich in polyphenols, especially Resveratrol, described for their anti-aging properties (FR2898493), used in combination with other extracts or active agents (FR2808682; FR2985906); or essential oils from the dregs to stimulate hair regrowth (EP0001860).

Specific extracts from winemaking already describe an anti-inflammatory activity combined with an antioxidant, detoxifying, anti-aging, regenerating activity in cosmetic applications; the composition of these extracts is very well characterized, singularly by the presence of 0.1 to 10% of proteins and 10 to 50% of polyphenols; it should be noted that their concentration is particularly high in organic matter. However, these extracts do not demonstrate inhibitory activity of sPLA2-V, no inhibition of lipoxygenase, no type I collagenase inhibiting activity, no stimulation of tyrosinase, and their Anti-wrinkle activity is not demonstrated. As we will see later, the extract described by the Applicant does not have the same chemical characteristics and biochemical and biological modes of action are singular and different. Other specific extracts from the vine show an ability to modulate the production of endothelin leading to a vasodilatory or vasoconstrictor action (FR 2955771).

In a recent publication, it has been shown that certain proanthocyanidins derived from grapes may have an inhibitory action on Phospholipase A2 Group III [3]. The trimeric or tetrameric form of a type A proanthocyanidin is particularly active because it can bind to the active site of Group III PLA2 and block it. To the knowledge of the applicant, it has never been described that an extract from the vine may have the ability to inhibit sPLA2-V, to inhibit lipoxygenase, to inhibit collagenase type I, stimulate Tyrosinase, and having the ability to reduce wrinkles. The extract according to the invention is derived from the vine, and more particularly from fermented grape juice, commonly called wine, in its entirety which has not undergone any clarification and / or filtration operation, ie which, after vinification, always contains its alcoholic ferments.

The settling legs of the wine tanks and the lees may also be considered as the raw material of the present invention.

The present invention specifically aims at the use of an extract which is advantageously obtained by maceration of vinified grape juice always containing its alcoholic ferments and / or an extract of decantation feet at the end of the vinification of the grape juice and / or an extract of the lees after the vinification of the grape juice, after a dehydration step. One of the main objects of the invention is an unhydrolyzed extract of uncleaned, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of lees. of wine which is used as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, said active ingredient and / or said composition being intended to act by inhibition of SPLA2-V, by inhibition of lipoxygenase, by inhibition of collagenase type I, by stimulation of tyrosinase, and so to reduce wrinkles. The invention also relates to the use of an unhydrolyzed extract of non-clarified, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of wine, as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, making it possible to avoid the stimulation of the enzyme Lipoxygenase, but on the contrary to inhibit the Lipoxygenase, a key enzyme in inflammation of the arachidonic cascade, as an active anti-inflammatory composition, limiting the production of leukotrienes or soothing or as an anti-inflammatory active ingredient limiting the production of leukotrienes or soothing, in order to limit aesthetic disorders related to the inflammatory state. The invention also relates to the use of an unhydrolyzed extract of non-clarified, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of wine, as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, for inhibiting type I collagenase (metallopeptidase 1) so as to limit the degradation cutaneous collagen and so enhance the integrity of the extra-cellular matrix of the skin as an active composition to maintain the firmness of the skin. The invention also relates to the use of an unhydrolyzed extract of non-clarified, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of wine, as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, for stimulating tyrosinase, the key enzyme in the production of melanin within melanocytes of the basal layer of the epidermis so as to promote the production of cutaneous melanin even in the absence of sun, as an active composition promoting tanning without sun. The invention also relates to the use of an unhydrolyzed extract of non-clarified, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of wine, as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, for modulating the activity of histones deacetylases (HDACs) and in particular type I sirtuins so as to activate them even at low concentration. Activation of Sirtuins type I is correlated with an anti-aging effect on cellular metabolism.

According to a particularly suitable embodiment, the invention relates to the use of a non-hydrolysed extract of unclarified, non-decanted wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of wine lees, as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions, making it possible to reduce and / or limit wrinkles on the skin. skin surface, as an active anti-wrinkle composition.

According to a particularly suitable mode of use, the invention aims at the use of an unhydrolyzed extract of unclarified wine, not decanted and / or an unhydrolyzed extract of settling feet at the end of the vinification, and or an unhydrolyzed wine lee extract making it possible to contribute to the stabilization of the oil-in-water (O / W) and water-in-oil (W / O) emulsion type compositions by encapsulation / coating of all or part of the phase dispersed.

Characterization of the non-hydrolysed extract of the vine according to the invention

Spectrometric profile: The invention refers to a singular active ingredient consisting of a non-hydrolysed non-clarified, non-decanted wine extract and / or a non-hydrolysed extract of settling feet at the end of vinification, and / or a non-clarified extract. hydrolysed wine lees corresponding to an infra-red profile as follows:

In the graph below the extract according to the invention is called "LDV ET"

Dry matter ratio:

The solids content is obtained by mass loss at 100 ° C. for a minimum hour until a constant mass is obtained. According to this method, the solids content of an active ingredient according to the invention is preferably between 1 and 3%, and preferably between 1.5 and 2.5%. _ρΗΗ

The pH of the active principle according to the invention is between 4 and 6.5, and preferably between 4.5 and 5.5.

Total sugar content:

The determination of the total sugar content can be carried out by the DUBOIS method (Dubois M. et al., 1956), Analytical Chemistry, 28, No. 3 p. 350-356. In the presence of concentrated sulfuric acid and phenol, the reducing sugars give an orange-yellow compound. From a standard range, the total sugar content of a sample can be determined. According to this method, the total sugar content of an active ingredient according to the invention may be between 0.01 and 0.5%, preferably between 0.1% and 0.2% (weight / mass), or between 5 and 10% of the dry matter of our extract according to the invention.

Protein content:

The total nitrogen content is determined by the method of Bradford (Bradford, J., (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal Biochem 72: 248-254). This method determines the protein content according to a colorimetric approach based on the change in absorbance around 595 nm. The color change is caused by the Coomassie blue, which becomes more complex with the basic amino acids and the hydrophobic residues of the amino acids constituting the proteins. According to this method, the protein content of an active principle according to the invention is preferably between 0.10 mg / ml and 1.00 mg / ml, and preferably between 0.15 and 0.40 mg / ml, or between 7.5 mg / g and 20 mg / g of the dry matter of our extract according to the invention.

Content of phenolic compounds:

The determination of total polyphenols by the colorimetric method using the Folin-Ciocalteu reagent was described in 1965 by Singleton and Rossi. The Folin Ciocalteau reagent is a yellow acid consisting of a mixture of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PM012O40). It is reduced, during the oxidation of phenols, in a mixture of blue oxides of tungsten and molybdenum (Ribereau, 1968). The color produced, whose maximum absorption is 760 nm, is proportional to the amount of polyphenols present in the plant extracts (Ghazi and Sahraoui, 2005). A standard curve is made in this experiment from catechin. According to this method, the content of polyphenols (catechin equivalents) of an active ingredient according to the invention is preferably between 0.01 and 0.5%, preferably between 0.1% and 0.2% (weight / mass). , or between 5 and 10% of the dry matter of our extract according to the invention. It should be noted that by gas chromatography coupled to mass spectrometry (GC / MS) on an extract according to the invention prepared with ethanol solvent 96 °, it is also pointed out that the dry matter according to the invention contained 0.17% phenyl acetaldehyde and 0.24% phenylethyl alcohol.

Fat content: The invention is characterized by a non-hydrolysed extract of uncleaned, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract of lees wine comprising in particular from 0.1 to 1 mg / ml of peptides and oligopeptides, from 0.01 to 0.05% of polyphenols, from 0.01 to 0.5% of sugars and by a specific polar fat composition detected by gas chromatography coupled to mass spectrometry (GC / MS) on an extract according to the invention prepared with ethanol solvent 96 °, it was thus demonstrated that the dry matter according to the invention contained different polar lipids for about 40 to 50% of its dry mass. The polar lipids are mainly esters of fatty substances, the latter represent approximately 30 to 35% of the dry mass of the extract according to the invention, then between 5 and 7% of free fatty acids, the latter are mainly represented by palmitic acid and decanoic acid. Below a table presents a typical profile of the polar fatty substances contained in the dry mass of the extract according to the invention obtained by extraction with ethanol solvent 96 °:

Esters of fatty acids:

Ethyl decanoate 11.22%

Ethyl Dodecanoate 11.15%

Ethyl caprylate 3.25%

Ethyl stearate 3.02%

Ethyl9-hexadecanoate 2.4%

Ethyl myristate 1.27%

Ethyl oletae 0.8%

Methyl Butyl decanoate 0.39%

Methyl linoleate 0.25%

Fatty acids :

Palmitic acid 3.84%

Decanoic acid 2.1%

Content of strict polar organic compounds: The invention is characterized by an unhydrolyzed extract of uncleaned, undecured wine and / or an unhydrolyzed extract of settling feet at the end of the vinification, and / or an unhydrolyzed extract wine lees comprising in particular from 0.1 to 1 mg / ml of peptides and oligopeptides, from 0.01 to 0.05% of polyphenols, from 0.01 to 0.5% of sugars and from 0.8 to 1% (40%). at 50% of its dry mass) of polar lipids and by the presence of strict polar compounds detected by GC / MS on the ethanolic extract (EtOH 96 °) according to the invention, at a level of 0.8 to 1%, ie 40 to 50% of the dry matter according to the invention and mainly comprises mainly acetic acid approximately 25 to 28% of the dry mass of the extract according to the invention, then between 15 and 17% of glycerol and finally 3 to 4% Butanediol.

Below a table presents a typical profile of the strict polar compounds contained in the dry mass of the extract according to the invention obtained by extraction with ethanol solvent 96 °:

Acetic acid 27.16%

Glycerol 15,81%

Butanediol 3.6%

Diester butanediol 1.42% The invention also relates to a particularly suitable embodiment as described below.

Method of production: Starting from unclarified wine, not decanted and / or from wine sedimentation foot and / or from wine lees, the starting material is concentrated by decantation and / or centrifugation, then by evaporation of the alcohol and then water until a concentrated paste containing a solids content of between 80 and 99.9%, and preferably between 90 and 98%. The extract according to the invention is thus obtained from raw material which may be unclarified wine, not decanted and / or settling feet at the end of the vinification, and / or lees of wine. This raw material once concentrated is then dried by dehydration in a furnace or a ventilated oven keeping a temperature below 60 ° C, or in a vacuum evaporator optionally at room temperature, or vacuum lyophilization after freezing of the material. first, or by any other method of non-denaturing drying and respecting a temperature below 60 ° C.

This dried, dry raw material is then advantageously macerated in a solvent of medium or low polarity, followed by filtration of the mixture. The solvent of the solution obtained may, if necessary, be evaporated to obtain a dry extract.

By solvent of medium or low polarity, it is considered a solvent having a polarity parameter less than or equal to 6, such index being defined according to the following bibliographic reference: High Performance Liquid chromatography, V.R. Meyer, 1988, p. 120-121.

Preferably the evaporation is conducted under reduced pressure. As solvents advantageously used, there are in particular: C12 carbon chain aliphatic hydrocarbons, such as hexane and heptane, chlorinated solvents such as dichloromethane; Ethers, such as ethyl or diisopropyl ether; - acetone; Esters such as ethyl acetate and butyl acetate; alcohols such as methanol, ethanol and isopropanol. The extract according to the invention can also be advantageously obtained by extraction with supercritical carbon dioxide from the dehydrated raw material. The evaporation of the alcohol is preferably carried out under vacuum with a condenser device, then the evaporation of the water is preferably carried out in a ventilated oven or in a vacuum reactor at a moderate temperature not exceeding 60.degree. preferentially around 45 ° C. The solids content is determined by the weight gain before and after, during a passage in a ventilated oven at 100 ° C for 1 hour of a representative sample.

The active ingredient according to the invention as described above is obtained by a process comprising a maceration step in a solvent having a medium or low polarity. The maceration is done by adding the dry matter obtained between 2% and 25% and preferably between 5 and 15% (weight / volume) in the chosen solvent. The maceration is preferably carried out with moderate agitation in a closed vessel protected from light and optionally under vacuum. The maceration lasts between 2 hours and 24 hours depending on the chosen solvent, the extract is then mechanically filtered, preferably under vacuum and titrated at 2% dry matter (mass / mass) by dilution with the solvent or by evaporation of the latter, preferentially under vacuum. A final step consists in transferring the dry matter to another cosmetically acceptable organic or inorganic support by evaporation, preferably under vacuum, of the solvent; in this case the organic or inorganic support mass is equal to that of the solvent. Thus, the active ingredient according to the invention is characterized by 2% of dry matter resulting from an unhydrolyzed extract of unclarified wine, not decanted and / or an unhydrolyzed extract of settling feet at the end of the vinification. and / or an unhydrolyzed extract of wine lees to reduce and / or limit wrinkles on the surface of the skin, as an active anti-wrinkle composition, fighting against wrinkles.

EXAMPLE 1 of composition of the extract according to the invention in liquid form:

EXAMPLE 2 Composition of the Extract According to the Invention in Liquid Form

EXAMPLE 3 of the composition of the extract according to the invention in liquid form:

EXAMPLE 4 Composition of the Extract According to the Invention in Solid Form

EXAMPLE 5 Composition of the Extract According to the Invention in Solid Form:

Example 6 of composition of the extract according to the invention in solid form:

Description of the potential for biological and / or biochemical activity: The extract according to the invention has an antioxidant action according to the In Tubo test with DPPH (1,1-diphenyl-2-picrylhydrazyl). This 2,2-diphenyl-1-picrylhydrazyl compound (DPPH) is a stable radical that has a characteristic (purple) color. It is in oxidized form. Reduction of the radical by an atom donor H (anti-oxidant - example: ascorbic acid) leads to the colorless compound 2,2-diphenyl-1-picrylhydrazine. This discoloration is measurable by spectrophotometry and estimated by absorbance at 490 nm and expressed in Optical Density (OD). The measurement points are made in triplicate and the results express the average of this triplicate, the statistical significance of the averages is evaluated by measuring the standard deviations around the mean for each triplicate, then validated with a tolerance threshold for the value. the standard deviation of less than 10% of the average value of OD (Optical Density Value) retained. The retained OD is calculated according to the following principle: The OD of the hydrolates alone without DPPH is deduced from the OD observed in the presence of DPPH so as to erase the impact of the basal color of the product to be tested on the OD. Below, table of absorbances obtained at 405 nm:

Assayed at 1%, the LDV ET extract according to the invention inhibits the radical activity by more than 91%; Dosed at 10%, the hydrolyzate according to the invention inhibits the radical activity by more than 100%, its anti-radical activity is therefore greater than that of the ascorbic acid control assayed at 0.33 mg / g. This activity appears dose-related, which is in favor of a specific interpretation of activity on this parameter. The extract according to the invention has In Tubo an inhibitory action on Phospholipase A2 of type V (sPLA2-V). This enzyme acts at the head of the arachidonic cascade, it is one of the first key enzymes of the inflammatory process. The purpose of this study is to evaluate the modulation by the extract according to the invention of the activity on the enzyme Phospholipase A2 type V (sPLA2-V) in an acellular In Vitro model.

Test system

A buffered solution of Phospholipase A2 reacts with a specific substrate (diheptanoyl thio-PC) for 15 minutes at about 25 ° C and transforms to form a compound that binds to a DTNB chromogen with simple stirring at room temperature. The activity of phospholipase A2 can thus be evaluated by measuring the absorbance at 405 nm.

The "LDV ET" product or the "Thioetheramide-PC" inhibitory reference product are contacted with the Phospholipase A2 solution together with the enzyme substrate for 15 minutes at 25 ° C., the substrate converted by the The enzyme is stained with a chromogen by simple stirring at room temperature. The activity

Phospholipase A2 in the presence / absence of the active ingredient, or the reference product is then evaluated by measuring the absorbance at 405 nm.

The modulation of this activity is expressed as a percentage inhibition or activation of the activity of Phospholipase A2 in the absence of an active ingredient, ie only in the presence of the substrate of the enzyme (diheptanoyl thio- PC).

A Phospholipase A2 enzyme solution is incubated in its substrate, diheptanoyl thio-PC, for 15 minutes at 25 ° C, in the absence (control) or presence of the reference product, or increasing concentrations of the products under test:

Extract preparation "LDV ET"; 1/100; 1/1000; 1/10000 (M / M)

Evaluation of the inhibitory or activating effects At the end of the incubation period, the activity of the enzyme Phospholipase A2 with and without a product under test or reference was evaluated by measuring the absorbance of the reaction media at 405. nm.

For each concentration tested, the modulation of the activity of the enzyme Phospholipase A2 by the test product is calculated according to the following formula:

Percent Modulation of Enzyme Activity Phospholipase A2 = 100 x [(DO405 Test or Reference Product) - (DO405 PLA2 alone)] DO405 PLA2 alone

If the result is negative, the percentage is expressed as inhibition of the enzyme; if the result is positive, the percentage is expressed as activation of the enzyme.

The results are given as percent inhibition of the reaction.

The statistical significance of the differences observed between the conditions "control" and "reference product" is evaluated by a Student's test (Student T test ***, p <0.001) and by a one-way analysis of the variance (One Way ANOVA), followed by a Holm-Sidak test (*, p <0.05).

Reference product

In reference control (-) a reference inhibitor is employed, it is Thioetheramide-PC 0.14 mM

An ethanolic extract according to the invention is tested at different concentrations in the presence of a positive control (= basal activity of the enzyme) and a negative control (Thioetheramide-PC 0.14 mM)

Table of absorbances obtained at 405 nm

In this acellular In Tubo model, at a dose of 2%, the extract according to the invention significantly inhibits the activity of Phospholipase A2 of type V (sPLA2-V) by 54.4%; the inhibitory activity is very powerful since the effect of the reference inhibitor is exceeded when the extract according to the invention is concentrated to 8%. The extract according to the invention has In Tubo an inhibitory action on lipoxygenase. This enzyme acts at the heart of the arachidonic cascade, it is a key enzyme of the inflammatory process.

The aim of this study is to evaluate the modulation by the extract according to the invention of the activity on the enzyme Lipoxygenase, in an acellular In Vitro model. Lipoxygenase is a key enzyme in the inflammatory process that occurs downstream of Phospholipase A2 by converting arachidonic acid to Leukotrienes.

Test system

A buffered solution of Lipoxygenase reacts with a specific substrate (arachidonic acid) for 5 minutes with orbital shaking at about 22 ° C and transforms to form a compound that binds to a chromogen with orbital shaking and at room temperature for 5 minutes again . The activity of the lipoxygenase can thus be evaluated by measuring the absorbance at 490 nm. The LDV ET extract according to the invention or the reference product inhibitor "Nordihydroguaiaretic acid" (NDGA at 0.16 mM) are brought into contact with the lipoxygenase solution at the same time as the substrate of the enzyme for 5 minutes at 22 °. C with orbital shaking, the substrate transformed with the enzyme is stained with a chromogen by orbital shaking for 5 minutes at room temperature. The activity of the lipoxygenase in the presence / absence of the active ingredient or reference product is then evaluated by measuring the absorbance at 490 nm.

The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of lipoxygenase in the absence of active agents, ie only in the presence of the substrate of the enzyme (arachidonic acid).

Reference product

In this test, the reference product is an inhibitor, namely a CURCUMA-EC EXTRACT (EPHYLA) 0.1%

Evaluation of the inhibitory or activating effects At the end of the incubation period, the activity of the enzyme Lipoxygenase with and without a product under test or reference was evaluated by measuring the absorbance (OD) of the reaction media. at 490 nm.

For each concentration tested, the modulation of the activity of the enzyme Lipoxygenase by the product under test is calculated according to the following formula:

Modulation of Lipoxygenase Enzyme Activity = 100x [(DO Test or Reference Product) - (OD Lipoxygenase Only)] DO Lipoxygenase Only Absorbance (OD) is measured at 490 nm in this test.

If the result is negative, the percentage is expressed as inhibition of the enzyme; if the result is positive, the percentage is expressed as activation of the enzyme.

The results are given as percent activation or inhibition of lipoxygenase. From the triplicate data, the statistical significance of the differences observed between the "control" conditions, that is to say the controls and "reference product", is evaluated by a Student's test (Student T test, p <0.05 ).

Table of absorbances obtained at 405 nm [T +] Lipoxygenase alone with its substrate (basal activity) [T '] Lipoxygenase in the presence of CURCUMA-EC extract (reference inhibition) The ethanolic extract according to the invention has an inhibitory action on the detectable and significant Lipoxygenase enzyme as early as the 1% dose. The extract according to the invention has In Tubo an inhibitory action on the type I collagenase activity. This conclusion results from a study whose purpose is to evaluate the anti-collagenase activity of the "LDV ET" product in a In Vitro acellular colorimetric model using a type I collagenase (Sigma Aldrich C0130), a substrate "Collagenase Substrate Kit" (Sigma Aldrich 27670 1EA) and a chromophore, Ninhydrin (Sigma Aldrich 72491).

Test system

Use of a collagenase type I buffered solution that reacts with a specific substrate for 5 minutes at 37 ° C and degrades it to form a compound capable of activating a chromophore by incubation at 80 ° C for 15 minutes. The maximum activity of collagenase can thus be evaluated by measuring the absorbance at 565 nm.

The "LDV ET" product is contacted with the collagenase solution together with the enzyme substrate for 5 minutes at 37.degree. C., the substrate degraded by the enzyme is capable of activating the chromophore by incubation at 80 ° C for 15 minutes. The activity of collagenase in the presence of the active is then evaluated by measuring the absorbance at 565 nm.

The modulation of this activity is expressed as a percentage of inhibition or activation of the maximum activity of collagenase in the absence of an active agent, that is to say only in the presence of the substrate of the enzyme.

To do this, a collagenase enzyme solution is incubated in its substrate for 15 minutes in the absence (control) or presence of the reference product, or increasing concentrations of the products under test:

The product "LDV ET" is tested at the following concentrations: 1%; 0.1%; 0.01% (V / V)

Then the solutions are brought into contact with the chromophore specific substrate degraded by the collagenase enzyme for 15 minutes at 50 ° C. At the end of the period of contact with the chromophore, the activity of the type I collagenase enzyme with and without test product was evaluated by measuring the absorbance of the reaction media at 565 nm.

For each concentration tested, the modulation of the activity of the type I collagenase enzyme by the product under test is calculated as a percentage according to the following formula:

Percentage modulation of collagenase enzyme activity = 100 X [(OD565 test or reference product) - (DO565 Collagenase alone)] DO565 Collagenase alone

If the result is negative, the percentage is expressed as inhibition of the enzyme; if the result is positive, the percentage is expressed as activation of the enzyme.

The results are given as percentages of inhibition of the radical reaction.

The statistical significance of the differences observed between the conditions "control" and "reference product" is evaluated by a Student's test (Student T test, p <0.001) and by a one-way analysis of variance (One Way ANOVA), followed by a Holm-Sidak test (p <0.05).

Summary table of the results obtained after reading the OD565_

The ethanolic extract according to the invention shows a significant inhibitory activity as soon as the dose of 0.01% with the score of 85.9% of inhibition of the basal activity of the enzyme. At 0.1% LDV AND inhibits by more than 100% the activity of collagenase. The product shows a dose-dependence effect. The extract according to the invention has In Tubo a stimulating action on the tyrosinase activity. The aim of this study is to evaluate the activity on the Tyrosinase enzyme of the product "LDV ET", in an acellular In Vitro model using a tyrosinase enzyme of fungal origin (Sigma Aldrich ref. substratum

L-Tyrosine (Sigma Aldrich Ref T3754) and a reference inhibitor hydroquinone (Sigma Aldrich Ref.H17902).

The product "LDV ET" corresponding to the ethanolic extract according to the invention is under study.

Test system

A Tyrosinase buffered solution reacts with a 2.5 mM L Tyrosine substrate for 60 minutes at 23 ° C and transforms to form a colored compound. The maximum activity of tyrosinase can thus be evaluated by measuring the absorbance at 475 nm.

The product "LDV ET" or the reference product "Hydroquinone" are brought into contact with the Tyrosinase solution at the same time as the substrate of the enzyme for 60 minutes at 23 ° C, the substrate transformed by the enzyme is naturally colored. The activity of Tyrosinase in the presence of the active is then evaluated by measuring the absorbance at 475 nm.

The modulation of this activity is expressed as a percentage of inhibition or activation of the maximum activity of Tyrosinase in the absence of an active agent, ie only in the presence of the substrate of the enzyme (L Tyrosine) .

Reference product

Inhibitor = Hydroquinone 2.5 mM

Incubation protocol

Tyrosinase enzyme solution is incubated in its L Tyrosine substrate for 60 minutes in the absence (control) or presence of the reference product, or increasing concentrations of the product under test:

LDV ET extract; 1/100; 1/1000; 1/10000 (V / V)

Evaluation of the Effects At the end of the incubation period, the activity of the Tyrosinase enzyme with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 475 nm.

For each concentration tested, the modulation of Tyrosinase enzyme activity by the test product is calculated according to the following formula: Percent Modulation of Tyrosinase Enzyme Activity = 100 X [(OD475 Tested Product or reference) - (DO475 Tyrosinase alone)] I DO475 Tyrosinase alone

If the result is negative, the percentage is expressed as inhibition of the enzyme; if the result is positive, the percentage is expressed as activation of the enzyme.

Statistics

The results are given as percentages of inhibition of the radical reaction. The statistical significance of the differences observed between the conditions "control" and "reference product" is evaluated by a Student's test (Student T test, ***, p <0.001) and by a one-way analysis of variance (One Way ANOVA), followed by a Holm-Sidak test (*, p <0.05).

Table of results (on average) on the products tested, the control (T +) and the reference product (T-) by measuring the absorbance at 475 nm

T + = Tyrosinase alone with its substrate (max activity) T '= Tyrosinase in the presence of Hydroquinone 2.5 mM (Reference max inhibition)

The results obtained on the LDV ethanol extract according to the invention show a Gausse curve effect on the studied parameter, which favors a specific activity of this extract on the activation of Tyrosinase. Peak activation peak is 0.1% LDV ET.

The extract according to the invention has in Tubo a stimulating action with regard to the deacetylase activity of histones deacetylases (HDACs) and in particular Sirtuins of type I. The deacetylase activity of Sirtuins type I (HDACs) is involved in the cellular mechanisms "anti-aging"; As a corollary, the hydrolyzate according to the invention has In Tubo anti-aging activity at a dose of 0.1%. The aim of this study is to evaluate the modulation of the deacetylating activity of HDACs and in particular of type I sirtuins by increasing concentrations of the hydrolyzate according to the invention, in an acellular In Vitro colorimetric model using an extract nuclear cell) "HeLa" rich in HDACS, and especially Sirtuines type I.

Test system

A buffered solution of HDACs, containing in particular Type I Sirtuins (nuclear extract of "HeLa" cells) reacts with a substrate for 20 minutes at 37 ° C. and transforms it into a compound which is colored in the presence of a developer after incubation at 37 ° C for 10 minutes. The maximum deacetylation activity of Type I Sirtuins can thus be evaluated by measuring the absorbance at 405 nm.

The different concentrations of the ethanolic extract (test products) according to the invention or the reference product "Inhibitor" are brought into contact with the solution of HDACs, containing in particular

Type I sirtuins together with the enzyme substrate for 20 minutes at 37 ° C, the substrate transformed with the enzyme is stained by the addition of a developer. The deacetylating activity of the HDACSs and in particular Sirtuines of type I in the presence of the active principle according to the invention is then evaluated by measuring the absorbance at 405 nm.

The modulation of this activity is expressed as a percentage inhibition or activation of the basal activity of HDACs, containing in particular Type I Sirtuins in the absence of active agents, that is to say only in the presence of the substrate of Sirtuines HDACs enzymes, including Type I Sirtuins.

Reference product

In this test, the reference product is an inhibitor namely Trichostatin A (TSA) 1 μΜ

Evaluation of the inhibitory or activating effects At the end of the incubation period, the activity of the HDACS enzymes, and in particular Sirtuines of type I with and without test or reference product, is revealed by staining with the aid of a developer solution (10 minutes at 37 ° C.) and evaluated by measuring the absorbance of the reaction media at 405 nm (OD 405).

For each concentration tested, the modulation of the deacetylating activity of the HDAC enzymes, and in particular of type I Sirtuins, by the test product is calculated according to the following formula:

Enzyme activity modulation percentage HDACs = 100 X [(DO405 test or reference product) - (DO405 HDACs)] DO405 HDACs

If the result is negative, the percentage is expressed as inhibition of the enzymatic reaction; if the result is positive, the percentage is expressed in activation of the enzymatic reaction.

The results are given in the form of percentages of activation or inhibition of HDACs, in particular type I sirtuins. From the triplicate data, the statistical significance of the differences observed between the "control" conditions, ie the controls and "reference product" is evaluated by a Student test (Student T test, p <0.05).

The extract according to the invention is in this test resulting from a 96 ° ethanolic solvent extraction, and is called LDV ET.

Table summarizing the results obtained after reading the DO405 T- = [T-] TSA1 μΜ (reference inhibitor) and [T] Basal activity of the enzyme

In this acellular In Tubo model, as of the dose of 0.01%, the extract according to the invention significantly increases the deacetylase activity of HDACs and in particular Type I sirtuins by 63.54%.

According to a physico-chemical aspect, unexpectedly beyond its biological activities, the extract according to the invention makes it possible to play an interface role in oil / water mixtures. Indeed, this extract makes it possible to stabilize as an interface agent a phase dispersed in a mobile phase.

Preparation 1: From 1% LDV ET dry matter, 9% sunflower oil is added to a laboratory test tube, the whole is vortexed at room temperature for 10 minutes and constitutes phase A.

Phase A is added to a 90% water beaker pH 5 containing 0.4% sodium benzoate and 0.2% potassium sorbate.

Shear force is used; a Silverson type homogenizer rotating at a speed of 5880 RPM is used for 10 minutes at room temperature. The emulsion 1 obtained is then observed under an optical microscope.

Observation preparation 1;

Photo a

This photograph was obtained by dilution to 1/6 of emulsion 1, during the observation with a high resolution optical microscope. In the light microscope,

we observe a dispersion of oily vesicles, and these vesicles are perfectly distinct, no coalescence phenomenon occurs between slide and coverslip during the observations under the microscope which are prolonged for 15 minutes. This observation highlights the good stability, and the resistance to the pressure exerted by the constraint of the crushing between blade and lamella. The vesicles formed are likely to accompany the stability of oil-in-water emulsions.

Photo b

In a dispersed system of oil in water, the dry matter of the extract according to the invention makes it possible to give rise to vesicles of vegetable oil by coating and / or coating the oily phase at the oil / water interface, the high-resolution optical microscope images make it possible to visualize these vesicles whose diameter is of the order of 5 microns. The dry matter according to the invention surrounds the microdroplets of vegetable oil, the dry matter is an interface agent capable of stabilizing a dispersed phase in a continuous phase in the manner of an encapsulation system. This oil / water interface property is reproducible when the dry matter / oil ratio is 1: 9. The extract according to the invention therefore makes it possible to film a

amount of vegetable oil at least nine times higher than its mass of dry matter. This feature allows the extract according to the invention to be useful for reinforcing the mobile phases in emulsions and for being also useful for carrying active substances that are sensitive in an aqueous medium, such as lipophilic vitamins, their derivatives, unsaponifiable compounds of oils, in particular vegetable oils, volatile lipophilic compounds such as perfumes, essential oils or flavorings, but also DHA (dihydroxyacetone), or to facilitate the solubility of tannins, anthocyanins, quinones, alkaloids, oligopeptides or amino acids, oses or polysaccharides.

According to another aspect, the present invention also covers the compositions, in particular the cosmetic and dermo-cosmetic compositions containing an unhydrolyzed extract of unclarified, non-decanted wine and / or an unhydrolyzed extract of settling feet at the end of the process. winemaking, and / or; an unhydrolyzed extract of wine lees comprising in particular from 0.1 to 1 mg / ml of peptides and oligopeptides, from 0.01 to 0.05% of polyphenols, from 0.01 to 0.5% of sugars and from 0.8 to 1% (40 to 50% of its dry mass) of polar lipids and polar compounds at a level of 0.8 to 1%, ie 40 to 50% of the dry matter according to the invention. In any cosmetic, dermo-) cosmetic or veterinary composition, the extract according to the invention may be used in the pure state of 0.001% to 100% of the total weight of the composition and preferably in a quantity representing 0.1 % to 10% of the total weight of the composition.

The preparation of a composition including the invention may be in all galenic forms, suitable both for topical application to the skin and / or the mucous membranes and / or the hair.

The composition including the invention is a cosmetic, dermo-cosmetic or veterinary composition as it is intended to improve the general cutaneous appearance of the individual or the animal that makes use of it.

For topical application to the skin, the hairs, the hair and / or the mucous membranes, the extract according to the invention or the composition including the invention comprises a cosmetically acceptable support, compatible with the skin, the mucous membranes, the nails , the hairs, the hair and can be in all galenical forms, especially in the form of an aqueous solution, hydroalcoholic or oily, an oil-in-water emulsion, a water-in-oil emulsion, an aqueous gel or oily, a liquid anhydrous product, pasty or solid. This composition may be more or less fluid and have the appearance of a white or colored cream, an ointment, a milk, a lotion, a serum, a paste, a mousse . It can optionally be applied to the skin in the form of an aerosol. It can also be in solid form, for example in the form of a stick.

Examples of formulations, below, allow to illustrate the compositions including the invention without however limiting the scope.

Example 1: BB cream cosmetic formula

* LDV ET = Extract according to the invention in ethanolic solution

Example 2: Shampoo & Shower Gel Treating 2 in 1

100

Example 3: Precious Oil Body and Face

100 * LDV MS = dry matter of the extract according to the invention

Example 4: Cosmetic Formula - SOOTHING Cream for the FACE

Example 5 Cosmetic Formula - Cleansing Water

Example 6: Cosmetic Formula - Anti-Aging Serum

Example 7 Cosmetic Formula - Face Scrub

LDV BE = extract according to the invention on bentonite support

Example 8: Inverse emulsion with precious oils

LDV BE = extract according to the invention on bentonite support

Example 9: Dessert Cream with Vanilla and Omegas 3

LDV MS = Dry matter of the extract according to the invention

Example 10: Guarana Energy Drink

LDV MS = Dry matter of the extract according to the invention

Bibliographical references [1] Ruiperez et al., J. immunol. 2007; 179: 631-638 [2] Oyoshi et al., PNAS, 2012, vol. 109; No. 13; 4992-4997 [3] Joshua et al., J. Agric. Food Chem., 2012, 60 (30): 7417-7420

Claims (11)

1) Use of a grape-derived cosmetic extract as a Tyrosinase activator capable of inhibiting the enzyme Phospholipase A2 (PLA2), in particular PLA2 group V, and the enzyme Lipoxygenase. 2) The use according to claim 1 of an extract derived from grapevine as a dermo-cosmetic active principle and its topical use in cosmetic, pharmaceutical or veterinary compositions which aim to stimulate tyrosinase by soothing on the cutaneous tissue. 3) Use according to claims 1 and 2 of an extract derived from the vine to improve the appearance of the skin by stimulating tyrosinase by avoiding the production of leukotrienes. 4) Use of an extract according to claims 1 and 2, characterized in that it is an unhydrolyzed extract of unclarified wine, not decanted or an unhydrolyzed extract of settling feet at the end of the vinification, or an unhydrolyzed extract of wine lees which is used as an active composition or as an active ingredient in pharmaceutical compositions intended for use in human or veterinary medicine, or also in cosmetic compositions. 5) Active ingredient according to claim 4 characterized by its ability to play an interface role in oil / water mixtures stabilizing O / W emulsions and E / H and its use in food composition, cosmetic, veterinary or pharmaceutical 6) active principle according to claim 4 characterized by its ability to act by inhibition of sPLA2-V, by inhibition of lipoxygenase, by inhibition of collagenase type I, by stimulation of tyrosinase, and by its ability to reduce wrinkles. 7) The use according to claim 4 of an active ingredient in topical cosmetic, pharmaceutical or veterinary compositions. 8) Use of an extract according to claims 1,2,5, 6 and 7, characterized in that it is composed in particular of a dry matter content of between 1 and 3%, and preferably between 1.5 and 2.5%. 9) Active ingredient according to claim 7, characterized in that its dry matter is characterized by a total sugar content of between 5 and 10%, with a protein content of between 7.5 mg / g and 20 mg / g, a content of phenolic compounds of between 5 and 10% of the dry matter of our extract according to the invention, a polar lipid content of between 30 and 35% and a content of polar organic compounds of between 40 and 50% . 10) Active ingredient according to claim 8 characterized in that the polar lipids of its dry matter are predominantly fatty esters, the latter represent about 30 to 35% of the dry mass of the extract according to the invention, and then between 5 and 7% of free fatty acids, the latter are mainly represented by palmitic acid and decanoic acid. 11) Active ingredient according to claims 1,2,5 to 10, characterized in that it is used in topical cosmetic, pharmaceutical or veterinary compositions between 0.001% to 100% of the total weight of the composition and preferably in a quantity representing from 0.1% to 10% of the total weight of the composition.