FR2938766A1 - Cosmetic composition, useful to prevent skin aging, comprises peptide hydrolyzate of vine leaves (Vitis vinifera) as aconitase activator and mitochondrial protector alone or n combination with other active ingredient, in medium - Google Patents

Abstract

Cosmetic or pharmaceutical composition comprises a peptide hydrolyzate of vine leaves ( Vitis vinifera) (I) as aconitase activator and mitochondrial protector, alone or in combination with at least one other active ingredient, in a medium. An independent claim is included for preparing the composition comprising dissolving (I) in one or more solvents comprising water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly and/or vegetable oil. ACTIVITY : Dermatological; Antidiabetic. MECHANISM OF ACTION : Aconitase activator.

Description

The present invention is in the field of cosmetics and pharmaceuticals, and more particularly in the field of dermatology. The subject of the present invention is a cosmetic and / or pharmaceutical and, in particular, dermatological composition comprising, in a physiologically acceptable medium, an active ingredient activating aconitase and protecting mitochondria, an effective amount of a hydrolyzate of vine leaves. (Vitis vinifera L.). Preferably, the active principle is a peptide hydrolyzate of grape leaves and contains mainly peptides. This active ingredient can be used alone or in combination with at least one other active ingredient. The present invention also relates to the use, in a cosmetic composition, of an effective amount of active ingredient derived from vine leaves (Vitis vinifera L.) to stimulate the functions of mitochondria and to increase the cellular energy level. The invention also relates to a cosmetic treatment method intended to protect the skin and the integuments from external aggressions and to fight against skin aging. Said active ingredient, activator of aconitase and protector of mitochondria, can also be used to prepare pharmaceutical compositions intended to prevent or combat pathologies related to mitochondrial dysfunctions; for example, certain neuromuscular or cardiac degenerations, type II diabetes or certain pathologies of aging.

Mitochondria are the organelles of eukaryotic cells specialized in the production of energy. They have their own DNA (mtDNA). The energy produced by the respiratory chain is stored as ATP. Under certain cellular stress conditions, electrons, released by the oxidative phosphorylation respiratory chain, produce free radicals that can damage mitochondrial structures. These stressors can be intrinsic or external. Among these are: UV radiation, toxins, radiation, food oxidants. Thus, it has been possible to show a progressive decline in mitochondrial functions with age, probably related to the accumulation of mutations on mtDNA (Singh, K., N.Y. Acad Sci, 1019, 2004). These mutations in mtDNA can be induced by repeated UV radiation exposures, and would even be a control of photo-aging (M. Bernburg et al., J. of Invest Dermatol 122 (5), 2004). Mitochondria also play a central role in apoptosis. Mitochondrial degradations can thus be a triggering factor of the reaction cascade leading to apoptosis in many cell types (G. Kroemer et al., FASEB J. 9: 1277-87, 1995). In mitochondria, natural mechanisms of repair have been identified. Aconitase (aconitate hydratase or EC Regulatory Protein, EC 4.2.1.3) thus proved to be essential for maintaining the integrity of mtDNA, regardless of its catalytic activity. Aconitase was first identified as a mitochondrial enzyme involved in the Krebs cycle; it catalyzes the stereospecific conversion of citrate to isocitrate and thus participates in the energy production of the cell. It has an active site with an iron-sulfur aggregate (4Fe-4S) with pseudo-cubic geometry. Recently, Dr. Butow's team (X.J. Chen et al., Science 307, 2005) has shown that aconitase plays a key role in preserving the integrity of mitochondrial DNA. It would interact with the DNA within regulatory regions suggesting a role in compaction of the mitochondrial genome in the manner of a protein of the HSP 70 family. The transition from the DNA binding form to the active form of the enzyme would be based on assembly or disassembly of the iron-sulfur cluster. Due to its dual functionality, aconitase establishes a direct link between the mitochondrial energy-generating activity and the conservation of mtDNA. Aconitase could thus constitute a therapeutic target to prevent or fight against pathologies related to mitochondrial dysfunctions, for example, type II diabetes, certain neuromuscular disorders, certain pathologies of aging.

The search for compounds that can stimulate or protect mitochondria is a concern of medical research and cosmetics. Thus, in order to combat the pathologies associated with the alteration of mitochondria or to combat the effects of cutaneous aging, solutions have been proposed such as the supply of substances involved in energy metabolism, and more particularly of intermediates or cofactors of the Krebs cycle such as fumarate, L-malate, acetyl CoA (WO 02064129) or the addition of substances capable of reducing free radicals, such as vitamin C (US 2004/0086526 ) or L-ergothioneine (WO 9836748). Furthermore, among the active ingredients for cosmetics extracted from the vine, we can find resveratrol extract of vine shoots rich in anti-aging properties (FR2898493), used in combination with other active agents. (FR2896990) or extracts of fresh vine cells grown in vitro with high polyphenol content (FR2808682) or a cosmetic composition containing 5 to 10% total extract of vine leaves (ES2172475). Nevertheless, the use of total extracts in cosmetic formulations has certain disadvantages. The complexity of their composition makes them difficult to characterize chemically. They may be responsible for certain intolerance reactions or allergic reactions, due, among other things, to the presence of certain native proteins. In addition, the presence of polyphenols, sensitive to oxidation, poses particular problems of cosmetic galenics. The object of the present invention is thus to overcome these disadvantages and to provide a cosmetic or pharmaceutical composition comprising a peptide-type hydrolyzate derived from grape leaves, capable of activating aconitase and protecting the mitochondria. Indeed, the peptides, and in particular the peptides of low molecular weight, do not cause allergic reactions and are therefore very interesting in dermato-cosmetic applications.

The main subject of the present invention is a cosmetic and / or pharmaceutical, and especially dermatological, composition comprising, in a physiologically acceptable medium, as active principle, an effective amount of a peptide hydrolyzate of grape leaves (Vitis vinifera L. .). Said active ingredient may be used alone or in combination with at least one other active ingredient. The inventors have indeed demonstrated that this active ingredient, when applied to the skin, significantly promotes mitochondrial activity, demonstrated by increased activity of the aconitase enzyme. The newly identified properties of this aconitase-activating active ingredient, capable of protecting the skin and the integuments from external aggressions, thus make it possible to open up new therapeutic and cosmetic perspectives. The term dander according to the invention encompasses all the keratinous appendages present on the surface of the body, in particular the hairs, the eyelashes, the eyebrows, the nails and the hair. Mitocondria is a protective active ingredient or capable of protecting the mitochondria, any substance of plant origin, and more particularly derived from the vine leaf (Vitis vinifera L.), capable of limiting functional alterations or of reducing the risk of damage. structural mitochondria of cells or tissues subjected to physicochemical or environmental stress. By active ingredient capable of activating the aconitase or activating active ingredient of aconitase, is meant any substance of vegetable origin, and more particularly derived from the vine leaf (Vitis vinifera L.), capable of increasing the cytoplasmic or mitochondrial aconitase, either by activation of protein synthesis of aconitase (by direct or indirect modulation of the gene expression of aconitase), or by increasing the enzymatic activity of aconitase or by other biological processes such as the stabilization of the aconitase protein or the stabilization of the messenger RNA transcripts. The term "hydrolyzate or derived from hydrolysis" refers to any substance or mixture of substances, or isolated preparation, obtained after hydrolysis of plant material. By peptide hydrolyzate is meant a hydrolyzate essentially consisting of peptides. The term peptide refers to a sequence of two or more amino acids bound together by peptide bonds or modified peptide bonds. Peptide-type compounds are understood to mean the protein fragments, the peptides and the free amino acids present in the mixture according to the invention. It is obvious that the invention is directed to mammals in general and more particularly to humans. The active ingredient according to the invention can be obtained by extraction of proteins of vegetable origin, followed by controlled hydrolysis which liberates biologically active peptide fragments. By biologically active expression is meant that which has an activity in vivo or in vitro characteristic of the activity of the active ingredient according to the invention. Many proteins present in plants are likely to contain biologically active peptide fragments within their structure. The controlled hydrolysis makes it possible to release these peptide fragments. It is possible, but not necessary to carry out the invention, to either extract the relevant proteins first and then hydrolyze them, or to perform the hydrolysis first on a crude extract and to purify the peptide fragments. then. It is also possible to use certain hydrolysed extracts without purifying the peptide fragments corresponding to the biologically active peptides according to the invention, but nevertheless ensuring the presence of said fragments by appropriate analytical means. More particularly according to the invention, one of the many plants of the family Vitaceae (or vitaceae), of the genus Vitis and preferably of the species Vitis vinifera L. Vitis vinifera L. is a sarmentous shrub of which there exists innumerable cultivated varieties, called grape varieties (cabernet, chardonnay, merlot, pinot, sauvignon, etc.). The webbed vein leaves have five main lobes more or less cut and are heart-shaped at the base. The main identified chemical components of the vine are tannins, quercetin, quercitrin, tartrates, sugars, inosites, acids, choline and carotene. Among the many products derived from the vine, mention may be made of grape seed oil, retinol from grapes or resveratrol, which has recently been used extensively in cosmetics. The traditional pharmacopoeia also describes the use of plant sap and leaves for their astringent and anti-inflammatory properties. To carry out the extraction according to the invention, it is possible to use the whole plant or a specific part of the plant (leaf, fruit, etc.). Preferably, the leaves are used. The proteins of the plant are then extracted according to the conventional method (Osborne, 1924) modified; the plant mash is suspended in an alkaline solution containing an insoluble polyvinylpolypyrrolidone adsorbent material (PVPP) (0.01-20%); in fact, it has been observed that the hydrolysis and subsequent purification operations are facilitated by this means. The concentration of phenolic-type substances interacting with the proteins is thus reduced. The soluble fraction is collected after centrifugation and filtration steps, this crude solution then constituting a first form of the extract containing proteins, carbohydrates and possibly lipids. The proteins are then precipitated by varying the ionic strength by acidifying the medium, which eliminates soluble components and nucleic acids. The precipitate is then washed with water, acidic pH buffer or an organic solvent such as, for example, ethanol or methanol, and the solvent is evaporated by drying in vacuo. The protein-rich precipitate is dissolved in water or other solvent and is then a more purified form of the hydrolyzate. The extraction can also be carried out in neutral or acidic medium always in the presence of polyvinylpolypyrrolidone. After a filtration step, the precipitation step is then carried out using a conventional precipitation agent such as salts (sodium chloride, ammonium sulfate) or an organic solvent (alcohol). acetone). The precipitate obtained can be separated from the precipitating agents by dialysis after redissolving in water or another solvent. The isolated protein fraction according to the invention is then hydrolyzed under mild conditions to generate soluble polypeptides and peptides. Hydrolysis is defined as a chemical reaction involving the cleavage of a molecule by water, this reaction being possible in a neutral, acidic or basic medium. According to the invention, the hydrolysis is carried out chemically and / or advantageously by proteolytic enzymes. The use of endoproteases of plant origin (papain, bromelain, ficin) and of microorganisms (Aspergillus, Rhizopus, Bacillus, etc.) can then be mentioned. For the same reasons as above, during this controlled hydrolysis step, an amount of polyvinylpolypyrrolidone is added to the reaction medium. After filtration, the solution obtained constitutes the active hydrolyzate. The vegetable hydrolyzate obtained according to the invention is analyzed qualitatively and quantitatively for its physicochemical characteristics and its content of protein and peptide compounds. Thus, according to an advantageous embodiment of the invention, the active plant hydrolyzate, which constitutes the active principle, has a pH of between 4 and 7, and preferably between 5 and 20 6, its solids content of 3 to 8 g / 1 and preferably from 4 to 6 g / l, its content of compounds of peptidic nature is between 0.5 and 5 g / l, and preferably between 1.5 and 3.5 g / l and its content in sugars between 0.5 and 1.5 g / 1. The active hydrolyzate can be further purified by tangential flow filtration steps to remove the high molecular weight proteins and select the low molecular weight peptides. Any of the more or less purified forms of the hydrolyzate is then solubilized in water or in any mixture containing water, and then sterilized by ultrafiltration. According to an advantageous embodiment of the invention, the active principle of peptide nature consists of peptides of low molecular weight, preferably of molecular weight less than 10 kDa, and even more preferably of molecular weight between 2 and 5 kDa. According to an advantageous embodiment of the invention, the active principle according to the invention is first solubilized in one or more cosmetically or pharmaceutically acceptable solvents, conventionally used by the person skilled in the art, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents. According to yet another advantageous embodiment of the invention, the active principle according to the invention is solubilized beforehand in a cosmetic or pharmaceutical vector such as liposomes or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilized in, or attached to, any cosmetically or pharmaceutically acceptable carrier. The effective amount of active ingredient according to the invention corresponds to the amount necessary to obtain the desired result, namely: activate the aconitase, protect the mitochondria and thus protect the skin from external aggression and fight against skin aging. According to an advantageous embodiment of the invention, the active principle is present in the compositions of the invention at a concentration of between 0.0001% and 20% approximately, and preferably at a concentration of between 0.05% and Approximately 5% relative to the total weight of the final composition.

The compositions according to the invention may be applied by any appropriate route, in particular oral, parenteral or external topical, and their formulation will be adapted by those skilled in the art, in particular for cosmetic or dermatological compositions. Advantageously, the compositions according to the invention are intended for topical administration to the skin. These compositions must therefore contain a cosmetically and / or dermatologically acceptable medium, that is to say compatible with the skin and superficial body growths, and cover all the cosmetic or dermatological forms. These compositions additionally comprise any additive commonly used in the intended field of application as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, agents and the like. self-tanners, pigments, fillers, preservatives, fragrances, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc. In all cases, those skilled in the art will ensure that these adjuvants and their proportions are chosen so as not to adversely affect the desirable properties of the composition according to the invention. These adjuvants may, for example, represent between 0.01 and 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent from 5 to 80% by weight and preferably from 5 to 50% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those conventionally used in the field under consideration. For example, they can be used in a proportion ranging from 0.3 to 30% by weight, relative to the total weight of the composition. The compositions which may be used according to the invention may be in the form of creams, oil-in-water or water-in-oil emulsions or multiple emulsions, solutions, suspensions, gels, milks, lotions, sticks or powders, suitable for an application on the skin, lips and / or integuments. The compositions that may be used according to the invention may also consist of a composition for hair care, and in particular a shampoo, a conditioner, a setting lotion, a treatment lotion, a cream or a styling gel, a restructuring lotion for hair, a mask, etc. The cosmetic composition according to the invention can be used in particular in treatments using an application that is followed or not followed by rinsing, or in the form of shampoo. The compositions according to the invention may also be in the form of dye or mascara to be applied by brush or comb, in particular on the eyelashes, the eyebrows or the hair.

It is understood that the active ingredient according to the invention may be used alone or in combination with at least one other active ingredient which promotes the action of said active ingredient. Advantageously, the compositions according to the invention further comprise various active principles intended, in particular, for the prevention and / or treatment of disorders associated with photoaging. For example, other active ingredients having an anti-radical or antioxidant action may be added, selected from vitamin C, vitamin E, coenzyme Q10 and polyphenolic extracts of plants. The composition according to the invention can also associate with the active principle according to the invention, other active ingredients stimulating the synthesis of dermal macromolecules (laminin, fibronectin, collagen), for example the collagen peptide marketed under the name Collaxyl ° by the company Vincience. The composition according to the invention can also associate with the active ingredient according to the invention, other active ingredients stimulating the energy metabolism, such as the active ingredient marketed under the name GP4G ° by the company Vincience. By its particular activities, the active ingredient according to the invention can be used advantageously in a cosmetic composition or for the preparation of a pharmaceutical composition. Thus, according to a particular aspect of the invention, the active ingredient can be advantageously used in a cosmetic composition to activate aconitase and protect mitochondria. In another aspect of the invention, the active ingredient is used in a cosmetic composition to protect the skin and integuments against all types of external aggression. The use of the active ingredient, or a composition containing it, allows the skin and integuments to be protected and to better withstand environmental stresses. By the term external aggression, we mean the aggressions that the environment can produce. By way of example, mention may be made of aggressions such as pollution, UV, or irritating products such as surfactants, preservatives or perfumes. Pollution is understood to mean both external pollution, due for example to diesel particles, ozone or heavy metals, as well as internal pollution which may be due in particular to the solvent emissions of paints, glues, or wallpaper (such as toluene, styrene, xylene or benzaldehyde), or even cigarette smoke.

According to one particular aspect of the invention, the active ingredient is used in a cosmetic composition for protecting the skin and the integuments against the particular type of external aggressions that are ultraviolet (UV) radiation. Thus, the active ingredient according to the invention can be advantageously used in a cosmetic composition as a photoprotective agent, and more particularly as a so-called secondary photoprotective agent. In fact, the primary photoprotective agents are distinguished from the secondary photoprotective agents. Primary photoprotective agents are substances that exert physical power: they are able to absorb UV radiation and return it in the form of heat to protect the skin. Secondary photoprotective agents are substances that usually have a biological effect; these are, for example, the agents capable of limiting damage to DNA and membranes by the penetration of UV radiation into the skin. According to this aspect of the invention the composition may be a solar composition, i.e. a composition aiding in the protection against solar radiation. Thus, it can be advantageously added to the composition according to the invention, assets assisting the sun protection such as, for example, sunscreens. These compositions may especially be in the form of an aqueous solution, hydroalcoholic or oily; an oil-in-water, water-in-oil emulsion or multiple emulsions; they may also be in the form of creams, suspensions or powders, suitable for application to the skin, mucous membranes, lips and / or integuments. These compositions may be more or less fluid and have the appearance of a cream, lotion, milk, serum, ointment, gel, paste or paste. a foam. They may also be in solid form, such as a stick, or applied to the skin as an aerosol. They can be used as a care product and / or as a make-up product for the skin.

According to another aspect of the invention the active ingredient can be used for the preparation of a pharmaceutical composition for preventing or treating damage to the skin by sun exposure or exposure to ionizing radiation during radiation therapy. .

The invention also relates to the use, in a cosmetic composition, of an effective amount of active ingredient according to the invention for preventing or treating damage caused to the skin and to the integuments by free radicals.

According to yet another aspect of the invention, said active ingredient is advantageously used in a cosmetic composition intended to fight in a preventive and / or curative manner against the manifestations of cutaneous aging, and more specifically, in order to fight against and / or prevent photo-induced aging (photo-aging). Skin manifestations of aging means any changes in the external appearance of the skin and skin growth due to aging such as, for example, wrinkles and fine lines, wilted skin, soft skin, thinned skin, lack of elasticity and / or tone of the skin, dull and lackluster skin or skin pigmentation spots, discoloration of the hair or spots on the nails, but also any internal changes in the skin that does not systematically result by a modified external appearance such as, for example, any internal degradation of the skin resulting from exposure to ultraviolet (UV) radiation. The active ingredient according to the invention, or the composition containing it, will make it possible, in particular, to combat the loss of elasticity and firmness of the skin.

According to yet another aspect of the invention, said active ingredient can be formulated to attenuate a pathology related to mitochondrial dysfunctions. Thus, the invention also consists in using an effective amount of said active ingredient to prepare a pharmaceutical composition for preventing or combating mitochondrial dysfunctions, such as neuromuscular degeneration, cardiac degeneration, type II diabetes. .

The invention also consists in a cosmetic treatment method intended to stimulate the defenses and to protect the skin and the integuments from external aggressions and to fight against skin aging, characterized by the topical application, on the skin or integuments to be treated. , a composition comprising an effective amount of active ingredient according to the invention.

The invention also consists in a cosmetic treatment method intended to prevent or treat cutaneous manifestations of aging and / or photo-aging, characterized by the topical application, on the skin or integuments to be treated, of a composition comprising a effective amount of active ingredient according to the invention.

Particular embodiments of this cosmetic treatment method also result from the foregoing description. Other advantages and characteristics of the invention will appear better on reading the examples given by way of illustration and not limitation. Example 1 Preparation of Active Ingredient from Grape Leaves (Vitis vinifera L.) In a first step, 1 kg of fresh leaves of vine is suspended in an aqueous alkaline solution (dilution 1/10) pH 10 containing 1% polyvinylpolypyrrolidone (Polyclar V ISP). The leaves are crushed with an ultra Turrax grinder. This mixture is stirred for a time long enough to allow the solubilization of the soluble fractions. The extraction temperature is variable (between 4 and 80 ° C), preferably the operation is carried out cold. After this extraction phase, the medium is clarified by centrifugation and then filtered through a plate filter. This filtrate which contains the soluble fractions of grape leaves is then subjected to precipitation of the proteins by varying the ionic strength in acid medium (pH 3.5). This operation largely eliminates soluble carbohydrate components, polyphenols and nucleic acids. The supernatant is removed and the precipitate is then washed with water. At this stage, the precipitate constitutes the crude protein extract, which is in the form of a brown-colored paste whose dry extract is characterized by: Proteins: 50 to 80% Carbohydrates: 10 to 30%

The protein-rich precipitate is redissolved in water. The crude protein extract is then subjected to a series of controlled and selective hydrolyses consisting of chemical and enzymatic hydrolyses in the presence of 0.5% PVPP (Polyclar V) and cysteine endopeptidases (Bromelain). After reaction, the hydrolyzate is filtered on a plate and then on a sterilizing cartridge (0.21 μm). An amber-colored hydrolyzate, titrating with 25 to 35 g / l of dry extract, is then obtained, which is then diluted so that the concentration of peptide-like compounds, determined by the method of Lowry, is between 0.degree. , 5 and 5g / l. The physico-chemical analysis of the vegetable hydrolyzate, which constitutes the active ingredient, shows that its pH is between 4 and 7, and preferably between 5 and 6, the solids content of 3 to 8 g / l and preferred manner of 4 to 6 g / 1, its content of peptide compounds is between 0.5 and 5 g / l, and preferably between 1.5 and 3.5 g / 1 and its sugar content between 0 , 5 and 1.5 g / l. A variant of the above protocol is to carry out the same sequence of mild and selective enzymatic hydrolysis in the presence of 0.5% PVPP. An amber-colored hydrolyzate grading 25 to 35 g / l of dry extract after sterilizing filtration is then obtained. The protein nature of this extract is evidenced by polyacrylamide gel electrophoresis. For this analysis, NuPAGE -Tricine 16% gels (Invitrogen) are used. The protein extract is heated at 70 ° C for 10 minutes under denaturing reducing conditions in NuPAGE LDS sample preparation buffer. An antioxidant NuPAGE solution is added to the lower vat (cathode) to prevent the reduced proteins from reoxidizing during electrophoresis. Protein migration is performed using the NuPAGE MES migration buffer with Mark 12 and / or Sharp standards as a molecular weight marker. Protein staining is performed using the SilverXpress silver staining kit (Invitrogen). Under these conditions, proteins with molecular weights between 70 kDa and less than 3 kDa are observed. This solution is then purified by removing the high molecular weight proteins by tangential flow filtration. The solution is ultrafiltered on a filter cartridge through a Pellicon support equipped with Pellicon 2 Biomax 50 kDa cassette. This ter filtrate is recovered and then filtered through a second Pellicon Biomax 30 kDa cassette. After this step, a second filtrate is obtained which is further eluted through a last Pellicon 2 Biomax 5 kDa cassette. At the end of purification, we obtain a plant extract of vine leaves of amber color, bright and limpid. It is characterized by a dry weight of 15 to 20 g / l, a protein content of 12 to 15 g / l and a sugar concentration of less than 0.5 g / l. The extract is then diluted so that the concentration of peptide compounds determined by the method of Lowry is between 0.5 and 5 g / l, and preferably between 1.5 and 3.5 g. / 1 and its sugar content less than 0.5 g / l. This solution is then analyzed in high pressure liquid chromatography using an HP1100 device controlled by the ChemStation software. The column used during the elution of the protein extract is a Nucleosil 300-5 C4 MPN (125 x 4 min). This column makes it possible to chromatograph, according to a suitable solvent gradient, proteins having molecular weights of 0.2 to 25 kDa. Under these chromatographic conditions, several peptide fractions were isolated. These various fractions were analyzed by mass spectrometry to identify their molecular peaks. Under these conditions, only peptides of low molecular weight, preferably peptides with a molecular weight of less than 10 kDa, and even more preferentially peptides with a molecular weight of between 2 and 5 kDa, are observed.

Another variant consists in carrying out a purification of the extract, obtained according to one of the above protocols, by ion exchange chromatography, on a TSK gel (TosoHaas) column with a pH 7 phosphate buffer.

EXAMPLE 2 Demonstration of the Activating Effect of the Active Principle According to Example 1 on the Enzymatic Activity of Aconitase The aim of this study is to determine the influence of the active ingredient according to Example 1 on the enzymatic activity of aconitase. For this, the total enzymatic activity (cytoplasmic and mitochondrial) and the mitochondrial enzymatic activity of aconitase were measured. Protocol: Normal human fibroblasts are treated with a 1% solution of the active ingredient according to Example 1 for 48 hours and then irradiated with UVB at 100mJ / cm 2. After irradiation, the cells are treated under the same conditions for another 24 hours. Negative controls are carried out under the same conditions but in the absence of active principle according to Example 1. To evaluate the enzymatic activity of aconitase, the fibroblasts are then harvested and centrifuged in PBS buffer. Part of the cells is then treated to perform the assay of the total enzyme activity, another part is used to isolate the mitochondrial fraction according to the following protocol: The cells are lysed using a Dounce type homogenizer and the The homogenate is centrifuged cold at 10,000 g for 10 minutes. The pellet, rich in mitochondria, is washed with TES buffer (10 mM Tris, 1 mM EGTA, 0.25 M sucrose) and resuspended in 0.2 mM trisodium citrate buffer before being used for the enzyme assay. The determination of the enzymatic activity of aconitase is carried out using the OXIS Bioxytech Aconitase-340 assay Kit (Oxys International), according to the following principle: the aconitase causes the isomerization of citrate to isocitrate. The isocitrate is then converted to alpha-ketoglutarate by the enzyme isocitrate dehydrogenase. This second reaction is accompanied by the transformation of NADP + into NADPH. It is the formation of NADPH which is quantified by spectrophotometry at 340 nm. Under the chosen experimental conditions, the rate of NADPH formation is proportional to the amount of aconitase. Analysis of the appearance kinetics of NADPH makes it possible to calculate the aconitase concentration of the sample (in mU / ml). Results: Assays for the total enzymatic activity of aconitase show an increase of more than 55% in activity in cells treated with the active ingredient according to Example 1 compared to untreated control cells. When the cells are irradiated with UVB, on the contrary, a decrease of more than 30% of this activity is observed. The treatment, before and after the UVB irradiation, with the active principle according to Example 1, makes it possible to limit the decrease observed in the untreated control cells. Assays of the enzymatic activity of mitochondrial aconitase show comparable results. Conclusions: The active ingredient according to Example 1 strongly stimulates the enzymatic activity of cytoplasmic and mitochondrial aconitases of cutaneous cells. The action of the active ingredient according to Example 1 protects cells and mitochondria against UVB stress.

EXAMPLE 3 Evaluation of the Protective Effect of the Active Principle According to Example 1 on Cutaneous Cells Subject to Ultraviolet Radiation (UVB) The aim of this study is to determine the protective effect of the active ingredient according to Example 1 to dermal fibroblasts subjected to stress by UVB radiation. For this, cell viability tests were performed by the MTT technique. Protocol: Human dermal fibroblasts are treated with a solution containing 1% of the active principle according to Example 1, for 24 hours, irradiated with UVB (from 25 to 100 mJ / cm 2) and then cultured for a further 24 hours in the presence of the same concentration of active principle according to Example 1. Untreated and non-irradiated controls and untreated but irradiated controls are carried out under the same conditions. At the end of the experiment, the cells are incubated in a solution containing 0.1 mg / ml of MTT (344.5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide). This compound is absorbed by living cells and then metabolized by the mitochondrial enzymes into a blue violet compound, formazan, which will be assayed spectrophotometrically at 540 nm. The optical density (O.D.) is then directly proportional to the mitochondrial enzymatic activity as well as to the number of living cells. Results: The evaluation of cell viability by the MTT technique shows, on the one hand, that the active ingredient according to Example 1, independently of any UVB radiation, increases the cell viability and secondly that the UVB irradiations have had a dose-dependent cytotoxic effect on human fibroblasts, but that this cytotoxic effect is not manifested in the presence of 1% of active principle according to Example 1. Conclusions: The active ingredient according to Example 1 increases cell viability and on the other hand, effectively protects the skin cells against the cytotoxic effects of UVB radiation.

EXAMPLE 4 Evaluation of the Protective Effect of the Active Principle According to Example 1 on Cutaneous Cells Under Oxidative Stress The aim of this study is to determine the protective effect of the active ingredient according to Example 1 with respect to dermal fibroblasts subjected to oxidative stress caused by hydrogen peroxide (H2O2) at 4 mM or 5 mM. For this, cell viability tests were performed by the technique at MIT. Protocol: The human dermal fibroblasts are treated with a 1% solution of the active principle according to Example 1, for 24 hours, subjected to oxidative stress caused by H 2 O 2 at 4 mM or 5 mM, for 30 minutes then grown for another 24 hours in the presence of the same concentration of active principle according to Example 1. Controls not treated with the active ingredient or with H 2 O 2 and treatment controls with the active principle alone are carried out under the same conditions . At the end of the experiment, the cells are incubated in a solution containing 0.1 mg / ml of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium, bromide), according to protocol described in Example 4. Results: The evaluation of the cell viability by the MTT technique shows, on the one hand, that the active ingredient according to Example 1, independently of oxidative stress, increases the viability on the other hand, that an oxidative stress caused by H202 at 4 mM or 5 mM for 30 minutes had a dose-dependent cytotoxic effect on human fibroblasts, but that this cytotoxic effect does not occur not in the presence of 1 active ingredient according to Example 1. Conclusions: The active ingredient according to Example 1 increases the cell viability and effectively protects the skin cells against the cytotoxic effects of oxidative stress. EXAMPLE 5 Evaluation of the cytoprotective effect of the active principle according to Example 1 on cutaneous cells subjected to ultraviolet radiation (UVB) A study of the cytoprotective effect of the active principle according to Example 1 was carried out performed by assessing the damage caused at the DNA level. This study was carried out using the comet cell assay (or Single Cell Gel Electrophoresis), a short and sensitive microelectrophoretic technique that allows the visualization and quantification of DNA breaks on individual cells. Protocol: Primary human fibroblasts are inoculated into dishes of diameters 100. When the cells have reached 70% confluence, they are treated with a 1% solution of active principle according to Example 1 for 24 hours. The cells are then subjected to UVB irradiation at 100 mJ / cm 2 and then cultured in the presence of the active ingredient for a further 24 hours. Boxes not treated with the active ingredient serve as control. A cell suspension at 100,000 cells / ml is then performed, 500 l are taken, included in an agarose solution and cast between slide and coverslip. The cells are cold lyced. The DNA is then denatured with an alkaline buffer and then subjected to a short electrophoresis (250 mA for 30 minutes) and evidenced by propidium iodide staining. The slides are then observed under a fluorescence microscope. An altered cell sees its DNA stretch toward the anode in proportion to the number of breaks in the DNA and form a comet. The highly degraded DNA is in the tail of the comet. An intact cell remains round and its DNA is then compacted at the head of the comet. The evaluation of DNA breaks is carried out using an image analysis software which makes it possible to determine the percentage of degradation of the DNA, by the quantitative evaluation of the Tail Moment, a parameter that defines itself. as the product of the length of the comet by the percentage of DNA in its distal part. Results: The results obtained show that the cells subjected to UVB irradiation, in the absence of the active principle according to Example 1 (UVB control), have the most important Tail Moment, that is to say the condition where DNA is the most damaged. In contrast, the cells treated with the active ingredient according to Example 1, have a Tail Moment, ie DNA damage, decreased by 25%. Conclusion: The active ingredient according to Example 1 plays an important role in protecting the DNA of cutaneous cells subjected to irradiation with UVB.

EXAMPLE 6 Preparation of compositions 1 - Sun protection cream: Trade names I INCI names I% by weight PHASE A Demineralised water Aqua (Water) qsp Pemulen TRI Acrylates / C10-30 Alkyl Acrylate 0.40 Crosspolymer Glycerine Glycerin 3.00 Nipastat Sodium Sodium Methylparaben (and) Sodium 0.15 Ethylparaben (and) Sodium Butyl paraben (and) Sodium Propylparaben (and) Sodium Isobutylparaben PHASE B Parsol MCX Ethylhexyl Methoxycinnamate 7.50 Eusolex 4360 Benzophenone-3 3.00 Parsol 1789 Butyl Methoxydibenzoylmethane 2.00 Myritol 318 Caprylic / Capric Triglyceride 4.00 Emulgade SEV Hydrogenated Palm Glycerides (and) 5.00 Ceteareth-20 (and) Ceteareth-12 (and) Cetearyl Alcohol Propylparaben Propylparaben 0.15 Nacol 16-98 Cetyl Alcohol 1.00 PHASE C TEA I Triethanolamine 0.20 PHASE D Active principle according to Example 1 3.00 Perfume Fragrance qsp Colorant qsp The constituents of phase A and phase B are separately heated between 70 ° C and 75 ° C. Phase B is emulsified in phase A with stirring. Phase C is added at 45 ° C, increasing stirring. Phase D is then added when the temperature is below 40 ° C. Cooling is continued up to 25 ° C with vigorous stirring. -18- After-sun milk: Trade names INCI Names I% by mass 1 PHASE A Montanov LC 14-22 Alcohols (and) C 12-20 Alkyl 3.00 Glucoside Waglinol 2559 Cetearyl Isononanoate 4.00 Tegosoft TN C12-15 Alkyl Benzoate 3.00 Apricot Kernel Oil Prunus Armeniaca (Apricot) Kernel 2.00 Oil Avocado Oil Persea Gratissima (Avocado) Oil 1.00 Abil 350 Dimethicone 1.00 PHASE B Demineralized Water Aqua (Water) qsp PHASE C Simulgel EG Sodium Acrylate / Acryloyldimethyl 0.4 Taurate Copolymer (and) Isohexadecane (and: Polysorbate 80 Copolymer (and) Polysorbate 80 PHASE D Phenonip Phenoxyethanol (and) Methylparaben 0.30 (and) Ethylparaben (and) Butylparaben (and) Propylparaben (and) ) Isobutylparaben Ethylparaben and Propylparaben and Buthylparaben Germail 115 Imidazolidinyl Urea 0.20 PHASE E Active principle according to Example 1 0,10 -19- 2938766 -20- Prepare phase A with stirring and stir in the xanthan gum gradually, with deflocculating stirring. Phases C and D will be incorporated u do not once the gel is finished. Phase E, prepared before perfect dissolution of the DHA, will be added later. Adjust the pH if necessary to 4 - 4.5. Color and perfume.

3 ûAnti-Aging Cream: Trade Names INCI Names J% Mass Phase A Montanov 68 Cetearyl Alcohol (and) Cetearyl Glucoside 6.00 Squalane Squalane 3.00 Cetiol SB 45 Butyrospermum Parkii (Shea Butter) 2.00 Waglinol 250 Cetearyl Ethylhexanoate 3.00 Amerchol L- 101 Ore Oil (and) Lanolin Alcohol 2.00 Abil 350 Dimethicone 1.50 BHT BHT 0.01 Coenzyme Q10 Ubiquinone 0.10 Phase B Avocado Oil Persea Gratissima (Avocado) Oil 1.25 Phenonip Phenoxyethanol (and) Methylparaben (and) 0.75 Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben Phase C Demineralized Water Aqua (Water) qsp Butylene Glycol Butylene Glycol 2.00 Glucam E10 Methyl Gluceth-10 1.00 Allantoin Allantoin 0.15 Carbopol Ultrez 10 Carbomer 0.20 Phase D 2938766 -21- Trade Names INCI Names% by mass TEA Triethanolamine 0.18 Phase E Active ingredient according to Example 1 0.50 GP4G Water (and) Artemia Extract 1.50 Collaxyl Water (and) ) Butylene Glycol (and) Hexapeptide- 3.00 9 Phase F Fragrance Perfume qsp Colorant qsp Prepare and melt phase A at 65-70 ° C. Heat phase C at 65-70 ° C. Phase B is added to phase A just before emulsifying A in B. At about 45 ° C, the carbomer is neutralized by addition of phase D. Phase E is then added with gentle stirring and cooling is continued up to 25 ° C. Phase F is then added if desired.

4 ûShield Day Cream: Trade Names INCI Names% by mass Phase A Emulium Delta Cetyl alcohol (and) Glyceryl Stearate (and) 4.00 PEG-75 Stearate (and) Ceteth-20 (and) Steareth-20 Lanette O Cetearyl Alcohol 1 , 50 DC 200 Fluid / 100cms Dimethicone 1.00 DUB 810C Coco Caprylate / Caprate 1.00 DPPG Propylene Glycol Dipelargonate 3.00 DUB DPHCC Dipentaerythrityl 1.50 Hex acylate / Hex acaprate 2938766 Cegesoft PS6 Vegetable Oil 1, 00 Vitamin E Tocopherol 0.30 Phenonip Phenoxyethanol (and) Methylparaben (and) 0.70 Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben Phase B Demineralized Water Aqua qs 100 Glycerine Glycerin 2.00 Carbopol EDT 2020 Acrylates / C 10-30Alkyl Acrylate 0.15 Crosspolymer Keltrol BT Xanthan Gum 0.30 Phase C Sodium Hydroxide (Sodium Hydroxide 0.30 10%) Phase D Demineralized Water Aqua 5.00 Stay-C 50 Sodium Ascorbyl Phosphate 0.50 Phase E Butylene Glycol Butylene Glycol 2.00 Dekaben CP Chlorphenesin 0.20 Phase F GP4G Water ( and) Artemia Extract 1.00 Active ingredient according to Example 1 5.00 Prepare phase A and heat to 75 ° C with stirring. Prepare phase B by dispersing the carbopol, then the xanthan gum with stirring. Let rest. Heat to 75 ° C. At temperature, emulsify A in B with rotor-stator stirring. Neutralize with phase C with rapid stirring. After cooling to 40 ° C., add phase D and then phase E. Cooling is continued with gentle stirring and phase F added.

Claims (16)

REVENDICATIONS1. Cosmetic or pharmaceutical composition characterized in that it comprises, as an activating active ingredient of the aconitase and protector of mitochondria, in a physiologically acceptable medium, a peptide hydrolyzate of grape leaves (Vitis vinifera L.), used alone or in combination with association with at least one other active ingredient. 2. Composition according to claim 1, characterized in that said active ingredient contains between 0.5 and 5 g / l, and preferably between 1.5 g / l and 3.5 g / l of peptide-like compounds. 3. Composition according to one of Claims 1 or 2, characterized in that the compounds of peptide nature contained in said active principle are peptides with a molecular weight of less than 10 kDa. 4. Composition according to claim 3, characterized in that the compounds of peptide nature contained in said active principle are peptides of molecular weight between 2 and 5 kDa. 20 5. Composition according to any one of the preceding claims, characterized in that the active ingredient is used in an amount representing from 0.0001% to 20% of the total weight of the composition, and preferably in an amount representing 0.05. % to 5% of the total weight of the composition. 25 6. Composition according to any one of the preceding claims, characterized in that it is in a form suitable for topical application comprising a cosmetically or dermatologically acceptable medium. 7. Composition according to any one of the preceding claims, characterized in that it further comprises at least one other active ingredient promoting the action of said active ingredient. 2938766 -24- 8. Composition according to claim 7, characterized in that the other active ingredient is selected from the antioxidant active ingredients and / or the active ingredients stimulating the synthesis of the extracellular matrix, and / or the active principles stimulating the energetic cellular metabolism. 5 9. Process for the preparation of a composition characterized in that said active ingredient is first solubilized in one or more cosmetically or pharmaceutically acceptable solvents, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petrolatum, a vegetable oil or any mixture of these solvents. 10. Use in a cosmetic composition, an effective amount of active ingredient, as defined in any one of claims 1 to 4, to protect the skin and integuments against all types of external aggression. 11. Use according to claim 10 characterized in that the external aggressions are UV radiation. 12. Use in a cosmetic composition of an effective amount of active ingredient as defined in any one of claims 1 to 4 for preventing or treating damage to the skin and integuments by free radicals. 13. Use in a cosmetic composition of an effective amount of active ingredient as defined in any one of claims 1 to 4 for preventing or treating cutaneous manifestations of aging and / or photo-aging. 14. Use of an effective amount of active ingredient, as defined in any one of claims 1 to 4, for preparing a pharmaceutical composition for preventing or combating mitochondrial dysfunctions, such as neuromuscular degenerations, cardiac degeneration, type II diabetes. 15 5 2938766 -25- 15. Cosmetic treatment process intended to stimulate the defenses and to protect the skin and the integuments from external aggressions, characterized in that a composition comprising an effective quantity of active principle is applied topically to the skin or integuments to be treated. as defined in one of claims 1 to 4. 16. A cosmetic treatment method for preventing or treating cutaneous manifestations of aging and / or photo-aging, characterized in that a composition comprising an effective amount of principle is applied topically to the skin or integuments to be treated. active agent as defined in one of claims 1 to 4.