FR2949781A1 - New peptide hydrolyzate enriched in bioactive peptide capable of activating the human transglutaminase useful e.g. to enhance the barrier function of the epidermis and stimulate the epidermal regeneration and differentiation - Google Patents

Abstract

Peptide hydrolyzate (A) enriched in bioactive peptide capable of activating the human transglutaminase, is new, where the bioactive peptide contains 4-6 amino acids having at least two arginine residues, glycine residue and glutamine residue. An independent claim is included for cosmetic composition comprising (A) (0.0001-20 wt.%, preferably 0.05-5 wt.%) in a medium. ACTIVITY : Dermatological. MECHANISM OF ACTION : Transglutaminase activator. The ability of (I) to activate transglutaminase was tested in normal human keratinocytes. The results showed that the transglutaminase activity of (I) was increased by 30% after 24 hours.

Description

The present invention is in the field of cosmetics and pharmaceuticals, and more particularly in the field of dermatology. The invention relates to a peptide hydrolyzate enriched in bioactive peptide, capable of activating transglutaminase in cutaneous cells. The bioactive peptide is characterized in that it comprises from 4 to 6 amino acids including at least two arginine residues, a glycine residue and a glutamine residue. Preferably, the active principle comes from the hydrolysis of plant proteins chosen from plants of the genus Zea, and more particularly from the species Zea maize L. The invention also relates to a cosmetic and / or pharmaceutical composition comprising, in a physiologically acceptable medium, said hydrolyzate as an active ingredient.

The invention also relates to the use, in a cosmetic composition, of the active ingredient according to the invention for reinforcing the cutaneous barrier function and stimulating regeneration and epidermal differentiation. The invention also relates to a pharmaceutical composition comprising this new active ingredient as a medicament. The invention also relates to a cosmetic treatment method intended to prevent and / or protect the skin and the integuments of external aggressions and to fight against the manifestations of skin aging, according to which an effective quantity of active ingredient is applied, or composition containing it, on the areas to be treated.

The primary function of the epidermis is to provide a barrier between the external environment and the internal environment. It is the outermost layer of the epidermis, the stratum corneum, which provides this function. It is composed of keratinocytes at the final stage of their differentiation, the corneocytes, sealed to each other by an intercellular cement, both flexible and impermeable. Thus, in the stratum corneum, there is a cell compartment consisting of corneocytes and an extracellular compartment consisting mainly of lipids, organized into multilamellar structures. Corneocytes are surrounded by a specific membrane, called the horny envelope, largely responsible for the resistance, insolubility and flexibility of the skin. The horny envelope consists of a mixture of structural proteins linked together by covalent bonds under the action of transglutaminase. The main constitutive proteins of the horny envelope are envoplakin, periplakine, involucrine, Small Prolin-Rich proteins (SPR protein) and loricrin. Transglutaminases (EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze the formation of peptide bridges between an E-amino of a lysine residue and an γ-carboxamide of a glutamine residue, these extra and intramolecular bridges are extremely resistant to degradation (Lorand et al., Nat Rev Mol Cell Biol Feb, 4 (2), 2003). In humans, 9 transglutaminases have been identified, 4 of which are expressed in the skin. Transglutaminase-1 (TG 1) is expressed in keratinocytes and is present in membrane-bound form. Transglutaminase-2 (TG2, type 2a or 2b) is present only in the basal layer of the epidermis. It is soluble and does not seem to have any role in the formation of the horny envelope. TG2 has the ability to induce covalent bonds between proteins, but it can also bind GTP or GDP and behave like a G protein, especially when the cell is in apoptosis or necrosis. TG2 can also be exported to the membrane and associate with the integrins to increase cell adhesion, cell spreading, and cell migration on fibronectin in the extracellular matrix. Because of its pleiotropic functions, TG2 is involved in other healing. Transglutaminase-3 (TG3) is expressed in hair follicles and in the late stages of keratinocyte differentiation. Transglutaminase-5 (TG5) is present in the upper layers of the epidermis and also plays a role in the early stages of epidermal differentiation. In the epidermis, TG1, TG3 and TG5 are involved in the formation of the horny envelope (Lorand et al., Nat Rev Mol Cell Biol Feb, 4 (2), 2003). TGs have very diverse substrates. Thus, they are able to crosslink the keratin with each other and with filaggrin, which leads to the stabilization and coordination of the keratin-filaggrin network in the horny cells. In the early stages of epidermal differentiation, TGs enhance anchoring of desmosomes, then in the granular layer these same TGs bind certain lipids to the horny envelope and the binding of loricrin to Small Proline Rich. Protein (SPR). In the upper granular layers, the lipids from golgi bodies are crosslinked by TG1 and TG5 to the already partially crosslinked envelope precursor proteins. Finally, the desquamation phase, which occurs at the outermost horny layers, involves additional crosslinking of loricrin and other proteins, involving TG1. Several studies show that the three TGs involved in the formation of the horny envelope in the epidermis have differentiated functions (Candi et al 1995). The key role of transglutaminases in stratum corneum formation, more broadly in epidermal differentiation and consequently in barrier function, is confirmed by some pathological models. Thus TG1 - / - homogenous TG1 - / - knockout mice suffer from lethal malformations of the stratum corneum and die rapidly after birth (Matsuki et al, Proc Nat Acad Sci 95, 1998). On the other hand, in humans, mutations carried by TG1 are responsible for more or less severe forms of ichthyosis (Huber et al., Science 267, 1995).

During skin aging, the integrity of the skin barrier and its repair capabilities deteriorate. An overall lipid deficiency is observed, resulting in a decrease of the lipid multilayers of the extracellular compartment of the stratum corneum. These functional changes correlate with increased susceptibility of older skin to external aggressions (Ghadially R. et al., J Clin Invest, 1995 (95 (5), pp. 2281-90.) Independently of intrinsic or induced, alterations of the cutaneous barrier can occur during external aggression.The term "external aggression" is understood to mean the aggressions that the environment can produce. pollution, UV, or irritating products such as surfactants, preservatives or perfumes, mechanical aggression, such as abrasions, shaving or depilation. diesel particulate matter, ozone or heavy metals, such as indoor pollution, which may be due to solvent emissions from paints, adhesives or paper-pe ints (such as toluene, styrene, xylene or benzaldehyde), or even cigarette smoke. Dryness of the atmosphere is also a major cause of skin aggression. These external aggressions result in an alteration of the barrier function which results in cutaneous discomfort, unpleasant sensory phenomena, such as tightness or itching, or even excessive fragility and redness. The persons particularly affected by this deterioration of the barrier function by external aggressions are the people with so-called "fragile" or "sensitive" skin, that is to say, particularly sensitive to changes in temperature or humidity. and / or particularly reactive vis-à-vis aggressive products (for example baby skins). People with "fragile" skin include people whose protective lipids of the stratum corneum become rare, as is the case for the elderly and very old (at least 75 years), people whose composition of protective lipids stratum corneum is modified, as is the case of people with diabetes, or dialysis, or with certain diseases. People with so-called sensitive skin have a lowered threshold of reactivity that can be linked to neurogenic hyperactivity. These sensitive skin will show clinical signs much more quickly and frequently than other types of skin. An alteration of the cutaneous barrier function may notably result in a disturbance of the hydration, in a loss of suppleness of the skin, in an alteration of the radiance of the complexion, in the appearance of roughness on the skin. and more generally by the appearance of cutaneous signs of aging.

It should then seek to prevent alteration or restore the barrier function of the epidermis. However, the key role of transglutaminase has made it a prime target for enhancing the barrier function of the epidermis. In the field of cosmetics, the activation of transglutaminase has already been described with the aim of improving the barrier function by using plant extracts, for example extracts of Asiasarum heterotropoides F., or Asiasarum sieboldi F., and of Apocynum venetum L, as cited in the patent application JP2004 1 1 545 1. The patent application US2007134172 describes the use of flavonoids preferentially extracted from Sidastrum to obtain a better resistance of the skin to environmental factors and in particular drying up. The inventors have now demonstrated a cosmetic and therapeutic, especially dermatological, activity of a peptide hydrolyzate enriched in bioactive peptide. In particular, it has been demonstrated that this peptide hydrolyzate, when applied to the skin, reinforces the barrier function of the epidermis and stimulates regeneration and epidermal differentiation. Therefore, the first subject of the present invention is a peptide hydrolyzate enriched in bioactive peptide capable of activating human transglutaminase, characterized in that it comprises from 4 to 6 amino acids including at least two arginine residues, a glycine residue and a glutamine residue. The inventors have in fact demonstrated a cosmetic and therapeutic, especially dermatological, activity of peptide hydrolysates containing certain particular peptides, hereinafter called bioactive peptides. By bioactive peptide according to the invention is meant a sequence of at least four amino acids linked together by peptide bonds or by modified peptide bonds, and which has an in vivo or in vitro activity characteristic of the activity of the peptide. active ingredient according to the invention. The characteristic biological activity according to the invention is defined in vitro by the ability of the peptide to activate transglutaminase, either by increasing the protein synthesis of transglutaminase (by direct or indirect modulation of transglutaminase gene expression). ), either by increasing the enzymatic activity of transglutaminase or by other biological processes such as the stabilization of the transglutaminase protein or the stabilization of messenger RNA transcripts. By skin is meant all the covering tissues constituting the skin and the mucous membranes, including the integuments (hair, eyelashes, hairs, eyebrows). By peptide hydrolyzate is meant a mixture of compounds predominantly represented by peptides or oligopeptides. According to the invention, the terms peptide hydrolyzate or active principle will be used interchangeably. The term "peptide-like compounds" means the protein fragments, the peptides and the free amino acids present in the peptide hydrolyzate according to the invention. By topical application is meant the application or spreading of the active ingredient according to the invention, or a composition containing it, on the surface of the skin or mucosa. Physiologically acceptable means that the peptide hydrolyzate according to the invention, or a composition containing it, is suitable for contact with the skin or mucosa without causing toxicity or intolerance reactions. According to a particularly advantageous method of carrying out the invention, the bioactive peptide contained in the hydrolyzate has a sequence of general formula (I): XI -X2-X3-X4-X5-X6 In which, XI is alanine, valine or no amino acid, X2 is alanine, valine or no amino acid, X3 is arginine, X4 is arginine, glycine or glutamine, X5 is arginine, glycine or glutamine, X6 is arginine, glycine or glutamine,

According to a very particularly preferred embodiment of the invention, the bioactive peptide is of the following sequence: (SEQ ID No. 1) Ala-Ala-Arg-Arg-Gly-Gln (SEQ ID No. 2) Val-Val- Arg-Arg-Gly-Gln (SEQ ID NO: 3) Ala-Val-Arg-Arg-Gln-Gly (SEQ ID NO: 4) Ala-Arg-Gly-Arg-Gln (SEQ ID NO: 5) Arg Arg-Arg-Gln-Gly (SEQ ID NO: 7) Arg-Gln-Gly-Arg (SEQ ID NO: 8) Arg-Gly-Arg-Gln 20 a particularly interesting embodiment, the biologically active peptide corresponds to the sequence SEQ ID No. 5.

Preferably, the active ingredient according to the invention is more particularly capable of activating human transglutaminases of type 1, 2a, 2b, 3 or 5.

The active ingredient according to the invention can be obtained by extraction of proteins of plant origin, followed by controlled hydrolysis which releases compounds of peptide nature, among which are the bioactive peptides. The use of peptide hydrolysates, and in particular of low molecular weight peptide hydrolysates, has many advantages in cosmetics. In addition to generating peptidic compounds which did not previously exist in the starting protein mixture, hydrolysis and purification make it possible to obtain more stable mixtures that are more easily standardized and do not cause allergic reactions. in dermato-cosmetics. It is also possible to use certain hydrolysed extracts without purifying the peptide-like compounds corresponding to the bioactive peptides according to the invention, but nevertheless ensuring the presence of said peptides by appropriate analytical means. Many plant proteins are likely to contain bioactive peptides within their structure. The controlled hydrolysis makes it possible to release these particular compounds of peptide nature. 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 hydrolysis first on a crude extract and purify the peptide nature next.

According to a preferred embodiment, said active principle comes from the hydrolysis of plant proteins chosen from plants of the genus Zea and preferably the species Zea mays L. According to the invention, the plant material used will be the grain and preferentially the grain removed from its envelope by a dehulling step. Preferably, the plants used are not subjected to prior fermentation. Any method of extraction or purification known to those skilled in the art can be used to prepare the hydrolyzate according to the invention. In a first step, the seeds are crushed using a plant grinder. The powder thus obtained may subsequently be "delipidated" with the aid of a conventional organic solvent (for example an alcohol, hexane or acetone). Protein extraction is then carried out by the conventional (Osborne, 1924) modified method; 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. In particular, the concentration of phenolic-type substances, interacting with proteins, is significantly reduced. The soluble fraction, containing proteins, carbohydrates and optionally lipids, is collected after centrifugation and filtration steps. This crude solution is then hydrolyzed under mild conditions to generate soluble 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) or of microorganisms (Aspergillus, Rhizopus, Bacillus, (Novozym alkalase, etc.) may be mentioned.) The hydrolysis conditions are chosen for promote the enrichment of bioactive peptide For the same reasons as above, that is to say the elimination of polyphenol substances, an amount of polyvinylpolypyrrolidone is added to the reaction medium during this mild hydrolysis step. In order to eliminate enzymes and polymers, the filtrate (solution) obtained is a first form of the active ingredient according to the invention.The hydrolyzate obtained at this stage can be further purified in order to select low molecular weight fractions. preferably less than 6 kDa, and the peptides generated according to their nature.The fractionation can be carried out advantageously by succinct ultrafiltration steps. essences through decreasing porosity filters, retaining the filtrates at each step and / or by a chromatographic-type method, in order to specifically enrich the hydrolyzate with bioactive peptide. A dilution phase is carried out in water or in any mixture containing water, then this dilution is sterilized by ultrafiltration in order to obtain a peptide hydrolyzate characterized by a protein content of 3.5 to 5, 5 g / 1. This peptide hydrolyzate corresponds to the most purified form of the active principle according to the invention.

The peptide hydrolyzate obtained according to the invention is analyzed qualitatively and quantitatively in high pressure liquid chromatography (HPLC), making it possible to analyze proteins having molecular weights of 0.2 to 25 kDa (according to a suitable solvent gradient). The different peptide fractions that could be isolated are then analyzed for their biological effectiveness. These various fractions are then analyzed by mass spectrometry to specifically identify the amino acid content of the peptides of each peak. Sequencing analysis was also performed to determine the peptide sequence of the bioactive peptide. The hydrolyzate obtained is composed of peptides with a molecular weight of less than 6 kDa and preferably less than 5 kDa, enriched with a bioactive peptide of 4 to 6 amino acids comprising at least two arginine residues, a glycine residue and a glutamine residue. The second object of the present invention is a composition comprising, in a physiologically acceptable medium, as active principle capable of activating human transglutaminase, the peptide hydrolyzate enriched in bioactive peptide according to the invention. According to an advantageous embodiment of the invention, the active principle according to the invention is present in the compositions of the invention at a concentration of between approximately 0.0001% and 20%, and preferably at a concentration of between 0.degree. , About 5% and 5% relative to the total weight of the final composition. According to an advantageous embodiment of the invention, the active ingredient according to the invention is solubilized in one or more physiologically acceptable solvents, conventionally used by those skilled in the art, such as water, glycerol, ethanol, propanediol, 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 physiologically acceptable vector. The composition that may be used according to the invention may in particular consist of a composition for hair care, and in particular a shampoo, a conditioner, a treatment lotion, a cream or a styling gel, a restructuring lotion for the hair, a mask, etc. The composition may also be in the form of a dye or mascara to be applied by brush or comb, in particular on eyelashes, eyebrows or hair. The composition that can be used according to the invention can be applied by any appropriate route, in particular oral, parenteral or topical, and the formulation of the compositions 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. These compositions must therefore contain a physiologically acceptable medium, that is to say compatible with the skin and superficial body growths, and cover all cosmetic or dermatological forms. It is understood that the active ingredient according to the invention can be used alone or in combination with other active ingredients. Advantageously, the compositions that can be used according to the invention also contain various protective or anti-aging active ingredients intended to promote and complete the action of said active ingredient. The following ingredients may be mentioned, in a non-limiting manner: healing, anti-aging, anti-wrinkle, soothing, antiradical, anti-UV agents, agents stimulating the synthesis of dermal macromolecules or energy metabolism, moisturizing agents, antibacterials, anti-fungal, anti-inflammatory, anesthetic, modulating agents differentiation, pigmentation or skin depigmentation, agents stimulating the growth of nails or hair. Preferably, an agent having an anti-wrinkle activity, such as an anti-radical or antioxidant agent, or an agent stimulating the synthesis of dermal macromolecules, an energy metabolism stimulating agent, a metalloproteinase inhibitor, will be used. For example, other active ingredients having anti-free radical or antioxidant action may be added, selected from vitamin C, vitamin E, coenzyme Q10 and polyphenolic plant extracts, retinoids. 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 sold under the name Collaxyl by the Vincience company. 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. It is intended for mammals in general, and more particularly for humans, and may be in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water, water-in-oil 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, a lotion, a milk, a serum, an ointment, a gel, a paste or a mousse. solid form, like u n stick 2949781 -11- or be 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. All of these compositions additionally comprise any additive commonly used in the intended field of application as well as adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, dyes , sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, other cosmetic 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 advantageous properties of the composition according to the invention. These adjuvants may, for example, correspond to a concentration ranging from 0.01 to 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 third object of the invention is a pharmaceutical composition comprising an effective amount of peptide hydrolyzate according to the invention as a medicament. For example, the pharmaceutical composition may be intended to prevent or treat pathologies characterized by impaired barrier function, such as hypersensitive, irritated, or reactive skin and atopic eczema. According to this form of the invention, the compositions will be suitable for oral administration for pharmaceutical use. Thus, the compositions may especially be in the form of tablets, capsules, capsules, chewable pastes, powders for consumption as they are or to be mixed extemporaneously with a liquid, syrup, gels, and any other form known to those skilled in the art. These compositions additionally comprise any additive commonly used in the intended field of application and the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, preservatives, other pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, etc.

The fourth subject of the invention is the use, in a cosmetic composition, of an effective amount of peptide hydrolyzate, as an active principle for reinforcing the cutaneous barrier function and for stimulating epidermal regeneration and differentiation. The term reinforces the cutaneous barrier function and stimulates epidermal regeneration and differentiation, the improvement of the structure of the stratum corneum, the increase of the signs of cellular regeneration, such as the density of the epidermal basal layers and the speed of migration of fibroblasts, and increased expression of keratinocyte differentiation markers.

The fifth subject of the invention is the use of a cosmetic composition comprising the peptide hydrolyzate, as an active ingredient, to fight in a preventive and / or curative manner against the signs of skin aging, and more particularly photo aging. -induced (photo-aging). Skin manifestations of aging are understood to mean any changes in the external appearance of the skin and skin appendages due to aging, such as, for example, the superficial roughness of the stratum corneum, wrinkles and fine lines, but also any internal modification of the skin. skin that does not systematically result in a modified external appearance such as, for example, the thinning of the dermis or any other internal degradation of the skin following exposure to ultraviolet (UV) radiation.

The sixth subject of the invention is the use of a cosmetic composition comprising the peptide hydrolyzate as active principle, for protecting the skin and integuments against any type of external attack. In particular, the subject of the invention is the use of the hydrolyzate according to the invention in a cosmetic composition intended to prevent or treat the damage caused to the skin by external aggressions of the skin, chosen from mechanical treatments such as shaving or waxing, excessive leaching by detergents, extreme weather conditions or sudden changes in temperature and humidity. The subject of the invention is a cosmetic treatment method characterized in that a composition containing an effective amount of active principle for preventing or treating the cutaneous signs of the skin is applied topically to the skin or integuments to be treated. aging or protect the skin and integuments against external aggressions. In particular, the invention relates to a cosmetic treatment method for protecting the skin and integuments against the aggressions due to UV radiation.

Particular embodiments of this cosmetic treatment method also result from the foregoing description. Other advantages and features of the invention will appear better on reading the examples given by way of illustration and not limitation.

List of Figures Figure 1: Example of chromatogram obtained by HPLC, with detection of the peak corresponding to the bioactive peptide in a maize hydrolyzate obtained according to Example 1. Figure 2: Evaluation of the protective effect vis-à-vis external aggression: Histological Hematoxylin-Eosin staining of human skin attacked by tape-stripping and treated with the hydrolyzate according to Example 1.

Example 1 Preparation of a peptide hydrolyzate from corn cake (Zea mars L.) The corn cake (Zea mays L.) is dissolved in 10 volumes of water in the presence of 2% of POLYCLAR 10 (polyvinylpyrrolidone - PVPP - insoluble). The mixture is adjusted to a pH of between 7.5 and 9.5 with a 1M aqueous sodium hydroxide solution. After adjusting the pH, a mixture of 2% bromelain and alkalase (Novozym) is added to the medium. reaction. The hydrolysis is obtained after 2 hours of stirring at 50 ° C. The enzyme is then inactivated by heating the solution at 80 ° C for 2 hours. After centrifugation, the supernatant aqueous solution corresponding to a crude maize hydrolyzate is recovered. The hydrolysis conditions were chosen to allow enrichment of the bioactive peptide of 4-6 amino acids containing the Arg, Gly and Gln residues. The process for purifying the crude hydrolyzate begins with successive filtrations using Seitz-Orion plate filters of decreasing porosity (up to 0.2 gm) in order to obtain a brilliant yellow solution. and limpid, qualified hydrolyzate 1. At this stage, the corn hydrolyzate 1 is characterized by a solids content of 20 to 30 g / kg, a protein content of 20 to 25 g / l and a rate of sugars of 2 to 5 g / l. The protein nature of the hydrolyzate 1 is demonstrated after analysis by NuPAGE Bis-Tris Pre-cast polyacrylamide gel electrophoresis (Invitrogen). The corn protein hydrolyzate is heated at 70 ° C for 10 minutes under denaturing reducing conditions in NuPAGE LDS sample preparation buffer. A solution of NuPAGE Antioxidant is added to the inner vessel (cathode) to prevent the reduced proteins from re-oxidizing during electrophoresis. Protein migration is performed in NuPAGE MES migration buffer with the SeeBlue Plus2 standard as a molecular weight marker. Protein staining is performed using Coomassie Blue R-250. Under these conditions, proteins with a molecular weight of less than 6 kDa are observed. The hydrolyzate 1 is then purified by removing the high molecular weight proteins by ultrafiltration using the Pellicon 2 Biomax 5 kDa cassette in order to retain only the peptide-like compounds with a molecular weight of less than 5 kDa. After this final purification, a dilution phase is carried out in order to obtain a peptide hydrolyzate characterized by a protein content of between 3.5 and 5.5 g / l. This peptide hydrolyzate corresponds to the active principle according to the invention. This peptide hydrolyzate is then analyzed by high pressure liquid chromatography (HPLC) using an HP1100 device controlled by the ChemStation software. The column used in the elution of the hydrolyzate is a Nucleosil 300-5 C4 MPN (125 x 4 min), for chromatography of proteins having molecular weights of 0.2 to 25 kDa under the following conditions: Methanol gradient: - Uptisphere OPB column 125 x 3 mm - Solvent A: HPLC grade water containing 0.1% heptafluorobutyric acid (HFBA) 30 - Solvent B: methanol HPLC grade - Gradient: 100% to 40% Solvent A in 13 minutes and then 40% to 10% in 5 minutes In these chromatographic conditions, several peptide fractions could be isolated. An example of a chromatogram obtained by HPLC (high pressure liquid chromatography) with the peak corresponding to the bioactive peptide is shown in FIG. 1. These various fractions are then analyzed by mass spectrometry in order to determine the peak. specifically identify the amino acid content of the peptides of each peak. Sequencing analysis was also performed to determine the peptide sequence of the bioactive peptide.

EXAMPLE 2 Demonstration of the Increase in the Overall Enzymatic Activity of Transglutaminases by the Hydrolyzate According to Example 1 The aim of this study is to determine the influence of the hydrolyzate according to Example 1 on the total transglutaminase activity in normal human keratinocytes (KHN). For this purpose, the total enzymatic activity of the transglutaminases is determined spectrophotometrically using a fluorescein-labeled amino acid donor substrate. Protocol: KHNs are seeded in 96-well black plates. After 4 days of culture, the KHNs are treated with a 1% hydrolyzate solution according to Example 1 for 24 or 48 hours (the active ingredient is added every 24 hours). A positive control is achieved by treating cells with EGCG at 20 g / ml (epigallocatechin gallate, the main polyphenol of green tea). The substrate used is fluorescein-labeled cadaverine (Invitrogen A10466) diluted to 100 M in culture medium. The substrate is incubated for 2 hours with the cells at 200% / well). The cells are then rinsed twice in HBSS buffer and fixed with a mixture of acetic acid-ethanol-water (1:49:50) for 20 minutes. After two rinses in ethanol and then three rinses in HBSS buffer, 100 l of PBS are added to each well and a spectrophotometer reading at excitation wavelengths of 485 nm and 530 nm emission. is realized. Under these conditions, the transglutaminase activity is proportional to the amount of fluorescence emitted (expressed in fluorescence units), relative to the total amount of proteins present in each well, previously assayed by the BCA technique. Results: Results are expressed as a percentage of untreated control. In the presence of 1% of hydrolyzate according to Example 1, the enzymatic activity is increased by more than 30% after 24 hours. The increase is even more marked after 48 hours of treatment. Conclusion: The hydrolyzate according to Example 1 significantly increases the total enzymatic activity of transglutaminases in normal human keratinocytes.

EXAMPLE 3 Demonstration of the Activating Effect of the Hydrolyzate According to Example 1 on the Expression of TG1, TG2, TG3 and TG5 The purpose of this study is to determine the influence of the hydrolyzate according to Example 1 on the expression of the different transglutaminases expressed in human skin. For this purpose, specific immunofluorescence markings were performed on normal human keratinocyte (KHN) culture and on skin biopsy. Immunofluorescence markings were also performed on normal human fibroblast cultures specifically for TG2, expressed in this type of cells. Protocol for immunostaining on normal human keratinocytes in culture: KHN in culture are treated with a 1% solution of hydrolyzate according to Example 1 for 24 hours. For anti-TG1 immunostaining, the cells are washed and fixed with 3.7% paraformaldehyde for 10 minutes. The cells are then incubated in the presence of an anti-TG1 specific antibody (Clinisciences BT-621, monoclonal mouse), followed by a suitable secondary antibody coupled to a fluorescent label. For the other immunolabelings, the cells are washed and fixed with cold methanol for 1 minute. The cells are then incubated in the presence of a specific antibody; anti-TG2 (Abcam ab2972, polyclonal rabbit), anti TG3 (Abcam ab53236, monoclonal mouse) or anti TG5 (Abcam ab26992, polyclonal rabbit). The slides are then observed under an epifluorescence microscope (Nikon Eclipse E 80i microscope), after mounting in an ad hoc environment. Protocol for immunolabeling on normal human fibroblasts in culture: Human dermal fibroblasts are treated and immunolabeled using an anti-TG2 antibody according to the same protocol as for KHNs. Protocol for immunostaining on skin biopsies: Human skin biopsies are cultured at the air / liquid interface. A 1% solution of hydrolyzate according to Example 1 is applied topically for 24 hours. For markings of TG1 and TG2, skin biopsies are then included in resin and frozen in nitrogen. Sections of about 6 m are then made by cryostat. Immunostaining is performed using a specific antibody; Anti-TG2 (AbcB ab2972, polyclonal rabbit) and then a suitable secondary antibody, coupled to a fluorescent marker. The skin sections are then examined under an Epi-fluorescence microscope (Nikon Eclipse E 80i microscope). For markings of TG3, skin biopsies are embedded in paraffin and histological sections of 3 μm thickness are performed. The slides are deparaffinized, hydrated and then subjected to immunolabeling with an antibody directed against TG3 (Abcam ab53236, monoclonal mouse) and then a suitable secondary antibody, coupled to a fluorescent marker. The skin sections are then examined under the Epi-fluorescence microscope (Nikon Eclipse E 80i microscope). Results: In all the conditions tested, more intense fluorescence was observed in the cultures and on the cuts of hides treated with the hydrolyzate according to Example 1 at 1% than under the control conditions, untreated. Conclusions: The hydrolyzate according to Example 1 stimulates the expression of TG1, TG2, TG3 and TG5 in normal human keratinocytes in culture, as well as the expression of TG2 in human fibroblasts. The hydrolyzate according to Example 1 stimulates the expression of TG1, TG2 and TG3 in skin biopsies cultured ex vivo.

EXAMPLE 4 Demonstration of the Activating Effect of Hydrolysate According to Example 1 on Epidermal Differentiation The aim of this study is to determine the influence of the hydrolyzate according to Example 1 on epidermal differentiation. For this, the expression of the main markers of epidermal differentiation, specifically expressed in the keratinocytes of suprabasal layers, was studied. The markers tested are transglutaminase 1, pankeratins, filaggrin, involucrine and loricrin. On the other hand, filaggrin, involucrine and loricrin are precursors of the horny envelope and substrates of transglutaminases. Immunotyping protocol for normal human keratinocytes in culture: Culture KHNs are treated with a 1% solution of a hydrolyzate solution according to Example 1 for 24 hours. The cells are then washed and fixed at 3.7% paraformaldehyde for 10 minutes. After delineation of the specific sites, the cells are incubated in the presence of a specific antibody directed against TG1 (Clinisciences BT-621, monoclonal mouse), or against loricrin (Abcam ab24722, polyclonal rabbit), or the involucrin (Novocastra NCL-INV, monoclonal mouse, clone SY5), and then incubated in the presence of a suitable secondary antibody coupled to a fluorescent label. For ease of observation, the nuclei of the cells can be counter-stained by DAPI (4 ', 6'-Di-Amidino-2-phenyl Indole), a blue fluorescent molecule capable of binding strongly to DNA) . The slides are then observed under an epifluorescence microscope (Nikon Eclipse E 80i microscope), after mounting in an ad hoc environment. Protocol for immunostaining on skin biopsies: Human skin biopsies are cultured at the air / liquid interface. A 1% solution of hydrolyzate according to Example 1 is applied topically for 24 hours. Skin biopsies are then embedded in the paraffin and histological sections 3 m thick are performed. The slides are deparaffinized, hydrated and then subjected to immunolabeling with an antibody directed against TG1 (Clinisciences BT-621, monoclonal mouse) or cyto-pankeratins (Novocastra (NCL-CK10, monoclonal mouse), or loricrin (Abcam ab24722 polyclonal rabbit), or involucrin (Novocastra NCL-1NV, clone SY5, monoclonal mouse) or filaggrin (Tebu Santa Cruz sc-58761, monoclonal mouse) .For greater ease of observation the nuclei of the cells can be counter-stained by DAPI (4 ', 6'-Di-2-Amidino-Phenyl Indole, a blue fluorescent molecule capable of strongly binding to DNA.) A suitable secondary antibody coupled to a fluorescent label is then used. After mounting in an ad hoc medium, the slides are observed under an epifluorescence microscope (Nikon Eclipse E 80i microscope) Immunoblot protocol: KHN in culture are treated with a 1% solution of hydrolyzate according to Example 1 during 24 hours. The cells are then rinsed and detached from the support by scraping in a RIPA buffer in the presence of a cocktail of protease inhibitors (Thermo Scientific, Rockford, USA). The lysed cells are centrifuged at 4 ° C at 10,000 rpm for 20 minutes and the supernatants collected. The samples are then standardized by protein assay using the BCA kit (Pierce, France). Samples are mixed and then electrophoresed on NuPAGE 3-8% Tris-acetate gel in NuPAGE Tris-acetate migration buffer and transferred to a nitrocellulose membrane using a transfer device (Invitrogen, Paisley). , UK). The membranes are saturated in 5% milk TBS, 2 hours at room temperature, then incubated at 4 ° C overnight with a primary antibody against TG1 (Clinisciences BT-621, monoclonal mouse) or against involucrin (Novocastra NCL-INV, clone SY5, monoclonal mouse) or against loricrin (Abcam ab24722, polyclonal rabbit). After washing with 0.05% TBS-Tween buffer, the membrane is incubated with a suitable secondary antibody. , coupled with peroxidase. The transfers are then developed using a chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate, Pierce, Brebiere, France). The specific protein bands thus revealed are quantified using a Chemi-Imager technology image analyzer (Alpha Innotech Corporation). Results: In all conditions tested by immunofluorescence, more intense fluorescence was observed in the cultures and sections of skins treated with the hydrolyzate according to Example 1 to 1% than under the control conditions, untreated. On ex vivo skin sections, there is epidermal differentiation with the presence of a thicker horny layer. The quantitative analysis of the immunoblots makes it possible to evaluate the increase in the expression of the markers tested. The increase in TG1 is of the order of 20%, the involucrin is greater than 25% and the loricrin is of the order of 15%. Conclusions: The hydrolyzate according to Example 1 stimulates the expression of pankeratins, involucrine, filaggrin, loricrin, as well as TG1 in normal human keratinocytes. The hydrolyzate according to Example 1 also improves the epidermal differentiation and in particular the morphology of the stratum corneum.

EXAMPLE 5 Demonstration of the protective effect vis-à-vis the external aggressions of hydrolyzate according to Example 1 The purpose of this study is to determine the protective effect on the skin of the hydrolyzate 25 according to US Pat. Example 1 vis-à-vis external aggression. For this, an ex vivo model of severe aggression of the cutaneous barrier was used. Protocol: The biopsies of human skin are subjected to an aggression provoked by successive stripping of layers of the stratum corneum with the help of adhesive tape (technique called tape stripping). The tearing step is repeated 20 times in succession on the same zone. The stripped human skin biopsies are then cultured and treated with the hydrolyzate according to Example 1 at 1% and 3% according to the protocol of Example 3, for 24 hours and 48 hours. The skin biopsies are then embedded in paraffin and histological sections of thickness 3 are made. The sections are deposited on Superfrost Plus slides (Menzel Glaser, Thermo Scientific), then deparaffinized in xylene and rehydrated in a series of alcohol-water solutions. The sections are then stained with 50% hematoxylin for 3 minutes, rinsed, then stained with 60% eosin, for 3 minutes and rinsed with water. The sections are dehydrated, mounted in Eukitt and examined by light microscopy. Results: The histological sections of skin treated with the hydrolyzate according to Example 1 at the 1% and 3% concentration show a greater neo-synthesis of the corneal layers. The whole epidermal layer has fewer vacuolated cells and a greater cell density. See Figure 2. Conclusion: The hydrolyzate according to Example 1 improves the reconstruction of the epidermis attacked.

EXAMPLE 6 Demonstration of the Regenerating Effect of the Hydrolyzate According to Example 1 The aim of this study is to determine the regenerating effect of the hydrolyzate according to Example 1 on dermal fibroblasts and on the epidermis. Protocol for the use of the in vitro Ibidi fibroblastic regeneration model: The Ibidi in vitro healing model was used (Biovalley, Marne la Vallée, France). Human fibroblasts are inoculated in two separate compartments of an Ibidi insert and then this is placed in a culture dish. At confluence, the insert is removed creating an acellular zone 400 m wide between the two cell mats. The hydrolyzate according to Example 1 diluted to 1% and 3% is then added to the culture medium, and the treatment is carried out for 48 hours with renewal of the active ingredient every 24 hours. Phase contrast microscopy observations (Olympus CK40 microscope, x5, connected to an Olympus E-510 camera) were made at different times (0 to 48 hours) during the migration process. Epidermal Regeneration Protocol: Human skin biopsies are cultured at the air / liquid interface. A 1% solution of the hydrolyzate according to Example 1 is applied topically, and then the samples are incubated for 24 hours. The skin biopsies are then embedded in paraffin and histological sections 3 m thick are made. The sections are deposited on Superfrost Plus slides (Menzel Glaser, Thermo Scientific), then deparaffinized in xylene and rehydrated in a series of alcohol-water solutions. The sections are then stained with 50% hematoxylin for 3 minutes, rinsed, then stained with 60% eosin, for 3 minutes and rinsed with water. The sections are then dehydrated, mounted in Eukitt and examined by light microscopy. Results: i) Involvement by fibroblasts in the acellular zone is faster when the cells are treated with the active ingredient at 1% and 3%, compared to the control conditions. The effect is dose-dependent. Ii) The histological sections of epidermis treated with the hydrolyzate according to Example 1 show a neo-synthesis of the stratum corneum which appears thicker and better hydrated. There is also a higher density of basal layer cells that appear better oriented in the vertical axis and more homogeneous. Conclusions: The hydrolyzate according to Example 1 ensures regeneration of the dermal fibroblasts as well as the epidermis. It induces greater cohesion of the horny layers.

EXAMPLE 7 Preparation of Compositions 1 - Sunscreen Cream: Trade Names I INCI Names% by mass PHASE A Demineralized 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 -22- Trade Names INCI Names% Massive 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 Hydrolyzate according to Example 1 0.5% Fragrance Fragrance qsp Colorant qsp The constituents of phase A and phase B are heated separately between 70 ° C. 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. Anti-Aging Cream: Names INCI Names% commercial mass A Phase 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 Q 10 Ubiquinone 0.10 Phase B 2949781 -23- Names INCI Names% commercial mass Avocado Oil Persea Gratissima ( Avocado) Oil 1.25 Phenonip Phenoxyethanol (and) Methylparaben (and) Ethylparaben 0.75 (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 TEA Triethanolamine 0.18 Phase E Hydrolyzate according to 1% Example 1 GP4G Water (and) Artemia Extract 1.50 Collaxyl Water (and) Butylene Glycol (and) Hexapeptide-9 3.00 Phase F Fragrance Perfume (Fragrance) qsp Col pouring 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. Protective Day Cream: Trade Names INCI Names% by mass Phase A Emulium Delta Cetyl alcohol (and) Glyceryl Stearate (and) PEG-4.00 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 Hexacaprylate / Hexacaprate 1.50 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 Glycerin Glycerin 2.00 Carbopol EDT 2020 Acrylates / C 10-30Alkyl Acrylate Crosspolymer 0.15 Keltrol BT Xanthan Gum 0.30 Phase C Sodium Hydroxide (Sodium Hydroxide Sodium 0.30 10%) Phase D Demineralized Water Aqua 5.00 Stay-C 50 Sodium Ascorbyl Phosphate 0.50 Phase E -24- 2949781 -25- Butylene Glycol Butylene Glycol 2.00 Dekaben CP Chlorphenesin 0.20 Phase F GP4G Water ( and) Artemia Extract 1.00 Hydrolyzate according to Example 1 2% 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 (15)

REVENDICATIONS1. Peptide hydrolyzate enriched in bioactive peptide capable of activating human transglutaminase, characterized in that said bioactive peptide contains from 4 to 6 amino acids including at least two arginine residues, a glycine residue and a glutamine residue. 2. A peptide hydrolyzate according to claim 1, characterized in that said bioactive peptide is of general formula (I) X1-X2-X3-X4-X5-X6 wherein XI is alanine, valine or no amino acid, X2 is alanine, valine or no amino acid, X3 is arginine, X4 is arginine, glycine or glutamine, X5 is arginine, glycine or glutamine, X6 is arginine, glycine or glutamine. 3. Peptide hydrolyzate according to claim 2, characterized in that said bioactive peptide is of sequence: (SEQ ID No. 1) Ala-Ala-Arg-Arg-Gly-Gln (SEQ ID No. 2) Val-Val-Arg -Arg-Gly-Gln (SEQ ID No. 3) Ala-Val-Arg-Arg-Gln-Gly (SEQ ID No. 4) Ala-Arg-Gly-Arg-Gln (SEQ ID No. 5) Arg-Arg -Gly-Gln (SEQ ID NO: 6) Arg-Arg-Gln-Gly (SEQ ID NO: 7) Arg-Gln-Gly-Arg (SEQ ID NO: 8) Arg-Gly-Arg-Gln 4. Peptide hydrolyzate according to one of the preceding claims, characterized in that the activated human transglutaminases are of type 1, 2a, 2b, 3 or 5. Peptide hydrolyzate according to one of the preceding claims, characterized in that it comes from the hydrolysis of plants selected from the genus Zea and preferably the species Zea mays L. (maize). 5 6. Peptide hydrolyzate according to one of the preceding claims, characterized in that it contains between 3.5 and 5.5 g / 1 of peptide compounds. 7. Peptide hydrolyzate according to one of the preceding claims, characterized in that it contains peptides with a molecular weight of less than 6 kDa and preferably of 5 kDa molecular weight. 8. Cosmetic composition comprising, in a physiologically acceptable medium, as active ingredient capable of activating human transglutaminase, the peptide hydrolyzate defined according to one of the preceding claims, in an amount representing from 0.0001% to 20%. % of the total weight of the composition, and preferably in an amount representing from 0.05% to 5% of the total weight of the composition. 9. Composition according to Claim 8, characterized in that the said peptide hydrolyzate is solubilized in one or more physiologically acceptable solvents, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, vegetable oil or any mixture of these solvents. 25 10. Composition according to one of claims 8 or 9, characterized in that it is in a form suitable for topical application. 11. Composition according to one of claims 8 to 10, characterized in that it further comprises at least one other active ingredient promoting the action of said peptide hydrolyzate. 2949781 -28- 12. A pharmaceutical composition comprising, in a physiologically acceptable medium, the peptide hydrolyzate defined according to one of claims 1 to 7, as a medicament. 5 13. Use of a cosmetic composition comprising the peptide hydrolyzate as defined according to one of claims 1 to 7 as an active ingredient, to enhance the barrier function of the epidermis and stimulate regeneration and epidermal differentiation. 10 14. Use of a cosmetic composition comprising the peptide hydrolyzate as defined according to one of claims 1 to 7 as an active ingredient, for preventing and combating cutaneous signs of aging and photo-aging. 15. Use of a cosmetic composition comprising the peptide hydrolyzate as defined according to one of claims 1 to 7 as an active ingredient, to protect the skin from external aggression.