FR2944796A1 - New peptide hydrolyzates, enriched in bioactive peptide having arginine residue, lysine residue and glycine residue, are proteasome activators, useful e.g. to prevent and/or treat spots of hyperpigmentation e.g. melasma and chloasma - Google Patents

Abstract

Proteasome activating peptide hydrolyzate (I) enriched in bioactive peptide containing 3-5 amino acid, where each of the bioactive peptide comprises at least one arginine residue, lysine residue, and glycine residue, is new. An independent claim is included for a cosmetic composition comprising (I). ACTIVITY : Dermatological. MECHANISM OF ACTION : Proteasome activator.

Description

The present invention relates to the field of depigmenting actives and their uses in cosmetics in particular. More particularly, the present invention relates to a peptide hydrolyzate enriched in bioactive peptides, said hydrolyzate being activator of the proteasome, as well as its use in cosmetics and / or pharmaceutical as lightening, depigmenting and / or whitening agent. Said hydrolyzate also makes it possible to prevent and / or treat cutaneous hyperpigmentation-related skin signs due to photo-aging, as well as hyperpigmentation spots of various origins.

In humans, the color of the hair and skin is related to individual factors (ethnic origin, gender, age, etc.) and environmental factors (including the seasons of the year, the area of habitation, etc.). It is mainly determined by the nature and concentration of melanin produced by melanocytes. Melanin has the property of protecting skin cells from the deleterious effects of UV radiation and slowing skin photoaging. Melanocytes are specialized cells that, via particular organelles, melanosomes, synthesize melanin. The synthesis of melanin or melanogenesis is a complex process whose precise mechanisms have not yet been elucidated and which schematically involves the following steps: Tyrosine Dopa, Dopaquinone, Dopachrome Melanine.

This melanin plays indeed a fundamental role in the determination of the color of the skin. It is synthesized by large dendritic cells: melanocytes, cells located in the basal layer of the epidermis. Melanin exists in two different forms: phaeomelanin, yellow pigment, and black eumelanin. It is the different proportions and the size of these pigments, not to mention the carotenoids and the micro-blood circulation, which give the skin its great diversity of colors. The production of melanin, as well as its transport, is regulated by various factors such as, for example, UV radiation, hormones or chemicals. Thus, an increase in exposure to UV radiation causes the synthesis of pigments and results in a darkening of the skin. Disturbances of this pigmentation, more or less benign, may appear. They are manifested, for example, by freckles, moles, diffuse spots such as pregnancy spots, chloasma, melasma, as well as other hyperpigmentary disorders such as lentigo. Other spots of hyperpigmentation may be due for example to poor healing especially in individuals with dark phenotype, or they may be due to the use of photosensitizing drugs etc. But aging can also modulate skin pigmentation. Thus, some people may see spots on the skin, more or less dark or colored, conferring areas of heterogeneous coloring forming spots of senescence or ephelides. Finally, in certain populations of Asian or African type, lightening cosmetic treatments are sought in order to obtain a clear and uniform complexion.

The use of inhibitors or regulators of melanin synthesis, as well as any other depigmenting and / or whitening product, is therefore particularly advantageous in cosmetology and / or dermatology. This use is not only interesting when a true depigmentation of the skin is sought, as in the case of bleaching heavily pigmented skin or when inhibiting hyperpigmented skin areas resulting in an unsightly appearance of the skin; It is also used in certain applications aimed at lightening the complexion, giving luminosity to the skin or giving radiance to the surface tissues. To date, many molecules have been proposed with more or less efficiency. Among these molecules, mention may be made of phenol derivatives such as hydroquinone and resorcinol which inhibit a series of reaction of the conversion of L-tyrosine to melanine by inhibition of the tyrosinase activity (Takano, 1984). There may also be mentioned L-ascorbic acid and its derivatives, magnesium ascorbyl acetate, kojic acid or lactic acid. But most of the products currently on the market are toxic and / or not sufficiently effective. For example, hydroquinone is irritating and cytotoxic to the melanocyte. Kojic acid for example is not stable in solution, etc. There is therefore a need for a new whitening agent which does not have the disadvantages of existing ones but which would be equally effective. Thus, other ways have been explored to find a new depigmenting agent having an action on both the enzyme tyrosinase and melanin.

Surprisingly, the Applicant has discovered that a peptide hydrolyzate enriched in bioactive peptides, said hydrolyzate being a proteasome activator could be used as a whitening agent and had a good depigmenting activity without being toxic.

The ubiquitin-proteasome pathway plays a fundamental role in a very large number of biological processes. Indeed, the mechanisms of protein degradation by the proteasome are involved in important cellular mechanisms such as DNA repair, the control of gene expression, the regulation of cell cycle progression, the control of quality of neosynthesized proteins, apoptosis or immune response (Glickman and Ciechanover, 2002).

The proteasome present in human cells is a very large multi-protein complex present in the cytoplasm and the nucleus. Purified forms of the proteasome include 2 large subunits; on the one hand, a proteolytic core called proteasome 2OS and, on the other hand, a 19S regulator complex that binds to each of the two ends of the 2OS proteasome (Coux et al., 1996, Glickman and Coux, 2001).

The 2OS proteasome is a hollow cylinder-shaped particle, composed of 28 alpha and beta subunits, distributed in 4 heptameric rings. The peptidase activities are present on the inner surface of the cylinder and influence allosterically. Three proteolytic activities (trypsin, chymotrypsin and caspase-like) have been associated with the 2OS proteasome and contribute to the destruction of proteins as inactive peptides of 3 to 20 amino acids. In addition to the 2OS proteasome, the 26S proteasome comprises the 19S regulator complex of 0.7 MDa consisting of about 20 subunits. Recent immunopurification studies have shown that other proteins may be associated with the 20S and 19S proteasome (eg the 11S regulatory complex). Given the diversity of cellular processes controlled by protein degradation, it is not surprising to find that alterations of the ubiquitinproteasome pathway are at the origin, or closely related, to several genetic diseases and to many human pathologies such as than colorectal cancers, lymphomas, inflammatory syndromes, neurodegenerative diseases such as Parkinson's or Alzheimer's ...

Many studies have been conducted in recent years on the role of the proteasome in aging skin. One of the recently explored pathways was the proteasome and its action on the degradation of proteins involved in melanogenesis. Experiments have shown that the degradation of the tyrosinase enzyme by the proteasome was activated by an algal extract on human melanocytes (Nizard et al., Antioxid Redox Signal, 2006, 1-2: 136-143). A composition comprising an extract of silybin, Bletilla striata and Iris sanguinea capable of increasing the activity of the proteasome and having an effect on the pigmentation of the skin has been disclosed in a patent application (US 12 / 088 919).

Thus, the Applicant has discovered that a peptide hydrolyzate enriched in bioactive peptides was capable of activating the proteasome and could thus depigment, lighten or whiten the skin and the integuments.

Therefore, the present invention firstly relates to a proteasome activator peptide hydrolyzate enriched in bioactive peptides of 3 to 5 amino acids, each bioactive peptide comprising at least one arginine residue, a lysine residue and a glycine residue. The subject of the present invention is a cosmetic composition comprising, as active principle, said enriched peptide hydrolyzate. In addition, the third subject of the present invention is the use of a cosmetic composition comprising said peptide hydrolyzate for improving the proteasome degradation of the damaged proteins and thus depigmenting the skin and / or the integuments and preventing and / or treating the hyperpigmentation spots. Finally, the fourth subject of the present invention is a process for cosmetic treatment of the skin or integuments to be treated with the aid of the composition comprising the said enriched peptide hydrolyzate. FIG. 1 represents an example of chromatogram obtained by HPLC, with detection of the peak corresponding to the bioactive peptide in a maize hydrolyzate. FIG. 2 represents an example of chromatogram obtained by HPLC, with demonstration of the peak corresponding to the bioactive peptide in a pea hydrolyzate. Figure 3 shows an example of a chromatogram obtained by HPLC, with the peak corresponding to the bioactive peptide in a rice hydrolyzate being demonstrated. FIG. 4 represents an example of chromatogram obtained by HPLC, with demonstration of the peak corresponding to the bioactive peptide in a soy hydrolyzate.

FIG. 5 represents an example of chromatogram obtained by HPLC, with demonstration of the peak corresponding to the bioactive peptide in a hydrolyzate of Saccharomyces cerevisiae.

The subject of the present invention is first and foremost a proteasome activator peptide hydrolyzate enriched in bioactive peptides of 3 to 5 amino acids, each bioactive peptide comprising at least one arginine residue, a lysine residue and a glycine residue. By peptide hydrolyzate is meant a mixture of compounds predominantly represented by peptides or oligopeptides.

By bioactive peptides is meant a fragment of protein composed by the sequence of at least 3 amino acids linked to each other by modified or unmodified peptide bonds and which exhibit an activity as activator of the proteasome. Present in an inactive form in the proteins, they become active after hydrolysis of the latter.

The said enriched peptide hydrolyzate according to the invention is characterized in that it is an activator of the proteasome. Peptide hydrolyzate (and / or bioactive peptides) proteasome activator is understood to mean any peptide hydrolyzate, or peptide or biologically active derivative capable of increasing the activity of the proteasome, either by increasing the protein synthesis of the proteasome subunits ( by direct or indirect modulation of gene expression), or by other biological processes such as the stabilization of the subunits constituting the proteasome or the stabilization of messenger RNA transcripts. The enriched peptide hydrolyzate according to the invention is characterized in that it makes it possible to activate the degradation by the proteasome of the damaged proteins. The term "damaged proteins" means proteins that have undergone oxidation reactions due to reactive oxygen species (free radicals), glycated proteins or conjugated with products derived from lipid peroxidation, etc.

Preferably, the peptide hydrolyzate is enriched in bioactive peptides of general formula (I): embedded image In which, XI is an isoleucine, a leucine, a proline, a valine, a glycine, an alanine, or is absent X2 is a cysteine, a glutamic acid, a glutamine, a histidine, a methionine, or is absent Preferably, the peptide hydrolyzate is rich in bioactive peptides of the following formula: (SEQ ID No. 1) Ile Arg-Lys-Gly-Glu (SEQ ID No. 2) Leu-Arg-Lys-Gly-Met (SEQ ID No. 3) Arg-Lys-Gly-His (SEQ ID No. 4) Pro-Arg-Lys In fact, these peptides have been identified as being particularly active as activators of the proteasome and are therefore of particular interest as lightening agents.

The enriched peptide hydrolyzate according to the invention can be obtained from various protein sources, whether of animal or vegetable origin. According to a first embodiment, the enriched peptide hydrolyzate is derived from the hydrolysis of plants selected from corn (Zea mayz L.), pea (Pisum sativum), rice (Oryza sativa L.), or soybean (Glycine). Max L.). Preferably, the plants used are not subjected to prior fermentation. In a second embodiment, the enriched peptide hydrolyzate is derived from the hydrolysis of yeasts of the genus Saccharomyces, and more particularly from the species Saccharomyces cerevisiae. Thus, the invention can be carried out using the seeds of one of the many 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 preferably the grain freed of its envelope by a dehulling step. The invention can also be carried out using one of the many plants of the pea family (Fabaceae). For example, the plants of the pea Pisum sativum L. species will be used. The term pea further refers to the seed, itself rich in proteins (25%). The plants of the rice family (Poaceae), in particular those of the genus Oryza and more preferably the species Orvza sativa L may be used to produce the hydrolyzate according to the invention. The plant material used will be the grain and preferentially the grain removed from its envelope by a dehulling step.

The invention can also be carried out using Fabaceae seeds, of the genus Glycine (soybean) or soybean meal, and preferentially Glycine Max species. L. According to the invention, the plant material used will be the grain and more preferably the grain removed from its envelope by a dehulling step.

Finally, the invention can also be carried out using yeasts of the genus Saccharomyces; and, preferably, yeasts of the Saccharomyces cerevisiae species.

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, or a specific part of the plant (leaves, tubers, roots, etc.) 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).

When it comes to yeasts, in a first step, they are cultured in a conventional manner in a medium adapted to their development, preferably in the presence of lactose. They are harvested by centrifugation and then suspended in a buffer solution, preferably a phosphate buffer. In a second step, these cells are exploded using a French press or using a ball mill, the majority of the insoluble membrane components being removed by centrifugation or by filtration. The proteins are then extracted by the conventional method (Osborne, 1924) modified; the plant mash or the yeast lysate 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 possibly 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. We can then mention the use of endoproteases of plant origin (papain, bromelain, ficin) and microorganisms (Aspergillus, Rhizopus, Bacillus, etc.). The hydrolysis conditions are chosen to promote the enrichment of bioactive peptides.

For the same reasons as above, that is to say the removal of the polyphenol substances, an amount of polyvinylpolypyrrolidone is added to the reaction medium during this mild hydrolysis step. After filtration, to remove enzymes and polymers, a first filtrate is obtained. The hydrolyzate obtained at this stage can be further purified in order to select the fractions of low molecular weight, preferably less than 6 kDa, as well as the peptides generated according to their nature. The purification is advantageously carried out by successive ultrafiltration steps through decreasing porosity filters, retaining the filtrates at each stage and / or by a chromatographic type method, in order to specifically enrich the hydrolyzate with bioactive peptides.

A dilution phase is carried out in water or in any mixture containing water and then sterilized by ultrafiltration in order to obtain an enriched peptide hydrolyzate characterized by a protein content of 0.5 to 5.5 g / ml. 1. This enriched 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), which makes 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 peptides. Finally, the enriched peptide hydrolyzate obtained is composed of peptides with a molecular weight of less than 6 kDa, and is enriched in bioactive peptides of 3 to 5 amino acids comprising at least one arginine residue, a lysine residue and a glycine residue.

This peptide hydrolyzate, according to the invention, enriched in bioactive peptides may also be used as a medicament.

The second subject of the present invention relates to a cosmetic composition comprising as active principle the peptide hydrolyzate enriched in bioactive peptides as described above. Preferably, the compositions according to the invention are in a form suitable for topical application comprising a cosmetically acceptable medium. By cosmetically acceptable means media that are suitable for use in contact with the skin or skin appendages, without risk of toxicity, incompatibility, instability, allergic response and others. Preferably, said peptide hydrolyzate is present in the composition 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. In the compositions according to the invention, hydrolysas. peptide enriched in bioactive peptides is solubilized in one or more solvents such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, vegetable oil or any mixture of these solvents. According to yet another advantageous embodiment, the peptide hydrolyzate according to the invention is solubilized in a cosmetic or pharmaceutical vector such as liposomes or adsorbed on organic powdery polymers, mineral supports such as talcs and bentonites, and more generally solubilized in or attached to any physiologically acceptable vector. The compositions intended to be applied to the skin may be in the form of aqueous or aqueous-alcoholic solution, water-in-oil or oil-in-water emulsion, microemulsion, aqueous or anhydrous gel, serum or dispersion of vesicles, patch, cream, spray, ointment, ointment, lotion, colloid, solution, suspension or other. The compositions may also be applied to the integuments in the form of shampoo, dye or mascara to be applied by brush or comb, in particular on eyelashes, eyebrows or hair, or care for nails such as varnishes .

In a particular embodiment, the composition according to the invention further contains at least one other active ingredient promoting the action of said enriched peptide hydrolyzate. The classes of ingredients having an activity in the field of lightening agents such as desquamating agents may be mentioned, in a nonlimiting manner; soothing agents, organic or inorganic photoprotective agents, moisturizing agents; other depigmenting agents, tyrosinase inhibitors; agents stimulating the synthesis of dermal or epidermal macromolecules and / or preventing their degradation; agents stimulating the proliferation of fibroblasts and / or keratinocytes or stimulating the differentiation of keratinocytes; agents acting on the energetic metabolism of cells; other depigmenting peptides, plant hydrolysates, anti-aging agents, and mixtures thereof. In addition, additives such as thickening agents, emulsifiers, humectants, emollients, perfumes, antioxidants, film-forming agents, chelating agents, sequestering agents, conditioning agents, etc. may be added to the composition. 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, be 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. Finally, the composition as described makes it possible to increase the activity of the proteasome and to improve the proteasome degradation of the damaged proteins involved in melanogenesis.

A third subject of the invention relates to the use of a cosmetic composition comprising said enriched peptide hydrolyzate and a cosmetically acceptable medium for depigmenting, lightening and / or whitening the skin. The composition according to the invention also makes it possible to prevent and / or treat hyperpigmentation spots such as melasma, chloasma, actinic lentigo, solar lentigines, ephelides, accidental hyperpigmentation spots, hyperpigmentation spots. post-cicatricial. Another subject of the invention relates to the use of a composition comprising said peptide compound for treating and / or preventing cutaneous signs of hyperpigmentary order due to photo-aging. Photo-aging is the premature aging of the skin caused by prolonged and cumulative exposure to the sun. Finally, another subject of the invention relates to the use of a cosmetic composition according to the invention for increasing the activity of the proteasome and improving the degradation by the proteasome of the damaged proteins involved in melanogenesis.

A final subject of the present invention relates to a cosmetic treatment method characterized in that a composition comprising an effective amount of peptide hydrolyzate according to the invention for depigmenting, is applied topically to the skin or integuments to be treated. lighten and / or whiten skin or integuments. Moreover, this cosmetic treatment method is also intended to prevent and / or treat the cutaneous signs of hyperpigmentary order due to photoaging.

The following examples describe and demonstrate the efficacy of peptide compounds as described according to the invention but should not be construed as a limitation of the present invention. Example 1 Preparation of a peptide hydrolyzate from corn cakes (Zea mavs L) The corn meal (Zea mays L.) is dissolved in 10 volumes of water in the presence of 2% of POLYCLAR 10 (polyvinylpolypyrrolidone) PVPP - insoluble). The mixture is adjusted to a pH of between 6 and 8 with a 1M aqueous sodium hydroxide solution. After adjusting the pH, papain 2% is added to the reaction medium.

The hydrolysis is obtained after stirring for 2 hours at 55 ° 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. Specific hydrolysis conditions are chosen so as to allow enrichment of bioactive peptides of 3 to 5 amino acids comprising at least one arginine residue, a lysine residue and a glycine residue. The purification process of the crude hydrolyzate starts with successive filtrations using Seitz-Orion plate filters of decreasing porosity (up to 0.2 m) in order to obtain a yellow, brilliant and clear solution, qualified as hydrolyzate 1.

At this stage, the maize 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 sugar content 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. 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 keep only those compounds of peptidic nature 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 enriched in bioactive peptides corresponds to the active principle according to the invention. This peptide hydrolyzate is then analyzed by high pressure liquid chromatography (HPLC) using an HP 1100 device controlled by the ChemStation software. The column used during the elution of the hydrolyzate is a Nucleosil 300-5 C4 MPN (125 x 4 min), allowing to chromatograph proteins having molecular weights of 0.2 to 25 kDa under the following conditions: - Methanol gradient - Uptisphere OPB 125 x 3 mm column - Solvent A: HPLC grade water containing 0.1% heptafluorobutyric acid (HFBA) - Solvent B: HPLC grade methanol containing 0.1% heptafluorobutyric acid (HFBA) - Gradient: 100% to 15% solvent A in 35 min An example of chromatogram obtained by HPLC (high-pressure liquid chromatography), with highlighting of the peak corresponding to the bioactive peptides is given in FIG.

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 peptides.

Example 2 Preparation of a peptide hydrolyzate enriched in bioactive peptides from peas (Pisum sativum L.) The peptide hydrolyzate is obtained from an extract of plants of the species Pisum sativum L. Of course the extract can be prepared from plants of at least one of many varieties and species belonging to the genus Pisum.

In a first step, 1 kg of husked peas are delipidated by the action of an organic solvent: hexane. The pea flour thus obtained is dissolved in 10 volumes of water in the presence of 2% of POLYCLAR 10 (polyvinylpolypyrrolidone - PVPP - insoluble). The mixture is adjusted to a pH of between 6 and 7 with a 1M aqueous sodium hydroxide solution. After adjusting the pH, 2% of flavourzym is added to the reaction medium. The hydrolysis is obtained after 2 hours of stirring at 50 ° C. The enzyme is inactivated by heating the solution at 80 ° C for 2 hours. The reaction mixture thus obtained corresponds to the pea extract. Specific hydrolysis conditions are chosen so as to allow enrichment of bioactive peptides of 3 to 5 amino acids comprising at least one arginine residue, a lysine residue and a glycine residue. The purification process begins with successive filtrations using decreasing porosity Seitz-Orion plate filters (up to 0.2 m) to obtain a bright and clear solution. At this stage, the pea hydrolyzate is characterized by a solids content of 70-80 g / kg, a protein content of 55-65 g / l, a sugar content of 2-5 g / l and a polyphenol of 1-3 g / l. The protein nature of this hydrolyzate is demonstrated by polyacrylamide gel electrophoresis. For this analysis, NuPAGE Bis-Tris Pre-cast (Invitrogen) gels are used. The pea peptide hydrolyzate is heated at 70 ° C for 10 minutes under denaturing reducing conditions in NuPAGE LDS sample preparation buffer. Protein migration is performed using the 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, two large families of proteins are observed: the first family corresponds to proteins of molecular weight of 20 to 25 kDa and the last family to proteins of molecular weight less than 5 kDa. This solution is then purified by removing the molecular weight proteins greater than 5 kDa using tangential flow filtration. For this, the pea hydrolyzate is pumped under pressure through a Pellicon support equipped with Pellicon 2 Biomax 30 kDa cassette. This first filtrate is recovered and then filtered through another Pellicon 2 Biomax 5 kDa cassette. At the end of purification, a yellow-beige pea peptide hydrolyzate is obtained which is brilliant and clear. It is characterized by a dryness of 50 to 55 g / kg, a protein content of 50 to 52 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 pea extract is a Nucleosil 300-5 C4 MPN (125 x 4 min). This column makes it possible to chromatograph proteins having molecular weights of 0.2 to 25 kDa (according to a suitable solvent gradient, identical to Example 1). In these chromatographic conditions, several peptide fractions could be isolated. 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 peptides. An example of a chromatogram obtained by HPLC (high-pressure liquid chromatography), showing the peak corresponding to the bioactive peptides is given in FIG. 2. The determination of the amino acid composition of the active ingredient according to the invention has also been carried out. This is carried out after acid hydrolysis and identification by high pressure liquid chromatography using pre-derivation with PICT (phenylisothiocyanate). Example 3 Preparation of a Peptide Hydrolysate Enriched with Bioactive Peptides from Soybean Meal (Glycine Max L.) The active principle is obtained from plants of the species Glycine max L. Of course, the extract can be prepared from plants of at least one of the many varieties and species belonging to the genus Glycine. The cakes are the solid residues obtained after extraction of the oil from the soya beans. They represent 50 to 75% of the seed mass.

In a first step, 1 kg of soybean meal is milled in a cereal mill. The flour obtained is delipidated by the action of an organic solvent, hexane. After filtration and drying under vacuum, the powder obtained is suspended in an aqueous alkaline solution (1/10 dilution) pH 10, containing 1% of polyvinylpolypyrrolidone (POLYCLAR V ISP). 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 will be performed 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 soybeans, is then precipitated by varying the ionic strength in a neutral or acidic medium to remove soluble carbohydrate components, lipids, and nucleic acids. The medium is brought to pH 3.5. The supernatant is removed and the precipitate is then washed with a solvent such as, for example, ethanol or methanol, and the solvent is evaporated by drying in vacuo. At this stage, about 50 grams of light yellow powder of crude protein extract are obtained containing: - Proteins: 75% - Carbohydrates: 20% - Lipids: 5% The precipitate rich in proteins is dissolved in water or another solvent. 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% polyvinylpolypyrrolidone (POLYCLAR V ISP) and cysteine endopeptidases (papain, ficin). After reaction, the hydrolyzate is filtered on a plate and then on a sterilizing cartridge (0.2 m). A light-colored hydrolyzate, titrating with 15 to 30 g / l of dry extract, is then obtained, which is then diluted so that the concentration of peptides determined by the method of Lowry is between 0, 1 and 5 g / l and preferably between 0.5 and 2 g / l. The physicochemical analysis of this peptide hydrolyzate shows that its pH is between 4 and 7, and preferably between 5 and 6, the solids content is 1 to 8 g / l and preferably 2 to 5 g. / 1, its content of peptide compounds is between 0.1 and 5g / l, and preferably between 0.5 and 2 g / l and its sugar content between 0.5 to 2.5 g / l. Specific hydrolysis conditions are chosen so as to allow enrichment of bioactive peptides of 3 to 5 amino acids comprising at least one arginine residue, a lysine residue and a glycine residue. The solution is then ultrafiltered on a Millipore Helicon filtration cartridge (1 kDa cutoff). The high molecular weights contained in the retentate are removed, the filtrate is preserved. 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 soy hydrolyzate is a Nucleosil 300-5 C4 MPN (125 x 4 min). This column makes it possible to chromatograph proteins having molecular weights of 0.2 to 25 kDa (according to a suitable solvent gradient, identical to Example 1). Under these chromatographic conditions, several peptide fractions were isolated. 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 peptides. An example of chromatogram obtained by HPLC (high-pressure liquid chromatography), with highlighting of the peak corresponding to the bioactive peptide, is given in FIG. 4. Determination of the amino acid composition of the active principle according to the invention was also carried out. This is carried out after acid hydrolysis and identification by high pressure liquid chromatography using pre-derivation with PICT (phenylisothiocyanate). One variant of the protocol consists in carrying out a purification of the active principle, obtained according to the preceding protocol, by ion exchange chromatography, on a TSK gel column (TosoHaas) with a pH 7 phosphate buffer, in order to enrich the hydrolyzate Peptide peptide bioactive.

EXAMPLE 4 Preparation of a Peptide Hydrolyzate Enriched with Bioactive Peptides from Saccharomyces cerevisiae yeasts The peptide hydrolyzate can be obtained from a yeast extract of the Saccharomyces cerevisiae species. The yeasts are cultured in a medium adapted to their development, preferably in the presence of lactose, and then centrifuged to recover a biomass. The Saccharomyces biomass is dissolved in 10 volumes of water in the presence of 2% of polyvinylpyrrolidone (POLYCLAR 10 - insoluble) and 0.2 of activated carbon. The mixture is adjusted to a pH of between 6 and 7.5 with a 1M aqueous sodium hydroxide solution. After adjusting the pH, 2% of papain is added to the reaction medium. The hydrolysis is obtained after stirring for 2 hours at 55 ° C. The enzyme is inactivated by heating the solution at 80 ° C for 2 hours. After centrifugation, the reaction mixture corresponding to the Saccharomyces extract is then obtained. Specific hydrolysis conditions are chosen so as to allow enrichment of bioactive peptides of 3 to 5 amino acids comprising at least one arginine residue, a lysine residue and a glycine residue.

The purification process begins with successive filtrations using decreasing porosity Seitz-Orion plate filters (up to 0.2 m) to obtain a bright and clear solution. At this stage, the Saccharomyces extract is characterized by a dryness of 25 to 35 g / kg, a protein level of 10 to 15 g / l and a sugar content of 5-10 g / l.

The protein nature of this extract is demonstrated by polyacrylamide gel electrophoresis. For this assay, NuPAGE Bis-Tris Pre-cast (Invitrogen) gels are used. The peptide 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 using the 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, three large families of proteins are observed: the first family corresponds to proteins of molecular weight greater than 75 kDa, the second family to proteins of 20 to 25 kDa and the last family to proteins of molecular weight less than 5kDa. This solution is then purified by removing proteins of molecular weight greater than 5 kDa using tangential flow filtration. For this purpose, the Saccharomyces peptide hydrolyzate is pumped under pressure through a Pellicon support equipped with a 50 kDa Pellicon 2 Biomax cassette. This first filtrate is recovered and then filtered through a second Pellicon 2 Biornax 10 kDa cassette. It is then recovered a second filtrate which is still eluted through a last cassette Pellicon 2 Biomax 5 kDa. At the end of purification, a vegetable extract of beige, brilliant and limpid saccharomyces is obtained. It is characterized by a dryness of 35 to 45 g / kg, a protein content of 30 to 40 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 saccharomyces hydrolyzate is a Nucleosil 300-5 C4 MPN (125 x 4 min). This column makes it possible to chromatograph proteins having molecular weights of 0.2 to 25 kDa (according to a suitable solvent gradient, identical to Example 1). Under these chromatographic conditions, several peptide fractions were isolated.

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 peptides. An example of chromatogram obtained by HPLC (high-pressure liquid chromatography), with detection of the peak corresponding to the bioactive peptides, is given in FIG. 5. Determination of the amino acid composition of the active principle according to the invention was also carried out. This is carried out after acid hydrolysis and identification by high pressure liquid chromatography using pre-derivation with PICT (phenylisothiocyanate).

EXAMPLE 5 Demonstration of the Depigmenting Effect of a Rice Peptide Hydrolyzate enriched in Bioactive Peptides by Ex Vivo Tests The depigmenting activity of a rice hydrolyzate enriched in bioactive peptides produced according to the invention was highlighted on a skin sample.

Biopsies 6 mm in diameter are taken from human skin samples. These biopsies are maintained in ex vivo survival by culture in the presence of a specific medium (DMEM 1 g / L, HAMF12, FCS and antibiotics) on inserts deposited in 6-well plates. The biopsies are then pretreated for 24 hours with the peptide hydrolyzate at a concentration of 3% at the rate of 2 applications per day.

Other skin samples will not be pretreated with this same hydrolyzate and will serve as a control condition. Subsequently, the biopsies are subjected to UVB irradiation at a rate of 100 mJ / cm 2. The pre-treated biopsies are treated again for 24 hours with the peptide hydrolyzate at a concentration of 3% at the rate of 2 applications per day. After this second treatment, a quantitative evaluation of the amount of melanin present in the epidermis of the skin samples is performed histologically, under an optical microscope, after staining according to the Fontana-Masson method. For this purpose, skin biopsies are included in the paraffin and histological sections of 4 μm thick are made. These sections are then stained by the Fontana-Masson technique: the slides are deparaffinized, hydrated and then treated with a solution of silver ammonia. After passing for two minutes in the microwave, the slides are rinsed, treated with sodium thiosulphate, rinsed again and counter-stained with hematoxylin before being dehydrated and mounted under lamellae thus allowing visualization under an optical microscope melanin present in the epidermis.

Results: Before UVB irradiation, the untreated skins have a higher level of pigmentation compared to the skins treated with the peptide hydrolyzate of rice. After UVB irradiation, the skins treated with said hydrolyzate have a significantly lower level of pigmentation compared to the untreated skins of the control condition. Therefore, these results allow us to conclude that in the absence of UVB irradiation, the rice peptide hydrolyzate enriched in bioactive peptides, decreases the melanin level compared to the untreated skin sample. Moreover, the melanin synthesis, induced by UVB irradiation, is very clearly attenuated when the skin samples are pretreated with our active ingredient. Thus, the peptide hydrolyzate according to the invention makes it possible to significantly reduce the pigmentation of the skin and makes it possible to obtain an inhibition of melanin synthesis. (Example 6: Depigmenting Cosmetic Composition with Solar Filter Trade Names I INCI Names I% Mass PHASE A Montanov 68 Cetearyl Alcohol (and) Cetearyl Glucoside 5.00 Isopropyl Palmitate Isopropyl Palmitate 7.00 Waglinol 250 Cetearyl Ethylhexanoate 3.00 Dow Corning 200 Dimethicone Polydimethylsiloxane 0.50 Parsol MCX Ethylhexyl Methoxycinnamate 3.00 Parsol 1789 Butyl Methoxydibenzoylmethane 1.00 Trade Names INCI Names% by mass Phenonip Phenoxyethanol (and) Methylparaben (and) 0.50 Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben Cegesoft PS6 Vegetable Oil 2.00 Jojoba Oil Simmondsia Chinensis (Jojoba) Seed Oil 5.00 PHASE B Water Demineralized Aqua (Water) qsp Glycerin Glycerin 3.00 Glucam E10 Methyl Gluceth-10 0.50 EDTA Tetrasodium EDTA 0.20 PHASE C Sepigel 305 Polyacrylamide (and) C13-14 Isoparaffin 0.35 (and) Laureth-7 Lemon Juice Citrus medica Limonum ( Lemon) Fruit 0.23 Extract PHASE D Peptide hydrolyzate of rice 2.00 Compo parfuma perfume (Fragrance) qsp Dye dye qsp

Claims (18)

REVENDICATIONS1. A peptide hydrolyzate activator of the proteasome, characterized in that the hydrolyzate is enriched in bioactive peptides of 3 to 5 amino acids, each bioactive peptide comprising at least one arginine residue, a lysine residue and a glycine residue. 2. An enriched peptide hydrolyzate according to claim 1, characterized in that the bioactive peptide is of general formula (I): ## STR2 ## wherein X 1 is an isoleucine, a leucine, proline, valine, glycine, alanine, or is absent X2 is cysteine, glutamic acid, glutamine, histidine, methionine, or is absent 3. An enriched peptide hydrolyzate according to the preceding claim, characterized in that the bioactive peptide corresponds to one of the following formulas: (SEQ ID No. 1) Ile ù Arg ù Lys ù Gly ù Glu (SEQ ID No. 2) Leu ù Arg ù Lys ù Gly ù Met 20 (SEQ ID No. 3) Arg ù Lys ù Gly ù His (SEQ ID No. 4) Pro ù Arg ù Lys ù Gly ùMet (SEQ ID No. 5) Arg ù Lys ù Gly 4. enriched peptide hydrolyzate according to one of claims 1 to 3, characterized in that it is derived from the hydrolysis of plants selected from corn (Zea mayz L.), pea (Pisum sativum), rice (Gold), za sativa L.), or soybean (Glycine Max L.). 5. Enriched peptide hydrolyzate according to one of claims 1 to 3, characterized in that it is derived from the hydrolysis of yeasts of the genus Saccharomyces, and more particularly from the species Saccharomyces cerevisiae. 6. Peptide hydrolyzate according to any one of the preceding claims, characterized in that said enriched peptide hydrolyzate contains between 0.5 and 5.5 g / l of peptide-like compounds. An enriched peptide hydrolyzate according to any one of the preceding claims, characterized in that it is used as a medicament. 8. Cosmetic composition comprising as active ingredient said enriched peptide hydrolyzate according to one of claims 1 to 6. 9. Cosmetic composition according to claim 8, characterized in that it is in a form suitable for topical application comprising a cosmetically acceptable medium. 10.Composition according to one of claims 8 or 9, characterized in that said enriched peptide hydrolyzate is present in the composition in an amount representing from 0.0001% to 20% of the total weight of the composition, and preferably in one amount representing from 0.05% to 5% of the total weight of the composition. 11.Composition according to any one of claims 8 to 10, characterized in that said hydrolyzate is solubilized in one or more 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. 12.Composition according to any one of claims 8 to 11, characterized in that it further contains at least one other active ingredient promoting the action of said enriched peptide hydrolyzate. 13.Composition according to claim 12, characterized in that said active ingredient is an agent having an activity in the field of lightening agents such as desquamating agents; soothing agents, organic or inorganic photoprotective agents, moisturizing agents; other depigmenting agents, tyrosinase inhibitors; agents stimulating the synthesis of dermal or epidermal macromolecules and / or preventing their degradation; agents stimulating the proliferation of fibroblasts and / or keratinocytes or stimulating the differentiation of keratinocytes; agents acting on the energetic metabolism of cells; depigmenting peptides, plant hydrolysates, anti-aging agents, and mixtures thereof. 14. Use of a cosmetic composition comprising said enriched peptide hydrolyzate as defined in one of claims 1 to 6 and a cosmetically acceptable medium, for increasing the activity of the proteasome and improving the degradation of the damaged proteins involved in melanogenesis. 15.Use of a cosmetic composition comprising said enriched peptide hydrolyzate as defined in one of claims 1 to 6 and a cosmetically acceptable medium for depigmenting, lightening and / or whitening the skin. 16.Use of a composition according to claim 15 for preventing and / or treating hyperpigmentation spots such as melasma, chloasma, actinic lentigo, solar lentigines, ephelides, accidental hyperpigmentation spots, post-scarring hyperpigmentation spots. 17.Use according to claim 15 for treating and / or preventing cutaneous signs of hyperpigmentary order due to photo-aging. 18. Cosmetic treatment process characterized in that a composition comprising an effective amount of enriched peptide hydrolyzate as defined in one of Claims 1 to 6 is applied topically to the skin or integuments to be treated in order to depigment , lighten and / or whiten the skin or integuments. 20 25