New use of peptides for improving dermal and / or mucosal extracellular matrix of skin

Abstract

The present invention relates to the non-therapeutic cosmetic use of a peptide of the sequence SEQ ID No.1 and / or a homolog and / or salt and / or derivative thereof, in particular a rice hydrolysate enriched in said peptide or a fermentation form enriched in said peptide, in order to improve the ECM fibres of the dermis and / or mucous membrane of the skin. The invention also relates to the use of said peptides in cosmetic and / or dermatological compositions.

Description

Technical Field

[0001] The present invention relates to the use of a peptide of sequence SEQ ID No. 1 and / or its homologues, derivatives and / or salts, optionally in the form of a microbial fermentation product or a protein hydrolyzate for preparing the peptide, to improve the flexibility and / or density of extracellular matrix fibers of the skin dermis and / or mucosa, especially the skin (including scalp) and / or mucosa, and / or the anchoring of appendages (preferably hair), especially to prevent and / or slow down shedding. The present invention also relates to the use of the peptide in cosmetic and / or dermatological compositions. Background Art

[0002] The extracellular matrix (ECM) plays a vital role in the tissue structure of the human and animal body, especially including functions such as providing support, adhesion and regulating exchange for cells. ECM is mainly composed of glycoproteins, proteins and glycosaminoglycans. These molecules are synthesized in natural form in cells that come into contact with the ECM. They are excreted from the intracellular compartment. After maturation, they are organized and arranged to form the fibrous network of the ECM. In this fibrous form, they are in their functional form.

[0003] Exposure of the skin to aggressive environmental factors, such as temperature changes (especially hot and cold), humidity, dry air, sunlight and / or pollution, activates proteases that lead to degradation of the dermal ECM, in particular by reducing the number of collagen fibers and elastic fibers in the dermis. This causes the skin, and in particular the dermis, to lose its conventional properties, such as density and flexibility. Therefore, in the cosmetic and pharmaceutical fields, ECM molecules have been particularly studied, with many ingredients aimed at preventing and compensating for this degradation in order to improve the overall condition of the dermis.

[0004] One of the conventional approaches is to inhibit proteases and thus prevent the degradation of elastic fibers. However, this purely preventive measure does not restore the already reduced ECM properties. A second approach consists in stimulating fibroblasts to synthesize procollagen I and elastin. However, the increase in this synthesis measured only in cells cultured in vitro does not necessarily mean that a greater amount of functional molecules, i.e. functionalized molecules in the ECM, is produced. Therefore, this approach is not enough to restore certain properties of the skin, in particular flexibility and density.

[0005] Restoring these properties requires obtaining a specific organization and arrangement of the dermal ECM fibers, which requires the coordination of multiple factors. In fact, in the case of collagen, the formation of the triple helical structure of collagen (tropocollagen) and its extracellular secretion in the extracellular matrix, as well as the assembly and organization of collagen triple helices into fibrils (the fibrils themselves organize into larger mature fibers), are necessary to maintain and restore functional ECM in the dermis and the properties of the dermis.

[0006] Likewise, the formation of mature elastic fibers with the ability to stretch is necessary to maintain a functional ECM within the dermis. Elastin is synthesized by fibroblasts as soluble tropoelastin and is cross-linked by enzymes of the lysyl oxidase family and deposited on fibrillin-rich microfibrils in the extracellular matrix to form mature elastic fibers, which acquire their physicochemical properties, especially stretchability and return to the original state after stretching. In addition to fibrillins, especially fibrillin-1, fibulin-5, EMILIN-1 and MFAP4 (microfibril-associated protein 4) are also particularly important in the formation of these mature elastic fibers.

[0007] The object of the present invention is therefore to provide a new cosmetic and / or dermatological active ingredient to improve the ECM fibers of the skin dermis, in particular the scalp and mucous membranes. The object of the present invention is also to provide a new cosmetic and / or dermatological active ingredient to improve the density and / or flexibility of the skin and / or mucous membranes. The object of the present invention is also to provide a new cosmetic and / or dermatological active ingredient to meet the special needs of caring for and / or treating skin and / or mucous membranes with weakened and / or altered dermal ECM, in particular under the action of aggressive substances, in particular in the case of sensitive and / or sensitized skin and / or mucous membranes.

[0008] Surprisingly and unexpectedly, the inventors have discovered that the peptide of SEQ ID No. 1, its homologues, salts and / or derivatives are capable of improving the fibrillar nature of the dermal ECM.

[0009] In patent application WO2017009484A1 (as SEQ ID No.349), among many peptides from rice and pea, the peptide of SEQ ID No.1 has been described in the field of cosmetics as an anti-inflammatory agent capable of inhibiting the secretion of TNFα by macrophages in the presence of lipopolysaccharide, and in patent application WO2017009490A1 (as SEQ ID No.245) as an active agent capable of increasing the proliferation of epidermal basal cells and having a mitogenic effect on the epidermal basal layer, thereby being able to treat visible signs of aging.

[0010] The peptide is naturally present in the proteins of rice grains of Oryza sativa, from which it can be obtained. However, the amount of its natural presence is insufficient to detect and produce the properties that are the subject of the present invention. Within the scope of the present invention, it is obtained by chemical synthesis and by a biotechnological preparation method of the yeast Saccharomyces cerevisiae. However, it can also be obtained by an enzymatic hydrolysis method of rice grains as described in patent applications WO2017009484A1 and WO2017009490A1.

[0011] Although there are a large number of rice peptide hydrolysates on the beauty and cosmetics market, their performance and composition vary greatly, especially they contain different protein fragments, which have a great influence on their biological properties. During protein hydrolysis, the bonds connecting different peptides and amino acids are actually cut by different conventional methods in the field (for example, by acid, base or catalysis). However, these different technologies lead to the production of protein hydrolysates with different components, resulting in the production of free amino acids. Due to its non-specificity, chemical hydrolysis using acid or base usually leads to the preparation of products of poor quality, especially in terms of color, smell and / or stability. Due to its specificity, enzymatic hydrolysis has the advantage of being able to be carried out under controlled conditions (especially pH, temperature and pressure). However, it is also necessary to select the enzyme to obtain the target peptide and obtain a specific hydrolyzate, which is very different in terms of the peptide fragments it contains, whether in the nature of the fragments, molecular weight or distribution.

[0012] All of these rice hydrolysates from different sources can be used for skin and / or hair care in the cosmetic field.

[0013] However, due to their non-specific preparation methods or also involving the acquisition of other peptides, all these hydrolysates have great differences in their composition and properties and none of them describes or contains a sufficient amount of the peptides of the invention to obtain the properties of the invention.

[0014] Therefore, the present invention relates to the non-therapeutic cosmetic use of the peptide of SEQ ID No. 1, its homologues, derivatives and / or salts for improving the ECM fibers of the healthy skin dermis and / or healthy mucosa.

[0015] This peptide has many other benefits. It can also increase the synthesis of collagen 1 by fibroblasts and reduce inflammation by inhibiting the release of TNFα by macrophages and interleukin-8 by keratinocytes.

[0016] The peptides according to the invention, through their complementary properties, offer a perfectly adapted and complete solution for the care and / or treatment of sensitive and / or sensitized skin and / or mucous membranes.

[0017] Furthermore, all these complementary properties also make it an excellent care and / or cosmetic treatment agent for the skin and / or mucous membranes and / or the health appendages of the body and face.

[0018] The peptide of SEQ ID No. 1 of the present invention can be obtained by chemical synthesis according to conventional methods. However, according to a preferred embodiment of the present invention, the peptide of SEQ ID No. 1 is obtained by recombinant microbial synthesis, preferably by bacteria such as Escherichia coli or yeast of the genus Pichia or Saccharomyces, preferably Saccharomyces cerevisiae. It is particularly interesting that the peptide of SEQ ID No. 1 is used together with the fermentation product of the microorganism that produces it.

[0019] Optionally, the peptide of the present invention is in the form of a hydrolyzate of rice protein from rice (Oryza sativa), advantageously, the peptide content is at least equal to or greater than 0.00001% (by dry matter weight) of the total weight of the hydrolyzate, preferably 0.0001% to 10% of the total weight of the hydrolyzate, more preferably 0.001% to 3% (by dry matter weight).

[0020] The peptide of the present invention is the peptide of SEQ ID No. 1: TVFDGVLRPGQL.

[0021] The peptide contains 12 amino acids: Tyr Val Phe Asp Gly Val Leu Arg Pro Gly Gln Leu, with an average molecular weight of 1301.51 g / mol.

[0022] The 12 amino acid sequence is naturally present in rice grain protein (Oryza sativa), and the peptide can be obtained by enzymatic hydrolysis of rice grains as described in patent applications WO2017009484A1 and WO2017009490A1, or by chemical or biotechnological synthesis according to conventional methods in the art. It can also be purified or obtained as shown in Example 1.

[0023] According to the present invention, the term "peptide" refers to any isolated, natural or synthetic amino acid sequence, which can be obtained, where appropriate, by chemical or biotechnological synthesis or by extraction from biological tissues (such as plants, animals or microorganisms, in particular yeast), which naturally express the amino acid sequence or express it after transduction, optionally after its translational modification.

[0024] The term "derivative" refers to a peptide modified by the addition of functional groups, such as coupling with hydrophilic or hydrophobic penetrants, stabilizers or protecting groups according to conventional methods known to those skilled in the art, in particular by the terminal -NH 2This may involve, in particular, grafting a carboxyl group containing 1 to 24 carbon atoms, saturated or unsaturated, linear or branched, onto the free -NH 2 The carboxyl group may also contain -OH, -SH, COOH or CONH 2 It can also involve grafting onto the free -COOH group of the last amino acid, converting the amide group (-CONH 2 ) or an alkoxy group is grafted onto the free -COOH group of the last amino acid, the group comprising 1 to 24 carbons, saturated or unsaturated, straight or branched, and may also contain -OH, -SH, COOH or CONH 2 Group.

[0025] Techniques for chemical synthesis of peptides are known to those skilled in the art, and for example, there can be mentioned the techniques described in the following references: JM Stewart and JD Yound, Solid phase peptide synthesis, 2nd editions, Pierce Chemical Company, Rockford, Illinois (1984), and M. Bodanzsky and A. Bodanzsky, The practive of peptide synthesis, Springer Verlag. New York (1984).

[0026] According to the present invention, the "homologue" of an amino acid sequence refers to an amino acid sequence that has at least 85%, more preferably 90%, particularly at least 95%, more particularly at least 98%, more preferably at least 99% sequence identity with the sequence and has the same biological activity. Sequence homology can be identified by any conventional technique in the art, for example, by the BLAST computer interface on the NCBI website: http: / / blast.ncbi.nlm.nih.gov, using the default settings configuration.

[0027] The homologue of an amino acid sequence may differ from the sequence, for example, by one or more deletions and / or insertions and / or one or more substitutions of an amino acid. According to an alternative embodiment, the homologue of an amino acid sequence may comprise one or more conservative amino acid substitutions. Conservative substitutions are substitutions of one amino acid with another amino acid in a sequence, wherein the another amino acid has a physicochemical property that is substantially similar to or sufficiently close to the original amino acid, so that the properties and functions of the peptide are not or are not substantially affected. The changes in the above-mentioned amino acid sequences may generally be referred to as "mutations". Therefore, the homologues of the peptides of the present invention also relate to mutants and variants of the amino acid sequences of the present invention having biological activities of the same nature. The "biological activity of the same nature" of the amino acid sequences of the present invention particularly refers to the ability thereof to exhibit the properties of the present invention.

[0028] According to the present invention, the term "salt" refers to a peptide containing a counter ion from an acid used in the cleavage process after its chemical synthesis. Salts of the peptides of the present invention include trifluoroacetate, acetate and hydrochloride.

[0029] Therefore, the peptide of the present invention may exist in the form of its homologue, its salt and / or its derivative. Preferably, the peptide of the present invention is the peptide of SEQ 1.

[0030] The amino acids constituting the peptides of the present invention may be in their L, D or DL ​​form. Preferably, they are all in the L configuration.

[0031] According to the present invention, "improving the fibers of the dermal extracellular matrix (ECM)" refers to improving the structure and / or organization and / or density of the collagen fibers and / or elastic fibers of the dermal extracellular matrix. This improvement is measured by an increase in the number of functional fibers in the dermal extracellular matrix and leads to an increase in the flexibility and / or density properties of the skin and / or mucous membranes. Therefore, advantageously, the use according to the present invention is to increase the density and / or flexibility of healthy skin and / or healthy mucous membranes. These properties can be measured by conventional methods known to those skilled in the art. As an example, the following techniques can be mentioned:

[0032] -Flexibility: This property can be measured according to conventional techniques, in particular by measuring the elastic modulus (or Young's modulus), for example on a reconstructed skin model - the improvement of the dermal ECM fibers after treatment with the peptides of the invention results in a decrease in the elastic modulus values ​​measured compared to the values ​​obtained without treatment. This method is described in Example 4.

[0033] - Density: This property can be measured according to conventional techniques, in particular by measuring the echogenicity of ultrasound in the dermis to determine the total density of the dermis. After treating the skin with the peptides of the invention, the improvement in the density of the dermal ECM fiber network leads to an increase in the measured total dermal density value compared to the value obtained without treatment. This method is described in Example 6.

[0034] According to the invention, the improvement of dermal extracellular matrix fibers does not include an increase in collagen and elastin synthesis, as this is not sufficient to induce an improvement of extracellular matrix fibers alone, especially for sensitive and / or sensitized skin. In addition, preferably, the use of the invention is not for preventing and / or treating signs of aging.

[0035] According to the present invention, the improvement of the dermal extracellular matrix fibers is measured by an increase in the amount of fibrillin-1 in the dermal ECM fibers and / or an increase in the amount of EMILIN-1 in the dermal ECM fibers and / or an increase in the amount of collagen fibers in the dermal extracellular matrix. Therefore, advantageously, the use of the present invention is to increase the amount of fibrillin-1 in the ECM fibers of the healthy dermis and / or to increase the amount of EMILIN-1 in the ECM fibers of the healthy dermis and / or to increase the amount of collagen fibers in the extracellular matrix of the healthy dermis. The use of the present invention is also to increase the amount of fibulin-5 synthesized by healthy dermal fibroblasts and / or to increase the amount of MFAP4 synthesized by healthy dermal fibroblasts, thereby increasing the synthesis of fibulin-5 and / or MFAP4 by healthy dermal fibroblasts.

[0036] The amount of fibrillin-1 in the dermal ECM can be measured by immunolabeling in a reconstructed skin model, for example as described in Example 2, and the increase in the presence of a peptide of the invention can be assessed by comparison with measurements made in the absence of the peptide.

[0037] The amount of EMILIN-1 in the dermal ECM can be measured by immunolabeling in a reconstructed skin model, for example as described in Example 2, and the increase in the presence of a peptide of the invention can be assessed by comparison with measurements made in the absence of the peptide.

[0038] The amount of collagen fibers in the ECM can be measured by measuring the intensity of the second harmonic signal by confocal optical microscopy, as described in Example 3.

[0039] The amount of fibulin-5 and / or MFAP4 synthesized by dermal fibroblasts can be measured by immunolabeling of dermal fibroblast lysates after culture, for example as described in Example 5, and the increased amount after culture in the presence of the peptide of the invention can be assessed by comparison with measurements made after culture in the absence of the peptide.

[0040] The peptides of the present invention are also suitable for improving the anchorage of appendages, in particular hair. Thus, the peptides of the present invention can prevent and / or reduce the loss of appendages, in particular hair loss.

[0041] For the purposes of the present invention, the term "appendages" refers to nails and "keratin fibers".

[0042] The term "keratin fibers" refers to hair fibers (hair), eyelashes, eyebrows, hair (especially nasal mucosal hair, ear hair, beard and / or moustache).

[0043] The peptides according to the invention are therefore particularly suitable for the care and / or treatment of sensitive or sensitized skin and / or mucous membranes.

[0044] Advantageously, the use according to the invention is therefore for caring for sensitive and / or sensitized skin and / or mucous membranes.

[0045] In general, "sensitive skin and / or mucous membranes" can be defined as healthy skin and / or healthy mucous membranes that have essentially very little tolerance to aggressive substances, in particular environmental substances (e.g. pollutants), climatic factors (wind, cold, heat), UV radiation, emotional factors (especially stress), and / or chemical agents (heavy metals, detergents, compounds contained in cosmetics, such as perfumes, preservatives, alcohol, pH, AHA or dermatological active agents, such as retinoic acid), and / or aggressive conditions, including sweating and mechanical aggression, such as hair removal, shaving, friction, and even water, in particular hard water. Sensitive skin is not pathological skin, unlike allergic skin. However, they may react to aggressive substances and / or conditions with unsightly and / or uncomfortable skin and / or mucous membrane manifestations, such as dryness of the skin and / or mucous membranes, loss of uniformity of the skin and / or mucous membrane color, in particular the appearance of redness, tightness, tingling, pins and needles, tension and / or itching, rough appearance of the skin and / or mucous membranes and / or loss of softness to the touch. Therefore, the nature of "sensitive skin" can be assessed by the individual himself based on subjective skin sensation or by a dermatologist based on objective skin reactions.

[0046] Most often, sensitive skin and / or mucous membranes occur in well-defined locations, such as the scalp, face, skin folds, baby’s buttocks, etc. Therefore, these are likely areas of sensitive skin and / or mucous membranes.

[0047] Similarly, "sensitized skin and / or mucous membranes" refers to healthy skin and / or healthy mucous membranes that are temporarily sensitized and therefore not pathological.

[0048] For the purposes of the present invention, "cosmetic products" are non-drug, non-therapeutic products that are not intended for the prevention and / or treatment of skin and / or mucous membranes and / or appendages that would be described as pathological by a specialist in the field (e.g. a dermatologist). They are therefore intended for use on healthy skin and / or mucous membranes and / or appendages.

[0049] The term "healthy skin (especially healthy scalp) and / or healthy mucous membranes and / or healthy appendages" refers to all or part of an area of ​​skin (especially scalp) and / or mucous membranes and / or healthy appendages (especially human appendages) to which the peptides of the present invention are applied and which is referred to by dermatologists as "non-pathological", i.e., there is no cancer, infection, scar, disease or skin condition, such as candidiasis, impetigo, psoriasis, eczema, acne, ichthyosis, gingivitis or dermatitis or wounds or injuries or oral ulcers or ulcers or burns and / or other skin diseases, or oral ulcers or inflammation (especially sunburn) or irritation, or urticaria or allergy, such as contact allergy or pathological allergies associated with a decrease in ECM fibers and / or a decrease in the content of fibrillin-1 and / or the content of EMILIN-1 and / or a decrease in the amount of collagen fibers and / or any combination thereof, in particular solar elastosis. Advantageously, the healthy skin and / or mucosa of the present invention is less likely to develop pathologies associated with reduced ECM fibers and / or reduced fibrillin-1 content and / or reduced EMILIN-1 content and / or reduced amount of collagen fibers and / or any combination thereof, in particular solar elastosis.

[0050] For the purposes of the present invention, the term "skin" refers to all or part of the skin of the body, in particular of the human body, including the scalp, selected from the group consisting of the face, hands, arms, chest, legs, neck, back, shoulders, abdomen, wrists, forearms, ankles, thighs, nape, scalp, joint and / or axillary folds, labial mucosa, preferably the face, in particular the area around the eyes and / or mouth and the forehead. In particular, the skin is a site that may be exposed to attacks, in particular those that may degrade the ECM fibers, in particular to areas that are exposed to aggressive environmental substances or chemicals, friction and / or immersion. This relates in particular to the face, scalp, hands, neck, upper chest, joint folds and / or axillary folds.

[0051] For the purposes of the present invention, the term "mucosa" refers to the ocular, nasal, vaginal, anal and / or oral mucosa, in particular the oral labial mucosa, preferably the labial mucosa, ocular and / or nasal mucosa.

[0052] Preference is given to topical application, advantageously to specific parts and / or areas of the body, selected from the scalp, face, hands, arms, upper chest, legs, neck, back, shoulders, abdomen, wrists, forearms, ankles, thighs, nape of the neck, joint folds and / or axillae, lip mucosa.

[0053] The peptides of the present invention are topically acceptable. For the purposes of the present invention, "topically acceptable" refers to ingredients suitable for topical application that are non-toxic, non-irritating to the skin and / or mucous membranes and / or appendages, do not induce allergic reactions and are not chemically unstable.

[0054] The peptides according to the invention can be obtained by chemical synthesis, by biotechnological synthesis using microorganisms (natural or recombinantly produced), and / or by enzymatic hydrolysis of biological material, in particular containing microorganisms of plant or animal nature.

[0055] Therefore, according to one embodiment of the present invention, the peptides are used in the form of rice protein hydrolysate (Oryza sativa), ie a peptide hydrolysate of rice (Oryza sativa), in particular a peptide hydrolysate of rice (Oryza sativa) enriched with the peptides of the present invention, preferably in liquid form.

[0056] According to an alternative embodiment of the present invention, the peptide is used in the form of a microbial ferment, the microorganism is preferably a recombinant yeast, more preferably a recombinant yeast selected from the genus Pichia or Saccharomyces, even more preferably Saccharomyces cerevisiae, in particular genetically modified to produce the sequence, and the ferment is preferably enriched in the peptide of the present invention.

[0057] According to an alternative embodiment of the invention, the peptide is used in a form purified from a hydrolysate or fermentation product by conventional peptide purification techniques, in particular selected from membrane filtration, chromatography, precipitation and / or immunoprecipitation.

[0058] According to the invention, the term "hydrolysate" refers to the chemical and / or enzymatic hydrolysis products of biological materials (eg animal, plant or microbial materials, in particular yeast) which contain and / or produce naturally or by genetic modification the peptides of the invention.

[0059] According to the present invention, the term "fermentation" refers to a product produced by fermentation of a microorganism (particularly yeast or bacteria) that produces the peptides of the present invention naturally or by genetic modification. Therefore, the fermentation corresponds to a culture medium containing the nutrients necessary for the growth and reproduction of the microorganism and a fermentation product excreted by the microorganism or released during the lysis of the microorganism.

[0060] According to the present invention, the term "hydrolysate or fermentation product rich in the peptide of the present invention" refers to a hydrolysate or fermentation product, in which the content of the peptide of SEQ ID No. 1 of the present invention is detected to exceed the detection limit threshold of sensitive technology in this field (e.g. LC-MS), that is, the content of the peptide is usually at least equal to or greater than 0.00001% (by dry matter weight) of the total weight of the hydrolysate or fermentation product.

[0061] According to an alternative embodiment of the present invention, the peptide of SEQ ID No. 1 is contained in the hydrolysate, preferably at least equal to or greater than 0.00001% (by dry matter weight) of the total weight of the hydrolysate, preferably 0.0001% to 10% (by dry matter weight) of the total weight of the hydrolysate, more preferably 0.001% to 1% (by dry matter weight).

[0062] According to a preferred embodiment, the rice peptide hydrolysate (or protein hydrolysate) is obtained by a one-step enzymatic hydrolysis. According to an alternative embodiment, the hydrolysate can be obtained by continuous hydrolysis using the same enzyme or a mixture of enzymes.

[0063] The enzymatic hydrolysis is performed by one or more proteolytic enzymes, which may be proteases of vegetable or microbial origin.

[0064] According to one embodiment, the enzymatic hydrolysis is carried out to completion, which can be determined by a person skilled in the art in a known manner, for example by determining the constant pH or by using a method for detecting free NH 2 The constant amount of peptide SEQ ID No. 1 is determined by photometry of the moiety, or by liquid chromatography and mass spectrometry.

[0065] The amount of enzyme used is not critical per se, but must be 0.05-5% by weight, preferably 0.1-2% by weight, relative to the weight of the peptide-containing starting material.

[0066] The hydrolysate obtained can finally be processed, for example by filtering the undissolved fraction.For better stabilization, the pH is preferably fixed at 3.0-7.5, preferably 3.5-5.5.

[0067] According to an alternative embodiment, the peptides of the present invention can be obtained by chemical synthesis according to conventional techniques in the art, in particular those described. Advantageously, the peptides of the present invention will have a purity level of at least 50%, preferably at least 70%, advantageously at least 80%, preferably at least 90%, more preferably at least 95% (w / w).

[0068] According to another alternative embodiment, the peptides of the invention may be obtained biotechnologically, in particular from recombinant bacteria or yeast strains, ie genetically modified to produce the peptides of the invention.

[0069] The yeast strain is preferably selected from the genera Pichia and Saccharomyces, in particular Saccharomyces cerevisiae.

[0070] Preparation of peptides by yeast strains generally involves the following steps:

[0071] - Transforming yeast using an expression system (usually plasmid-based) of the peptide of the invention coupled to an export signal sequence;

[0072] - growing the transgenic yeast in a culture medium;

[0073] - Batch and / or fed-batch fermentation;

[0074] - recovering the fermentation broth and removing the yeast biomass, for example by centrifugation;

[0075] - Concentration and / or separation of peptides.

[0076] Preferably, the peptides according to the invention are prepared as described in Example 1c).

[0077] In a particularly advantageous embodiment, the peptide is contained in the fermentation product and is used directly in this form.

[0078] In one embodiment, the hydrolyzate or fermentate obtained is in liquid form, in particular an aqueous solution, and can be used directly or in concentrated form; they preferably have a dry matter content of 1 to 50% by weight, preferably 5 to 30% by weight, based on the total weight of the hydrolyzate or fermentate. Auxiliary agents such as polyols, diols, acids (citric acid, sorbic acid, sulfuric acid, benzoic acid or its salts, etc.) can be added.

[0079] The hydrolysate or fermentate obtained at this stage can be further concentrated and / or purified to select the molecular weight fraction of interest, by successive ultrafiltration or nanofiltration steps through filters of variable porosity, or by chromatography, to obtain a fermentate or hydrolysate specifically enriched in these peptides.

[0080] The peptide hydrolysate or fermentate of the present invention can also be prepared in powder form by drying (atomization, freeze drying, etc.) with or without a carrier such as mannitol, maltodextrin, cyclodextrin according to conventional formulation techniques known to those skilled in the art.

[0081] According to one embodiment, the preferred hydrolysate or fermentation product of the present invention contains the peptide of SEQ ID No. 1, and its total content is at least equal to or greater than 0.00001% by weight of the total weight of the dry matter of the hydrolysate or fermentation product, preferably 0.0001% to 10%, more preferably 0.001% to 3%, even more preferably 0.01% to 1%.

[0082] The average molecular weight expressed in Daltons (or g / mol) is determined by size exclusion chromatography methods known to those skilled in the art.

[0083] The rice peptide hydrolysate of the present invention can be obtained by the following steps:

[0084] - dissolving rice protein or rice protein isolate containing at least 70%, preferably at least 80% protein in water and pasteurizing at a temperature above 70°C;

[0085] - protein hydrolysis; preferably the hydrolysis is carried out enzymatically by proteolytic enzymes, preferably of vegetable origin or derived from microorganisms, by selecting one or more enzymes and adjusting the temperature and pH conditions to obtain the correct degree of hydrolysis and molecular weight distribution;

[0086] - inactivation of the enzyme, preferably by heat treatment: this inactivation is carried out according to the enzyme supplier's technical instructions;

[0087] - separating the soluble phase and the insoluble phase by centrifugation and / or filtration and recovering the soluble phase containing the peptide;

[0088] - a concentration step may be performed to increase the dry matter content, followed by a filtration step to recover the soluble phase containing the peptides;

[0089] - The filtrate is obtained, ie the peptide-rich rice protein hydrolysate of the present invention, which is in liquid form and also constitutes an embodiment of the present invention.

[0090] The steps of the above method, taken individually, are common in the field of protein hydrolysates, and those skilled in the art can adjust the reaction parameters according to their common sense to obtain the rice protein hydrolysate of the present invention.

[0091] The peptides of the present invention are used for topical application, either in pure form or in the form of peptide-enriched hydrolysates or peptide-enriched ferments.

[0092] According to one embodiment, it is possible to use:

[0093] - in the form of a cosmetic or pharmaceutical ingredient, intended for incorporation into a cosmetic or pharmaceutical composition, and also comprising a suitable cosmetic or pharmaceutical carrier, or

[0094] - in the form of a cosmetic or pharmaceutical composition comprising it and advantageously also comprising a suitable cosmetic or pharmaceutical carrier.

[0095] For the purposes of the present invention, the term "topical route" means applying the peptides of the present invention, preferably in the form of a peptide-enriched hydrolyzate or fermentate, and / or the compositions and / or ingredients of the present invention to the skin surface, in particular the scalp and / or mucous membranes, and / or appendages, preferably the hair, in particular by direct application or by evaporation.

[0096] For the purposes of the present invention, the term "cosmetic and / or pharmaceutical ingredient" refers to one or more plant extracts and / or one or more natural or synthetic molecules and / or mixtures thereof for cosmetic and / or pharmaceutical applications. Cosmetic ingredients are specifically defined by the International Nomenclature of Cosmetic Ingredients (INCI).

[0097] For purposes of the present invention, the term "suitable cosmetic or pharmaceutical carrier" means that the composition or components thereof are suitable for contact with human skin and / or mucous membranes without undue toxicity, incompatibility, instability, allergic response, and the like.

[0098] A cosmetic or pharmaceutical composition in liquid form containing the peptide of the present invention, in particular a dermatological composition, in particular a fermentation product rich in the peptide of the present invention and in particular the fermentation product of Example 1C, can be used in a cosmetic or pharmaceutical composition, in particular a dermatological composition, preferably with a dry matter weight content of 0.01 to 10%, advantageously 0.1 to 5%, in particular 0.2 to 3%, based on the total weight of the composition.

[0099] In one embodiment of the present invention, the peptide of the present invention (the peptide of SEQ ID No. 1 and / or its homologues, derivatives and / or salts) is in the form of a cosmetic composition comprising the same, and further advantageously comprises a suitable cosmetic carrier.

[0100] In another embodiment of the present invention, the peptide of the present invention, in particular the peptide of SEQ ID No. 1, is preferably present in the cosmetic or pharmaceutical composition in the form of a peptide-rich fermentation product, and its content is 1×10 of the total weight of the composition. -6 % to 10%, preferably 1×10 -5 % to 0.1%, more preferably 1×10 -4 % to 0.01% (by dry matter weight).

[0101] Compositions of the present invention may contain any suitable solvent and / or any suitable carrier and / or any suitable excipient, optionally in combination with other target compounds. They may also contain cosmetic or dermatologically acceptable excipients and / or suitable cosmetic or pharmaceutical carriers, selected from surfactants, preservatives, buffers, swelling agents, chelating agents, biocides, denaturants, sunscreens, pH regulators, reducing agents, stabilizers, emulsifiers, thickeners, gelling agents, film-forming polymers, solvents, fillers, bactericides, odor absorbers, matting agents, conditioners, texture improvers, glossing agents, pigments, dyes, spices and chemical or mineral sunscreens, trace elements, essential oils, sweeteners, flavoring agents. These combinations also belong to the scope of the present invention. The CTFA Cosmetic Ingredient Handbook, the second edition (1992) describes a variety of cosmetic and pharmaceutical ingredients commonly used in cosmetic and pharmaceutical industries, and is particularly suitable for oral administration and / or topical application.

[0102] Advantageously, the excipient and / or carrier is selected from polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose-based stabilizers, vitamin E and its derivatives, xanthan gum, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiables, phytosterols, silicones, protein hydrolysates, betaine, amine oxides, plant extracts, sucrose esters, titanium dioxide, glycine and parabens, more preferably selected from steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, butylene glycol, caprylyl glycol, natural tocopherols, glycerine, sodium dihydroxycetyl phosphate, isopropyl hydroxycetyl ether , glycol stearate, triisononanoin, caprylyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, hexylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide, PEG-30 dipolyhydroxystearate, capric / caprylic triglyceride, cetearyl caprylate, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulfate, mineral waxes and oils, isostearyl isostearate, propylene glycol dipelargonate, propylene glycol isostearate, PEG-8, beeswax, hydrogenated palm heart glycerides, lanolin oil, sesame oil, cetyl lactylate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline, and mixtures thereof.

[0103] The cosmetic or pharmaceutical composition or peptide according to the invention, preferably a hydrolyzate or fermentation product rich in peptides, optionally in the form of a cosmetic or pharmaceutical ingredient, can be in all galenic forms customary for topical application, for example in liquid or solid form, or even in the form of a pressurized liquid. They can be formulated in particular in the form of an aqueous or oily solution, an aqueous cream or gel or an oily gel, in particular in a jar or tube, in particular a shower gel, a shampoo, a milk, an emulsion, a hydrogel, a microemulsion or a nanoemulsion, in particular an oil-in-water or water-in-oil or a multiphase or silicone, a serum, a lotion, in particular in a glass or plastic bottle or a dosage bottle, or an aerosol, an ampoule, a liquid soap, a paste, a dermatological soap, an ointment, a foam, an aerosol, a mask, a patch, an anhydrous product, preferably a liquid, a paste or a solid, for example in the form of a stick, in particular a stick or a powder, in particular a cosmetic. In particular, the composition is in the form of a serum, lotion, cream, milk, ointment, paste, foam, emulsion, hydrogel, shower gel, mask, stick, patch or cosmetic powder, advantageously a cream or lotion. Preferably, the composition is in the form of a cream, lotion or shampoo, conditioner (rinsable or non-rinsable type) for skin and / or hair.

[0104] The peptides according to the invention, preferably the peptide-rich ferments according to the invention, have the advantage of not attacking and / or stripping the skin and / or mucous membranes, allowing the sebum level to be maintained, and are particularly suitable for the care and / or treatment of sensitive and / or sensitized skin and / or mucous membranes.

[0105] In case of administration via the mucosal route, the cosmetic or pharmaceutical composition or peptide of the invention, optionally in the form of a cosmetic or pharmaceutical ingredient, may be in the form of eye drops, lotions, aerosols, gels or mucoadhesive compositions.

[0106] The cosmetic or pharmaceutical composition may also contain other cosmetic or pharmaceutical ingredients active for the treatment of the skin and / or mucous membranes and / or appendages, which induce complementary or synergistic effects with the peptides of the invention, for example selected from known anti-aging active cosmetic ingredients.

[0107] Among the anti-aging active cosmetic ingredients, we can cite:

[0108] - those used to prevent the appearance of pigmentation and / or to increase the radiance of the complexion, in particular those marketed by the Applicant under the name Inolixir TM The extract of the mushroom Inonotus obliquus sold by the applicant under the name Arganyl TM Argania spinosa oil extract sold by the applicant under the name Purisoft TM The applicant sells Moringa oleifera seed extract under the name CollRepairTM The combination of Salvia miltiorrhiza extract and niacinamide marketed by the applicant under the name Neurobiox TM The extract of Achillea millefolium sold by the applicant under the name DN-Age TM Cassia alata leaf extract is marketed and / or marketed by the applicant under the name Litchiderm TM The lychee extract (as an antioxidant active ingredient) sold by the applicant under the name Lox-Age TM Chicory extract is sold under the name Vitacell TM Yeast extract sold under the name Perlaura TM Polygonum bistorta extract is marketed under the name Hyalufix TM Galangal extract sold under the name Deliner TM Corn extract sold under the name Epigenist TM Voandzeia subterranea extracts for sale;

[0109] - those used to promote skin firming, in particular by acting on collagen, such as those under the name Dermican TM A synthetic tetrapeptide sold under the name Linefactor TM Hibiscus abelmoschus extract is marketed under the name Proteasyl TM A purified pea extract is marketed under the name Elestan TM The extract of Manilkara multinervis is sold under the name Collalift TM 18African neem (Khaya senegalensis) extract sold under the name Argassential TM Argan nut pulp extract sold by the applicant under the name Sqisandryl TM Schizandra chinensis extract, retinol, vitamin C, and Collguard TM Davilla rugosa extract is sold under the name Phytokine TM Soy protein hydrolysate extract for sale.

[0110] In a particularly advantageous embodiment, the cosmetic or pharmaceutical composition will comprise the peptides of the invention, preferably in the form of a peptide-enriched ferment, in combination with at least one further ingredient selected from:

[0111] - retinol, preferably in a dosage of 0.01% to 1% by weight of the total weight of the composition (by weight of dry matter),

[0112] - Vitamin C, preferably in a dosage of 0.5% to 25% by weight of the total composition (by weight of dry matter),

[0113] - Vitamin E, preferably in a dosage of 0.02% to 0.5% by weight of the total composition (by weight of dry matter),

[0114] - nicotinamide, preferably in a dosage of 0.1% to 10% by weight of the total composition (by weight of dry matter),

[0115] - hyaluronic acid, preferably in a dosage ranging from 0.01% to 3% by weight of the total weight of the composition (by weight of dry matter),

[0116] - a hydrolyzate extracted from protein-rich Hibiscus esculentus seed powder described in Example 1 of patent application EP0975322B1 and sold by BASF under the brand Myoxinol in a dextrin carrier, preferably in a dosage ranging from 0.5% to 2% by weight of the total composition (by weight of dry matter).

[0117] In particular, the subject of the present invention is such a cosmetic composition comprising a peptide according to the invention and one of the above ingredients chosen from retinol, vitamin C, vitamin E, niacinamide, hyaluronic acid, hydrolyzate extracted from ambrette seeds powder and mixtures thereof.

[0118] Such compositions are particularly useful by topical application to healthy skin and / or healthy mucous membranes to prevent and / or reduce signs of skin and / or mucous membrane aging due to time and / or UV rays and / or to improve the extracellular matrix fibers of the healthy skin dermis and / or healthy mucous membranes.

[0119] The peptides of the present invention can also be used in combination with cosmetic ingredients for sensitive skin, particularly those sold under the trade name Protein extract of de-oiled ungerminated seeds of Moringa oleifera is marketed under the name Symbiocell TM The plant extract of Cestrum latifolium is sold under the name Irwinol TM Butter extracted from the fruit of the Gabonese mango (Irvingia gabonensis), sold under the name EperulineTM An extract of the root of Eperua falcata is marketed under the name Skinasensyl TM The peptide N-acetyl-L-tyrosyl-L-prolyl-L-phenylalanyl-L-phenylalaninamide (INCI: Acetyl Tetrapeptide 15) is marketed by the applicant under the name Inolixir TM An extract of Inonotus obliquus is marketed.

[0120] The peptides of the invention can also be used in combination with active ingredients acting on the skin and / or mucosal microflora and / or ingredients acting on the skin barrier function, in particular moisturizing and / or soothing active ingredients, including those marketed by the company Solabia under the name BioEcolia TM Oligosaccharides obtained by enzymatic synthesis are marketed or by the same company under the name Ecoskin TM Alpha-oligoglucose complex, Alisma plantago-aquatica extract, Argania spinosa extract (Lipofructyl TM Argan), Ceramide Blend (Sphingoceryl TM VEG, Boldo tree (Betapur TM ), inulin- or oligofructose-based products, bifidobacterium extracts or Orthosiphon stamineus extracts for oily skin (MAT-XS TM Bright), the applicant used the name Melhydran TM A natural honey extract marketed by the applicant under the name Oligolin TM Flax extract sold by the applicant under the name Relipidium TM Biotechnologically modified yeast extract marketed by the applicant under the name Inhipase TM Pueraria lobata extract is sold by Mibelle under the name CM-Glucan Forte TM Baker's yeast beta-glucan derivatives and / or Sederma are marketed under the name Pacifeel TM Mirabilis jalapa extract for sale.

[0121] The present invention also relates to a non-therapeutic cosmetic care and / or treatment method, comprising topical application (particularly daily application) of a peptide of the present invention and / or its homologues, salts and / or derivatives to at least one area of ​​healthy skin (advantageously a healthy scalp), and / or healthy mucous membranes and / or healthy appendages, preferably in the form of a ferment enriched with the peptide of the present invention or a cosmetic composition of the present invention, in order to improve the structure and / or organization and / or density of the ECM fibers of the dermis of healthy skin and / or healthy mucous membranes, in particular the collagen fibers and / or elastic fibers of the extracellular matrix of the dermis, and more particularly, to increase the density and / or flexibility of healthy skin and / or healthy mucous membranes.

[0122] Advantageously, the area of ​​skin and / or mucous membranes is selected from the group consisting of the scalp, face, hands, arms, upper chest, legs, neck, back, shoulders, abdomen, wrists, forearms, ankles, thighs, nape, joint folds and / or axillae, lip mucosa.

[0123] Advantageously, the present invention also relates to a cosmetic treatment method for improving the ECM fibers of the dermis of healthy skin and / or healthy mucous membranes of an individual in need / desire, comprising the following steps:

[0124] - Identify healthy skin and / or healthy mucosal areas in individuals who wish to improve dermal ECM fibers, and

[0125] - Topically applying the peptide of the present invention or the cosmetic composition comprising the peptide of the present invention to the area of ​​healthy skin and / or healthy mucosa in an amount effective for improving the ECM fibers of the dermis, in particular the peptide of SEQ ID No. 1 and / or its homologues, salts and / or derivatives in an amount of 1×10 of the total weight of the composition. -6 % to 10%, preferably 1×10 -5 % to 0.1%, still advantageously 1×10 -4 % to 0.01% (by dry matter weight).

[0126] The present invention also relates to the peptide of SEQ ID No. 1 of the present invention and / or its homologue and / or its salt and / or its derivative, preferably in the form of a fermentation product enriched in the peptide of the present invention, for use alone or in a pharmaceutical composition, in particular a dermatological composition, comprising it, for the treatment and / or prevention of at least one pathology associated with a decrease in ECM fibers and / or a decrease in the content of fibrillin-1 and / or a decrease in the content of EMILIN-1 and / or a decrease in the amount of collagen fibers and / or any combination thereof, such as solar elastosis.

[0127] Advantageously, the peptide is present in the pharmaceutical composition, in particular the dermatological composition, in an amount of 1×10 -6 % to 10%, preferably 1×10 -5 % to 0.1%, more preferably 1×10-4 % to 0.01% (by weight of dry matter), the composition further advantageously comprises a suitable pharmaceutical carrier, advantageously a suitable dermatological carrier.

[0128] Other objects, features and advantages of the present invention will become apparent to those skilled in the art after reading the illustrative description with reference to the embodiments and drawings, which are given by way of illustration only and in no way limit the scope of the present invention.

[0129] The embodiments are an integral part of the invention and any feature which appears novel in relation to any prior art in its function and generality, seen from the entire description (including the embodiments), is an integral part of the invention.

[0130] Therefore, each embodiment has a general scope.

[0131] On the other hand, in the Examples, unless otherwise indicated, the temperature is expressed in degrees Celsius and the pressure is atmospheric pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1A-1B-1C 3 photographs representing reconstructed skin tissue sections obtained by confocal microscopy after immunolabeling for filaggrin (green): 1A - untreated control; 1B - after treatment with 0.00001% (0.1 μg / mL) of the peptide of the present invention; 1C - after treatment with 0.0001% (1 μg / mL) of the peptide of the present invention.

[0133] Figure 2A-2B-2C Three photographs representing reconstructed skin tissue sections obtained by confocal microscopy after immunolabeling for EMILIN-1 (green): 2A - untreated control; 2B - after treatment with 0.00001% (0.1 μg / mL) of the peptide of the present invention; 2C - after treatment with 0.0001% (1 μg / mL) of the peptide of the present invention.

[0134] Figure 3A-3B Two photographs representing the reconstructed dermis obtained by multiphoton confocal microscopy and visualization of the second harmonic generation signal (SHG) (white): 3A - untreated control; 3B - after treatment with 0.0001% (1 μg / mL) of the peptide of the invention. Example

[0135] Example 1: Different methods for obtaining SEQ ID No. 1 TVFDGVLRPGQL peptide

[0136] 1.A) Plant hydrolysates:

[0137] The 12 amino acid peptide can be obtained from rice (Oryza sativa) protein hydrolysate obtained by enzymatic hydrolysis with a single protease, or as described in patent application WO2017009484A1 for obtaining sequence SEQ ID No. 349 or as described in patent application WO2017009490A1 for obtaining sequence SEQ ID No. 245.

[0138] 1.B) By chemical synthesis:

[0139] The peptide of the present invention was obtained by chemical synthesis with a purity of 99.4%, as determined by HPLS-UV. The identity of the peptide of the present invention was confirmed by LC-MS through a peptide spectrum containing 1305.5 (M+H+) and 651 (M+2H+) ions.

[0140] 1.C) Recombinant microbial fermentation products as raw materials

[0141] A system for expressing the peptides of the present invention coupled to an export signal sequence was constructed in the yeast Saccharomyces cerevisiae for producing the peptides and secreting them into the culture medium. The transgenic yeast was cultured in a standard culture medium (SY-2) containing glucose as a carbon source, ammonium sulfate as a nitrogen source, and minerals (KH 2 PO 4 MgSO 4 、Na 2 SO 4 , NaCl, CaCl 2 ) and vitamins, pH 4.1. After pre-cultivation in standard SY-2 medium for 12-16 hours and reaching an optical density (OD) of 5-8, the bioreactor was inoculated into standard SY-2 medium at an inoculation rate of 7% (weight / weight) of the total weight of yeast and medium. The culture was carried out at 30°C, the pH was adjusted to 4, and the oxygen partial pressure (pO 2) was adjusted to a minimum of 30% and a maximum aeration of 0.3 VVM. After an 11-hour batch fermentation phase, glucose and ammonium sulfate were added for a 37-hour fed-batch fermentation phase. After 48 hours of culture, the fermentation broth was collected and centrifuged to recover the supernatant and remove the yeast biomass. The supernatant was concentrated 5 to 10 times by vacuum evaporation at 25°C, and activated carbon was added at a concentration of 25% (weight / weight) relative to the dry mass of the concentrate. The activated carbon was removed by pre-filtration, and after filtering through a 0.22 μm filter, the resulting liquid was freeze-dried to obtain a powder. The peptide content of the present invention was 2984 μg / g as determined by HPLC-UV. The identity of the peptide was confirmed by LC-MS using a peptide spectrum containing ions 1305.5 (M+H+) and 651 (M+2H+).

[0142] Example 2: Evaluation of the properties of the peptides of the invention on fibrillin-1 and EMILIN-1

[0143] A 3D reconstructed skin model (Mimeskin) was used to evaluate the performance of the peptides of the invention obtained by chemical synthesis as described in Example 1.B). 6 Fibroblasts were seeded on dermal culture supports containing a mixture of collagen, glycosaminoglycans (GAGs), and chitosan (Mimedisc) and cultured at 37°C, 5% CO 2 The dermis was then inoculated for 56 days as a dermis construction stage. 6 keratinocytes and cultured for 7 days as the keratinocyte proliferation stage. The model was then placed at an air-liquid interface for the keratinocyte differentiation stage and cultured for 14 days. Starting from the 2nd day and every 2 days (except when cells were inoculated), the cultures were treated or not treated (untreated control) with a dose of 0.1 μg / mL or 1 μg / mL of the peptide of the present invention in aqueous solution. Each condition was performed with n=4 (4 replicates). For each condition and each replicate, a cryostat was used to make tissue sections. After fixation, the sections were immunolabeled to fluorescently detect fibrillin-1 and EMILIN-1 ( Figure 1A-1B-1C and 2A-2B-2C , green). Evans blue was used as a counterstain to visualize the gross morphology (red). Confocal laser scanning microscopy (TCS-SP2, Leica) was used for observation, and the surface occupancy of the target protein on the dermis surface was quantified by image analysis. The results are expressed as the mean percentage compared to the untreated control, which was normalized to 100%. After verifying the normality of the distribution of values ​​using the Student's t test (Shapiro-Wilk test), statistical analysis was performed compared to the untreated control group. The significance threshold was 5%.

[0144] After treatment with a dose of 0.1 μg / mL and 1 μg / mL, the peptide of the present invention stimulated fibrillin-1 by +69% and +87%, respectively. After treatment with a dose of 0.1 μg / mL and 1 μg / mL, the peptide of the present invention stimulated EMILIN-1 by +19% and +27%, respectively.

[0145] exist Figure 1A-1B-1C This increase in the fibrillin-1 microfibril network in the extracellular matrix can be clearly observed in the

[0146] Figure 2A-2B-2C An increase in EMILIN-1, a ligand for microfibrils involved in their proper organization and function in the extracellular matrix, is shown.

[0147] Therefore, the peptides of the present invention can increase the content of fibrillin-1 and Emilin-1, thereby increasing the organization of elastic microfibrils.

[0148] Example 3: Evaluation of the properties of the peptides of the present invention on collagen fibers

[0149] A 3D reconstructed dermis model (Mimederm) was used to evaluate the performance of peptides obtained by chemical synthesis. 6 Fibroblasts were seeded on dermal culture supports containing a mixture of collagen, glycosaminoglycans (GAGs), and chitosan (Mimedisc) and cultured at 37°C, 5% CO 2 The culture was immersed in water for 56 days. Starting from the second day and every two days, the culture was treated with a peptide of the present invention obtained by chemical synthesis (as described in Example 1.B) in an aqueous solution at a dose of 1 μg / mL or not treated (untreated control). Each condition was performed with n=3 (repeated 3 times). For each condition and each repetition, analysis was performed by multiphoton confocal microscopy to generate a second harmonic signal (SHG).

[0150] SHG imaging is a specific technique for observing collagen fibrils and fibers in the extracellular matrix. Collagen organized into fibrils has a non-centrosymmetric structure, which is necessary for the generation of SHG signals. Figure 3A-3B SHG signals are shown in white (autofluorescence of surface cells and reconstructed skin support is shown in green).

[0151] Results are expressed as mean percentages compared to untreated controls, which were normalized to 100%. Statistical analysis was performed compared to untreated controls after verifying the normality of the distribution of values ​​using the Student's t test (Shapiro-Wilk test). The significance threshold was 5%.

[0152] Figure 3A-3BShown is the presence of a large number of organized collagen fibers in the dermal extracellular matrix. In Fig. 3B, a higher SHG signal intensity was observed, showing a higher density of collagen fibers in the extracellular matrix when the peptide of the present invention was present. Analysis of the volume occupied by the SHG signal also showed that the amount of these fibers in the extracellular matrix increased by +68% in the presence of the peptide of the present invention compared to the untreated control.

[0153] Example 4: Evaluation of the properties of the peptides of the invention on dermal flexibility

[0154] A 3D reconstructed skin model (Mimeskin) was used to evaluate the performance of the peptides of the invention obtained by fermentation of recombinant yeast as described in Example 1.C). 6 Fibroblasts were seeded on a dermal culture support containing a mixture of collagen, glycosaminoglycans (GAGs), and chitosan (Mimedisc) and incubated at 37°C with 5% CO. 2 The dermis was constructed by immersion culture for 57 days and then inoculated for 10 6 Keratinocytes were placed in the air-liquid interface for the keratinocyte differentiation stage and cultured for 7 days as the keratinocyte proliferation stage. The model was then placed at an air-liquid interface for the keratinocyte differentiation stage and continued to be cultured for 14 days. From the second day and every 2 days (except when cells were inoculated), the cultures were treated or not treated (untreated control) with a dose of 0.8 μg / mL or 3 μg / mL of the peptide of the present invention in aqueous solution. Each condition was carried out with n=4 (4 repetitions). For each condition and each repetition, 3 histological sections were made using a cryostat, and the elastic modulus of the dermis was measured by atomic force microscopy (AFM) using a 0.5N / m conical probe. The results are expressed as the average of different frozen sections. The elastic modulus (or Young's modulus) is the amount of elastic deformation that a material resists when subjected to force. The higher the elastic modulus of a material, the greater its stiffness. On the contrary, the lower the value, the more flexible the material. A Student't test was performed, and the results showed that for the two test doses, the results were significantly different from the measurements of the untreated model (p<0.001). The Young's modulus values ​​in the absence (untreated) or presence of the peptides of the invention are summarized in Table 1 below.

[0155] [Table 1]

[0156] condition Average value (Pa) Unprocessed 15.1KPa 0.8 μg / mL peptide 7.7KPa 3 μg / mL peptide 3.4KPa

[0157] The results showed that the flexibility of the dermis was improved.

[0158] Example 5: Evaluation of the properties of the peptides of the invention on fibulin-5 and MFAP4

[0159] Mature functional elastic fibers in the ECM require the assembly of multiple glycoproteins, including fibulin-5 and MFAP4 (microfibril associated protein 4). Human dermal fibroblast culture assays were used to evaluate the performance of the peptides of the invention synthesized as described in Example 1.B) in the synthesis of these glycoproteins.

[0160] Human dermal fibroblasts were cultured at 5×10 4 cells / cm 2 The cells were inoculated at a density of 1.5 and incubated at 37°C with 5% CO 2 The cells were immersed in a defined medium (FGM) until confluence. When confluence was reached, the cells were incubated for 48 hours in the presence or absence (untreated control) of the peptide of the present invention (Example 1.B) in an aqueous solution at a dose of 0.1 μg / mL and 1 μg / mL. Then, the cells were collected and lysed to measure the fibulin-5 level in the lysate. The culture medium was collected to measure the MFAP4 level. The concentration in the sample was determined by the BCA assay to adjust the deposition of each sample to the same protein concentration for Western blot analysis. After incubation with a primary antibody, immunolabeling with a secondary antibody, and measurement of chemiluminescence, Fibulin-5 and MFAP4 were analyzed by capillary electrophoresis. Each condition was performed with n=4 to 6 (4 to 6 replicates). The results are expressed as the average percentage compared to the untreated control, and the untreated control is standardized to 100%. After checking the normality of the numerical distribution (Shapiro-Wilk test) using a one-way analysis of variance (Dunnett method), statistical analysis was performed with the untreated control. The significance threshold was 5%.

[0161] Compared to untreated controls, at a 0.1 μg / mL peptide dose, fibulin-5 and MFAP4 levels increased by +22% and +38%, respectively. Thus, the peptides of the invention can increase the amount of fibulin-5 and MFAP4 synthesized by fibroblasts, thereby contributing to the organization of elastic microfibrils.

[0162] Example 6: Evaluation of the properties of the peptides of the invention on dermal density

[0163] A group of 28 women aged 45-65 years with skin type I, II or III according to the Fitzpatrick scale applied a cream in the form of an emulsion on half of their face, the emulsion containing the peptide according to Example 1.C) at a final weight concentration of 0.1% (w / w) of the total weight of the preparation (Formulation Example 7.c), or without the peptide and replaced with water (placebo), twice a day for 2 months. The total dermal density of the cheek was assessed using a device called DUB Skin Scanner, which measures the fiber network density of the dermal extracellular matrix using the ultrasound echo principle. The efficacy of the composition containing the peptide according to Example 1.C) was compared with the efficacy of the so-called placebo emulsion.

[0164] Total dermal density was measured at the beginning of the study (D0), after 1 month of application (D28) and finally after 2 months of application (D56). The efficacy of the peptide on total dermal density was determined by comparing the % change relative to D0 28 and 56 days after application of the formulation containing the peptide of Example 1.C) and the placebo formulation. These values ​​are expressed as the mean of the volunteers analyzed. Statistical analysis was performed after checking the normality of the distribution of values ​​using the Student's t test (Shapiro-Wilk test). The significance threshold was 5% (p<0.05).

[0165] After 28 and 56 days, the total density of the dermis increased by 4.9% (p>0.05) and 12.9% (p<0.05) using the preparation containing the peptide of Example 1.C), respectively. Thus, the peptides of the invention can increase the density of the fiber network in the ECM.

[0166] Example 7: Formulation Example

[0167] Proportions are expressed as % by weight of the total and ingredient names in capital letters correspond to the INCI names.

[0168] 7.a)

[0169] [Table 2]

[0170]

[0171]

[0172] 7.b) [Table 3]

[0173] INCI name Amount (% total weight) water Appropriate amount up to 100.00 Xanthan gum 10.00 Coco-Caprylate / Caprate 5.00 Cetearyl Alcohol 3.50 glycerin 3.00 Caprylic / capric triglyceride 3.00 Pentylene glycol 2.00 Sodium Polyacrylate (and) Dicaprylyl Carbonate (and) Polyglyceryl-3 Caprate 1.20 Sucrose Polystearate (and) Cetyl Palmitate 1.00 Phenoxyethanol (and) Ethylhexylglycerin 0.50 Dimethicone 0.50 Disodium Cetearyl Sulfosuccinate 0.20 The peptide of the present invention of Example 1b 0.0001

[0174] 7.c) [Table 4]

[0175]

[0176]

[0177] The peptides used in the present invention are shown in Table 5 below: [Table 5]

[0178] Sequence number sequence SEQ ID No.1 TVFDGVLRPGQL

Claims

1. Non-therapeutic cosmetic use of the peptide of SEQ ID No. 1 and / or its homologues, derivatives and / or salts for improving the dermis of healthy skin and / or the extracellular matrix fibers of healthy mucous membranes.

2. The use according to claim 1, for improving the structure and / or organization and / or density of collagen fibers and / or elastic fibers of the dermal extracellular matrix.

3. Use according to any one of the preceding claims for increasing the density and / or flexibility of healthy skin and / or healthy mucous membranes.

4. Use according to any of the preceding claims for improving the anchoring of appendages, preferably for preventing and / or slowing down shedding, more preferably hair shedding.

5. The use according to any one of the preceding claims, for increasing the amount of fibrillin-1 in the ECM fibers of the healthy dermis and / or for increasing the amount of EMILIN-1 in the ECM fibers of the healthy dermis and / or for increasing the amount of collagen fibers in the extracellular matrix of the healthy dermis, for increasing the synthesis of fibulin-5 and / or MFAP4 by fibroblasts of the healthy dermis.

6. Use according to any one of the preceding claims, wherein the peptide is in the form of the peptide of SEQ ID No. 1 and has a purity of at least 50% (w / w).

7. Use according to any one of claims 1 to 6, wherein the peptide of SEQ ID No. 1 is in the form of a peptide hydrolysate of rice (Oryza sativa), in particular enriched in the peptide of SEQ ID No. 1, preferably in liquid form.

8. The use according to any one of claims 1 to 6, wherein the peptide of SEQ ID No. 1 is in the form of a fermentation product of a microorganism that produces it, preferably a recombinant yeast selected from the genus Pichia or Saccharomyces.

9. Use according to any one of the preceding claims, wherein the yeast is Saccharomyces cerevisiae.

10. The use according to any one of the preceding claims, Features The peptide of SEQ ID No. 1 and / or its homologues, derivatives and / or salts are in the form of a cosmetic composition comprising it and advantageously also comprising a suitable cosmetic carrier.

11. The use according to claim 10, Features The content of the peptide of SEQ ID No. 1 in the cosmetic composition is 1×10 of the total weight of the composition. -6 % to 10%, preferably 1×10 -5 % to 0.1%, more preferably 1×10 -4 % to 0.01% by weight of dry matter.

12. The use according to any one of the preceding claims, It is characterized in that It is for topical application.

13. The use according to any one of the preceding claims, It is characterized in that It is used to care for sensitive and / or sensitized skin and / or mucous membranes.

14. Methods of non-therapeutic cosmetic care and / or treatment, It is characterized in that The method comprises topically applying a peptide of SEQ ID No. 1 and / or its homologues, salts and / or derivatives to at least one area of ​​healthy skin, advantageously a healthy scalp, and / or a healthy mucous membrane to improve the structure and / or organization and / or density of the fibers of the dermal extracellular matrix of healthy skin and / or healthy mucous membranes, in particular the collagen fibers and / or elastic fibers of the dermal extracellular matrix.

15. Use according to any of the preceding claims for increasing the density and / or flexibility of healthy skin and / or healthy mucous membranes.

16. The cosmetic care method according to any one of claims 14 or 15, wherein the peptide of SEQ ID No. 1 and / or its homologues and / or salts and / or derivatives are as defined in any one of claims 6 to 11.

17. The method according to any one of claims 14 to 16, wherein the area of ​​skin and / or mucous membranes is selected from the group consisting of the scalp, face, hands, arms, upper chest, legs, neck, back, shoulders, abdomen, wrists, forearms, ankles, thighs, nape of the neck, joint folds and / or axillae, lip mucosa.

18. A peptide of SEQ ID No. 1 and / or its homologues and / or salts and / or derivatives for use in the treatment and / or prevention of pathologies associated with a decrease in ECM fibers and / or a decrease in the content of fibrillin-1 and / or a decrease in the content of EMILIN-1 and / or a decrease in the amount of collagen fibers and / or any combination thereof, in particular solar elastosis.

19. The peptide of SEQ ID No. 1 for use according to claim 18, It is characterized in that The peptide of SEQ ID No. 1 and / or its homologues are as defined in any one of claims 6 to 9.

20. The peptide of SEQ ID No. 1 for use according to any one of claims 18 or 19, It is characterized in that The content of the peptide in the pharmaceutical composition is 1×10 -6 % to 10%, preferably 1×10 -5 % to 0.1%, more preferably 1×10 -4 % to 0.01% by weight of dry matter, the composition also advantageously comprises a suitable pharmaceutical carrier.

Images