CN116322637A

CN116322637A - Novel use of peptides for improving skin and/or mucous membrane comfort and/or integument appearance

Info

Publication number: CN116322637A
Application number: CN202180065694.5A
Authority: CN
Inventors: C·博诺德-罗赛; L·达努; D·赫劳特; P·穆苏
Original assignee: BASF Beauty Care Solutions France SAS
Current assignee: BASF Beauty Care Solutions France SAS
Priority date: 2020-10-02
Filing date: 2021-10-01
Publication date: 2023-06-23
Also published as: WO2022069844A1; FR3114744A1; BR112023006002A2; KR20230074492A; EP4221678A1; FR3114744B1

Abstract

The present invention relates to the use of peptides having the sequence SEQ ID No.1 and/or homologues thereof, optionally in the form of or in combination with the peptide SEQ ID No.2, in particular in the form of rice hydrolysates enriched in this/these peptides, and optionally in combination with the peptides SEQ ID No.3 and/or SEQ ID No.4, for improving the comfort of the skin, in particular the scalp, and/or mucous membranes, and/or the appearance of the integuments, in particular of keratin fibres. The invention also relates to the use of such peptides in cosmetic and/or dermatological compositions.

Description

Novel use of peptides for improving skin and/or mucous membrane comfort and/or integument appearance

Technical Field

Background

Skin sensitivity, most particularly scalp sensitivity, is a condition that is often ignored. However, skin sensitivity is not limited to the face, and it is estimated that one third to half of the population has scalp known as "sensitive". Trigger factors having a significant influence on the manifestation of sensitive skin, in particular of sensitive scalp, are temperature changes, in particular heat and cold, humidity, dry air, sunlight, polluting and aggressive and/or repetitive shampoos and aggressive hair treatments (dyeing, straightening hair, etc.).

The stratum corneum, also known as the Stratum Corneum (SC), typically has a pH in the range of 4 to 6. This acidic character (and thus making it a so-called "acid coat") is considered a prerequisite for healthy skin. This creates a pH gradient of 2 to 3 units between SC and the underlying layers (i.e., epidermis and dermis containing epithelial cells, which have an almost neutral pH).

This pH gradient within the skin helps to coordinate epidermal differentiation and keratinocyte shedding through the involvement of several pH-dependent enzymes. Thus, the alpha-glucose, a key component of the stratum corneum barrier, is involved in ceramide

The enzymes synthesized by the enzyme phospholipase (α -glucorebrosidase) and sphingomyelinase have an acidic pH optimum.

Due to the increased activity of serine proteases and kallikrein 5 and 7 and decreased activity of ceramide synthase, the pH increase in normal skin thus affects the integrity, cohesion and desquamation of the stratum corneum. Thus, maintaining or restoring an acidic skin pH is necessary to maintain and/or restore healthy skin and to avoid uncomfortable manifestations such as itching, tightness and sensitivity that are typically caused by it.

Sensitive scalp exhibits a higher pH than non-sensitive scalp and is more susceptible to irritation. Such elevated pH may lead more rapidly to uncomfortable and/or unpleasant scalp manifestations, in particular itchiness, and to dandruff formation, in part by affecting sensitivity to malassezia metabolites.

The present invention relates to the provision of novel cosmetic and/or dermatological active agents for improving the comfort of the skin, in particular the scalp and mucous membranes and/or improving the appearance of body covers, in particular keratin fibres, as a substitute for existing cosmetic and dermatological ingredients. The invention also relates to the provision of novel cosmetic and/or dermatological active agents for the specific care and/or treatment needs of sensitive scalp.

Surprisingly and unexpectedly, the inventors have found that peptides having SEQ ID No.1 are capable of improving the comfort of skin (including scalp and/or mucous membranes) and/or improving the appearance of bodily integuments (particularly keratin fibers, preferably hair).

Peptides with SEQ ID No.1 have been described in the cosmetic field in a number of peptides derived from rice and pea in patent application WO 2017/009484 A1 (as SEQ ID No. 423) as anti-inflammatory agents capable of inhibiting the secretion of TNF- α by macrophages in the presence of lipopolysaccharides, and in patent application WO 2017/009490 A1 (as SEQ ID No. 248) as agents capable of increasing the proliferation of human skin fibroblasts and thus treating visible signs of ageing.

The peptide is naturally present in rice (Oryza sativa), a protein of rice grains from which the peptide can be obtained. However, the naturally occurring amounts thereof are insufficient to be detected and to produce the characteristics of the inventive subject matter. In the context of the present invention, it is obtained by enzymatic hydrolysis.

Although there are a large number of rice peptide hydrolysates in the cosmetic market, they vary greatly in their characteristics and composition, in particular the different protein fragments they contain, which have a great influence on their biological properties. For proteolysis, the bridges linking the various peptides and amino acids are cleaved by various methods conventional in the art, for example by acid, base or by catalysis. However, these different techniques result in the production of protein hydrolysates of very different composition, which may lead to the production of free amino acids. Given that it is non-specific, chemical hydrolysis by the use of acids or bases generally results in the production of "lower quality" products, in particular in terms of colour, odour and/or stability. Due to its specificity, enzymatic hydrolysis offers the advantage of being able to be carried out under controlled conditions, in particular pH, temperature and pressure. Again, however, the enzymes are selected in such a way that the peptides of interest are obtained and result in the production of specific hydrolysates, which are very different in terms of the peptide fragments they comprise, whether in terms of the nature of the fragments, their molecular weight or their distribution.

These various rice hydrolysates are used in skin care and/or hair care applications in the cosmetic field. Thus, patent application WO 03/039496A1 describes octanoyl glutamate and/or octanoyl salt of rice protein hydrolysates as self-protecting and moisturizing agents, as well as sebum regulators, antidandruff agents and deodorants. Patent KR 20040108226B describes a black rice protein hydrolysate obtained by hydrolysis with papain for moisturizing, increasing collagen synthesis and inhibiting melanin production.

However, due to their non-specific production methods or methods for producing other peptides, all of these hydrolysates differ greatly in their composition and properties, and none of them describe or contain a peptide according to the invention in an amount sufficient to provide the properties according to the invention.

Disclosure of Invention

Thus, one subject of the present invention is the use of a peptide having SEQ ID No.1 and/or a homologue thereof for its cosmetic properties selected from improving the comfort of healthy skin, in particular healthy scalp and/or healthy mucous membranes, and/or improving the appearance of healthy body covers, preferably hair. This peptide has many additional advantages, since it does not attack or remove sebum present on the skin, in particular the scalp, which makes it particularly suitable for caring for and/or treating sensitive skin, in particular sensitive scalp, in particular in the form of a mild shampoo.

Furthermore, the peptides according to the invention have the advantage of inhibiting the release of TNF- α by macrophages. In addition, it can inhibit elastase as described in example 10.1.

The peptides according to the invention, due to their complementary properties, provide a complete adaptation and a complete solution to the specific needs of sensitive hair and sensitive scalp.

All of these complementary properties also make them excellent sedatives and/or cares for sensitive skin, especially sensitive scalp.

Furthermore, the peptides according to the invention are particularly suitable for preparing complete compositions for cosmetic care and/or body treatment, due to their complementary properties in terms of hardness as shown in example 10.2.

Advantageously, the peptide of SEQ ID No.1 is in the form of the peptide of SEQ ID No.2 and/or a homologue thereof.

Preferably, the peptide according to the invention is in the form of a rice-rice protein hydrolysate, advantageously at a content of at least equal to or greater than 0.00001%, preferably between 0.0001% and 0.03%, more preferably between 0.001% and 0.01% by weight of dry matter relative to the total weight of the hydrolysate.

More preferably, the peptide according to the invention is used in combination with at least one peptide selected from the peptides of SEQ ID No.2 and/or SEQ ID No.3 and/or SEQ ID No.4, advantageously in combination with the peptides of SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, preferably in the form of a hydrolysate enriched in the peptides according to the invention.

According to a preferred mode, the peptide according to the invention is the peptide of SEQ ID No. 1:

SEQ ID No.1:GYYGEQQQQPGMTR

the peptide contains 14 amino acids:

Gly-Tyr-Tyr-Gly-Glu-Gln-Gln-Gln-Pro-Gly-Met-Thr-Arg and has an average molecular weight of 1642.72 daltons.

It naturally occurs in rice (rice) grain proteins and can be obtained by enzymatic hydrolysis of rice grains as described in patent applications WO 2017/009484 A1 and WO 2017/009490 A1, or by chemical synthesis according to methods conventional in the art. It can also be purified or obtained as shown in example 1.

According to one variant of the invention, the peptide according to the invention is a peptide with SEQ ID No.2, which contains the peptide SEQ ID No.1, preceded by three additional amino acids Ser-Glu-Glu.

SEQ ID No.2:SEEGYYGEQQQQPGMTR

Thus, peptide SEQ ID No.2 contains 17 amino acids and has an average molecular weight of 1988.01 daltons. It is also naturally present in rice (rice) grain proteins and can be obtained by enzymatic hydrolysis of rice grains as described in patent applications WO 2017/009484 A1 and WO 2017/009490 A1, or by chemical synthesis according to methods conventional in the art. It can also be purified or obtained as shown in example 1.

Particularly advantageously, the peptide according to the invention is in the form of SEQ ID No.1 and/or SEQ ID No.2, preferably in the form of a mixture of the peptides SEQ ID No.1 and SEQ ID No.2, in particular comprised in the enriched hydrolysate according to the invention.

According to one embodiment, the peptide of SEQ ID No.1 and/or SEQ ID No.2 may be combined with the peptide of SEQ ID No.3 and/or SEQ ID No.4, optionally in the form of a hydrolysate, for example those according to example 2.

According to one embodiment, the peptide of SEQ ID No.1 is used in combination with the peptide of SEQ ID No.2 and/or in combination with the peptide of SEQ ID No.3 and/or in combination with the peptide of SEQ ID No. 4.

Peptide SEQ ID No.3: IYGPDTGVDYKDNQMR

The peptide SEQ ID No.3 contains 16 amino acids and has a molecular weight of 1971.97 daltons. It may be obtained from rice (rice) protein hydrolysates according to the method described in example 3, or may be obtained as described in patent application WO2017/009484 to produce the sequence SEQ ID No.218 or obtained by chemical synthesis. It can also be purified or obtained as shown in example 1.

Peptide SEQ ID No.4: FYNEGDAPVVAL

The 12 amino acid peptide has a molecular weight of 1294.37 daltons; it may be isolated from rice (rice) protein hydrolysates according to the method described in example 3, or may be obtained as described in patent application WO2017/009484 to produce the sequence SEQ ID No.194 or obtained by chemical synthesis. It can also be purified or obtained as shown in example 1.

According to the invention, the term "peptide" refers to any isolated natural or synthetic amino acid sequence, which can be synthesized by chemical or biological techniques where appropriate or obtained by extraction from biological tissues (e.g. plants, animals or microorganisms, in particular yeasts), which expresses the amino acid sequence naturally or after transduction, optionally after post-translational modification thereof. Can be prepared according to conventional methods known to the person skilled in the art, in particular by acylating the terminal-NH ₂ A group, or amidated or alkylated terminal-COOH group, the peptide is modified by adding a functional group, for example, coupling with a hydrophilic or hydrophobic penetrant, a stabilizer, or coupling with a protecting group.

Techniques for peptide chemical synthesis are known to those skilled in the art, and examples that may be mentioned include the references J.M.Stewart and J.D.Young, solid Phase Peptide Synthesis [ solid phase peptide synthesis ], 2 nd edition, pierce Chemical Company, rockford, illinois [ Pierce chemical Co., illinois Rockword ] (1984), and M.Bodanzsky and A.Bodanzsky, the Practice of Peptide Synthesis, springer Verlag. New York [ peptide synthesis practice, springer, new York ] (1984).

According to the invention, the term "homologue" of an amino acid sequence refers to a biologically active amino acid sequence having at least 85%, more preferably 90%, especially at least 95%, more especially at least 98%, more preferably at least 99% sequence identity and having the same properties as said sequence. Sequence homology can be identified by any conventional technique in the art, e.g., via BLAST computer interface http:// blast.ncbi.lm.nih.gov on the NCBI website, using default parameter configurations.

The homology of an amino acid sequence may differ from the sequence, for example, by one or more deletions and/or insertions of amino acids and/or one or more substitutions. According to one embodiment variant, the homologue of the amino acid sequence may comprise one or more conservative amino acid substitutions. A conservative substitution is a substitution in the sequence of one amino acid with another amino acid that has substantially similar physicochemical properties or properties sufficiently close to those of the original amino acid such that the properties and function of the peptide are not affected or substantially unaffected. Such amino acid sequence changes are commonly referred to as "mutations". Thus, homologs of the peptides according to the invention also relate to mutants and variants of the amino acid sequences of the invention having the same properties of biological activity. The term "biological activity of the same nature" of the amino acid sequence according to the invention refers in particular to its ability to exhibit the properties according to the invention.

According to the present invention, the term "improving the comfort of skin and/or mucous membranes" refers to reducing unpleasant and/or uncomfortable manifestations of healthy skin and/or mucous membranes (in particular manifestations due to a decrease in the steady state of skin and/or mucous membranes and/or due to a change in the stratum corneum, in particular due to a decrease in the content of endo-and/or filaggrin and/or an increase in the pH of the skin), selected from the group consisting of skin and/or mucous membrane dryness (in particular dry scalp), itching, redness, skin tightness, desquamation (in particular desquamation of scalp), dandruff, cracking and skin sensitivity (in particular scalp sensitivity), and/or providing a pleasant feel of skin and/or mucous membranes and/or making skin and/or mucous membranes softer and/or reducing their roughness.

Improvement in skin comfort can be measured by conventional methods known to those skilled in the art. Examples that may be mentioned include the following techniques:

measuring the increase in the synthesis of endo-capelin of epidermal keratinocytes as shown in example 5,

measurement of the increase in silk fibroin synthesis in the reconstituted epidermis as shown in example 6,

measuring redness, in particular of the scalp,

measuring the amount of shedding, in particular of dander shedding,

skin hydration is measured, in particular with a corneometer (skin moisture tester), for example as described in example 8,

measuring the skin pH, in particular with a pH meter.

As demonstrated in example 8, the peptides according to the invention have the advantage of providing a durable, sustained hydration even after they have been taken out of service.

Similarly, improvement in scalp comfort may be measured, for example, by:

measuring redness, in particular of the scalp,

the amount of shedding, in particular the amount of dandruff shedding, was measured according to the method shown in example 7.

The term "decrease in skin and/or mucosal homeostasis" refers to an imbalance in the skin and/or mucosal barrier due to a decrease in water homeostasis and/or a change in pH, in particular an increase in pH. According to the invention, the loss of skin homeostasis of healthy skin and/or healthy mucosa is not caused by damage of epithelial cells, which may be the case for example with damage to dermatological problems.

Thus, the peptides according to the invention are particularly suitable for caring for and/or treating dry mucous membranes, dry skin (in particular dry scalp), sensitive skin, irritated skin and/or mucous membranes, and/or skin and/or mucous membranes exhibiting reduced homeostasis (in particular water) and/or elevated pH.

Thus, advantageously, the use according to the invention is intended for caring for dry and/or sensitive skin, and/or dry and/or sensitive scalp.

Preferably, the unpleasant and uncomfortable manifestations of healthy skin and/or healthy mucosa are independent of age or epithelial cell damage.

According to the present invention, the term "improving the comfort of the scalp" refers to reducing unpleasant and/or uncomfortable manifestations of healthy scalp (in particular those manifestations due to a decrease in skin homeostasis and/or to a change in the stratum corneum, in particular due to a decrease in the content of endogenous toglobin and/or a decrease in the content of filaggrin and/or an increase in skin pH), selected from the group consisting of dry scalp, itchy scalp, redness of the scalp, tightness of the scalp, flaking of the scalp, dandruff, cracking and scalp sensitivity, and/or providing a pleasant feel to the skin and/or making the scalp softer and/or reducing its roughness.

Improvement in scalp comfort can be measured particularly according to conventional methods. For example, it may be measured by measuring scalp redness and/or by measuring scalp pH and/or by assessing dandruff shedding according to the method given in example 7.

According to the invention, the term "improving the appearance of the body cover" means increasing the strength and/or resistance of the body cover, and/or protecting the body cover from cosmetic oxidation treatments and/or aggressive agents, reducing the breakage rate, repairing damaged body covers, improving the formation and/or volume, increasing the growth and/or gloss of keratin fibres and/or reducing their loss. The peptide according to the invention, in particular the hydrolysate according to the invention containing it, has the advantage of not significantly reducing the amount of sebum present on the scalp.

The improvement in appearance of the body cover, in particular hair, can be measured according to conventional techniques. For example, as described in example 12.1, there may be mentioned improvements in body quilt strength and resistance by measuring improvements in keratin fiber structure. Keratin fibre growth can be measured according to the method described in example 12.2.

For the purposes of the present invention, the term "cosmetic" refers to a non-pharmaceutical, non-therapeutic use, which is not intended for the prevention and/or treatment of skin and/or mucous membranes and/or bodily integuments identified as pathological by an expert in the field (for example, a dermatologist). Thus, it is intended for healthy skin and/or mucous membranes and/or integuments.

The term "healthy skin, in particular healthy scalp and/or healthy mucosa and/or healthy body quilt" refers to all or part of an area of healthy skin, in particular scalp and/or mucosa and/or body quilt, in particular a human, wherein the peptide according to the invention is applied, and the dermatologist refers to "non-pathological", i.e. without any infection, scar, disease or skin condition, such as candidiasis, impetigo, psoriasis, eczema, acne, ichthyosis, gingivitis or dermatitis or wounds or lesions or sores or ulcers or burns and/or other skin diseases, or aphtha or inflammation or irritation, or urticaria or allergies such as contact allergies.

For the purposes of the present invention, the term "skin" refers to all or part of the skin of the body, in particular the human body, including the scalp, selected from the group consisting of scalp, face, hands, arms, neckline, legs, neck, back, shoulders, stomach, wrists, forearms, ankles, thighs, nuchal, joint folds and armpits. In particular, the skin is skin that may have uncomfortable and/or unpleasant appearance, particularly in exposed, rubbed and/or impregnated areas. And thus more particularly the face, scalp, hands, neck, collar, scalp, joint folds and armpits.

For the purposes of the present invention, the term "mucous membrane" refers to ocular, nasal, vaginal, anal and/or oral mucous membranes, in particular labial mucous membranes, preferably labial, ocular and/or nasal mucous membranes.

The term "body quilt" refers to nails and "keratin fibers.

The term "keratin fibres" refers to hair fibres (hair), eyelashes, eyebrows, body hair, in particular nasal mucosa, ears, beard and/or beard, in particular hair fibres (hair).

Preferably, the application is topical, advantageously on specific parts and/or areas of discomfort of the body, in particular selected from scalp, face, hands, arms, neckline, legs, neck, back, shoulders, stomach, wrist, forearm, ankle, thigh, nape, joint folds and armpits, mucous membranes of the lips, body hair, eyelashes, eyebrows and/or hair, preferably scalp and/or hair.

The peptides according to the invention are topically acceptable. For the purposes of the present invention, the term "topically acceptable" refers to an ingredient suitable for topical application, which is non-toxic, non-irritating to the skin and/or mucous membranes and/or bodies, which does not induce any allergic reactions, and which is not chemically unstable.

According to a preferred mode of the invention, the peptides, alone or in combination, are used in the form of rice (rice) protein hydrolysates, in particular enriched with the peptides according to the invention.

According to the invention, the term "peptide-enriched hydrolysate according to the invention" refers to a hydrolysate in which the peptides of SEQ ID No.1 and/or SEQ ID No.2 according to the invention are detected in a content exceeding the detection limit threshold by sensitive techniques in the art, such as LC-MS/MS, i.e. a content generally at least equal to or greater than 0.00001% by weight relative to the total dry matter weight of the hydrolysate.

According to a preferred mode of the invention, the peptide SEQ ID No.1, optionally in the form of SEQ ID No.2 or in combination with the peptide SEQ ID No.2, is contained in the rice protein hydrolysate, preferably in an amount at least equal to or greater than 0.00001%, preferably between 0.0001% and 0.03%, more preferably between 0.001% and 0.01% by weight of dry matter relative to the total weight of the hydrolysate.

According to a preferred embodiment, the rice peptide hydrolysate (or protein hydrolysate) is obtained by a single step enzymatic hydrolysis. According to alternative embodiments, the hydrolysate can be obtained by continuous hydrolysis using the same enzyme or mixture of enzymes.

The enzymatic hydrolysis is performed with one or more proteolytic enzymes, which may be plant-derived proteases or proteases obtained from microorganisms.

According to one embodiment, enzymatic hydrolysis occurs until it is completed, which can be determined by one skilled in the art in a manner known, for example, by determining a constant pH or by detecting free NH ₂ Photometry of the groups or determination of the constant amounts of peptides SEQ ID No.1 and SEQ ID No.2 by liquid chromatography and mass spectrometry.

The amount of enzyme used is not critical per se, but should be in the range of 0.05% to 5%, preferably 0.1% to 2% by weight relative to the weight of the starting material containing the peptide.

The hydrolysate obtained can finally be processed, for example by filtration of the undissolved fraction. For better stabilization, the pH is preferably set to a value between 3.0 and 7.5, preferably 3.5 to 5.5.

In one embodiment, the hydrolysate obtained is in liquid form, in particular in aqueous solution, and can be used directly or in concentrated form; they preferably have a dry matter content of from 1% to 50%, preferably from 5% to 30% by weight. Adjuvants may be added, such as polyols, diols, acids (citric acid, sorbic acid, sulfuric acid, benzoic acid or salts thereof, etc.).

The hydrolysates obtained at this stage may be further concentrated and/or purified to select the target molecular weight fraction, by successive ultrafiltration or nanofiltration steps of a variable porosity filter, or by chromatographic methods, such as specifically enriching the hydrolysates in these peptides. Enriched hydrolysates according to the invention are shown in example 2.

The peptide hydrolysates according to the invention in powder form can also be produced by drying (nebuliser, freeze-drying, etc.) with or without carriers such as mannitol, maltodextrin or cyclodextrin according to conventional formulation techniques known to the person skilled in the art.

According to one embodiment, the preferred hydrolysates according to the invention contain the peptide of SEQ ID No.1 and/or the peptide of SEQ ID No.2, preferably both peptides, in a total content of at least equal to or greater than 0.00001%, preferably between 0.0001% and 0.03%, more preferably between 0.001% and 0.01% by weight relative to the total dry matter weight of the hydrolysates.

According to a preferred embodiment, the hydrolysate also has at least one, preferably at least two and more preferably all of the following features:

-in liquid form;

-its dry matter content is 15% to 25% by weight;

-its nitrogen content is from 1.5% to 3% by weight, preferably from 1.9% to 2.8% by weight;

-having an average molecular weight ranging from 1000 to 2500 daltons, preferably from 1250 to 2000 daltons, more preferably 1500 daltons;

it contains at least 50%, more preferably 60% of peptides having a molecular weight greater than 1000 daltons;

it contains at least 70%, more preferably 80% of peptides having a molecular weight of less than 2500 daltons.

Preferably, the preferred protein hydrolysates according to the invention are characterized by the presence of all these features.

The average molecular weight in daltons (or g/mol) is determined by size exclusion chromatography as known to those skilled in the art.

The molecular weight distribution was measured according to the invention as described in example 3.

The rice peptide hydrolysate according to the present invention can be obtained by performing the steps of:

-dissolving rice proteins or rice protein isolates containing at least 70%, preferably at least 80% of proteins in water having a temperature higher than 70 ℃ for pasteurization;

-hydrolysis of proteins; preferably, the hydrolysis is facilitated by proteolytic enzymes (preferably of plant origin or derived from microorganisms) enzymes, by selecting enzymes and adjusting the temperature and pH conditions to obtain a suitable degree of hydrolysis and molecular weight distribution;

inactivation of the enzyme, preferably by heat treatment: inactivation was performed according to the technical recommendations of the enzyme suppliers;

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

-optionally a concentration step to increase the dry matter content, followed by a filtration step to recover a soluble phase containing peptides;

-generating a filtrate, which is in liquid form and also constitutes an embodiment of the invention, of the peptide-enriched rice protein hydrolysate according to the invention.

The individual steps of the above-described process are common in the field of protein hydrolysates, and the person skilled in the art will be able to adjust the reaction parameters according to his general knowledge to obtain a rice protein hydrolysate according to the invention.

Although rice protein hydrolysates are already present in the cosmetic market, they do not contain the peptides of the invention and therefore lack the characteristics of the subject matter of the invention. Thus in example 4 a commercially available product is described and its properties are tested by comparison with the peptides according to the invention in examples 5, 6, 10 and 12.2.

Of particular interest, the hydrolysate also contains the peptides SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, each in an individual amount of at least equal to or greater than 0.00001% by weight relative to the total dry weight of the hydrolysate, the sum of these four peptides being at least equal to 0.00004% by weight relative to the total dry weight of the hydrolysate, preferably greater than 0.0001%. Examples of such peptide-rich hydrolysates according to the invention are shown in examples 2 a), 2 b) and 2 c) and constitute preferred modes of the invention.

The peptides of the invention are preferably used topically in the form of a peptide-rich hydrolysate.

According to one embodiment, one may employ:

in the form of a cosmetic or pharmaceutical ingredient intended to be incorporated in a cosmetic or pharmaceutical composition, and further comprising a suitable cosmetic or pharmaceutical vehicle, or

In the form of a cosmetic or pharmaceutical composition comprising it, and advantageously also a suitable cosmetic or pharmaceutical vehicle.

For the purposes of the present invention, the term "topical route" means that the peptide according to the invention is applied to the skin surface, in particular the scalp and/or mucous membrane and/or body, preferably the hair, preferably in the form of a peptide-rich hydrolysate and/or composition and/or ingredient according to the invention, in particular by direct application or by spraying.

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 defined in particular by the International Nomenclature of Cosmetic Ingredients (INCI).

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

Cosmetic or pharmaceutical, in particular dermatological, ingredients comprising the peptide according to the invention, in particular in the form of a peptide-enriched hydrolysate according to the invention and in particular in the form of the hydrolysate of example 9, may be used in cosmetic or pharmaceutical, in particular dermatological, compositions, preferably in a content of from 0.01% to 10%, advantageously from 0.1% to 5%, in particular from 0.2% to 3%, by weight of dry matter relative to the total weight of the composition.

In one embodiment of the invention, the peptide, preferably in the form of a peptide-rich hydrolysate, will be 1X 10 by weight of dry matter relative to the total weight of the composition ^-5 % to 20%,preferably 1X 10 ^-4 % to 10%, more advantageously 1X 10 ^-3 The content of% to 3%, more preferably 0.001% to 0.1%, is comprised in a cosmetic or pharmaceutical composition.

The composition according to the invention may contain any suitable solvent and/or any suitable vehicle and/or any suitable excipient, optionally in combination with other compounds of interest. They may in particular contain cosmetically or dermatologically acceptable excipients and/or suitable cosmetic or pharmaceutical vehicles selected from surfactants, preservatives, buffers, swelling agents, chelating agents, biocides, denaturants, opacifiers, pH adjusting agents, reducing agents, stabilizers, emulsifiers, thickeners, gelling agents, film forming polymers, solvents, fillers, bactericides, odor absorbers, matting agents, conditioning agents, texturing agents, gloss agents, pigments, colorants, fragrances, chemical or mineral sunscreens, trace elements, essential oils, sweeteners and flavor modifiers. The present invention also encompasses these combinations. CTFA cosmetic ingredients handbook, second edition (1992) describes different cosmetic and pharmaceutical ingredients commonly used in the cosmetic and pharmaceutical industry, which are particularly suitable for oral and/or topical use.

Advantageously, the excipient and/or vehicle is selected from the group comprising polyglycerol, esters, cellulosic 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, betaines, amine oxides, plant extracts, sucrose esters, titanium dioxide, glycine and parabens, more preferably from the group consisting of: stearyl polyether-2, stearyl polyether-21, glycol-15 stearyl ether, cetostearyl alcohol, phenoxyethanol, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, butylene glycol, octylglycol, natural tocopherols, glycerol, sodium dihydrooctyl phosphate, isopropyl hydroxycetyl ether, ethylene glycol stearate, triisononine, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, hexylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide, PEG-30 dimer hydroxystearate, caprylic/capric triglyceride, cetylstearyl octanoate, dibutyl adipate, grapeseed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulfate, mineral waxes and oils, isostearyl alcohol isostearate, propylene glycol di-acid ester, propylene glycol isostearate, PEG8, glycerol esters from hydrogenated palm kernel oil, lanolin oil, sesame oil, cetyl esters, lactic acid, sucrose, low density and the like.

The cosmetic or pharmaceutical composition or peptide, preferably the peptide-enriched hydrolysate according to the invention, optionally in the form of a cosmetic or pharmaceutical ingredient, may be in any presentation form conventionally used for topical administration, 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 aqueous or oily solutions, creams or aqueous or oily gels, in particular in the form of tanks or tubes, in particular shower gels, shampoos, milks, emulsions, hydrogels, microemulsions or nanoemulsions, in particular oil-in-water or water-in-oil or multiple or silicone-based emulsions, slurries, lotions, in particular in glass or plastic bottles or measuring bottles or aerosol bottles, vials, liquid soaps, pastes, dermatological sticks, ointments, foams, aerosols, masks, patches, anhydrous products, preferably in liquid, pasty or solid form, for example in stick form, in particular stick or powder form, in particular in the form of a dusting powder. In particular, the composition is in the form of a slurry, 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 for skin and/or hair conditioner, whether or not it is flushable (leave-on).

The peptides according to the invention, preferably the peptide-enriched hydrolysates according to the invention, have the advantage of not attacking and/or exfoliating the scalp, allowing to maintain sebum levels, and are most particularly suitable for caring for and/or treating sensitive scalp and/or scalp susceptible to irritation.

In the case of administration by the mucosal route, the cosmetic or pharmaceutical composition or peptide according to 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.

The cosmetic or pharmaceutical composition may also comprise other cosmetic or pharmaceutical ingredients active for the treatment of the skin and/or mucous membranes and/or integuments, inducing a complementary or synergistic effect with the peptides of the invention, for example selected from the group consisting of active ingredients for sensitive skin and/or sensitive mucous membranes, active ingredients for the scalp, antidandruff agents and/or active ingredients for preventing hair loss.

Among the active ingredients for sensitive skin, mention will be made of the trade names

Protein extract of de-oiled unmalted moringa seed sold under the trade name symbriocell ^TM Plant extract sold under the trade name Irwilol of Cestrum latifolium ^TM Butter extracted from fruit of African mango (Irvingia gabonensis) tree sold under the trade name Eperuline ^TM Extracts of the roots of Fusarium falcatum (Eperua falcata) sold under the trade name Skinasenyl ^TM The peptide N-acetyl-L-tyrosyl-L-prolyl-L-phenylalanyl amide (INCI: acetyl tetrapeptide 15) sold by the applicant under the trade name Inolixir ^TM An extract of Inonotus obliquus (Inonotus obliquus) is sold.

Among the ingredients for preventing alopecia, there will be mentioned thiopeptides, amino acids, amino sugars, vitamins of the B group, zinc and/or Trichogen under the trade name of applicant ^TM Extracts and/or hair protectants of ginseng and American fern sold by LS 8960, e.g. under the trade name Litchiderm by the applicant ^TM Extracts of Litchi (litchei chinensis) pericarp, and/or sedative and antipruritic actives, for example sold by the applicant under the trade name Phytosooth ^TM Rapeseed plant sterols sold by LS9766, and/or agents for enhancing hair follicles, such as those sold by AmorepacificRambutan (Nephelium lappaceum) and/or minoxidil (minoxidil), valproic acid extract and/or Silab sold as stemoxidine and/or aminoxil and/or Hairgenyl and/or Rednensyl (sold by Givaudan-Induchem and/or Anagaain sold by Mibelle and/or sold by Provital)

Anti-dandruff agents such as zinc pyrithione and/or octopirox and/or ketoconazole glycolipid and/or Elestab from yeasts sold by BASF under the trade name biotailfe ^TM Hexamidine dibasic sold by HP 100 and/or a complex containing black pepper (Piper nigrum) berry extract, ingaalba bark extract and sodium lauroyl lactylate sold by the applicant under the trade name Sanicapyl, and/or an anti-dandruff active agent, such as piroctone olamine salt in particular in combination with salicylic acid.

Among the ingredients active on the scalp, mention may be made of the compositions under the trade name DN-Age ^TM Extracts of leaves of the sold Cassia tora (Cassia alata) as antioxidant active agents, particularly for hair care, under the trade name CollRepair ^TM A combination of Saviae Miltiorrhizae radix (Salvia miltiorhizza) extract and nicotinamide as degradation agent is sold under the trade name MAT-XS ^TM Clerodendranthus spicatus (Orthosiphon stamineus) extract sold by Bright under the trade name Scalposine ^TM Sarcosine sold by the applicant. These combinations of active agents are capable of enhancing hair follicles and thus help reduce loss of keratin fibers, preferably hair.

In the context of body care compositions, mention may also be made of combinations with cosmetically active ingredients for promoting skin firmness, for example under the trade name Dermican ^TM Synthetic tetrapeptides sold under the trade name lineactor ^TM An extract of Abelmoschus manihot (Hibiscus abelmoschus) sold under the trade name Proteasyl ^TM Purified pea extract sold under the trade name Elestan ^TM Manilkara multinervis extract sold under the trade name Collalift ^TM 18 (African chinaberry Khaya senegalensis) extract sold under the trade name Argassential ^TM Sold extract of Argin pulpUnder the trade name Sqisandryl by the applicant ^TM Fructus Schisandrae chinensis (Schizandra chinensis) extract is sold.

The peptides according to the invention may also be used in combination with ingredients active against the skin and/or mucosal microbial flora and/or active against the skin barrier function, in particular moisturizing and/or sedative actives, including those sold under the trade name bioecola by solasia company ^TM Oligosaccharides obtained by enzymatic synthesis or sold by the same company under the trade name Ecoskin ^TM Complexes of alpha-glucan oligosaccharides sold, extracts of Alisma plantago-aquatica (Alisma plantago-aquatica) and extracts of Argania spinosa (Lipofructyl) ^TM Argan), ceramide (Sphingoceryl) ^TM VEG), purified extract of the basilar membrane (Betapur ^TM ) Inulin-or fructooligosaccharide-based product, bifidobacterium extract or Clerodendranthus spicatus (Orthosiphon stamineus) extract (MAT-XS) for combating oily skin ^TM Bright), under the trade name Melhydro by the applicant ^TM Natural honey extract sold under the trade name Oligolin by the applicant for its moisturizing properties ^TM Flax extracts sold by the applicant under the trade name Relipidium ^TM Biotechnologically modified yeast extracts sold by the applicant under the trade name Inhipase ^TM Pueraria lobata root extract sold under the trade name CM-Glucan form by Mibelle ^TM Beta-glucan derivatives from baker's yeast and/or under the trade name Pacifel by Sederm ^TM Mirabilis jalapa (Mirabilis jalapa) extract is sold.

The subject of the present invention is also a method of care and/or cosmetic treatment comprising the topical application, in particular daily, of a peptide according to the invention to healthy skin, advantageously to healthy scalp, and/or to healthy mucous membranes, and/or to at least one area of healthy integuments, preferably in the form of a peptide-enriched hydrolysate according to the invention or a cosmetic composition according to the invention, for improving the comfort of healthy skin, in particular healthy scalp, and/or to healthy mucous membranes, and/or the appearance of integuments (in particular keratin fibres).

Advantageously, the subject of the present invention is also a cosmetic treatment method for improving the comfort of healthy skin, in particular healthy scalp, and/or healthy mucous membranes, and/or the appearance of healthy body cover (in particular healthy keratin fibres) of an individual in need/desiring thereof, comprising the steps of:

-identifying on the individual healthy skin, in particular healthy scalp and/or healthy mucosal areas, where improvement of comfort is desired, and/or healthy body quilt areas, where improvement of appearance is desired, and

-topical application of a cosmetic composition comprising peptides according to the invention in an amount effective to improve the appearance of healthy skin, in particular healthy scalp, and/or healthy mucous membranes and/or healthy body covers, in particular healthy keratin fibres, in an amount effective to improve the comfort of healthy skin, in particular healthy scalp, and/or healthy mucous membranes and/or healthy body covers, in particular healthy keratin fibres, in particular in a peptide content of 1 x 10 by weight relative to the total weight of the composition ^-5 % to 20%, preferably 1X 10 by weight ^-4 % to 10%, more advantageously 1X 10 by weight ^-3 % to 3%, more preferably 0.001% to 0.1% by weight.

The subject of the present invention is also the peptide of SEQ ID No.1 and/or a homologue thereof according to the invention, preferably in the form of a hydrolysate enriched in the peptide according to the invention, for its use alone or in pharmaceutical compositions, in particular dermatological compositions, for the treatment and/or prevention of at least one pathology (in particular ichthyosis and alopecia) associated with a decrease in skin homeostasis and/or a decrease in endo-and/or filaggrin content and/or an increase in skin pH and/or any combination thereof, and desquamation associated with certain pathologies such as acute radiation, sunburn and burns. The pharmaceutical, in particular dermatological, composition preferably comprises 1 x 10 by weight, relative to the total weight of the composition ^-5 % to 20%, preferably 1X 10 by weight ^-4 % to 10%, more advantageously 1X 10 by weight ^-3 % to 3%, more preferably 0.001% to 0.1% by weight of the peptide according to the invention.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art upon reading the explanatory description, with reference to the examples and drawings, given purely by way of illustration, and without in any way limiting the scope of the invention.

Examples form part of the present invention and any features that are novel with respect to any prior art are, in their functional and general nature, apparent from the description including examples, constitute part of this invention.

Thus, each instance has a generic scope.

Further, in the examples, unless otherwise indicated, temperatures are in degrees celsius and pressures are atmospheric.

Drawings

Fig. 1A is a photograph of the scalp surface prior to application of the composition according to the present invention, and fig. 1B is a photograph of the scalp surface of a person after one week of application of the composition according to the present invention under the conditions of example 1.

Fig. 2A is a photograph of the scalp surface prior to application of the composition of the present invention, and fig. 2B is a photograph of the scalp surface of a person after 3 weeks of application of the composition of the present invention under the conditions of example 1.

Fig. 3A is a photograph of the scalp surface prior to application of the composition of the present invention, and fig. 3B is a photograph of the scalp surface of a human after 3 weeks of application of the composition of the present invention under the conditions of example 1 and after 1 week of discontinuation of the application.

Fig. 4 is a confocal micrograph of a tissue section of reconstructed epidermis after silk fibroin immunostaining (green): 4A-untreated control; 4B-after treatment with 0.01% (w/v) of rice protein hydrolysate of example 4 without the peptide of the invention; 4C-after treatment with 0.01% (w/v) of the peptide according to the invention under the conditions of example 6, in particular in the form of a hydrolysate according to example 2 b.

Examples

Example 1: raw peptides having SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.4 Production of

Peptide SEQ ID No.1: GYYGEQQQQPGMTR

Such a 14 amino acid peptide may be obtained from rice (rice) protein hydrolysates as described in example 2 or as described in patent application WO 2017/009484 A1 to produce the sequence SEQ ID No.423 or obtained by chemical synthesis. Its molecular weight is 1642.72 daltons.

Peptide SEQ ID No. 2: SEEGYYGEQQQQPGMTR

Such 17 amino acid peptides may be obtained from rice (rice) protein hydrolysates as described in example 2, or may be obtained as described in patent application WO 2017/009484 to produce the sequence SEQ ID No.424 or obtained by chemical synthesis. Its molecular weight is 1988.01 daltons.

The peptide of SEQ ID No.1 and/or SEQ ID No.2 may be combined with the peptide of SEQ ID No.3 and/or SEQ ID No.4, optionally in the form of a hydrolysate according to example 2.

Peptide SEQ ID No. 3: IYGPDTGVDYKDNQMR

Such a 16 amino acid peptide may be obtained from rice (rice) protein hydrolysates as described in example 2, or may be obtained as described in patent application WO 2017/009484 to produce the sequence SEQ ID No.218 or obtained by chemical synthesis. Its molecular weight is 1971.97 daltons.

Peptide SEQ ID No.4: FYNEGDAPVVAL

Such a 12 amino acid peptide may be obtained from rice (rice) protein hydrolysates as described in example 2, or may be obtained as described in patent application WO 2017/009484 to produce the sequence SEQ ID No.194 or obtained by chemical synthesis. Its molecular weight is 1294.37 daltons.

Example 2: production of hydrolysates enriched in peptides SEQ ID No.1 and/or SEQ ID No.2 according to the invention

2a) Is rich in peptide SEQ Rice hydrolysates of ID No.1 and SEQ ID No.2

The rice hydrolysate is obtained by enzymatic hydrolysis with a single protease and has the following characteristics:

-a liquid hydrolysate having a dry matter content of 20.4% by weight relative to the total weight of the hydrolysate;

-nitrogen content of 2.5% by weight relative to the total weight of the hydrolysate;

Average molecular weight: 1540g/mol;

-distribution: 64.2% of the peptides have a molecular weight greater than 1000 daltons and 84.5% of the peptides have a molecular weight less than 2.5 kDa;

-the pH of the hydrolysate is 4.2;

-the hydrolysate contains by weight, relative to the dry weight of the hydrolysate:

0.0031% peptide SEQ ID No.1

And 0.0028% peptide SEQ ID No.2.

2b) Rice hydrolysate enriched in peptides SEQ ID No.1 and SEQ ID No.2

-a liquid hydrolysate having a dry matter content of 20.0% by weight relative to the total weight of the hydrolysate;

-nitrogen content of 2.6% by weight relative to the total weight of the hydrolysate;

average molecular weight: 1500g/mol;

-distribution: 62.4% of the peptides have a molecular weight greater than 1000 daltons and 85.5% of the peptides have a molecular weight less than 2.5 kDa.

-the pH of the hydrolysate is 4.1;

0.0033% of peptide SEQ ID No.1

And 0.0111% of peptide SEQ ID No.2.

2c) Rice hydrolysate enriched in peptides SEQ ID No.1 and SEQ ID No.2

-a liquid hydrolysate having a dry matter content of 15.5% by weight relative to the total weight of the hydrolysate;

-nitrogen content of 2.0% by weight relative to the total weight of the hydrolysate;

average molecular weight: 1440g/mol;

-distribution: 61.5% of the peptides have a molecular weight greater than 1000 daltons and 87.1% of the peptides have a molecular weight less than 2.5 kDa.

-the pH of the hydrolysate is 4.2;

peptide SEQ ID No.1 with a content of 0.0016%

And 0.0012% peptide SEQ ID No.2.

Example 3: measurement of average molecular weight and molecular weight distribution of peptides

The average molecular weight and molecular weight distribution, defined in daltons (Da), was determined by size exclusion chromatography.

To determine the average molecular weight and molecular weight distribution, an Agilent 1260Infinity liquid chromatography device with a binary pump and degasser was used in combination with PSS WinGPC UniChrom. P55 WinGPC UniChrom is a macromolecular chromatographic data system with real-time data acquisition independent of manufacturer for comprehensive macromolecular analysis and suitable for analysis of protein hydrolysates.

The column used was a specific column for high resolution separation of proteins and peptides (Superdex Peptide 10/300GL from GE Healthcare Life Science, pore width 100A, particle size 5 tm). Such a column is chosen because it is particularly suitable for the determination of biomolecules having a molecular weight of 100 to 7000 daltons. As eluent, dilute hydrochloric acid (0.05M) was used at a flow rate of 0.5 ml/min. Detection is performed using a Refractive Index Detector (RID).

Samples were prepared by filtration through a 0.2 μm filter prior to injection.

Analysis of peptides SEQ ID No. 1 and SEQ ID No.2 in rice protein hydrolysates

First, 5 to 15mg of rice protein hydrolysate was suspended in acetonitrile/water (1/1, v/v) and then diluted with 0.1% formic acid aqueous solution to 2mg.mL ^-1 Final concentrations of rice protein and 5% acetonitrile. After centrifugation, 100. Mu.L of supernatant was mixed with an equal amount of internal standard solution. By liquid chromatography-tandem massThe spectrum (LC-MS/MS) analyzed 20 μl of the mixture with a detection limit of 0.00001% by weight of dry matter.

Liquid chromatography was performed on an aeies 1.7 μm peptide XB-C18X 2.1mm reverse phase column (Phenomenex) coupled to a QTRAP 5500 mass spectrometer (AB Sciex) using a 13 minute gradient of 0-95% acetonitrile. The chromatographic peak corresponding to the predicted peptide was integrated using skyline version 3.5 (university of washington). Isotopically labeled peptides with 13C-, 15N-labeled C-terminal arginine residues were synthesized and used as internal standards and calibration standards. The detection Limit (LOD) of each peptide was estimated using the signal-to-noise method.

EXAMPLE 4 Prior Art hydrolysates

The composition of rice protein hydrolysates sold by BASF under the trade name gludin R Benz and their effect on skin and hair are studied in the following examples.

The prior art hydrolysate has the following molecular weight distribution:

an average molecular weight of-1430 g/mol

-distribution: 59.6% of the peptides have a molecular weight greater than 1000 daltons and 79.6% of the peptides have a molecular weight less than 2.5 kDa.

The peptides of SEQ ID No.1 and SEQ ID No.2 were not present in the hydrolysates (not detected at the detection threshold of 0.00001%)

-pH 4.3.

The comparison was chosen for its similar properties in terms of peptide distribution.

Example 5: effect of the product according to the invention on endo-protein synthesis

Endo-coat proteins are proteins synthesized by keratinocytes from the first stratum spinosum to the stratum granulosum of the human epidermis. During the differentiation of keratinocytes into keratinocytes, the endo-capelin proteins polymerize with other proteins to form a keratinized envelope deposited on the inner surface of the cell membrane. In several pro-inflammatory skin conditions, endo-coat protein expression is disrupted; several chemical stimuli such as SDS (sodium dodecyl sulfate) and DNCB (dinitrochlorobenzene) alter the expression of endo-togenic proteins by inducing TNF- α.

Cell culture and treatment:

normal human epidermal keratinocytes obtained from abdominal biopsies of 26 and 30 year old females were inoculated into DMEM growth medium (Invitrogen, france) containing fetal bovine serum (Dutscher, france) on collagen-coated microplates, then at 37 ℃ and 5% co ₂ Incubate for 3 days.

The cells were then incubated for a further 3 days in standard medium containing 1mM calcium, 10ng/mL TNF-. Alpha. (Gibco Invitrogen, france) and the hydrolysate of the invention of example 2b, either compared to the rice protein hydrolysate of example 4 or without any product (untreated control was called Ctrl). The content tested is expressed as the dry matter weight of the hydrolysate relative to the total volume of the culture medium containing the hydrolysate.

Quantification of endo-cape proteins

The cell layer was washed with saline solution (called HBSS-Hanks balanced salt solution) and the level of endo-toglobin on the cell homogenate was measured by ELISA (ELISA Human involucrin BT601 kit, clinic france) according to the manufacturer's protocol. Cell MTT assays were also performed simultaneously to verify that the product was not cytotoxic at the dose tested (MTT content >75% relative to the control).

And (3) statistical inspection:

results are expressed as a percentage relative to untreated control Ctrl.

Results:

TABLE 1

	Concentration (p/vol)	Average value of
			Untreated control (Ctrl)	0	100
Example 4	0.05％	155
			Example 2b	0.05％	172

The peptide-rich hydrolysates of the invention stimulate keratinocytes to synthesize endogenous toglobin. At equal doses, the inventive hydrolysates containing the inventive peptides were 1.3 times more effective than the comparative hydrolysates of example 4. The presence of the peptides SEQ ID No.1 and SEQ ID No.2 leads to this difference.

EXAMPLE 6 Effect of the product according to the invention on the Synthesis of filaggrin

The effect of the product of the invention on the synthesis of filaggrin (late markers of differentiation) was evaluated on epidermis reconstituted from primary human keratinocytes. After 7 days of keratinocyte proliferation and differentiation, the reconstituted epidermis is treated or not with the peptide of the invention (untreated control), in particular in the form of the hydrolysate of example 2b or the rice protein hydrolysate of example 4, which does not contain the peptide of the invention, after 2 days of incubation 10ng/ml TNF- α is added and the incubation is continued for 2 days. The reconstructed epidermis was then embedded in paraffin and sectioned for silk fibroin immunostaining and image analysis by confocal microscopy.

The results are shown in FIG. 4.

The peptide according to the invention stimulates the synthesis of silk fibroin, in particular in the form of a hydrolysate according to example 2b, with respect to the untreated control, and with respect to the rice protein hydrolysate of example 4, which does not contain the peptide according to the invention.

Example 7: clinical trials of hair and scalp

In the case of clinical trials on human volunteers, the effect of the treatment with the hydrolysate enriched in the present invention, containing 2% (w/w) in the following shampoo composition, was evaluated and compared with the effect of the treatment with the shampoo without the hydrolysate of the present invention (referred to as placebo).

The compositions tested in this clinical study were as follows (percentages are by weight):

TABLE 2

The study was performed on 44 female and male volunteers 18 to 55 years old, who had sensitive scalp and scattered dry sloughing. Volunteers were divided into two groups: one group tested the composition of the invention in shampoo form containing 2% (w/w) rice hydrolysate and the second group tested the composition without the hydrolysate of the invention, called placebo. Shampoo was applied three times per week for 21 days.

The ability to improve scalp comfort was assessed by measuring scalp redness (DermoGenius Ultra), scalp pH (pH meter), scalp Sebum (Sebum meter) and by assessing scalp loss (by expert scoring).

All measurements and clinical scores were also performed immediately after the first administration, 1 and 3 weeks after the use of the product, and then also 1 week after the discontinuation of the treatment (post-treatment stage).

After distribution normality is verified through Shapiro-Wilk test, statistical analysis is performed on the change of instrument parameters along with time: initial values were compared by paired Student t-test or Wilcoxon test, and products were compared by unpaired Student t-test or Mann-Whitney test. For the scoring grid, the change in parameters over time was determined by a non-parametric Wilcoxon or Mann-Whitney test. The significance level was set at 5% (p < 0.05).

The results are shown in FIGS. 1-3.

The first use of shampoo containing 2% rice protein hydrolysate of the present invention resulted in immediate significant improvement of scalp redness, scalp pH and exfoliation score. Shampoo containing 2% rice peptide hydrolysate according to the invention also provided a milder washing effect on the scalp, a significantly lower reduction of scalp sebum levels compared to placebo shampoo.

After 1 and 3 weeks of use, shampoos containing 2% rice peptide hydrolysates according to the invention significantly reduced scalp redness, reduced scalp pH and reduced shedding scores. Shampoo containing 2% rice protein hydrolysate according to the present invention also provided a mild washing effect on scalp after 1 and 3 weeks of use without significantly changing normal scalp sebum levels, while placebo shampoo simultaneously reduced the scalp sebum levels.

In a notable manner, even after one week of discontinuation of the test shampoo, scalp redness in the volunteer group with shampoo containing 2% rice protein hydrolysate according to the invention was still significantly better than in the placebo group, and the pH of their scalp was significantly lower than in the volunteer group with placebo shampoo.

EXAMPLE 8 clinical study of skin moisturization

In clinical studies on human volunteers, the effect of treatment with body wash containing 2% (w/w) of the rice protein hydrolysate of the invention on skin moisturization was tested in control placebo treatment. The body washes used consisted of the following (weight percent)

TABLE 3 Table 3

The study was performed on 24 female volunteers between 40 and 65 years old, with dry leg skin. Volunteers applied one body wash composition per leg. The body wash was applied twice daily for a total of 28 days.

Moisture retention was assessed using a corneometer (skin moisture tester). Measurements were made at the time corresponding to the initial value, and at 7, 14 and 28 days of treatment, and 7 days after stopping using the product (35 days after initial value). Consumer conscious questionnaires were also completed by volunteers at 28 days.

Statistical analysis of the instrument parameters over time was performed after verifying the normality of the distribution by either paired Student t-test or Wilcoxon signed rank test by shape-Wilk test. The significance level was set at 5% (p < 0.05). For consumer self-questionnaires, the frequency statistical difference (%) between approving (consent) and disapproving (disagreement) opinions was evaluated at 5% by a two-tailed binomial test.

Body washes containing 2% rice protein hydrolysate of the invention significantly increased skin moisturization after 2 and 4 weeks of use relative to placebo body washes after 1, 2 and 4 weeks of use.

In a notable manner, skin moisturization of the legs treated with body washes containing 2% rice protein hydrolysate according to the invention was still significantly higher relative to the initial value and placebo even one week after discontinuation of the use of the composition.

After 4 weeks of use of body washes containing 2% rice protein hydrolysate according to the present invention, most volunteers agreed that their skin was deeply moisturized, the skin appeared and felt nourished, they felt that the skin barrier of the legs was strengthened, and the skin was significantly smoother and softer, not rough, more comfortable and more comfortable.

Example 9: formulation of a cosmetic ingredient according to the invention

The cosmetic composition according to the invention comprises, in weight percent (w/w):

benzoic acid 0.4% as preservative

Rice hydrolysate 20% according to example 2

Proper amount of water 100%

Such ingredients according to the invention are liquids and may be formulated according to conventional techniques known to those skilled in the art for preparing cosmetic compositions.

Example 10: other advantages/Properties of peptides according to the invention

10.1 anti-elastase Activity: HLE (human leukocyte elastin)Enzyme) inhibition assay

Aging of Hair Follicle Stem Cells (HFSC) results in aging and progressive shrinkage of the hair follicle. This aging of the hair follicle is induced by proteolytic transepidermal elimination of stem cells by the hemidesmin COL17A1 (collagen BP180/XVII type). This proteolytic action of the hemidesmosomes anchoring the hair follicle stem cells to the basement membrane results in senescent asymmetric cell division and hair follicle aging. This proteolysis is triggered by neutrophil elastase, which is induced in aged HFSC but not detectable in young HFSC, resulting in COL17A1 depletion, hair follicle shrinkage or shedding, and age-related hair loss.

In particular the peptide of the invention in the form of the hydrolysate of example 2b showed a significant inhibition of elastase activity at a dose of 0.1% (w/w) relative to the untreated control, i.e. 2.2-fold more effective than the reference hydrolysate of example 4 without the peptide of the invention.

10.2 Activity on collagen Synthesis

The synthesis of the deposited collagen I was evaluated according to the method described in patent application WO 2012/175454 A2.

In particular the peptide of the invention in the form of the hydrolysate of example 2b significantly stimulated collagen I synthesis at a dose of 0.05% (w/w) relative to the untreated control, i.e. 2.4 times more efficient than the rice protein hydrolysate of example 4 without the peptide of the invention.

Example 11: clinical study of skin softness and firmness

The study was performed on 34 female volunteers 40 to 65 years old. Volunteers applied a body wash composition similar to example 7 on each side of the abdomen. The body wash was applied twice daily for a total of 28 days.

The effect on stiffness was evaluated using a cutometer and a Skinfibre gauge. After 2 and 4 weeks of use, body washes containing 2% (w/w) of the rice protein hydrolysate of the present invention significantly increased skin firmness relative to placebo body wash. The skin extensibility was also significantly increased relative to placebo after 28 days of use of the product.

In a notable manner, the skin firmness of the abdominal side treated with body washes containing 2% of the rice protein hydrolysate of the invention was still significantly higher relative to the starting and placebo even one week after the cessation of the use of the lotions.

After 4 weeks of use of the body wash containing 2% rice protein hydrolysate of the invention, most volunteers agreed that their abdominal skin appeared younger and more comfortable.

Example 12: effect of the peptides of the invention on hair appearance

12.1 Effect on the structure of keratin fibers):

the hair restoration properties of the hydrolysates according to the invention were evaluated by the DSC (differential scanning calorimetry) method according to conventional protocols.

DSC processes are common thermal analysis processes for measuring the amount of heat absorbed by/emitted from a sample during heating. Due to the different heat fluxes between the sample and the reference during the temperature change procedure (heating rate), conclusions can be drawn about the denaturation temperature of the treated hair (sample) relative to the untreated hair (reference). A TA Instruments Q100 DSC machine with an auto sampler was used. The heating rate was 2℃per minute as measured according to the method of F.J.Wortmann et al, J.Cosmet.Sci. [ J.J.cosmetic science ],2002,53,219-228. As a sample, caucasian hair was subjected to super bleaching and then treated three times with an aqueous solution containing 1% (expressed as dry matter, w/v) of the rice protein hydrolysate of example 2 of the present invention. Treatment with water alone served as a negative control.

Damaged hair treated with the protein hydrolysate according to the invention of example 2 shows a higher decomposition temperature, i.e. an improvement of the hair denaturation temperature, due to an improvement of the hair structure.

TABLE 4 Table 4

12.2 Effects on proliferation of dermal papilla fibroblasts

The method used was based on a 3D model of hair follicle pseudopapilla from aggregation cultured skin papilla fibroblasts to evaluate the effects on activation of hair follicles and on increasing hair growth.

Cell culture and treatment:

the process is based on the aggregation of human hair papilla fibroblasts by gentle centrifugation of a human hair papilla fibroblast suspension. Human skin fibroblasts were prepared as a suspension in basal medium containing rice peptide in the form of hydrolysate of the invention of example 2c and transferred to 96-well microplates, as compared to rice protein hydrolysate of example 4 or without any product (untreated control). The cell suspension was centrifuged at 200 Xg for 5 minutes, and the formed aggregates were then centrifuged at 37℃and 5% CO ₂ And incubating for 5 days at a relative humidity of greater than 95%.

DNA quantification

The aggregates were rinsed with balanced saline solution (PBS) and then destroyed by incubation with a mixture of protease (collagenase a, trypsin) and EDTA for about 1 hour. Then, a portion of the cell suspension was centrifuged and the pellet was recovered for measuring the DNA (deoxyribonucleic acid) level. DNA of cells dissolved in a specific buffer was measured according to the kit manufacturer's (CyQUANT NF cell proliferation assay kit, invitrogen, france).

Results are expressed as a percentage relative to untreated control and as an average.

Results:

TABLE 5

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The peptides of the invention in the form of hydrolysates stimulated total DNA content in pseudopapilla fibroblasts relative to untreated controls and relative to reference rice protein hydrolysates (example 4). The peptides of the invention in the form of hydrolysates were 5-fold more effective than the rice protein hydrolysates of example 4 at the same dose of 0.01% (dry weight).

Example 13:examples of formulations

O/W polymer gel

TABLE 6

Suitable formulations are also those described in BASF patent application US 2019/0216709 examples B3 to B9, substituting the peptides of example 2 with the inventive hydrolysates described in example 2 a).

Peptides used in the context of the present invention are collated in table 7 below:

TABLE 7

Sequence number	Sequence(s)
		SEQ ID No.1	GYYGEQQQQPGMTR
SEQ ID No.2	SEEGYYGEQQQQPGMTR
		SEQ ID No.3	IYGPDTGVDYKDNQMR
SEQ ID No.4	FYNEGDAPVVAL

Sequence listing

<110> Pasteur beauty treatment French company (BASF BEAUTY CARE SOLUTIONS FRANCE SAS)

Novel use of <120> peptides for improving skin and/or mucosal comfort and/or appearance of integuments

<130> 1H197810 0141 PCT

<150> FR2010090

<151> 2020-10-02

<160> 4

<170> BiSSAP 1.3.6

<210> 1

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> MW 1642.72

<400> 1

Gly Tyr Tyr Gly Glu Gln Gln Gln Gln Pro Gly Met Thr Arg

1 5 10

<210> 2

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> MW 1988.01

<400> 2

Ser Glu Glu Gly Tyr Tyr Gly Glu Gln Gln Gln Gln Pro Gly Met Thr

1 5 10 15

Arg

<210> 3

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> MW 1971.97

<400> 3

Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys Asp Asn Gln Met Arg

1 5 10 15

<210> 4

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> MW 1294.37

<400> 4

Phe Tyr Asn Glu Gly Asp Ala Pro Val Val Ala Leu

1 5 10

Claims

1. Use of a peptide having SEQ ID No.1 and/or a homologue thereof for its cosmetic properties selected from improving the comfort of healthy skin, in particular healthy scalp and/or healthy mucous membranes, and/or improving the appearance of a healthy body cover.

2. Use according to claim 1 for reducing and/or preventing unpleasant and/or uncomfortable manifestations of healthy skin, in particular healthy scalp and/or healthy mucous membranes due to a decrease in skin and/or mucous membrane homeostasis and/or a decrease in endo-and/or filaggrin content and/or an increase in skin pH.

3. Use according to claim 2, characterized in that the unpleasant and/or uncomfortable manifestations of healthy skin and/or healthy mucous membranes are independent of age or epithelial cell damage.

4. Use of a peptide having SEQ ID No.1 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, for its cosmetic properties selected from the group consisting of

-improving the comfort of healthy skin, in particular healthy scalp and/or healthy mucous membranes to reduce and/or prevent unpleasant and/or uncomfortable manifestations of healthy skin, in particular healthy scalp and/or healthy mucous membranes due to a decrease in skin and/or mucous membrane homeostasis and/or a decrease in endo-and/or filaggrin content and/or an increase in skin pH;

-and/or improving the appearance of a healthy quilt;

these unpleasant and uncomfortable manifestations of healthy skin and/or healthy mucosa are independent of age or epithelial cell damage.

5. Use according to any one of the preceding claims for reducing and/or preventing skin and/or mucous membrane dryness, in particular scalp dryness, itching, redness, skin tightness, desquamation, in particular scalp exfoliation, dandruff, cracking and skin sensitivity, in particular scalp sensitivity; and/or to provide a pleasant feel to the skin and/or mucous membranes and/or to make the skin and/or mucous membranes softer and/or to reduce their roughness.

6. Use according to any one of the preceding claims, wherein improving the appearance of the body cover, preferably hair, is selected from increasing its strength and/or resistance, and/or protecting it from cosmetic oxidation treatments and/or aggressive agents, reducing the breakage rate, repairing damaged body covers, improving the shaping and/or volume, increasing the growth and/or gloss of keratin fibres and/or reducing the loss thereof.

7. The use according to any one of the preceding claims, wherein the bodies are hair.

8. Use according to any one of the preceding claims, wherein the peptide and/or homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, is in the form of a peptide having SEQ ID No.2 and/or homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence.

9. Use according to any one of the preceding claims, wherein the peptide having SEQ ID No.1 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and identical properties to said sequence, is in the form of a peptide hydrolysate of rice (Oryza sativa), in particular enriched in the peptide having SEQ ID No.1, preferably in liquid form.

10. Use according to any one of the preceding claims, wherein the hydrolysate has at least two characteristics selected from the group consisting of:

-its dry matter content is 15% to 25% by weight;

-its nitrogen content is 1.5% to 3% by weight;

11. Use according to any one of the preceding claims, wherein the peptide having SEQ ID No.1 and/or homologues thereof, in particular biologically active homologues having at least 85% sequence identity and having the same properties as said sequences, is used in combination with:

-a peptide having SEQ ID No.2 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, and/or

-a peptide having SEQ ID No.3 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, and/or

-a peptide having SEQ ID No.4 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence.

12. Use according to any one of the preceding claims, characterized in that the peptide having SEQ ID No.1 and/or homologues thereof, in particular biologically active homologues having at least 85% sequence identity and identical properties to said sequence, are in the form of a cosmetic composition comprising same and advantageously also comprising a suitable cosmetic vehicle.

13. Use according to claim 12, characterized in that the peptide having SEQ ID No.1 and/or homologues thereof, in particular biologically active homologues having at least 85% sequence identity and identical properties with said sequence, is present in an amount of 1 x 10 by weight of dry matter relative to the total weight of the cosmetic composition ^-5 % to 20%, preferably 1X 10 ^-4 % to 10%, more advantageously 1X 10 ^-3 % to 3%, more preferably 0.001% to 0.1% is present in the composition.

14. Use according to any one of the preceding claims, characterized by being a topical use.

15. Use according to any one of the preceding claims, characterized in that it is intended for caring for dry and/or sensitive skin, and/or dry and/or sensitive scalp.

16. Use according to any one of the preceding claims, characterized in that the homolog of the peptide has at least 85% sequence identity and biological activity of the same nature as said sequence.

17. Cosmetic care and/or treatment method, characterized in that it comprises the topical application of the peptide having SEQ ID No.1 and/or homologues thereof, in particular biologically active homologues having at least 85% sequence identity and identical properties to said sequence, to healthy skin, advantageously to at least one region of healthy scalp and/or healthy mucous membrane and/or healthy body cover, for improving the comfort of healthy skin, in particular healthy scalp and/or healthy mucous membrane, and/or improving the appearance of healthy body cover, in particular keratin fibres.

18. Cosmetic care and/or treatment method, characterized in that it comprises the topical application of the peptide having SEQ ID No.1 and/or its homologs, in particular biologically active homologs having at least 85% sequence identity and identical properties to said sequence, to healthy skin, advantageously to at least one region of healthy scalp and/or healthy mucous membrane and/or healthy body quilt, for

and/or improving the appearance of a healthy body cover, in particular keratin fibres,

19. Cosmetic care method according to claim 17 or 18, wherein the peptide having SEQ ID No.1 and/or homologues thereof, in particular biologically active homologues having at least 85% sequence identity and having the same properties as said sequences, are as defined in any of claims 8 to 13 or 16.

20. The method according to any one of claims 17 to 19, wherein the area of skin and/or mucous membrane and/or integument is selected from scalp, face, hands, arms, collarband, legs, neck, back, shoulders, stomach, wrist, forearm, ankle, thigh, nape, joint folds and armpits, lip mucous membrane, body hair, eyelashes, eyebrows and/or hair.

21. A peptide having SEQ ID No.1 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, for use in the treatment and/or prophylaxis thereof of pathologies associated with a reduction in skin homeostasis and/or a reduction in endo-and/or filaggrin content and/or an increase in skin pH and/or any combination thereof, in particular ichthyosis and alopecia, and desquamation associated with certain pathologies such as acute radiation, sunburn and burns.

22. Peptide having SEQ ID No.1 and/or a homologue thereof, in particular a biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, for use according to claim 21, characterized in that the peptide having SEQ ID No.1 and/or a homologue thereof is as defined in any of claims 8 to 11 or 16.

23. Peptide having SEQ ID No.1 and/or homologue thereof, in particular biologically active homologue thereof having at least 85% sequence identity and having the same properties as said sequence, for use according to any of claims 21 and 22, characterized in that the peptide and/or homologue thereof, in particular biologically active homologue having at least 85% sequence identity and having the same properties as said sequence, is present in an amount of 1 x 10 by weight of dry matter relative to the total weight of the pharmaceutical composition ^-5 % to 20%, preferably 1X 10) ^-4 % to 10%, more advantageously 1X 10 ^-3 Levels of% to 3%, more preferably 0.001% to 0.1% are present in the composition, which advantageously also comprises a suitable pharmaceutical vehicle.