Combination of cosmetic active ingredients to maintain or rebalance the skin microbiota

Abstract

The invention relates to a cosmetic combination comprising a red clover extract and a peptide for preventing or treating imbalance of the skin microbiota.

Description

Title of the invention: Combination of cosmetic active ingredients to maintain or rebalance the skin microbiota Technical field

[0001] The present invention relates to the field of cosmetic active ingredients and their uses, in particular for preventing dysbiosis of the skin microbiota, or maintaining homeostasis of the skin microbiota. Technological background

[0002] The skin is the first barrier protecting the body from external aggressions. This organ is composed of several layers of tissue. We distinguish (i) the epidermis which is the outermost part of the skin, (ii) the dermis, a connective tissue made up of fibroblasts and an extracellular matrix which ensures the functions of cohesion and nutrition of the skin, and (iii) the hypodermis made up of adipocytes.

[0003] The epidermis is made up of several cellular strata of keratinocytes. We distinguish, among others, the germinative layer of the epidermis, called the basal layer, containing, in particular, the cutaneous stem cells, the spinous layer, Stratum spinosum, made up of several layers of polygonal cells, the granular layer, Stratum granulosum, comprising one to three layers of flattened cells containing cytoplasmic inclusions, the keratohyaline grains, and finally, the horny layer, Stratum corneum which is composed of anucleated and keratin-rich cells called corneocytes which correspond to the terminal stage of differentiation of keratinocytes.

[0004] The outermost cells of the stratum corneum are continually shed and replaced by cells from a lower layer, in a process called desquamation. Cell regeneration of the stratum corneum is based on a process of cell maturation in which cells in the basal layer of the epidermis differentiate and gradually migrate through the different strata of the epidermis until they reach the stratum corneum in the form of corneocytes.

[0005] Furthermore, the cells of the stratum corneum are linked together by epidermal lipids. These lipids create a protective barrier and have a water-retaining power. In the event of a lipid deficiency, the skin can become dry, tight and rough.

[0006] The epidermis is, in addition, covered with an emulsion of water and lipids (fats), called the hydrolipidic film. This film, renewed by the secretions of the sebaceous and sweat glands, helps to keep the skin supple and acts as an additional barrier against pathogens and irritants.

[0007] Much more than an external envelope, the skin is a real organ ensuring a barrier function that protects our organism against multiple external aggressions, whether microbiological, chemical or mechanical. The skin is supported in this protective function by the cutaneous microbiota (also known as cutaneous flora or commensal flora) made up of various types of microorganisms including bacteria, fungi and viruses.When a pathogen attempts to penetrate the body via the epidermis, the skin and the skin microbiota can act in concert to counteract this aggression: the skin microbiota prevents the pathogen from attaching and can secrete bacteriocins and other toxins while the innate immune system of the skin is activated, involving the release of pro-inflammatory cytokines, the recruitment of immune cells such as neutrophils, NK cells and macrophages and the secretion of antibacterial peptides.

[0008] The balance between the skin and the skin microbiota is fragile.

[0009] This balance can be altered over time due to intrinsic factors such as age (for example hormonal modulation in adolescence or menopause) but also due to repeated stress or external aggressions, for example due to the climate (temperature, ambient humidity, UV), medical treatment (antibiotic, corticosteroid), cosmetic treatment (frequent washing, peeling, etc.), clothing worn (synthetic or cotton), or lifestyle habits (diet, alcohol, tobacco). These different aggressions can modify the relative and / or quantitative abundance of the different communities of microorganisms, in particular the relationships between the different populations of commensal bacteria: the balance of the skin microbiota is altered, which ultimately impacts the cooperation between the skin and the microbiota.

[0010] An imbalance in the skin microbiota can alter the visual appearance and properties of the skin, in particular its ability to ensure its barrier function, and make it more reactive to external aggressions.

[0011] It has thus been shown that sensitive skin is associated with a clear imbalance of the skin microbiota, in particular with regard to the populations of Bacteriodetes, Actinobacterium and S. epidermidis (overabundant) and the populations of Firmicutes and S. aureus (underabundance) (Byrd et al., 2018, Nat. Rev. Microbiol. 16, 143-155)

[0012] An imbalance of the skin microbiota can thus make the skin more reactive, more sensitive and cause it to respond disproportionately or inappropriately to external aggressions, whether of bacterial or environmental origin. =.

[0013] There is therefore a need for new cosmetic agents capable of preserving or rebalancing the skin microbiota. Summary of the invention

[0014] The invention relates to the cosmetic use of a peptide of general formula (I):

[0015] AX-Gly-His-Lys-Y (I) in which:

[0016] X represents a chain of 1 to 3 lysine residues, optionally methylated,

[0017] Y represents OH or NH2, and

[0018] A represents H or a radical RC(O)- in which

[0019] R represents a saturated or unsaturated hydrocarbon chain, of 1 to 24 carbons, linear or branched, or RC(O)- represents the radical of lipoic acid or the reduced form thereof, the radical of dihydrolipoic acid or the radical of retinoic acid,

[0020] in combination with a red clover extract as an agent for maintaining or rebalancing healthy skin microbiota.

[0021] In a preferred embodiment, the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb):

[0022] - A-MeLys-Gly-His-Lys-Y (lia) or

[0023] - A-Lys-Gly-His-Lys-Y (Ilb)

[0024] A and Y being as defined in claim 1.

[0025] In some embodiments,

[0026] - the peptide is such that Y is NH2 and A is H or an RC(O)- radical in which R re has a hydrocarbon chain, saturated or unsaturated, of 1 to 6 carbon atoms, and

[0027] - the extract is a hydroalcoholic extract of red clover flowers.

[0028] In a particular embodiment, the peptide of formula (I) in combination with the red clover extract is used to maintain or rebalance the healthy scalp microbiota, in particular to promote scalp health.

[0029] In another embodiment, the peptide of formula (I) and the red clover extract are used in combination to rebalance, or maintain the balance of, bacterial populations of S. epidermidis, C. acnes, and S. aureus in the microbiota of healthy skin, including the scalp.

[0030] The peptide of formula (I) and the red clover extract can be used in any type of subject, in particular in an individual with fragile skin, preferably a fragile scalp.

[0031] Preferably, the peptide of formula (I) and the red clover extract are present as active agents in a cosmetic product.

[0032] The cosmetic product is typically chosen from a makeup product, a care product or a body hygiene product.

[0033] For example, the cosmetic product may be a hair product chosen from the group consisting of a shampoo including a dry or solid shampoo, a conditioner, a hair balm, a hair oil, a hair serum, a hair lacquer, a hair fixing gel, and a hair spray.

[0034] In certain embodiments, the peptide of formula (I) and the red clover extract are incorporated into the cosmetic product in the form of a functional ingredient comprising (i) said peptide, (ii) said red clover extract and a cosmetically acceptable carrier.

[0035] The cosmetically acceptable vehicle may be selected from triethyl citrate, glycerol (also called glycerin), C2-C8, preferably C2-C5 alkanediols such as propanediol, butylene glycol, pentylene glycol and isomers thereof, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate, water, and mixtures thereof.

[0036] For example, the functional ingredient comprises a butylene glycol / water mixture as a cosmetically acceptable carrier, and optionally dextran.

[0037] In certain embodiments the peptide in combination with the red clover extract is used to treat, alleviate or prevent one or more non-pathological alterations caused by dysbiosis in healthy skin, preferably selected from a decrease in the barrier function of the skin, a loss of skin suppleness, a loss of skin elasticity, a loss of skin hydration, and a loss of evenness of skin tone. Legend of the figures

[0038] [Fig-1] shows the modulation of the populations of S. epidermidis, C. acnes and S. aureus before (day 0) and after 90 days of scalp treatment (day 90) with the combination according to the Invention (see Example 1).

[0039] [Fig.2] shows the ratio of S. epidermidis versus C. acnes before (day 0) and after 90 days of treatment (day 90) with the combination according to the Invention (see Example 1). Figures [Fig.l] and [Fig.2] show that the combination according to the invention makes it possible to modulate the populations of bacteria whose abundances are altered in sensitive or weakened skin. In particular, the combination according to the invention makes it possible to increase the ratio of the population of S. epidermidis versus C. acnes which is reduced in sensitive skin.

[0040] [Fig.3] shows the overall absolute populations of bacteria of the Actino- family bacterium, Proteobacterium and Firmicutes before (day 0) and after 90 days of scalp treatment (day 90) with the combination according to the Invention (see Example 1).

[0041] [Fig.4] shows the overall absolute populations of bacteria of different genera before (day 0) and after 90 days of scalp treatment (day 90) with the com- combination according to the Invention (see Example 1).

[0042] [Fig.5] shows the ratios of C. acnes / S. epidermidis, C. acnes / S. aureus and S. epidermidis / S. aureus before treatment (day 0) and after 90 days of scalp treatment (day 90) with the combination according to the Invention (see Example 1).

[0043] Figures [Fig.3], [Fig.4] and [Fig.5] show that the combination according to the invention is capable of modulating the abundance of commensal bacteria involved in sensitive or weakened scalp. Detailed description of the invention

[0044] The Applicant has shown that the combined use of a peptide of formula (I) as described below and a red clover extract is capable of maintaining or rebalancing the skin microbiota, when the extract and the peptide are applied topically, in particular in the form of a rinse-off cosmetic product.

[0045] Indeed, as shown in the examples, the repeated application of a shampoo and a conditioner comprising the combination according to the invention in individuals presenting an unbalanced microbiota makes it possible to rebalance the bacterial populations.

[0046] As illustrated in Example 1, repeated topical application of the combination according to the invention makes it possible to increase the abundance of S. epidermidis and to decrease the abundance of S. aureus while maintaining the population of C. acnes. This modulation results in a significant increase in the ratio of the population of S. epidermidis versus C. acnes ([Fig.l] and [Fig.2]). However, sensitive or weakened skin is characterized by a deficiency in S. epidermidis, an overabundance of S. aureus and a deficient ratio of S. epidermidis versus C. acnes. Thus, the combination according to the invention is capable of specifically modulating the populations of S. epidermidis, S. aureus and C. acnes to values characteristic of healthy skin.

[0047] Furthermore, the combination according to the invention is capable of modulating the abundance of commensal bacteria involved in sensitive or weakened scalp.

[0048] Example 1 shows that the composition according to the invention makes it possible to increase the overall population of bacteria belonging to the Proteobacteria and to increase those of bacteria belonging to the genus Firmicutes ([Fig.3]). However, it is known that weakened or sensitive scalp is characterized by a deficit in bacteria of the genus Firmicutes and an overabundance of bacteria of the genus Bacteriodetes.

[0049] Example 1 also shows that the combination according to the invention is capable of reducing the abundance of different bacterial genera (Corynebacterium, Prevotella, Rothia and Acinetobactef) and of increasing the overall population of bacteria of the genus Staphylococcus, these bacterial genera being known to be involved in dysbiosis of weakened or sensitive scalp. ..

[0050] Overall, Example 1 shows that the combination makes it possible to correct the relative abundance of the populations of C. acnes versus S. aureus and S. epidermidis versus S. aureus, the ratios between these bacterial populations being generally altered in weakened or sensitized scalps.

[0051] Thus, Example 1 shows that the combination according to the Invention makes it possible to modulate in a relative and quantitative manner the bacterial populations involved in the phenomena of sensitive or weakened skin, in particular at the level of the sensitive or weakened scalp, so as to tend towards the characteristic state of healthy skin.

[0052] The peptides of formula (I) according to the invention are described in patent EP 1 663 285 for use in the treatment of alopecia. This patent makes no mention of any effect of the peptides on the skin microbiota.

[0053] The literature suggests various medicinal activities of red clover extracts, notably to relieve menopausal disorders or certain skin diseases. Some red clover extracts are said to have anti-inflammatory properties.

[0054] The Applicant markets a functional ingredient comprising a red clover extract in combination with a tetrapeptide as an agent for densifying hair and reducing scalp inflammation (Capixyl™). To the Applicant's knowledge, this functional ingredient has never been proposed to act on the skin microbiota.

[0055] To the Applicant's knowledge, the literature does not suggest that the combination of a peptide of formula (I) and a red clover extract, as described below or as present in Capixyl™, can exert any action on the skin microbiota.

[0056] Thus, the subject of the invention is the use of a peptide of formula (I) in combination with a red clover extract as a cosmetic agent for maintaining or restoring the balance of the microbiota of healthy skin.

[0057] In other words, the invention relates to a combination of active ingredients, namely a peptide of formula (I) as described below and a red clover extract, for use in maintaining the balance of the skin microbiota or in preventing an imbalance of the skin microbiota.

[0058] The invention also relates to the use of a peptide of formula (I) in combination with a red clover extract as a cosmetic agent for preventing an imbalance (or dysbiosis) of the skin microbiota and / or for alleviating an imbalance (or dysbiosis) of the skin microbiota and / or for restoring the balance of the skin microbiota in healthy skin, including in healthy scalps.

[0059] The invention also relates to a combination comprising a peptide of formula (I) as described below and a red clover extract, for use in the prevention of an imbalance (or dysbiosis) of the skin microbiota and / or for the alleviation of an imbalance (or dysbiosis) of the skin microbiota and / or in the restoration of the balance of the skin microbiota.

[0060] The combination according to the invention can further be used to restore or strengthen the barrier property of the skin, including the scalp.

[0061] The combination according to the invention can also be used to prevent or treat alterations in the mechanical or visual properties of the skin induced by an imbalance in the skin microbiota.

[0062] The invention also relates to the use of a peptide of formula (I) in combination with a red clover extract as a cosmetic agent for maintaining the health of healthy skin, including the health of the scalp. The peptide of formula (I) in combination with a red clover extract may further be used to improve or maintain the health of the appendages, in particular the hair.

[0063] According to a particular aspect, the invention also relates to the use of a peptide of formula (I) in combination with a red clover extract as a cosmetic agent for rebalancing the microbiota of weakened skin, including a weakened scalp.

[0064] In a further aspect, the invention relates to a cosmetic method for maintaining or restoring the balance of the microbiota of healthy skin, or for preventing an imbalance (or dysbiosis) of the skin microbiota and / or for alleviating an imbalance (or dysbiosis) of the skin microbiota and / or for restoring the balance of the skin microbiota in healthy skin including the scalp, said cosmetic method comprising the application of a peptide of formula (I) in combination with a red clover extract to the area of skin to be treated.

[0065] In a further aspect, the invention relates to the use of a combination comprising a peptide of formula (I) and a red clover extract as described herein in the manufacture of a cosmetic product for preventing an imbalance (or dysbiosis) of the skin microbiota and / or for alleviating an imbalance (or dysbiosis) of the skin microbiota and / or for restoring the balance of the microbiota of the skin, in particular of the scalp.For the purposes of the invention, the terms "skin" and "cutaneous" refer to any part of the skin of the human body, in particular the skin of the face, including the lips and eyelids, but also the scalp. Preferably, this is the scalp.

[0066] In the context of the present invention, the combination of the peptide and the extract according to the invention is typically applied topically to healthy skin, that is to say to skin (e.g. the scalp) not presenting wounds or skin pathologies.

[0067] In other words, the area of skin (eg the scalp) on which the combination according to the invention is applied is healthy, the subject not suffering from any derma- tological at least at the level of the application area.

[0068] Thus, the combination according to the invention does not produce a therapeutic effect on the skin, when it is used according to the invention.

[0069] The term "cutaneous microbiota", "skin microbiota" or "cutaneous flora" means all the bacterial, fungal, and viral germs present on the skin surface, and the sebaceous and sweat glands. This term includes in particular the resident flora and the transient flora. The cutaneous microbiota includes in particular bacteria belonging to the Actinobacteria (such as Coryne-bacterium), Firmicutes (such as Clostridium), Proteobacterium (such as Pseudomonas) and Bacteriodetes as well as fungi, mites (Demodex), phages and viruses, this list not being exhaustive. For example, the following bacterial species of the commensal flora may be mentioned: Staphylococcus epidermidis, S. aureus, and Corynebacterium acnes

[0070] “Preventing an imbalance of the skin microbiota” means preventing the appearance, or delaying or delaying the appearance, of an imbalance of the skin microbiota.

[0071] The term "imbalance of the skin microbiota" means a quantitative alteration (i.e. an alteration of the absolute abundance) of one or more microbial populations, preferably bacterial, and / or an alteration of one or more ratios of the microbial populations to each other, preferably bacterial, compared to a basal state characteristic of healthy skin. The term "alteration" includes an increase or a decrease in a parameter (absolute abundance or ratio) compared to this same parameter in the basal state.

[0072] An imbalance of the skin microbiota can alter the barrier property of the skin. It can also alter the visual or mechanical properties of the skin. Non-pathological alterations of dysbiosis on the skin include but are not limited to:

[0073] a decrease in the barrier function of the skin,

[0074] a loss of skin suppleness,

[0075] a loss of skin elasticity,

[0076] less hydrated skin,

[0077] a loss of uniformity of skin tone

[0078] an increase in redness, particularly following exposure to external stress.

[0079] For the purposes of the present invention, the term "maintaining the balance of the skin microbiota" means maintaining or preserving the normal balance of the skin flora, i.e. maintaining and preserving the different commensal communities on a qualitative and / or quantitative level. Maintaining the balance of the skin microbiota also includes maintaining a ratio between two mi- crobial, preferably bacterial.

[0080] This term also includes a preventive effect against an imbalance of the microbiota caused for example by age or a physiological hormonal variation (menopause, puberty) or external stress or aggression.

[0081] External stresses and aggressions include, but are not limited to, cosmetic treatments (e.g. frequent washing, peeling, scrubbing, hair removal), chemical treatments (e.g. exposure of the skin to aggressive agents such as detergents, or to chorea water or sea water), exposure to climatic conditions (high or low temperatures, humidity, dryness, wind, UV), or to climate change (e.g. change of season), etc.

[0082] The imbalance of the skin microbiota can also be induced by certain lifestyle habits such as a diet that is too rich, particularly too fatty, the consumption of alcohol or tobacco, a tiring lifestyle (night work, jet lag, too short a sleep period), a stressful work or private environment.

[0083] These stresses and / or lifestyle habits can also make the skin, including the scalp, more fragile. This is called “fragile skin”.

[0084] In certain embodiments, the combination according to the invention is used in a subject having sensitive skin or weakened skin.

[0085] In a particular embodiment, the combination of active ingredients according to the invention makes it possible to increase, restore or maintain the abundance of S. epidermidis in the skin.

[0086] In a particular embodiment, the combination of active ingredients according to the invention makes it possible to reduce, restore or maintain the abundance of S. aureus in the skin.

[0087] In certain embodiments, the combination of active ingredients according to the invention makes it possible to maintain or restore the ratio of the S. epidermidis versus C. acnes population in the skin. In particular, the combination of active ingredients according to the invention makes it possible to increase the ratio of the S. epidermidis versus C. acnes population in sensitive or weakened skin, in particular in sensitive or weakened scalp.

[0088] In a particular embodiment, the combination of active ingredients according to the invention makes it possible to increase, restore or maintain the overall abundance of bacteria belonging to the genus Staphylococcus on the skin, in particular on the scalp, preferably on a sensitive or weakened scalp.

[0089] In a particular embodiment, the combination of active ingredients according to the invention makes it possible to reduce, or restore the overall abundance of bacteria belonging to a genus chosen from Acinetobacter, Bacteriodetes, Corynebacterium, Prevotella, Rothia, and combinations thereof, at the level of the skin, in particular at the level of the scalp, preferably at the level of a sensitive or weakened scalp.

[0090] In another particular embodiment, the combination of active ingredients according to the invention makes it possible to reduce or restore the overall abundance of bacteria belonging to the Proteobacteria and to increase or restore the overall abundance of bacteria belonging to the Firmicutes at the level of the skin, in particular at the level of the scalp, preferably at the level of a sensitive or weakened scalp.

[0091] In a particular embodiment, the combination of active ingredients according to the invention makes it possible to maintain or restore the ratio of the S. acnes versus S. epidermidis and / or C. acnes versus S. aureus population in the skin. In particular, the combination of active ingredients according to the invention makes it possible to reduce the ratio of the S. acnes versus S. epidermidis population and / or the C. acnes versus S. aureus ratio in sensitive or weakened skin, preferably in sensitive or weakened scalp.

[0092] In certain embodiments, the combination of active ingredients according to the invention makes it possible to maintain or restore the ratio of the C. acnes population versus the S. epidermidis population, and / or the ratio of the C. acnes population versus the S. aureus population and / or the S. epidermidis versus S. aureus ratio in the cutaneous microbiota of healthy skin.

[0093] In certain embodiments, the combination of active ingredients according to the invention is used to prevent or treat a non-pathological alteration of a mechanical or visual property of the skin caused by an imbalance of the microbiota. Such alterations include, but are not limited to, a decrease in the barrier function of the skin, a loss of suppleness of the skin,

[0094] a loss of skin elasticity, less hydrated skin, a loss of evenness of skin tone, and an increase in redness, particularly following exposure to external stress. The peptide according to the invention

[0095] The peptide according to the invention is a peptide of formula (I)

[0096] AX-Gly-His-Lys-Y (I)

[0097] in which:

[0098] X represents a chain of 1 to 3 lysine residues, optionally methylated,

[0099] Y represents OH or NH2, and

[0100] A represents H or a radical RC(O)- in which

[0101] R represents a saturated or unsaturated hydrocarbon chain of 1 to 24 carbon atoms or of 1 to 18 carbon atoms, linear or branched, optionally substituted by one or more hydroxyls, or

[0102] RC(O)- represents the radical of lipoic acid or the reduced form thereof, the dihydrolipoic acid radical, or retinoic acid radical.

[0103] In some embodiments, R is a hydrocarbon chain having from 1 to 6 unsaturations, preferably 1 to 3 double bonds.

[0104] “A is H” means that the NH2 group of the N-terminal amino acid is not amended.

[0105] “A is RC(O)-” means that the NH2 group of the N-terminal amino acid is modified to “RC(O)-NH-.

[0106] “Y is OH” means that the C-terminal group is -COOH and therefore that the lysine is not modified at the C-terminus.

[0107] “Y is NH2” means that the C-terminal group is -CONH2 and therefore that the C-terminal -COOH group was replaced by -CONH2 (amide function).

[0108] Such peptides are described in patent EP 1 663 285.

[0109] In a particular embodiment, the peptide of formula (I) is chosen from the peptides of sequence A-[SEQ ID NO:X]-Y in which SEQ ID NO:X is chosen from any one of the sequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, preferably of SEQ ID NO:1 or SEQ ID NO:4

[0110] In a preferred embodiment, the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb):

[0111] - A-MeLys-Gly-His-Lys-Y (lia) (or A-[SEQ ID NO:4]-Y) or

[0112] - A-Lys-Gly-His-Lys-Y (Ilb) (or A-[SEQ ID NO: 1]-Y)

[0113] A and Y being as defined in formula (I).

[0114] MeLys denotes methyl-lysine, i.e. a lysine comprising a methyl substituent on the amine function of its side chain.

[0115] In a particular embodiment, the peptide of formula (I), (IIa) or (IIb) is such that Y is NH2 and A is H or an RC(O) radical - in which R represents a saturated or unsaturated hydrocarbon chain of 1 to 10 carbon atoms, preferably of 1 to 6 carbon atoms. Preferably R is a saturated chain. For example, in the peptide of formula (I), (IIa) or (IIb), R is a saturated chain, preferably linear, comprising of 1 to 6 carbon atoms.

[0116] In a preferred embodiment, RC(O)- corresponds to the acetyl radical CH3 C(O)-.

[0117] The amino acids present in the peptide of formula (I), (IIa) or (IIb) can independently be of D or L configuration, preferably L.

[0118] In a preferred embodiment, the peptide according to the invention is Acetyl Te-trapeptide-3 of CAS Number: 827306-88-7 or a cosmetically acceptable salt thereof.

[0119] The peptide of formula (I), (IIa) or (IIb) may be in free form or in salt form, preferably cosmetically acceptable.

[0120] It may be an acid addition salt or a base addition salt.

[0121] The acid addition salt may be formed by reaction with an inorganic acid, or " mineral acid”, such as hydrochloric, hydrobromic, iodic, sulfuric, sulfamic, bisulfuric, phosphoric or nitric acid, for example, or with an organic acid, such as formic, acetic, pyruvic, trifluoroacetic, pro-pionic, butyric, hexanoic, heptanoic, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, this list not being exhaustive.

[0122] In other embodiments, the compound of the invention is in the form of a base addition salt. Bases that can be used to obtain such salts are, for example, alkali metal hydroxides, in particular potassium hydroxide, sodium hydroxide and lithium hydroxide; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; and organic bases, such as piperidine, diethanolamine and N-methylglucamine, this list not being exhaustive.

[0123] Suitable salts include in particular hydrochloride salts, acetate salts, citrate salts, potassium salts or sodium salts.

[0124] For example, when Y is NH2, the peptide can be in the form of an acetate salt.

[0125] The peptide according to the invention can be prepared by any method known to those skilled in the art, for example by peptide synthesis on a solid support using techniques well known to those skilled in the art. Red Clover Extract according to the Invention

[0126] The red clover extract (Trifoliumpratense) according to the invention is typically obtained from an aerial part of the plant, namely the stems, leaves and / or flowers, preferably the flowers. The extract is prepared by extraction using an organic solvent optionally mixed in water.

[0127] Preferably, the red clover extract is a hydroalcoholic extract.

[0128] For the purposes of the invention, a hydroalcoholic extract refers to an extract whose manufacturing process comprises a hydroalcoholic extraction step, i.e. an extraction using a water / alcohol mixture.

[0129] For the purposes of the invention, the term “alcohol” includes lower alcohols, in particular C2-C5, glycerin, C2-C5 alkanediols and mixtures thereof.

[0130] For example, the alcohol may be selected from glycerin, ethanol, propanol, butanol, isomers thereof such as isopropanol, tert-butanol, pentanol, ethylene glycol, 1,3-propanediol, and mixtures thereof. Preferred solvents are ethanol, isopropanol, glycerol, and mixtures thereof.

[0131] The “alcohol / water” volume ratio in the hydroalcoholic mixture is typically in a range from 0.01 to 20.0, for example from approximately 0.5 to approximately 10.

[0132] Typically, the red clover plant material is ground and macerated in a hydroalcoholic mixture. This maceration step can be carried out at room temperature or in the presence of heating. In certain embodiments, the maceration step is carried out with stirring or in the presence of ultrasound. At the end of the maceration step, the liquid phase is recovered and then concentrated.

[0133] In some embodiments, the concentrated extract may undergo an additional processing step, for example a chromatography step.

[0134] At the end of the extraction process, the extract is typically freeze-dried in the form of a powder.

[0135] In a preferred embodiment, the red clover extract is enriched with flavonoids, in particular biochanin A, i.e. it contains at least 5% by weight of flavonoids. Subject of interest

[0136] The combination of the peptide of formula (I) and the red clover extract as described herein may be used topically in any type of human subject. The human subject is preferably at least 15 years old. The subject may be male or female. In some embodiments, the subject is a male or female at least 35 years old, preferably at least 40 or 45 years old.

[0137] In a particular embodiment, the subject has mature skin.

[0138] In some embodiments, the subject is a pre-menopausal (eg, between 45 and 55 years old) or menopausal woman.

[0139] In certain embodiments, the subject is at risk of exhibiting skin dysbiosis. The risk of dysbiosis may be linked to the subject's exposure to external stress and / or to a life condition, for example as described above and / or to a physiological hormonal change (puberty, menopause, aging).

[0140] In another embodiment, the subject has sensitive skin or weakened skin, in particular a weakened or sensitive scalp.

[0141] Mode of use of the combination according to the invention

[0142] The red clover extract and the peptide according to the invention are applied topically, i.e. to the skin. They can be applied directly to the skin or incorporated into a cosmetic product beforehand. Preferably, the clover extract and the peptide according to the invention are applied by means of one or more cosmetic products.

[0143] The red clover extract and the peptide according to the invention are used in combination, that is to say they are applied to the skin sequentially, consecutively or simultaneously.

[0144] Preferably, the red clover extract and the peptide of formula (I) are applied simultaneously to the skin. They are thus typically contained in the same cosmetic product that is applied to the skin. Cosmetic products suitable for application to the skin, especially to the scalp, are described below.

[0145] The mass ratio of the red clover extract to the peptide of formula (I) is generally in a range from 0.1 to 10.0, preferably from 0.1 to 5.0.

[0146] The combination of red clover extract and the peptide of formula (I) according to the invention together generally represents from 0.0001% to 10%, preferably from 0.0001% to 5% by weight of the cosmetic product.

[0147] In the cosmetic methods and uses according to the invention, the dose to be administered and the frequency of administration of the extract according to the invention vary according to the cosmetic effect sought, the characteristics of the individual, in particular their sex, their age and their skin type.

[0148] Typically, the extract can be applied at least once every two weeks, at least once a week, or even 2 to 3 times a week and even daily to the skin, including the scalp.

[0149] The combination or cosmetic product according to the invention can be applied without rinsing or for a few seconds or even a few minutes before rinsing, on the skin, including the scalp.

[0150] By way of example, the combination of the peptide of formula (I) and the red clover extract according to the invention can be applied at least once a week to the scalp in the form of a shampoo or conditioner or any other hair product with rinsing. The hair product is left for a few seconds (e.g. at least 30 s) or even a few minutes (e.g. 2 to 5 min) before being rinsed. This treatment can be carried out at least once a week, for example at least two or three times a week.

[0151] As a further example, the combination according to the invention can be applied in the form of an oil, a serum or a hair lotion to the scalp without rinsing.

[0152] The treatments and uses according to the invention can last several weeks, or even several months. Cosmetic ingredient according to the invention

[0153] In certain embodiments, the peptide and the red clover extract according to the invention are preformulated together in the form of a functional ingredient before being integrated into a cosmetic composition or into a cosmetic product.

[0154] Thus in certain embodiments, the combination of the peptide and the red clover extract is in the form of a functional ingredient. The functional ingredient comprises (i) the peptide according to the invention, (ii) the red clover extract according to the invention and (iii) a cosmetically acceptable vehicle.

[0155] The vehicle can be of any kind, it can be a solid support such as maltodextrin or a cosmetically acceptable solvent. For example, the peptide and the red clover extract according to the invention may be suspended, or even diluted in a solvent chosen from triethyl citrate, glycerol (also called glycerin), C2-C8, preferably C2 C5 alkanediols such as propanediol, butylene glycol, pentylene glycol and their isomers, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate or glycerol ketal levulinate, water, and mixtures thereof.

[0156] Preferably, the vehicle is a mixture of water and alkanediol (e.g., water and C2-C5 alkanediols), e.g., water and butylene glycol.

[0157] In some embodiments, the functional ingredient may comprise one or more additional cosmetically acceptable excipients. The one or more additional excipients may be selected from a thickening agent, an antioxidant agent, a preservative agent, a viscosity agent, a surfactant, a carrier agent, a buffering agent, and combinations thereof.

[0158] In one embodiment, the additional excipient is a viscosity agent such as dextran.

[0159] By way of example, the functional ingredient comprises the combination according to the invention ((i) a peptide of formula (I) according to the invention and (ii) red clover extract) and (iii) dextran in a butylene glycol / water mixture.

[0160] An example of a suitable functional ingredient is, for example, the Capixyl™ product marketed by the Applicant.

[0161] The functional ingredient is preferably diluted in a suitable carrier or integrated into a cosmetic product. The functional ingredient according to the invention generally represents from 0.01% to 10%, preferably from 0.1% to 5% by weight of the cosmetic product.

[0162] Composition or cosmetic product according to the invention

[0163] The combination of red clover extract and peptide of formula (I) according to the invention is typically integrated into a cosmetic composition to be applied to the skin.

[0164] The cosmetic composition according to the invention generally comprises from 0.0001% to 10%, preferably from 0.0001% to 5% by weight of the combination according to the invention.

[0165] As indicated above, the combination according to the invention is integrated into said cosmetic composition in the form of a functional ingredient as described above. The functional ingredient according to the invention generally represents from 0.1% to 10%, preferably from 0.5% to 5% by weight of the cosmetic composition.

[0166] The cosmetic composition may further comprise one or more cosmetically acceptable excipients, typically from 50% to 99.99% by weight, preferably from 60% to 99.90% by weight, the percentages referring to the total weight of the cosmetic composition.

[0167] The cosmetically acceptable excipient(s) present in the cosmetic composition according to the invention may be chosen from vehicles such as an aqueous solution, demineralized water, mineral, thermal or floral water, solvents, in particular lower alcohols, in particular ethanol, isopropanol, dipropylene glycol, butylene glycol, propanediol, pentylene glycol and propylene glycol, dispersing agents, gelling or thickening agents, emollients, vectorizing agents such as phospholipids, preservatives, odor absorbers, pH regulating agents or neutralizing agents, washing agents or surfactants, in particular anionic, cationic, amphoteric or non-ionic surfactants, sequestering agents, foam stabilizing agents, foaming agents, humectants such as glycerin or sorbitol, wetting agents, dispersing agents, perfumes,pigments and other coloring agents, pearlescent agents, opacifying agents, softening agents, oily agents such as oils or fats of vegetable origin such as essential oils, silicone oils, fatty alcohol esters, fixing or plasticizing agents and / or other substances commonly used in formulation in the cosmetic or pharmaceutical field.

[0168] The cosmetic composition according to the invention may further comprise, optionally, one or more cosmetic active agents, typically between 0.0001% and 10.0% by weight of one or more cosmetic active agents. These may be anti-dandruff agents, UV filters, soothing agents, sebum-regulating agents, moisturizing agents, smoothing agents, anti-aging agents, and combinations thereof.

[0169] The cosmetic composition may be in any form suitable for topical application to the skin, in particular to the scalp. The cosmetic composition may be in the form of aqueous solutions, dispersions, oil-in-water (O / W) or water-in-oil (W / O) or multiple (triple: W / O / W or O / W / O) emulsions, nanoemulsions, in particular O / W nanoemulsions, the droplet size of which is less than 100 nm, aqueous gels, dispersions, powders, lacquers, aerosols, sprays, oils, serums, mousses, this list not being exhaustive.

[0170] Typically, the cosmetic composition is in the form of a cosmetic product. It may be a rinse-off or leave-on product.

[0171] Examples of cosmetic products include makeup products, care products, and body hygiene products. These may include, in particular, a lotion, a milk, a serum, an aqueous or oily gel, an emulsion, a cream, in particular a day cream or a night cream, a gel-cream, a treatment water, an ointment, a balm, a foundation, a spray, an eye shadow, a stick, a lipstick, a gloss, a lip balm, a mousse, a deodorant, a nourishing mask, a shower gel, a shampoo, a conditioner, a serum, an ointment and an exfoliating or scrubbing product.

[0172] In certain embodiments, the cosmetic composition is in the form of a hair product. The hair product may be a rinse-off or leave-in product. Preferably, said hair product is chosen from a shampoo including a dry or solid shampoo, a conditioner, a hair mask, a hair balm, a hair cream, a hair oil, a hair serum, a hair lacquer, a fixing gel, a hair spray, a sun spray, or a hair lotion.

[0173] Other aspects and advantages of the present invention will appear on reading the following examples, which must be considered as illustrative and in no case as limiting. Example 1: Evaluation of the Active Ingredient

[0174] Microorganisms present on the surface of the skin play an essential role in protecting against the invasion of pathogens but also in educating the immune system. Like the skin, the microbiome acts in concert with the skin to ensure a barrier function against external aggressions. Dysbiosis of the microbiota can alter its interactions with the skin. In the long term, the barrier function of the skin, but also its visual appearance and its sensitivity to external aggressions can be altered. The scalp is an extension of the skin with the same requirements in maintaining the microbiota. Indeed, dysbiosis of the scalp microbiota is associated with a sensitive scalp which can ultimately lead to hair fragility. It has been discovered that 25% of the skin microbial population is found in the scalp.Thus, we were able to demonstrate that the use of Capixyl™ as a rinse aid allows the modulation of bacteria which are involved in the development of sensitive skin and scalp. Objective

[0175] The objective of the study was to evaluate the variation of bacterial strains at the scalp, being a niche representative of the main bacteria present elsewhere on the skin, following repeated use of hair products comprising the combination according to the Invention.

[0176] The combination according to the Invention was introduced into the hair products implemented in this study in the form of the ingredient Capixyl™. Capixyl™ comprises a hydroalcoholic extract of red clover flowers and Acetyl tetrapeptide of CAS No. 827306-88-7 in a butylene glycol / water mixture as a carrier. Protocol -Panel

[0177] 23 volunteers (men, women) aged 18 to 65 showing scalp sensitive or weakened due to stress, fatigue or the season. - Formulas of rinse-off products

[0178] Shampoo Formula INCI Active Formula % Decyl glucoside 8.00 Betaine 3.50 Sodium Benzoate 0.30 Levulinic Acid (and) sodium levulinate (and) Glycerin (and) water 1.00 water 84.60 Xanthan Gum 1.10 Butylene glycol 0.00 Butylene Glycol, Water, Dextran, Acetyl Tetrapeptide-3, Hydroalcoholic extract of Trifolium Pratense flowers (Capixyl™) -Combination according to the invention 1.50

[0179] Conditioner formula INCI Active Formula % Tetrasodium EDTA 0.10 Xanthan Gum 0.30 Cetearyl Alcohol (and) Ceteareth-20 5.00 Cetearyl Alcohol 4.00 Capric / Caprylic Triglycerides 9.00 Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben 0.80 Tocopherol (and) Helianthus Annuus Seed Oil 0.20 Cocamidopropyl Betaine 3.00 Water 76.02 Butylene Glycol 0.00 Water (and) Citric Acid 0.08 Butylene Glycol, Water, Dextran, Acetyl Tetrapeptide-3, Trifolium Pratense Flower Hydroalcoholic Extract 1.50 (Capixyl™) - Combination according to the invention - Treatment

[0180] Volunteers applied the shampoo and conditioner combination every other day for 90 days. The scalp was massaged with the shampoo for at least one minute, before being rinsed thoroughly with water. Then the conditioner was applied and massaged for at least two to three minutes, before being rinsed thoroughly with water. - Collection of microbiome samples

[0181] Scalp microbiota sampling was performed on Day 0, before the application of the active rinsing formulas, and 90 days after their applications according to the established protocol. Samples were collected using the Sequential Skin kit used according to the manufacturer's instructions (Sequential Skin Ltd., UK). Briefly, a Sequential Skin adhesive patch was applied to the participant's scalp for 10 s, then placed in a preservation solution to preserve the microbial DNA, which was then extracted according to the Sequential Skin DNA extraction protocol (Sequential Skin Ltd., UK). After extraction of total microbial DNA, the purity and quality of the samples were assessed before proceeding with bacterial detection. To do this, a high-throughput qPCR was performed using the 16S RNA marker, which allows the quantification of the bacterial community by sequencing. - Evaluation of the main bacteria

[0182] Absolute abundance was calculated for the main bacteria present on the skin and scalp, including S. epidermidis, S. aureus and C. acnes, to highlight the ratios C. acnesl S. epidermidis, C. acnes / S. aureus, S. epidermidis / S. aureus and S. epidermidis!C. acnes. The percentage of families and genera of bacteria at Day 90 was obtained by normalizing the average from day 0 to 100%. - Statistical method

[0183] According to the normality test in the variance homogenization result, the paired T test or the Wilcoxon test were used to quantify the bacteria present on the scalp. The statistical significance value is p<0.05 (#p<0.1; *p<0.05, **p<0.01, ***p<0.001). Results

[0184] Studies of the absolute abundances of bacterial species whose modulation is associated with sensitive skin.

[0185] The results are illustrated in [Fig.l]. After 90 days of application of the shampoo and conditioner comprising Capixyl™, the absolute number of S. epidermidis is significantly increased, that of S. aureus tends to significantly decrease while the absolute quantity of C. acnes does not vary. The combination according to the Invention has the ability to specifically modulate the absolute quantity of bacteria associated with sensitive skin and promotes the reestablishment of a healthy microbiota in the skin.

[0186] Study of the specific bacterial ratio of sensitive skin

[0187] The S. epidermidis / C. acnes ratio is decreased in sensitive skin (Keum et al., Microorganisms, 2020 (Structures of the Skin Microbiome and Mycobiome Depending on Skin Sensitivity).

[0188] The result is illustrated in [Fig.2]. Compared to day 0, the S. epidermidis / C. acnes ratio increases significantly after 90 days of application of the combination according to the invention. The combination according to the invention makes it possible to restore the S. epidermidis / C. acnes ratio and promotes the rebalancing of the skin microbiota.

[0189] Results regarding bacterial populations associated with scalp dysbiosis

[0190] - Study of the main bacterial families of the scalp

[0191] The results are illustrated in [Fig.3].

[0192] Compared to day 0, after 90 days of application of the combination according to the invention, as expected the absolute number of Actinobacterium does not vary. On the other hand, the absolute abundance of Proteobacterium decreases significantly and that of Firmicutes increases significantly.

[0193] The application of the combination according to the invention to a sensitive scalp acts specifically on the bacterial families involved in dysbiosis. The combination according to the invention makes it possible to restore the balance of the microbiota and thus promotes the health of the scalp.

[0194] - Studies of the different types of bacteria present on the scalp.

[0195] The results are illustrated in [Fig.4].

[0196] Compared to day 0, the application of the combination according to the invention for 90 days does not significantly modify the absolute abundance of bacteria of the genus Cutibacterium. On the other hand, the overall bacterial populations of the genera Acinetobacter, Bacteriodetes, Corynebacterium, Prevotella, Rothia and Acinetobacter decrease significantly. Conversely, the absolute population of bacteria of the genus Staphylococcus increases significantly.

[0197] The combination according to the invention is capable of modulating the populations of certain bacterial genera and of allowing the reestablishment of a healthy microbiota at the level of the scalp.

[0198] Study of the main ratios of scalp bacteria

[0199] The results are illustrated in [Fig.5]

[0200] Compared to day 0, after 90 days of application of the combination according to the invention, the C. acnes / S. epidermidis ratio is and that of C. acnes / S. aureus are significantly decreased. As expected, the S. epidermidis / S. aureus ratio does not vary from significantly.

[0201] The application of the combination according to the invention to a sensitive scalp presenting dysbiosis makes it possible to restore the balance of the microbiota, thus promoting the health of the scalp.

Claims

Claims

1. Cosmetic use of a peptide of general formula (I): AX-Gly-His-Lys-Y (I) in which: X represents a chain of 1 to 3 lysine residues, optionally methylated, Y represents OH or NH2, and A represents H or an RC(O)- radical in which: - R represents a hydrocarbon chain, saturated or unsaturated, of 1 to 24 carbons, linear or branched, or RC(O)- represents the radical of lipoic acid or the reduced form thereof, the radical of di-hydrolipoic acid or the radical of retinoic acid, in combination with a red clover extract as an agent for maintaining or rebalancing the microbiota of healthy skin.

2. Use according to claim 1 in which the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb): - A-MeLys-Gly-His-Lys-Y (IIa) (A-[SEQ ID NO:4]-Y) or - A-Lys-Gly-His-Lys-Y (A-[SEQ ID NO:1]-Y (IIb) A and Y being as defined in claim 1.

3. Use according to claim 1 or 2 in which - the peptide is such that Y is NH2 and A is H or an RC(O) radical - in which R represents a saturated or unsaturated hydrocarbon chain of 1 to 6 carbon atoms, and - the extract is a hydroalcoholic extract of red clover flowers.

4. Use according to any one of the preceding claims wherein the peptide of formula (I) in combination with the red clover extract is used to maintain or rebalance the healthy scalp microbiota, in particular to promote scalp health.

5. Use according to any one of the preceding claims wherein the peptide of formula (I) in combination with the red clover extract is used to rebalance, or maintain the balance of, bacterial populations of S. epidermidis, C. acnes, and S. aureus in the microbiota of healthy skin, including the scalp.

6. Use according to any one of the preceding claims in which the peptide of formula (I) and the red clover extract are used in an individual having weakened or sensitive skin, preferably a weakened or sensitive scalp.

7. Use according to any one of the preceding claims in in which the peptide of formula (I) and the red clover extract are present as active agents in a cosmetic product.

8. Use according to claim 7 in which the cosmetic product is chosen from a makeup product, a care product or a body hygiene product.

9. Use according to claim 7 or 8 wherein the cosmetic product is a hair product selected from the group consisting of a shampoo including a dry or solid shampoo, a conditioner, a hair balm, a hair oil, a hair serum, a hair lacquer, a hair fixing gel, and a hair spray.

10. Use according to any one of claims 7 to 9 wherein the peptide of formula (I) and the red clover extract are incorporated into the cosmetic product in the form of a functional ingredient comprising (i) said peptide, (ii) said red clover extract and a cosmetically acceptable carrier.

11. Use according to claim 10 wherein the cosmetically acceptable vehicle is selected from triethyl citrate, glycerol (also called glycerin), C2-C8, preferably C2-Cs alkanediols such as propanediol, butylene glycol, pentylene glycol and isomers thereof, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate, water, and mixtures thereof.

12. Use according to claim 10 or 11 wherein the functional ingredient comprises a butylene glycol / water mixture as a cosmetically acceptable carrier, and optionally dextran.

13. Use according to any one of the preceding claims, wherein the peptide in combination with the red clover extract is used to treat, alleviate or prevent one or more non-pathological alterations caused by dysbiosis in healthy skin, preferably chosen from a decrease in the barrier function of the skin, a loss of skin suppleness, a loss of skin elasticity, a loss of skin hydration, and a loss of evenness of skin tone.

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