Combination of cosmetic surfactants for maintaining or rebalancing the skin microbiome

Abstract

This invention relates to cosmetic combinations containing red clover extract and peptides for the prevention or treatment of imbalances in the skin microbiome.

Description

Technical Field

[0001] This invention relates to the field of cosmetic active agents and their uses, particularly for preventing ecological imbalance of the skin microbiome or for maintaining the homeostasis of the skin microbiome. Background Technology

[0002] The skin is the first line of defense protecting the body from external attacks. This organ is composed of several layers of tissue. These are: (i) the epidermis, which is the outermost layer of the skin; (ii) the dermis, which is connective tissue composed of fibroblasts and extracellular matrix, performing the cohesive and nutritional functions of the skin; and (iii) the subcutaneous tissue, which is composed of fat cells.

[0003] The epidermis is composed of several layers of keratinocytes. Specifically, it is divided into: the germinal layer (called the basal layer), which specifically contains skin stem cells; the stratum spinosum, which consists of several layers of polygonal cells; the stratum granulosum, which contains one to three layers of flattened cells containing cytoplasmic inclusions (hyalinous granules); and finally, the stratum corneum, which is composed of anucleate cells rich in keratin (called corneocytes), which correspond to the late stage of keratinocyte differentiation.

[0004] The outermost layer of cells in the stratum corneum continuously sheds and is replaced by cells from the underlying layers; this process is called desquamation. Cell regeneration of the stratum corneum is based on the process of cell maturation, in which cells from the basal layer of the epidermis differentiate and gradually migrate through different layers of the epidermis until they reach the stratum corneum as keratinocytes.

[0005] In addition, the cells of the stratum corneum are connected by epidermal lipids. These lipids form a protective barrier and have water-retention capabilities. In the absence of lipids, the skin becomes dry, tight, and rough.

[0006] In addition, the epidermis is covered with an emulsion of water and lipids (fat), called the hydrolipidic film. This film is renewed through the secretion of sebaceous and sweat glands, helping to keep the skin soft and acting as an additional barrier against pathogens and irritants.

[0007] The skin is not merely an external covering; it is a true organ that provides a barrier function, protecting our bodies from a variety of external aggressors, whether microbial, chemical, or mechanical. This protective function of the skin is supported by the skin microbiome (also known as the skin flora or symbiotic flora), which consists of various types of microorganisms, including bacteria, fungi, and viruses. When pathogenic microbes attempt to invade the body through the epidermis, the skin and the skin microbiome can work together to defend against this attack: the skin microbiome prevents pathogenic microbes from attaching and can secrete bacteriocins and other toxins, while the skin's innate immune system 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 microbiome is fragile.

[0009] This balance can change over time due to internal factors such as age (e.g., hormonal regulation during puberty or menopause), but also due to stress or repeated external aggressors, such as climate (temperature, humidity, UV radiation), medication (antibiotics, corticosteroids), cosmetic care (frequent washing, peeling, etc.), clothing (synthetic fibers or cotton), or lifestyle habits (diet, alcohol, smoking). These different aggressors can alter the relative and / or quantitative abundance of various microbial communities, particularly the ratios between multiple symbiotic bacterial groups: the balance of the skin microbiome is disrupted, which ultimately affects the collaboration between the skin and the microbiome.

[0010] Imbalances in the skin microbiome can alter the skin’s visual appearance and properties, particularly its ability to perform its barrier function and make it more responsive to external aggressors.

[0011] Therefore, it has been shown that sensitive skin is associated with a significant imbalance in the skin microbiome, particularly involving an excess of Bacteroides, Actinobacterium, and Staphylococcus epidermidis populations and an underabundance of Firmicutes and Staphylococcus aureus populations (Byrd et al., 2018, Nat. Rev. Microbiol. 16, 143-155).

[0012] Therefore, an imbalance in the skin microbiome can make the skin more reactive and sensitive, leading to a disproportionate or inappropriate response to external aggressors, whether of bacterial or environmental origin.

[0013] Therefore, there is a need for new cosmetic substances that can maintain or rebalance the skin microbiome. Summary of the Invention

[0014] One object of the present invention is the cosmetic use of a combination of peptide of general formula (I) and hydro-alcoholic extract of red clover as a substance for maintaining or rebalancing the microbiome in healthy skin:

[0015] ,in:

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

[0017] Y represents OH or NH2, and

[0018] A represents H or the group RC(O)-, where

[0019] R represents a straight or branched hydrocarbon chain of 1 to 24 carbon atoms, saturated or unsaturated, or RC(O)- represents a lipoic acid group or its reduced form, a dihydrolipoic acid group or a retinoic acid group.

[0020] In a preferred embodiment, the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb):

[0021] - or

[0022] -

[0023] A and Y are as defined in claim 1.

[0024] In some implementation schemes:

[0025] - The peptide is such that Y is NH2 and A is H or the group RC(O)-, where R represents a saturated or unsaturated hydrocarbon chain of 1 to 6 carbon atoms, and

[0026] - The extract is an aqueous-alcoholic extract of red clover flowers.

[0027] In one specific implementation, the combination of the peptide of formula (I) with red clover extract is used to maintain or rebalance the scalp microbiome.

[0028] In another embodiment, the peptide of formula (I) and red clover extract are used in combination to rebalance or maintain the balance of the bacterial communities of Staphylococcus epidermidis, Propionibacterium acnes, and Staphylococcus aureus in the microbiome of healthy skin (including scalp).

[0029] The peptides and red clover extract of formula (I) can be used on any type of object, especially individuals with fragile skin (preferably fragile scalp).

[0030] Preferably, the peptide of formula (I) and red clover extract are present in the cosmetic product as active agents.

[0031] The cosmetic products mentioned are typically selected from makeup products, skincare products, or personal hygiene products.

[0032] As an example, the cosmetic product may be a hair product selected from the group consisting of: shampoos, including dry or solid shampoos; conditioners; hair balms; hair oils; hair serums; hair lacquers; hair styling gels; and hair sprays.

[0033] In some embodiments, the peptide of formula (I) and red clover extract are incorporated into a cosmetic product as functional ingredients comprising (i) the peptide, (ii) the red clover extract, and a cosmetically acceptable carrier.

[0034] Cosmetic-acceptable carriers may be selected from triethyl citrate; glycerin (also known as glycerol); C2-C8 and preferably C2-C5 alkyl diols such as propylene glycol, butanediol, pentanediol and their isomers; ketals, such as ketals of levulinate such as methyl levulinate or ethyl levulinate; water; and mixtures thereof.

[0035] As an example, the functional ingredient comprises a butylene glycol / water mixture as a cosmetically acceptable carrier, and optionally dextran.

[0036] In some embodiments, the combination of peptides with red clover extract is used to treat, alleviate, or prevent one or more non-pathological changes caused by ecological imbalances in healthy skin, preferably selected from decreased skin barrier function, reduced skin suppleness, decreased skin elasticity, decreased skin hydration, and decreased skin tone evenness.

[0037] Another object of the present invention is a cosmetic method for rebalancing the skin microbiome, such as that on the scalp, comprising applying a combination of a peptide of formula (I) as defined herein and an aqueous-alcoholic extract of red clover to a skin area to be treated. The peptide and extract may be applied to the skin in the form of a cosmetic product, particularly a hair product. Attached Figure Description

[0038] Figure 1The regulation of the Staphylococcus epidermidis, Propionibacterium acnes, and Staphylococcus aureus populations is shown before treatment of the scalp with the combination according to the invention (day 0) and after 90 days of treatment (day 90) (see Example 1).

[0039] Figure 2 The ratio of Staphylococcus epidermidis to Propionibacterium acnes is shown before treatment with the combination according to the invention (day 0) and after 90 days of treatment (day 90) (see Example 1). Figure 1 and Figure 2 The combination according to the invention demonstrates the ability to modulate bacterial communities with impaired abundance in sensitive or fragile skin. In particular, the combination according to the invention enables an increase in the ratio of Staphylococcus epidermidis to Propionibacterium acnes, a ratio that is reduced in sensitive skin.

[0040] Figure 3 The absolute total population size of bacteria of the Actinobacterium, Proteobacteria, and Firmicutes families is shown before treatment of the scalp with the combination according to the invention (day 0) and after 90 days of treatment (day 90) (see Example 1).

[0041] Figure 4 The absolute total population size of different genera of bacteria is shown before treatment of the scalp with the combination according to the invention (day 0) and 90 days after treatment (day 90) (see Example 1).

[0042] Figure 5 The ratios of Propionibacterium acnes / Staphylococcus epidermidis, Propionibacterium acnes / Staphylococcus aureus, and Staphylococcus epidermidis / Staphylococcus aureus are shown before treatment of the scalp with the combination according to the invention (day 0) and after 90 days of treatment (day 90) (see Example 1).

[0043] Figure 3 , 4 5 indicates that the combination according to the invention can regulate the abundance of symbiotic bacteria involved in sensitive or fragile scalps. Detailed Implementation

[0044] The applicant has demonstrated that when extracts and peptides are applied topically, particularly in the form of rinse-off cosmetic products, the combination of peptides of formula (I) as described below with red clover extract can maintain or rebalance the skin microbiome.

[0045] In fact, as illustrated in the examples, repeatedly applying a shampoo and revitalizing formula containing the combination according to the invention to an individual with an imbalanced microbiome enables the bacterial community to be rebalanced.

[0046] As shown in Example 1, the repeated surface application according to the combination of the present invention enables an increase in the abundance of Staphylococcus epidermidis and a decrease in the abundance of Staphylococcus aureus, while maintaining the population of Propionibacterium acnes. This regulation is reflected in a significant increase in the ratio of Staphylococcus epidermidis to Propionibacterium acnes. Figure 1 and Figure 2 However, sensitive or fragile skin is characterized by a lack of Staphylococcus epidermidis, an excess of Staphylococcus aureus, and an insufficient ratio of Staphylococcus epidermidis to Propionibacterium acnes. Therefore, the combination according to the invention can specifically modulate the populations of Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes to the characteristic values ​​of healthy skin.

[0047] Furthermore, the combination according to the invention can regulate the abundance of symbiotic bacteria involved in sensitive or fragile scalps.

[0048] Example 1 shows that the composition according to the invention enables an increase in the total population of bacteria belonging to the genus *Proteobacteria* and an increase in the total population of bacteria belonging to the genus *Firmwallis*. Figure 3 However, known characteristics of fragile or sensitive scalps include a lack of Firmicutes bacteria and an excess of Bacteroides bacteria.

[0049] Example 1 also shows that the combination according to the invention can reduce the abundance of multiple bacterial genera (Corynebacterium, Prevotella, Rothia, and Acinetobacter) and increase the total population of genus Staphylococcus, which are known to be involved in the dysbiosis of fragile or sensitive scalps.

[0050] Overall, Example 1 demonstrates that this combination enables the correction of the relative abundance of Propionibacterium acnes and Staphylococcus aureus, as well as Staphylococcus epidermidis and Staphylococcus aureus populations, which are typically impaired in fragile or sensitive scalps.

[0051] Therefore, Example 1 shows that the combination according to the invention enables the relatively and quantitative regulation of the bacterial populations involved in sensitive or fragile skin phenomena (especially on sensitive or fragile scalps) to tend toward a characteristic state of healthy skin.

[0052] The peptide of formula (I) according to the invention is described in patent EP 1 663 285 for the treatment of hair loss. The patent does not mention any effect of this peptide on the skin microbiome.

[0053] Literature indicates the various pharmacological activities of red clover extract, particularly for relieving menopausal symptoms or certain skin conditions. Some extracts of red clover are also said to possess anti-inflammatory properties.

[0054] The applicant sells a functional ingredient comprising a combination of red clover extract and a tetrapeptide, as a substance for promoting hair growth and reducing scalp inflammation (Capixyl). TM To the best of the applicant's knowledge, this functional ingredient has never been proposed to act on the skin microbiome.

[0055] To the best of the applicant's knowledge, the literature does not indicate that the following description or presence exists in Capixyl. TM The combination of the peptide of formula (I) with red clover extract can exert any effect on the skin microbiome.

[0056] Therefore, one object of the present invention is the use of the combination of the peptide of formula (I) and red clover extract as a cosmetic substance for maintaining or restoring the microbiome balance of healthy skin.

[0057] In other words, one object of the present invention is a combination of active agents (i.e., peptides of formula (I) and red clover extract as described below) for maintaining the balance of the skin microbiome or for preventing imbalances in the skin microbiome.

[0058] Another object of the present invention is the use of the combination of the peptide of formula (I) with red clover extract as a cosmetic substance for preventing imbalance (or dysbiosis) in the skin microbiome and / or for alleviating imbalance (or dysbiosis) in the skin microbiome and / or for restoring the balance of the skin microbiome in healthy skin (including healthy scalp).

[0059] Another object of the present invention comprises a combination of a peptide of formula (I) and red clover extract, which is used to prevent imbalance (or dysbiosis) in the skin microbiome and / or to alleviate or reduce imbalance (or dysbiosis) in the skin microbiome and / or to restore the balance of the skin microbiome.

[0060] The combinations according to the invention can also be used to restore or enhance the barrier properties of the skin (including the scalp).

[0061] Another object of the present invention is the use of a combination of peptide of formula (I) as described below with red clover extract as a cosmetic substance for rebalancing the skin microbiome (e.g., the skin microbiome on the scalp).

[0062] Another object of the present invention is to comprise a combination of a peptide of formula (I) as described below and a red clover extract, which is used as a substance for rebalancing the skin microbiome (e.g., the skin microbiome on the scalp).

[0063] Preferably, the combination according to the invention is used on healthy skin as defined below.

[0064] In some implementations, healthy skin is considered to be sensitive or fragile skin, such as sensitive scalp.

[0065] As described below, red clover extract can be an aqueous-alcoholic extract of red clover, particularly an aqueous-alcoholic extract of red clover flowers and stems.

[0066] The combinations according to the invention can also be used to prevent or treat changes in the mechanical or visual properties of the skin induced by imbalances in the skin microbiome.

[0067] The combination according to the invention is preferably applied to the surface, typically in the form of a cosmetic composition.

[0068] The combination of the peptide of formula (I) with red clover extract can also be used to improve or maintain the appearance of skin appendages, especially hair.

[0069] Preferably, the combination according to the invention is used on healthy skin as defined below.

[0070] In some implementations, healthy skin is considered to be sensitive or fragile skin, such as sensitive scalp.

[0071] In one specific aspect, another object of the present invention is the use of the combination of the peptide of formula (I) with red clover extract as a cosmetic substance for rebalancing the microbiome of fragile skin (including fragile scalp).

[0072] In another aspect, the present invention relates to a cosmetic method for maintaining or restoring the balance of the skin microbiome in healthy skin, or for preventing imbalance (or dysbiosis) in the skin microbiome and / or for alleviating imbalance (or dysbiosis) in the skin microbiome and / or for restoring the balance of the skin microbiome in healthy skin (including the scalp), the cosmetic method comprising applying a combination of a peptide of formula (I) with red clover extract to a skin area to be treated.

[0073] As an example, one object of the present invention is a cosmetic method for rebalancing the skin microbiome (e.g., the skin microbiome on the scalp), the cosmetic method comprising applying a combination of a peptide of formula (I) and red clover extract to a skin area to be treated.

[0074] In another aspect, the present invention relates to the use of a combination of a peptide of formula (I) as described herein and red clover extract in the preparation of cosmetic products for preventing (or dysbiosis) of the skin microbiome and / or for alleviating (or dysbiosis) of the skin microbiome and / or for restoring the balance of the skin microbiome, particularly the scalp microbiome.

[0075] The present invention also relates to the use of a combination of a peptide of formula (I) as described herein and red clover extract in the preparation of cosmetic products for rebalancing the skin microbiome, particularly the scalp microbiome.

[0076] Within the meaning of this invention, the terms "skin" and "of skin" refer to any part of human skin, particularly facial skin, including the lips and eyelids, but also including the scalp. Preferably, the scalp is involved.

[0077] In the context of this invention, the combination of peptides and extracts according to the invention is typically applied topically to healthy skin, that is, to skin without wounds or skin pathologies (e.g., scalp).

[0078] In other words, the skin area (e.g., scalp) to which the combination according to the invention is applied is healthy, and the subject does not suffer from any skin condition, at least in the area to which it is applied.

[0079] Therefore, when the combination according to the invention is applied to healthy skin, it does not produce a therapeutic effect on the skin.

[0080] The terms "skin microbiota," "skin microbiome," or "skin flora" refer to all bacteria, fungi, and viruses present on the skin surface and on the sebaceous and sweat gland accessory glands. This term specifically includes resident flora and transient flora. The skin microbiota specifically includes bacteria belonging to Actinobacteria (e.g., Corynebacteria), Firmicutes (e.g., Clostridium), Proteobacteria (e.g., Pseudomonas), and Bacteriodetes, as well as fungi, mites (Demodex), bacteriophages, and viruses; this list is not restrictive. For example, Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes can be listed as symbiotic bacterial species.

[0081] The term "prevention of imbalance in the skin microbiome" refers to preventing the occurrence of imbalance in the skin microbiome, or delaying or postponing its occurrence.

[0082] The term "imbalance in the skin microbiome" refers to a quantitative change (i.e., a change in absolute abundance) of one or more microbial communities (preferably bacterial communities) relative to the baseline characteristics of healthy skin, and / or a change in one or more ratios between microbial communities (preferably bacterial communities). The term "change" includes an increase or decrease in a parameter (absolute abundance or ratio) compared to the same parameter in the baseline state.

[0083] Imbalances in the skin microbiome can impair the skin's barrier properties. They can also damage the skin's visual or mechanical properties. Non-pathological changes in skin ecology include, but are not limited to:

[0084] Decreased skin barrier function

[0085] Reduced skin flexibility

[0086] Reduced skin elasticity

[0087] Low skin hydration

[0088] Reduced skin tone evenness

[0089] Increased redness, especially after exposure to external stimuli.

[0090] Within the meaning of this invention, the expression "maintaining the balance of the skin microbiome" refers to maintaining or protecting the normal balance of the skin flora, that is, qualitatively and / or quantitatively maintaining and protecting multiple symbiotic communities. Maintaining the balance of the skin microbiome also includes maintaining the ratio between two microbial communities (preferably bacterial communities).

[0091] The term also includes preventative measures against non-pathological imbalances in the microbiome caused by factors such as age or physiological hormonal changes (menopause, puberty) or by stimulation or external aggression.

[0092] Irritation and external aggression include, but are not limited to, cosmetic care (such as frequent washing, exfoliation, scrubbing, hair removal), chemical treatments (such as exposing the skin to abrasives such as detergents, or to chlorinated water or seawater), and exposure to climatic conditions (high or low temperatures, humidity, dryness, wind, UV) or climate change (such as seasonal changes).

[0093] Imbalances in the skin microbiome can also be induced by certain lifestyle habits, such as an overly rich (especially high-fat) diet, drinking or smoking, a strenuous lifestyle (working at night, jet lag, lack of sleep), and a stressful work or personal environment.

[0094] These irritants and / or lifestyle habits can also make the skin (including the scalp) more vulnerable. This is then referred to as "fragile skin".

[0095] In some embodiments, the combination according to the invention is used for objects with sensitive or fragile skin.

[0096] The term "sensitive or fragile skin" refers to skin characterized by an increased (non-pathological) responsiveness to external factors, which can lead to skin discomfort and impaired visual appearance of the skin and its appendages. This increased responsiveness can be caused by a non-pathological decrease in the skin's barrier function to external stimuli. Sensitive skin can be exacerbated by certain non-pathological intrinsic causes (such as hormonal changes during menopause or pregnancy) or exogenous factors (such as irritation, lack of sleep and poor diet, climatic conditions (wind, cold, solar radiation) or pollution (nanoparticles, ozone)).

[0097] In one specific embodiment, the combination of active agents according to the invention enables the enhancement, restoration, or maintenance of the abundance of Staphylococcus epidermidis in the skin.

[0098] In one specific embodiment, the combination of active agents according to the invention enables the reduction, restoration, or maintenance of the abundance of Staphylococcus aureus in the skin.

[0099] In some embodiments, the combination of active agents according to the invention enables the maintenance or restoration of the ratio of Staphylococcus epidermidis to Propionibacterium acnes in the skin. In particular, the combination of active agents according to the invention enables the improvement of the ratio of Staphylococcus epidermidis to Propionibacterium acnes in sensitive or fragile skin, especially sensitive or fragile scalp.

[0100] In one specific embodiment, the combination of active agents according to the invention enables the enhancement, restoration, or maintenance of the overall abundance of Staphylococcus bacteria on the skin, particularly on the scalp, preferably on sensitive or fragile scalps.

[0101] In one specific embodiment, the combination of active agents according to the invention enables the reduction or restoration of the overall abundance of bacteria belonging to genera selected from Acinetobacter, Bacteriodetes, Corynebacterium, Prevotella, and Rothia, and combinations thereof, on the skin, particularly on the scalp, preferably on sensitive or fragile scalps.

[0102] In another specific embodiment, the combination of active agents according to the invention enables the reduction or restoration of the overall abundance of bacteria belonging to Proteobacteria on the skin, particularly on the scalp, preferably on sensitive or fragile scalps, and the enhancement or restoration of the overall abundance of bacteria belonging to Firmicutes on the skin, particularly on the scalp, preferably on sensitive or fragile scalps.

[0103] In one specific embodiment, the combination of active agents according to the invention enables the maintenance or restoration of the ratio of *Propionibacterium acnes* to *Staphylococcus epidermidis* and / or *Propionibacterium acnes* to *Staphylococcus aureus* populations in the skin. In particular, the combination of active agents according to the invention enables the reduction of the ratio of *Propionibacterium acnes* to *Staphylococcus epidermidis* and / or the ratio of *Propionibacterium acnes* to *Staphylococcus aureus* on sensitive or fragile skin, preferably on sensitive or fragile scalp.

[0104] In some embodiments, the combination of active agents according to the invention enables the maintenance or restoration of the ratio of Propionibacterium acnes to Staphylococcus epidermidis in the skin microbiota of healthy skin, and / or the ratio of Propionibacterium acnes to Staphylococcus aureus, and / or the ratio of Staphylococcus epidermidis to Staphylococcus aureus.

[0105] In some embodiments, the combination of active agents according to the invention is used to prevent or treat non-pathological damage to the mechanical or visual properties of the skin caused by an imbalance in the microbiome. Such damage includes, but is not limited to, reduced skin barrier function, reduced skin suppleness, reduced skin elasticity, lower skin hydration, reduced skin tone evenness, and increased redness, especially after exposure to external stimuli.

[0106] peptides according to the present invention

[0107] The peptide according to the present invention is a peptide of formula (I).

[0108]

[0109] in:

[0110] X represents a chain of 1 to 3 lysine residues, which may optionally be methylated.

[0111] Y represents OH or NH2, and

[0112] A represents H or the group RC(O)-, where

[0113] R represents a straight-chain or branched, saturated or unsaturated hydrocarbon chain with 1 to 24 carbon atoms or 1 to 18 carbon atoms, optionally substituted with one or more hydroxyl groups, or

[0114] RC(O)- represents a lipoic acid group or its reduced form, a dihydrolipoic acid group, or a retinoic acid group.

[0115] In some embodiments, R is a hydrocarbon chain containing 1 to 6 degrees of unsaturation, preferably 1 to 3 double bonds.

[0116] "A is H" means that the NH2 group of the N-terminal amino acid is not modified.

[0117] "A is RC(O)-" means that the NH2 group of the N-terminal amino acid has been changed to RC(O)-NH-.

[0118] "Y is OH" means that the C-terminal group is -COOH, and therefore lysine is not modified at the C-terminus.

[0119] "Y is NH2" means that the C-terminal group is -CONH2, and therefore the C-terminal -COOH group is replaced with -CONH2 (amide functional group).

[0120] Such compounds are described in patent EP 1 663 285.

[0121] In one specific embodiment, the peptide of formula (I) is selected from peptides of sequence A-[SEQ ID NO: X]-Y, wherein SEQ ID NO: X is selected 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 the sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

[0122] In a preferred embodiment, the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb):

[0123] - (or A-[SEQ ID NO: 4]-Y) or

[0124] - (or A-[SEQ ID NO: 1]-Y)

[0125] A and Y are defined as in equation (I).

[0126] MeLys refers to methyl-lysine, which is lysine whose amine functional group on its side chain contains a methyl substituent.

[0127] In one specific embodiment, the peptide of formula (I), (IIa), or (IIb) is such that Y is NH2 and A is H or the group RC(O)-, wherein R represents a saturated or unsaturated hydrocarbon chain of 1 to 10 carbon atoms (preferably 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 containing 1 to 6 carbon atoms, preferably a straight chain.

[0128] In a preferred embodiment, RC(O)- corresponds to acetyl CH3C(O)-.

[0129] The amino acids present in peptides of formula (I), (IIa) or (IIb) may independently be of D or L configuration, preferably L.

[0130] In a preferred embodiment, the peptide according to the invention is acetyl tetrapeptide-3 or a pharmaceutically acceptable salt thereof having CAS number: 827306-88-7.

[0131] The peptides of formula (I), (IIa) or (IIb) may be in free or salt form, preferably cosmetically acceptable.

[0132] It can be an acid addition salt or a base addition salt.

[0133] Acid addition salts can be formed by reacting with, for example, inorganic acids (or "mineral acids") or with organic acids, such as hydrochloric acid, hydrobromic acid, iodic acid, sulfuric acid, aminosulfonic acid, bisulfuric acid, phosphoric acid, or nitric acid, and such organic acids as formic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, or adipic acid, and this list is not exhaustive.

[0134] In other embodiments, the compounds of the present invention are in the form of base addition salts. Bases that can be used to obtain such salts are, for example, alkali metal hydroxides, particularly 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-methylglucosamine, to name a few.

[0135] Suitable salts include, in particular, hydrochloride, acetate, citrate, potassium salts, or sodium salts.

[0136] As an example, when Y is NH2, the peptide can be in the form of acetate.

[0137] The peptides according to the invention can be prepared by any method known to those skilled in the art, such as peptide synthesis on a solid support using techniques known to those skilled in the art.

[0138] Red clover extract according to the present invention

[0139] The red clover (Trifolium pratense) extract according to the present invention is generally obtained from the above-ground parts of the plant, namely the stems, leaves, flowering tops and / or flowers, preferably the flowering tops and / or stems.

[0140] In some embodiments, red clover extract is prepared from the top of red clover. Preferably, red clover extract is prepared from the stems and flowers of red clover.

[0141] The extract is prepared by extraction using an organic solvent optionally mixed in water.

[0142] Preferably, the red clover extract is a water-alcohol extract.

[0143] Within the meaning of this invention, a water-alcohol extract refers to an extract whose preparation process includes a water-alcohol extraction step, that is, an extract made using a water / alcohol mixture.

[0144] Within the meaning of this invention, the term "alcohol" includes lower alcohols, particularly C2-C5 alcohols, glycerol, C2-C5 alkanediols, and mixtures thereof.

[0145] As an example, the alcohol may be selected from glycerol, ethanol, propanol, butanol, its isomers (e.g., isopropanol, tert-butanol), pentanol, ethylene glycol, 1,3-propanediol, and mixtures thereof. Preferred solvents are ethanol, isopropanol, and glycerol, and mixtures thereof.

[0146] The volume ratio of alcohol to water in a water-alcohol mixture is typically from 0.01 to 20.0, for example from about 0.5 to about 10.

[0147] Typically, red clover plant material is ground and impregnated in a water-alcohol mixture. This impregnation step can be carried out at room temperature or in the presence of heat. In some embodiments, the impregnation step is carried out under stirring or in the presence of ultrasound. At the end of the impregnation step, the liquid phase is recovered and subsequently concentrated.

[0148] In some implementations, the concentrated extract may undergo additional processing steps, such as chromatographic steps.

[0149] At the end of the extraction process, the extract is usually freeze-dried into powder form.

[0150] As an example, red clover extract is an aqueous-alcoholic extract prepared from the stems and flowers of red clover.

[0151] In a preferred embodiment, the red clover extract is rich in flavonoids, particularly chickpea sprout A, meaning it contains at least 5% by weight of flavonoids.

[0152] As an example, red clover extract is a water-alcohol extract prepared from red clover stems and flowers with a flavonoid content of at least 5% by weight.

[0153] Examples of the purpose combination according to the present invention

[0154] In some embodiments, the combination according to the invention is characterized by:

[0155] - The peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb), wherein preferably, Y is NH2 and A is an acetyl group, such as acetyl tetrapeptide-3 having CAS number: 827306-88-7 or a cosmetically acceptable salt thereof, and

[0156] Red clover extract is a water-alcohol extract, preferably an extract of the stems and flowers.

[0157] Such a combination is particularly suitable as a substance for cosmetic purposes to rebalance the skin microbiome of the scalp (especially on healthy, sensitive, or fragile scalps).

[0158] Target

[0159] The combination of the peptide of formula (I) and red clover extract as described herein can be surface-applied to any type of human subject. The human subject is preferably at least 15 years old. It can be male or female. In some embodiments, the subject is a male or female who is at least 35 years old, preferably at least 40 or 45 years old.

[0160] In one specific implementation, the object has mature skin.

[0161] In some implementations, the target group is premenopausal (e.g., 45 to 55 years old) or postmenopausal women.

[0162] In some implementations, the subject is at risk of skin dysbiosis. The risk of dysbiosis may be related to the subject's exposure to external stimuli and / or living conditions (e.g., as described above) and / or physiological hormonal changes (puberty, menopause, aging).

[0163] In another implementation, the subject has sensitive or fragile skin, particularly a fragile or sensitive scalp.

[0164] Using the combined method according to the invention

[0165] The red clover extract and peptides according to the present invention are surface-applied, i.e., applied to the skin. They can be applied directly to the skin or pre-incorporated into cosmetic products. Preferably, the clover extract and peptides according to the present invention are applied through one or more cosmetic products.

[0166] The red clover extract and peptides according to the present invention are used in combination, i.e., they are applied to the skin sequentially, sequentially or simultaneously.

[0167] Preferably, red clover extract and the peptide of formula (I) are applied to the skin simultaneously. Therefore, they are often contained in the same cosmetic product applied to the skin. Cosmetic products suitable for application to the skin, particularly the scalp, are described below.

[0168] The mass ratio of red clover extract to the peptide of formula (I) is typically from 0.1 to 10.0, preferably from 0.1 to 5.0.

[0169] The combination of the red clover extract and the peptide of formula (I) according to the invention generally accounts for 0.0001% to 10% by weight, preferably 0.0001% to 5% by weight, in cosmetic products.

[0170] In the cosmetic methods and cosmetic uses according to the invention, the dosage and frequency of application of the extract according to the invention vary depending on the desired cosmetic effect and individual characteristics (especially gender, age and skin type).

[0171] Typically, the extract can be applied to the skin (including the scalp) at least once every two weeks, at least once a week, or even two to three times a week, or even daily.

[0172] The combination or cosmetic products according to the invention can be applied to the skin (including the scalp) without rinsing, or can be applied to the skin (including the scalp) and rinsed off after a few seconds or even minutes.

[0173] As an example, the combination of the peptide of formula (I) according to the invention and red clover extract can be applied to the scalp and rinsed at least once a week in the form of shampoo or conditioner or any other hair product. The hair product is left on for several seconds (e.g., at least 30 seconds) or even several minutes (e.g., 2 to 5 minutes) before rinsing it off. This treatment can be performed at least once a week, for example, at least two or three times a week.

[0174] As another example, the combination according to the invention can be applied to the scalp in the form of oil, serum or hair lotion and not rinsed off.

[0175] The processing and use according to the present invention can last for weeks or even months.

[0176] Cosmetic ingredients according to the present invention

[0177] In some embodiments, the peptides and red clover extract according to the invention are preformed together as functional ingredients before being incorporated into a cosmetic composition or cosmetic product.

[0178] Therefore, in some embodiments, the combination of peptides and 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 carrier.

[0179] The carrier can be of any kind: it can be a solid support such as maltodextrin or a cosmetically acceptable solvent. For example, the peptides and red clover extracts according to the invention can be suspensions, or even diluted in solvents selected from: triethyl citrate, glycerol (also known as glycerol), C2-C8 and preferably C2-C5 alkyl diols (e.g., propylene glycol, butanediol, pentanediol and their isomers), ketals (e.g., ketals of levulinate, such as methyl levulinate or ethyl levulinate or glyceryl levulinate ketals), water, and mixtures thereof.

[0180] Preferably, the carrier is a mixture of water and alkanediol (e.g., water and C2-C5 alkanediol), such as a mixture of water and butanediol.

[0181] In some embodiments, the functional ingredient may include one or more additional cosmetically acceptable excipients. These additional excipients may be selected from thickeners, antioxidants, preservatives, viscosity agents, surfactants, vectorizing agents, buffers, and combinations thereof.

[0182] In one embodiment, the additional excipient is a viscosity agent such as dextran.

[0183] As an 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.

[0184] An example of a suitable functional ingredient is, for instance, Capixyl, a product sold by the applicant. TM .

[0185] The functional ingredients are preferably diluted in a suitable carrier or integrated into the cosmetic product. The functional ingredients according to the invention typically comprise 0.01% to 10% by weight, preferably 0.1% to 5% by weight, of the cosmetic product.

[0186] Cosmetic compositions or products according to the present invention

[0187] The combination of red clover extract and peptide of formula (I) according to the present invention is typically incorporated into cosmetic compositions to be applied to the skin.

[0188] The cosmetic compositions according to the invention typically contain 0.0001% to 10% by weight, preferably 0.0001% to 5% by weight of the composition according to the invention.

[0189] As described above, the combination according to the invention is incorporated into the cosmetic composition in the form of the functional ingredients described above. The functional ingredients according to the invention typically comprise 0.1% to 10% by weight, preferably 0.5% to 5% by weight, of the cosmetic composition.

[0190] The cosmetic composition may also contain one or more cosmetically acceptable excipients, typically from 50% to 99.99% by weight, preferably from 60% to 99.90% by weight, referring to the total weight of the cosmetic composition.

[0191] Cosmetic-acceptable excipients present in cosmetic compositions according to the invention may be selected from carriers, such as aqueous solutions, demineralized water, mineral water, thermal water, or floral water; solvents, particularly lower alcohols, especially ethanol, isopropanol, dipropylene glycol, butylene glycol, propylene glycol, pentanediol, and propylene glycol; dispersants; gelling agents or thickeners; emollients; carriers such as phospholipids; preservatives; odor absorbers; pH adjusters or neutralizers; detergents or surfactants, particularly anionic, cationic, amphoteric, or nonionic surfactants; chelating agents; foam stabilizers; foaming agents; humectants such as glycerin or sorbitol; wetting agents; dispersants; fragrances; pigments and other colorants; pearlescent agents; opacifiers; softeners; oily agents such as plant-derived oils or fats, such as essential oils, silicone oils, and fatty alcohol esters; fixatives or plasticizers; and / or other substances commonly used in formulations in the cosmetic or pharmaceutical fields.

[0192] The cosmetic compositions according to the invention may optionally contain one or more cosmetic active agents, typically from 0.0001% to 10.0% by weight. These may include anti-dandruff agents, UV screening agents, soothing agents, sebum regulators, moisturizers, smoothing agents, anti-aging agents, and combinations thereof.

[0193] Cosmetic compositions can be in any form suitable for surface application to the skin, particularly the scalp. Cosmetic compositions can be in the form of aqueous solutions, dispersions, oil-in-water (O / W) emulsions, water-in-oil (W / O) emulsions, or multiple emulsions (triple: W / O / W or O / W / O), nanoemulsions (especially O / W nanoemulsions with droplet sizes less than 100 nm), aqueous gels, dispersions, powders, lacquer agents, aerosols, sprays, oils, serums, or foams; this list is not exhaustive.

[0194] Typically, cosmetic compositions are in the form of cosmetic products. They can be rinse-off or leave-on products.

[0195] Examples of cosmetic products include makeup products, skincare products, and personal hygiene products. Specifically, these can be lotions, lotions, serums, water-based or oil-based gels, emulsions, creams (especially day or night creams), cream-gel products, care water, ointments, balms, foundations, sprays, eyeshadows, sticks, lipsticks, lip glosses, lip balms, foaming agents, deodorants, moisturizing masks, shower gels, shampoos, conditioners, serums, ointments, and exfoliating or scrubbing products.

[0196] In some embodiments, the cosmetic composition is in the form of a hair care product. The hair care product may be a rinse-out or leave-on product. Preferably, the hair care product is selected from shampoos (including dry or solid shampoos), conditioners, hair masks, hair creams, hair lotions, hair serums, hair styling agents, setting gels, hair sprays, sunscreen sprays, or hair lotions.

[0197] Other aspects and advantages of the invention will become apparent from the following embodiments, which should be considered illustrative rather than limiting.

[0198] Example 1: Evaluation of active ingredients

[0199] The microbes present on the skin's surface play a crucial role in defending against pathogen invasion and in immune system education. Like the skin, the microbiome works synergistically with the skin to provide a barrier against external aggressors. Dysbiosis of the microbiome can alter its interaction with the skin. In the long term, the skin's barrier function, its visual appearance, and its sensitivity to external aggressors can all be impaired. The scalp, being an extension of the skin, has the same requirements in maintaining its microbiome. In fact, dysbiosis of the scalp microbiome is associated with sensitive scalps, which can ultimately lead to brittle hair. It has been found that 25% of the skin microbiome resides on the scalp. Therefore, it can be argued that the use of Capixyl... TM As a rinse-off product, it can regulate the bacteria involved in the development of sensitive skin and scalp.

[0200] Purpose

[0201] The aim of this study is to evaluate changes in bacterial strains on the scalp (a representative ecological niche for the major bacteria present on other parts of the skin) after repeated use of hair products containing the combination according to the invention.

[0202] The combination according to the present invention will be based on the ingredient Capixyl TM Capixyl was incorporated into the hair products used in this study. TM It contains an aqueous-alcoholic extract of red clover flowers in a butylene glycol / water mixture (as a carrier) and an acetyl tetrapeptide with CAS number 827306-88-7.

[0203] plan

[0204] - Group

[0205] 23 volunteers (male and female) aged 18 to 65 years who exhibited sensitive or fragile scalps due to irritation, fatigue, or seasonal factors.

[0206] - Rinse-off product formulation

[0207] Shampoo formulation

[0208]

[0209] Revitalizing agents

[0210]

[0211] - deal with

[0212] Volunteers applied the shampoo and revitalizing formula combination every other day for 90 days. They massaged the scalp with the shampoo for at least one minute, then rinsed thoroughly with water. They then applied the revitalizing formula and massaged it in for at least two to three minutes, then rinsed thoroughly with water.

[0213] - Collection of microbiome samples

[0214] Following established protocols, scalp microbiota samples were collected on day 0 (before application of the active rinse agent) and 90 days after application. Samples were collected using the Sequential Skin kit (Sequential Skin Ltd., UK), used according to the manufacturer's instructions. In short, a Sequential Skin adhesive patch was applied to the participant's scalp for 10 seconds and then placed in a preservation solution to preserve microbial DNA, which was subsequently extracted according to the Sequential Skin (SequentialSkin Ltd., UK) DNA extraction protocol. After total microbial DNA extraction, the purity and quality of the samples were evaluated prior to bacterial assays. For this purpose, high-throughput qPCR was performed using a 16S RNA marker, which enabled quantification of the bacterial community via sequencing.

[0215] - Evaluation of major bacteria

[0216] The absolute abundance of major bacteria present on the skin and scalp (specifically Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes) was calculated to highlight the ratios of Propionibacterium acnes / Staphylococcus epidermidis, Propionibacterium acnes / Staphylococcus aureus, Staphylococcus epidermidis / Staphylococcus aureus, and Staphylococcus epidermidis / Propionibacterium acnes). The percentages of bacterial families and genera on day 90 were obtained by normalizing the mean from day 0 to 100%.

[0217] - Statistical methods

[0218] Based on the normality test in the homogeneity of variance results, paired t-tests or Wilcoxon tests were used to quantify the bacteria present on the scalp. Statistical significance was p < 0.05 (#p < 0.1). p < 0.05, p < 0.01, p < 0.001).

[0219] result

[0220] Research on its regulation of the absolute abundance of bacterial species associated with sensitive skin

[0221] The result is Figure 1 As shown in the diagram. When applying Capixyl... TM After 90 days, the shampoo and revitalizing formula showed a significant increase in the absolute number of Staphylococcus epidermidis, a significant decreasing trend in the absolute number of Staphylococcus aureus, while the absolute number of Propionibacterium acnes remained unchanged. The combination according to the invention has the ability to specifically regulate the absolute number of bacteria associated with sensitive skin and promote the restoration of a healthy skin microbiome.

[0222] Research on the specific bacterial ratio of sensitive skin

[0223] In sensitive skin, the ratio of Staphylococcus epidermidis / Propionibacterium acnes is reduced (Keum et al., Microorganisms, 2020 (Structures of the Skin Microbiome and Mycobiome Depending on Skin Sensitivity)).

[0224] The result is Figure 2 As shown in the figure, compared to day 0, after 90 days of application of the combination according to the invention, the Staphylococcus epidermidis / Propionibacterium acnes ratio was significantly increased. The combination according to the invention enables the restoration of the Staphylococcus epidermidis / Propionibacterium acnes ratio and promotes the rebalancing of the skin microbiota.

[0225] Results regarding bacterial communities associated with scalp dysbiosis

[0226] - Research on the main bacterial families of the scalp

[0227] The result is Figure 3 As shown in the image.

[0228] Compared to day 0, after 90 days of applying the combination according to the invention, as expected, the absolute number of Actinobacterium remained unchanged. On the other hand, the absolute abundance of Proteobacterium decreased significantly, and the absolute abundance of Firmicutes increased significantly.

[0229] Applying the combination according to the invention to a sensitive scalp has a specific effect on bacterial families involved in dysbiosis. The combination according to the invention enables the restoration of the microbiome balance and thus promotes scalp health.

[0230] - Research on the various genera of bacteria present on the scalp

[0231] The result is Figure 4 As shown in the image.

[0232] Compared to day 0, applying the combination according to the invention for 90 days did not significantly alter the absolute abundance of *Propionibacterium* bacteria. On the other hand, the overall bacterial populations of *Acinetobacter*, *Bacteroides*, *Corynebacterium*, *Prevotella*, *Rochetomyces*, and *Acinetobacter* were significantly reduced. Conversely, the absolute population of *Staphylococcus* bacteria was significantly increased.

[0233] The combination according to the invention can modulate the population of certain bacterial genera and enable the restoration of a healthy microbiome on the scalp.

[0234] - A study on the main ratios of bacteria on the scalp

[0235] The result is Figure 5 As shown in the image.

[0236] Compared to day 0, after 90 days of application of the combination according to the invention, the ratios of Propionibacterium acnes / Staphylococcus epidermidis and Propionibacterium acnes / Staphylococcus aureus were significantly reduced. As expected, the ratio of Staphylococcus epidermidis / Staphylococcus aureus did not change significantly.

[0237] Applying the combination according to the invention to a sensitive scalp exhibiting ecological imbalance enables the restoration of the microbiome balance, thereby promoting scalp health.

Claims

1. The cosmetic use of a combination of peptide of general formula (I) and hydro-alcoholic extract of red clover as a substance for maintaining or rebalancing the microbiome in healthy skin: ,in: X represents a chain of 1 to 3 lysine residues, which may optionally be methylated. Y represents OH or NH2, and A represents H or the group RC(O)-, where: - R represents a straight or branched chain of 1 to 24 carbon atoms, a saturated or unsaturated hydrocarbon chain, or RC(O)- represents a lipoic acid group or its reduced form, a dihydrolipoic acid group, or a retinoic acid group.

2. The use according to claim 1, wherein the peptide of formula (I) is a tetrapeptide of formula (IIa) or (IIb): - (A-[SEQ ID NO:4]-Y) or - (A-[SEQ ID NO:1]-Y)(IIb) A and Y are as defined in claim 1.

3. The use according to claim 1 or 2, wherein: - The peptide is such that Y is NH2 and A is H or the group RC(O)-, where R represents a saturated or unsaturated hydrocarbon chain of 1 to 6 carbon atoms, and - The extract is an aqueous-alcoholic extract of the flowers and stems of red clover.

4. The use according to any one of the preceding claims, wherein the combination of the peptide of formula (I) with the red clover extract is used to rebalance the scalp microbiome.

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

6. The use according to any one of the preceding claims, wherein the peptide of formula (I) and the red clover extract are used on an individual with fragile or sensitive skin, preferably a fragile or sensitive scalp.

7. The use according to any one of the preceding claims, wherein the peptide of formula (I) and the red clover extract are present in the cosmetic product as active agents.

8. The use according to claim 7, wherein the cosmetic product is selected from makeup products, care products or personal hygiene products.

9. The use according to claim 7 or 8, wherein the cosmetic product is selected from hair products including: shampoos, including dry or solid shampoos; conditioners; hair creams; hair oils; hair serums; hair styling agents; hair setting gels; and hair sprays.

10. The 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 as a functional ingredient, the functional ingredient comprising (i) the peptide, (ii) the red clover extract and a cosmetically acceptable carrier.

11. The use according to claim 10, wherein the cosmetically acceptable carrier is selected from triethyl citrate; glycerin (also known as glycerol); C2-C8 and preferably C2-C5 alkyl diols such as propylene glycol, butanediol, pentanediol and their isomers; ketals, such as ketals of levulinate such as methyl levulinate or ethyl levulinate; water; and mixtures thereof.

12. The 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. The use according to any one of the preceding claims, wherein the combination of the peptide and the red clover extract is used to treat, alleviate or prevent one or more non-pathological changes caused by ecological imbalance in healthy skin, wherein the non-pathological changes are preferably selected from decreased skin barrier function, decreased skin suppleness, decreased skin elasticity, decreased skin hydration and decreased skin tone evenness.

14. A cosmetic method for rebalancing the skin microbiome, such as the skin microbiome on the scalp, the cosmetic method comprising applying a combination of a peptide of formula (I) as defined in any one of claims 1 to 3 and an aqueous-alcoholic extract of red clover to a skin area to be treated.

15. The cosmetic method according to claim 14, wherein the peptide of formula (I) and the aqueous-alcoholic extract of red clover are applied to the skin in the form of a cosmetic product.

16. The cosmetic method according to claim 14 or 15, wherein the peptide of formula (I) and the aqueous-alcoholic extract of red clover are applied to the scalp in the form of a hair product.

17. The beauty method of claim 16, wherein the hair care product is selected from shampoos, including dry or solid shampoos; conditioners; hair creams; hair oils; hair serums; hair styling agents; hair setting gels; and hair sprays.

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