Hair care activator

Abstract

The present invention provides a hair care active agent comprising taxifolin glucoside and N-acetyltyrosine, and a hair care composition comprising the hair care active agent. The hair care agent can also reduce the occurrence of gray and white hair in an individual.

Description

[Technical Field]

[0001] This invention relates to a hair care activator comprising taxifolin glucoside and N-acetyltyrosine, a hair care composition comprising the same, and the use and application thereof. [Background technology]

[0002] The graying of hair, or loss of pigment in the hair shaft leading to white hair, is one of the most obvious signs of aging. It is also one of the major cosmetic concerns that arise with age. In fact, between the ages of 45 and 65, approximately 74% of the world's population is affected by gray hair. The loss of pigment typically appears gradually between the ages of 35 and 45. Although it affects both men and women, men appear to exhibit a greater proportion of gray hair. Hair graying differs by geographical and ethnic origin, being less common in Asian and African populations than in Caucasians.

[0003] Human hair is composed of three distinct layers: the cuticle (outer shell), the cortex (inner layer containing melanin particles, which is responsible for hair color), and the medulla (a soft core present only in mature white hair). Human hair color is determined by its structure and the type and percentage of melanin it contains.

[0004] Melanin production in hair is controlled by melanocytes located in the hair follicle matrix. Melanocyte activity is regulated by the normal hair cycle: during the growth phase, melanin produced by active melanocytes is transferred to cortical keratinocytes, resulting in pigmentation throughout the hair shaft. During the regression phase, melanocytes enter apoptosis, and they disappear during the resting phase. To produce pigmented hair during the new growth phase, a new pool of melanocytes migrates and differentiates from the hair follicle stem cell reservoir, which includes, among other things, outer root sheath cells (ORSc), into the hair follicle, naturally pigmenting the new hair.

[0005] Hair graying is explained by age-related functional changes in the stimulation and migration of stem cells from ORSc, but also by environmental factors. Indeed, the accumulation of reactive oxygen species (ROS) in melanocytes during aging leads to the accumulation of mutations, decreased antioxidant protection, inflammation, hair loss, and graying. ROS produced in the scalp reduces the differentiation of melanoblasts into melanocytes, resulting in a deficiency of mature melanocytes in the globular region. ROS also reduces melanin production at the globular level, leading to a decrease in the transfer of melanin pigment to cortical keratinocytes. When loss of pigmentation begins, three types of hair can be identified: colored hair (high melanin content), gray hair (low melanocyte count and dilute melanin content), and white hair (complete deficiency of melanocytes).

[0006] In an aging society, anti-aging products that alleviate the signs of aging are in increasing demand. For this reason, many people choose to color their gray or white hair. However, chemical hair dyes are often aggressive and damage the hair. Moreover, users frequently complain about a lack of tolerability (itching, redness, pain) and unsustainability (the need for regular recoloring).

[0007] To overcome these difficulties, it is desirable to find ways to positively influence the natural pigmentation process of hair. To this end, US 2012 / 0114583 describes a hair treatment comprising (a) dihydroquercetin and / or a dihydroquercetin derivative; and (b) at least one amino acid. A preferred embodiment comprises a mixture of six amino acids: taurine, proline, valine, arginine, lysine, and glycine.

[0008] Dihydroquercetin (DHQ), or taxifolin, is a flavonoid found in certain coniferous trees. It is an antioxidant and a stimulant of stem cell proliferation and maintenance. Taxifolin has been used in cosmetics to date for a variety of purposes, including skin whitening (e.g., US 2011 / 0038968) and stimulating hyperpigmentation. Furthermore, taxifolin is known to inhibit melanin production (Sang Mi An et al. Phytother. Res. 22, 1200-1207 (2008)).

[0009] The object of the present invention is to provide a highly efficient hair care activator for reducing the occurrence of gray and white hair in individuals and for restoring the color of natural hair. This objective is achieved by the hair care activator of the present invention and the hair care composition containing the same. [Overview of the Initiative]

[0010] In a first aspect, the present invention provides a hair care activator comprising taxifolin glucoside and N-acetyltyrosine. In a second aspect, the present invention provides a hair care composition comprising the hair care activator and a suitable carrier of the present invention.

[0011] In a third aspect, the present invention refers to the non-therapeutic use of the hair care activators of the present invention for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, re-pigmenting hair, and / or reducing the proportion and / or density of gray or white hair.

[0012] In a fourth aspect, the present invention provides a method for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, re-pigmenting hair, and / or reducing the proportion and / or density of white or gray hair, the method comprising the step of topically applying the hair care activator of the present invention to human hair.

[0013] The combination of taxifolin glucoside and N-acetyltyrosine provides optimal anti-aging activity for hair by stimulating melanin synthesis and thereby enhancing the natural coloration of hair. Within four months, the hair care activator of the present invention was found to significantly reduce the percentage and density of gray and white hair (up to 50% less gray hair). The mode of action is completely independent of gender, hair type, or hair color, and it ideally addresses graying of hair, which is its universal target.

[0014] The effects of the hair care activator and hair care composition of the present invention have been studied in several in vitro and in vivo studies (see examples below). The hair care active agent of the present invention has been found to stimulate the proliferation of stem cells, stimulate the production of active melanocytes in hair follicles, activate the protective action of antioxidants in hair follicles, protect melanocytes against oxidative stress, reactivate melanin production in gray hair, reduce the density of gray hair / white hair, and cause repigmentation of hair. Therefore, it is specifically useful as an anti-aging agent for hair, and an anti-gray hair agent and an anti-white hair agent.

[0015] Although the stimulation of melanin production by taxifolin and / or taxifolin derivatives in combination with at least one amino acid has been previously described (US 2012 / 0114583), the hair care active agent of the present invention has been found to be even more effective. Surprisingly, while the hair care active agent of the present invention can significantly stimulate melanin synthesis in melanocytes, taxifolin glucoside alone has no effect, and a mixture of taxifolin glucoside and L-tyrosine has been found to cause only a slight and non-significant increase (see Example 7 below). Furthermore, the hair care active agent of the present invention also exhibited improved solubility.

[0016] In a preferred embodiment, at least a part of the taxifolin glucoside is taxifolin alpha-D-glucoside. A suitable method for preparing taxifolin alpha-D-glucoside is described, for example, in WO 2007 / 144368. In the hair care active agent of the present invention, taxifolin glucoside and N-acetyltyrosine may be used alone or in combination with other active ingredients, adjuvants and / or solvents suitable for cosmetic use.

[0017] In a preferred embodiment, the hair care active agent of the present invention further contains taxifolin. Surprisingly, it has been found that the solubility of taxifolin is improved in the presence of taxifolin glucoside: the solubility of taxifolin in water is typically about 1 g / L, but can be increased to 20 g / L or more in the presence of a suitable amount of taxifolin glucoside. As the solubility of taxifolin in water increases, the concentration of taxifolin glucoside in the aqueous solution also increases, typically having a molar ratio of about 1:1. This enables the preparation of a more concentrated and thus more active formulation. Furthermore, due to the presence of taxifolin, the initial activity of the hair care active agent is also increased.

[0018] The mixture of taxifolin and taxifolin glucoside can be prepared, for example, by stopping the reaction according to WO 2007 / 144368 after approximately half of the taxifolin has been converted, as an example, prior to complete conversion, and a roughly 1:1 mixture may be obtained. Taxifolin glucoside and taxifolin may be present in a weight ratio of 100:0 to about 40:60. Preferably, the weight ratio of taxifolin glucoside to taxifolin is 90:10 to 40:60, more preferably 70:30 to 50:50, and most preferably about 60:40.

[0019] In a preferred embodiment, the hair care active agent of the present invention further contains glycine and / or epigallocatechin gallate and / or epigallocatechin gallate glucoside. Glycine is the simplest amino acid possible. Glycine is typically used as a buffering agent in cosmetics. Furthermore, glycine is a collagen precursor. Epigallocatechin gallate (EGCG, also known as epigallocatechin-3-gallate) is an ester of epigallocatechin and gallic acid, and is a type of catechin. Epigallocatechin gallate is known to stimulate hair growth. A preferred method for preparing epigallocatechin gallate alpha-D-glucoside is described, by example, in WO 2007 / 144368. The hair care activator of the present invention may further contain other beneficial activators, such as zinc chloride. Zinc is an enzyme cofactor that is beneficial for hair growth.

[0020] In the hair care activator of the present invention, taxifolin glucoside and N-acetyltyrosine may be used in any suitable concentration range and ratio. In particular, it is desirable to adapt them to a concentration range that ensures sufficient activity and solubility. The concentration of taxifolin glucoside in the hair care activator of the present invention may, as an example, be 0.01 to 0.50 wt%, more preferably 0.05 to 0.25 wt%, and most preferably 0.7 to 0.15 wt%, for example, about 0.10 wt%.

[0021] The concentration of N-acetyltyrosine in the hair care activator of the present invention may, as an example, be 1.0 to 30 wt%, more preferably 10 to 20 wt%, and most preferably 13.5 to 16.5 wt%, for example, about 15.0 wt%. In the hair care activator of the present invention, the weight ratio of taxifolin glucoside to N-acetyltyrosine is preferably 1:5'100 to 1:3.4, more preferably 1:340 to 1:70, and most preferably 1:200 to 1:100, for example, about 1:150.

[0022] Furthermore, all other active ingredients, adjuvants, and / or solvents may be used in the hair care activators of the present invention within any suitable concentration range and ratio. In particular, it is desirable to adapt the concentration range to ensure sufficient activity, solubility, stability, and ease of formulation. As an example, glycine may be used at concentrations of 0.01 to 0.50 wt%, more preferably 0.10 to 0.30 wt%, and most preferably 0.12 to 0.18 wt%, for example, about 0.15 wt%.

[0023] As an example, epigallocatechin glucoside gallate can be used at concentrations of 0.001 to 0.60 wt%, more preferably 0.010 to 0.060 wt%, and most preferably 0.015 to 0.045 wt%, for example, about 0.03 wt%. As an example, sodium metabisulfite can be used at concentrations of 0.01 to 1.00 wt%, more preferably 0.10 to 0.750 wt%, and most preferably 0.45 to 0.55 wt%, for example, about 0.50 wt%. Sodium metabisulfite prevents the oxidation of polyphenols.

[0024] In a preferred embodiment, the hair care activator of the present invention comprises 13.5 to 16.5 wt% N-acetyltyrosine, 0.12 to 0.18 wt% glycine, 0.05 to 0.09 wt% zinc chloride, 0.08 to 0.12 wt% taxifolin glucoside, 0.5 to 0.08 wt% taxifolin, 0.015 to 0.045 wt% epigallocatechin glucoside gallate, 0.45 to 0.55 wt% sodium metabisulfite, and 47.5 to 52.5 wt% glycerol.

[0025] In a specifically preferred embodiment, the hair care activator of the present invention comprises about 15.0 wt% N-acetyltyrosine, about 0.15 wt% glycine, about 0.07 wt% zinc chloride, about 0.10 wt% taxifolin glucoside, about 0.07 wt% taxifolin, about 0.03 wt% epigallocatechin glucoside gallate, about 0.50 wt% sodium metabisulfite, and about 50.0 wt% glycerol.

[0026] In the hair care activator of the present invention, a portion of the taxifolin glucoside may be replaced with taxifolin. In particular, up to 60%, preferably up to 50%, and more preferably up to 40%, of the taxifolin glucoside may be replaced with taxifolin.

[0027] In a preferred embodiment, the hair care activator of the present invention comprises 13.5 to 16.5 wt% N-acetyltyrosine, 0.12 to 0.18 wt% glycine, 0.05 to 0.09 wt% zinc chloride, 0.14 to 0.20 wt% a 60:40 mixture of taxifolin glucoside and taxifolin, 0.015 to 0.045 wt% epigallocatechin glucoside gallate, 0.45 to 0.55 wt% sodium metabisulfite, and 47.5 to 52.5 wt% glycerol.

[0028] In a specifically preferred embodiment, the hair care activator of the present invention comprises about 15.0 wt% N-acetyltyrosine, about 0.15 wt% glycine, about 0.07 wt% zinc chloride, about 0.17 wt% a 60:40 mixture of taxifolin glucoside and taxifolin, about 0.03 wt% epigallocatechin glucoside gallate, about 0.50 wt% sodium metabisulfite, and about 50.0 wt% glycerol.

[0029] Alternatively, taxifolin may be added to the hair care activator of the present invention in addition to the indicated amount of taxifolin glucoside. In this case, taxifolin may be added in an amount of up to 0.24 wt%, more preferably 0.07 to 0.20 wt%, and most preferably 0.10 to 0.15 wt%. Preferably, the weight ratio of taxifolin to taxifolin glucoside is 1.5:1 or less.

[0030] In a further aspect, the present invention also provides a hair care composition comprising the hair care activators and suitable carriers described above. When used herein, "hair" means human hair, including scalp hair, facial hair, and body hair, specifically including eyelashes, beards, and mustaches, and refers to the hair on the head and scalp of a human being. The term "hair care composition" encompasses both leave-on products such as shampoos, sprays, and lotions, as well as rinse-off products.

[0031] Examples include hair cleansing compositions, hair conditioning compositions, and hair styling compositions such as shampoos, conditioners, sprays, treatments, masks, toners, pre-shampoos, lotions, serums, creams, foams, mousses, and gels. Specific examples, though not limited to these, include anti-gray hair lotions, anti-gray hair shampoos, natural re-pigmentation hair masks, anti-gray hair agents for beards and mustaches, hair color restoration sprays, and prematurely matured gray hair / gray hair treatment gels. Many of these known compositions are water-based formulations.

[0032] Hair cleansing compositions are generally effective in removing dirt (soil) from hair. This dirt includes natural exudates from the scalp, environmental substances, and styling products. The dirt coats or accumulates on the hair and scalp. Hair coated with such dirt typically looks greasy, feels heavy, may have a bad odor, and generally cannot maintain the desired style. Known cleansing compositions typically include a combination of water and a surfactant such as soap or a synthetic surfactant, and may also include a non-aqueous blend of starch. The combination of water and surfactant emulsifies the dirt from the hair and scalp, allowing it to be washed away.

[0033] The cleansing composition may also contain conditioning agents that adhere to the hair and scalp while being rinsed with water. Such conditioning agents may include polymers, oils, waxes, protein hydrolysates, silicones, and mixtures and derivatives thereof. In addition, the conditioning composition may be a separate and distinct product from the cleansing composition.

[0034] Conditioning compositions known in the art are typically water-based formulations. However, conditioning compositions are also known that include at least one of silicones; animal oils, mineral oils, or vegetable oils; waxes; petrolatum; and fats and oils. Water-based conditioning compositions typically include substituted cationic waxes, fatty alcohols, cationic polymers, hydrolyzed proteins and their derivatives, and fragrances. Such conditioning formulations impart combability and manageability to the treated hair, thereby minimizing breakage during the styling process and resulting in shiny, healthy, and manageable hair. Conditioning compositions may also be effective in moisturizing the hair. The subsequent drying and styling process may include air drying or heating.

[0035] A suitable carrier must be cosmetically acceptable. "Cosmetically acceptable" means a carrier suitable for use in contact with human keratin tissue, which, when used herein, is free from excessive toxicity, incompatibility, instability, allergic reactions, etc. All compositions described herein that are intended to be applied directly to keratin tissue are limited to those that are cosmetically acceptable.

[0036] Hair care compositions typically contain a carrier at a level of about 20 wt% to about 99 wt%. The carrier may include water, organic solvents (miscible or immiscible with water), silicone solvents, and / or mixtures thereof. The solvent should be dermatologically acceptable. The carrier usually does not contain more than about 2 wt% of non-volatile solvents, as significantly higher concentrations increase hair weigh-down and greasy feel. Water, organic, and silicone solvents with boiling points below 250°C are considered volatile solvents. Suitable carriers include aqueous solutions of water, lower alkyl alcohols such as monohydric alcohols with 1 to 6 carbon atoms (e.g., ethanol and / or isopropanol), and polyhydric alcohols such as glycols, glycerin, and other diols.

[0037] The hair care composition according to the present invention may further contain one or more materials selected from the group consisting of solvents, surfactants, thickeners, styling polymers, anti-dandruff activators, antimicrobial materials, skin and scalp activators, vitamins, salts, buffers, hair growth agents, conditioning materials, hair fixing polymers, fragrances, colorants / colorants, dyes, pigments, opacifiers, pearlescent additives, oils, waxes, preservatives, sensates, sunscreens, drugs, defoamers, antioxidants, binders, biological additives, buffering agents, fillers, chelating agents, chemical additives, film-forming agents or materials, pH adjusters, propellants, oxidizing agents, and reducing agents.

[0038] All additives should be physically and chemically compatible with the essential components of the hair care composition and should not excessively impair its stability, aesthetics, or performance if they are not. Most importantly, they should also be cosmetically acceptable. As a thickening agent, the hair care composition may contain rheological modifiers to improve the feel, usability, and suspension stability. For example, the rheological properties may be adjusted so that the hair care composition remains unchanged during its storage and transport, and so that it does not unnecessarily drip onto the body, clothing, or other areas of furniture during use. Any suitable rheological modifier may be used. Typically, this includes about 0.01 to about 3 wt% of the thickening agent. Examples of suitable thickening agents are disclosed in WO 2015 / 03516 and US 2001 / 0043912.

[0039] The hair care composition of the present invention may also contain any suitable optional ingredients as desired. Such optional ingredients should be physically and chemically compatible with the components of the hair care composition and should not excessively impair stability, aesthetics, or performance if they are not. The CTFA Cosmetic Ingredient Handbook, Tenth Edition (published by the Cosmetic, Toiletry, and Fragrance Association, Inc., Washington DC) (2004) describes a wide variety of non-limiting materials that may be added to the hair care composition of the present invention.

[0040] As an example, the hair care composition of the present invention may include a styling polymer. The styling polymer may be selected from the group consisting of acrylate polymers and their esters, methacrylate polymers and their esters, acrylate copolymers and their esters, methacrylate copolymers and their esters, polyurethane polymers and copolymers, polyvinylpyrrolidone (PVP), PVT-polyvinyl acetate copolymer, PVP-polyvinyl alcohol copolymer, polyester, and other polymers.

[0041] The hair care composition of the present invention may contain a sensory stimulant. As used herein, the term “sensory stimulant” refers to a substance that, when applied to the skin, causes a change in state, a perceived sensation such as, but not limited to, heating, cooling, or refreshing. The sensory stimulant is preferably used at a level of about 0.001 to about 10 wt% in the consumer product. Examples of suitable sensory stimulants include camphor, menthol, L-isoplegol, ethylmenthanecarboxamide, and trimethylisopropylbutanamide.

[0042] The hair care compositions of the present invention may also contain any components that modify the physical and performance characteristics. Such components include surfactants, salts, buffers, thickeners, solvents, emulsions, pearlescent enhancers, preservatives, fragrances, colorants, dyes, pigments, chelating agents, sunscreens, vitamins, and pharmaceuticals. Any components included herein that are useful are disclosed in US 4,387,090.

[0043] The hair care composition of the present invention may also contain an anti-dandruff agent to provide antimicrobial activity. The anti-dandruff agent may be particulate or soluble. Preferred anti-dandruff agents include, but are not limited to, particulate crystalline anti-dandruff agents such as heavy metal salts of sulfur, selenium sulfide, and pyridinethione. Zinc pyridinethione is particularly preferred. Possible anti-dandruff agents such as ketoconazole are also known in the art. The anti-dandruff agent is preferably present at a concentration of about 0.1 to 4 wt%.

[0044] The hair care compositions of the present invention may also optionally contain hair growth agents such as zinc pyridinethione. The compositions and consumer products of the present invention may also optionally contain compounds useful for regulating hair growth and loss. Such compounds known in the art include lupin triterpenes and their derivatives, oleanan triterpenes and ursan triterpenes, and their salts and mixtures, minoxidil (6-(1-piperidinyl)-2,4-pyrimidinediamine 3-oxide), or finasteride.

[0045] The hair care composition of the present invention may also optionally contain salts and / or buffers to modify the rheology. For example, salts such as potassium chloride and sodium chloride may be added at levels of about 0.001 to about 1 wt%. Buffers such as citrate buffer or phosphate buffer may also be used. Preferably, the pH of the consumer product is modified to a pH of about 3 to about 10, preferably about 3 to about 7.

[0046] The hair care composition of the present invention may also optionally contain additional conditioning polymers, particularly cationic conditioning polymers. If present, these are preferably used at a level of about 0.5 to about 10 wt%. Suitable cationic conditioning polymers are disclosed in US 2001 / 0043912.

[0047] A wide variety of other additional components may be incorporated into this hair care composition. These include: other conditioning agents such as hydrolyzed collagen, vitamin E, panthenol, panthenyl ethyl ether, hydrolyzed keratin, proteins, plant extracts, and nutrients; hair fixing polymers such as amphoteric, nonionic, cationic, and anionic fixing polymers, and silicone graft copolymers; preservatives such as benzyl alcohol, methylparaben, propylparaben, and imidazolidinyl urea; pH adjusters such as glutamic acid, citric acid, sodium citrate, succinic acid, phosphoric acid, lactic acid, sodium hydroxide, and sodium carbonate; common salts such as potassium acetate and sodium chloride; colorants; hair oxidizing (bleaching) agents such as hydrogen peroxide, pervolate, and persulfates; hair reducing agents such as thioglycolate; fragrances; chelating agents such as ethylenediaminetetraacetate disodium; ultraviolet and infrared blocking and absorbing agents such as octyl salicylate; and mixtures thereof.

[0048] Additional optional ingredients may include, but are not limited to, skin and scalp surfactants, oils, waxes, defoamers, antioxidants, binders, biological additives, fillers, chelating agents, chemical additives, film-forming agents or materials, and propellants.

[0049] In a further aspect, the present invention relates to the non-therapeutic use of the hair care activator of the present invention for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, re-pigmenting hair, and / or reducing the proportion and / or density of gray or white hair.

[0050] In a further aspect, the present invention relates to a method for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, and reducing the re-pigmentation of hair and / or the proportion and / or density of gray or white hair, wherein the method comprises the step of topically applying a hair care activator according to the present invention.

[0051] The hair care activator of the present invention may be applied to either wet or dry hair, depending on the formulation. Preferably, the hair care activator of the present invention is applied in the form of the hair care composition of the present invention described above. The beneficial effects of the hair care activator of the present invention have been confirmed by numerous in vitro, ex vivo, and clinical studies, some of which are described in the examples below. This patent application includes the following figures: [Brief explanation of the drawing]

[0052] [Figure 1] Melanin content produced by co-culture of normal human melanocytes (NHM) and normal human epidermal keratinocytes (NHEK) after 72-hour incubation in the absence or presence of 0.01% of the test solution from Example 1 in 1:1 NHM / NHK medium. Student's t-test: **p-value < 0.01 and *p-value < 0.05. [Figure 2] Evaluation of melanin content in microsectional hair follicles of Gray treated with a 1% standard solution from Example 1 via Fontana-Masson staining. [Figure 3] Evaluation of melanin content in microsectional hair follicles of Gray treated with a 1% standard solution from Example 1 via Fontana-Masson staining. Student's t-test *p-value < 0.05.

[0053] [Figure 4]Gene expression analysis after induction of oxidative stress in the entire scalp in ex vivo. ANOVA using Fisher's LSD test: ***p<0.01, *p<0.05, and #p<0.1. [Figure 5] Evaluation of ROS production after 1 hour of pretreatment with a standard solution from Example 1, followed by 1 hour using 50 μM cumene hydroperoxide. One-way ANOVA with rearrangement test, F(2,33)31.33; *p-value < 0.05 and **p-value < 0.01, followed by a rearrangement t-test. [Figure 6] Representative photographs of ROS production analysis in microsectional hair follicles after induction with 50 μM cumene hydroperoxide, with or without treatment using a 1% standard solution from Example 1.

[0054] [Figure 7] Percentage of NKI / beteb-positive cells after treatment with oxidative stress followed by treatment with a 1% standard solution from Example 1. One-way ANOVA with rearrangement test, F(2,33) 7.91;**p-value<0.01, followed by t-test with rearrangement. [Figure 8] Representative photographs of NKI / beteb (red) immunostaining representing HF melanocytes and melanoblasts, as well as DAPI counterstaining (blue). The different conditions presented are microsections of hair follicles after induction with 50 μM cummenperoxide, with or without treatment using a standard solution from 1% Example 1.

[0055] [Figure 9] A. Scoring of the percentage of gray hair (T4M in %) B. Representative image of a hair lotion containing 1% of the active ingredient of the present invention for reducing the percentage of gray hair [Figure 10] A. Scoring of gray hair density (T4M) B. Representative photographs showing reduction in gray hair density [Figure 11] Melanin content in % of untreated conditions

[0056] Therefore, the present invention can be further described by the following non-limiting examples: Example 1: Hair care activator according to the present invention Standard solution The following standard solutions may be used to prepare the hair care activator of the present invention: [Table 1]

[0057] The hair care activator of the present invention may consist of the standard solution or may include the standard solution in combination with other components. Therefore, the standard solution can be used as is. Alternatively, the standard solution may also be diluted in a solvent, buffer, or culture medium.

[0058] Test solution For some of the examples below (see the notation in the examples), the test solutions were prepared by diluting the above standard solutions in William Medium E to a final concentration of 0.01–1% v / v. INCI The hair care composition containing the hair care activator of the present invention may be described by the following INCI (International Nomenclature for Cosmetic Ingredients) designation: Water, Glycerin / Acetyl Tyrosine / Sodium Metabisulfite / Larix Europaea Wood Extract / Glycine / Zinc Chloride / Epigallocatechin Glucoside Gallate, Sodium Benzoate, Citric Acid, Sodium Citrate, PPG-26 Buteth-26, PEG-40 Hydrogenated Castor Oil, Aqua / Water, Fragrance, Butylphenyl, Methylpropional, D-Limonene, Alpha-Isomethyl Ionone.

[0059] Example 2: Evaluation of melanin production: in vitro co-culture model Design of cultures and experiments The cells used in this study were primary cultures of normal human keratinocytes (NHK) and normal human melanocytes (NHM) extracted after skin surgery (foreskin) from an 8-year-old Caucasian male donor (phenotype III / IV). Well 200,000 NHK / 9.6cm 2 and 50,000 NHM / 9.6cm 2 The cells were inoculated and grown for 24 hours in supplement medium (1:1 NHM supplement medium / NHK supplement medium). After 24 hours of culture, the cells were treated three times for 24 hours each time (conditions: untreated; 0.01% test solution from Example 1 in a 1:1 mixture of untreated NHM supplement medium and NHK supplement medium; and 1 μM rapamycin).

[0060] Melanin colorimetric assay In parallel, a protein assay was performed to determine the melanin content in the cell pellet. The cell pellet was placed in NaOH at 60°C for 30 minutes. The concentration of melanin in the supernatant and the standard range of melanin synthesis were determined by lecture at 405 nm, using synthetic melanin as a control.

[0061] Protein assay (BCA) Total protein assays in the cell pellet were performed in parallel using a bicinchoninic acid-based colorimetric method. A standard range was prepared using BSA (bovine serum albumin). The cell pellet was immersed in NaOH at 60°C for 30 minutes. Dosage was recognized by adding a reagent mixture (bicinconinic acid + CuSO4) to an aliquot of the lysate (lysed cell pellet). The plate was incubated at 37°C for 30 minutes, followed by lecture at 570 nm.

[0062] Manifestation of Results For both melanin and protein doses, the raw data, i.e., OD measurements obtained for each standard, were plotted on a graph to obtain standard rigid curves. The measured amount / concentration of protein or melanin in the sample was then determined. The quantitative values ​​for each condition were averaged. The data were plotted as volume / concentration (μg / mL). The results obtained for each condition were also expressed relative to an untreated control set to 100%. %≪試料≫ =(Average OD ≪試料≫ / Average OD ≪対照≫ ) × 100 To obtain the melanin concentration (μg / mL / mg of protein) relative to the total protein amount, each concentration value (μg / mL) was divided by the protein data (mg) for each well. Then, the values ​​for each condition were averaged. The statistical significance of the results was determined by a Student's t-test.

[0063] result 72 hours after treatment, we found that a positive reference (1 μM rapamycin) significantly stimulated melanin production in the co-culture model (+282%). * The model was confirmed to be robust and responsive (Figure 1). It was also demonstrated that the presence of a 0.01% test solution from Example 1 stimulated an effect on melanin production. In fact, the amount of melanin increased by 363%. ** It increased.

[0064] Example 3: Evaluation of melanin production: hair follicles of ex vivo gray Tissue sample Microscopically segmented hair follicles (HF) were obtained from healthy human hair follicle unit skin from the occipital region of a healthy female donor (35 years old; Donor 1) who underwent surgical hair transplantation after informed consent and ethical approval, or from scalp biopsies from a male donor (53 years old; Donor 2) (University of Muenster, n.2015-602-fS).

[0065] Hair follicle organ culture Microsections of growing human VI scalp HF (60 and 26 HF / experiment, respectively) were cultured at 37°C with 5% CO2 in a minimal amount of William's medium E supplemented with 2 mM L-glutamine (Gibco), 10 ng / mL hydrocortisone (Sigma-Aldrich), 10 μg / mL insulin (Sigma-Aldrich), and 1% penicillin / streptomycin mix (Gibco) to prepare William's complete medium (WCM; J Cell Sci. 1990 Nov; 97(Pt 3):463-71. Human hair growth in vitro. Philpott MP1, Green MR, Kearey T; Exp Dermatol. 2010 Mar; 19(3):305-12).

[0066] Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Kloepper JE1, Sugawara K, Al-Nuaimi Y, Gapar E, van Beek N, Paus R; Exp Dermatol. 2015 Dec;24(12):903-11. Human hair follicle organ culture: theory, application and perspectives. Langan EA, Philpott MP, Kloepper JE, Paus R). After 24 hours, the culture medium was replaced with fresh medium containing either WCM (vehicle) or a 1% final concentration of the test solution diluted in WCM. 11 "Gray" (defined by weak pigmentation) growth-stage VI HF was cultured for each experimental group. HF was cultured for a total of 3 days.

[0067] Freezing hair follicle processing Frozen samples were divided using a cryostat (CM3050S, Leica Bio Systems), and 6 μm sections were collected. The HF was carefully oriented to obtain intact hair follicle sections and open dermal papillae. Serial sections of hair follicles were collected, and the slides were stored at -80°C.

[0068] Masson-Fontana histochemical staining To evaluate melanin as a marker for HF pigmentation, Masson-Fontana (MF) staining was performed on frozen slides as previously described (Exp Dermatol. 2010 Mar;19(3):305-12. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Kloepper JE1, Sugawara K, Al-Nuaimi Y, Gaspar E, van Beek N, Paus R). In short, melanin was stained with an ammonia-based solution of silver nitride (Caesar & Loretz, Hilden, Germany) and fixed with 5% aqueous sodium thiosulfate (Merck Millipore, Darmstadt, Germany) (developed).

[0069] Quantitative (immunological) tissue morphometry The images were captured using Keyence Biozero Microscope 8100 and 9000 at the original magnification of 200x. Melanin content in VI hair follicles during the growth phase Three areas of 100×175 or 113×159 pixels on Auber's line were measured using ImageJ software (Rasband, WS, imageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / ) to assess the intensity of pigmentation in donor 1 or 2 during the growing HF stage.

[0070] Data Management All data were expressed as mean ± mean or magnification change of SEM. Gaussian distribution was tested using the Shapiro-Wilk normality test. Outlier analysis was performed using a two-tailed Grubbs test on pooled data for donors 1 and 2. Significant outliers were removed, and statistical analysis was then performed using the Student's t-test, comparing the results of each test group to the vehicle using GraphPad Prism 6 (GraphPad software). A p-value of <0.05 was considered statistically significant (*).

[0071] result The induction of melanin production was evaluated via Fontanamasson staining and quantification. As can be seen from Figures 2 and 3, the test solution from Example 1 was able to induce melanin production in gray hair follicles, stimulating melanin production by 15% (p<0.05). These results demonstrate that the hair care activator of the present invention can stimulate melanin production not only in melanocytes but also in gray hair follicles.

[0072] Example 4: Transcriptome analysis of the entire scalp of ex vivo subjects Culture of skin explants The tests were performed on NativeSkin®, a skin explant that is a full-thickness skin biopsy encapsulated in a solid and nutrient-rich matrix, with its dermal surface exposed to air. The skin biopsy is tightly encapsulated in a matrix that prevents diffusion of any aspect of the locally applied formulation.

[0073] The study was conducted on lift explants from three donors, each possessing a sufficient number of hair follicles and comparable numbers among samples from the same donor: - Donor 1: A 66-year-old Caucasian woman with 18 hair follicles for untreated hair, 19 hair follicles for oxidation treatment, and 19 hair follicles for oxidation treatment using a test solution from Example 1 in 1% Carbopol®. - Donor 2: A 58-year-old Caucasian woman with 10 hair follicles for untreated hair, 10 hair follicles for oxidation treatment, and 9 hair follicles for oxidation treatment using a test solution from Example 1 in 1% Carbopol®. - Donor 3: A 64-year-old Caucasian woman with 30 hair follicles for untreated hair, 30 hair follicles for oxidation treatment, and 31 hair follicles for oxidation treatment using a test solution from Example 1 in 1% Carbopol®. Carbopol® is composed of acrylic acid crosslinked with allyl clucrose or allyl pentaerythritol.

[0074] Product evaluation and research design The study was conducted on three donors (one explant per condition). Oxidative stress was applied to the skin surface by adding 9 mg of hypoxanthine + 10 units of xanthine oxidase once daily for 3 days, during a 1-hour reaction period. - It produced radicals. - Explant + oxidative stress + placebo (Carbopol®) 48 hours - Explant + oxidative stress + 1% test solution from Example 1 for 48 hours After processing, the dermis of each explant was removed by microsurgery, and the focus was on quantifying the genes of the epidermal cells.

[0075] Method of Analysis We used qPCR microfluidic technology following the Fluidigm® protocol (BMC Genomics. 2011 Mar 9;12:144. Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. Jang JS1, Simon VA, Feddersen RM, Rakhshan F, Schultz DA, Zschunke MA, Lingle WL, Kolbert CP, Jen J). Microfluidic technology arises from combining the world of nanotechnology with gene analysis using q-PCR. System miniaturization has led to the development of a chip that currently makes it possible to analyze 48 conditions for 48 genes.

[0076] The target genes encompass the following functions: - Hair Physiology - Antioxidant activity - Pigmentation Skin explants were collected in a specific lysis solution for mRNA extraction. The lysates were transferred to plates for mRNA purification. A reverse transcription system was then used. Specific steps for 48×48 chip preparation were initiated according to the Fluidigm® protocol. A pre-amplification step was performed using the primers used in the chip. The pre-amplified cDNA / PCR mix and primers were deposited onto the chip. The mixtures were blended using an IFC controller for real-time PCR, and the chips were then placed in a BioMark® system.

[0077] To confirm the activating or inhibitory effects, the values ​​were compared with control explants treated with Carbopol® alone. The results were expressed as relative expression rates.

[0078] result The effects of oxidative stress were investigated at the transcriptome level. To mimic the oxidative stress experienced by hair follicles during human lifespan, the scalp was repeatedly stimulated with a mixture of xanthine and hypoxanthine. Subsequently, the test solution was applied as a therapeutic treatment for 48 hours. The results presented in Figure 4 show the differences in relative expression compared to the oxidative stress control.

[0079] It was found that the test solution from Example 1 induced positive responses to various biological functions important for the preservation of melanin content. Indeed, the test solution from Example 1 stimulated the expression of proteins involved in melanosome biosynthesis and transport, such as AP3B1 (+37.8%#), CTNS (+130.4% *** ), HPS5 (+105.14 ** ), KRT5 (+58.7% * ), and MYO5A (+55.07% ** ). It was also found that the test solution from Example 1 stimulated the expression of major genes involved in the regulation of melanin synthesis, such as EDN1 (+165.56% *** ), MC1R (+72.64%#), MITF (+80.51% * ), and POMC (+130.51% ** ).

[0080] Furthermore, the test solution from Example 1 had an impact on the expression of genes involved in regeneration and autophagy, such as FST (150.95% ** ), KRT19 (+119.28% ** ), and MAP1LC3A (+73.47%#). Finally, the test solution from Example 1 induced the expression of genes that participate at various levels in the antioxidant response, such as HMOX1 (+110.47% ** ), GLRX (+37.47% * ), GSS (+39.70% ** ), MGST1 (+118.63% ** ), and NRF2 (+30.41%#).

[0081] Example 5: Evaluation of the effects of oxidative stress on microscopically segmented hair follicles. Microscopic sectioning and hair shaft elongation Human hair follicles (HF) in the growth phase VI were excised from the scalp (donor: female, born in 1962). HFs were selected after 18 hours of pre-culture. The following parameters were evaluated during the selection procedure: growth rate (>0.18 mm / 18 hours), and morphology (no signs of nutrient deficiency). After selection on day 0, the process began on day 1. The test compound was dissolved in the test medium and cultured until the HF (High Frequency) endpoint was set. Twelve hair follicles were used for each parameter to be read. The culture medium was replaced every other day.

[0082] ROS evaluation After selection, the HFs were incubated for 1 hour with a standard solution from Example 1 dissolved in William Medium E. Subsequently, the HFs were cultured for 30 minutes in the presence of dichlorofluorescein diacetate (DCFH-DA), a probe that reacts with ROS to produce fluorescence. Following the DCFH-DA incubation, the HFs were washed in PBS and incubated with cumene hydroperoxide at 50 μM (oxidative stimulation) for 1 hour. At the end of the experimental stage, the HFs were collected, cryo-fixed, and sectioned using a cryomicrotome for image acquisition of the results and image analysis of fluorescence within the sections. Slides for each HF were processed by image acquisition and related analysis (i.e., 12 images for each treatment).

[0083] NKI / Beteb rating Total melanocyte quantification was obtained by NKI / beteb-DAPI double immunostaining of the following cryo-sections. The NKI / beteb antibody (Monosan #MON7006-1) recognizes (pre)melanosome antigens (i.e., both active melanocytes and melanoblasts) present in all melanocytes. Image analysis was performed on immunohistochemical sections to quantify the number of NKI-beteb-positive cells in each hair follicle. The obtained values ​​were normalized by comparing them to the total number of cells in the area considered.

[0084] Image and statistical analysis Image analysis was performed using ImageJ software (NIH, USA). All quantitative data were summarized in terms of mean score, mean standard deviation, and standard error for each processing step. Differences between groups were evaluated using a one-way ANOVA with a rearrangement test, followed by Tukey's test and a t-test using rearrangement.

[0085] Results: Limitation of oxidative stress in microsectional hair follicles using standard solution from Example 1 Treatment with 50 μM cumene hydroperoxide induced a significant increase in ROS production due to oxidative stress (a 256% increase compared to the untreated condition). ** Pretreatment with the standard solution from Example 1 significantly reduced ROS production by 53%. Therefore, the reduction was significant compared to cumene hydroperoxide, along with a p<0.01 indicating the inhibitory effect of the activator (Figure 5). Representative photographs are shown in Figure 6.

[0086] Results: Effects of oxidative stress on melanocytes in microsectional hair follicles We confirmed that cumene hydroperoxide significantly reduced the number of melanocytes by 78% compared to the untreated condition. When treated with the standard solution from Example 1, oxidative stress induced a slight decrease in melanocytes, but the presence of the activator mitigated this harmful effect. Indeed, compared to the cumene hydroperoxide control, the percentage of NKI / beteb-positive cells increased by 189% with the standard solution from Example 1. ** This increased level was confirmed to have a significant impact on hair follicle melanocytes and melanoblast protection (Figure 7). Representative photographs are shown in Figure 8.

[0087] Example 6: Clinical Survey introduction To demonstrate the in vivo efficacy of the lotion containing the hair care activator of the present invention compared to a placebo lotion, two clinical evaluation methods were performed: - Reduction of the percentage of gray hair using a scoring method - Reduction of the percentage of gray hair using a method for counting the density of gray hair (number of gray hairs / cm²) 2 ).

[0088] Description of the composition used Hair lotion containing 1% of the hair care activator of the present invention: Water, Glycerin / Acetyl Tyrosine / Sodium Metabisulfite / Larix Europaea Wood Extract / Glycine / Zinc Chloride / Epigallocatechin Glucoside Gallate, Sodium Benzoate, Citric Acid, Sodium Citrate, PPG-26 Buteth-26, PEG-40 Hydrogenated Castor Oil, Aqua / Water, Fragrance, Butylphenyl, Methylpropional, D-Limonene, Alpha-Isomethyl Ionone.

[0089] Placebo hair lotion: Water, sodium benzoate, citric acid, sodium citrate, PPG-26 buteth-26 PEG-40 hydrogenated castor oil, aqua / water, fragrance, butylphenyl, methylpropional, D-limonene, alpha-isomethyl ionone.

[0090] Panel and research conditions A double-blind, between-individual, and placebo-controlled clinical evaluation was conducted in 44 Caucasian male volunteers (18 years of age or older) with gray hair. A placebo hair lotion was tested by a first group of 22 volunteers, and a hair lotion containing 1% of the hair care activator of the present invention was tested by a second group of 22 volunteers. The treatment was applied once a day for four months by massaging the scalp.

[0091] Results: Reduction in the percentage of gray hair (scored in percentage) Scalp photographs were taken using a Nikon D7100 in combination with the Canfield Epiflash® system on the first day of the trial and after 4 months of daily application of the product. The area of ​​hair parting was determined according to the localization of gray hair. Blinded scoring was performed to evaluate the percentage of gray hair in the photographs.

[0092] The results are shown in the table below: [Table 2]

[0093] After 4 months of application, a reduction of -17% in the percentage of gray hair was observed, which is 2.1 times greater than that of the placebo group. The proportion of gray hair was noticeably reduced, with the best response showing a 50% reduction in gray hair (Figure 9).

[0094] Results: Reduction in gray hair density (several times / cm²) 2 ) Before processing, 1 cm 2 Images were taken two days after shaving the scalp area. The equipment used was a Nikon D7100 digital camera combined with a Canfield Epiflash® system equipped with contact lenses. The contact lenses allow for the flattening of the hair on the scalp. Next, the number of gray hairs was determined from 0.7 cm in the image. 2 The test was performed on a test area (1 x 0.7 cm) using a specific tool in Photoshop. All hairs with white roots within the zone were counted. The size and location of the studied area remained the same for all evaluations. In cases of offset, the location of the test area was adjusted. The results are shown in the table below:

[0095] [Table 3]

[0096] A hair lotion containing 1% of the hair care activator of the present invention is used in cm 2 The number of gray hairs per person was significantly reduced, more than 2.8 times compared to the placebo group. After 4 months, the density of gray hair was noticeably reduced, and for the best response, cm 2 Each reduction was accompanied by a decrease of -55.7 in gray hair (Figure 10).

[0097] Example 7: Evaluation of melanin production using an in vitro co-culture model: Comparison introduction The purpose of this study was to compare the effects of the hair care activator of the present invention, which contains taxifolin glucoside and N-acetyltyrosine, with the effects of L-tyrosine (positive reference), the effects of taxifolin glucoside, and the effects of a mixture of taxifolin glucoside and L-tyrosine. The model used for this comparison was a co-culture of normal human epidermal keratinocytes (NHEK) and normal human melanocytes (NHM).

[0098] cell culture Cell culture was actually performed using primary cells isolated from human skin biopsies. Normal human keratinocytes (NHK) were seeded at a rate of 60,000 cells per well in a 6-well plate pre-coated with collagen I. Four hours later, normal human melanocytes (NHM) were seeded at a rate of 60,000 cells per well in the same 6-well plate. The cells were incubated for 48 hours in complete medium (EpliLife® supplemented with HKGS = Human Kerationcyte Growth Supplement).

[0099] After 48 hours of culture, the cells were stimulated with the following composition for 5 days: -450 μg / mL of L-tyrosine (Sigma) -0.13 μg / mL of taxifolin glucoside -0.13 μg / mL taxifolin glucoside + 15 μg / mL N-acetyltyrosine (same as Example 1) -0.13 μg / mL taxifolin glucoside + 15 μg / mL L-tyrosine (Sigma) The composition was diluted in basal medium (EpiLife®) without supplementation. The treatment was renewed every two days.

[0100] Melanin extraction and dosage Five days after treatment, the cells were washed with PBS. Then, 200 μL of 0.5N NaOH solution was added to each well. Cell lysates were collected in 1.5 mL microtubes equipped with secure caps. In parallel, a standard range of melanin was prepared (100 to 0 μg / mL). The samples and standard range microtubes were heated in a dry bath at 80°C for 1 hour. After heating, 100 μL of each sample was transferred to a 96-well plate. The optical density was measured at 405 nm to determine the melanin content.

[0101] statistical analysis All experiments were performed at least three times. We actually performed the Xaviro-Wilk normality test to evaluate whether the data conformed to Gauss's law. If the results were not statistically significant, the data were evaluated using statistical parametric tests (ANOVA, followed by Dunnett's multiple comparison test). If the results were not statistically significant, the data were evaluated using statistical nonparametric tests (Kruskal-Wallis ANOVA, followed by Mann-Whitney U test).

[0102] result The results are shown in Figure 11. A positive reference (450 μg / ml L-tyrosine) induced melanin synthesis by 35% (p<0.001) compared to the untreated condition. This confirms that the co-culture model corresponds to melanin production and is capable of producing melanin. However, taxifolin glucoside alone was unable to induce melanin synthesis. Treatment with a combination of taxifolin glucoside and N-acetyltyrosine resulted in an 18% increase in melanin content (p<0.001) compared to the untreated condition.

[0103] On the other hand, the combination of taxifolin glucoside and L-tyrosine resulted in only slight and non-significant increases in melanin content. Significant differences also exist between stimulation using taxiphorin glucoside and N-acetyltyrosine, and stimulation using taxiphorin glucoside and L-tyrosine.

Claims

1. A hair care activator containing taxifolin glucoside and N-acetyltyrosine.

2. The hair care activator according to claim 1, wherein at least a portion of the taxifolin glucoside is taxifolin alpha-D-glucoside.

3. A hair care activator according to claim 1 or 2, further comprising taxifolin.

4. The hair care activator according to claim 3, wherein taxifolin is added in an amount of up to 0.24 wt%.

5. The hair care activator according to claim 3 or 4, wherein the weight ratio of taxifolin glucoside to taxifolin is 90:10 to 40:

60.

6. A hair care activator according to any one of claims 1 to 5, further comprising glycine and / or epigallocatechin gallate and / or epigallocatechin glucoside gallate.

7. A hair care activator according to any one of claims 1 to 6, comprising 0.01 to 0.50 wt% of taxifolin glucoside.

8. A hair care activator according to any one of claims 1 to 7, comprising 1.0 to 30 wt% N-acetyltyrosine.

9. A hair care activator according to any one of claims 1 to 8, wherein the weight ratio of taxifolin glucoside to N-acetyltyrosine is 1:5100 to 1:

2.

10. A hair care composition comprising a hair care activator according to any one of claims 1 to 9 and a cosmetically acceptable carrier.

11. Non-therapeutic use of the hair care activator according to any one of claims 1 to 10 for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, for re-pigmentation of hair, and / or for reducing the proportion and / or density of white or gray hair.

12. A method for stimulating the proliferation of hair follicle stem cells, stimulating the production of active melanocytes, stimulating melanin production, stimulating the restoration of hair pigmentation, reactivating melanin production in gray hair, activating the protective action of antioxidants in hair follicles, protecting melanocytes against oxidative stress, and reducing the rate and / or density of hair re-pigmentation and / or gray hair, the method comprising the step of topically applying a hair care activator according to any one of claims 1 to 9 to human hair.

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