Uses of urolithiasis to prevent / slow down hair thinning

Abstract

The present invention relates to the cosmetic use of at least one of a compound of formula (I) and / or its salt and / or solvate and / or its glucuronidated derivative for preventing / slowing down hair thinning and / or promoting an increase in hair thickness.

Description

Technical Field

[0001] The present invention relates to the cosmetic use of at least one of the compounds of formula (I) and / or its salts and / or solvates and / or its glucuronic acid-conjugated derivatives for preventing and / or slowing down hair thinning or promoting an increase in hair thickness. Background Technology

[0002] In the hair care industry, users are increasingly looking for products that can improve hair quality.

[0003] In particular, the demand for continuous thickening of fine hair is growing, especially in regions with aging populations such as Europe, the United States, and Japan.

[0004] In fact, several studies have shown that after age 40, especially in women, the heterogeneity of hair diameter on the same scalp increases, and the average thickness tends to decrease.

[0005] Therefore, it is now necessary to seek a technological solution that enables the acquisition of new compounds and compositions for use in the field of hair, thereby improving hair quality, particularly by increasing hair diameter and thus hair mass.

[0006] Hair fibers consist of a cortex covered by a finely overlapping layer of keratin. Cortical cells are needle-shaped, tightly packed, and aligned along the hair shaft. They contain macrofibrils, composites of keratin intermediate filaments integrated into a matrix composed of keratin-associated proteins (KAPs) and primarily stabilized by disulfide bonds. Within macrofibrils, keratin IFs exhibit a double-helix structure because they are oriented both axially and follow a helical shape around the shaft. The pitch (so-called double-helix strength) of macrofibrils in hair from the human scalp varies among macrofibrils and is negatively correlated with their diameter, allowing for the classification of macrofibrils as either "low-diameter and high-helix" or "high-diameter and low-helix." Human hair may also contain medulla, composed of large vacuolated cells with a unique keratin expression profile located in the center of the cortex. The presence and size of the medulla vary by individual and body location, and the presence of medulla in hair fibers from the human scalp is correlated with the diameter of the hair fiber.

[0007] Hair fibers are gradually formed within the hair follicle by keratinization of the matrix keratinocytes at the base of the follicle, during which keratinization increases the stiffness of the hair fiber. This process occurs in the early stages of hair growth and is based on complex molecular interactions between fibroblasts in the dermal papilla and keratinocytes of the hair. These interactions within the hair follicle largely determine the shape and diameter of the hair fiber. In an individual's scalp, scalp hair exhibits significantly variable morphology, such as the coexistence of small-diameter and large-diameter hairs. Small-diameter hairs are notably characterized by increased expression of keratin 38 (Baltenneck's keratin 38) compared to other keratins in the hair cortex and KAP. et al. (2022), Age-associated thinhair displays molecular, structural and mechanical characteristic changes, J. Struc. Biol ., vol. 214, issue 4, doi: 107908).

[0008] However, little is still known about the characteristics of small-diameter hairs.

[0009] It is evident that it would be advantageous to obtain compositions and / or active ingredients capable of preventing and / or slowing down hair thinning or promoting an increase in hair thickness. This would enable the satisfaction of consumers, particularly those with fine hair, of their desire for improved hair thickness.

[0010] The purpose of this invention is precisely to meet this need. Summary of the Invention

[0011] The applicant has now surprisingly discovered that natural urolithin can regulate the expression of K38 in hair to promote the expression of cortical keratin, a characteristic of coarse hair.

[0012] Therefore, the present invention relates to the cosmetic use of at least one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates for preventing / reducing hair thinning or promoting hair thickening: [Chemical Formula 1]

[0013] in: R1 = -OH, and R2 to R5 may be the same or different, and are independently selected from H and -OH.

[0014] The present invention also relates to a cosmetic treatment method for increasing the diameter of hair fibers. This method is, of course, non-therapeutic. The cosmetic treatment method includes at least one step of applying one of at least one compound of formula (I) and / or one of its salts and / or its glucuronic acid-conjugated derivatives and / or solvates to fine hair or hair lacking coarseness.

[0015] The present invention also relates to cosmetic compositions, particularly cosmetic compositions for topical application to the scalp and / or hair fibers, comprising: at least one of a compound of formula (I) and / or a salt thereof and / or a glucuronic acid-conjugated derivative thereof and / or a solvate, and one of vitamin C or a derivative thereof.

[0016] The present invention also relates to a method for evaluating the efficacy of a compound in preventing and / or slowing down hair thinning or promoting hair thickening, the method comprising at least the following steps: a. Obtain at least one hair follicle from a scalp sample; b. In the presence of the test compound, particularly at a temperature of 30°C to 38°C, preferably 37°C, in an atmosphere of 2% to 7% CO2, preferably 5% CO2, the hair follicle is incubated in a culture medium for at least 1 day, preferably at least 2 days, and particularly at least 5 days. c. Measure the expression levels of keratin K38 and K35 in the incubated samples from step b; d. Calculate the expression level ratio of keratin K38 / K35 based on the expression levels measured in step c; and e. When the K38 / K35 ratio is significantly lower than that obtained from untreated hair follicles, the compound is identified as effectively preventing and / or slowing down hair thinning or promoting an increase in hair thickness.

[0017] First, the present invention relates to the cosmetic use of at least one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates for preventing / reducing hair thinning or promoting hair thickening: [Chemical Formula 1]

[0018] in: R1 = -OH, and R2 to R5 may be the same or different, and are independently selected from H and -OH.

[0019] Preferably, the compound of formula (I) is natural urolithin.

[0020] More preferably, the compound of formula (I) is selected from urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, isourolithin A, and mixtures thereof.

[0021] According to a preferred embodiment, the compound of formula (I) is a compound containing hydroxyl groups (-OH), such as R1=OH and R2=R3=R4=R5=H.

[0022] According to another preferred embodiment, the compound of formula (I) is a compound containing two hydroxyl groups (-OH) such that R1=OH, R2, R3, R4, R5=OH or H, provided that one of the groups R2 to R5 represents a hydroxyl group (-OH), preferably R1=R2=OH, and R3=R4=R5=H.

[0023] According to another preferred embodiment, the compound of formula (I) is a compound containing three hydroxyl groups (-OH) such that R1=OH, R2, R3, R4, R5=OH or H, provided that two of the groups R2 to R5 represent hydroxyl groups (-OH), preferably R1=R2=R3=OH and R4=R5=H.

[0024] According to another preferred embodiment, the compound of formula (I) is a compound containing four hydroxyl groups (-OH) such that R1=OH, R2, R3, R4, R5=OH or H, provided that three of the groups R2 to R5 represent hydroxyl groups (-OH), preferably R1=R2=R4=OH.

[0025] According to a preferred embodiment, the compound of formula (I) is selected from urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, isourolithin A, and their salts and / or solvates.

[0026] Urolithiasis A has the following CAS number and molecular formula:

[0027] Urolithiasis A is especially sold by Sigma-Aldrich under the name SML1791-5MG.

[0028] Urolithin B has the following CAS number and molecular formula:

[0029] Urolithin B is especially marketed by Sigma-Aldrich under the commercial brand name SML1649-50mg.

[0030] Urolithin C has the following CAS number and molecular formula:

[0031] Urolithin C is especially marketed by Sigma-Aldrich under the commercial brand name SML3047-50mg.

[0032] Urolithin D has the following CAS number and molecular formula:

[0033] Urolithiasis E has the following CAS number and molecular formula:

[0034] Isourolithin A is an isomer of urolithin A. Its chemical name is 3,9-dihydroxy-6H-dibenzo[c]chromene-6-one. Isourolithin A is particularly marketed by Astress under the commercial name ATE638637155.

[0035] Preferably, the compound of formula (I) is one of urolithin A and / or its salt and / or its glucuronic acid-conjugated derivatives and / or its solvates.

[0036] "Salt" specifically refers to alkaline salts.

[0037] "Alkaline salt" refers to an organic or mineral alkaline salt, particularly selected from salts derived from alkaline hydroxides, such as sodium hydroxide which produces sodium salts or potassium hydroxide which produces potassium salts.

[0038] "Glucuronate-conjugated derivatives" or "glucuronides" refer to urolithin derivatives containing a glucuronic acid molecule linked to urolithin via an O-glycosidic bond. Thus, glucuronic acid condenses with one or more hydroxyl groups of the urolithin molecule to form an ether bond (RO-R'). Specifically, urolithin produced by glucuronidation is formed by the transfer of the glucuronic acid portion of uridine diphosphate glucuronide to urolithin via any of several types of UDP-glucuronyltransferases. Preferably, the glucuronate-conjugated derivatives are selected from urolithin A 3-O-glucuronide, urolithin A 8-O-glucuronide, isourolithin A 3-O-glucuronide, urolithin B 3-O-glucuronide, and mixtures thereof.

[0039] More preferably, the glucuronic acid conjugated derivative is selected from urolithin A-8-β-D-glucuronide, urolithin A-3-β-D-glucuronide, isourolithin A-3-β-D-glucuronide, urolithin B-3-β-D-glucuronide, and mixtures thereof.

[0040] Urolithin is a metabolite produced by the gut microbiota from ellagic acid and ellagitannins. Studies have shown that urolithin has positive biological effects. To date, several mechanisms of action of urolithin have been proposed.

[0041] "Hair getting finer" refers to a decrease in hair diameter, particularly characterized by the expression of keratin K38 in at least 50% of the cells in the cortex of fine hair. This distinguishes it from the loss of hair density caused by hair loss. "Hair getting thicker" refers to an increase in hair diameter, characterized by the expression of keratin K38 in less than 50% of the cells in the cortex of coarse hair.

[0042] For example, the average diameter of fine hair in Caucasians (especially women) is less than or equal to 70 μm, preferably about 60 μm. Preferably, the average diameter of coarse hair in Caucasians (especially women) is about 80 μm to about 90 μm.

[0043] Hair thinning worsens with age, particularly affecting people over 40 and older. Therefore, preferably, hair thinning is associated with the ratio of small-diameter hairs to the total number of hairs as age increases.

[0044] In particular, one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates is formulated in a cosmetic composition.

[0045] In particular, one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates is used to increase the volume or amount of hair.

[0046] Preferably, one of the compounds of formula (I) and / or their salts and / or their glucuronic acid-conjugated derivatives and / or solvates is applied topically, especially to the scalp and / or hair fibers.

[0047] The present invention also relates to a method for evaluating the efficacy of a compound in preventing and / or slowing down hair thinning or promoting hair thickening, the method comprising at least the following steps: a. Obtain at least one hair follicle from a scalp sample; b. In the presence of the test compound, particularly at a temperature of 30°C to 38°C, preferably 37°C, in an atmosphere of 2% to 7% CO2, preferably 5% CO2, the hair follicle is incubated in a culture medium for at least 1 day, preferably at least 2 days, and particularly at least 5 days. c. Measure the expression levels of keratin K38 and K35 in the incubated samples from step b; d. Calculate the expression level ratio of keratin K38 / K35 based on the expression levels measured in step c; and e. When the K38 / K35 ratio is significantly lower than that obtained from untreated hair follicles, the compound is identified as effectively preventing and / or slowing down hair thinning or promoting an increase in hair thickness.

[0048] Preferably, the culture medium is William's complete medium. This medium (William's Medium E) is generally free of phenol red and contains 40 ng / mL (i.e., 2 mM) of hydrocortisone, 10 μg / mL of insulin, 2 mM of L-glutamine, and 1% of an antibiotic / antifungal agent.

[0049] "Expression level" refers to the amount of mRNA or protein produced through the expression of a gene. This amount can be expressed in concentration or ratio form. Expression level can be measured using common techniques well known to those skilled in the art, such as RT-qPCR, Western blot, or immunolabeling.

[0050] "Scalp sample" refers to scalp fragments, particularly those obtained from a subject's scalp via biopsy or from a reconstructed scalp model.

[0051] As demonstrated in the examples, the applicant has demonstrated that one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates can reduce the increase in K38 gene expression levels in hair follicles (an increase in K38 expression levels is characteristic of fine hair) and promote the expression of keratin profiles characteristic of coarse hair. Preferably, the K38 expression level in hair samples treated with urolithin A is reduced by at least 20%, preferably at least 40%, and most preferably at least 50%, compared to the K38 expression level in untreated hair samples.

[0052] Preferably, one of the compounds of formula (I) and / or their salts and / or their glucuronic acid conjugate derivatives and / or solvates has no effect on the expression level of the K35 gene in hair follicles.

[0053] The present invention also relates to a non-therapeutic cosmetic treatment method for increasing the diameter of hair fibers, comprising: applying at least one of a compound of formula (I) or a salt thereof and / or a glucuronic acid-conjugated derivative thereof and / or a solvate.

[0054] The present invention also aims at a cosmetic treatment method comprising: at least one step of applying one of at least one compound of formula (I) and / or its salt and / or its glucuronic acid-conjugated derivative and / or solvate to fine hair or hair lacking coarseness.

[0055] The method preferably includes: a first step of evaluating the expression levels of proteins K38 and K35 in hair follicle samples, and then comparing the obtained K38 / K35 ratio with the characteristic K38 / K35 ratio of fine hair.

[0056] The "K38 / K35 ratio" refers to the ratio between the expression levels of K38 and K35 in a hair follicle sample. For example, in Caucasians (especially women), the characteristic K38 / K35 ratio of fine hair corresponds to a ratio of approximately 0.070 for RNA expression levels. This differs from coarse hair, particularly in women, where the characteristic K38 / K35 ratio corresponds to a ratio of approximately 0.045 for RNA expression levels.

[0057] The cosmetic treatment method of the present invention is carried out, in particular, by topically applying one of at least one compound of formula (I) and / or its salt and / or its glucuronic acid-conjugated derivative and / or solvate to fine hair or hair lacking coarseness. For example, one of the compounds of formula (I) or its salt or solvate is formulated into a suspension, gel, emulsion, shampoo, unrinsed lotion, or foam.

[0058] Therefore, the cosmetic treatment method according to the present invention can be implemented by daily topical application of one of the compounds of formula (I) and / or their salts and / or their glucuronic acid-conjugated derivatives and / or solvates.

[0059] The method according to the invention may include a single application. According to another embodiment, it may be repeatedly applied, for example, two to three times a day for a day or longer, and typically for at least four weeks, or even an extended period of four to fifteen weeks, with one or more interruptions if desired.

[0060] This invention relates to cosmetic compositions, particularly cosmetic compositions for topical application to the scalp and / or hair fibers, which particularly comprise, in a physiologically acceptable medium, at least one of a compound of formula (I) and / or its salt and / or its glucuronic acid-conjugated derivatives and / or solvates, and one of vitamin C and / or its derivatives. Preferably, the compound of formula (I) is urolithin A.

[0061] The compositions according to the invention also contain one of vitamin C (i.e., ascorbic acid) or a derivative thereof.

[0062] In this invention, ascorbic acid preferably corresponds to L-ascorbic acid or vitamin C. It has the structure of formula (II): [Chemical Formula 2] (II).

[0063] L-Ascorbic acid is specifically marketed by Reckon Organics under the name Ascorbic Acid (L).

[0064] "Ascorbic acid derivatives" preferably refers to compounds selected from 5,6-di-O-dimethylsilyl ascorbate (particularly sold by Exsymol under the brand name PRO-AA), DL-α-tocopherol-DL-ascorbic acid-potassium phosphate (also known as ascorbic acid tocopherol potassium phosphate (sold by SEPPIC under the brand name SEPIVITAL EPC)), magnesium ascorbic acid phosphate, sodium ascorbic acid phosphate (sold by DSM under the brand name Stay-C5Q), disodium ascorbic acid sulfate, sulfinyl-L-ascorbic acid, glucopyranosyl-L-ascorbic acid, and ascorbic acid glucoside.

[0065] "Ascorbate glucoside" refers to the condensation product of D-form (i.e., α- or β-glucanopyranose or α- or β-furanose) or L-form glucose with ascorbic acid (preferably L-form). Preferably, ascorbate glucoside is 2-O-α-D-glucanopyranoside-L-ascorbic acid, and is particularly marketed by Macrocare Tech under the name VG2.

[0066] Preferably, the composition according to the invention comprises ascorbic acid, more preferably L-ascorbic acid.

[0067] Preferably, the composition contains at least 1% by weight, preferably at least 2% by weight, more preferably at least 5% by weight, more preferably at least 7% by weight, and even more preferably at least 10% by weight of ascorbic acid or a derivative thereof, relative to the total weight of the composition.

[0068] Preferably, the composition contains 2% to 20% by weight, more preferably 5% to 15% by weight, or even more preferably 7% to 12% by weight of ascorbic acid or a derivative thereof, relative to the total weight of the composition.

[0069] Preferably, the composition, particularly a cosmetic composition, is intended for topical application, especially to the scalp and / or hair fibers, particularly hair.

[0070] "Physiologically acceptable medium" means a medium that is compatible with hair fibers, especially the scalp and / or hair. A physiologically acceptable medium is generally suitable for the nature of the matrix of the composition to be applied and the form in which the composition is conditioned.

[0071] The physiologically acceptable medium for the implemented composition may be an aqueous medium, such as water or a mixture of water and at least one physiologically acceptable organic solvent.

[0072] As organic solvents, examples include: (C2-C4) alcohols, such as ethanol and isopropanol; polyols, particularly polyols having 2 to 6 carbon atoms, such as glycerol, propylene glycol, butanediol, pentanediol, hexanediol, dipropylene glycol, and diethylene glycol; ethers of polyols, such as 2-butoxyethanol, propylene glycol monomethyl ether, monoethyl ether, and diethylene glycol monomethyl ether; and mixtures thereof. Preferably, organic solvents are particularly C2 to C6 monohydric alcohols, such as ethanol.

[0073] Preferably, compositions according to the invention comprising at least one compound of formula (I) or its salt and / or its glucuronic acid-conjugated derivatives and / or solvates in a physiologically acceptable medium may further comprise one or more conventional cosmetic additives, particularly selected from nonionic surfactants, anionic surfactants, cationic surfactants and amphoteric surfactants, vitamins and provitamins (including panthenol), anti-hair loss agents (such as stemoxydine and 2,4-diaminopyrimidine-3-oxide), sunscreens, fillers, coloring materials, pearlescent agents, sunblocking agents, multivalent chelating agents, polymers (especially film-forming agents), conditioning agents, plasticizers, thickeners, oils, antioxidants, defoamers, moisturizers, emollients, penetrants, fragrances and preservatives.

[0074] According to one embodiment of the invention, one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates is present in the cosmetic composition at a level of at least 0.0001% by weight, particularly about 0.0001% by weight to about 20% by weight, relative to the total weight of the composition, and in particular, one of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates is present in the cosmetic composition at a level of about 0.001% by weight to about 2% by weight, relative to the total weight of the composition.

[0075] The cosmetic compositions suitable for implementing the present invention may be in the form of suspension, gel, lotion, shampoo, unrinsed lotion, or foam.

[0076] The composition can be any form suitable for caring for hair fibers, especially hair and / or scalp, particularly the following forms: hair care shampoos for daily or bi-weekly application, especially shampoos or conditioners for weekly or bi-weekly application, liquid or solid scalp cleansing soaps for daily application, hair styling products (hair gel, styling gel), hair care masks, hair cleansing creams or foaming gels.

[0077] Compositions suitable for carrying out the present invention may be aqueous, alcoholic or water-alcoholic solutions or suspensions, or oily suspensions, emulsions or multiple emulsions of varying fluid consistency obtained by dispersing a fatty phase in an aqueous phase (O / W) or an aqueous phase in a fatty phase (W / O), or multiple emulsions, aqueous, water-alcoholic or oily gels, loose or compacted powders to be used as is or incorporated into physiologically acceptable media, microcapsules or microparticles, ionic or nonionic dispersions.

[0078] According to a specific embodiment of the invention, in the context of the invention, the composition used is anhydrous. "Anhydrous" means that the composition contains less than 5% by weight of water relative to the total weight of the composition, preferably less than 3% by weight, even more preferably less than 1% by weight, or even completely anhydrous (0%).

[0079] Advantageously, one of the compounds of formula (I) according to the invention and / or their salts and / or their glucuronic acid-conjugated derivatives and / or solvates is administered in a formulation that promotes the penetration of the agent at the hair follicle level. As examples, noncyclic monohydric and dihydric alcohols, ethyl acetate, butyl acetate, isopropyl myristate, fatty acids, phospholipids, terpenes, azones and their derivatives, propylene glycol and its derivatives, cyclodextrins, octyl salicylate, cyclopentadecanolactone, polysorbate, polyvinylpyrrolidone and its derivatives may be mentioned; this list is not limiting.

[0080] According to one embodiment, the composition comprises a nonionic, anionic, cationic, or amphoteric surfactant, wherein alkyl sulfates / esters, alkylbenzene sulfates / esters, alkyl ether sulfates / esters, alkyl sulfonates / esters, quaternary ammonium salts, alkyl betaines, oxyethyleneized alkylphenols, fatty acid chain alkanolamides, oxyethyleneized fatty acid esters, and other nonionic surfactants of the hydroxypropyl ether type may be mentioned.

[0081] When the composition contains at least one surfactant, it is typically present at a maximum concentration of 30% by weight relative to the total weight of the composition, preferably at a concentration of 0.1% to 5% by weight.

[0082] To improve the cosmetic properties of hair or even reduce or prevent its deterioration, the composition may also contain cationic, anionic, nonionic or amphoteric treatment agents.

[0083] In the treatment agents, volatile or non-volatile, linear or cyclic siloxanes and mixtures thereof may be specifically mentioned, polydimethylsiloxanes, quaternized polyorganosiloxanes (such as those described in French Patent Application No. 2 535 730), polyorganosiloxanes having aminoalkyl groups modified with alkoxycarbonyl groups (such as those described in US Patent No. 4 749 732), polyorganosiloxanes (such as polydimethylsiloxane-polyoxyalkyl copolymers of the type of polydimethylsiloxane copolyols, polydimethylsiloxanes containing stearoxy end groups (stearoxydimethylsiloxane), polydimethylsiloxane-dialkylammonium acetate copolymers or polydimethylsiloxane-polyalkyl β-olefin copolymers, as described in application GB 2 197 352), and organopolysiloxanes modified with mercapto or mercaptoalkyl groups (such as those in French Patent No. 1 530 369 and European Patent Application No. 0 295). Those described in 780), as well as silanes (such as stearoxytrimethylsilane).

[0084] The compositions according to the invention may also contain other ingredients, such as cationic polymers, basic amino acids (such as lysine and arginine) or acidic amino acids (such as glutamic acid and aspartic acid), peptides and their derivatives, protein hydrolysates, waxes and other compounds (such as fatty alcohols and lanolin derivatives), active ingredients (such as pantothenic acid), anti-hair loss agents, anti-dandruff agents, thickeners, suspending agents, chelating or complexing agents, sunscreens, antioxidants, fragrances, and preservatives. Attached Figure Description

[0085] The present invention will now be described with reference to the accompanying drawings and the following embodiments.

[0086] Figure 1 These are the measurement results of proteins K35 and K38 in in vitro and in vitro hair follicle extracts.

[0087] Figure 2 This is an evaluation of the relative expression of KRT38 and KRT35 in isolated and in vitro hair follicles.

[0088] Figure 3 It is a measurement of K38 markers in the cortex of isolated and in vitro hair follicles.

[0089] Figure 4 It is a measurement of K38 markers in the cortex of hair follicles of human subjects. Detailed Implementation

[0090] Example Example 1 - Regulatory Model of K38 Expression in Human Hair Follicles Materials and methods 1. Sample collection Biopsy samples obtained through scalp surgery were scraped and cut into small pieces. The subcutaneous-dermal junction was incised with a scalpel. Hair follicles were removed from the subcutaneous tissue and examined for integrity under a microscope. They were then placed in 500 μL of Williams' complete medium (Williams' medium E, without phenol red-hydrocortisone 40 ng / ml - 2 mM insulin 10 μg / mL - L-glutamine 2 mM - 1% antibiotic / antifungal agent) in 24-well plates. The plates with hair follicles were incubated at 37°C and 5% CO2 for 5 days, and the entire medium was replaced after 2-3 days. Urolithin A (CAS No.: 1143-70-0, SML1791-5MG, Sigma-Aldrich) was used at the start of culture and when changing the medium. One 24-well plate was used for each experimental condition, i.e., 24 hair follicles per condition. The cultured hair follicles were observed daily using a Zeiss microscope, and photographs were taken using Archimed software (Microvision) to measure the vitality and elongation of each follicle.

[0091] Hair follicles were collected on days 0 and 5 to analyze K38 and K35 expression by Western blotting, RT-qPCR, or immunofluorescence.

[0092] 2. Immunofluorescence of cross-section and longitudinal section of hair follicle Treated and untreated ex vivo hair follicles and in vitro hair follicles (cultured for 5 days) were incorporated into the Tissue-Tek® OCT™ compound, frozen in a dry ice / ethanol mixture, and cryosectioned with a 10 μm cross section and a 5 μm longitudinal section. All sections were retained each time and placed on three consecutive glass slides. The first slide was used to label K35, the second slide was used to label K38, and the third slide was kept as a backup.

[0093] Sections were infiltrated in PBS-0.05% Tween 20 for 15 minutes and blocked in PBS-10% goat serum-0.05% Tween 20 for 15 minutes. Sections were then incubated with anti-K35 (1:500) or anti-K38 (1:500) antibody for 30 minutes. After washing three times in PBS-0.05% Tween 20, sections were incubated with secondary antibody (anti-guinea pig Cy3 1:200) for 30 minutes. Sections were then washed again and counterstained with Hoechst (1:5000) for 1 minute for nuclear staining, and fixed with Prolong Gold Antifade reagent (Life Technologies). Photographs of longitudinal sections of hair follicles were taken using a Nikon microscope and NIS Element software. For quantitative evaluation of transverse sections, photographs were taken at the same distance from the bulb of each hair follicle using a Leica microscope. K38 region labeling was analyzed using ImageJ software.

[0094] 3. protein imprinting of hair follicle proteins Hair follicles collected in vitro and in vitro were placed in test tubes containing 100 μL of TP3 buffer (Ready Prep Reagent 3 Sequential Extraction Kit, Biorad) and stored at -80°C. The samples were then ground with a pestle for a few minutes, followed by vortexing several times at room temperature and incubation for 30 minutes. After centrifugation at 10000 g for 10 minutes, the supernatant was collected and protein concentration was determined using the Pierce 660 nm method.

[0095] For Western blotting, samples were prepared in NuPAGE LDS sample buffer (4X) and NuPAGE reducing agent (10X), then heated to 95°C for 1 minute. 10 μg of total protein was separated by 4–15% SDS-PAGE (Criterion™ TGX Stain-Free™ Precast Gels Bio-rad) using 1X Tris / glycine / SDS operation buffer and transferred to a nitrocellulose membrane (Trans-Blot Turbo Midi Format Bio-rad) via semi-dry blot. The membrane was then blocked for 1 hour at room temperature in PBS-1% skim milk-0.05% Tween 20. The membrane was then probed for 1 hour at room temperature with a primary antibody (anti-K35 or anti-K38) diluted in blocking buffer. After washing in PBS-1%-0.05% Tween 20, the membrane was incubated with a secondary antibody labeled with horseradish peroxidase at room temperature for 30 minutes. Targeted proteins were detected using the SuperSignal West Dura extended-duration substrate (Thermofisher). Peroxidase activity was detected using a CDD camera imager (ChemiDoc Bio-Rad). Quantification and normalization were performed using ImageLab software (Bio-Rad). The expression water of K35 and K38 was normalized to the total protein in the electrophoretic gel trajectory.

[0096] 4. Purification and extraction of total RNA Hair follicles collected in vitro and in vitro were placed in tubes containing 200 μL of mercaptoethanol buffer (RLT-1%β) and stored at -80°C. For in vitro conditions, 24 hair follicles were collected; for in vitro conditions, only viable hair follicles were collected for gene expression studies (approximately 20 follicles, depending on conditions). A homogenization step was required before RNA extraction from the hair follicles. First, the hair follicles were ruptured with a pestle, then the lysate was passed through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe at least 10 times. Finally, the lysate was loaded onto a QIAshredder rotating column and centrifuged at maximum speed for 2 minutes.

[0097] Total RNA was purified and extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol, followed by DNase digestion using an RNase-free DNase kit. RNA was then eluted with 30 μL of RNase-free water.

[0098] RNA concentration was determined by nanodrops, and RNA quality was determined using the 6000 Nano RNA Kit (Agilent) on an Agilent 2100 Bioanalyzer, following the manufacturer's protocol.

[0099] 5. cDNA synthesis and real-time quantitative PCR (RT-qPCR) Reverse transcription was performed using a 50 μL iScript cDNA Synthesis Kit (Bio-Rad), and the cDNA was stored at -80°C until use. qPCR was performed on a CFX384 system (BioRad) and CFX Maestro software, with a total volume of 10 μL containing 5 ng cDNA, 5 μL Syber Green Mix (Sso Advanced SYBR Green Supermix Biorad), 1 μL 10X primers (QuantiTect Primer Assay, Qiagen, Table 3), and 1.5 μL water. The reaction was seeded in 384-well plates, and PCR cycles consisted of initial pre-denaturation at 95°C for 30 seconds, 45 denaturation cycles at 95°C for 10 seconds each, and annealing at 60°C for 30 seconds. At the end of PCR, melting curves were plotted to control the proliferation of individual PCR products in each well. Gene normalization was performed using the geometric mean of RPL13A, RRN18S, and TBP as reference genes, and relative mRNA expression levels were then calculated using the 2-method (ΔΔCt). Therefore, the relative expression of genes in the treatment groups was reported as a fold change from the mean control value. Statistical analysis was performed using the MyStat application and the Wilcoxon test.

[0100] The cited introduction to qRT-PCR Hs_RPL13A_1_SG QuantiTect primer assay QT00089915Qiagen Hs_RRN18S_1_SG QuantiTect primer assay QT00199367Qiagen Hs_TBP_1_SG QuantiTect primer assay QT00000721Qiagen Hs_KRT35_1_SG QuantiTect primer assay QT00012397Qiagen Hs_KRT38_1_SG QuantiTect primer assay QT00042000Qiagen result 1. Measurement of K35 and K38 proteins in in vitro and ex vivo hair follicle extracts Hair follicles were held in vitro for 5 days, and then keratin expression levels were measured. Under these conditions, K38 expression increased, while K35 expression decreased. Figure 1 (a and b in the original text). Therefore, in hair follicles maintained in vitro, the K38 / K35 ratio increases ( Figure 1 c), which behaves in a manner similar to that observed in fine hair, relative to coarse hair.

[0101] 2. Evaluation of the relative expression of KRT38 and KRT35 in isolated and in vitro hair follicles This change in hair keratin expression was confirmed at the mRNA level using qRT-PCR. The results showed that K38 expression was significantly increased in in vitro hair follicles compared to isolated follicles. Figure 2 (a and b in the text). At least 100% increases were systematically observed, but the observed magnitudes ranged from 2 to 10-fold, depending on the sample. Simultaneously, K35 expression was significantly reduced when the hair follicles were held in vitro. Figure 2 (c and d in the text). For the latter, the reduction was also variable, showing a reduction of more than 50% in 6 out of 9 experiments.

[0102] 3. To evaluate the distribution of K38 and K35 in hair follicles in vitro.

[0103] To control the distribution of two keratins in in vitro maintained hair follicles, K38 and K35 were immunolabeled on isolated hair follicles and in vitro control hair follicles. K38 was labeled only in the cortex of isolated and in vitro control hair follicles exhibiting a discontinuous pattern of positive cells. K35 was labeled across the entire surface of the human hair follicle cortex and then gradually decreased both in vitro and in vitro. Therefore, the distribution of K35 and K38 in in vitro maintained hair follicles remained confined to the hair cortex and did not exhibit a pattern of loss of regulation.

[0104] 4. Measurement of K38 markers in the cortex of isolated and in vitro hair follicles In one subject, the percentage of cortical area in hair follicles labeled with K38 was evaluated using ImageJ software. Analysis showed that the percentage of K38 labeling was significantly higher in the hair follicle cortex after 5 days of in vitro storage. Figure 3 ).

[0105] 5. Measurement K38 markers in human hair follicle cortex Also, 129 hair follicles from 5 subjects ( Figure 4The values ​​were assigned to the "Series 1" and the percentage of K38 labeling in the cortex of isolated and in vitro hair follicles obtained from a single subject. All values ​​for isolated hair follicles followed a normal distribution (Series 1). Conversely, for a given hair diameter, in vitro hair follicles showed an off-normal distribution with an increased percentage of K38 labeling. The percentage of K38-labeled hair in the cortex (80–90%) was similar to that evaluated in fine hairs.

[0106] 6. in conclusion: The results shown above demonstrate the use of an in vitro human hair follicle model that reproduces the characteristic changes in hair keratin expression in fine hair. This model can be used to evaluate the properties of a given product to restrict the development of small-diameter hairs and promote keratin expression in coarse hairs, thereby increasing hair thickness.

[0107] Example 2 - Evaluation of the effect of urolithin A on K38 and K35 expression in in vitro hair follicle assays Materials and methods From day 1 of in vitro maintenance, 5 μM urolithin A (CAS No.: 1143-70-0, SML1791-5MG, Sigma-Aldrich) was added to the culture and replaced with culture medium. For each experimental condition, 24-well plates were produced, i.e., 24 hair follicles / condition.

[0108] Hair follicles were collected on days 0 and 5 under in vitro conditions to analyze K38 and K35 expression by qRT-PCR. Keratin expression was normalized to the in vitro (“native”) state and compared with untreated (“control”) and urolithin A (“Uro-A”) conditions by Wilcoxon test.

[0109] result 1. K38 expression was evaluated by qRT-PCR. Data represent the mean expression level compared to expression in isolated hair follicles (native, normalized mean equal to 1).

[0110] [Table 1]

[0111] * The ratio of 2-control or 3-Uro-A expression levels to 1-native expression levels The results indicate that, compared with the untreated control, urolithin A reduced the increase in K38 expression in treated human hair follicles by approximately 50%.

[0112] 2. Evaluation of K35 expression using qRT-PCR Data represent the mean expression level compared to expression in isolated hair follicles (native, normalized mean equal to 1).

[0113] [Table 2]

[0114] * The ratio of 2-control or 3-Uro-A expression levels to 1-native expression levels The above results indicate that, compared with the untreated control, urolithin A does not significantly alter the expression of K35 in the treated human hair follicles.

[0115] 3. Conclusion: Therefore, urolithin A reduced the increase in K38 expression observed in human hair follicles in vitro, while having no significant effect on K35 expression. Thus, treatment with urolithin A counteracted the changes in keratin K38 expression in hair cortex cells (a characteristic of small-diameter hair) and promoted the expression of keratin associated with coarse hair. Urolithin A could be used to promote the production of large-diameter hair.

Claims

1. Cosmetic use of at least one of a compound of formula (I) and / or a salt thereof and / or a glucuronidated derivative thereof and / or a solvate thereof to prevent and / or slow down hair thinning or promote hair thickening. in: R1 = -OH, and R2 to R5 may be the same or different, and are independently selected from H and -OH.

2. The cosmetic use according to claim 1, wherein, The compounds of formula (I) are selected from urolithin A, urolithin B, urolithin C, urolithin D, urolithin E, isourolithin A, and mixtures thereof.

3. The cosmetic use according to any one of the preceding claims, wherein, The compound of formula (I) is urolithin A.

4. The use according to any one of the preceding claims, wherein, The thinning of hair and the ratio of the number of small-diameter hairs to the total number of hairs are related to increasing age.

5. The use according to any one of the preceding claims, wherein, One of the compounds of formula (I) and / or its salts and / or its glucuronic acid-conjugated derivatives and / or solvates are applied topically, particularly to the scalp and / or hair fibers.

6. A makeup application method, comprising: At least one step of applying at least one compound of formula (I) and / or one of its salts and / or its glucuronic acid-conjugated derivatives and / or solvates to fine hair or hair lacking coarseness: in: R1 = -OH, and R2 to R5 may be the same or different, and are independently selected from H and -OH.

7. A cosmetic composition, particularly a cosmetic composition for topical application to the scalp and / or hair fibers, comprising: At least one of the compounds of formula (I) and / or one of its salts and / or one of its glucuronic acid-conjugated derivatives and / or solvates: in: R1 = -OH, and R2 to R5 may be the same or different, and are independently selected from H and -OH; and One of vitamin C or its derivatives.

8. The cosmetic composition according to claim 7, wherein, The vitamin C or one of its derivatives is present in an amount of at least 1% by weight, preferably at least 2% by weight, more preferably at least 5% by weight, more preferably at least 7% by weight, and even more preferably at least 10% by weight, relative to the total weight of the composition; preferably, the vitamin C or one of its derivatives is present in an amount of 2% to 20% by weight, relative to the total weight of the composition, particularly in an amount of 5% to 15% by weight, preferably 7% to 12% by weight, relative to the total weight of the composition.

9. The cosmetic composition according to claim 7 or 8, wherein, The compound of formula (I) and / or one of its salts and / or one of its glucuronic acid conjugated derivatives and / or solvates are present in an amount of at least 0.00001% by weight, particularly about 0.0001% by weight, to about 20% by weight, particularly about 0.001% by weight, relative to the total weight of the composition.

10. The cosmetic composition according to any one of claims 7 to 9, characterized in that, The composition is in the form of a suspension, gel, emulsion, shampoo, no-rinse wash, or foam.

11. A method for evaluating the efficacy of a compound in preventing and / or slowing down hair thinning or promoting hair thickening, comprising at least the following steps: (a) Obtain at least one hair follicle derived from a scalp sample; (b) The hair follicles were incubated in a culture medium for at least one day in the presence of the test compound, particularly at a temperature of 30°C to 38°C and an atmosphere of 2% to 7% CO2; (c) Measure the expression levels of keratin K38 and K35 in the incubated samples from step b; (d) Calculate the ratio of keratin K38 / K35 expression levels based on the expression levels measured in step c; (e) When the K38 / K35 ratio is significantly lower than the ratio obtained from hair follicles not treated with the compound, the compound is identified as effectively preventing and / or slowing down hair thinning or promoting an increase in hair thickness.

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