HAIR CARE ACTIVE AGENT

Abstract

The present invention provides a hair care active agent comprising taxifolin glucoside and N-acetyl tyrosine and a hair care composition comprising said hair care active agent. The hair care agent is able to reduce the occurrence of grey and white hair in an individual.

Claims

1. A hair care active agent, comprising taxifolin glucoside and N-acetyl tyrosine.

2. The hair care active agent of claim 1, wherein at least part of the taxifolin glucoside is taxifolin alpha-D-glucoside.

3. The hair care active agent of claim 1, further comprising taxifolin.

4. The hair care active agent of claim 3, wherein the weight ratio of taxifolin glucoside to taxifolin is from 90:10 to 40:60.

5. The hair care active agent of claim 1, further comprising glycine and/or epigallocatechin gallate and/or epigallocatechin gallatyl glucoside.

6. The hair care active agent of claim 1, comprising from 0.01 to 0.50 wt %, of taxifolin glucoside.

7. The hair care active agent of claim 1, comprising from 1.0 to 30 wt %, of N-acetyl tyrosine.

8. The hair care active agent of claim 1, wherein the weight ratio of taxifolin glucoside to N-acetyl tyrosine is from 1:5100 to 1:2.

9. A hair care composition comprising the hair care active agent according to claim 1 and a suitable carrier.

10. A non-therapeutic method of stimulating the proliferation of hair follicle stem cells, and/or stimulating the active melanocytes production, and/or stimulating the melanogenesis, and/or stimulating the recovery of hair pigmentation, and/or reactivating the melanin production in grey hair, and/or activating the antioxidant defenses in hair follicles, and/or protecting the melanocytes against oxidative stress, and/or hair repigmentation, and/or reducing the proportion and/or density of white or grey hair, the method comprising the step of topically applying the hair care active agent to wet or dry hair.

11. A method of stimulating the proliferation of hair follicle stem cells, and/or stimulating the active melanocytes production, and/or stimulating the melanogenesis, and/or stimulating the recovery of hair pigmentation, and/or reactivating the melanin production in grey hair, and/or activating the antioxidant defenses in hair follicles, and/or protecting the melanocytes against oxidative stress, and/or hair repigmentation, and/or reducing the proportion and/or density of white or grey hair, said method comprising the step of topically applying the hair care active agent according to claim 1 to human hair.

12. The hair care active agent of claim 2 further comprising taxifolin.

13. The hair care active agent of claim 2, wherein the weight ratio of taxifolin glucoside to taxifolin is from 90:10 to 40:60.

14. The hair care active agent according to claim 2 further comprising glycine and/or epigallocatechin gallate and/or epigallocatechin gallatyl glucoside.

15. The hair care agent of claim 2 comprising from 0.01 to 0.50 wt % of taxifolin glucoside.

16. The hair care agent of claim 2 comprising from 1.0 to 30 wt. % of N-acetyl tyrosine.

17. The hair care active agent of claim 2, wherein the weight ratio of taxifolin glucoside to N-acetyl tyrosine is from 1:5100 to 1:2.

18. A hair care composition comprising the hair care active agent according to claim 2 and a suitable carrier.

19. A hair care composition comprising the hair care active agent according to claim 12 and a suitable carrier.

Description

EXAMPLE 1: HAIR CARE ACTIVE AGENT ACCORDING TO THE PRESENT INVENTION

[0079] Standard Solution

[0080] The following standard solution may be used to prepare a hair care active agent of the present invention:

TABLE-US-00001 Standard Solution Substance Concentration N-acetyl tyrosine 15.0 wt % glycine 0.15 wt % zinc chloride 0.07 wt % taxifolin glucoside 0.17 wt % epigallocatechin gallatyl glucoside 0.03 wt % sodium metabisulfite 0.50 wt % glycerol 50.0 wt %

[0081] The hair care active agent of the present invention may consist of said standard solution, or may comprise said standard solution in combination with other ingredients.

[0082] Thus, the standard solution may be used as such.

[0083] Alternatively, the standard solution may also be diluted in a solvent, buffer or culture medium.

[0084] Test Solution

[0085] For some of the examples below (see indications in examples), a test solution was prepared by diluting the above standard solution in William's E medium to a final concentration of 0.01 to 1% v/v.

[0086] INCI

[0087] A hair care composition comprising the hair care active agent of the present invention may be described by the following INCI (International Nomenclature of Cosmetic Ingredients) indications:

[0088] WATER, GLYCERIN/ACETYL TYROSINE/SODIUM METABISULFITE/LARIX EUROPAE WOOD EXTRACT/GLYCINE/ZINC CHLORIDE/EPIGALLOCATECHIN GALLATYL GLUCOSIDE, SODIUM BENZOATE, CITRIC ACID, SODIUM CITRATE, PPG-26 BUTETH-26, PEG-40 HYDROGENATED CASTOR OIL, AQUA/WATER, FRAGANCE, BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYLIONONE.

EXAMPLE 2: EVALUATION OF MELANIN PRODUCTION: IN VITRO CO-CULTURE MODEL

[0089] Culture and Experimental Design

[0090] The cells used in this study were primary cultures of normal human keratinocytes (NHK) and normal human melanocytes (NHM) extracted after skin surgery (foreskin) of an 8 years old Caucasian male donor (phenotype III/IV).

[0091] The wells were plated with 200′000 NHK/9.6 cm.sup.2 and 50′000 NHM/9.6 cm.sup.2 and grown for 24 hours in supplemented medium (1:1 NHM supplemented medium/NHK supplemented medium).

[0092] After 24 hours of culture, cells were treated (conditions: untreated; 0.01% of the test solution from Example 1 in a 1:1 mixture of NHM supplemented medium and NHK supplemented medium without supplements; and 1 μM Rapamycin) 3 times 24 hours.

[0093] Colorimetric Assay of Melanin

[0094] Melanin content in cells pellets was performed in parallel with proteins assay.

[0095] The cell pellets were taken up in NaOH at 60° C. for 30 minutes. The concentration of melanin in supernatants and in standard range of melanin synthetic were determined by lecture at 405 nm, using synthetic melanin as control.

[0096] Protein Assay (BCA)

[0097] Total proteins assay in cells pellets was performed in parallel by colorimetric method based on bicinchoninic acid. Standard range was prepared with BSA (Bovine Serum Albumin).

[0098] The cell pellets were taken up in NaOH at 60° C. for 30 minutes. Dosage was realized by adding a mixture of the reagents (bicinchoninic acid+CuSO.sub.4) to aliquots of lysates (cells pellets lysed). The plates were incubated at 37° C. for 30 minutes and then lecture is performed at 570 nm.

[0099] Expression of Results

[0100] For both melanin and protein dosages, raw data, i.e. OD measurements obtained for respective standards were plotted on graphics to obtain standard calibration curves. Then, the amount/concentrations of proteins or melanin measured in the samples were determined.

[0101] Quantitative values of each condition were averaged. Data were graphically presented as amount/concentrations (μg/mL). Results obtained for each condition was also expressed relatively to the untreated control, set to 100%:

%.sub.«sample»=(Mean OD.sub.«sample»/Mean OD.sub.«control»)×100

[0102] To obtain melanin concentrations reported to the total proteins amount (μg/mL/mg of proteins), each concentration value (μg/mL) was divided by each proteins data (mg), well by well. Values of each condition were then averaged.

[0103] The statistically significant effects of the results were determined by the Student's t-test.

[0104] Results

[0105] After 72 hours of treatment, the positive reference (1 μM Rapamycin) was found to stimulate significantly the production of melanin in the co-culture model (+282%*) confirming that the model was robust and respondent (FIG. 1).

[0106] The presence of the test solution from Example 1 at 0.01% also demonstrated a stimulating effect on the melanin production. Indeed, the quantity of melanin was increased by 363%**.

EXAMPLE 3: EVALUATION OF MELANIN PRODUCTION: EX VIVO GREY HAIR FOLLICLES

[0107] Tissue Samples

[0108] Micro-dissected hair follicles (HFs) were obtained from occipital healthy human follicular units skin obtained from a healthy female donor (35 years old; donor 1) undergoing hair transplantation surgery or from scalp biopsy from a male donor (53 years old; donor 2) after informed consent and ethics approval (University of Muenster, n. 2015-602-f-S).

[0109] Hair Follicles Organ Culture

[0110] Microdissected human anagen VI scalp HFs (60 and 26 HFs/experiment, respectively) were cultured at 37° C. with 5% CO.sub.2 in a minimal media of William's E media (Gibco, Life Technologies) supplemented with 2 mM of L-glutamine (Gibco), 10 ng/mL hydrocortisone (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich) and 1% penicillin/streptomycin mix (Gibco) to make Williams Complete Media (WCM; J Cell Sci. 1990 November; 97 (Pt 3):463-71. Human hair growth in vitro. Philpott M P1, Green M R, Kealey T; Exp Dermatol. 2010 March; 19(3):305-12. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Kloepper J E1, Sugawara K, Al-Nuaimi Y, Gaspar E, van Beek N, Paus R; Exp Dermatol. 2015 December; 24(12):903-11. Human hair follicle organ culture: theory, application and perspectives. Langan E A, Philpott M P, Kloepper J E, Paus R). After 24 hrs, the medium was replaced with fresh medium containing either WCM (vehicle) or the Test Solution at final concentrations of 1% diluted in WCM. 11 ‘grey’ (defined by being low pigmented) anagen VI HFs were cultured per experimental group. HFs were cultured for a total of 3 days.

[0111] Frozen Hair Follicles Processing

[0112] Frozen samples were sectioned with a cryostat (CM3050S, Leica Biosystems) and 6 μm sections were collected. The HFs were carefully orientated to obtain intact hair follicle sections and open dermal papillae. Consecutive sections of hair follicles were collected and slides were stored at −80° C.

[0113] Masson Fontana Histochemical Staining

[0114] To evaluate melanin as a marker for HF pigmentation, Masson Fontana (MF) staining was performed as previously described (Exp Dermatol. 2010 March; 19(3):305-12. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Kloepper J E1, Sugawara K, Al-Nuaimi Y, Gaspar E, van Beek N, Paus R) on frozen slides. In short, melanin was stained with silver nitrate (Caesar & Loretz, Hilden, Germany) ammonia-based solution and developed with 5% aqueous sodium thiosulphate (Merck Millipore, Darmstadt, Germany).

[0115] Quantitative (Immuno) Histomorphometry

[0116] Pictures were taken with a Keyence Biozero Microscope 8100 and 9000 at an original magnification of 200×.

[0117] Melanin Content in Anagen VI Hair Follicles

[0118] Three areas of 100×175 or 113×159 pixel were measured above the Auber's line to assess pigmentation intensity for donor 1 or 2 in anagen HFs, respectively, using the ImageJ software (Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, Md., USA, https://imagej.nih.gov/ij/).

[0119] Data Management

[0120] All data were expressed as mean or fold change of Mean±SEM. Gaussian distribution was tested with Shapiro-Wilk normality test. Outlier analysis was performed by two-sided Grubbs' test of the pooled data for donor 1 and 2. Significant outliers were removed and subsequently statistical analysis was performed using Student's t test comparing the results of each tested group to vehicle using GraphPad Prism 6 (GraphPad Software). A p value of <0.05 was considered statistically significant (*).

[0121] Results

[0122] The induction of melanin production was evaluated through Fontana Masson staining quantification.

[0123] As can be seen in FIGS. 2 and 3, the test solution from Example 1 was able to induce melanogenesis in greying hair follicles and stimulate the melanin production by 15% (p<0.05).

[0124] These results show that the hair care active agent of the present invention is not only able to stimulate melanogenesis in melanocytes, but also in greying hair follicles.

EXAMPLE 4: TRANSCRIPTOMIC ANALYSIS IN A FULL EX VIVO SCALP

[0125] Skin Explants Culture

[0126] The test was carried out on skin explants NativeSkin®, a full-thickness skin biopsy embedded in a solid and nourishing matrix while its epidermal surface is left in contact with air. The skin biopsy is firmly embedded in the matrix that prevents any lateral diffusion of topically applied formulations.

[0127] The study was conducted on lift explant from 3 donors with sufficient hair follicles and equivalent number between the samples from a same donor: [0128] Donor 1: 66 years old Caucasian Woman with 18 follicles for the untreated, 19 follicles for oxidative treatment and 19 follicles for oxidative treatment with 1% of the test solution from Example 1 in Carbopol®. [0129] Donor 2: 58 years old Caucasian Woman with 10 follicles for the untreated, 10 follicles for oxidative treatment and 9 follicles for oxidative treatment with 1% of the test solution from Example 1 in Carbopol®. [0130] Donor 3: 64 years old Caucasian Woman with 30 follicles for the untreated, 30 follicles for oxidative treatment and 31 follicles for oxidative treatment with 1% of the test solution from Example 1 in Carbopol®.

[0131] Carbopol® consists of acrylic acid crosslinked with allyl sucrose or allyl pentaerythritol.

[0132] Product Evaluation and Design of Study

[0133] The study was conducted on 3 donors (1 explant per condition).

[0134] Oxidative stress was applied for 3 days, once a day, by adding 9 mg of Hypoxanthin+10 units of Xantin oxidase during 1 h of reaction to produce free O.sub.2..sup.− radicals at the skin surface. [0135] Explant+oxidative stress+placebo (Carbopol®) for 48 h [0136] Explant+oxidative stress+test solution from Example 1 at 1% for 48 h

[0137] After treatment, the dermis of each explant was removed by microsurgery to focus gene quantification on epidermal cells.

[0138] Method of Analysis

[0139] qPCR microfluidic technology according to the protocol of Fluidigm® (BMC Genomics. 2011 Mar. 9; 12:144. Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. Jang J S1, Simon V A, Feddersen R M, Rakhshan F, Schultz D A, Zschunke M A, Lingle W L, Kolbert C P, Jen J) was used. Microfluidic technology comes from crossing the world of nanotechnology and gene analysis by q-PCR. System miniaturization has led to the development of a chip that currently allows analysing 48 conditions versus 48 genes.

[0140] The targeted genes cover the following functions: [0141] Hair physiology [0142] Antioxidant activities [0143] Pigmentation

[0144] Skin explants were collected in specific lysis solution for mRNA extraction. Lysates were transferred on plate in order to purify mRNA. Afterwards, a reverse transcription system was used. According to the Fluidigm® protocol, specific stages for 48×48 chip preparation were started. A pre-amplification step was carried out with the primers used in the chip. Pre-amplified cDNA/PCR mix and primers were deposited on the chip. The mixture blending was undertaken by the IFC Controller and then the chip was placed in the BioMark™ system in order to carry out real time PCR.

[0145] To confirm an activating or inhibitory effect, the values were compared to the control explant treated with Carbopol® only. The results are expressed as relative expression rate.

[0146] Results

[0147] The impact of oxidative stress was investigated at the transcriptomic level. The scalps were repeatedly stimulated by a mixture of xanthin and hypoxanthin in order to mimic the oxidative stress experienced by the hair follicles during human life. Then, the Test Solution was applied for 48 hours as a curative treatment.

[0148] The results presented in FIG. 4 show the difference of the relative expression compared to the oxidative stress control.

[0149] It was found that the test solution from Example 1 induced a positive response on various biological functions important in the preservation of melanin content.

[0150] Indeed, the test solution from Example 1 was found to stimulate the expression of proteins involved in melanosome biogenesis and transport, such as AP3B1 (+37.8%#), CTNS (+130.4%***), HPS5 (+105.14**), KRT5 (+58.7%*) and MYO5A (+55.07%**).

[0151] It was also found to stimulate the expression of major genes involved in the regulation of the melanin synthesis, such as EDN1 (+165.56%***), MC1R (+72.64%#), MITF (+80.51%*) and POMC (+130.51%**).

[0152] Furthermore, the test solution from Example 1 had an impact on the expression of genes involved in renewal and autophagy, such as FST (150.95%**), KRT19 (+119.28%**) and MAP1LC3A (+73.47%#).

[0153] Finally, the test solution from Example 1 induced the expression of genes participating of the antioxidant response at various level such as HMOX1 (+110.47%**), GLRX (+37.47%*), GSS (+39.70%**), MGST1 (+118.63%**) and NRF2 (+30.41%#).

EXAMPLE 5: EVALUATION OF THE IMPACT OF OXIDATIVE STRESS ON MICRO-DISSECTED HAIR FOLLICLES

[0154] Microdissection and Hair Shaft Elongation

[0155] Anagen VI human hair follicles (HFs) were dissected from the scalp (Donor: female born in 1962). The HFs used were selected after 18 hours of pre-culture. The following parameters were evaluated in the selection procedure: growth rate (>0.18 mm/18 hours), morphology (no signs of dystrophy).

[0156] Starting from day 1, after the selection at day 0, the test compounds were dissolved in the culture medium and the HFs were cultured up to planned endpoints. Twelve hair follicles were used per treatment for each read out parameter. The culture medium was renewed every other day.

[0157] ROS Evaluation

[0158] After the selection, the HFs were incubated for 1 hour with the standard solution from Example 1 dissolved in William's E medium. Afterwards, the HFs were cultured for 30 min in the presence of dichlorofluoerescein diacetate (DCFH-DA), a probe that reacts with ROS to become fluorescent. Following the DCFH-DA incubation, the HFs were rinsed in PBS and incubated with cumene hydroperoxide at 50 μM (oxidative stimulus) for 1 hour. At the end of the experimental phase, the HFs were harvested, cryo-fixed and cut at the cryo-micro-tome for consequent image acquisition and image analysis of fluorescence within the sections. A slide for each HF was processed by image acquisition and related analysis (i.e. 12 images for each treatment).

[0159] NKI/Beteb Evaluation

[0160] Total melanocytes quantification was obtained following NKI/beteb-DAPI double immunostaining of the cryo-sections. NKI/beteb antibody (#MON7006-1 by Monosan) recognizes a (pre-) melanosomal antigen present in all melanocytes (i.e. both active melanocytes and melanoblasts).

[0161] On the immunostained sections, image analysis was performed in order to quantify the number of NKI-beteb positive cells within each hair follicle. The obtained value was normalized relative to the total number of cells of the considered area.

[0162] Image and Statistical Analysis

[0163] Image analysis was performed using ImageJ software (NIH, USA).

[0164] All quantitative data was summarized in terms of the mean score, standard deviation and standard error of the mean for each treatment.

[0165] Differences between groups were evaluated by One-way ANOVA with permutation test followed by Tukey and t-test with permutation.

[0166] Results: Limitation of Oxidative Stress by the Standard Solution from Example 1 in Micro-Dissected Hair Follicles

[0167] The oxidative stress represented by the treatment with 50 μM of cumene hydroperoxide induced a significant increase of ROS production (+256%** compared to the untreated condition). The pre-treatment with the standard solution from Example 1 reduced considerably the ROS production by 53%. Thus, the reduction was significant compared to the cumene peroxide control with p<0.01 showing a preventive effect of the active (FIG. 5).

[0168] Representative pictures are presented FIG. 6.

[0169] Results: Impact of Oxidative Stress on Melanocytes in Micro-Dissected Hair Follicles

[0170] It was observed 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, the oxidative stress induced a slight decrease of melanocytes, but the presence of the active mitigated considerably its deleterious effect. Indeed, compared to the cumene hydroperoxide control, the standard solution from Example 1 increased the percentage of NKI/beteb positive cells by +189%**, confirming a major impact on hair follicles melanocytes and melanoblasts protection (FIG. 7).

[0171] Representative pictures are presented in FIG. 8.

EXAMPLE 6: CLINICAL INVESTIGATION

[0172] Introduction

[0173] Two clinical evaluation methods were performed in order to demonstrate the in vivo efficacy of a lotion containing the hair care active agent of the present invention versus a placebo lotion: [0174] reduction of the proportion of white hair using the scoring method [0175] reduction of the proportion of white hair using the counting method of white hair density (number of hair/cm.sup.2).

[0176] Description of Compositions Used

[0177] Hair lotion containing 1% of the hair care active agent of the present invention:

[0178] WATER, GLYCERIN/ACETYL TYROSINE/SODIUM METABISULFITE/LARIX EUROPAE WOOD EXTRACT/GLYCINE/ZINC CHLORIDE/EPIGALLOCATECHIN GALLATYL GLUCOSIDE, SODIUM BENZOATE, CITRIC ACID, SODIUM CITRATE, PPG-26 BUTETH-26, PEG-40 HYDROGENATED CASTOR OIL, AQUA/WATER, FRAGANCE, BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYLIONONE.

[0179] Placebo Hair Lotion:

[0180] WATER, SODIUM BENZOATE, CITRIC ACID, SODIUM CITRATE, PPG-26 BUTETH-26, PEG-40 HYDROGENATED CASTOR OIL, AQUA/WATER, FRAGANCE, BUTYLPHENYL, METHYLPROPIONAL, D-LIMONENE, ALPHA-ISOMETHYLIONONE.

[0181] Panel and Study Conditions

[0182] A double blind, inter-individual and placebo-controlled clinical evaluation was performed on 44 Caucasian male volunteers (18 years old and more) with white hair.

[0183] A first group of 22 volunteers tested the placebo hair lotion and a second group of 22 volunteers tested the hair lotion containing 1% of the hair care active agent of the present invention.

[0184] The treatment was applied by massage on the scalp, once a day for four months.

[0185] Results: Reduction of the proportion of white hair (scoring in %)

[0186] Pictures of the scalp were taken using a Nikon D7100 in combination with the system Canfield Epiflash®, on the first day of the test and after 4 months of daily application of the product. The hair parting area is defined according to the white hair localization. A blind scoring was performed to evaluate the proportion of while hair in the picture.

[0187] The results are shown in the following table:

TABLE-US-00002 Hair lotion containing 1% of the hair care Placebo hair lotion active agent of the present invention mean +/− Δ% p Mean +/− Δ% p SEM mean value SEM mean value T.sub.0 54.8 ± 5.3 59.3 ± 4.6 ns T.sub.4 M 50.2 ± 5   −8% p < 0.05 49.1 ± 4   −17% p < 0.0001 (T0) p < 0.5 (placebo)

[0188] After 4 month of application, a significant reduction of the proportion of white hair by −17%, i.e. 2.1 times more than for the placebo, was observed.

[0189] The proportion of white hair was visibly reduced, with a reduction of white hair by −50% for the best respondent (FIG. 9).

[0190] Results: Reduction of White Hair Density (Number/Cm.sup.2)

[0191] Before treatment, an image was taken two days after the shaving of a 1 cm.sup.2 scalp zone. The instrument used was a Nikon D7100 digital camera in combination with the system Canfield Epiflash® equipped with a contact lens. The contact lens allows flattening hair on the scalp.

[0192] A count of white hair was then done with a specific tool of Photoshop on a 0.7 cm.sup.2 test area (1×0.7 cm) defined on the image. All hairs with a white root within the zone were counted.

[0193] The size and the position of the studied area was the same for all evaluation time. In case of offset, the position of the test area was adjusted.

[0194] The results are shown in the following table:

TABLE-US-00003 Hair lotion containing 1% of the hair care Placebo hair lotion active agent of the present invention Mean +/− Δ% p Mean +/− Δ% p SEM mean value SEM mean value T.sub.0 41.1 ± 6.1 49.1 ± 7.3 ns T.sub.4 M 38.8 ± 4.6 −5% ns 43.4 ± 5.3 −14% p < 0.05 (T0) p < 0.1 (placebo)

[0195] The hair lotion containing 1% of the hair care active agent of the present invention significantly decreased the number of white hair per cm.sup.2, namely by 2.8 times more than the placebo.

[0196] After 4 months, the density of white hair was visibly reduced with a decrease of white hair of −55.7 per cm.sup.2 for the best respondent (FIG. 10).

EXAMPLE 7: EVALUATION OF MELANIN PRODUCTION BY IN VITRO CO-CULTURE MODEL: COMPARISON

[0197] Introduction

[0198] The aim of this study was to compare the effect of the hair care active agent of the present invention, which comprises taxifolin glucoside and N-acetyl tyrosine, with those of L-tyrosine (positive reference), of taxifolin glucoside, and of a mixture of taxifolin glucoside and L-tyrosine. The model used for this comparison was a co-culture of Normal Human Epidermal Keratinocytes (NHEKs) and Normal Human Melanocytes (NHMs).

[0199] Cell Culture

[0200] The cell culture was realized with primary cells isolated from human skin biopsies.

[0201] Normal Human Keratinocytes (NHKs) were seeded at 60000 cells per well in 6-wells plates pre-coated with collagen I. 4 hours later, Normal Human Melanocytes (NHMs) were seeded at 60 000 cells per well in the same 6-wells plates. The cells were incubated for 48 hours in complete medium (EpiLife® supplemented with HKGS=Human Kerationcyte Growth Supplement).

[0202] After 48 hours of culture, the cells were stimulated for 5 days with the following compositions: [0203] L-tyrosine at 450 μg/mL (Sigma) [0204] Taxifolin glucoside at 0.13 μg/mL [0205] Taxifolin glucoside at 0.13 μg/mL+N-acetyl tyrosine at 15 μg/mL (similar to Example 1) [0206] Taxifolin glucoside at 0.13 μg/mL+L-tyrosine at 15 μg/mL (Sigma)

[0207] The compositions were diluted in a basal medium without supplements (EpiLife®). Every 2 days, the treatments were renewed.

[0208] Melanin Extraction and Dosage

[0209] After 5 days of treatment, cells were rinsed off with PBS. Then, in each well, 200 μL of a 0.5N NaOH solution was added. The cell lysates were collected in 1.5 mL microtubes with secure caps. In parallel, a standard range of melanin was prepared (1000 μg/mL). Microtubes of samples and standard range were heated for 1 hour at 80° C. in a dry bath. After the heating, 100 μL of each sample was transferred to a 96-wells plate.

[0210] Optical density was measured at 405 nm to determine the melanin content.

[0211] Statistical Analysis

[0212] All experiments were conducted at least in triplicate.

[0213] A Shapiro-Wilk normality test was realized to evaluate if the data followed the Gaussian Law.

[0214] If it was not significant, the data was evaluated with statistical parametric tests (ANOVA followed by Dunnett's multiple comparisons test).

[0215] If it was significant, the data was evaluated with statistical non parametric tests (ANOVA of Kruskall-Wallis followed by a Mann-Whitney U test).

[0216] Results

[0217] Results are shown in FIG. 11.

[0218] The synthesis of melanin was induced by the positive reference (L-tyrosine at 450 μg/ml) by +35% (p<0.001) compared to the untreated condition. This confirms that the co-culture model was responding and able to produce melanin.

[0219] Taxifolin glucoside alone, however, was not able to induce synthesis of melanin.

[0220] Treatment with a combination of taxifolin glucoside and N-acetyl tyrosine led to an increase in melanin content by +18% (p<0.001) compared to the untreated condition.

[0221] The combination of taxifolin glucoside and L-tyrosine, on the other hand, only led to a slight and non-significant increase in melanin content.

[0222] There is also a significant difference between the stimulation with taxifolin glucoside and N-acetyl tyrosine and that with taxifolin glucoside and L-tyrosine.