SESTRIN ACTIVATORS FOR PREVENTING AND/OR ATTENUATING SKIN AGEING AND/OR HYDRATING THE SKIN AND/OR FOR REGULATING SKIN PIGMENTATION

Abstract

The disclosure relates to the identification and the use of compounds which regulate the expression of at least one Sestrin gene, for preventing and/or attenuating skin ageing, and/or for hydrating the skin and/or for regulating skin pigmentation. The method includes the following steps: a. bringing at least one test compound in contact with a sample of human keratinocytes or melanocytes; b. measuring the expression of at least one Sestrin gene chosen from SESN3, SESN2 and SESN1 in the keratinocytes or melanocytes; c. selecting the compounds for which an activation of at least 1.6 fold of the expression of at least one of the genes is measured in the keratinocytes treated in a. compared with the untreated keratinocytes, or for which a significant modulation of the expression of at least one of the genes is measured in the melanocytes treated in a. compared with the untreated melanocytes.

Claims

1-11. (canceled)

12. In vitro method for screening candidate compounds for preventing and/or attenuating skin ageing, and/or for hydrating the skin, comprising the following steps: a. bringing at least one test compound in contact with a sample of human keratinocytes; b. measuring the expression of at least one Sestrin gene chosen from SESN3, SESN2 and SESN1 in said keratinocytes; c. selecting the compounds for which an increase of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. compared with the untreated keratinocytes.

13. In vitro method for screening candidate compounds for regulating skin pigmentation, comprising the following steps: a. bringing at least one test compound in contact with a sample of human melanocytes; b. measuring the expression of at least one Sestrin gene chosen from SESN3, SESN2 and SESN1 in said melanocytes; c. selecting the compounds for which a significant modulation of the expression of at least one of said genes is measured in the melanocytes treated in a. compared with the untreated melanocytes.

14. Method according to claim 12, wherein step b. is performed before and after step a.

15. Method according to claim 12, wherein it comprises the following steps: a′. preparing at least two samples of human keratinocytes; a. bringing one of the samples into contact with at least one test compound; then b. measuring the expression of at least one Sestrin gene in said samples; and c. selecting the compounds for which an increase of at least 1.6 fold of the expression of at least one of said genes is measured in the keratinocytes treated in a. as compared to the untreated keratinocytes.

16. Method according to claim 13, wherein it comprises the following steps: a′. preparing at least two samples of human melanocytes; a. bringing one of the samples into contact with at least one test compound; then b. measuring the expression of at least one Sestrin gene in said samples; and c. selecting the compounds for which a significant modulation of the expression of at least one of said genes is measured in the melanocytes treated in a. as compared to the untreated melanocytes.

17. Method according to claim 12, wherein the expression of at least one Sestrin gene is measured by quantifying the mRNA of the corresponding gene or by quantifying the protein encoded by the corresponding gene.

18. Method according to claim 13, wherein that the expression of at least one Sestrin gene is measured by quantifying the mRNA of the corresponding gene or by quantifying the protein encoded by the corresponding gene.

19. Method according to claim 12, wherein the test compounds are chosen from chemical products or botanical extracts.

20. Method according to claim 12, wherein the increase of the expression of at least one of said genes measured in step c. is of at least 2 fold as compared to untreated keratinocytes.

21. A method for preventing and/or attenuating skin ageing and/or for hydrating the skin, comprising the administration of an effective amount of a regulator of the expression of at least one Sestrin gene which can be obtained according to the method of claim 12.

22. A method for regulating skin pigmentation comprising the administration of an effective amount of a regulator of the expression of at least one Sestrin gene, which can be obtained according to the method of claim 13.

23. Method according to claim 13, wherein step b. is performed before and after step a.

24. Method according to claim 13, wherein the test compounds are chosen from chemical products or botanical extracts.

25. The method of claim 13, wherein the method also inhibits skin pigmentation.

26. The method of claim 13, wherein the significant modulation of expression is a significant increase of expression.

27. The method of claim 25, wherein the significant modulation of expression is a significant increase of expression.

Description

EXAMPLE

Example 1

Test of the Expression Level of SESN1, SESN2 and SESN3 Genes in Normal Human Keratinocytes Depending on the Differentiation Status

[0086] Protocol:

[0087] Normal Human Epidermal Keratinocytes (PromoCell) derived from juvenile donors were cultivated in 6-well plates in supplemented Keratinocyte Growth Medium (KGM2 basal medium+SupplementMix, Promocell) at 37° C., 5% CO.sub.2. After 24 hours, cells were incubated for additional 48 hours in supplemented medium (proliferating keratinocytes) or in high calcium (1 mM) supplemented medium (differentiated keratinocytes).

[0088] Total RNA was extracted with RNeasy kit (Qiagen), according to manufacturer's instructions. The recovered RNA was quantified with Quant-iT RiboGreen RNA Assay kit (Invitrogen) and reverse transcribed using iScript Reverse Transcription SuperMix Kit (BioRad), according to the manufacturer's instructions. The cDNA generated was then subjected to quantitative real-time PCR (qRT-PCR) for the analysis of gene expression using appropriate Taqman primers corresponding to target and housekeeping genes (Applied Biosystems) and the iQ Supermix (BioRad). The reaction was carried out in a BioRad CFX96 Touch Real-Time PCR Detection System. Results were normalized relative to the expression of housekeeping genes (B2M and RPLPO). Results were expressed in terms of the fold change in expression of the target gene in the treated sample versus the untreated control sample.

[0089] Results:

[0090] Data were obtained from two donors of keratinocytes.

[0091] Evaluation of SESN1 expression level in proliferating (1.0±0.22) versus differentiated (1.53±0.09) keratinocytes shows no major variation.

[0092] Evaluation of SESN2 expression level in proliferating (1.0±0.38) versus differentiated (0.99±0.12) keratinocytes shows no variation with cell differentiation.

[0093] Evaluation of SESN3 expression in proliferating (1.0±0.16) versus differentiated (1.93±0.19) keratinocytes shows an increase in the expression of SESN3 gene with cell differentiation.

Example 2

Test of the Expression Level of SESN1, SESN2 and SESN3 Genes in Normal Human Keratinocytes After UVA/UVB Exposure

[0094] Protocol:

[0095] Normal Human Epidermal Keratinocytes (PromoCell) derived from juvenile donors were cultivated in 6-well plates in supplemented Keratinocyte Growth Medium (KGM2 basal medium+SupplementMix, Promocell) at 37° C., 5% CO.sub.2. At 70% confluency, cells were washed with PBS buffer (Life Technologies), then irradiated or not with UVA (10 J/cm.sup.2) or UVB (20 mJ/cm.sup.2) using a BioSun irradiator (Vilber Lourmat) and further incubated for 24 hours in supplemented Keratinocyte Growth Medium (Promocell).

[0096] A test similar to that of Example 1 was performed to determine Sestrin gene expression levels.

[0097] Results:

[0098] Evaluation of SESN1 expression in keratinocytes from a donor shows no major variation after UV irradiation. In untreated control keratinocytes, the relative expression level was 1.0 (±0.03), after UVA irradiation, 1.09 (±0.05) and after UVB irradiation, 1.26 (±0.08).

[0099] Evaluation of SESN2 expression in keratinocytes from a donor shows an increased expression in irradiated keratinocytes. In untreated control keratinocytes, expression level was 1.0 (±0.03), after UVA irradiation, 1.69 (±0.07) and after UVB irradiation, 1.74 (±0.11).

[0100] Evaluation of SESN3 expression in keratinocytes from a donor shows no major variation after UV irradiation. In untreated control keratinocytes, expression level was 1.0 (±0.02), after UVA irradiation, 1.01 (±0.06) and after UVB irradiation, 0.94 (±0.06).

[0101] Increasing the expression of SESN2 gene in keratinocytes thus participates in the first line of the skin defense against UV-irradiation. Therefore, compounds which stimulate the expression of SESN2 in keratinocytes should also protect against oxidative stress.

Example 3

Test of the Expression Level of SESN1, SESN2 and SESN3 Genes in Normal Human Melanocytes After UVA/UVB Exposure

[0102] Protocol:

[0103] Normal Human Epidermal Melanocytes (Promocell) derived from juvenile donor were cultivated in 6-well plates in Melanocyte Growth Medium (MGM2 basal medium+SupplementMix, PromoCell) at 37° C., 5% CO.sub.2. After culturing for 48 hours, the cells were washed with PBS buffer (Life Technologies) and then irradiated or not with UVA (10 J/cm.sup.2) or UVB (50 mJ/cm.sup.2) using a BioSun irradiator (Vilber Lourmat) and finally incubated for 24 hours in supplemented Melanocyte Growth Medium (Promocell).

[0104] Sestrin gene expression analysis was performed as described in Example 1.

[0105] Results:

[0106] Evaluation of SESN1 expression in melanocytes from a donor shows an increased expression in UVB-irradiated melanocytes. In untreated control melanocytes, expression level was 1.0 (±0.06), after UVA irradiation, 0.77 (±0.05) and after UVB irradiation, 2.91 (±0.23).

[0107] Evaluation of SESN2 expression in melanocytes from a donor shows an increased expression in UVB-irradiated melanocytes. In untreated control melanocytes, expression level was 1.0 (±0.06), after UVA irradiation, 1.04 (±0.09) and after UVB irradiation, 4.66 (±0.18).

[0108] Evaluation of SESN3 expression in melanocytes from a donor shows a decreased expression in UVB-irradiated melanocytes. In untreated control melanocytes, expression level was 1.0 (±0.08), after UVA irradiation, 0.93 (±0.04) and after UVB irradiation, 0.16 (±0.01).

[0109] It emerges from this test that UV irradiation modulates the Sestrin genes expression by the melanocytes and thus probably impacts on melanocyte response towards environmental insults.

Example 4

Assessment of the Expression Profile of SESN1, SESN2 and SESN3 Genes in the Human Skin with Age.

[0110] Protocol:

[0111] The Distribution of Sestrin Proteins in Human Skin was Evaluated by Immunofluorescence, on Paraffin-Embedded Skin Samples from Donors of Various Age Groups.

[0112] Tissue array with sections of 4 months (4 m), 30 years (30 y), 35 years (35 y), 39 years (39 y), 49 years (49 y), 50 years (50 y) and 69 years (69 y) old female skin biopsies was used (Cybrdi). Sections were deparaffinated in xylene, rehydrated in ethanol baths, rinsed in deionised water. Antigen retrieval in 10 mM citrate buffer pH6 at 90° C. for 20 minutes was performed before blocking reaction in goat serum 3% for 1 hour. Slides were incubated overnight at 4° C. with primary antibodies: mouse pAb anti-human SESN1 (Abcam), rabbit pAb anti-human SESN2 (Sigma), rabbit pAb anti-human SESN3 (Abcam). Secondary antibodies: goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 488 (Abcam) were applied 1 hour at room temperature to reveal SESNs staining. DAPI was used for nuclei staining.

[0113] Results:

[0114] Immunofluorescent staining shows that SESN1 is uniformly present in all epidermal cell layers, and that there is no major variation of SESN1 expression with age.

[0115] Concerning SESN2, there is no detectable SESN2 staining in neonatal (4 m) and aged (69 y) skin. A patchy distribution is observed in the basal layer of the epidermis of individuals between 30 and 50 years old.

[0116] SESN3 immunofluorescent staining shows an increasing gradient intensity from basal to granular layers. SESN3 is detected at all ages but varies in intensity with age: its expression is stronger in the 30-39 y old skin samples, as compared to younger (4 m) or older (69 y) skin samples.

Example 5

Assessment of the Expression Profile of SESN1, SESN2 and SESN3 Genes in Human Skin Equivalents After UVB Exposure

[0117] Protocol:

[0118] The Distribution of Sestrin Proteins in UVB-Irradiated Skin Equivalents was Evaluated by Immunofluorescence on Paraffin-Embedded Skin Sections.

[0119] Human Skin Equivalents Protocol:

[0120] Keratinocytes and fibroblasts from juvenile donors were purchased from Promocell. The dermal equivalent consists of a collagen solution containing rat tail collagen type I (BD Biosciences), 10× DMEM medium (Gibco/Invitrogen), sodium bicarbonate (Gibco/Invitrogen) and fibroblasts added into 6 well-culture inserts (BD) and placed in deep 6-well culture plates (BD). After 2 hours, of polymerisation at 37° C., dermal equivalents were equilibrated in supplemented Keratinocyte Growth Medium (KGM2 basal medium+SupplementMix, Promocell) and placed at 37° C., 5% CO.sub.2. After 24 hours, suspension of keratinocytes was added over the gel and submerged for 3 days in supplemented Keratinocyte Growth Medium. The inserts were placed at the air liquid interface in Serum-free Keratinocyte Defined Medium (SKDM: SKDM is a high Ca.sup.2+ medium consisting of KGM2 basal medium, transferrin (Sigma), BSA (Sigma) and L-ascorbic acid (Sigma)) for 10 days. 24 hours before culture arrest, skin equivalents were irradiated, in duplicates, with 100 mJ/cm.sup.2 UVB.

[0121] Immunofluorescence Protocol:

[0122] Human skin equivalents were fixed in 10% formalin before embedding in paraffin and cutting into 5 μm-thick sections. Sections were deparaffinized in xylene, rehydrated in ethanol baths, rinsed in deionised water. Antigen retrieval in 10 mM citrate buffer pH6 at 90° C. for 20 minutes was performed before blocking reaction in goat serum 3% for 1 hour. Slides were incubated overnight at 4° C. with the rabbit anti-human SESN2 antibody (Sigma) or the rabbit anti-human SESN3 antibody (Abcam). The secondary goat anti-rabbit Alexa 488 antibody (Abcam) was applied 1 hour at room temperature to reveal SESN2 or SESN3 staining. DAPI was used for nuclei staining.

[0123] Results:

[0124] In non-irradiated skin equivalents, SESN2 immunofluorescent staining is detected in basal keratinocytes. After UVB exposure, an increased signal intensity is observed in basal keratinocytes and SESN2-positive keratinocytes are also detected in suprabasal layers. These observations demonstrate the stimulation of the SESN2 protein expression in UVB-stressed skin equivalents.

[0125] In non-irradiated skin equivalents, SESN3 immunofluorescent staining is strongly detected in suprabasal layers, notably in granular layers. After UVB exposure, an overall decreased signal intensity is observed.

Example 6

Effect of Sestrin Gene Silencing in Cultured Keratinocytes.

[0126] Protocol:

[0127] Normal Human Epidermal Keratinocytes (PromoCell) derived from juvenile donors were transfected with a silencer RNA specific for SESN2 or SESN3 (Dharmacon) using the Nucleofector® Solution (Amaxa nucleofection kit, Lonza) according to the transfection protocol described by the supplier. Cells transfected with the siRNA to SESN2 or SESN3 and scrambled siRNA (negative control) were cultured for 48 hours in Keratinocyte Growth Medium (KGM2 basal medium+SupplementMix, Promocell) at 37° C., 5% CO.sub.2.

[0128] Samples were then analyzed by quantitative real-time PCR using the same method described in Example 1 to confirm the targeted gene knockdown and also to assess its impact on the expression of autophagy (LC3) and differentiation (Loricrin) markers.

[0129] Results:

[0130] Evaluation of the expression level of the SESN2 gene in keratinocytes after silencing with siRNA to SESN2 indicates up to 79% decrease in SESN2 expression. Evaluation of the expression level of the SESN3 gene in keratinocytes after silencing with siRNA to SESN3 indicates up to 93% decrease in SESN3 expression.

[0131] The results obtained from a donor of keratinocytes show that inactivation of SESN2 expression through the specific silencer RNA decreased the LC3 gene expression level. In control keratinocytes, the expression level was 1.0 (±0.06) and in keratinocytes silenced for SESN2, the expression level was 0.55 (±0.02).

[0132] Inactivation of SESN3 expression through the specific silencer RNA decreased the Loricrin gene expression level. In control keratinocytes, the expression level was 1.0 (±0.08) and in keratinocytes silenced for SESN3, the expression level was 0.65 (±0.05).

Example 7

Test of Stimulation of the Expression of Sestrin Genes in Normal Human Keratinocytes with a Botanical Extract

[0133] Protocol:

[0134] Botanical Extract 1 (Solidago):

[0135] Solidago extract is prepared as follows: the extract of Solidago virgaurea is obtained by extraction of crushed dried aerial parts with ethanol (or any alcoholic solvent), discoloration with activated charcoal, filtration and dilution with 1,3-propanediol (or other appropriate cosmetic solvent) so as to obtain a final extract on a liquid form.

[0136] Normal Human Epidermal Keratinocytes (PromoCell) derived from juvenile donors were cultivated in 6-well plates in supplemented Keratinocyte Growth Medium (KGM2 basal medium+SupplementMix, Promocell) at 37° C., 5% CO.sub.2. At 70% confluency, cells were incubated for 24 hours with KGM2 basal medium (Promocell) containing the botanical extract tested, at various non-toxic concentrations, in triplicates. The cytotoxicity of the botanical extract was evaluated in human cultured keratinocytes before testing the activity.

[0137] Samples were then analyzed by quantitative real-time PCR using the same method described in Example 1.

[0138] Results:

[0139] The results obtained from a donor of keratinocytes are given in Table 1 below:

TABLE-US-00001 TABLE 1 Expression Expression Expression level level level Concentration of SESN1 of SESN2 of SESN3 Untreated —  1.0 ± 0.25  1.0 ± 0.21  1.0 ± 0.25 control Botanical 0.025% 1.74 ± 0.21 1.29 ± 0.20 2.14 ± 0.18 extract 1

[0140] The botanical extract 1 makes it possible to stimulate the expression of SESN1 and SESN3 in normal human keratinocytes.

Example 8

Test of Stimulation of Sestrin Genes Expression in Human Skin Equivalents with a Botanical Extract 2

[0141] Protocol:

[0142] The Effect of a Botanical Extract 2 on the Expression of SESN3 Gene was Evaluated on Human Skin Equivalents by Immunofluorescence

[0143] Synthetic Compound (Retinol) and Botanical Extract 2 (Fenugreek):

[0144] Fenugreek extract is prepared as follows: the extract of Trigonella foenum-graecum is obtained by extraction of crushed seeds with hexane, fractionation of the resulted oil using supercritical extraction process and dilution with 1,3-propanediol (or other appropriate cosmetic solvent) so as to obtain a final extract on a liquid form.

[0145] Human skin equivalents were performed as described in Example 5. Skin equivalents were placed at the air liquid interface for 10 days in serum-free keratinocyte defined medium. Botanical extract was diluted in culture medium for the last 7 days and changed by fresh dilutions every 2 days. Synthetic compound (retinol) was diluted in culture medium for the last 4 days. Each condition was done in duplicates.

[0146] Immunostaining of skin equivalents was performed as described in Example 5.

[0147] Results:

[0148] Immunofluorescence experiment reveals that the SESN3 staining intensity is decreased in skin equivalents treated with the synthetic compound retinol as compared to untreated (control) sample. Differentiation markers evaluated (Cytokeratin 10 and Loricrin) are also decreased after retinol treatment as compared to control. Inversely, SESN3 and Loricrin immunostainings are increased in skin equivalents treated with the botanical extract.

[0149] This observation, combined with those from Examples 4, 5 and 6, strongly suggests that SESN3 expression is linked to the differentiation process of keratinocytes in the epidermis.

[0150] The botanical extract 2 makes it possible to stimulate the expression of SESN3 in human skin equivalents.

Example 9

Effect of Sestrin Gene Silencing in Cultured Melanocytes—Expression Level of TYR gene

[0151] Protocol:

[0152] Normal Human Epidermal Melanocytes (Invitrogen) derived from neonate donors were transfected with a silencer RNA specific for SESN1, SESN2 or SESN3 (Dharmacon) using the Lipofectamine® 3000 Reagent (Lipofectamine® 3000 Transfection Reagent, Thermo Fisher Scientific) according to the transfection protocol described by the supplier. Cells transfected with the siRNA to SESN1, SESN2 or SESN3 and scrambled siRNA (negative control) were cultured for 5 days in Melanocyte Growth Medium M2 (MGM2 basal medium+SupplementMix, Promocell) at 37° C., 5% CO.sub.2.

[0153] The knockdown of the targeted genes and their impact on the expression of Tyrosinase (TYR), a key enzyme controlling the production of melanin, was then analyzed by quantitative real-time PCR using the following method.

[0154] Total RNA was extracted with RNeasy kit (Qiagen), according to manufacturer's instructions. The recovered RNA was quantified by spectrophotometry (Multiskan GO+μdrop plate, Thermo Scientific) and reverse transcribed using iScript Reverse Transcription SuperMix Kit (BioRad), according to the manufacturer's instructions. The cDNA generated was then subjected to quantitative real-time PCR (qRT-PCR) for the analysis of gene expression using appropriate Taqman primers corresponding to target and housekeeping genes (Applied Biosystems) and SsoAdvanced™ Universal Supermix (BioRad). The reaction was carried out in a BioRad CFX96 Touch Real-Time PCR Detection System. Results were normalized relative to the expression of housekeeping genes (B2M, GUSB and TBP). Results were expressed in terms of the mean fold change in expression of the target gene in the treated samples versus the untreated control samples.

[0155] Results:

[0156] Evaluation of the expression level of the SESN genes in melanocyte after silencing with specific siRNA indicates a decrease up to 74% in SESN1, 77% in SESN2 and 89% in SESN3 expression. Data were obtained from two donors of melanocytes.

[0157] The results show that inactivation of SESN1 has no major impact on the TYR gene expression level. In control melanocytes, the relative expression level was 1.0 (±0.00) and in melanocytes silenced for SESN1, the expression level was 0.98 (±0.06).

[0158] Inactivation of SESN2 increases the TYR gene expression level. In control melanocytes, the expression level was 1.0 (±0.00) and in melanocytes silenced for SESN2, the expression level was 1.40 (±0.02).

[0159] Inactivation of SESN3 increases the TYR gene expression level. In control melanocytes, the expression level was 1.0 (±0.00) and in melanocytes silenced for SESN3, the expression level was 1.39 (±0.06).

[0160] This observation suggests that SESN2 and SESN3 expression may influence the process of melanogenesis in human melanocytes.

Example 10

Cosmetic Compositions

[0161] The following compositions are prepared according to conventional methods.

[0162] The amounts of components are indicated in percentage by weight as compared to the total weight of the composition.

[0163] O/W Emulsion:

TABLE-US-00002 INCI/TRADE NAME SUPPLIER (% W/W) Jojoba esters  1-10 Camellia seed oil  1-10 Butyrospermum Parkii Butter (LIPEX SHEA)  1-10 Butyrospermum parkii butter (LIPEX SHEASOFT)  1-10 Shea Butter Ethyl Esters (LIPEX SHEALIGHT)  1-10 Butyrospernum parkii butter extract (LIPEX SHEA TRIS)  1-10 Moringa oil/hydrogenated moringa oil esters & tocopherol (FLORALIPIDS 0.5-5   MORINGA BUTTER) Hydrogenated coconut oil 0.1-7   phytosteryl/octyldodecyl lauroyl glutamate (ELDEW PS-203) 1-5 cetearyl alcohol & cetearyl glucoside (MONTANOV 68 EC) 1-5 hydrogenated lecithin & C12-16 alcohols & palmitic acid (BIOPHILIC H) 1-5 PEG-8 BEESWAX (APIFIL CG) 1-5 polyglycery1-6 distearate & jojoba esters 1 polyglycery1-3 beeswax & 1-5 cetyl alcohol (EMULIUM MELLIFERA) Ammonium Acryloyldimethyltaurate/VP Copolymer (ARISTOFLEX AVC) 1-5 methyl methacrylate crosspolymer (SEPIIVIAT H 10) 1-5 silica & lauroyl lysine (AMILON) 0.1-10  methyl methacrylate crosspolymer (MAKIBEADS 150) 0.1-10  synthetic fluorphlogopite & titanium dioxide & tin oxide (HELIOS R10Y)  1-10 Sodium hyaluronate 0.01-3   Glycerin  1-30 Polyquaternium-51  1-10 Adenosine 0.1-0.5 Niacinamide 0.1-5   Palmitoyl Tripeptide-1 & Palmitoyl Tetrapeptide-7 1-5 Secale Cereale (Rye) Seed Extract 1-5 Ascorbyl glucoside 0.001-5    Solidago extract of example 7 0.001-5    Glycols (Caprylyl Glycol and/or Pentylene Glycol and/or Butylene 0.1-10  Glycol and/or propanediol) Water Qs 100

[0164] O/W Emulsion:

TABLE-US-00003 INCI/TRADE NAME SUPPLIER (% W/W) Jojoba esters 1-10 Camellia seed oil 1-10 Butyrospermum Parkii Butter (LIPEX SHEA) 1-10 Butyrospermum parkii butter (LIPEX SHEASOFT) 1-10 Shea Butter Ethyl Esters (LIPEX SHEALIGHT) 1-10 Butyrospernum parkii butter extract (LIPEX SHEA TRIS) 1-10 PHYTOSQUALAN 0.5-7   cetyl dimethicone (ABIL WAX 9801) 0.1-7   isostearyl isostearate (CRODAMOL ISIS-LQ) 1-5 cetyl alcohol & glyceryl stearate & peg-75 stearate & 1-5 ceteth-20 & steareth-20 (EMULIUM DELTA) sodium polyacrylate (COVACRYL MV 60) 1-5 methyl methacrylate crosspolymer (SEPIIVIAT H 10) 1-5 silica & lauroyl lysine (AMILON) 0.1-10  methyl methacrylate crosspolymer (MAKIBEADS 150) 0.1-10  Sodium hyaluronate 0.01-3   Glycerin  1-30 Polyquaternium-51  1-10 Adenosine 0.1-0.5 Niacinamide 0.1-5   Palmitoyl Tripeptide-1 & Palmitoyl Tetrapeptide-7 1-5 Secale Cereale (Rye) Seed Extract 1-5 Ascorbyl glucoside 0.001-5    Fenugreek extract according to example 8 0.001-5    Glycols (Caprylyl Glycol and/or Pentylene Glycol 0.1-10  and/or Butylene Glycol and/or propanediol) Water Qs 100