BERTHOLLETIA EXCELSA EXTRACT AND USE THEREOF

Abstract

The present invention relates to an extract of Bertholletia excelsa seeds obtained from extracting an at least partly deoiled press residue of Bertholletia excelsa seeds and to a method for preparing such extract. Furthermore, the present invention refers to a cosmetic or pharmaceutic composition comprising such extract as active ingredient and to the use of the extract or the composition for skin care.

Claims

1. An extract of Bertholletia excelsa seeds obtainable from extracting an at least partly deoiled press residue of Bertholletia excelsa seeds having an oil content lower than the oil content in Bertholletia excelsa seeds before pressing with at least one alcoholic or hydroalcoholic solvent, wherein the extract comprises at least 1.5% by weight, referred to the total weight of the dry content of the extract, of one or more polyphenols, wherein the residual oil content is more than 15% and less than 50% by weight referred to the Bertholletia excelsa seeds before pressing.

2. The extract of claim 1, wherein the solids content in the extract represents at least 0.1% by weight, referred to the total weight of the extract.

3. The extract of claim 1, wherein the at least partly deoiled press residue is obtained from pressed fresh Bertholletia excelsa seeds.

4. The extract of claim 1, wherein the at least one alcoholic or hydroalcoholic solvent is selected from the group consisting of ethanol, methanol, propanol, butanol, pentanol, phenol, glycerol, 1,3-butylene glycol, propane diol, and mixtures of two or more thereof, and mixtures of one or more thereof with water or an aqueous buffer.

5. The extract of claim 1, wherein the at least one alcoholic or hydroalcoholic solvent is ethanol, a mixture of ethanol and water, or a mixture of ethanol and an aqueous buffer.

6. The extract of claim 1, wherein the solvent is an alcohol/water mixture containing between 10 and 90% (v/v) alcohol, or containing between 25 and 85% (v/v) alcohol, or containing between 50 and 82% (v/v) alcohol, or containing between 60 and 80% (v/v) alcohol, or containing between 65 and 75% (v/v) alcohol, or containing approximately 70% (v/v) alcohol.

7. The extract of claim 1, wherein the at least partly deoiled press residue comprises a residual oil content more than 15 to 45% by weight, referred to the Bertholletia excelsa seeds before pressing.

8. A method for preparing an extract of Bertholletia excelsa seeds of claim 1 comprising the following successive steps: (i) providing an at least partly deoiled press residue of Bertholletia excelsa seeds, comprising a residual oil content more than 15% and less than 50% by weight referred to the Bertholletia excelsa seeds before pressing, wherein the at least partly deoiled press residue is optionally dried; (ii) contacting the deoiled press residue with at least one alcoholic or hydroalcoholic solvent; (iii) enabling homogenization of the deoiled press residue within the at least one alcoholic or hydroalcoholic solvent for 30 minutes to 2 hours, at 15 to 30? C.; (iv) separating the extract from the solid residuals of step (iii), by means of filtration or centrifugation; and (v) concentrating the extract by removing the at least one solvent or parts thereof.

9. An extract obtainable from a method of claim 8.

10. A cosmetic or pharmaceutical composition comprising: (A) the extract of Bertholletia excelsa seeds of claim 1 as an active ingredient; and (B) at least one further cosmetically and/or pharmaceutically acceptable ingredient other than the extract of Bertholletia excelsa seeds, wherein the composition is a composition for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, a shampooing, and a spray, or wherein the composition is a nutraceutical composition which is administered orally.

11. The cosmetic or pharmaceutic composition of claim 10, wherein the composition comprises 0.0001 to 20% by weight, referred to the total weight of the composition, of the at least one extract of Bertholletia excelsa seeds.

12. A method for reinforcing, for preserving, and/or for restoring the skin barrier function, for improving the skin hydration, for preventing and/or mitigating skin aging, and/or for alleviating and/or for preventing skin disorders or damages, comprising administering a therapeutically effective amount of an extract of Bertholletia excelsa seeds of claim 1, to a patient in need thereof.

13. The method of claim 12, wherein the method is for tissue care, wherein the method is for ameliorating the tissue condition selected from the group consisting of dryness, redness, aberrant pigmentation and sebum-deregulation, itching and inflammation, atopic dermatitis, psoriasis, vitiligo, systemic lupus erythematosus, ichthyosis, and skin aging.

14. The method of claim 12, wherein: (a) the tissue is selected from a group consisting of a tissue present in the skin and in the scalp, in particular a dermal tissue, an epidermal tissue, a subcutaneous tissue, and a combination of two or more thereof; and/or (b) the skin is selected from compromised skin, dry skin, healthy skin, normal skin and oily skin.

15. The method of claim 12, wherein the use is for: (a) stimulating skin lipids metabolism, preferably cholesterol, fatty acids, sphingolipids and/or ceramides metabolism; (b) promoting epidermal proliferation and/or epidermal differentiation; (c) regulating keratinocytes cohesion and/or cell cytoskeleton; and/or (d) regulating inflammatory processes by inhibiting the activation of the inflammasome and the proinflammatory molecules.

16. A method of using an extract of Bertholletia excelsa seeds obtainable from extracting at least a partly deoiled press residue of Bertholletia excelsa seeds having a residual oil content of 0 to 50% by weight than the oil content in Bertholletia excelsa seeds before pressing with at least one alcoholic or hydroalcoholic solvent, wherein the extract comprises at least 1.5% by weight, referred to the total weight of the dry content of the extract, of one or more polyphenols, for reinforcing, for preserving and/or for restoring the skin barrier function, for improving the skin hydration, for preventing and/or mitigating skin aging, and/or for alleviating and/or for preventing skin disorders or damages.

17. The extract of claim 1, wherein the extract comprises at least 3% by weight, referred to the total weight of the dry content of the extract, of one or more polyphenols.

18. The extract of claim 17, wherein the solids content in the extract represents at least at least 1.5% by weight, referred to the total weight of the extract.

19. The extract of claim 18, wherein the at least partly deoiled press residue comprises a residual oil content more than 20 to 35% by weight, referred to the Bertholletia excelsa seeds before pressing.

Description

BRIEF DESCRIPTION OF FIGURES

[0106] FIG. 1 (A, B) demonstrates the modulation of epidermal lipids genes and proteins expressions of the reconstructed human skin models treated with an extract of Bertholletia excelsa seeds (seed cake). The set of cellular factors depicted in FIG. 1A comprises STARD2 (StAR-related lipid transfer protein 2), STARD3 (StAR-related lipid transfer protein 3), STARD4 (StAR-related lipid transfer protein 4), APOL1 (apolipoprotein L1), MVK (mevalonate kinase), SGPP1 (sphingosine-1-phosphate phosphatase 1), and FITM2 (fat-inducing protein 2). The set of cellular factors depicted in FIG. 1B comprises DHCR7 (7-dehydrocholesterol reductase), CPT2 (carnitine O-palmitoyltransferase 2), ACOT11 (acyl-coenzyme A thioesterase 11), and TECR (very-long-chain enoyl-CoA reductase).

[0107] FIG. 2 shows the effect of an extract of Bertholletia excelsa seeds of the present invention (EBE) on a reconstructed human skin model on modulation of global lipids content.

[0108] FIG. 3 shows the effect of modulation of the genes and proteins expressions involved in the epidermal differentiation process of the reconstructed human skin models treated with an extract of the present invention. The set of cellular factors comprises FGF7 (fibroblast growth factor 7), LCESA (late cornified envelope protein 5A), KLK14 (kallikrein-14), EGR1 (early growth response protein 1), BTC (probetacellulin), RUNX3 (runt-related transcription factor 3), ZFPM2 (zinc finger protein), and CRNN (cornulin).

[0109] FIG. 4 demonstrates the modulation of the genes and proteins expressions involved in regulation of keratinocytes cohesion and cytoskeleton of the reconstructed human skin models treated with an extract of the present invention. The set of cellular factors comprises EML1 (echinoderm microtubule-associated protein-line 1), EML2 (echinoderm microtubule-associated protein-line 2), WAS (Wiskott-Aldrich syndrome protein), WASF2 (Wiskott-Aldrich syndrome protein family member 1), ARHGAP6 (Rho GTPase-activating protein 6), NEDD9 (enhancer of filmentation 1), SERBS1 (sorbin and SH3 domain-containing protein 1), and CLDN12 (claudin 12).

[0110] FIG. 5 shows the modulation of the keratin 10 (K10) protein expression in the reconstructed human skin models treated with an extract of the present invention.

[0111] FIG. 6 shows the modulation of the genes expressions involved in the regulation of the inflammatory pathways of the reconstructed human skin models treated with an extract of the present invention. The set of cellular factors comprises IL18RAP (interleukin 18 receptor accessor protein), IL26 (interleukin 26), IL4 (interleukin 4), CXCL13 (C-X-C motif chemokine ligand 13), CXCL11 (C-X-C motif chemokine ligand 11), and AIM2 (absent in melanoma 2).

[0112] FIG. 7 relates to the barrier recovery of forearm's skin of volunteers disrupted with tape stripping to reach a TransEpidermal Water Loss (TEWL) value of at least and then treated with placebo formulation or a formulation containing an extract of Bertholletia excelsa seeds of the present invention (EBE).

EXAMPLES

1 Preparation of Extract of the at Least Partly Deoiled Press Residue (Seed Cake) of Bertholletia excelsa

[0113] The seeds of Bertholletia excelsa were harvested. Two days later, the seeds were pressed for recovering the majority of their oil content. The obtained press residues contained up to 30% by weight of oil. Prior to the extraction, the press deoiled residues were decontaminated by heating for obtaining dried press deoiled residues reduced in number of bacteria, mold and yeasts.

[0114] a. Extraction Step:

[0115] Extraction was conducted on the dried and decontaminated at least partly deoiled press residue (hereinafter press residue) as defined above. Approximately 40 kg of each press residues were used.

[0116] Extraction was performed by using solvent extractor according to a standard procedure. For example, extraction was conducted by the means of 70% (v/v) ethanol in water (i.e., ethanol/water ratio of 70:30, (v/v)). The extraction was performed by means of complete homogenization of the at least partly deoiled press residue within the extractor solvent and constant stirring (maximum 500 rpm) during 1 hour at ambient temperature (i.e., 18 to 25? C.).

[0117] b. Filtration Step:

[0118] Subsequently, the obtained hydroethanolic extract was filtered according to a standard procedure. For example, filtration was conducted by using filters with increased pores, starting from tissue having pore diameter from 3 to 10 ?m to avoid microbial contamination. The obtained filtered liquid was limpid and had less than 10 CFU/g (colony forming unit per gram) of bacteria and less than 10 CFU/g of mold and yeast.

[0119] c. Concentration Step:

[0120] The filtered liquid extract obtained in step b) was further concentrated according a standard procedure. For example, concentration was performed with reduced pressure in a constant vacuum, in a continuous stirring at temperature between 50 to 80? C., until complete evaporation of ethanol for obtaining a concentrate, having acceptable microbial content, which preferably constitutes the extract according to the present invention. The total polyphenols content of the said concentrated extract obtained, measured according to the Folin-Ciocalteu method, represented 3% by weight, based on the total weight of the dry extract. For the following, the extract of the at least partly deoiled press residue of Bertholletia excelsa of the present invention obtained at the end the step c) is diluted in a mixture of glycerol:water 80/20 (v/v) and will be called EBE, it has a dry content of 20 g per 1 L of glycerol:water.

[0121] The EBE does not contain in terms of polyphenol components the gallic acid, protocatechuic acid, 2,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, p-coumaric acid, catechin and sinapic acid.

2. In Vitro Studies

[0122] In the following studies, the skin barrier function improvement, the skin hydration effect and the skin anti-inflammatory effect of EBE were demonstrated.

[0123] a. EBE Stimulates Lipids Metabolism and Results in a Global Upregulation of Genes Expressions Involved in Skin Lipids Metabolism

[0124] The stimulatory effect of EBE on lipids metabolism was assessed in vitro by means of a 3D full thickness reconstructed human skin model FTS (Phenion, Henkel). Briefly summarized, EBE formulated at 0.25% by weight or the placebo as described in Table 1, were topically applied at 2 mg/cm.sup.2 on full thickness reconstructed human skin cultured for 24 h and five days at 37? C. with 5% CO.sub.2.

TABLE-US-00001 TABLE 1 Gel cream containing EBE at 0.25% by weight, and placebo EBE at 0.25% Composition of gel cream in (% weight) Placebo by weight Water 96.00 95.75 Ammonium 0.80 0.80 acryloyldimethyltaurate/VP crosspolymer Dicaprylyl ether 1.00 1.00 Aqueous buffer of citric acid 1.00 1.00 and sodium citrate EBE 0.00 0.25 Phenoxyethanol and 1.10 1.10 methylparaben and ethylparaben Fragrance 0.10 0.10

[0125] After 24 hours of tissue culture, total RNAs were extracted using RNeasy Mini kit from Qiagen. Their concentrations and integrities were analysed by spectrophotometry and capillary electrophoresis. Transcriptomic analysis was performed on Affymetrix human Clariom S arrays according to the Affymetrix user manual.

[0126] After five days of culture, three tissues per conditions were sampled for proteomic analysis and three other tissues per conditions were sampled for histologic analyses. Proteins were extracted using a lysis buffer. Protein concentrations were determined using the Bradford method and quality control of the extraction was validated by high resolution SDS-PAGE. Mass spectrometry (MS) analysis were performed on a Dionex U3000 RSLC nano-LC system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). For histology, tissues were divided in two parts: one half were snap-frozen, and the other half were paraffin embedded.

[0127] To analyse data from transcriptomic and proteomic studies, all significantly deregulated genes were analysed using the DAVID bioinformatics resources. The tool identifies functional regulated pathways from large genes or proteins datasets. This analysis showed an enrichment of transcripts involved in lipids metabolism pathway.

[0128] Results:

[0129] The FIG. 1 shows upregulation of genes due to EBE, analysed by Affymetrix technology on FTS models treated with 0.25% by weight of EBE compared to a placebo. All analyses were performed in triplicate. Lipids metabolism was highlighted by DAVID bioinformatics resources as modulated functional pathway, and the list of significantly modulated genes involved in was displayed. Statistical analyses were performed in comparison to the placebo.

[0130] As shown in FIG. 1A, an upregulation of the transcription of genes involved in cholesterol metabolism and its transport (MVK, STARD3, STARD4), ceramides metabolism via activation of sphingolipids pathway (SGPP1), phosphatidylcholine metabolism and transport (STARD2), lipids storage and their transports (FITM2 and APOL1) were demonstrated in the cells tissues treated by EBE. StarD family of proteins that have steroidogenic acute regulatory protein-related lipid transfer domains and function in the transport and metabolism of lipids. StarD3 and 4 are involved in cholesterol and sterols transport.

[0131] Moreover, as shown in FIG. 1B, the proteomic analysis revealed also a stimulation of cholesterol biosynthesis pathway as we found that the protein DHCR7 was upregulated. This enzyme is involved in the production of cholesterol by reduction of C7-C8 double bond of 7-dehydrocholesterol (7-DHC). The proteomic analysis showed also an upregulation of biological targets involved in fatty acids metabolism (CPT2, ACOT11 and TECR). CPT2 is involved in fatty acid beta-oxidation pathway. ACOT11 is an enzyme harboring an acyl-CoA thioesterase activity with a preference for the long chain fatty acyl-CoA thioesters hexadecanoyl-CoA/palmitoyl-CoA and tetradecanoyl-CoA/myristoyl-CoA which are the main substrates in the mitochondrial beta-oxidation pathway. TECR is an enzyme involved in both the production of very long-chain fatty acids for sphingolipid synthesis and the degradation of the sphingosine moiety in sphingolipids through the sphingosine 1-phosphate metabolic pathway. Fatty acids and sphingosine moiety are crucial for ceramides (CERs) production.

[0132] Disruption of the skin's barrier function results in a rapid and marked increase in epidermal cholesterol and fatty acids synthesis. Furthermore, inhibitors of these pathways delay the recovery of the barrier function. The increase of sphingolipids synthesis, which precedes ceramides synthesis, is more delayed than cholesterol and fatty acids, but is equally important for the restoration of skin's barrier function.

[0133] These results provide evidence that skin lipids content, particularly cholesterol, free fatty acids and ceramides were surprisingly effectively upregulated. Consequently, the barrier function is plausibly reinforced. Indeed, the skin barrier function relies on stratum corneum lipids composition mainly dominated by these three aforementioned lipids classes.

[0134] b. EBE Stimulates Lipids Metabolism and Results in a Global Upregulation of Global Skin Lipids Content:

[0135] In order to investigate whether the global skin lipids content is effectively increased, snap frozen samples from day five timepoint were processed for lipids content visualization and quantification using known Nile Red Staining as previously described. FIG. 2 represents the modulation of global lipid content of FTS models treated with 0.25% by weight of EBE or with placebo. All measurements were performed in triplicate. Lipids content was estimated by fluorescence quantification of red fluorescence (polar lipids) and yellow-gold fluorescence (neutral lipids) with ImageJ. Statistical analysis (unpaired t-tests) were performed in comparison to the placebo, *: p<0.05. Nile Red is a fluorescent probe which is yellow-gold in the presence of non-polar lipids (esterified cholesterol and triglycerides) and which is red in the presence of polar lipids (phospholipids and other amphipathic lipids). Fluorescence intensity was quantified using Image J software by measuring the RawIntDen parameter in the epidermis region of interest (ROI), as shown in FIG. 2.

[0136] Results:

[0137] It was demonstrated that the upregulation of lipids metabolism highlighted in the transcriptomic and proteomic study and occurring in a variety of metabolic stages and in a variety of lipids molecules is reflected at the level of epidermis. As demonstrated, EBE upregulates both polar and non-polar lipids content of epidermis and consequently reinforces the barrier function of epidermis.

2.2. EBE Promotes a Better Skin Barrier Function Through the Promotion of the Epidermal Differentiation Process

[0138] Because skin barrier function is largely insured by the stratum corneum, it is closely related to the quality of the epidermal differentiation process underwent by keratinocytes. An interesting effect is the differentiation process, the strongest is the stratum corneum and the better is the skin barrier function.

[0139] FIG. 3 relates to the modulation of gene expression analysed by Affymetrix technology on FTS models treated with 0.25% by weight of EBE or with a placebo (Table 1). All analyses were performed in triplicate. Epidermal differentiation was highlighted by DAVID bioinformatics resources as modulated functional pathway, and the list of significantly modulated genes involved in is presented. Statistical analyses were performed in comparison to the placebo. Statistical test based on the moderated t method implemented in the R Limma 3.26.8 package ***: p<0.001, *: p<0.05. In the same type of in vitro 3D skin model as described above, it was demonstrated that gel cream containing EBE at 0.25% by weight according to Table 1 upregulates genes involved in epidermal differentiation (FIG. 3).

[0140] Results:

[0141] It was demonstrated that by promoting the upregulation of growth factors such as FGF7, EGR1 or BTC, the extract of the present invention (EBE) promotes the renewal of epidermal cells and the promotion of their differentiation. Moreover, the upregulation of CRNN and LCESA confirm the efficiency of keratinocytes proliferation and differentiation processes. Interestingly, it was confirmed at the protein level the significant upregulation of CRNN observed at the transcription level.

2.3. EBE Reinforces Skin Barrier Function by Regulating Keratinocytes Cohesion, and Cytoskeleton

[0142] As the viable epidermis plays also a crucial role in the skin barrier function, it was studied the effect of EBE on this level. Indeed, the viable epidermis exerts the skin barrier function thanks to the cell-cell cohesion, and the cell cytoskeleton, which is a real scaffold for the cell. Moreover, three cytoskeleton structural components the microtubules, actin microfilaments, and intermediate filaments are directly linked to cell junctions, showing their peculiar interest for skin barrier function.

[0143] FIG. 4 relates to the modulation of gene expression analysed by Affymetrix technology on FTS models treated with 0.25% by weight of EBE or by a placebo (table 1). All analyses were performed in triplicate. Cell junctions and cytoskeleton were highlighted by DAVID bioinformatics resources as modulated functional pathways, and the list of significantly modulated genes involved in is presented. Statistical analyses were performed in comparison to the placebo. Statistical test based on the moderated t method implemented in the R Limma 3.26.8 package. **: p<0.01, *: p<0.05.

[0144] FIG. 5 relates to the modulation of keratin 10 (K10) expression analysed by immunofluorescence on FTS models treated with 0.25% by weight of EBE or by a placebo (Table 1). All measurements were performed in triplicate. K10 expression was estimated by fluorescence quantification with ImageJ. Statistical analysis (Mann-Whitney) was done in comparison to the placebo (**: p<0.01).

[0145] Results:

[0146] Interestingly, it was surprisingly found that the EBE upregulates the transcriptional expression of tight junction's components (such as SORBS 1, and NEDD9) (FIG. 4). SORBS1 is the gene coding for Ponsin protein, which is crucial in the cell adhesion process, due to its interaction with numerous proteins located in tight junctions' structures. These tight junction components may all have a role in cytoskeleton organization. For example, it is known that Ponsin mediates the organization of actin cytoskeleton and NEDD9 is involved in the assembly of actin fibers. It was demonstrated that EBE upregulates also the expression of Claudin-12. The family of claudins proteins are the main constituents of tight junctions and interact also with the cytoskeleton. Moreover, EBE upregulates also the expression of EML1 and EML2, two members of EMLs family. The EMLs are a conserved family of microtubule-associated proteins (MAPs) involved in microtubule binding, assembly and regulation. While, it is known that EML1 modulates the assembly and organization of the microtubule cytoskeleton, EML2 binds microtubules and promotes microtubules dynamics to allow the evolution of the cytoskeleton. It was observed also a significant modulation of mRNA transcripts of WAS and WASF1 by EBE. It is known that these two transcripts belong to the WASP family proteins, required for normal cytoskeletal function. WAS and WASF1 (are implicated in actin polymerization and thus formation of actin filaments. In parallel, EBE stimulates also a Rho GTPase-activating protein the ARHGAP6, which is known to be a very important in cytoskeletal organization. Interestingly, this Rho GTPase-activating protein ARHGAP6 is a cytoskeletal protein that promotes actin remodelling.

[0147] Finally, it was demonstrated that EBE upregulates the translation of K10, a cytoskeleton crucial protein in epidermis differentiation process (FIG. 5). Keratins belong to intermediate filaments, a cytoskeleton component. It is agreed that keratins confer mechanical resilience on epidermis, forming intracellular filament meshworks linked into desmosome cell junction. K10 is one of the most keratin found in intermediate filaments of differentiated keratinocytes in the stratum spinosum and stratum granulosum. It was demonstrated that lacking K10 exhibits larger-than-normal suprabasal keratinocytes and defective flattening. Defects in K10 are known to contribute to barrier defect proving, consequently, the importance of K10 in cytoskeleton, cell junction and the resulting barrier functionality.

2.4. EBE Regulates the Inflammatory Pathways in the Skin

[0148] In order to investigate whether EBE is enabling to alleviate skin inflammation, which is part of a skin disorder, it was performed the same studies by using the transcriptomic method, the same material (reconstituted skin) in the same conditions as set forth in paragraph 2.3 above. FIG. 6 relates to the modulation of the genes expressions related to inflammatory pathways analysed by Affymetrix technology on FTS models treated with 0.25% by weight of EBE or by placebo (Table 1). All analyses were performed in triplicate. The list of the significantly modulated genes involved in inflammation is presented. Statistical analyses were performed in comparison to the placebo. Statistical test based on the moderated t method implemented in the R Limma 3.26.8 package **: p<0.01, *: p<0.05.

[0149] Results:

[0150] It was surprisingly found that EBE down regulates the mRNA expression of several biological markers involved in inflammasome activation (AIM-2) and IL-18 dependent signaling, such as IL18RAP, IL-26, IL-4 and CXCL13, but also in JAK/STAT signaling pathway where the CXCL11 chemokine is stimulated (C-X-C motif chemokine ligand 11), two signaling pathways upregulated in dermatological diseases such as psoriasis, atopic dermatitis presenting a well-described defect in barrier function. Additionally, it was also demonstrated a down regulation of granulocyte-macrophage colony-stimulating factor 1 expression in the proteomic analysis.

3. Cosmetic Formulation Example

[0151] A gel cream comprising an extract of the at least partly deoiled press residue of Bertholletia excelsa, according to the Table 1, suitable for a topical application is prepared according to a conventional method.

4. In Vivo Study

[0152] The following study aims to demonstrate the ability of extract of Bertholletia excelsa seeds of the present invention (EBE) to restore the skin barrier function.

4.1 EBE Promotes the Skin Barrier Recovery

[0153] The stimulatory effect of EBE on barrier recovery was assessed by the mean of in vivo study on 15 healthy women aged from 29 to 65 years old, with a mean age of 44?11. In order to generate a skin barrier default, tape stripping was realized on the forearm as many as necessary to reach a TransEpidermal Water Loss (TEWL) value of 20, with a limit of 20 strips. This skin barrier disruption was done in three areas. Mean value of TEWL before tape stripping was 9.88?2.64. One of the areas received an application of EBE formulated at 1%, another area received the placebo as described in the Table 2 below, and a third area was non-treated. TEWL measurements were performed on the three areas, three hours after to evaluate the skin barrier recovery.

[0154] As used herein, TEWL is the amount of water that passively evaporates from inside the skin to outside due to water gradient differences on both side of the skin barrier. TEWL was the parameter used to evaluate skin barrier recovery. An integer barrier is able to retain water whereas a disrupted barrier is responsible for a higher water loss.

[0155] TEWL measurements were performed using a Tewameter TM300? (Courage & Khazaka electronics) which measured the water evaporated from skin in g/cm.sup.2. Twenty consecutive values were measured, the mean value was calculated.

TABLE-US-00002 TABLE 2 Gel cream containing EBE at 1% by weight, and placebo EBE at 1% Composition of gel cream in (% weight) Placebo by weight Water 96.00 95.00 Ammonium 0.80 0.80 Acryloyldimethyltaurate/VP Crosspolymer Dicaprylyl Ether 1.00 1.00 Aqueous buffer of Citric Acid 1.00 1.00 and Sodium Citrate Glycerin, Water, EBE 0.00 1.00 Phenoxyethanol and 1.10 1.10 Methylparaben and Ethylparaben Fragrance 0.10 0.10

[0156] The skin barrier is able to recover naturally in three hours as skin has an intrinsic repair metabolism to protect the body.

[0157] FIG. 7 shows that the skin barrier recovery of the area treated with placebo was essentially not different from the non-treated one, showing that the empty formulation has no effect on skin barrier recovery. As a consequence, it was surprisingly found that EBE promotes the recovery of the skin barrier function as TEWL value is significantly lowest than the one obtained without any formula in 3 hours after disruption. This result confirms that EBE is able to improve the skin barrier function by promoting a better recovery.