COSMETIC USE OF KHAYA SENEGALENSIS EXTRACT

Abstract

The present invention relates to the cosmetic use of a Khaya senegalensis extract, obtained by aqueous extraction, for maintaining and/or increasing the expression of collagen XVIII in the skin and/or the mucosae and/or the scalp and/or the skin appendages, for maintaining and/or increasing the firmness and/or the elasticity of the skin and/or the mucosae and/or the scalp and/or for reducing the visibility of the skin's pores and/or making the skin smoother and/or for limiting and/or reducing perspiration and/or limiting and/or reducing loss of head hair and/or body hair and/or increasing the growth of head hair and/or body hair and/or reducing or limiting sebum production. Finally, the present invention relates to a cosmetic care method comprising the application of a Khaya senegalensis extract or of a cosmetic composition comprising same, for maintaining and/or increasing the expression of collagen XVIII in the skin and/or the mucosae and/or the scalp and/or the skin appendages, for maintaining and/or increasing the firmness and/or the elasticity of the skin and/or the mucosae and/or the scalp and/or for reducing the visibility of the skin's pores and/or making the skin smoother and/or for limiting and/or reducing perspiration and/or limiting and/or reducing loss of head hair and/or body hair and/or increasing the growth of head hair and/or body hair and/or reducing or limiting sebum production.

Claims

1-14. (canceled)

15. A cosmetic method for maintaining and/or increasing the expression of collagen XVIII in the skin and/or the mucous membranes and/or the scalp and/or the skin appendages comprising administering to a patient in need thereof of an effective amount of a Khaya senegalensis extract obtained by aqueous extraction.

16. The method as claimed in claim 15, for maintaining and/or increasing the expression of collagen XVIII in the dermoepidermal junction of the skin, and/or the mucous membranes and/or the scalp and/or the sebaceous glands, and/or the basal laminae of adipocytes, endothelial cells, sudoriparous glands and/or hair follicles.

17. The method as claimed in claim 15, wherein the expression of collagen XVIII is protein expression.

18. The method as claimed in claim 15, wherein the collagen XVIII is expressed by keratinocytes, adipocytes, endothelial cells, epithelial cells of the sudoriparous glands and/or stern cells of hair follicles.

19. The method as claimed in claim 15 for decreasing the visibility of cutaneous pores and/or making the skin smoother and/or for maintaining and/or increasing the firmness and/or elasticity of the skin and/or the mucous membranes and/or the scalp and/or for limiting and/or reducing perspiration and/or limiting and/or reducing the loss of head hair and/or body hair and/or increasing the growth of head hair and/or body hair and/or reducing or limiting the production of sebum.

20. The method as claimed in claim 15, wherein the Khaya senegalensis extract is a bark extract.

21. The method as claimed in claim 15, wherein the extract is applied topically.

22. The method as claimed in claim 15, wherein the Khaya senegalensis extract is present in a cosmetic composition comprising at least one cosmetically acceptable excipient at a concentration from 110.sup.4% to 10% by weight relative to the total weight of the composition.

23. The method as claimed in claim 21, wherein the extract is applied onto the skin and/or the mucous membranes and/or the scalp.

24. The method as claimed in claim 23, wherein the topical application of the Khaya senegalensis extract obtained by aqueous extraction is carried out over all or part of the body and/or the face and/or the scalp.

25. The method as claimed in claim 22, wherein the cosmetic composition comprises the Khaya senegalensis extract according to the invention at a concentration from 110.sup.4% to 5% by weight relative to the total weight of the composition.

26. A method for preventing and/or treating diseases involving a loss of gene and/or protein expression of type XVIII collagen comprising the administration to a patient in need thereof of an effective amount of a Khaya senegalensis extract.

27. The method as claimed in claim 26, wherein the extract is in the form of a dermatological and/or pharmaceutical composition comprising at least one dermatologically and/or pharmaceutically acceptable excipient.

28. The method as claimed in claim 27, wherein the extract is present in the pharmaceutical, composition at a concentration from 110.sup.4% to 10% by weight, relative to the total weight of the composition.

29. The method as claimed in claim 24, wherein the topical application of the Khaya senegalensis extract obtained by aqueous extraction is carried out over all or part of the legs, thighs, arms, stomach, bust, neck, armpits and/or the lips.

30. The method as claimed in claim 24, wherein the topical application of the Khaya senegalensis extract obtained by aqueous extraction is carried out over all or part of the cheeks, forehead, chin, lips and/or the area around the eyes.

31. The method as claimed in claim 24, wherein the topical application of the Khaya senegalensis extract obtained by aqueous extraction is carried out over all or part of the T zone of the face.

32. The method as claimed in claim 25, wherein the cosmetic composition comprises the Khaya senegalensis extract according to the invention at a concentration from 110.sup.3% to 3% by weight relative to the total weight of the composition.

33. The method as claimed in claim 32, wherein the cosmetic composition comprises the Khaya senegalensis extract according to the invention at a concentration from 0.001% to 0.1% by weight relative to the total weight of the composition.

34. The method as claimed in claim 26, wherein the diseases involving a loss of gene and/or protein expression of type XVIII collagen are chosen in the group consisting of the diseases involved in eye development, the diseases involved in cerebral development, the diseases involved in the nervous system and certain glomerulopathies.

Description

LIST OF THE FIGURES

[0113] FIG. 1: Evaluation of the biomechanical properties of the skin, expression of the residual deformation of the skin and of the viscoelastic component.

[0114] FIG. 2: Coefficients of in vivo measurement of skin elasticity.

EXAMPLE 1

[0115] Preparation of different Khaya senegalensis extracts

Example 1a

[0116] An amount of 20% by weight of bark from the Khaya senegalensis plant, relative to the total weight of bark and of water, was milled then extracted in water as sole solvent at a temperature of 50 C. during a period of 4 hours, then at a temperature of 85 C. during a period of 1 hour. The extract was then centrifuged and the pellet was eliminated.

[0117] The extract was then cooled to a temperature of 4 C. The liquid extract obtained was filtered and maltodextrin was added (in an amount such that the maltodextrin represents 50% by weight relative to the total weight of the mixture of liquid extract and maltodextrin obtained after dehydration).

[0118] The whole mixture was UHT sterilized then spray-dried.

Example 1b

[0119] An amount of 10% by weight of bark from the Khaya senegalensis plant, relative to the total weight of bark and of water, was milled then extracted in water as sole solvent at a temperature of 50 C. during a period of 4 hours, then at a temperature of 85 C. during a period of 1 hour. The extract was then centrifuged, then cooled to a temperature of 4 C. The liquid extract obtained was filtered, and maltodextrin was added. The extract was UHT sterilized then spray-dried.

Example 1c

[0120] An amount of 10% by weight of leaves from the plant, relative to the total weight of leaves and of water, was milled then extracted in water as sole solvent at a temperature of 50 C. during a period of 4 hours, then at a temperature of 85 C. during a period of 1 hour. The extract was then centrifuged, then cooled to a temperature of 4 C. The liquid extract obtained was filtered, dried, and maltodextrin was added.

[0121] The extract was UHT sterilized then spray-dried.

Example 1d

[0122] An amount of 20% by weight of bark from the Khaya senegalensis plant, relative to the total weight of bark and of the solvent mixture below was milled then extracted in a water/ethanol mixture (70/30; v/v) at a temperature of 85 C. during a period of 1 hour. The extract was then centrifuged and the pellet was eliminated. The maltodextrin was added. The extract was spray-dried.

Example 1e

[0123] An amount of 20% by weight of bark from the Khaya senegalensis plant, relative to the total weight of bark and of water, was milled then extracted in water as sole solvent at a temperature of 50 C. during a period of 4 hours, then at a temperature of 85 C. during a period of 1 hour. The extract was then centrifuged and the pellet is eliminated.

[0124] The extract is then cooled to a temperature of 4 C. The liquid extract obtained was filtered and maltodextrin was added (in an amount such that the maltodextrin represents 50% by weight relative to the total weight of the mixture of liquid extract and maltodextrin obtained after dehydration).

[0125] The whole mixture was UHT sterilized then spray-dried. The extract is then formulated in the form of a cosmetic active ingredient as follows: The spray-dried extract is thus diluted in a mixture of water and glycerin at an amount of 1.5% by weight relative to the total weight of the water/glycerin/extract mixture and 80% of glycerin by weight relative to the total weight of the water/glycerin/extract mixture, then filtered. The extract obtained is a liquid extract.

Example 2

Demonstration of the Decreased Expression of Collagen XVIII over the Course of Aging of the Skin

[0126] Protocol: Samples of facial skin from 6 donors of different ages (from 10 to 69 years), referred to as normal, that is to say not having a disease, were collected then frozen at 80 C. The samples were cut up then fixed with a mixture of methanol/acetone during a period of 10 minutes and rinsed with PBS (phosphate-buffered saline) buffer. The samples were then incubated in the presence of a primary anti-collagen XVIII antibody (an antibody that specifically targets the collagenic part of collagen XVIII and not the endostatin part) at ambient temperature. After rinsing with PBS buffer, the samples were incubated with a secondary rabbit antibody containing a fluorescent Cy3 marker. After rinsing, the cell nuclei were stained with DAPI reagent (4,6-diamidino-2-phenylindole). The decrease in the protein expression of collagen XVIII present at the dermoepiderrnal junction is observed and quantified using a confocal microscope and quantified by image analysis (3 biopsies per donor (n=3) and 5 fields per biopsy). (SD: standard deviation).

[0127] Results:

TABLE-US-00001 TABLE 1 MEAN SD Donors aged 10 years 36.81 11.79 Donors aged 15 years 27.07 4.29 Donors aged 30 years 20.60 8.79 Donors aged 44 years 16.51 3.19 Donors aged 60 years 14.26 2.72 Donors aged 69 years 13.34 3.98

[0128] Conclusion: The results showed a decrease in the expression of collagen XVIII with age of the donors, demonstrating the link between the expression of collagen XVIII and the aging of the skin.

Example 3

Increasing the Expression of Collagen XVIII in the Presence of a Khaya senegalensis Extract

Example 3a

Increasing the Expression of Collagen XVIII in Keratinocytes

[0129] Protocol: Normal human keratinocytes, that is to say not having a disease, originating from the breast of a healthy 36-year-old female donor (female, breast) were cultured in a defined medium (KSFM) during a period of 48 hours in the presence of 2 different final concentrations of the Khaya senegalensis extract prepared according to example 1a), then the culture medium was eliminated. The cell layer obtained was fixed (phosphate-buffered saline (PBS) buffer/paraformaldehyde) then permeabilized (PBS buffer/Triton).

[0130] The collagen XVIII was assayed with an anti-collagen XVIII antibody, diluted to 1/500 in a PBS buffer solution containing bovine serum albumin (BSA). After a period of 90 minutes, a secondary antibody coupled to fluorescein diluted to 1/200 is applied for a period of 2 hours in darkness.

[0131] After drying, the mixture was resolubilized with a solution of ammonium hydroxide. The fluorescence was measured (ENVision, PerkinElmer). The fluorescence results were standardized relative to the fluorescence obtained with the same cell medium in the absence of Khaya senegalensis extract (control) and were related to the cell viability obtained under each condition. The results presented correspond to the mean of 6 tests (n=6). (SD: standard deviation).

[0132] Results:

TABLE-US-00002 TABLE 2 MEAN SD Control 100 14.0 K. senegalensis extract 3 10.sup.3 % (w/v medium) Ex. 1a) 154.0 18.6 K. senegalensis extract 1.5 10.sup.3 % (w/v medium) 149.9 11.1 Ex. 1a)

[0133] Conclusion: the Khaya senegalensis extract according to the invention significantly increased protein expression of collagen XVIII by human keratinocytes by at least +49% versus the untreated control (Dunnett's statistical analysis test, p<0.001).

Example 3b

Increasing the Expression of Collagen XVIII in Adipocytes

[0134] Protocol: Subcutaneous human pre-adipocytes that are referred to as normal, that is to say not having a disease, originating from a female donor, were cultured in a specific growth medium during a period of 3 days at a temperature of 37 C. under 5% CO.sub.2 atmosphere. When the stage of saturation of the cell layer was reached, the medium was replaced by a differentiation medium, into which had been added the Khaya senegalensis extract according to the invention, prepared under the conditions described in example 1a), at different final concentrations in the medium (w/v), and cultured during a period of 6 days.

[0135] The protein expression of collagen XVIII was evaluated by immunocytochemistry. The above cultured cells were fixed with acetone at 20 C. for a period of 10 minutes then rinsed (PBS buffer) and placed in a serum for a period of 30 minutes at 37 C. and rinsed (PBS buffer). A primary anti-collagen XVIII antibody diluted to 1/200 was then incubated during a period of 2 hours at ambient temperature. After rinsing, a secondary antibody diluted to 1/200 was incubated during a period of 45 minutes at ambient temperature and away from light. The medium was then eliminated, then the cells were counterstained (Evans Blue) during a period of 10 minutes, then rinsed (PBS). The observations were carried out by confocal microscopy. The collagen XVIII was quantified after image analysis. The increase in the protein expression of collagen XVIII is proportional to the percentage occupation of said collagen on the images.

[0136] The results of table 3 are expressed as the mean of the percentage occupation of the collagen XVIII (n=6). (SD: standard deviation) Results:

TABLE-US-00003 TABLE 3 MEAN SD Control 4.1 0.4 K. senegalensis extract 1 10.sup.4 % (w/v medium) Ex. 1a) 12.9 0.8 K. senegalensis extract 3 10.sup.5 % (w/v medium) Ex. 1a) 8.9 0.8

[0137] Conclusion: the Khaya senegalensis extract according to the invention significantly increased protein expression of collagen XVIII by human adipocytes by at least +117.1% and up to +214.6% versus the untreated control (Shapiro-Wilk statistical analysis test, p<0.001).

Example 3c

Increasing the Expression of Collagen XVIII in Endothelial Cells

[0138] Protocol:

[0139] Human endothelial cells that are referred to as normal, that is to say not having a disease, originating from a healthy 62-year-old donor, were cultured in a specific growth medium (EMB-2) during a period of 5 days at a temperature of 37 C. under 5% CO.sub.2 atmosphere. The cells were then seeded on 8-well Labtecks EZ slide), still in a specific growth medium, into which had been added the Khaya senegalensis extract according to the invention, prepared under the conditions described in example 1a), at different final concentrations in the medium (w/v), and cultured during a period of 3 and 7 days.

[0140] The protein expression of collagen XVIII was evaluated by immunocytochemistry. The cultured cells below were fixed with acetone at 20 C. for a period of 10 minutes then rinsed (PBS buffer) and placed in a serum for a period of 30 minutes at 37 C. and rinsed (PBS buffer). An anti-collagen XVIII antibody diluted to 1/500 was then incubated for a period of 2 hours at ambient temperature. A secondary antibody (Anti-rabbit Alexas 488, Invitrogen) diluted to 1/250 was incubated during a period of 45 minutes at ambient temperature and away from light. The medium was then rinsed, then stained (Evans Blue) during a period of 10 minutes, then rinsed (PBS). The observation was carried out by confocal microscopy.

[0141] The quantification of the collagen XVIII was obtained after image analysis.

[0142] The increase in the protein expression of collagen XVIII is proportional to the percentage occupation of said collagen on the images.

[0143] Results:

[0144] The results obtained showed a significant increase in the expression of collagen XVIII by the endothelial cells.

Example 4

In Vitro Demonstration of the Improvement in the Biomechanical Properties of the Skin in the Presence of a Khaya senegalensis Extract

Example 4a

Improvement in the Residual Deformation of Reconstructed Skin Models

[0145] Protocol: During each step of renewing the medium, the Khaya senegalensis extract according to the invention, prepared under the conditions described in example 1a), was added at a concentration of 0.00075% (w/v). Human keratinocytes that are referred to as normal, that is to say not having a disease, obtained from a breast biopsy from a healthy 30-year-old female donor, and human fibroblasts that are referred to as normal, that is to say not having a disease, obtained from a breast biopsy from a healthy 18-year-old female donor, were obtained. The fibroblasts were cultured in a specific DMEM (Dulbecco's modified Eagle's medium) medium containing 10% calf serum and antibiotics (Normocin, 100 g/ml). The keratinocytes were cultured in a K-FSM medium (Life Technologies) containing antibiotics, until sub-confluence. Dermal fibroblasts were seeded onto a substrate consisting of collagen, chitosan and glycosaminoglycans, and these dermis equivalents were cultured during a period of 28 days at a temperature of 37 C. under 5% CO.sub.2 atmosphere. The medium containing the fibroblasts cultured on specific DMEM medium was supplemented with 10% of fetal calf serum, ascorbic acid (50 g/ml) and antibiotics.

[0146] The keratinocytes were then seeded on dermis equivalents and were cultured during an additional period of 21 days in a simple DMEM medium containing bovine serum albumin (BSA) (0.8%), insulin, hydrocortisone, ascorbic acid and antibiotics. In order to trigger the differentiation process, the reconstructed skins were placed at an air-liquid interface 7 days after the seeding of the keratinocytes.

[0147] The biomechanical properties of the reconstructed skin models were measured via a method based on evaluating the deformation of the skin under a controlled air stream. The deformation of the skin is monitored by virtue of a laser coupled to the equipment. A deformation curve is then obtained (cf. FIG. 1). The residual value reflects the residual deformation of the skin in mm (Residual, FIG. 1). The smaller this residual coefficient is, the more the skin is elastic and resumes its initial form. The results are expressed in mm (n=13) (SD: standard deviation). Results:

TABLE-US-00004 TABLE 4 Residual (mm) SD Control 0.0451 0.0131 Khaya senegalensis extract 7.5 10.sup.4 % Ex. 1a) 0.0318 0.0189

[0148] Conclusion: the results showed that reconstructed skins cultured in the presence of the Khaya senegalensis extract according to the invention have a significantly lower residual deformation rate than when they are cultured in the absence of the extract (Mann-Whitney statistical analysis test p<0.05). The extract according to the invention therefore does indeed make it possible to increase the elasticity of the skin.

Example 4b

Improvement of the Viscoelastic Component of Reconstructed Skins in the Presence of the Khaya senegalensis Extract

[0149] Protocol: The reconstructed skins were cultured under the conditions described above. The evaluation of the viscoelastic component of the skins is reflected via the parameter (Y2Ue)/Y2 (FIG. 1). When said parameter decreases, the elasticity of the skin increases, implying that the skin returns more easily to its initial state. The results are presented in table 5 and are expressed by the mean of 13 tests (n=13).

[0150] Results:

TABLE-US-00005 TABLE 5 MEAN SD Control 0.333 0.031 Khaya senegalensis extract 7.5 10.sup.4 % Ex. 1a) 0.256 0.062

[0151] Conclusion: the results showed a significant decrease (Mann-Whitney statistical analysis test p<0.001) of the parameter (Y2Ue)/Y2 by at least 23.1% relative to the control in reconstructed skins cultured in the presence of the Khaya senegalensis extract according to the invention, reflecting an increase in the elasticity of the reconstructed skins.

Example 5

In Vivo Demonstration of the Effects on the Skin of a Khaya senegalensis Extract

[0152] Protocol: The cream described in example 6 comprising the Khaya senegalensis extract prepared according to example 1e) at a final concentration by weight of 1% relative to the total weight of the cream, and the same placebo cream without extract, were applied every day each to a half face of 25 female Caucasian volunteers aged from 53 to 65 years, having skin with visible pores and having a loss of elasticity, during a period of 2 months.

Example 5a

In Vivo Measurement of the Skin Elasticity

[0153] The skin elasticity was evaluated by cutometry, taking into account the R2 and R7 coefficients. The R2 coefficient corresponds to the Ua/Uf ratio (part between the maximum amplitude and the capacity for deformation of the skin) (cf. FIG. 2), reflecting overall elasticity. The R7 coefficient corresponds to the Ur/Uf ratio, reflecting the immediate elasticity of the skin (FIG. 2). The results are expressed as % increase in elasticity at the times T28 and T56 days, compared to the control (cream without K. senegalensis extract).

[0154] Results:

TABLE-US-00006 TABLE 6 R2 coefficient (Ua/Uf) % improvement vs placebo Ua/Uf coefficient (R2) T56 1% K. senegalensis extract (w/w) +10.70%

[0155] Conclusion: The results showed a significant increase (p<0.05, Student's t test) in the overall elasticity of the skin of the half faces treated with the cream comprising the Khaya senegalensis extract after 56 days of daily application, compared to the skin of the half faces treated with the placebo cream.

TABLE-US-00007 TABLE 7 R7 coefficient (Ur/Uf) % improvement vs placebo Ur/Uf coefficient (R7) T28 T56 1% K. senegalensis extract (w/w) +9.60 +11.60

[0156] Conclusion: The results showed a significant increase (p<0.05, Student's t test) of 9.6% in the immediate elasticity of the skin of the half faces treated with the cream comprising the Khaya senegalensis extract after 28 days of daily application, compared to the skin of the half faces treated with the placebo cream, and of 11.60% in the immediate elasticity of the skin of the half faces treated with the cream comprising the Khaya senegalensis extract after 56 days of daily application, compared to the skin of the half faces treated with the placebo cream.

Example 5b

In Vivo Measurement of the Density of the Cutaneous Pores

[0157] The pore density was evaluated by the fringe projection method, by analyzing the cutaneous microrelief via the parameter referred to as curvature. The larger this parameter is, the greater the surface homogeneity (pores and cutaneous microrelief). Since this measurement was carried out on the cheeks, it can be directly correlated to a measurement of the pore density. A decrease in this parameter therefore reflects a decrease in the density of the cutaneous pores and of the cutaneous microrelief.

[0158] Results: The results are expressed as % reduction in the curvature parameter compared to the placebo.

TABLE-US-00008 TABLE 8 T28 T56 1% K. senegalensis extract (w/w) 22.8% 38.2%

[0159] Conclusion: The results showed a decrease in the parameter of interest, reflecting a significant decrease (p<0.05, Student's t test) in the pore density of 22.80% in the presence of the cream comprising the K. senegalensis extract according to the invention after 28 days of application.

Example 5c

In Vivo Measurement of Skin Wrinkles

[0160] The effectiveness of the extract according to the invention on crow's feet wrinkles was measured at the time T28 (28 days) by measuring the volume of said wrinkles by the fringe projection method under the conditions already described above (protocol for example 5b). The results are expressed as % reduction in the volume of the wrinkles vs. control (cream without extract), in the presence of the cream comprising the K. senegalensis extract according to the invention.

[0161] Results:

TABLE-US-00009 TABLE 9 T56 1% K. senegalensis extract (w/w) 16.2

[0162] Conclusion: The K. senegalensis extract decreased the volume of the wrinkles detected at the time T56 days by 16.2% compared to the control (p<0.05, Student's t test).

Example 6

Example of Cosmetic Composition Comprising a Khava senegalensis Extract

[0163] Methods known to those skilled in the art are used to mix together the different phases A, B, C and D below in order to prepare a composition according to the present invention. The proportions are expressed as %.

TABLE-US-00010 Phase A Glyceryl stearate, PEG-100 Stearate 4.00 Pentaerythrityl distearate 1.50 Cetearyl Isononanoate 3.00 Propylheptyl caprylate 5.00 Coco caprylate 2.00 Dicaprylyl carbonate 3.00 Dimethicone 1.00

TABLE-US-00011 Phase B Water 64.43 Propylene glycol, phenoxyethanol, chlorphenesin, methylparaben 1.00 Glycerin 1.57 Xanthan gum 0.20 Butylene glycol 2.00 Sodium hydroxide, water 0.15

TABLE-US-00012 Phase C Carbomer 0.15 Water 10

TABLE-US-00013 Phase D Khaya senegalensis extract obtained according to example 1 e) 1.00