Use of a <i>Nephelium lappaceum </i>extract for increasing the firmness of the skin and/or of the mucous membranes

Abstract

The invention relates to the use of various extracts of the Nephelium lappaceum plant for increasing the firmness and/or elasticity of the skin and/or mucous membranes, by increasing type I and/or type V collagen and/or LOX-L, fibulin-5, emiline-1 and/or fibrillin-1 gene and/or protein expression, and/or by decreasing CYR61 expression, in the skin and mucous membranes. Another subject of the invention relates to the use of said extracts in a cosmetic composition comprising at least one cosmetically acceptable excipient. Another subject further relates to a cosmetic care method comprising the application of the extract according to the invention or of a cosmetic composition comprising it, for increasing the firmness and/or elasticity of the skin and/or mucous membranes, by increasing type I and/or type V collagen, fibrillin-1 and/or LOX-L, fibulin-5, emiline-1 and/or fibrillin-1 gene and/or protein expression, and/or by decreasing CYR61 expression, in the skin and mucous membranes. A final subject relates to an N. lappaceum extract for use thereof, alone or in a dermatological or pharmaceutical composition, in the prevention and/or treatment of pathological conditions involving a loss of collagen protein and/or gene expression and/or a pathological loss of firmness of the skin and/or mucus membranes, such as rosacea and telangiectasia.

Claims

1. A method of increasing the firmness and/or elasticity of the skin and/or mucous membranes comprising administering an effective amount of a Nephelium lappaceum extract or a cosmetic composition comprising the Nephelium lappaceum extract to a human subject in need thereof, wherein the Nephelium lappaceum extract is a leaf extract obtained by extracting with water as sole solvent in an amount of 1-20% by weight based on the total weight of the water, at a temperature of 60 to 90° C. for a time of 30 minutes to 12 hours followed by drying, wherein the Nephelium lappaceum extract or the cosmetic composition is topically applied, and wherein the Nephelium lappaceum extract is present in the cosmetic composition at a concentration of from 1×10.sup.−4% to 3% by weight, relative to the total weight of the composition.

2. The method of claim 1, wherein the firmness and/or elasticity of the skin and/or mucous membranes is increased.

3. The method of claim 1, wherein the firmness of the skin and/or mucous membranes is increased.

4. The method of claim 1, wherein the Nephelium lappaceum extract increases gene and/or protein expression of type I and/or type V collagen.

5. The method of claim 1, wherein the Nephelium lappaceum extract increases gene and/or protein expression of LOX-L, fibulin-5, emilin-1 and/or fibrillin-1.

6. The method of claim 1, wherein the Nephelium lappaceum extract and/or the cosmetic composition comprising the Nephelium lappaceum extract is topically applied on all or part of a human body selected from the group consisting of leg, thigh, arm, stomach, bust, neck, face, cheek, forehead, chin, lips, area around the lips, area around the eyes, the T zone of the face, and combinations thereof.

7. The method of claim 1, wherein the Nephelium lappaceum extract is obtained by aqueous extraction under subcritical conditions.

8. The method of claim 1, wherein the extract is spray-dried in the presence of a concentration by weight of maltodextrins of between 20% and 90%, between 40% and 80%, or from 70% to 80%, relative to the total weight of the powder obtained.

9. The method of claim 1, wherein the cosmetic composition comprising the Nephelium lappaceum extract further comprises at least one acceptable cosmetic excipient.

10. The method of claim 1, wherein the Nephelium lappaceum extract is obtained by aqueous extraction at 70° C. to 85° C. for 1 to 5 hours.

Description

EXAMPLES

Example 1: Preparation of Various N. lappaceum Extracts According to the Invention

(1) Example 1a) The extract was obtained by maceration in water as sole solvent, of an amount of 10% by weight of dried leaves of the N. lappaceum plant relative to the total weight of leaves and water as sole solvent, at a temperature of 80° C., for a period of 1 hour. The crude extract was decanted, centrifuged and then filtered. This extract may then be subsequently dried.

(2) Example 1b) The extract vas obtained by maceration starting from an amount of 5% by weight of leaves of the N. lappaceum plant relative to the total weight of dried leaves and water as sole solvent, at a temperature of 80° C., for a period of 1 hour. The crude extract was centrifuged and then filtered.

(3) Example 1c) The extract was obtained by maceration starting from an amount of 10% by weight of dried branches of the N. lappaceum plant relative to the total weight of branches and water as sole solvent, at ambient temperature, that is to say at a temperature of 20° C., for a period of 2 hours in the presence of a concentration by weight of 1% of caprylyl/capryl glucoside (Plantacare® 810 UP). The crude extract was decanted, centrifuged and then filtered.

(4) The extracts obtained in examples 1a) to 1c) were then concentrated by evaporation of the solvent and dried by spray drying in the presence of maltodextrins. These extracts are in powder form.

(5) Example 1d) The extraction was carried out by maceration starting from an amount of 10% by weight of dried leaves of N. lappaceum in a propanediol/water mixture (80, 20; w/w) at a temperature of 80° C. for a period of 1 hour. The crude extract was decanted, centrifuged and then filtered.

(6) Example 1e) The extraction was carried out starting from an amount of 10% by weight of dried leaves of N. lappaceum in water under subcritical conditions, in a pressure extraction autoclave, at a temperature of 250° C., under a pressure of 250 bar. The crude extract was decanted, centrifuged and then filtered.

(7) Example 1f) The extraction was carried out by maceration in water as sole solvent, starting from an amount of 10% by weight of fruit pulp of N. lappaceum relative to the total weight of the pulp and water, at a temperature of 80° C. for a period of 1 hour. The crude extract was decanted, centrifuged and then filtered.

(8) Example 1g) The extraction was carried out by maceration in water as sole solvent of an amount of 10% of seeds by weight relative to the total weight of the seeds and water, at ambient temperature, that is to say 20° C., for a period of 2 hours. The crude extract was decanted, centrifuged and then filtered.

(9) Example 1h) The extract was obtained by maceration starting from an amount of 10% by weight of dried branch of the N. lappaceum plant relative to the total weight of branch and water as sole solvent, at a temperature of 80° C., for a period of 1 hour. The crude extract was decanted, centrifuged and then filtered.

Example 2: Increase in Type I Collagen Expression in the Presence of Various N. lappaceum Extracts According to the Invention

(10) Protocol: “normal” human fibroblasts, that is to say fibroblasts exhibiting no pathological condition, from a 34-year-old healthy female donor, were cultured in a defined medium (FGM) for a period of 48 hours in the presence of 2 different final concentrations of various N. lappaceum extracts, then the cell medium was removed. The same culture medium without addition of extract according to the invention was used as a control (Control). The cell layer obtained was lyzed with an ammonium hydroxide solution.

(11) The type I collagen was assayed using the lysate obtained with an anti-collagen I antibody, used at 500 ng/ml in a buffer solution (PBS). After a period of 60 minutes, a secondary antibody used at 40 ng/ml was applied for a period of 60 minutes. After washing, a revealing solution was added and 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 the N. lappaceum extract (Control) and were related to the amount of DNA obtained under each condition. The results presented correspond to the mean of 6 assays (n=6). (SD: Standard deviation).

(12) Results:

(13) TABLE-US-00001 TABLE 1 MEAN SD Control 100 14 Extract of N. lappaceum leaves a 1% (w/v medium 290 72 prepared according to ex. 1a) Extract of N. lappaceum seeds at 10% (w/v medium) 103 5 prepared according to ex. 1g) Extract of N. lappaceum branch at 10% (w/v medium) 94 10 prepared according to ex. 1h) Extract of N. lappaceum pulp at 10% (w/v medium) 95 8 prepared according to ex. 1f)

(14) Conclusion: the extract of N. lappaceum leaves increased the type I collagen protein expression by at least 104% and up to 686% in the fibroblasts analyzed.

Example 3) Increase in Type V Collagen Expression in the Presence of Various N. lappaceum Extracts

(15) Protocol: “normal” human fibroblasts, that is to say fibroblasts exhibiting no pathological condition, from a 34-year-old healthy female donor, were cultured in a defined medium (FGM) for a period of 48 hours in the presence of different final concentrations of various N. lappaceum extracts, then the cell medium was removed. The same culture medium without addition of extract according to the invention was used as a control (Control). The cell layer obtained was lyzed with ammonium hydroxide solution, and then the type V collagen was assayed with an anti-collagen V antibody, diluted to 1/4000 in buffer solution (PBS). After a period of 60 minutes, a secondary antibody diluted to 1/25000 was applied for a period of 60 minutes. After washing, a revealing solution was added and 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 the N. lappaceum extract (Control) and were related to the amount of DNA obtained under each condition. The results presented correspond to the mean of 3 assays (n=3) (SD: Standard deviation).

(16) Results:

(17) TABLE-US-00002 TABLE 2 MEAN SD Control 100 5 Extract of N. lappaceum leaves 1% (w/v medium) 231 39 prepared according to ex. 1a) Extract of N. lappaceum branch 0.1% (w/v medium) 176 24 prepared according to ex. 1c) Extract of N. lappaceum seeds 1% (w/v medium) 134 25 prepared according to ex. 1g) Extract of N. lappaceum pulp 0.5% (p/v medium) prepared 131 10 according to example 1f)

(18) Conclusion: the N. lappaceum extracts increased type V collagen protein expression by at least 4% and up to at least 400% in the normal fibroblasts analyzed, demonstrating their properties of increasing the firmness of the skin and mucous membranes.

Example 4: Increase in Fibrillin-1 Expression in the Presence of Various N. lappaceum Extracts

(19) Protocol: “normal” human fibroblasts, that is to say fibroblasts exhibiting no pathological condition, from a 34-year-old healthy female donor, were cultured in a defined medium (FGM) for a period of 48 hours in the presence of 2 different final concentrations of the N. lappaceum extract, then the cell medium was removed. The same culture medium without the addition of extract was used as a control (Control). The cells were subsequently harvested and then lyzed with a specific lysis buffer in order to carry out the immunolocalization (Western Blot). The protein concentration was determined by the BCA method. The proteins were identified by capillary electrophoresis (ProteinSimple, USA) using an anti-fibrillin primary antibody and immunolocalized using a peroxidase-coupled conjugated secondary antibody. The results were quantified using the Compass Software (version 2.7.1 (ProteinSimple)).

(20) Results:

(21) TABLE-US-00003 TABLE 3 MEAN SD Control 100 9 Extract of N. lappaceum leaves 1% (p/v medium) prepared 239 56 according to example 1a) Extract of N. lappaceum branches 0.1% (p/v medium) 148 1 prepared according to example 1c) Extract of N. lappaceum seeds 1% (p/v medium) prepared 272 95 according to example 1g) Extract of N. lappaceum pulp 0.5% (p/v medium) prepared 166 18 according to example 1f)

(22) Conclusion: the results showed an increase of at least 30% and up to at least 200% of fibrillin-1 protein expression in the normal human fibroblasts, showing its ability to increase the elasticity of the skin and/or mucous membranes.

Example 5: Decrease in CYR61 Protein Expression in the Presence of an N. lappaceum Extract

(23) Protocol: “normal” human fibroblasts, that is to say fibroblasts exhibiting no pathological condition, from a 34-year-old healthy female donor, were cultured in a defined medium (FGM) for a period of 48 hours in the presence of different final concentrations of N. lappaceum extracts, then the cell medium was removed. The same culture medium without addition of extract according to the invention was used as a control (Control). The supernatants were then recovered for analysis. The protein concentration was determined by BCA assay (BiCinchoninic acid Assay). The protein expression was measured by Western Blot (n=4) (SallySue®, ProteinSimple®). The CYR61 protein of interest was detected by capillary electrophoresis (Antibody AbCam (ab24448)e diluted to 1/50) then revealed with a horseradish peroxidase-coupled secondary antibody and a chemiluminescent substrate. The chemiluminescent signal was then detected and quantified (Compass® version 23.1 (ProteinSimple®)).

(24) Results:

(25) The results are expressed as relative percentages with respect to the level of CYR61 protein expression in the supernatants of non-treated fibroblasts (Control).

(26) TABLE-US-00004 TABLE 4 MEAN SD Control 100 7.8 Dried extract of N. lappaceum leaves prepared according 79.4 8.2 to example 1a) (4 × 10.sup.−3 w/v medium) Dried extract of N. lappaceum leaves prepared according 52.4 7.9 to example 1a) (8 × 10.sup.−3 w/v medium)

(27) Conclusion: the extract of N. lappaceum leaves showed its ability to decrease CYR61 protein expression. The extract is therefore active on the firmness of the skin and/or mucous membranes.

Example 6: Example of Cosmetic Ingredient

(28) The amounts indicated are as percentage by weight relative to the total weight of the cosmetic ingredient.

(29) Example 6a)

(30) TABLE-US-00005 N. lappaceum extract Ex. 1a) to 1e)  1-20% (w/w) Maltodextrins 80-99% (w/w)

(31) Example 6b)

(32) TABLE-US-00006 N. lappaceum extract Ex. 1a) to 1e) 1% Glycerin 79% Biopropanediol 10% Water 10%

Example 7: Examples of Cosmetic Compositions

(33) The compositions below are prepared according to methods known to those skilled in the art, in particular as regards the various phases to be mixed together.

(34) The cosmetic ingredient is prepared according to example 6 above. The amounts indicated are as percentage by weight relative to the total weight of the composition.

(35) 7a)

(36) TABLE-US-00007 Cosmetic ingredient* 0.2-0.3 EDTA 0.05 Steareth-2 2.00 Steareth-21 2.50 Cetearyl alcohol 1.00 Propylheptyl caprylate 15.00 Sodium hydroxide (30% in solution) 0.10 Mixture of phenoxyethanol, chlorphenesin, benzoic acid, 1.25 Butylene glycol, sorbic acid (Germazide ™ PBS) Mixture of polyacrylate-X, isohexadecane and polysorbate 60 4.00 (Sepigel ™ SMS 60) Water qs 100

(37) 7b)

(38) TABLE-US-00008 Cosmetic ingredient* 0.2-0.3 EDTA 0.05 Steareth-2 2.00 Steareth-21 2.50 Cetearyl alcohol 1.00 Propylheptyl caprylate 15.00 Sodium hydroxide (30% in solution) 0.10 Mixture of phenoxyethanol, chlorphenesin, benzoic acid, 1.25 Butylene glycol, sorbic acid (Germazide ™ PBS) Mixture of polyacrylate-X, isohexadecane and polysorbate 60 4.00 (Sepigel ™ SMS 60) Water qs 100

(39) 7c)

(40) 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 %.

(41) Phase A

(42) TABLE-US-00009 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

(43) Phase B

(44) TABLE-US-00010 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

(45) Phase C

(46) TABLE-US-00011 Carbomer 0.15 Water 10

(47) Phase D

(48) TABLE-US-00012 N. lappaceum extract obtained according to example 6a) 1.00