COSMETIC COMPOSITION COMPRISING ROSE EXTRACTS

Abstract

The present invention relates to a cosmetic composition for topical application to the skin comprising, in a physiologically acceptable medium, at least an effective amount of at least one aqueous extract of rose and of at least one oily extract of rose, and the use thereof in particular for promoting the natural rhythmic process of skin cells and/or improving the micro-nutritional balance of the skin.

Claims

1. Cosmetic composition for topical application to the skin comprising, in a physiologically acceptable medium, at least an effective amount of at least one aqueous extract of rose and at least one oil extract of rose.

2. Cosmetic composition according to claim 1, characterized in that it comprises at least one aqueous phase comprising said aqueous extract of rose and at least one fat or oil phase comprising said oil extract of rose.

3. Cosmetic composition according to claim 1, characterized in that the aqueous extract of rose and the oil extract of rose are extracts of rose flower, preferably extracts of rose flower of the Evanrat or Jardin de Granville® rose variety.

4. Cosmetic composition according to claim 1, characterized in that the cosmetic composition is in the form of a dispersion of a fat or oil phase in an aqueous phase, in particular selected from the group consisting of emulsions, macroemulsions, nanoemulsions, microemulsions, pickering emulsions, solid dispersions of fat substances, or dispersions of oil droplets stabilized by a polymeric membrane.

5. Cosmetic composition according to claim 1, characterized in that the oil extract of rose comprises an extract of rose flowers in a vegetable oil, preferably sunflower oil, in particular in a weight ratio of 1:99 to 10:90 (plant extract to oil) and is present in the composition in a content ranging from 0.1% to 10%, in particular from 0.5% to 5%, and according to a particular embodiment from 1% to 2% by weight of raw material based on the total weight of said composition.

6. Cosmetic composition according to claim 1, characterized in that the aqueous extract of rose comprises an extract of rose flowers in a polar solvent, in particular in a weight ratio of 0.5:99.5 to 10:90 (plant extract to polar solvent) and is present in the composition in a content ranging from 0.1% to 10%, in particular from 0.5% to 5%, and according to a particular embodiment from 1% to 4% by weight of raw material based on the total weight of said composition.

7. Cosmetic composition according to claim 1, characterized in that it is in the form of a dispersion of oil droplets comprising the oil extract of rose of the Evanrat or Jardin de Granville® rose variety in a gelled aqueous phase comprising the aqueous extract of rose of the Evanrat or Jardin de Granville® rose variety.

8. Cosmetic process to promote the natural rhythmic process of skin cells and/or the micronutritional balance of the skin, comprising the application to the skin, in particular of the body, face and/or neck and in particular of the face and/or neck, of a cosmetic composition as defined in claim 1.

9. Non-therapeutic cosmetic use of at least an effective amount of at least one aqueous extract of rose and at least one oil extract of rose in a cosmetic composition, as an agent to promote and/or improve the natural rhythmic process of skin cells, the micronutritional balance of the skin, the lipid metabolism of the skin, the skin barrier function, the hydration and/or regeneration of the skin.

10. Non-therapeutic cosmetic use according to claim 9, wherein the effective amount of at least one aqueous extract of rose and at least one oil extract of rose stimulates the expression of epidermal clock genes and genes involved in lipid metabolism, the skin barrier, cell differentiation, cell communication and/or cell cohesion.

Description

FIGURES

[0202] FIG. 1: Activation of CRY2 gene transcription in NHK treated with rose extracts.

[0203] FIG. 2: Activation of PER 1 gene transcription in NHK treated with rose extracts.

[0204] FIG. 3: Activation of PER 3 gene transcription in NHK treated with rose extracts.

EXAMPLES

Materials and Methods

[0205] The plant material used to obtain the aqueous and oil extracts of rose illustrated in these examples is rose flowers (petals) of the Evanrat variety, in particular Jardin de Granville® rosebush flowers, available from nurseries.

Aqueous Extract of Rose (‘Cryoextract’)

[0206] The plant material used is rose flowers (petals) of the Evanrat variety, in particular Jardin de Granville® rosebush flowers available from nurseries.

[0207] The aqueous extract of rose flower is obtained by the cryoextraction process described above, in particular by the process described in patent application EP0425391. A cryoextract is obtained comprising 0.5% by weight dry matter (active matter), 49-50% by weight water, 49% by weight glycerol and preservatives qs 100%. The INCI name of this aqueous extract of rose is Water, Glycerine, Rose Extract or Rosa Hybrid Flower Extract, Water, Glycerine.

Oil Extract of Rose (‘Satin Oil’)

[0208] The plant material used is rose flowers (petals) of the Evanrat variety, in particular Jardin de Granville® rosebush flowers available from nurseries.

[0209] The oil extract of rose flower is obtained by the dynamic enfleurage process described above, in particular by the process described in patent application WO2010/112760. The result is an oil extract ‘satin oil’ comprising 0.5-1.5% by weight rose dry matter (active matter), and 98.5-99.5% by weight organic deodorized oleic sunflower oil.

[0210] The INCI name of this oil extract of rose is Rose extract and Helianthus annuus (sunflower) seed oil or Rosa Hybrid Flower Extract, Helianthus annuus (sunflower) seed oil.

Example 1: Effect of the Aqueous Extract of Rose Flower on the Expression of Epidermal Clock Genes (PER1, PER 3, CRY2)

[0211] A study was carried out on the impact of the aqueous extract of rose on the expression of genes involved in the molecular clock in normal human keratinocytes, in particular the genes Period 1 (PER1), Period 3 (PER3), Cryptochrome Circadian Clock 2 (CRY2), involved in metabolic pathways for skin resistance, skin nutrition such as lipid metabolism, glucose metabolism, calcium homeostasis (involved in epidermal differentiation).

[0212] Normal human keratinocytes (NHK) are cultured and then treated with the 2 rose extracts. After NHK treatment, a TaqMan low-density array (TLDA) study on the genes studied is carried out using cDNA obtained after reverse transcription of the total extracted RNA.

[0213] The oil extract of rose is tested at 25 μg/mL and 10 μg/mL (DMSO excipient for compatibility of the oil extract with the cell culture medium).

[0214] The aqueous extract of rose is tested at 1% and 3% (water excipient).

NHK Culture

[0215] Normal human keratinocytes are derived from a skin sample from plastic surgery. The cells are cultured in the complete EpiLife medium at P5 with a seeding density of 50,000 cells per well, in 12-well plates. At subconfluence, the cells are treated 24 hours with the doses of rose extracts described above.

Real-Time Quantitative RT-PCR

[0216] Obtaining Total RNA

[0217] The cell culture medium is removed, and 250 μL of RLT lysis buffer (provided in the NucleoSpin RNA Trace Kit, Macherey-Nagel) is added. The cells are scraped with a cell scraper and then the cell lysate is recovered in a 1.2 mL deep-well (provided in the NucleoSpin RNA Kit). Total RNA is extracted according to the defined protocols.

[0218] The total RNA solutions obtained are measured and their quality verified using a microplate reader, the SPECTROstar Nano (BMG Labtech) coupled to the Microlab STAR. This device is connected to the computer controlling the automated system and has the specific software for the analysis of results (MARS software). The technique requires a 384-well microplate (LoBase), a positive control (RNA 250, AM7155, Thermo Fisher) to validate the pipetting performed by the robotic system as well as the values generated by the SPECTROstar Nano reader.

[0219] Synthesis of Complementary DNA

[0220] The reverse transcription (RT) kit used is the High Capacity Reverse Transcription Kit (Thermo Fisher). It was used according to the protocol provided. Total RNA (500 ng) is diluted in water to a final volume of 25 μL. It is then incubated for 10 minutes at 25° C. and then 2 hours at 37° C. in the presence of 25 μL of High Capacity Reverse Transcription Kit 2× reaction mixture previously prepared as indicated below. The different incubations are done within the TRobot (Biométra).

TABLE-US-00001 TABLE 1 High Capacity Reverse Transcription Kit 2X reaction mixture for 1 reaction Reagents RT buffer dNTP Primer RNase OUT RT H2O Volume 5 μL 2 μL 5 μL 0.5 μL 2.5 μL 10 μL

[0221] PCR-TaqMan Low-Density Array

[0222] Each RT (15 μL) is mixed with 60 μL of water and then 75 μL of TaqMan Gene Expression Master Mx (Thermo Fisher item 4369510), containing the DNA polymerase, is added. After homogenization, 100 μL is deposited on microfluidic cards containing the probes corresponding to the clock genes tested (Table 2 below), the latter are centrifuged and then sealed. The CD-ROM corresponding to the profile of the genes deposited on the plates is loaded into the SDS 2.3 software, specifying the location of each gene on the card. The control gene (or “endogenous” gene) to be used for normalizing the results should be indicated before the start of PCR. This is done according to the protocol provided by Applied Biosystems in the ABI Prism 7900HT Sequence Detection System. The qPCR steps are 2 min at 50° C., 10 min at 94.5° C. then 30 s at 97° C. and 1 min at 59.7° C. for 40 cycles.

TABLE-US-00002 TABLE 2 List of genes on the microfluidic card. RefSeq GenBank Gene TaqMan item no. accession number Per1 Hs01092603_m1 NM_002616.2 Per3 Hs00997925_m1 NM_016831.2 Cry2 Hs00323654_m1 NM_021117.3 Beta-2-M Hs00187842_m1 NM_004048.2 (control gene)

Statistical Analyses

[0223] Real-time quantitative PCR can be used if its effectiveness is between 90% and 110%. For each sample, the number of cycles at which the signal appears is determined by the SDS 2.3 software. For the same test, the expression levels of the transcripts of interest obtained are normalized to the value obtained for the housekeeping gene beta-2-microglobulin. This gene, whose expression is constitutive and invariant, makes it possible to avoid any variations induced during the experiment (total RNA assay, pipetting, reverse transcription step, PCR in the apparatus).

[0224] In the RT-PCR TLDA method, quantification is performed using the ΔΔCt comparative method. The relative quantification (RQ) values obtained correspond to the amplitude level (x times more or less than the control) of the expression compared to our control, here the non-irradiated one. The RQ is obtained by the following calculation where the control is equal to 1:

RQ=2.sup.−ΔΔCt=2.sup.−(ΔCt treated−ΔCt untreated) [0225] ΔCt treated=Ct target gene treated−Ct housekeeping gene treated [0226] ΔCt untreated Ct target gene untreated−Ct housekeeping gene untreated

[0227] In order to evaluate statistically significant changes in transcriptional activity, we will use Student's t-test. Each condition is carried out in triplicate (3 untreated and 3 treated under the same conditions). Fischer's F-test is first applied by comparing the two data matrices. When the value is greater than α=0.05 then the variance for Student's t-test is 2, when the Fischer F-test is less than α=0.05 then the variance will be 3. The transcriptional variations selected will be those with a Student's t-test lower than α=0.05.

[0228] The results are presented as an average with n=3. Student's t-test was used to compare the effect between treated and untreated cells.

[0229] The results are considered significant for p<0.05(*) or p<0.01(**).

Effect on CRY2

[0230] FIG. 1 shows a significant increase in transcriptional activity for the aqueous extract of rose flower at 3% which increases expression of the CRY 2 gene by 28%, and the oil extract of rose flower at 25 μg/mL which increases expression of the CRY 2 gene by 12%.

Effect on PER 1

[0231] FIG. 2 shows a significant increase in the expression of the PER 1 gene.

Effect on PER 3

[0232] FIG. 3 shows a significant increase in transcriptional activity for the aqueous extract of rose flower at 3% which increases gene expression by 38%.

[0233] The aqueous extract of rose flower alone therefore increases the effect of the CRY2, PER1, and PER3 genes.

Example 2: Effect of the Oil Extract of Rose Flower on the Expression of the Clock Gene PER 2

[0234] An oil extract of rose flower at 1 μg/mL (DMSO excipient) was tested on NHK according to a protocol similar to that described in example 1.

TABLE-US-00003 TABLE 3 List of genes on the microfluidic card. RefSeq GenBank Gene TaqMan item no. accession number Per2 Hs00256143_m1 NM_022817.2 Beta-2-M Hs00187842_m1 NM_004048.2 (control gene)

[0235] A 21% increase in the expression of the PER 2 gene is observed.

[0236] The oil extract of rose flower therefore has an effect complementary to the aqueous extract of rose on the expression of clock genes.

Example 3: Effect of Oil Extract of Rose Flower on Gene Expression in Normal Human Keratinocytes

[0237] An oil extract of rose flower at 1 μg/mL (DMSO excipient) was tested on NHK according to a protocol similar to that described in example 1.

TABLE-US-00004 TABLE 4 List of genes present on the microfluidic card. RefSeq GenBank Gene TaqMan item no. accession number Differentiation/ CERS3/LASS3 Hs00698859_m1 NM_178842.4 Skin barrier/ CALM3 Hs00270914_m1 NM_005184.2 Lipids KRT1 Hs00196158_m1 NM_006121.3 Accession/ GJA1/CX43 Hs00748445_s1 NM_000165.4 Cohesion/ DSC3 Hs00170032_m1 NM_001941.4 Communication Beta-2-M Hs00187842_m1 NM_004048.2 (control gene)

[0238] The oil extract of rose flower at 1 μg/mL, after 24 hours of treatment showed significant stimulation on the expression of the following target genes: [0239] ceramide synthase (CERS3): +33% [0240] calmodulin 3 (CALM 3): +28% [0241] keratin 1 (KRT1): +14% [0242] gap junction alpha-1 protein or connexin 43 (GJA1/CX43): +23% [0243] desmocollin 3 (DESC3): +23%

[0244] The oil extract of rose flower according to the invention therefore has an effect on epidermal clock target genes, involved in lipid metabolism, the skin barrier, cell differentiation, cell communication and/or cell cohesion.

Example 4: Effect of the Aqueous Extract of Rose Flower on Gene Expression in Normal Human Keratinocytes

[0245] An aqueous extract of rose flower at 0.08% was tested on NHK according to a protocol similar to that described in example 1.

[0246] A butylene glycol/water excipient is used as a control.

TABLE-US-00005 TABLE 5 List of genes present on the microfluidic card. RefSeq GenBank Gene TaqMan item no. accession number Cell Keratin 10 Hs00166289_m1 NM_000421.3 differentiation KRT10 Epidermal Desmoglein 1 Hs00170047_m1 NM_001942.3 cohesion DSG1 Beta-2-M Hs00187842_m1 NM_004048.2 (control gene)

[0247] The aqueous extract of rose flower at 0.08%, after 24 hours of treatment showed significant stimulation on the expression of the following target genes: [0248] keratin 10 (KRT10): +88% [0249] desmoglein 1 (DSG1): +29%

[0250] The aqueous extract of rose flower according to the invention therefore has an effect on epidermal clock target genes, involved in cell differentiation, and/or epidermal cohesion.

[0251] All these biological results show that the aqueous extract of rose flower and the oil extract of rose flower according to the invention stimulate, in a complementary manner, clock genes and target genes involved in lipid metabolism, the skin barrier, cell differentiation, cell communication and/or cell cohesion.

[0252] Their combined use in a cosmetic composition is therefore advantageous in that it stimulates complementary clock genes and thus activates several metabolic pathways of interest.

[0253] Non-limiting examples of formulations are described below. Percentages are weight percentages expressed by weight based on the total weight of the composition.

Example 5: Composition in Emulsion Form

Aqueous Phase:

[0254]

TABLE-US-00006 Demineralized water qs 100% Glycols  20% Preservatives 0.6% Chelator 0.04%  Carbomer (Carbopol ® 981) 0.3% Sodium polyacrylate (Covacryl ® MV60) 0.2% Sodium hydroxide 0.15%  Rose cryoextract* .sup. 3%

Fat Phase:

[0255]

TABLE-US-00007 Vegetable oil, esters, silicones  16% Satin oil* .sup. 1% Antioxidant 0.2% Fragrance concentrate 0.4% Steareth-2 0.8% Steareth-21 1.5%
*as described in the Materials and Methods section above.

[0256] The composition is prepared according to the following procedure: [0257] the gelling agents are dispersed in the aqueous phase (excluding rose cryoextract and sodium hydroxide) which is heated to 70° C.; [0258] the fat phase (excluding fragrance concentrate, antioxidant and satin oil) is heated to 70° C.; [0259] satin oil is added extemporaneously just before the emulsion; [0260] the emulsion is produced by introducing the fat phase into the aqueous phase under strong stirring; [0261] the gelling agents are neutralized by adding sodium hydroxide and the emulsion is cooled under moderate stirring with the introduction of the fragrance concentrate, the antioxidant and the rose cryoextract at low temperature.

[0262] Applying this composition to the skin of the face promotes the rhythmic process of skin cells and thus improves lipid metabolism, the skin barrier, hydration and regeneration of the skin.

Example 6: Composition in the Form of a Solid Dispersion of Fat, of Spherical or Spheroidal Shape

Aqueous Phase

[0263]

TABLE-US-00008 Demineralized water qs 100% Rose cryoextract* 3.00% Phenoxyethanol 0.42% Xanthan gum 0.36%

Fat Phase

[0264]

TABLE-US-00009 C.sub.10-18 triglycerides 38.84% Satin oil*    1% Antioxidant  0.1%
*as described in the Materials and Methods section above.

[0265] The composition is prepared according to the following procedure: [0266] the wax (C.sub.10-18 triglycerides=Gatefossé Lipocire) is heated above its melting point (a few degrees) with the antioxidant and the oil extract of rose ‘satin oil’; [0267] the melted fat phase is poured under stirring into water previously heated to the same temperature as the fat phase; [0268] the whole is kept under stirring by a rotating system for a few minutes until the desired droplet size is obtained; [0269] the dispersion obtained is rapidly cooled by adding previously cooled glycol water (approximately −4° C.) to solidify the lipid spheroids; [0270] stirring is stopped when the spheroids are solidified and then recovered from the surface or filtered; [0271] the aqueous phase is prepared by mixing water, xanthan gum, preservative and rose cryoextract; [0272] the spheroids are recovered from the surface and incorporated into the xanthan gel containing the rose cryoextract.

[0273] Applying this composition to the skin of the face promotes the rhythmic process of skin cells and thus improves lipid metabolism, the skin barrier, hydration and regeneration of the skin.

Example 7: Composition in the Form of Oil Droplets Stabilized by a Polymeric Membrane on the Surface, in a Gelled Aqueous Phase

Aqueous Phase:

[0274]

TABLE-US-00010 Demineralized water qs 100% Glycols  20% Preservatives 0.6% Chelator 0.04%  Acrylates/C10-30 Alkyl acrylate crosspolymer (Pemulen ® TR2) 0.3% Sodium polyacrylate (Covacryl ® MV60) 0.2% Sodium hydroxide 0.15%  Rose cryoextract* .sup. 3%

Fat Phase:

[0275]

TABLE-US-00011 Vegetable oil, esters, silicones  16% Satin oil* .sup. 1% Antioxidant 0.2% Fragrance concentrate 0.4% Amodimethicone (Dow Corning 2-8566 Amino Fluid ®) 0.07% 
*as described in the Materials and Methods section above.

[0276] The composition is prepared according to the following procedure: [0277] the gelling agents are dispersed in the aqueous phase except cryoextract and sodium hydroxide, [0278] the emulsion is prepared at room temperature by introducing the fat phase into the aqueous phase under strong stirring, [0279] the gelling agents are neutralized by adding sodium hydroxide before introducing the rose cryoextract.

[0280] Applying this composition to the skin of the face promotes the rhythmic process of skin cells and thus improves lipid metabolism, the skin barrier, hydration and regeneration of the skin.