Cosmetic Composition Comprising Vetiver Root Extract

Abstract

A cosmetic composition is provided, which comprises a carrier and a Vetiver root extract, in particular an extract from exhausted Vetiver root. This composition provides a stimulation of sebum production, stimulation of sebum antimicrobial, lipids production, activation of adipocytes volume increase, skin hydration, skin tonicity booster, skin fatigue reduction, perilabial wrinkles reduction, skin replumping, and fragrance long-lastingness enhancement.

Claims

1. A cosmetic composition comprising a carrier and at least one active cosmetic ingredient, wherein the at least one active cosmetic ingredient comprises a Vetiver root extract.

2. The cosmetic composition according to claim 1, wherein the cosmetic composition is a skin care composition.

3. The cosmetic composition according to claim 1, wherein the at least one active cosmetic ingredient comprises an aqueous extract of Vetiver root.

4. The cosmetic composition according to claim 1, wherein the at least one active cosmetic ingredient comprises an extract of exhausted Vetiver root.

5. A method of preparing an active cosmetic ingredient, comprising the step of: extracting Vetiver root.

6. The method according to claim 5, comprising the steps of: (i) providing exhausted Vetiver root; and (ii) extracting the exhausted Vetiver root.

7. The method according to claim 5, wherein the extraction is performed using water.

8. (canceled)

9. A method of stimulating the sebum production, of stimulating sebum antimicrobials, of stimulating the lipids production, of activating the adipocytes volume increase, of improving the skin hydration, of boosting the skin tonicity, of reducing skin fatigue, of reducing perilabial wrinkles, of replumping skin, and/or of enhancing fragrance long-lastingness, the method comprising the step of: applying the cosmetic composition of claim 1 to human skin.

10. A chemical substance selected from the group consisting of: ##STR00009## ##STR00010##

Description

EXAMPLE 1: PREPARATION OF VETIVER ROOT EXTRACT FOR IN VITRO AND EX VIVO STUDIES

[0083] Exhausted Vetiveria zizanioides roots from Haiti (75 g) were suspended in water (2000 g). Extraction was performed under stirring at 60 C. for 4 h. The pH was monitored during extraction and varied from about 7.4 to about 7.8.

[0084] After filtration, the solution was concentrated by water removal until it reached a final dry matter content of 0.7%. The product is typically a limpid dark yellow to amber liquid with no smell, and has a pH of about 7.7 to 7.8. This extract is typically used for in vitro and ex vivo studies.

[0085] For the studies described in the subsequent examples, aqueous solutions containing 1%, 2% and 3% of the extract, respectively, were prepared.

EXAMPLE 2: PREPARATION OF VETIVER ROOT EXTRACT FOR IN VIVO STUDIES

[0086] Exhausted Vetiveria zizanioides roots from Haiti (75 g) were suspended in water (2000 g). Extraction was performed under stirring at 60 C. for 4 h. The pH was monitored during extraction and varied from about 7.4 to about 7.8.

[0087] After filtration, 1,3-propanediol was added to the solution, and the resulting mixture concentrated by water removal until it reached a final dry matter content of 0.7%. The product is typically limpid dark yellow to amber liquid with no smell, and has a pH of about 6.5 to 7.5 This extract is typically used for in vivo studies.

[0088] For the studies described in the subsequent examples, aqueous solutions containing 2% of the Vetiver extract, respectively, were prepared.

[0089] For examples 8 and 9:

TABLE-US-00001 INCI Name Quantity (%) AQUA/WATER 88.65 Vetiveria zizanoides root extract 2.0 CAPRIC/CAPRYLIC TRIGLYCERIDE 4.5 CETEARYL WHEAT STRAW GLYCOSIDES, 4.0 CETEARYL ALCOHOL PHENOXYETHANOL, METHYL PARABEN, 0.4 PROPYL PARABEN, ETHYL PARABEN DIMETHICONE 0.3 FRAGRANCE, HEXYL CINNAMAL, 0.1 BUTYLPHENYL METHYLPROPIONAL, CITRONELLOL, ALPHA ISOMETHYL IONONE, HYDROXYISOHEXYL 3-CYCLOHEXENE CARBOXALDEHYDE SODIUM HYDROXIDE 0.05

[0090] For example 11:

TABLE-US-00002 INCI Name Quantity (%) AQUA/WATER 96.01 ACRYLATES/C10-30 ALKYL 0.5 ACRYLATE CROSSPOLYMER PHENOXYETHANOL 1 VETIVERIA ZIZANOIDES ROOT 2 EXTRACT, PROPANEDIOL, WATER SODIUM HYDROXIDE 0.49

EXAMPLE 3: INCREASING LIPIDS PRODUCTION FOR BARRIER FUNCTION (EX VIVO)

[0091] A proteomic analysis was performed by LC-MS/MS on human skin explants of three donors (54, 56 and 69 years old women) on which the 1% solution from the extract of example 1 was applied every day for six days. The results are shown in the following table:

TABLE-US-00003 Cellular x fold function Protein expression Lipids ORM1 - Alpha 1 acid glyco-protein 1 0.57* metabolism & RTN4 - Reticulon-4 1.33* transport CLTA - Clathrin light chain A 3.77* SNX5 - Sorting nexin 5 1.72* CERT - Ceramide transport protein 2.69* TFG - Protein TFG 1.34** Epidermis INV - Involucrin 1.59 differentiation KRT36 - Keratin type I cuticular Ha6 1.73** DMKN - Dermokine 1.53** LOR - Loricrin 3.39* *p < 0.05 Student's t-test **p < 0.01 Student's t-test

[0092] These results show that the extract of example 1 stimulates the expression of a complete set of proteins involved in the multiple pathways targeting lipids production, lipids transport, and epidermis differentiation. The composition improves the epidermal barrier through the modulation of lipid metabolism of the skin.

EXAMPLE 4: BOOSTING SEBUM QUANTITY AND QUALITY

a) Sebocytes Stimulation (In Vitro)

[0093] Sebocytes were pre-incubated with the 1% solution from the extract of example 1 for 4 hours. Then, a lipogenic mix (vitamin C, vitamin D3, insulin and calcium) was added for another 7 days of incubation. Treatment was renewed after 3 days. The lipid content was then evaluated using Bodipy fluorescent labeling (lipids were made visible in green).

[0094] The results are shown in FIG. 2. It was found that the extract of example 1 shows a significant stimulation of sebocytes, with an increase of sebum lipids accumulation of up to +31% versus lipogenic mix only.

b) Antimicrobial Lipids (AML) Content Increase (In Vitro)

[0095] Sebocytes were cultivated in 3D culture and treated in systemic with the 1% solution from the extract of example 1 for 7 days. The content of free fatty acids was then assessed by GC/MS to analyse their content in AML.

[0096] The results are shown in FIG. 3. It was found that the extract of example 1 significantly increases the content of antimicrobial lipids in sebum (lauric acid and sapienic acid) by up to +23% compared to no treatment.

[0097] The same experimental procedure was repeated without lipogenic mix stimulation. It was found that the Vetiver root extract is able to induce the production of anti-microbial lipids without any other lipogenic stimulation on sebaceous cells model (FIG. 4).

[0098] In particular, the Vetiver root extract significantly induced the production of lauric acid (+42%) and sapienic acid (+43%), both of which are considered as key AMLs.

EXAMPLE 5: BARRIER FUNCTION RECONSTRUCTION (IN VITRO): REACTIVATION OF LIPIDS PRODUCTION (IN VITRO/EX VIVO)

[0099] a) Ceramides and their Precursors Synthesis

[0100] Reconstructed Human Epidermis (RHE) was incubated with the 1% solution from the extract of example 1 for 7 days. Lipids contained in RHE were then extracted and studied by thin layer chromatography and densitometric analysis to assess the quantity of ceramide precursors in the RHE.

[0101] The results are shown in the following table:

TABLE-US-00004 p value Untreated Vetiver 1% (versus untreated) Sphingomyelins 100 142 p < 0.01 Phosphoglycerides 100 138 p < 0.01 Ceramides series 1, 100 132 p < 0.01 polar lipids

[0102] It was found that the extract of example 1 significantly increases the production of the ceramides and their various precursors in the epidermis by up to +42% compared to untreated RHE. Thus, the Vetiver extract has the ability to stimulate the neosynthesis of specific lipids in RHE.

b) Ceramides Transport Stimulation

[0103] Normal Human Keratinocytes (NHEKs) were stimulated for 5 days with the 1% solution from the extract of example 1. Ceramide Transport Protein (CERT) was then quantified by immunofluorescence.

[0104] The results are shown in FIG. 5. It was found that the extract of example 1 significantly increases the expression of CERT by up to +124.5% compared to untreated NHEKs, enabling a better transport of the lipids in the epidermis.

c) Boosting Lipids Content

[0105] Skin explants from a donor aged 69 were topically treated with the 1% solution from the extract of example 1 for 6 days. The neutral lipids from skin were stained using LipidTOX. The immunostaining was quantified by microscopical observation (green fluorescence) to assess stimulation of keratinisation.

[0106] The results are shown in FIG. 6. It was found that, as a result of boosting lipids production and transport, the extract of example 1 significantly increases the lipid content in the cornified envelope by up to +30% compared to untreated samples.

EXAMPLE 6: BARRIER FUNCTION RECONSTRUCTION (IN VITRO): REACTIVATION OF SKIN BARRIER PROTEINS (EX VIVO)

[0107] Human skin explants from three donors (54, 56 and 69 years old women) on which the 1% solution from the extract of example 1 was applied every day for 6 days were assessed by proteomic using LC-MS/MS for quantification of dermokine, involucrin and loricrin, three characteristic proteins of the cornified envelope.

[0108] The results are shown in FIG. 7. It was found that the extract of example 1 significantly increases the expression of these three major proteins of the stratum corneum, by +53% up to +239% in average. Thus, the Vetiver extract has a clear impact on the improvement of the epidermal barrier by reactivating the expression of these proteins, making the cornified envelope of the skin more cohesive.

EXAMPLE 7: BOOSTING SKIN ADIPOCYTES DIFFERENTIATION AND SIZE

a) Improving Adipocytes Differentiation (In Vitro)

[0109] Pre-adipocytes were cultured for 13 days with the 3% solution from the extract of example 1. Lipid droplets, a marker of adipocytes differentiation, were then labelled using AdipoRed (a fluorescent lipid probe) to quantify the differentiation.

[0110] It was found that the extract of example 1 significantly increases the adipocytes differentiation, with an effect up to +3413% compared to untreated samples.

b) Increasing Adipocytes Volume (Ex Vivo)

[0111] Explants of full skin (epidermis, dermis, and hypodermis) were treated with the 3% solution from the extract of example 1. On D0, D1, D4 and D6, the product was topically applied. On D8, explants were stained for microscopical analysis. Adipocytes size determination was then performed by image analysis, by measuring their equivalent circular diameter.

[0112] It was found that the extract of example 1 significantly increases the average size of adipocytes in the hypodermis, as observed by microscopy.

[0113] It is also noticeable that a shift in the population of adipocytes can be observed after the use of the extract of example 1: [0114] The number of small adipocytes (20-60 m) increases by +26%, reflecting the differentiation boost observed in vitro (appearance of new small adipocytes from pre-adipocytes) [0115] The number of large adipocytes (100-140 m) increases by +189%, as a consequence of the average size increase and a better fat storage capacity (accumulation of lipids in the existing adipocytes)

[0116] During aging, the loss of adipose tissue volume is the consequence of an alteration of the fat tissue functions, such as the reduction of adipocyte size and changes in pre-adipocyte dynamics. As demonstrated by these two studies, the Vetiver extract has a beneficial impact on volume redefinition and acts as a plumping agent by reactivating the differentiation of pre-adipocytes and by stimulating the lipogenesis in mature adipocytes.

EXAMPLE 8: VISIBLE SKIN FATIGUE REDUCTION AND DECREASE OF PERILABIAL WRINKLES (CLINICAL TEST)

[0117] A double blind clinical evaluation was performed on two groups of 21 volunteers with sagging face and wrinkles (women from 52 to 69 years old, average of 60). Volunteers applied a placebo or the 2% solution from the extract of example 2 on their face for 56 days, twice a day (morning and evening).

a) Improvement of Tonicity/Firmness and Anti-Fatigue Effect

[0118] At D0, D28 and D56, skin biomechanical properties were measured by cutometry on cheekbones, focusing on the R6 and R9 parameters, respectively representative of the viscoelastic balance and of the fatigue effect. At D0, D28 and D56, face of the volunteers was analysed using AEVA HE, a patented projection unit combined with stereo imaging.

[0119] The results are shown in FIG. 8. The extract of example 2 enables a time-progressive and significant recovery of the skin biomechanical properties on aged volunteers, with up to +11.2% recovery in tonicity and firmness and 17.8% of skin fatigue decrease versus placebo. These results are also clearly visible on the 3D face images of the volunteers (FIG. 9).

b) Smoothing of Perilabial Wrinkles

[0120] At D0 and D56 a particular focus was put on the perilabial wrinkles of the volunteers to quantify them using AEVA HE.

[0121] The results are shown in FIG. 10. The daily application of the extract of example 2 enables a significant effect for 100% of the volunteers versus placebo in 2 months, with an average reduction of 18%*** of the perilabial wrinkles.

***p<0.001 Student's t-test

c) Self-Assessment of the Skin Benefits

[0122] All volunteers were asked to assess the benefits of the extract of example 2 for their skin at D56: [0123] 76% of the volunteers felt that their skin was more hydrated. [0124] 72% of the volunteers felt that their skin was more pleasant and comfortable. [0125] 71% of the volunteers felt that their skin was nourished.

EXAMPLE 9: SKIN COMPOSITION AND HYDRATION BENEFITS (CLINICAL TEST)

[0126] A double blind clinical evaluation was performed on 20 volunteers (women from 50 to 70 years old, average of 63), with dry legs (corneometry value<35). Volunteers applied the 2% solution from the extract of example 2 or a placebo on their legs twice a day (morning and evening) for 28 days.

a) Improvement of Lipids Organisation

[0127] At D0 and D28, Raman spectroscopy was used to assess the quality of lipids conformation in the skin.

[0128] The results are shown in FIG. 11. The extract of example 2 shows a significant improvement of the lipids organisation in the skin, of up to +21%, therefore increasing their compactness, and ultimately the barrier function.

b) Increase of Skin Hydration

[0129] At D0 and D28, skin hydration was analysed by corneometry.

[0130] The results are shown in FIG. 12. The extract of example 2 shows a significant increase of skin hydration of up to +7.3% on volunteers with dry skin.

EXAMPLE 10: FRAGRANCE BOOSTING BENEFITS (CLINICAL TEST)

[0131] To assess the long-lastingness of a fragrance, a clinical test was performed on 20 female volunteers. Volunteers applied twice a day a placebo cream on one of their forearms and the 2% solution from the extract of example 2 on the other forearm for 1 month. Then, on the last day, a fine fragrance (Tom Ford Neroli Portofino) was applied to both of their forearms.

a) Self-Assessment of Long-Lastingness

[0132] Volunteers were asked to rank the olfactive intensity of the fine fragrance 2 hours and 4 hours after its application on their skin.

[0133] Volunteers observed a significant decrease of fragrance intensity on the forearm treated with placebo while those treated with the extract of example 2 maintained the fragrance intensity after 2 h and 4 h of fragrance application.

[0134] Comparative results are shown in FIG. 13. The extract of example 2 demonstrates a boosting effect of fragrance long-lastingness by significantly increasing the fragrance olfactive intensity hours after application, by up to +25%.

b) Professional Fragrance Evaluator Opinion

[0135] To confirm the benefits of the extract of example 2 in terms of fragrance boosting properties, a professional fragrance evaluator was asked to assess the differences in terms of olfactive properties between the two forearms of the volunteers.

[0136] The evaluator came to the conclusion that, the extract of example 2 is emphasizing the fragrance heart and base notes over time, and therefore functions as a fragrance sensuality booster.

EXAMPLE 11: IMPACT ON SEBUM PRODUCTION (CLINICAL TEST)

[0137] A double blind and placebo controlled clinical evaluation was carried out on 30 female volunteers (aged 63 to 70, mean age 672). The volunteers presented a low level of sebum on their face. All volunteers gave their informed consent sign date the beginning of the study.

[0138] The volunteers applied the 2% solution from the extract of example 2 on one side of their face and a placebo on the other side twice a day (morning and evening) for 28 days. At D0 and D28, sebum production on forehead and cheek was analyzed using Sebumeter (ex CourageKhazaka Electronic).

Results

[0139] It was found that the Vetiver extract induced a significant increase of sebum production after 28 days of application on forehead and cheek with +99% and +78% respectively.

[0140] The detailed results are as follows:

TABLE-US-00005 Forehead Cheek Placebo D 0: Mean +/ SEM 33.71 19.49 14.29 11.53 (g/cm.sup.2) D 28: Mean +/ SEM 50.42 33.15 19.81 15.76 (g/cm.sup.2) % D 28 D 0 (mean) +50% +39% Student t-test versus p < 0.5 p < 0.1 D 0 (p) 2% Vetiver D 0: Mean +/ SEM 33.96 19.03 17.02 14.16 extract (g/cm.sup.2) D 28: Mean +/ SEM 67.58 35.08 30.23 28.12 (g/cm.sup.2) % D 28 D 0 (mean) +99% +78% Student t-test versus p < 0.001 p < 0.05 D 0 (p) Student t-test 2% p < 0.05 p < 0.05 Vetiver extract versus placebo (p)

[0141] Therefore, Vetiver extract at 2% is able to reactivate the sebum production that is otherwise drastically reduced on old people.

EXAMPLE 12: ANALYSIS OF EXHAUSTED VETIVER ROOT EXTRACT

Extract Preparation

[0142] Exhausted vetiver roots were washed, dried and ground before extraction with water at 60 C. (37.5 g per 1000 g of water) during 6 h, followed by filtration and concentration to obtain 500 g of aqueous extract. The aqueous extract was extracted three times with ethyl acetate (ratio of aqueous extract to total ethyl acetate=1:6). The ethyl acetate extract was evaporated to obtain 2.081 g of a yellow oil.

Fractionation by Centrifugal Partition Chromatography (CPC)

[0143] The ethyl acetate extract was fractionated on a CPC instrument FCPE300 (Rousselet Robatel Kromaton) using a column of 303 mL and a two-phase solvent system (n-hexane/EtOAc/methanol/water (10/8/10/8, v/v)).

[0144] The CPC chromatogram was monitored at 254, 280, 300, and 360 nm. Fractions of 20 mL were collected over the whole experiment, and combined according to their thin layer chromatography profiles.

NMR Analyses and Identification of the Major Metabolites

[0145] The CPC fractions were analyzed by .sup.13C NMR at 298 K on a Bruker Avance AVIII-600 spectrometer (Karlsruhe, Germany) equipped with a TXI cryoprobe and compared to metabolites previously reported in the species Chrysopogon zizanoides. Additional 2D NMR experiments (HSQC, HMBC, and COSY) were performed on fractions containing putatively identified compounds to confirm the molecular structures proposed by the database at the end of the dereplication process. Also, where necessary, a second CPC fractionation was performed.

[0146] The following major metabolites were identified:

##STR00006## ##STR00007## ##STR00008##

[0147] Of the above metabolites, zizanoic acid (1), oplopanone (6), solanerianone A (8), vetiverianine B (10), valencene-11,12-diol (12), isovalencenic acid (13), and teuhetenone (15) have been previously described.

[0148] Substances (2), (3), (4), (5), (7), (9), (11), and (14), on the other hand, have never been described before.