Cosmetic Composition

Abstract

A cosmetic composition is provided, which comprises a carrier and a Patchouli stem extract, in particular an extract from exhausted Patchouli stems. This composition provides anti-oxidant, anti-elastase, anti-hyaluronase, and anti-tyrosinase activities.

Claims

1. A cosmetic composition comprising a carrier and at least one active cosmetic ingredient, which is a first active cosmetic ingredient comprising a Patchouli stem extract.

2. The cosmetic composition of claim 1, wherein the cosmetic composition is a skin care, scalp care or body care composition.

3. The cosmetic composition of claim 2, wherein the cosmetic composition is a serum for dry skin, an anti-aging serum, and anti-aging night or day cream, an anti-dry dandruff product, a dry scalp lotion or is a body care lotion.

4. The cosmetic composition according to claim 1, wherein the first active cosmetic ingredient comprises an aqueous and/or alcoholic extract of Patchouli stems.

5. The cosmetic composition according to claim 1, wherein the first active cosmetic ingredient comprises an extract of exhausted Patchouli stems.

6. A method of preparing an active cosmetic ingredient, comprising the step of extracting Patchouli stems.

7. The method of claim 6, comprising the steps of (i) providing exhausted Patchouli stems; and (ii) extracting the exhausted Patchouli stems.

8. The method of claim 6, wherein the extraction is performed using water and/or ethanol.

9. A method of treating human skin, comprising the use of an extract from Patchouli stems in treating skin.

10. A method of stimulating sebum production, of stimulating an antioxidant property and/or of inhibiting wound healing, the method comprising the step of: applying the cosmetic composition of claim 1 to human skin.

11. The method of claim 9, wherein the extract is an aqueous and/or alcoholic extract from Patchouli stems.

12. The method of claim 9, wherein the extract is an extract from exhausted Patchouli stems.

13. The method of claim 10, wherein the extract is an aqueous and/or alcoholic extract from Patchouli stems.

14. The method of claim 10, wherein the extract is an extract from exhausted Patchouli stems.

Description

[0039] The present invention is further illustrated by means of the following non-limiting examples:

EXAMPLE 1: PREPARATION OF PATCHOULI STEM EXTRACT

[0040] Dried hydrodistilled Patchouli stems were crushed to obtain a powder. 25 g of powder were extracted at room temperature in 500 g of ethanol (70% in water) for 30 min under stirring. The extract (around 420 g) was filtered over an Eaton KDS15 filter (123 cm.sup.2). The filtrate was concentrated by a factor of 5.2 by evaporation under vacuum to obtain about 80 g of concentrated product.

[0041] One batch of the concentrated product (Batch A) was used as such. This non decolorized product was black.

[0042] A second batch of the concentrated product (Batch B) was decolorized on charcoal filters (24 cm.sup.2). This decolorization process efficiently decreased colour. It decreased dry matter around 2 fold.

[0043] The two batches were finally filtered over an Eaton S60 filter (24 cm.sup.2) and sterile PES filtration unit (VWR) to store the final products without preservatives.

[0044] The characteristics of the two batches are shown in the following table:

TABLE-US-00001 Amount of Dry matter final product content Gardner pH Batch A 56 g 3.80% 15.2 5.1 Batch B 40 g 1.57% 5.6 4.6

[0045] For comparison of different solvents, extractions were also performed with water, 100% ethanol, ethanol 70% and ethanol 50%. Color (Gardner) and dry matter content were measured on raw extracts. The results are shown below:

TABLE-US-00002 Water 50% Ethanol 70% Ethanol 100% Ethanol Gardner 8.0 9.0 8.5 5.6 Dry matter 0.52% 0.53% 0.54% 0.0% content

[0046] Essentially no difference of color or even dry matter content was observed (except for 100% ethanol), but the yield in dry matter was in general very low.

EXAMPLE 2: ANALYSIS OF PATCHOULI STEM EXTRACT

[0047] Dried hydrodistilled Patchouli stems were crushed to obtain a powder. 75 g of powder were extracted at 20 C. in 550 g of ethanol (70% in water) for 60 min under stirring. Extract was filtered through a 0.7 m cellulosic filter, concentrated five times to remove ethanol and finally filtered through a 0.35 m cellulosic filter. The extract was then freeze dried.

Fractionation of the Crude Extract by Centrifugal Partition Chromatography (CPC)

[0048] The crude extract (1.078 g) was dissolved in 30 ml a biphasic solvent system consisting of methyl-tert-butylether (MTBE), acetonitrile and water in a 3:3:4 ratio (v/v). Centrifugal Partition Chromatography (CPC) was performed using an FCPE300 instrument (Rousselet Robatel Kromaton) with a column of 303 ml, a column rotation speed of 1200 rpm and a flow rate of 20 ml/min.

[0049] An isocratic elution of the mobile phase (lower phase of the two-phase solvent system) was performed in the ascending mode for 75 min (with an initial flow rate ramp from 0 to 20 ml/min during 5 min). The column was finally extruded by switching the mode selection valve for 20 min. The CPC chromatogram was monitored at 220 nm. Fractions of 20 ml were collected over the whole experiment, and combined according to their thin layer chromatography (TLC) profiles. TLC was performed on pre-coated silica gel 60 F254 Merck plates with the migration solvent system EtOAc/toluene/formic acid/acetic acid (70/30/11/11; v/v), visualized under UV light at 254 nm and 360 nm and revealed by spraying the dried plates with 50% H.sub.2SO.sub.4 and vanillin followed by heating. As a result, 16 sub-fractions were collected.

NMR Analyses and Identification of the Major Metabolites

[0050] An aliquot of each fraction from F1 to F16 (up to 20 mg) was dissolved in 700 l DMSO-d6 and analyzed by .sup.13C NMR at 298 K on a Bruker Avance AVIII-600 spectrometer (Karlsruhe, Germany) equipped with a TXI cryoprobe. Spectra were manually phased and baseline corrected using the TOPSPIN 3.2 software (Bruker) and calibrated on the central resonance of DMSO-d6 ( 39.80 ppm). The absolute intensities of all .sup.13C NMR signals were automatically collected and binned across the spectra of the fraction series by using a locally developed computer script. The resulting table was imported into the PermutMatrix version 1.9.3 software (LIRMM, Montpellier, France) for Hierarchical Clustering Analysis (HCA). The resulting .sup.13C chemical shift clusters were visualized as dendrograms on a two-dimensional map. For metabolite identification, each .sup.13C chemical shift cluster obtained from HCA was manually submitted to the structure search engine of the database management software ACD/NMR Workbook Suite 2012 software, ACD/Labs, Ontario, Canada) comprising the structures and predicted chemical shifts of low molecular weight natural products (n 2950 in March 2018). In parallel, a literature survey was performed to obtain the names and chemical structures of a maximum of metabolites already reported in the species Pogostemon cablin (n70). Additional 2D NMR experiments (HSQC, HMBC, and COSY) were performed on fractions containing putatively identified compounds in order to confirm the molecular structures proposed by the database at the end of the dereplication process.

[0051] The following major metabolites were identified: [0052] Verbascoside [0053] Apigenin-7-O-glucuronide [0054] Succinic acid [0055] Glucosyl-cytosporone V (2 isomers) [0056] Lactic acid [0057] Choline [0058] -D-fructose [0059] -D-fructose [0060] -D-glucose [0061] -D-glucose [0062] -D-fructopyranose [0063] 7,3-dimethyleriodictyol [0064] Pachypodol [0065] Cytosporone V [0066] p-hydroxybenzoic acid [0067] Protocatechuic acid [0068] Syringaresinol [0069] Vanillin [0070] Rhamnocitrin [0071] Luteolin-7-O-glucuronide [0072] Hydroxymethylglutaric acid [0073] 12-hydroxyjasmonic acid [0074] a caffeoyl derivate [0075] apigenin-7-sugars

[0076] The composition of the CPC fractions was as follows (Maj=major; Med=medium; Min=minor):

TABLE-US-00003 CPC- Mass % crude Fractions (mg) extract Composition 1 7.9 0.7 7,3-dimethyleriodictyol (Maj); Pachypodol (Maj); Rhamnocitrin Elution (Min) 2 16.7 1.5 7,3-dimethyleriodictyol (Maj); Pachypodol (Maj); Cytosporone V Elution (Med); Rhamnocitrin (Min) 3 7.7 0.7 p-hydroxybenzoic acid (Maj); Syringaresinol (Min); Vanillin (Med) Elution 4 10.2 0.9 Protocatechuic acid (Maj); Syringaresinol (Min); Vanillin (Med) Elution 5 14.0 1.3 12-hydroxyjasmonic acid (Maj) + caffeoyl derivatives and Elution Apigenin-7-sugars 6 11.8 1.1 Apigenin-7-O-glucuronide (Min); Succinic acid (Min); caffeoyl Elution derivatives and Apigenin-7-sugars 7 10.6 1.0 Verbascoside (Min); Apigenin-7-O-glucuronide (Maj); Succinic Elution acid (Maj); Glucosyl-cytosporone V (Min) 8 16.0 1.5 Verbascoside (Med); Apigenin-7-O-glucuronide (Med); Succinic Elution acid (Maj); Glucosyl-cytosporone V (Min) 9 26.7 2.5 Verbascoside (Maj); Apigenin-7-O-glucuronide (Min); Succinic Elution acid (Med); Glucosyl-cytosporone V (Med); Luteolin-7-O- glucuronide (Min) 10 26.9 2.5 Verbascoside (Maj); Apigenin-7-O-glucuronide (Min); Succinic Elution acid (Min); Glucosyl-cytosporone V (Med); Luteolin-7-O- glucuronide (Min); Hydroxymethylglutaric acid (Med) 11 56.0 5.2 Apigenin-7-O-glucuronide (Min); Succinic acid (Min); Lactic acid Elution (Maj); Luteolin-7-O-glucuronide (Min); Hydroxymethylglutaric acid (Maj) 12 97.5 9.0 Choline (Min); -D-fructose (Min); -D-fructose (Med); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Min) 13 329.4 30.3 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 14 306.0 28.2 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 15 68.0 6.3 Choline (Min); -D-fructose (Med); -D-fructose (Maj); -D- Extrusion glucose (Maj); -D-glucose (Maj); -D-fructopyranose (Med) 16 81.1 7.5 Choline (Min); -D-fructose (Min); -D-fructose (Med); -D- Extrusion glucose (Med); -D-glucose (Med); -D-fructopyranose (Min)

EXAMPLE 3: SCREENING OF BIOLOGICAL ACTIVITY

[0077] The two batches A and B from Example 1 were tested for antioxidant (DPPH), anti-elastase, anti-hyaluronidase, and anti-tyrosinase activities. Results are presented in the following table:

TABLE-US-00004 IC50 DPPH Elastase Hyaluronidase Tyrosinase Batch A 0.07 mg/ml 16.60 mg/ml 37.38 mg/ml 6.11 mg/ml Batch B 0.20 mg/ml n.a. n.a. 9.53 mg/ml

[0078] The non-decolorized Batch A clearly presented high biological activities.

[0079] The decolorized Batch B only displayed a slight antioxidant activity, but similar anti-tyrosinase activity as Batch A.

EXAMPLE 4: WOUND HEALING

[0080] Normal Human Keratinocytes (NHEKs) were seeded at 200000 cells per well pre-coated with Type I collagen in 12-wells culture plates in the presence of keratinocyte growth medium (KGM, Lonza) supplemented with growth factors such as hydrocortisone, transferrin, epinephrine, bovine pituitary extract (BPE), recombinant human epidermal growth factor (rhEGF) and insulin. At confluence, cells were pre-incubated with the Patchouli stem extract of Example 1 at a concentration of 0.5% (v/v) or heparin-binding epidermal growth factor (HB-EGF) at 1 ng/ml (positive control) overnight in keratinocyte basal medium without supplement. After this pre-conditioning phase, the cell monolayer sticking to the bottom of the well was scratched with a P200 sterile cone, followed by two washes with phosphate buffered saline (PBS). The NHEK were then again stimulated with Patchouli extract at 0.5% or HB-EGF at 1 ng/ml for 8 h in basal medium.

[0081] The wound healing process was analyzed by pictures recording at T.sub.0 and T.sub.8h using inverting optical microscope (Zeiss). After image analysis, the percentage of scratch closing was determined relative to untreated condition.

[0082] It was found that the Patchouli stem extract significantly inhibited wound healing by about 50% in the NHEK scratch assay, while HB-EGF led to an enhancement by about +60%. An inhibition of wound healing is particularly important in conditions involving a hyperproliferation of keratinocytes, e.g. hyperkeratosis or dry dandruff.

EXAMPLE 5: ANTI-OXIDANT ACTIVITY

[0083] NHEKs were seeded in a black plate with a glass bottom at 20000 cells per well pre-coated with type I collagen. Cells were incubated at 37 C. with 5% CO.sub.2 for 24 h. On the next day, the cells were incubated for 24 h with the Patchouli stem extract of Example 1 at a concentration of 0.5% (v/v) or Resveratrol (positive control) at 200 M. After this pre-incubation, the cells were incubated with dichloro-dihydro-fluorescein (DCFH) probe at 50 M for 30-45 min. The cells were then rinsed two times with PBS and incubated with PBS alone or with PBS containing 5 mM tert-butyl peroxide (TBP) to induce an oxidative stress. Fluorescence reading was performed every 10 min for 1 h, exciting at 488 nm and emitting at 525 nm.

[0084] It was found that the Patchouli stem extract led to a reduction in reactive oxygen species (ROS) production by 28%, evidencing anti-oxidant properties.

EXAMPLE 6: REGULATION OF SEBUM PRODUCTION

[0085] Human sebocytes were seeded in 96-wells plates (50000 cells/well) and cultured for 24 h in keratinocyte serum free medium (SFM) supplemented with gentamycin at 25 g/ml. The culture medium was then remove and replaced by the Patchouli stem extract of example 1 at a concentration of 1% (v/v) or the reference, olumacostat glasaretil at 1 M. The cells were pre-incubated for 4 h. Then, a lipogenic mix containing vitamin C, vitamin D3, insulin and calcium (without androgens) was added and the cells were incubated for 7 days. After 3 days of incubation, half of the culture medium was removed and the treatments were renewed (including lipogenic mix stimulation). A non-stimulated control condition was performed in parallel. At the end of the incubation, the cells were rinsed, fixed and permeabilized. The lipid droplets contained in the cells were then labeled using a specific Bodipy fluorescent lipid probe labelling mainly neutral lipids. In parallel, the cell nuclei were stained using a Hoechst 33258 solution. The acquisition of the images was performed using INCell Analyzer 2200. Five photos were taken per well (20 objective lens). The labelling was quantified by fluorescence intensity measurement normalized to the total number of cells.

[0086] It was found that the Patchouli stem extract significantly induced the sebum production from sebocytes under lipogenic stimulation by +39%. A stimulation of sebum production is particularly useful in the treatment of dry skin and for restoring the skin barrier and skin permeability.