Cosmetic composition comprising royal jelly of the Ouessant black bee

Abstract

The invention relates to a cosmetic composition comprising royal jelly of the Ouessant black bee in a cosmetically acceptable medium, and to its use for regenerating and/or healing the skin.

Claims

1. A cosmetic composition comprising royal jelly of the Ouessant black bee in a cosmetically acceptable medium, wherein: said royal jelly is at a concentration between 0.001% and 1% by weight of the total composition, and said composition further comprises one or more cosmetically acceptable excipient selected from the group consisting of: polymers, surfactants, rheology agents, fragrances, electrolytes, pH adjusters, antioxidants, preservatives, dyes, mother-of-pearl, pigments, and mixtures thereof, and said cosmetic composition is in the form of an oil-in-water emulsion or a water-in-oil emulsion.

2. The cosmetic composition of claim 1, wherein said royal jelly is at a concentration between 0.005% and 0.5% by weight of the total composition.

3. The cosmetic composition of claim 1, wherein said royal jelly is at a concentration between 0.05% and 0.2% by weight of the total composition.

4. A method for stimulating skin regeneration and/or healing comprising the administration of an effective amount of the cosmetic composition of claim 1 to a patient in need thereof.

5. The cosmetic composition of claim 1, further comprising unifloral or polyfloral honey or honey extract.

6. The cosmetic composition of claim 1, further comprising clover (Trifolium repens) or euphorbia honey.

Description

FIGURES

(1) FIG. 1: TIEG-1 protein expression in an in vitro healing model in NHF. A. Diagram of areas of interest where an immunofluorescent signal is quantified in a healing model. area A: remote from the injured area; area B: mixed area including the area bordering the wound and a control area on the cell monolayer; area C: wound area corresponding to the area scraped by a pipette tip; B. TIEG-1 protein expression in fibroblasts 48 hours after injury.

(2) FIG. 2: Effect of Ouessant black bee royal jelly on the TIEG1 protein level in cultured NHF in injured (or healing) and non-injured areas.

(3) FIG. 3: Effect of Ouessant black bee royal jelly and Buckfast bee royal jelly on the synthesis of type V collagen in cultured NHF.

(4) FIG. 4: Effect of Ouessant black bee royal jelly and Buckfast bee royal jelly on elastin synthesis in cultured NHF.

EXAMPLES

Example 1

Expression of TIEG-1 in an In Vitro Healing Model

(5) Cell culture can be used to obtain a surface uniformly covered with cells. This cell monolayer can be injured and torn by scraping a pipette tip across its surface.

(6) On this model, three different areas are distinguished: the wound area corresponding to the area scraped by the pipette tip (area C), a mixed area i.e. the area comprising the area bordering the wound (area B) and a control area on the cell monolayer, at a distance from the wounded area (area A) (FIG. 1, A). TIEG-1 expression was revealed by immunolabeling and quantified by image analysis in the fibroblasts present in these three areas when the healing process is initiated.

(7) This study shows that TIEG-1 is significantly overexpressed in injury area C by 47% compared to mixed area B and 53% compared to control area A (FIG. 1, B).

(8) This indicates that TIEG-1 has a major role in the mechanism of cell mobility, particularly fibroblastic, a major mechanism in the healing process.

Example 2

Effect of Ouessant Black Bee Royal Jelly on TIEG Protein Expression in Cultured Normal Human Fibroblasts (NHF) Subjected to a Healing Test

(9) TIEG 1 expression is evaluated under the effect of a treatment with Ouessant black bee royal jelly (0.01%) in an in vitro healing model of cultured NHF.

2.1. Cell Treatments

(10) NHF from a 37-year-old Caucasian woman's abdominoplasty are seeded (80,000 cells per Petri dish) 35 ibidi treat in low-glucose DMEM medium implemented with 10% FBS and a mixture of antibiotics (penicillin/streptomycin) and maintained in culture for 48 hours, then the medium is changed and replaced by FBS-depleted medium. After 24 hours of culture without FBS, the NHF are treated with Ouessant black bee royal jelly at a dose of 100 μg/ml.

(11) After 15 hours of treatment, the NHF monolayer of each Petri dish is injured using a cross-shaped pipette tip.

(12) Twenty-four hours after the injury, the culture medium is removed and TIEG1 immunolabeling is performed.

(13) The cells are fixed in formalin and then permeabilized with Triton (0.1%) for 10 minutes. They are then saturated with 1% phosphate-buffered saline (PBS)/bovine serum albumin (BSA) for 30 minutes. The primary antibody (rabbit anti-KLF10; Abcam) is diluted 1:200 in 1% PBS-BSA buffer and incubated on the cells at room temperature for 1 hour. After rinsing with PBS, the secondary antibody (Alexa Fluor® 568 Goat Anti-Rabbit IgG) is diluted 1:200 in 1% PBS-BSA buffer and deposited at room temperature for 1 hour in the dark. Labeling of nuclei with DAPI (diluted 1:100) and of actin filaments with phalloidin 488 (diluted 1:200) is carried out in parallel with the incubation of the secondary antibody.

(14) After rinsing with PBS, a few drops of aqueous mounting medium are added. The dishes are finally stored at 4° C. waiting for image acquisition under a fluorescence microscope.

2.2. Image Acquisition and Analysis Under a Confocal Microscope

(15) Images are taken with a Leica SP5 II confocal microscope with an ×20 air objective at 1024×1024 pixel resolution. Three images are taken in the uninjured area and three or more images in the injured area, with the same acquisition parameters. Images are taken after excitation of the fluorochromes by a specific laser: 1. argon laser (488 nm), 2. laser diode (405 nm) and 3. helium-neon laser (633 nm).

(16) Once the acquisitions have been made, the images are analyzed one by one using Leica QWin software to obtain a quantitative description.

(17) An image analysis program allows specific detection of nuclei and TIEG-1 labeling. The detection of TIEG-1 labeling is performed in the cytoplasmic compartment and in the nucleus of the cells. For each condition, the quantification of TIEG-1 is measured in the healing area and in the uninjured area. The values of this quantification are systematically weighted by the number of nuclei or cell surface area.

2.3 Results

(18) The results (FIG. 2) are grouped in Table 1 below for the uninjured and injured areas (healing area).

(19) TABLE-US-00001 TABLE 1 Nuclear expression of TIEG1 in the uninjured area and in the healing area treated with Ouessant black bee royal jelly or untreated. Nuclear TIEG expression/cell Standard Treatment conditions Mean deviation Uninjured area Control 12.9 0.6 untreated Ouessant black bee royal 102.7 6.5 jelly (100 μg/ml) Healing area Control 14.9 2.1 untreated Ouessant black bee royal 126.8 12.5 jelly (100 μg/ml)

(20) Ouessant black bee royal jelly induces a significant increase in the nuclear expression of TIEG1 compared to the untreated control by +700% in the uninjured area and +751% in the healing area (FIG. 2).

(21) In addition, it is noted that treatment with Ouessant black bee royal jelly increases TIEG1 expression in the nuclear compartment of cells in the healing area by 23% compared to the expression of TIEG-1 after treatment of cells in the uninjured area, compared to 16% for the untreated control.

(22) Thus, treatment with Ouessant black bee royal jelly acts more effectively on a healing area, which makes it particularly attractive for use as an anti-aging agent in cosmetic skin care compositions designed to prevent or slow the appearance of signs of intrinsic or extrinsic skin aging.

Example 3

Study of the Effect of Ouessant Black Bee Royal Jelly and Buckfast Bee Royal Jelly on the Expression of Collagen V and Elastin in Cultured Normal Human Fibroblasts (NHF)

3.1 Cell Treatments

(23) NHF are seeded in T75 vials (75 cm.sup.2 flask, BD Biosciences) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

(24) The NHF are trypsinized at confluence with 0.05% trypsin-EDTA solution and neutralized with medium containing serum. Then they are seeded in 2 24-well plates at 38,000 cells per well.

(25) The NHF are then optionally treated with royal jelly of the Ouessant black bee or of the Buckfast bee tested at 100 μg/ml concentrations for 6 days. The Buckfast bee is a cross between the bee subspecies Apis mellifera mellifera and Apis mellifera ligustica. The treatment is performed twice for each condition and repeated 3 times during these 6 days. For the untreated (UT) control conditions), the medium is replaced by new medium.

(26) After 6 days of treatment, the cells are rinsed twice with phosphate buffer, then fixed in formalin (10% solution) for 10 minutes. After 2 rinses with PBS, the cell membranes are permeabilized with 0.1% PBS/Triton X-100 solution (Sigma), then rinsed twice with phosphate buffer.

(27) The cells are covered with 1% bovine serum albumin solution in phosphate buffer for 30 minutes at room temperature.

(28) The PBS/BSA solution is then replaced by a primary antibody solution corresponding to each labelled protein (see Table 2 below) diluted 1:100 in 1% PBS/BSA.

(29) TABLE-US-00002 TABLE 2 Summary of antibodies used Primary Antibody Secondary Antibody Collagen V Novotec 20511, Rabbit Alexa Fluor 568 Goat Anti Rabbit 1:500 (Molecular Probes) 1:200 Elastin Novotec 25011, Rabbit 1:200

(30) The plates are incubated for 2 hours at room temperature.

(31) The cells are then rinsed with phosphate buffer and covered with a solution of secondary antibody according to the primary antibody to be targeted (Table 2) and DAPI (4′,6′-diamidino-2-phenylindole, dihydrochloride) diluted respectively 1:200 and 1:100 in 1% bovine serum albumin solution in 1% phosphate buffer. The plates are stored for one hour in the dark and at room temperature.

(32) The secondary antibody solution is then aspirated, and the cells rinsed with PBS and then distilled water. A few drops of mounting medium (Aqua-Mount, Lab Vision Thermo Scientific) are deposited in each well.

3.2. Image Acquisition and Analysis by High-Content Screening (HCS)

(33) The plates are scanned with the “ArrayScan XTi” (Thermo Cellomics).

(34) Acquisition conditions:

(35) Detection: DAPI: filter XF53_386_23 Alexa Fluor 568: filter XF53_572_15 Resolution: 1104×1104 Objective: 10× dry Number of images: 49 per well (i.e. 2×49=98 per condition)

(36) The images are analyzed using the Spot Detector image analysis software, which detects the red labeling of the target protein, corresponding to its expression. The surface of the measurement area corresponds to the entire surface of the image. The number of cells is determined by counting the nuclei by detecting the blue labeling.

3.3. Results

(37) a. Type V Collagen Expression

(38) Ouessant black bee royal jelly significantly increases the synthesis of type V collagen on NHF compared to the untreated control (FIG. 3).

(39) Ouessant black bee royal jelly also has a significantly higher effect than that observed for Buckfast bee royal jelly (FIG. 3).

(40) b. Elastin Expression

(41) Ouessant black bee royal jelly more significantly increases (+66.8%) elastin synthesis on NHF (FIG. 4).

(42) The results obtained on different targets involved in the synthesis of the extracellular matrix in NHF show that Ouessant black bee royal jelly has an activating effect on the expression of type V collagen and elastin, which is significantly higher than that measured for Buckfast bee royal jelly. No particular effects on the expression of type V collagen and elastin are observed in NHF treated with Buckfast bee royal jelly.

(43) On the contrary, Buckfast bee royal jelly showed that it significantly decreased (−13.2%) the synthesis of type V collagen on NHF compared to the untreated control.

Example 4

SPF 15 Day Cream

(44) The composition below is an oil-in-water emulsion.

(45) Ouessant black bee royal jelly is identified by a high 10-hydroxy-2-decenoic acid content compared to the content of that compound in a Buckfast bee jelly.

(46) Percentages are expressed by weight with respect to the final composition:

(47) TABLE-US-00003 Ouessant black bee royal jelly 0.01 Octocrylen 2.0 Octyl methoxycinnate 7.5 Ethyl 2 hexyl palmitate 4.0 Phenyl trimethicone 0.5 Glyceryl monostearate 0.2 Cetearyl alcohol/dicetylphopshate/ceteth-10 phosphate 2.0 Glyceryl stearate/PEG-100 stearate 2.0 Cetyl alcohol 2.0 Beeswax Polyglyceryl-3 0.4 Benzophenone-3 1.5 Caprylic/caric triglyceride 3.5 Butylene glycol dicaprylate/dicaprate 2.5 C12-15 alkyl benzoate/titanium dioxide/polyhydroxystearic 2.5 acid/aluminum stearate/alumina Sodium polyacrylate 0.6 Butylene glycol 2.0 Diglycerin 1.0 Glycerol 3.5 Sodium hyaluronate <0.1 Xanthan gum 0.2 Ammonium acryloyldimethyltaurate/VP copolymer 0.4 Citric acid <0.1 Trisodium citrate 0.1 Aluminum starch octenylsuccinate 1.5 Rosemary leaf extract solution 2.0 Silk tree bark extract solution (PRODIZIA ®) 2.0 Tocopheryl acetate 0.2 Adjuvants (fragrances, alkalinizers, preservatives) qs Purified water qs 100

(48) Ouessant black bee royal jelly is a paste that is dissolved in the continuous aqueous phase of the cream.

(49) Each phase is prepared separately and then the fat phase is added under stirring in the aqueous phase in order to obtain a homogeneous dispersion of fat phase droplets in the continuous phase.

(50) The cream has a particularly pleasant texture when applied. It is applied upon awakening, on the face, by a light massage, focusing on areas showing signs of aging such as wrinkles or fine lines or areas with sagging skin.

REFERENCES

(51) Biofactors, 2010, 36, 8-18) Gumez L. et al., J. Appl. Physiol., 2010, 108, 1706-1710 Paul Martin, Wound Healing-Aiming for Perfect Skin Regeneration, Science, 1997, 276, 5309, pp. 75-81. Snyder L. R. Journal Of Chromatography, vol. 92, 1974, pages 223-230 Subramaniam M. et al. Mol. Cell. Biol., 2005, 1191-1199 Subramaniam M. et al., Biofactors, 2010, 36, 8-18 Taguchi M. et al., J. Musculoskelet. Res., 2008, 11, 63-69