Patent Application
20110268812
This application claims the benefit of Serial No. 1051708, filed 9 Mar. 2010 in France and which application is incorporated herein by reference. A claim of priority, to the extent appropriate is made.
The subject of the invention is a cosmetic composition comprising the combination of honey, or royal jelly, and a peptide, comprising at least the amino acid sequence Gly-X1-X2-Pro-Gly.
As is well known, skin aging is the result of genetic factors, but also environmental factors such as, for example, exposure to the sun, and manifests itself at the same time through molecular, cellular, histological and clinical modifications at the level of the epidermis and the dermis.
Among these skin-aging-related modifications, a decrease is observed in the thickness of the epidermis and in the size of the epidermal ridges, inducing a flattening of the dermal-epidermal junction which produces less cohesion at the interface of the epidermis and the dermis. A decrease in fibroblast density is also observed in the dermis.
This skin atrophy is also gradually reflected in changes to the extracellular matrix, linked to a decrease in the amount of collagen produced and to a decline in the levels of proteoglycans, glycosaminoglycans and fibronectin which are constitutive elements of said extracellular matrix, and in the levels of elastin fibres in the skin.
These phenomena lead to fragility of the skin, impairment of its mechanical and functional properties and of its protective and barrier functions, and, finally, a loss of elasticity which makes the expression wrinkles more visible.
Conversely, healing is itself a tissue repair process which comprises several steps, including a step of repair of the dermis and of the epidermis, resulting in re-epithelialisation of the wound (Ann. Dermatol. Venereol. 2005, 132:8549-68). The tissue repair phase corresponds to fibroblast proliferation and to extracellular matrix synthesis. The epithelialisation phase comprises the migration of epithelial cells (keratinocytes), and then the differentiation thereof so as to reform the epidermis. The extracellular matrix is gradually remodeled.
The main defect of skin which heals and repairs itself alone is often its lack of plasticity and elasticity. This is also true for skin which ages or which has been exposed to the sun (photoaging/elastosis). However, this quality is essential to the biomechanical properties and the beauty of the skin.
The main purpose of the present invention is to provide a novel cosmetic agent, which can in particular be used in the context of a cosmetic composition, capable of accelerating tissue repair processes, by stimulating the fibroblasts which behave as real skin construction accelerators, so as to result in a tissue repair process that acts as an internal-growth and matrix-reorganization factor, for a more dense and more elastic dermis.
Another main purpose of the invention is to provide a cosmetic composition comprising such a cosmetic agent.
Another main purpose of the invention is to provide a cosmetic care method which comprises the application of such a cosmetic agent.
The inventors have presently discovered that a combination of honey, or royal jelly, and a peptide comprising a particular sequence makes it possible to accelerate tissue repair processes, by stimulating the fibroblasts which behave as real skin reconstruction accelerators.
The abovementioned combination thus allows a tissue repair process that acts as an internal-growth and matrix-reorganization factor for a more dense and more elastic dermis.
a, 1b and 2a to 2d are examples illustrating the methodology used for demonstrating the activity of the combination of the invention.
a and 1b are two examples of observations, made under a phase contrast microscope, of an artificially “damaged” zone of the well in which normal human fibroblasts (NHFs) are cultured. The image in
a to 2d represent the steps of the captured image analysis in blue fluorescence and in red fluorescence, making it possible to characterize the colonization, by the fibroblasts, of the zone of the support artificially damaged by means of the abovementioned culture inserts.
A first subject of the present invention is thus directed towards a cosmetic agent, or a cosmetic composition comprising at least one cosmetically acceptable excipient, characterized in that it comprises honey or royal jelly, and a peptide, or a derivative of said peptide, comprising the sequence Gly-X1-X2-Pro-Gly, X1 and X2 being amino acids selected from natural or synthetic amino acids, and derivatives thereof. The present invention thus relates to a combination of honey or royal jelly, and a peptide, or a derivative of said peptide, comprising the sequence Gly-X1-X2-Pro-Gly and to a cosmetic composition comprising this combination.
The abovementioned peptide of the combination advantageously comprises 5 to 8 amino acids, advantageously selected from natural or synthetic amino acids, or a derivative of these amino acids.
X1 and X2 are preferably selected from valine (Val), proline (Pro), alanine (Ala), glycine (Gly), lysine (Lys), serine (Ser), aspartic acid (Asp), arginine (Arg) and isoleucine (Ile), or a salt or derivative thereof.
Among the preferred peptides, mention is made of those comprising or formed by a sequence selected from:
The peptide of the combination can be advantageously esterified with at least one linear or branched, saturated or unsaturated, hydroxylated or nonhydroxylated, sulphur-containing or non-sulphur-containing fatty acid comprising from 2 to 22 carbon atoms.
According to one preferred embodiment, the peptide is esterified with a fatty acid comprising between 8 and 22 carbon atoms, that may advantageously be a palmitoyl, stearoyl, elaidoyl, lauroyl, myristoyl, stearoyl, oleoyl, arachidyl or linoleoyl group.
The particularly preferred peptide of the combination is a peptide comprising or formed by the sequence Val-Gly-Val-Ala-Pro-Gly, or a cosmetically acceptable salt, said peptide also being advantageously esterified with a palmitoyl or stearoyl group, or a cosmetically acceptable salt.
A preferred peptide is palmitoyl-Val-Gly-Val-Ala-Pro-Gly sold in the form of an aqueous-glycolic solution under the trade name Biopeptide EL by the company Sederma.
The honey may be a unifloral or polyfloral honey.
The honey is preferably a clover (Trifolium repens) honey, a thyme (Thymus vulgaris) honey or alternatively a manuka (Leptospernum scoparium) honey.
The cosmetic agent according to the present invention can be incorporated into a cosmetic composition.
Thus, the present invention is also directed towards a cosmetic composition comprising a cosmetic agent as defined above or as resulting form the following description.
The concentration of honey or royal jelly which is used as one of the two components of the cosmetic agent according to the invention could be
In the examples, all the percentages are given by weight, the temperature is in degrees Celsius, and the pressure is atmospheric pressure, unless otherwise indicated.
This experiment is based on a model of biocompatible cell culture inserts allowing quantitative and perfectly reproducible measurement of the phenomena of healing in vitro.
1. Cell Culture
The cells used in this study are normal human fibroblasts (NHFs).
The cells are seeded in 175 cm2 flasks at a density of 1 million per flask in DMEM (Dulbecco's Modified Eagle's Medium) culture medium supplemented with 10% of foetal calf serum and 1.3 mM L-glutamine. Having reached confluence, the cells are trypsinized and re-seeded for the healing test.
2. Healing Test
Seeding of the Cells
Before seeding of the cells, inserts sold in the form of kits under the name Culture-Insert (ref. 80209) by the company IBIDI (Germany) are placed at the bottom of six wells of a plate. The NHFs are seeded in a proportion of 20 000 cells/cm2 in DMEM medium containing 10% of foetal calf serum, and cultured for 48 h.
When the cells have reached confluence, the culture medium is changed and replaced with DMEM alone. The culture is continued for 24 h.
Treatments
Two wells are used per treatment (substance tested) in this study.
The substances tested are the following (the concentrations indicated are the final concentrations):
A cell culture is also carried out in DMEM medium alone, without the addition of test substance, in two wells, in order to carry out a nontreated control (NT).
The Pal-VGVAPG peptide is in the form of an aqueous-glycolic solution, sold by the company Sederma under the trade name Biopeptide EL.
The active agents tested are prepared in the following way:
A stock solution at 100 mg/ml of clover honey in PBS is prepared and sterilized by filtration through a 0.22 μm filter. The final dilution ( 1/1000th) is then made in DMEM alone.
The commercial Biopeptide EL solution is diluted to 1/100th in the culture medium.
Healing
The culture insert is first of all removed with sterile tweezers. Using a phase contrast microscope, a “damaged” zone, devoid of any cells, is observed at the place where the insert was located (
The initial culture medium is then replaced in each well with the culture medium containing the active agents tested according to the conditions described above.
The cells are maintained in culture for 16 h (optimum measurement time determined previously), in an incubator at 37° C. and in an atmosphere containing 5% CO2. The degree of recolonization of the cicatricial zone according to the treatments is observed by means of an image analysis technique (
Cell Labelling
After rinsing with PBS, the cells are fixed for 10 minutes with 10% formalin solution and then rinsed with PBS. The cells are then permeabilized with a 0.1% Triton X100 solution for 10 minutes.
A solution containing DAPI (′,6′-diamidino-2-phenylindole), a fluorescent molecule used for labelling cell nuclei, and phalloidin 546, used for labelling the actin cytoskeleton, is deposited onto the cells and incubated at ambient temperature, in the dark for 1 h.
The cells are then rinsed with PBS and covered with a drop of aqueous mounting medium.
Images are immediately taken on a Nikon TE2000 video microscope in blue fluorescence (cell nuclei) and red fluorescence (actin filaments), at a rate of 2 photos per well at the damaged zone.
Image Analysis
Image analysis is used to visualize and quantify the colonization by fibroblasts of the artificially “damaged” zone after removal of the insert. The Leica QWIN software is used for the analysis.
The analysis comprises the following four steps:
1—capture of an image before treatment (time 0), using the Nikon TE2000 video microscope equipped with the NIS software (
2—determination of the measurement zone by means of a computer tool-zone in yellow (
3—capture of a new image after the steps of treatment and labelling of the cell nuclei (
4—automatic selection of the cell nuclei labelled in the zone defined in step 2, and counting of said labelled cell nuclei.
The number of cells that have recolonized the damaged zone, per unit area (cm2), is calculated. The measurement is reproduced on 4 images per active agents tested.
It should be noted here that
3. Results
The effect of the honey tested on the recolonization of the wound is presented in
Clover honey (+190% compared with the nontreated control) and Pal-VGVAPG (+150% compared with the nontreated control) have a significant effect on recolonization of the damaged zone. The combination of clover honey and Pal-VGVAPG (+224% compared with the nontreated control) exhibits an effect that is significantly greater than that observed for the treatments with clover honey or Pal-VGVAPG alone. The combination of honey and the Pal-VGVAPG peptide makes it possible to potentiate the action of each of these two active agents, which constitutes an unexpected result.
This justifies the selection of clover honey as a cosmetic active agent that is particularly effective for preventing or delaying the appearance of the signs of skin aging or slowing down the effects thereof.
The compositions below represent examples of embodiment of the invention. The percentages are expressed by weight relative to the weight of the final composition.
Antiwrinkle Serum Improving Firmness of the Skin
Centella asiatica
Vigna aconitifolia extract
The serum is applied in the morning to the areas of the face showing signs of aging.
Soothing Anti-Aging Repair Night Cream
Cyathea medullaris extract
The cream is applied to the face at bedtime.
Antiwrinkle Repair Essence
Bertholletia excelsa extract
Malva sylvestris extract
The preparation is applied to the wrinkles and fine lines of the face.
Anti-Aging Foundation
The preparation is applied to the face.
Correspondence for the Sequence Listing
| Number | Date | Country | Kind |
|---|---|---|---|
| 1051708 | Mar 2010 | FR | national |
