Cosmetic use of dedifferentiated plant cells

Abstract

The present invention relates to the cosmetic use of dedifferentiated plant cells of a plant of the Rosa sp. genus, or of an extract or a lyophilizate of said cells, as an active agent for caring for aged skin or aged hair. It also relates to rose dedifferentiated cells, in particular Lancme rose dedifferentiated cells.

Claims

1. A cosmetic composition comprising, as active agent, at least one dedifferentiated plant cell of a plant of the Rosa sp. genus, or an extract or a lyophilisate of said cells, said dedifferentiated plant cells being obtained by placing cells of a plant of the Rosa sp. genus in culture in a culture medium comprising at least NH.sub.4NO.sub.3; KNO.sub.3; CaCl.sub.2.2H.sub.2O; MgSO.sub.4; KH.sub.2PO.sub.4; MnSO.sub.4.4H.sub.2O; ZnSO.sub.4.7H.sub.2O; KI; Na.sub.2MoO.sub.4.2H.sub.2O; CuSO.sub.4.5H.sub.2O; FeSO.sub.4.7H.sub.2O; myo-inositol; nicotinic acid; pyridoxine HCl; thiamine HCl; and sucrose; and optionally kinetin, Na.sub.2EDTA.2H.sub.2O, and/or naphthaleneacetic acid, wherein the extract is an aqueous solution obtained by grinding the at least one dedifferentiated plant cell in order to obtain a suspension, and then removing the particles from the suspension.

2. A cosmetic method for caring for aged skin or aged hair, comprising administering, to an individual in need thereof, the composition according to claim 1.

3. The method according to claim 2 for treating and/or preventing an esthetic defect subsequent to a deficiency in the tissue renewal of the skin or of the hair.

4. The method according to claim 2 for preventing and/or reducing and/or treating signs of skin aging.

5. The method according to claim 2, wherein the signs of skin aging are chosen from thinning of the skin, a loss of firmness, a loss of elasticity, a loss of density or a loss of tonicity of the skin, dryness of the skin, an alteration of the surface appearance of the skin, the appearance of a marked microrelief of the skin, the appearance of roughness, the formation and/or presence of fine lines and/or of wrinkles, a modification of the radiance of the skin complexion, a wizened appearance of the skin, a modification of the odor of the skin, sagging of the skin, or withering of the skin.

6. The method according to claim 2 for preventing and/or reducing and/or treating thin, lifeless, brittle, weak hair with no hold, no radiance and/or which is graying.

7. The method according to claim 2 for preventing and/or reducing and/or treating partial or total loss of hair fibers.

8. The method according to claim 2 for preventing and/or reducing and/or treating whitening of the hair.

9. The method according to claim 2 wherein the dedifferentiated plant cells are derived from a leaf, a stem, a flower, a petal, a sepal or a root.

10. The method according to claim 2 wherein the dedifferentiated plant cells are derived from a Lancme delboip rosebush.

11. The method according to claim 2 wherein the dedifferentiated plant cells, or an extract or a lyophilisate of said cells, are formulated in water or a water-soluble organic solvent, or a mixture thereof.

12. The method according to claim 2 wherein the dedifferentiated plant cells, or an extract or a lyophilisate of said cells, are formulated in the presence of a gelling agent and/or of a C.sub.4 to C.sub.6 sugar.

13. The method according to claim 2 wherein the dedifferentiated plant cells, or an extract or a lyophilisate of said cells, are formulated in the presence of water, of 1,3-propanediol and/or of xanthan gum.

14. The method according to claim 2 wherein the dedifferentiated plant cells, or an extract or a lyophilisate of said cells, are used in an amount representing from 0.01% to 30% relative to the total weight of the composition containing them.

15. The composition according to claim 1 wherein a concentration of the dedifferentiated plant cell of a plant of the Rosa sp. genus, or the extract or the lyophilisate of said cell, in the composition is from 0.01% to 50% by weight of dry matter relative to the total weight of the composition.

16. The composition according to claim 1, wherein the active agent is at least one dedifferentiated plant cell derived from a Lancme delboip rosebush, or an extract or a lyophilisate of said cell.

17. Dedifferentiated plant cells derived from a Lancme delboip rosebush, or a lyophilisate of said cells.

18. The dedifferentiated plant cells according to claim 17, wherein they are obtained from plant material derived from whole plants or from plant parts.

19. The dedifferentiated plant cells according to claim 17, which cells are obtained from plant material derived from Lancme delboip rosebush leaves.

20. The dedifferentiated plant cells according to claim 17, which are obtained by means of a method comprising the following steps: a) providing Lancme delboip rose leaves, or cells derived from said leaves, b) cultivating the leaves or the cells provided in step a) in the presence of at least one plant hormone, so as to generate dedifferentiated cells, and c) recovering the dedifferentiated cells obtained at the end of step b).

21. The rose dedifferentiated cells belonging to the cell line deposited according to the Treaty of Budapest on Jul. 4, 2012, under the reference 2-6307 with the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) [German Collection of Microorganisms and Cell Cultures].

22. The composition according to claim 1, wherein the extract is sterilized.

23. A cosmetic composition comprising, as active agent, at least one dedifferentiated plant cell of a plant of the Rosa sp. genus, or an extract or a lyophilisate of said cells, said dedifferentiated plant cells being obtained by placing cells of a plant of the Rosa sp. genus in culture in a culture medium comprising at least NH.sub.4NO.sub.3; KNO.sub.3; CaCl.sub.2.2H.sub.2O; MgSO.sub.4; KH.sub.2PO.sub.4; MnSO.sub.4.4H.sub.2O; ZnSO.sub.4.7H.sub.2O; KI; Na.sub.2MoO.sub.4.2H.sub.2O; CuSO.sub.4.5H.sub.2O; FeSO.sub.4.7H.sub.2O; myo-inositol; nicotinic acid; pyridoxine HCl; thiamine HCl; and sucrose; and optionally kinetin, Na.sub.2EDTA.2H.sub.2O, and/or naphthaleneacetic acid, wherein the composition is in the form of a gel, a cream, a foam, a capsule, an emulsion, a tablet, an aerosol composition comprising a pressurized propellant, or an ionic or nonionic vesicular dispersion.

24. The composition according to claim 23, wherein the active agent is at least one dedifferentiated plant cell derived from a Lancme delboip rosebush, or an extract or a lyophilisate of said cell.

25. A cosmetic method for caring for aged skin or aged hair, comprising administering, to an individual in need thereof, a composition comprising, as active agent, the dedifferentiated plant cells or lyophilisate of claim 17.

26. A cosmetic method for caring for aged skin or aged hair, comprising administering, to an individual in need thereof, the composition according to claim 23.

Description

FIGURES

(1) FIG. 1: represents photographs of models of reconstructed skins with low regenerative potential, in section, after treatment for 10 days in the absence (control) or in the presence of Lancme rosebush dedifferentiated cells at 0.43 mg/ml or 0.043 mg/ml and culturing up to 13 (top photograph) or 20 (bottom photograph) days.

(2) FIG. 2: represents the amount of keratinocyte growth factor (KGF) determined in the culture media of models of reconstructed skins with low regenerative potential, treated in the absence or in the presence of Lancme rosebush dedifferentiated cells (0.43 mg/ml). The analyses were carried out on D3, D5, D7, D10, D13, D17 and D23. Light gray histogram: control. Dark gray histogram: Lancme rosebush dedifferentiated cells at 0.43 mg/ml. White histogram: Lancme rosebush dedifferentiated cells at 0.0043 mg/ml.

(3) FIG. 3: represents photographs of models of reconstructed skins with low regenerative potential, in section, which are untreated (control) or treated in the presence of rosebush dedifferentiated plant cells at 0.43 mg/ml or in the presence of retinol at 10 M.

(4) In the description and in the examples that follow, unless otherwise indicated, the percentages are percentages by weight and the ranges of values written in the form between . . . and . . . include the specified lower and upper limits. The ingredients are mixed, before being formed, in the order and under conditions that are easily determined by those skilled in the art.

(5) The examples hereinafter are presented as nonlimiting illustrations of the field of the invention.

EXAMPLES

Example 1

Culture of Rosebush (Lancme Rose) Dedifferentiated Plant Cells Using Plant Tissue

(6) Leaves of a rosebush (Lancme Rose) were decontaminated by means of the usual techniques of those skilled in the art, and placed on the following culture medium:

(7) TABLE-US-00001 mg/l MACRONUTRIENTS NH.sub.4NO.sub.3 1650 KNO.sub.3 1900 CaCl.sub.22H.sub.2O (CX41) 440 MgSO.sub.4 180.8 KH.sub.2PO.sub.4 170 MICRONUTRIENTS MnSO.sub.44H.sub.2O 22.3 ZnSO.sub.47H.sub.2O 8.6 KI 0.83 Na.sub.2MoO.sub.42H.sub.2O 0.25 CuSO.sub.45H.sub.2O 0.025 Na.sub.2 EDTA2H.sub.2O 37.3 FeSO.sub.47H.sub.2O 27.8 VITAMINS MYO-INOSITOL 100 NICOTINIC ACID 0.5 PYRIDOXINE HCl (B.sub.6) 0.5 THIAMINE HCl (B.sub.1) 0.1 HORMONES Naphthaleneacetic acid 10 Kinetin 0.06 CARBON SOURCE Sucrose 30000

(8) The hormones used are hormones naturally present in plants.

(9) Following successive subculturings, dedifferentiated plant cells that can be cultivated in a fermenter and are therefore industrializable were obtained. The parameters used for the control of the culture in a bioreactor are the following:

(10) T: from 24 C. to 30 C., ideally 26-27 C.

(11) Aeration: pO.sub.2 10%, regulated by the sterile air entering and/or via agitation while avoiding any shear stress on the cells.

(12) The batchwise production lasts from 6 to 14 days. The cells are then harvested by filtration on a cloth with a porosity of 80 m.

(13) The Lancme rosebush dedifferentiated cells obtained are formulated in water, in the presence of 1,3-propanediol and of xanthan gum in a proportion of 10% by weight of fresh biomass relative to the total weight of the composition.

Example 2

Demonstration of the Pro-Regenerating Effects of the Lancme Rosebush Dedifferentiated Plant Cells on a Model of Reconstructed Skin with Low Regenerative Potential

(14) aPreparation of the Models of Reconstructed Skin with Low Regenerative Potential

(15) Keratinocytes were isolated from a human skin specimen.

(16) After removal of the subcutaneous tissue with a scalpel, the skin specimen was cut into fragments and then decontaminated by means of an antibiotic-antimycotic treatment in DMEM culture medium (Life Technologies Ltd, Scotland).

(17) In order to enable the dermis to be separated from the epidermis, the specimen was subsequently subjected to a proteolytic treatment (Dispase II)/Trypsin 0.25%) overnight at 4 C. The fragments of epidermis separated from the dermal tissue were placed in a 1 trypsin-EDTA solution for 20 minutes at 37 C. The effect of the trypsin was neutralized by adding a culture medium containing 10% of fetal calf serum (FCS). The cell suspension was homogenised and then washed in keratinocyte culture medium (keratinocyte growth medium, KGM) (KGM Bullet Kit, BioWhittaker, Clonetics Corp., San Diego, Calif., USA). The cell suspenion was then placed in culture flasks pre-coated with collagen type I (Sigma Chemical Co Ltd, Irvine, UK). After 15 minutes, the keratinocytes which had not adhered were recovered by washing in PBS buffer. The nonadherent cells thus selected are depleted of stem and progenitor cells of the basal layer.

(18) This subpopulation of keratinocytes was seeded, in a proportion of from 150 000 to 450 000 cells, in culture medium (N Fortunel et al (2011). Eur J Dermatol. 21(52):12-20) onto the upper part of a dermal support placed beforehand in an insert.

(19) The culture substrate used is a devitalized human dermis free of epidermis or a live dermis comprising fibroblasts within a collagen lattice (stretched model, production Episkin). Medium is then added below and above each insert and this medium is renewed every two days. After 6-7 days at 37 C. in an incubator at 5% CO.sub.2, the medium on the upper part of the sample was completely drawn off and the medium below the insert was replaced with the same medium mentioned below but devoid of transferrin, of triiodothyronine and of adenine. This begins the emersion phase necessary for inducing the stratification and differentiation of the epidermal compartment. The samples were cultivated for 1-3 weeks at 37 C. and 5% CO.sub.2, the medium is renewed every two days.

(20) bTreatments with the Dedifferentiated Plant Cells

(21) The rose dedifferentiated cells obtained in example 1 were lyophilized, then resuspended by successive passage in a syringe. The cell debris was removed by centrifugation. The resulting active agent with the cell debris removed was used in a proportion of 0.43 mg/ml and 0.043 mg/ml. The active agent was applied systemically (in the culture medium below the inserts) as soon as the dermal support was seeded with keratinocytes. The application of the active agent was renewed on days 3, 6, 8 and 10. Histological analyses: thickness of the epidermis, general organization, stratification, quality of the basal layer, etc.,
were carried out on day 13 and on day 20.

(22) The control corresponds to culture medium devoid of rose dedifferentiated cells.

(23) As shown in FIG. 1, in the presence of rose dedifferentiated cells, thickening of the epidermis was observed. Furthermore, the basal layer is more dense, the morphological quality is improved and correct differentiation, namely stratification of the living layers and a semblance of a granular layer, is observed.

(24) These results demonstrate a restoration of the regenerative capacity of the models of reconstructed skins with low regenerative potential, following the treatments performed with dedifferentiated cells of a rosebush (Lancme Rose) and support the use of such an ingredient for treating the skin.

Example 3

Titration of the Secretion of Keratinocyte Growth Factor (KGF) Induced by Treating a Model of Reconstructed Skin, with Low Regenerative Potential, with Lancme Rosebush Dedifferentiated Plant Cells

(25) The culture media of the experiments described in example 2 were collected on days 3, 5, 7, 11, 13, 17 and 20. The soluble amounts of KGF secreted by the reconstructed skins and present in these media were then analysed by ELISA (Enzyme-linked immunosorbent assay) (R&D Systems, Catalog N.sup.oDKG00).

(26) As shown in FIG. 2, an increase in secretion of KGF was observed in comparison with the control (untreated), especially on day 13 following treatments with the rose dedifferentiated cells at 0.43 mg/ml.

(27) This growth factor is produced by the fibroblasts and it is known for its mitogenic effects on kratinocytes (Finch P W et al. Science 1989 245:752; Marchese et al. J Cell Physiol 1990 144:326). This result shows that the rose (Lancme Rose) dedifferentiated plant cells induce secretion of diffusible factors, produced at least in part by the fibroblasts, which have a beneficial effect on epidermal regeneration. A pro-regenerating dialogue is therefore created between the dermis and the epidermis.

Example 4

Demonstration of the Beneficial Effects on the Self-Renewal of Dermal Stem Cells in Vitro

(28) Isolation of Dermal Stem Cells Derived from Human Biopsy

(29) Stem cells derived from the dermal compartment (SKPs) have the capacity to proliferate and to self-renew in the form of spheres in suspension when they are cultivated in vitro in the presence of FGF2 and EGF. They are isolated from the skin and the hair follicles according to the protocol described in the publication Nature Protocols (Biernaskie et al. 2006). The feeding medium used consists of DMEM/F12 (Invitrogen) supplemented with antibiotics: penicillin (Lonza) and streptomycin (Lonza), with an antifungal agent Fungizone (Invitrogen), with the growth factors EGF (BD) and FGF2 (BD), and with serum substitute supplement B27 (Invitrogen).

(30) The SKPs used are derived from skin specimens and in particular from the foreskins from 3 different donors (8 years old, 10 years old, 22 years old). After a first passage in culture in flasks, the spheres are associated with collagenase and are then put back in culture in the form of individualized cells in the presence of 50% of feeding medium and 50% of conditioned medium (conditioned by the cells themselves).

(31) Evaluation of the Level of Self-Renewal in the Presence or in the Absence of the Rose Dedifferentiated Cells

(32) The method used to evaluate the levels of self-renewal is similar to that used during the passages of the cells. The SKPs are first dissociated with collagenase, before being seeded in a proportion of 3000 cells per well (in 96-well plates). The cells are cultured in a mixture containing half conditioned medium and half feeding medium, as described previously. The cells are cultivated at 37 C. (21% of O.sub.2-5% CO.sub.2) and the treatments are performed in the absence (control) or in the presence of 0.43 mg/ml of the active agent derived from Lancme rosebush dedifferentiated cells, obtained as described in example 2. After the days of culture, the spheres formed are counted in order to evaluate the self-renewal capacity of these cells after treatment.

(33) The results are given in table I hereinafter.

(34) TABLE-US-00002 TABLE I Mean number of spheres per well, per condition, and per biopsy on D10 (mean of 4 wells) Treatment Donor 1 Donor 2 Donor 3 Mean SD +rose 88.67 55.75 183.50 109.31 54.16 dedifferentiated cells 52.50 33.50 112.25 66.09 33.55

(35) The self-renewal of the dermal stem cells is significantly increased after a treatment with the active agent derived from the rose dedifferentiated cells (+treatment) compared with the control (treatment).

(36) This study makes it possible to conclude that a treatment with the active agent derived from the rose dedifferentiated cells significantly increases the self-renewal capacities of the dermal stem cells in vitro. Given the importance of the role played by these cells in biological processes such as hair follicle regeneration, in the control of the shape of the hair shaft and in skin repair, this result emphasises the advantage of this ingredient as a technical solution for treating aging of the skin and of the hair.

Example 5

Comparison of the Pro-Regenerating Effects of the Rose Dedifferentiated Cells with Those of Another Reference Anti-Aging Active Agent on a Model of Reconstructed Skin with Low Regenerative Potential

(37) The model of reconstructed skin was prepared as indicated in example 2. The active agent derived from the rose dedifferentiated cells was prepared as indicated in example 2. The model of reconstructed skin was treated in the absence (control-culture medium alone) or in the presence of the active agent derived from the rose dedifferentiated cells in a proportion of 0.43 mg/ml, or of retinol in a proportion of 10 M, for 12 days.

(38) The histological observations were made as indicated in example 2.

(39) As shown in FIG. 3, in the presence of the active agent derived from rose dedifferentiated plant cells, thickening of the epidermis was observed. Furthermore, the basal layer is more dense, the morphological quality is improved and correct differentiation (stratification of the living layers, a semblance of a granular layer) is observed.

(40) On the other hand, treatment with retinol did not improve the epidermal quality and even appears to inhibit differentiation.