WO2015001524A2

WO2015001524A2 - Stabilization of rose dedifferentiated cells

Info

Publication number: WO2015001524A2
Application number: PCT/IB2014/062860
Authority: WO
Inventors: Cécile SAQUET-GOUVILLE; Marie OURCIVAL; Dang-Man Pham; Martine Dubois
Original assignee: L'oreal
Priority date: 2013-07-05
Filing date: 2014-07-04
Publication date: 2015-01-08
Also published as: FR3007982A1; WO2015001524A3; FR3007982B1

Abstract

Use of a metal-ion-chelating agent as a stabilizing agent in a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

Description

STABILIZATION OF ROSE DEDIFFERENTIATED CELLS

FIELD OF THE INVENTION

The present invention relates to the use of chelating agents for stabilizing a composition comprising dedifferentiated cells of a plant of the Rosa sp. species.

More particularly, the use of a metal-ion-chelating agent makes it possible to stabilize a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

PRIOR ART

The use of dedifferentiated plant cells for cosmetic purposes is a concept which emerged about twenty years ago.

Dedifferentiated plant cells arose from the work by Haberland in 1902. During the past 40 years, plant cell cultures have been used to produce metabolites of interest or for identical plant multiplication (somatic embryogenesis).

This plant biotechnology is based on the concept of "cell totipotency", which proposes that any plant cell is capable of differentiating and of regenerating another individual identical to that from which it is derived.

A dedifferentiated plant cell can be obtained from a plant cell originating from a differentiated organ, such as, for example, the leaf, the stem, the root or the petal. When this differentiated cell is placed in culture, it can return to a dedifferentiated state, i.e. it loses its leaf, stem, root or petal cell specificity, and again becomes optionally capable of generating the whole plant.

A dedifferentiated plant cell is the equivalent of an actual plant stem cell, derived from meristematic plant cells, and not having an organ-specific biological past.

WO 2009/151302 and KR 2009-0118877 describe anti-ageing or antioxidant compositions containing undifferentiated plant cells derived from the cambium of Panax ginseng or of a plant of the Taxus genus.

EP 1 985 280 describes the use of dedifferentiated plant cells derived from a plant of the family Rosaceae, and in particular of the apple tree Malus domestica, for protecting stem cells of the skin faced with various intrinsic and extrinsic stresses, and in particular for treating age-related damage to the skin or to the hair.

EP 1 699 423 describes the use of a lyophilisate of dedifferentiated plant cells derived from a halophilic plant, for rejuvenating the appearance of the skin. DE-A-102 009 027 361 and EP 2 266 529 describe the use of dedifferentiated plant cells, in particular derived from a plant of the Mal genus, for treating keratin hair fibres, and in particular protecting them against UV radiation, or for treating aged skin.

Finally, EP 0 909 556 describes the use of an extract of a plant of the family Rosaceae, in particular of a rose, optionally obtained from undifferentiated plant cells, as a bradykinin antagonist for the treatment of various conditions, in particular skin conditions.

The inventors have observed that rose dedifferentiated plant cells, or an extract or a lyophilisate of said cells, have proved to be particularly suitable and effective for treating aged skin or aged hair.

However, the inventors have observed that compositions comprising rose dedifferentiated plant cells, and in particular compositions comprising rose dedifferentiated cells obtained by means of a process comprising a step of freezing and then thawing or a lyophilization step, have reduced stability over time.

The applicant has observed that, over time during storage, a composition comprising rose dedifferentiated plant cells or comprising an extract or a lyophilisate of said cells, has a yellowish colouration, or even a brownish colouration, and may contain black- coloured aggregates. These characteristics, which embody a deterioration of the composition with time, appear a few hours to a few days after the composition has been obtained. In particular, these characteristics of yellowing of the composition, of browning of the composition or of the formation of black-coloured aggregates appear a few hours to a few days after the composition has been obtained by means of a process comprising a step of freezing and then thawing.

These visible modifications of the composition over time signify a reduced stability thereof, with a deterioration that is accentuated with the duration of storage.

This degradation of the visual appearance, that would result from a change in colour, via a change in texture that would possibly result from the the presence of aggregates, or via a combination of several of these types of degradation, is incompatible with formulation of the composition in a cosmetic product.

In particular, these changes in colour and/or in texture of a composition comprising dedifferentiated cells of a plant of the Rosa sp. species or an extract or a lyophilisate of said cells signify the occurrence of a physicochemical deterioration of the composition, these changes being capable of reducing the cosmetic effect(s) of said composition as storage time increases. There is therefore a need to identify technical solutions which make it possible to increase the stability properties of compositions comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

More particularly, there is a need to confer a preservation over time of the textural uniformity of a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, i.e. a need to delay, or to reduce, or to block the formation of aggregates of plant material.

There is also a need to maintain over time the visual appearance of a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, i.e. a need to delay, or to reduce, or to block the yellowing or the browning of the composition.

SUMMARY OF THE INVENTION

The present invention relates to the use of a metal-ion-chelating agent as a stabilizing agent in a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

In another aspect, the invention relates to a process for stabilizing a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, which process comprises at least one step of bringing said dedifferentiated cells, or said extract or said lyophilisate of said cells, into contact with a metal- ion-chelating agent.

Another aspect of the invention relates to a stable composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, said composition comprising a metal-ion-chelating agent as stabilizing agent.

According to another aspect, the invention relates to the cosmetic use of a stable composition as defined in the present description, as an active agent for caring for aged skin or aged hair. FIGURE LEGEND

Figure 1: Photon microscopy images of compositions of rose dedifferentiated plant cells after storage at ambient temperature. Figure 1 A: non-polarised light microscopy image, at magnification (^χ 10), of a Lancome Rose cell composition obtained according to a process comprising a step of freezing and thawing.

Figure 1 B: polarized light microscopy image, at magnification (^χ 10), of a Lancome Rose cell composition obtained according to a process comprising a step of freezing and thawing.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have shown that the addition of a metal-ion-chelating agent, for instance EDTA or an EDTA salt, makes it possible to stabilize a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells. It is thus shown according to the invention that the addition of a metal-ion-chelating agent, for instance EDTA or an EDTA salt, makes it possible to maintain a uniform texture, without visible deterioration of the original appearance of said composition during storage time.

For the purposes of the invention, the term "stabilization" is intended to mean the fact that the composition, during preservation or storage thereof, retains a visual appearance, in particular its colour, and a texture identical or close to that of the composition freshly obtained by means of the process, i.e. a uniform texture and a colourless, or slightly whitish, visual appearance.

The presence of a metal-ion-chelating agent in the composition has the effect of delaying, of reducing or blocking the formation of aggregates of plant material, which encompasses aggregates visible to the naked eye and aggregates visible only by means of an optical microscopy observation. Thus, with a composition comprising a metal-ion-chelating agent as defined in the present description, an absence of aggregates of plant material is observed, or at least the presence of a reduced amount of aggregates is visible after optical microscopy observation.

The presence of a metal-ion-chelating agent in the composition also has the effect of delaying, or reducing or blocking the yellowing or the browning of the composition, as can be observed directly with the naked eye or by photon microscopy.

Owing to the absence of aggregates of plant material, or owing to the presence of a reduced amount of aggregates of plant material, and owing to the absence of detectable yellowing or browning, the visual appearance and the texture of the composition are identical or similar to the original appearance of the composition, i.e. to the visual appearance and the texture of the composition as immediately resulting from the process used to prepare it.

For the purposes of the invention, the term "dedifferentiated plant cell" is intended to mean any cell strain derived from organs of a rosebush of the Rosa sp. species and obtained by means of specific in vitro culture conditions, no longer exhibiting any specialization character and capable, under the effect of induction, of any differentiation in accordance with its genome and of generating by itself a whole plant of a plant from which it originates. Such cells are capable of living by themselves and not in a dependency relationship with other cells.

Dedifferentiated plant cells are therefore distinct from undifferentiated plant cells which naturally exist in plants.

Under normal conditions, plant cells express approximately 20% of their genome, the remaining 80% being expressed only in response to particular environmental conditions. The in vitro culturing of these cells under particular culture conditions makes it possible to reprogram the cells and thus to gain access to an unexpressed part of this genome. Some compounds, which are difficult to obtain by extraction from plants, become more accessible in cell cultures.

Thus, advantageously, the dedifferentiated plant cells make it possible to gain access to new compounds not present in the whole plant or to significantly increase the expression of molecules which are known but are found in very small amount in the whole plant.

Dedifferentiated plant cells can be obtained from any plant of the Rosa sp. species. These cells can be obtained from a plant material derived from whole plants or from plant parts, such as the leaves, stems, flowers, petals, sepals or roots cultivated in vivo or in vitro.

The term "in vivo culture" is intended to mean any culture of standard type, i.e. in soil in the open air or in a greenhouse, or alternatively out of the soil.

The term "in vitro culture" is intended to mean all the techniques known to those skilled in the art for artificially obtaining a plant or a plant part. The selection pressure imposed by the physicochemical conditions during the growth of plant cells in vitro makes it possible to obtain a standardized plant material that is available throughout the year, in contrast with plants cultivated in vivo.

Preferentially, according to the invention, a plant derived from in vivo culture is used.

Preferably, dedifferentiated plant cells of the invention are obtained from at least one leaf of a plant of the Rosa sp. species. The Rosa genus comprises more than 1000 species, among which mention may be made of Rosa alba, Rosa alpina, Rosa canina, Rosa cinnamonea, Rosa gallica, Rosa repens, Rosa rubrifolia, Rosa rubiginosa, Rosa sempervirens, Rosa spinosissima, Rosa stylosa, Rosa tomentosa or Rosa villosa.

More preferably, the dedifferentiated plant cells of the invention are obtained from a Lancome Rose rosebush, and more particularly from a Lancome delboip Rose rosebush. Such a rosebush is commercially available, for example, from the company Delbard.

The Lancome rosebush is a tea hybrid, the female parent of which is a hybrid obtained by crossing of the varieties Dr. Albert Schweitzer ^χ [Michele Meilland ^χ Bayadere] and which has been pollinated by the variety Melmet.

Depending on the culture method chosen, and in particular depending on the culture medium chosen, it is possible to obtain, from the same explant, dedifferentiated plant cells which have different characteristics.

A culture medium suitable for obtaining dedifferentiated plant cells of the invention is well known to those skilled in the art and can comprise NH₄NO₃ KNO₃; CaCl₂.2H₂0; MgS0₄; KH₂P0₄; MnS0₄.4H₂0; ZnS0₄.7H₂0; KI; Na₂Mo0₄.2H₂0; CuS0₄.5H₂0; FeS0₄.7H₂0; myoinositol; nicotinic acid; pyridoxine HC1; thiamine HC1; sucrose; kinetin; Na₂EDTA.2H₂0; naphthaleneacetic acid,and mixtures thereof.

The culturing is carried out at a temperature ranging from 24 to 30°C for a partial 0₂ pressure of approximately 10%. The culturing is carried out in batches for 7 to 10 days.

It can be carried out by fed-batch fermentation or continuous fermentation.

After having been cultured in a suitable medium, the dedifferentiated plant cells of the invention are harvested by filtration. The dedifferentiated plant cells thus harvested can then be brought into contact with a metal-ion-chelating agent.

At at this stage, the dedifferentiated plant cells can undergo, prior to any subsequent treatment step, a freezing step followed by a thawing step. The thawed dedifferentiated plant cells can then be brought into contact with a metal-ion-chelating agent.

The dedifferentiated plant cells can then be lyophilized or be subjected to an extraction process, these methods being well known to those skilled in the art.

According to the present invention, uses made of dedifferentiated plant cells which are fresh, or lyophilized, or extracts thereof, or which are formulated in stable compositions, i.e. in the presence of a metal-ion-chelating agent.

Within the scope of the invention, "fresh" dedifferentiated plant cells have not undergone any freezing/thawing steps. In one embodiment, the dedifferentiated plant cells are preferably fresh dedifferentiated plant cells, or an extract thereof.

The lyophilized dedifferentiated plant cells can be re-extracted in a mixture comprising a metal-ion-chelating agent. The lyophilization of the dedifferentiated plant cells advantageously makes it possible to separate the active agents from the biopolymer fillers, such as cell wall polysaccharides.

The dedifferentiated cells can be lyophilized, and then resuspended in a mixture comprising a metal-ion-chelating agent. The cell debris which may be present in the suspension obtained is removed, for example by passing through a syringe and then centrifugation. The suspension with the cell debris removed can be used as an active agent derived from dedifferentiated plant cells according to the invention.

Any extraction method known to those skilled in the art may be used to prepare an extract of dedifferentiated plant cells according to the invention.

An "extract", according to the present invention, relates to all or part of a fraction comprising the cell content of the dedifferentiated cells of a plant of the Rosa sp. species. The cell content comprises, in a non-limiting manner, the membrane fraction, the cytoplasmic fraction, the cytosolic fraction, the organelles such as the mitochondria, the chloroplasts or the protoplasts, and a mixture thereof.

An extract may be a total extract, i.e. it contains all of the cell fractions, and may be obtained by any method well-known to those skilled in the art, for example by milling the plant material using balls or grains of sand, freezing/thawing, sonication, passing through a French press, enzymatic or chemical lysis of the plant wall, etc.

By way of an extract suitable for the invention, mention may be made of aqueous extracts, alcoholic extracts or extracts using an organic solvent, advantageously an alcoholic organic solvent.

The term "aqueous solvent" is intended to mean any solvent consisting totally or partially of water. Mention may thus be made of water itself, aqueous-alcoholic solvents in any proportion or else solvents consisting of water and a compound such as propylene glycol in any proportion. Among the alcoholic solvents, mention may particularly be made of methanol, ethanol, propanol, propanediol, butanol and butanediol.

An extraction method suitable for the invention may comprise a first step of milling the dedifferentiated plant cells, for example in an aqueous solution under cold conditions, then a second step of removing the particles in suspension from the aqueous solution resulting from the first step, and a third step of sterilizing the aqueous solution resulting from the second step. This aqueous solution corresponds to an extract.

The extract obtained can then be lyophilized by any conventional lyophilization methods. A powder is thus obtained, which can be used directly or else mixed into an appropriate solvent before use.

The dedifferentiated plant cells of the invention may also be used in the form of a lyophilisate. Such a lyophilisate can be obtained by any lyophilization method known to those skilled in the art.

It it is understood that these various treatment methods involve the presence of a metal-ion-chelating agent, in particular EDTA or a salt thereof, as an agent for stabilizing a solution comprising dedifferentiated cells of a plant of the Rosa sp. species.

Uses and processes

The present invention relates, in a first aspect, to the use of a metal-ion-chelating agent as a stabilizing agent in a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

According to one preferred embodiment of the invention, the process for stabilizing a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, comprises at least one step of bringing said dedifferentiated cells, or said extract or said lyophilisate of said cells, into contact with a metal- ion-chelating agent.

This bringing of the dedifferentiated plant cells into contact with the metal-ion- chelating agent is carried out at any step of a process for obtaining said composition or said extract or lyophilisate.

In certain embodiments, the bringing of the metal-ion-chelating agent into contact with the dedifferentiated plant cells is carried out in the final step of a process for the obtaining of a non-stabilized composition of dedifferentiated cells of the Rosa sp. species, or of an extract or lyophilisate of said cells. By way of illustration, the metal-ion-chelating agent may be added to the non-stabilized final composition of dedifferentiated cells of a plant of the Rosa sp. species, or to an extract or a lyophilisate of said cells, in order to have a stable or stabilized composition as defined in the present invention.

In certain embodiments, the metal-ion-chelating agent is brought into contact with the dedifferentiated plant cells of Rosa sp. in a plurality of steps, among the steps included in the process for obtaining the composition of said dedifferentiated cells, or an extract or a lyophilisate of said cells.

In certain embodiments, the process comprises at least the following steps:

a) providing a composition, in liquid form, comprising dedifferentiated plant cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, and b) adding a metal-ion-chelating agent to the composition in liquid form provided in step a).

Generally, the composition in liquid form can be chosen from a suspension and a solution.

In one preferred embodiment, the dedifferentiated plant cells of a plant of the Rosa sp. species, or an extract or a lyophilisate of said cells, may be in the form of a suspension.

In another preferred embodiment, an extract or a lyophilisate of dedifferentiated plant cells of a plant of the Rosa sp. species can be used in the form of a solution.

Advantageously, the metal-ion-chelating agent comprises a mono-, di-, tri- or tetraamine group, to which one or more acetic acid-derived group(s) is (are) covalently attached.

Thus, advantageously, the chelating agent is chosen from the compounds of general formula (I):

RRN-((CH₂)₂-NRR)x (formula I),

for which x is equal to 1, 2, 3 or 4;

and for which R is advantageously CH₂-COOR', for which R' is chosen from H and Na.

In certain embodiments, the metal-ion-chelating agent, for implementing the uses or the processes in accordance with the present invention, is chosen from EDTA

(ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), MGDA (methylglycine diacetic acid), GLDA (glutamic acid diacetic acid), NTA (nitrilotriacetic acid),

TTHA (irieihyleneieiraaminehexaaceiic acid), EDDS (ethylenediaminedisuccinic acid), and salts thereof.

In one advantageous embodiment, the metal-ion-chelating agent is a sodium salt.

In certain embodiments, the metal-ion-chelating agent is an EDTA derivative, for example an EDTA derivative chosen from the disodium salt of EDTA and a tetrasodium salt of EDTA (for example the tetrasodium EDTA salt sold under the name Versene 220 Crystals Chelating Agent® by the company Dow Chemicals).

In certain embodiments, the metal-ion-chelating agent is chosen from the tetrasodium glutamate diacetate salt (C H₁₃NO₈, for example sold under the name Dissolvine GL-47® by the company Akzo Nobel), the trisodium ethylenediamine disuccinate compound (C1₀H1₃N2O₈, for example sold under the name Natrlquest E30® by the company Innospecactive Chemicals).

In one preferential embodiment, the metal-ion-chelating agent is used at a concentration ranging from 0.01% w/w (weight/weight) to 10% w/w, preferably at a concentration ranging from 0.1% w/w to 5% w/w, relative to the total weight of said composition.

Preferably, the metal-ion-chelating agent is EDTA or a salt thereof, and is used at a concentration ranging from 0.01% w/w (weight/weight) to 10% w/w, preferably at a concentration ranging from 0.1% w/w to 5% w/w, relative to the total weight of said composition.

In one preferential embodiment, the invention relates to the use of a metal-ion- chelating agent, preferably at a concentration ranging from 0.01% w/w to 0.25% w/w, relative to the total weight of said composition, as a stabilizing agent in a composition comprising fresh dedifferentiated cells of a plant of the Rosa sp. species, or an extract thereof.

In one preferential embodiment, the invention relates to a process for stabilizing a composition comprising fresh dedifferentiated cells of a plant, in particular of the Rosa sp. species, or an extract thereof, said process comprises at least the following steps:

a) providing a composition, in liquid form, comprising fresh dedifferentiated plant cells of a plant of the Rosa sp. species, or an extract thereof, and

b) adding a metal-ion-chelating agent to the composition in liquid form provided in step a), at a concentration ranging from 0.01% w/w to 0.25% w/w, relative to the total weight of said composition.

Compositions

The present invention relates to a composition, in particular a composition for topical use, comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, said composition comprising a metal-ion-chelating agent. Such a composition is preferentially a cosmetic composition, for example a cosmetic composition for topical use.

The present invention also relates to a stable composition of dedifferentiated cells of a plant of the Rosa sp. species, or of an extract or a lyophilisate of said cells, said composition comprising a metal-ion-chelating agent as stabilizing agent. Advantageously, the metal-ion-chelating agent is chosen from EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), MGDA (methylglycine diacetic acid), GLDA (glutamic acid diacetic acid), NTA (nitrilotriacetic acid), TTHA (irieihyleneieiraaminehexaaceiic acid), EDDS (ethylenediaminedisuccinic acid), and salts thereof.

According to one particular embodiment of the invention, the metal-ion-chelating agent may be EDTA or a salt thereof.

Advantageously, the chelating agent, and in particular the EDTA or a salt thereof, may be present at a concentration ranging from 0.01% w/w (weight/weight) to 10% w/w, preferably at a concentration ranging from 0.1% w/w to 5% w/w, relative to the total weight of said composition.

Dedifferentiated plant cells of the invention, or a lyophilisate or an extract of said cells, may be formulated in any compositions suitable for cosmetic or pharmaceutical use.

A composition according to the invention comprises a physiologically or pharmaceutically acceptable medium.

After having been cultured in a suitable medium, the dedifferentiated plant cells of the invention are harvested by filtration and can be lyophilized or extracted or placed in solution.

Advantageously, the dedifferentiated plant cells of the invention, or an extract or a lyophilisate of said cells, can be formulated or placed in solution in water or a water-soluble organic solvent, or a mixture thereof.

A water-soluble organic solvent suitable for the invention can be chosen from C₂ to Cg, preferably C3 to C₆, hydrocarbon-based compounds comprising from 2 to 6 hydroxyl groups, preferably from 3 to 5 hydroxyl groups, and mixtures thereof.

Among the water-soluble organic solvents suitable for the invention, mention may in particular be made of glycols having from 2 to 8 carbon atoms, such as ethylene glycol, propylene glycol or 1,3 -propanediol, 1,3-butylene glycol, dipropylene glycol, glycerol, sorbitol, and mixtures thereof. Preferably, propylene glycol or 1,3 -propanediol are quite particularly suitable for the invention.

A water-soluble organic solvent may constitute from 20% to 99.9% by weight of the composition containing it, preferably from 30% to 90%, preferably from 40% to 80%, and more preferably from 50% to 70% by weight of the composition containing it.

A water suitable for the invention may be a spring and/or mineral water, chosen in particular from Vittel water, waters from the Vichy basin and Roche Posay water. The water may constitute from 20% to 99.9% by weight of the composition containing it, preferably from 30% to 90%, preferably from 40% to 80%, and more preferably from 50% to 70% by weight of the composition containing it. Advantageously, the water constitutes up to 50% by weight of the composition containing it.

According to one preferred embodiment, a preparation comprising dedifferentiated plant cells of the invention, or comprising an extract or a lyophilisate of said cells, may comprise at least one hydrophilic gelling agent. Such an agent makes it possible to avoid or reduce setting out of the cells.

A hydrophilic gelling agent suitable for the invention can be chosen from polysaccharide polymers, and mixtures thereof.

For the purposes of the present invention, the term "polysaccharide polymer" is intended to denote any carbohydrate macromolecule formed by the linking of a large number of elementary sugars, for instance xylose, glucose, galactose, rhamnose, mannose, fucose, arabinose, and their respective acids. The polysaccharide polymers particularly suitable for the present invention are preferably chosen from polymers having molecular weights ranging from 10 to 250 kDa. By way of polysaccharide polymers suitable for the invention, mention may in particular be made of pectin; guar gum; cellulose; dextrin; maltodextrin; starch; tara gum; locust bean gum; inulin; acacia gum; gum arabic; fucose-rich polymers, such as fucogel; carrageenans; konjac gum; xanthan gum; dextran; chitosan; gum tragacanth; ghatti gum; karaya gum; tamarin gum; agar-agar; alginate; gellan gum; and mixtures thereof. Xanthan gum, locust bean gum, guar gum, gellan gum, agar-agar, alginate, and mixtures thereof are preferably used.

According to one preferred embodiment, a preparation comprising dedifferentiated plant cells of the invention, or comprising an extract or a lyophilisate of said cells, may comprise a xanthan gum.

A gelling agent may be present in a proportion ranging from 0.01% to 10% by weight and preferably from 0.1% to 5% by weight of polysaccharide polymer(s), relative to the total weight of the composition.

According to another preferred embodiment, a preparation comprising dedifferentiated plant cells of the invention, or comprising an extract or a lyophilisate of said cells, may comprise at least one sugar.

A sugar suitable for the invention may be a C4 to Ce monosaccharide and may in particular be chosen from inositol, mannitol, glucose, sucrose, trehalose, maltose, xylitol, fructose, and mixtures thereof. The sugar content in the composition of the invention may range from 1% to 80% by weight, for example from 5% to 75% by weight and in particular from 10% to 70% by weight relative to the total weight of the composition.

According to one preferred embodiment, the dedifferentiated plant cells of the invention, or an extract or a lyophilisate of said cells, may be formulated in the presence of water, of 1,3 -propanediol, of a metal-ion-chelating agent and/or of xanthan gum.

The dedifferentiated plant cells of the invention, or an extract or a lyophilisate of said cells, may be used in an amount representing from 0.001% to 50% by weight of solids relative to the total weight of the composition containing them, and preferentially in an amount representing from 0.01% to 30% by weight of solids relative to the total weight of the composition.

In one preferred embodiment, the dedifferentiated plant cells of the invention, or an extract or a lyophilisate of said cells, may be used in an amount representing from 0.1% to 30% by weight of solids relative to the total weight of the composition containing them, and advantageously in an amount representing from 0.1% to 20% by weight of solids relative to the total weight of the composition.

Dedifferentiated plant cells of Rosa sp. of the invention, or a lyophilisate or an extract of said cells, may be formulated in any cosmetic composition, in particular intended to be ingested, injected or applied to the skin, the hair, the nails or the mucosae (oral, jugal, gingival, genital, conjunctival). According to the embodiment retained, a composition of the invention may be in any of the galenical forms normally used.

For topical application to the skin, a composition may be in the form, in particular, of an aqueous or oily solution or of a dispersion of the lotion or serum type, of emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (oil-in-water or O/W emulsion) or vice versa (water-in-oil or W/O emulsion), or of suspensions or emulsions of soft consistency of the aqueous or anhydrous gel or cream type, or else of microcapsules or microparticles, or of vesicular dispersions of ionic and/or non-ionic type. These compositions are prepared according to the usual methods.

These compositions may also consist of solid preparations constituting soaps or cleansing bars or may be packaged in the form of an aerosol composition also comprising a pressurized propellant.

A composition intended to be applied to the hair may be in the form of aqueous, alcoholic or aqueous-alcoholic solutions, or in the form of creams, gels, emulsions or mousses, or alternatively in the form of aerosol compositions also comprising a pressurized propellant. Such a composition can be provided in the form of a haircare composition, such as a shampoo, a hairsetting lotion, a treating lotion, a styling cream or gel, a dyeing composition (in particular an oxidation dyeing composition), a dyeing shampoo, a restructuring lotion, a permanent-wave composition (in particular a composition for the first step of a permanent wave), a lotion or a gel for combating hair loss.

For subcutaneous or intradermal administration, a composition may be in the form of an aqueous or oily lotion or in the form of a serum.

A composition suitable for ingestion may be in the form of capsules, granules, syrups or tablets.

The amounts of the various constituents in the compositions according to the invention are those conventionally used in the fields under consideration.

When a composition is an emulsion, the proportion of the fatty phase may range from 5% to 80% by weight and preferably from 5% to 50% by weight relative to the total weight of the composition. The oils, waxes, emulsifiers and co-emulsifiers used in a composition in emulsion form are chosen from those conventionally used in the cosmetics field. The emulsifier and the co-emulsifier may be present in a composition in a proportion ranging from 0.3% to 30% by weight and preferably from 0.5% to 20% by weight relative to the total weight of the composition.

When the composition is an oily solution or gel, the fatty phase may represent more than 90% of the total weight of the composition.

In a known manner, a cosmetic composition as defined by the present invention may also comprise adjuvants normally used in the cosmetics field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, fragrances, fillers, screening agents, odour absorbers and colorants. The amounts of these various adjuvants are those conventionally used in the cosmetics field, and are between, for example, 0.01% and 10% of the total weight of the composition. Depending on their nature, these adjuvants may be introduced into the fatty phase, into the aqueous phase and/or into lipid spherules.

As oils or waxes that may be used in the invention, mention may be made of mineral oils (liquid petroleum jelly), plant oils (liquid fraction of shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils (purcellin oil), silicone oils or waxes (cyclomethicone) and fluoro oils (perfluoropolyethers), beeswax, carnauba wax or paraffin wax. Fatty alcohols and fatty acids (stearic acid) may be added to these oils. As emulsifiers that may be used in the invention, mention may be made, for example, of glyceryl stearate, polysorbate 60, and the PEG-6/PEG-32/glycol stearate mixture sold under the name Tefose^R 63 by the company Gattefosse.

As solvents that may be used in the invention, mention may be made of lower alcohols, for instance ethanol, isopropanol and propylene glycol.

As hydrophilic gelling agents that may be used in the invention, mention may be made of carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums, preferably xanthan gum, and clays, and, as lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids, for instance aluminium stearates, and hydrophobic silica, ethylcellulose and polyethylene.

Cosmetic use

According to one preferred embodiment, a composition according to the present invention may be used cosmetically as an active agent for caring for aged skin or aged hair.

For example, a composition according to the present invention can be used for treating and/or preventing an aesthetic defect subsequent to a tissue renewal defect of aged skin or aged hair in an individual in need of same, or for reinforcing, improving or even restoring a youthful appearance of the skin and/or of the head of hair.

This composition may be administered, in particular, orally, subcutaneously, intradermally or topically, and preferably topically.

This composition may be administered daily for example, at a rate, for example, of a single administration per day or of an administration twice a day, for example once in the morning and once in the evening.

A composition in accordance with the present invention may be formulated, for example, in a form suitable for topical administration, for instance in the form of gel, a lotion, cream or a foam.

Moreover, a composition in accordance with the invention may be formulated, for example, in a form suitable for oral administration, for instance in the form of a gel capsule, a dragee, an emulsion, a tablet, capsule or an oral ampoule.

An effective amount of an active agent of the invention may be administered in a single dose per day or in doses fractionated over the course of the day, for example 2 or 3 times per day. A cosmetic composition according to the invention may advantageously be used over a time period ranging from one week to several weeks, or even several months, this period moreover possibly being repeated after periods without treatment, for several months or even several years.

By way of example, the administration of an active agent according to the invention may be repeated, for example, 2 or 3 times per day, or more, and generally over an extended period of at least 4 weeks, or even 4 to 15 weeks, with, where appropriate, one or more periods of interruption.

For the purposes of the invention, the term "skin" is intended to mean the entire surface area of skin of an individual, including the scalp.

For the purposes of the invention, the term "hair" is intended to mean an assembly comprising the hair shaft and the hair follicle.

The term "aged skin" is intended to mean a general aesthetic state of the skin resulting from chronological ageing and/or photoinduced ageing.

More particularly, the composition according to the present invention is directed towards preventing and/or reducing and/or treating signs of skin ageing.

The term "signs of skin ageing" is intended to mean all the modifications of the external appearance of the skin due to ageing, whether the latter is of chronological and/or photoinduced origin.

By way of example of these modifications considered in the invention, mention may be made of a surface which is not very uniform and less smooth, an epidermis which is thinned and/or less effective in terms of barrier function (which is repaired less quickly following attacks), wrinkles and fine lines, withered skin, a lack of elasticity and/or of tonicity of the skin, thinning of the dermis and/or the degradation of collagen fibres, which leads to the appearance of flaccid, wrinkled skin.

It is also intended to mean all the internal modifications of the skin which are not systematically reflected by a modified external appearance, for instance all the internal degradations of the skin, and more particularly the degradation of elastin fibres, or elastic fibres, subsequent to exposure to ultraviolet radiation.

In particular, the signs of skin ageing targeted by the invention 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, a deterioration 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 wrinkles, a deterioration of the radiance of the skin complexion, a wizened appearance of the skin, a modification of the odour of the skin, sagging of the skin or withering of the skin.

The modifications of the quality of the hair that appear with age consist in particular of a change in appearance of the fibre (thin, lifeless, weak hair with no hold), with no radiance and/or greying, more readily brittle, and also a loss of hair density.

The present composition is thus directed towards restoring/maintaining good hair density, and/or improving the quality of the head of hair and/or of the hair fibres, and in particular aged hair. More particularly, the dedifferentiated plant cells of the invention, or an extract or a lyophilisate of said cells, make it possible to prevent a loss of hair density and to protect, reinforce or improve the quality of aged hair in order to confer a youthful and dense appearance on a head of hair.

EXAMPLES

EXAMPLE 1: Stability of a composition comprising a plant extract of rose dedifferentiated cells in the presence of a metal-ion-chelating agent

1) Culturing of (Lancome Rose) rosebush dedifferentiated plant cells from a plant tissue

Leaves of a (Lancome Rose) rosebush were decontaminated using the normal techniques of those skilled in the art, and placed on the following culture medium:

The hormones used are hormones that are naturally present in plants.

Following successive subculturings, dedifferentiated plant cells that can be cultivated in a fermenter and can therefore be industrialized were obtained. The parameters used for controlling the in-bioreactor culturing are the following: Temperature: from 24°C to 30°C, and preferentially 26-27°C

Aeration: 10% partial oxygen pressure (pC^), regulated by sterile entering air

(WM: volume of air / volume of fermenter / minute and/or by agitation while avoiding any stress due to shear forces on the cells.

The production by batchwise culturing lasts from 8 to 14 days. The cells are then harvested by filtration on a cloth with a porosity of 80 μιτι.

2) Treatment of the various batches of rosebush dedifferentiated plant cells

The Lancome Rose dedifferentiated cells obtained in part 1) are separated into 2 distinct batches.

Dedifferentiated-cells batch No. 1 ("bl") is subjected to a freezing step, at -20°C (on dry ice), then to a first slow thawing step at 4°C overnight (12 hours) and then a second thawing step in a water bath at 30-40°C for 8 hours. After these freezing/thawing steps, the dedifferentiated plant cells are subjected to a step of resuspension of the thawed batch in a mixture comprising water, in the presence of 1,3 -propanediol (60% by weight of the mixture) and of xanthan gum (1% by weight of the mixture), in a proportion of 10% by weight of fresh biomass relative to the total weight of the composition. For the purposes of the process, the combination of the freezing and then thawing steps can also be denoted step of "lyophilization by freezing then thawing".

Dedifferentiated-cells batch No.2 ("b2"), which has not undergone freezing/thawing steps, is formulated in a mixture of water, in the presence of 1,3 -propanediol (60% by weight of the mixture), of xanthan gum (1% by weight of the mixture) and of a disodium EDTA salt (0.25% by weight of the mixture), in a proportion of 10% by weight of fresh biomass relative to the total weight of the composition.

EDTA is known to be a very powerful chelater of calcium, zinc, magnesium, copper and manganese ions, and also of some ferric species.

3) Analysis of the visual appearance and of the texture of the solutions obtained according to batches No.1 and No.2

The appearance of the solutions was determined visually (colouration) and by optical microscopy (aggregation) on solutions at TO (immediately after resuspension of the lyophilisate), 1 day (T-ld), 15 days (T-15d), 1 month (T-30d) and 2 months (T-60d) after resuspension of the lyophilisate and storage at ambient temperature in the dark.

The storage step was also tested at other temperatures, such as 4°C, 40°C or 45°C. The optical microscopy observations were carried out in non-polarized light and in the polarized light (Leica DM LB2, Leica Microsystems, Germany).

4) Results

4.1) Appearances of the solutions comprising rose dedifferentiated cells at ambient temperature

A composition comprising rose dedifferentiated cells according to batch No. l is, just after it has been prepared, in the form of a translucent gel in which beige/brown-coloured cells in suspension are observed. This composition remains stable up to T-15d after it has been prepared and exhibits considerable yellowing at T-30d and T-60d, when it is stored at ambient temperature (see Table 1).

On the other hand, in the presence of the disodium salt of EDTA, the composition according to batch No.2 is in the form of a translucent gel with white cells, or even light off- white- coloured cells, just after production. The occurrence of the yellowing is both less intense and delayed over time, since a slight yellowish colouration is observed only after T-30d (see Table 1).

Table 1: Assessment of the visual appearance (colouration) of the solutions of batches No.1 and No.2 at ambient temperature

Observation with the naked eye of the compositions of rose dedifferentiated cells according to batches No. l and No.2 shows that they are free of aggregates at T-Od (see Table 2).

A few microaggregates are formed in rose dedifferentiated cells batch No.2 as early as the 1st day of storage (T-ld) at ambient temperature (see Table 2).

However, although the number of aggregates considerably increases in the composition according to batch No. l after 15, 30 and 60 days of storage (T-15d, T-30d and T- 60d), the composition according to batch No.2, which contains the metal-ion-chelating agent disodium EDTA, does not show any aggregate, even after 60 days of storage at ambient temperature (T-60d) (see Table 2).

Table 2: Assessment of the texture, through the presence of aggregates, of the solutions of batches No.1 and No.2 at ambient temperature

The optical microscopy observation confirms the observations made with the naked eye.

Indeed, a composition according to batch No. l, i.e. comprising rose dedifferentiated cells having undergone freezing/thawing steps without the addition of a metal - ion-chelating agent, reveals the presence of aggregates (Figure 1). It it should be noted that these aggregates are visible both by microscopy in non-polarized light (Figure 1A) and microscopy in polarized light (Figure IB).

On the other hand, aggregates are non-existent when the rose dedifferentiated plant cells are suspended in a solution comprising a metal-ion-chelating agent (disodium EDTA salt; not shown).

4.2) Observations of the aggregates in solutions comprising rose dedifferentiated cells at temperatures of 4°C. 40°C and 45°C

The formation of aggregates is observed in batch No.l, at all the temperatures tested, as early as one day after production (T-ld).

For batch No.2, no aggregates were observed for the three storage temperatures tested, at the times tested (T-ld and T-15d). 5) Conclusions

It thus seems that the disodium EDTA salt is capable of conferring stability on a composition comprising rosebush dedifferentiated plant cells.

The presence of disodium EDTA in the composition therefore improves the homogeneity, preventing the formation of aggregates of plant material, and also makes it possible to maintain the initial white/off-white colour of the rose cells, during the process of preservation or storage of the composition over time.

EXAMPLE 2: Formulation examples

1) Day cream

The topical application of a day cream or of a lotion in accordance with the present invention provides a beneficial effect in the treatment and/or prevention of an aesthetic defect subsequent to a tissue renewal defect of aged skin or of aged hair, and/or in the reinforcement, improvement or even restoration of a youthful appearance of the skin and/or of the head of hair.

EXAMPLE 3: Stability of compositions comprising a plant extract of rose dedifferentiated cells, treated or not by freezing/thawing steps, in the presence of a metal-ion- chelating agent 1) Materials and methods

1.1) Culturing and treatment of (Lancome Rose) rosebush dedifferentiated plant cells from a plant tissue

Leaves of a (Lancome Rose) rosebush were cultured and subjected or not to a freezing and thawing steps, as in example 1.

Each batch, i.e. dedifferentiated-cells batch No. 3 (batch No.3, "b3"), which has undergone freezing/thawing steps, and dedifferentiated-cells batch No. 4 (batch No.4, "b4"), which has not undergone freezing/thawing steps were further treated or not with disodium EDTA.

In practice, dedifferentiated-cells from batch No.3 or No.4 were subsequently suspended in a solution comprising water, 1,3 -propanediol (60% by weight of the mixture), xanthan gum (1% by weight of the mixture), in the presence or absence of disodium EDTA, in a proportion of 10% by weight of fresh biomass relative to the total weight of the composition (raw material).

1.2) Formulated suspensions

"Formulated batches" were obtained by mixing 10% (w/w) of raw material (in water, 1,3-propanediol and xanthane gum, see 1.1) above) in a composition according to Example 2 (referred as day cream).

1.3) Analysis of the visual appearance and of the texture of the suspensions

Analysis of the visual appearance and of the texture of the raw material and formulated suspension obtained after treatment of batches No.3 and No.4 was performed accordingly to example 1, at the temperature of 4°C, room temperature (RT), 40°C and 45°C, the same day of preparation.

2) Results

2.1) visual appearance and texture of the raw material

The coloration of the suspensions obtained with the raw material made up of dedifferentiated-cell batches No. 3 and No.4 suspended in water, 1,3-propanediol and xathane gum, in the presence or in the absence of EDTA, at the 4 indicated temperatures is presented in Table 3 below. Table 3: coloration of the suspensions (raw material)

Batch No. I -EDTA I +EDTA¹

3 yellow/beige white

4 beige white

¹ Batch No.3 was treated with 1% EDTA; Batch No.4 was treated with 0.25%

EDTA.

In the presence of disodium EDTA, dedifferentiated-cells batches No. 3 and No. 4

(raw material) present a translucent gel with a white coloration, which corresponds to the "original" coloration obtained just after the mixing of the ingredients, for each of the tested temperatures.

By contrast, in the absence of disodium EDTA, suspensions of both batches No.3 and No.4 showed an alteration of the colour, which is the hallmark of a decreased stability, for each of the tested temperatures.

The texture of the suspensions obtained with dedifferentiated-cells batches No. 3 and No.4 suspended in water, 1,3 -propanediol, xanthane gum, in the presence or in the absence of disodium EDTA, at the 4 indicated temperatures was further assessed and presented in Table 4 below.

Table 4: texture of the suspensions (raw material)

¹ Batch No.3 was treated with 1% EDTA; Batch No.4 was treated with 0.25%

EDTA.

In the presence of EDTA, dedifferentiated-cells batches No. 3 and No. 4 (raw material) do not present any black-coloured aggregates.

However, in the presence of 1% EDTA, the suspension of batch No.3 displays some EDTA crystals.

In conclusion, the presence of EDTA stabilizes the rose dedifferentiated-cells in suspension referred as raw material, i.e. allows for maintaining the original visual aspect and the original texture of the suspensions obtained after suspending the rose dedifferentiated cells in a mixture comprising water, 1,3 -propanediol, xanthane gum and disodium EDTA.

2.2) visual appearance and texture of "formulated batches"

The coloration of the suspensions, at the 4 indicated temperatures was assayed. In the presence of disodium EDTA, formulated dedifferentiated-cells batches No. 3 and No. 4 present the original white coloration for all the tested temperatures, which do not vary with time, i.e. do not become yellowish, even after a 2 month period of storage. Formulated batches behaved similarly as compared to suspensions referred as raw material.

The texture of the suspensions obtained with dedifferentiated-cells batches No. 3 and No.4 formulated in a day cream composition according to Example 2at the 4 indicated temperatures was further assessed and presented in Table 5 below.

Table 5: texture of the suspensions

¹ Batch No.3 was treated with 1% EDTA; Batch No.4 was treated with 0.25%

EDTA.

In the presence of disodium EDTA, formulated dedifferentiated-cells batches No. 3 and No. 4 do not present any black-coloured aggregates.

In conclusion, the presence of EDTA stabilizes the rose dedifferentiated-cells in a formulated suspension, as they form a gel with a white coloration and the suspensions do not show any aggregates. EDTA provides a homogeneous texture, without further visible alteration of the original appearance of the suspension.

Claims

1. Use of a metal-ion-chelating agent as a stabilizing agent in a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells.

2. Process for stabilizing a composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or an extract or a lyophilisate of said cells, which process comprises at least one step of bringing said dedifferentiated cells, or said extract or said lyophilisate of said cells, into contact with a metal-ion-chelating agent.

3. Process according to claim 2, comprising at least the following steps: a) providing a composition, in liquid or gelled form, of dedifferentiated plant cells of a plant of the Rosa sp. species, or of an extract or a lyophilisate of said cells, and b) adding a metal-ion-chelating agent to the composition in liquid or gelled form provided in step a).

4. Use according to claim 1 or process according to either of claims 2 and 3, in which the metal-ion-chelating agent is chosen from EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), MGDA (methylglycine diacetic acid), GLDA (glutamic acid diacetic acid), NTA (nitrilotriacetic acid), TTHA (triethylenetetraaminehexaacetic acid), EDDS (ethylenediaminedisuccinic acid), and salts thereof.

5. Use according to one of claims 1 and 4 or process according to one of claims 2 to 4, in which the metal-ion-chelating agent is EDTA or a salt thereof.

6. Use according to one of claim 1 and 4 to 5 or process according to one of claims 2 to 5, in which the metal-ion-chelating agent, preferably EDTA or a salt thereof, is used at a concentration ranging from 0.01% w/w to 10% w/w, preferably at a concentration ranging from 0.1% w/w to 5% w/w, relative to the total weight of said composition.

7. Use according to claim 1, in which the metal-ion-chelating agent represents a concentration ranging from 0.01% w/w to 0.25% w/w, relative to the total weight of said composition, as a stabilizing agent in a composition comprising fresh dedifferentiated cells of a plant of the Rosa sp. species, or an extract thereof.

8. Process according claims 2 and 3, comprising at least the following steps:

a) providing a composition, in liquid form, comprising fresh dedifferentiated plant cells of a plant of the Rosa sp. species, or an extract thereof, and b) adding a metal-ion-chelating agent to the composition in liquid form provided in step a), at a concentration ranging from 0.01% w/w to 0.25% w/w, relative to the total weight of said composition.

9. Composition comprising dedifferentiated cells of a plant of the Rosa sp. species, or comprising an extract or a lyophilisate of said cells, said composition comprising a metal- ion-chelating agent.

10. Composition according to claim 9, in which the metal-ion-chelating agent is chosen from EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), MGDA (methylglycine diacetic acid), GLDA (glutamic acid diacetic acid), NTA (nitrilotriacetic acid), TTHA (triethylenetetraaminehexaacetic acid), EDDS (ethylenediaminedi succinic acid), and salts thereof.

11. Composition according to claim 9, in which the metal-ion-chelating agent is EDTA or a salt thereof.

12. Composition according to one of claims 9 to 11, in which the metal-ion- chelating agent, preferably EDTA or a salt thereof, is present at a concentration ranging from

0.01% w/w to 10% w/w, preferably at a concentration ranging from 0.1% w/w to 5% w/w, relative to the total weight of said composition.

13. Cosmetic use of a composition according to one of claims 9 to 12, as an active agent for caring for aged skin or aged hair.