WO2016113659A1 - Use of plant cells of leontopodium alpinum for a cosmetic treatment and corresponding active ingredient - Google Patents

Abstract

The invention provides the use of undifferentiated or undifferentiated plant cells of Leontopodium alpinium obtained by in vitro cell culture for a non-therapeutic cosmetic treatment for restoring the homeostasis of the cells of aged skin and increase their metabolic and energetic activity. With this treatment a tensor and smoothing restorative effect is obtained on an already aged skin, particularly pronounced on the sagging neck skin and tear trough.

Description

USE OF PLANT CELLS OF LEONTOPODIUM ALPINUM FOR A COSMETIC TREATMENT AND CORRESPONDING ACTIVE INGREDIENT

TECHNICAL FIELD

The present invention relates to the use of the Leontopodium nivale subsp. alpinum Cass plant, hereinafter referred to Leontopodium alpinum, for a cosmetic treatment (topical or oral route administration).

The present invention thus relates to the cosmetics and hygiene and personal care products industries, for skin and its appendages (such as hair, eyelashes, eyebrows, nails, body hair) of mammals, animals or humans.

BACKGROUND ART

Leontopodium alpinium, also called Edelweiss, is an herbaceous plant of the Asteraceae family. It grows above an elevation of 1500m in the Pyrenees, Alps and Himalayas mountains, in fairly hostile places such as ravines, rocky, cold, and very exposed to UV areas. Subsequently, this plant is excellent at adapting to extreme conditions because it contains a wide variety of molecules of interest and has protective hair on its flower and leaves. However, this plant remains relatively not well distributed and therefore quite rare.

Products from the plant may be obtained by conventional extraction methods directly from the whole plant or parts thereof or by in vitro culture methods either by cell culture or tissue culture from cell or tissue lines derived from different organs of the plant.

The present invention relates more particularly to the products obtained as a result of in vitro cells or tissue culture.

The preparation by in vitro culture of an extract of plant origin has many advantages over the agro- industrial way (plants growing in open fields and subsequent extraction in facilities). Because of the total control of culture conditions, the extracts obtained by in vitro culture are free of toxic substances (herbicides, pesticides, fertilizers, heavy metals and other contaminants, such as those derivable from plant parasites). Furthermore, the strict control of the in vitro culture conditions reduces the risk of spontaneous variation of the line and guarantees a reproducible profile of secondary metabolites which correspond to the desired molecules of interest, in contrast to culture in open fields where the variability problem exists, linked to the climate, weather and geographical conditions and their hazards. Still further, this technology overcomes barriers such as the natural biological cycle of the plant and the seasonality of production of secondary metabolites, allowing better security and rapidity of supply. In addition, the environmental impact is minimal because substantially limiting water consumption, avoiding consumption of arable land, and preventing pollution of the soil. Besides, biodiversity is preserved since one plant or even one seed is enough to initiate a new in vitro culture. Finally, this technology offers the possibility to direct the cellular metabolism toward the production of molecules of interest (elicitation of the cultures) and to achieve controlled and relatively rapid protocols to increase the yields of certain molecules including those produced ordinarily in low amount in the plant.

Among the existing techniques to date in the in vitro plant cell culture domain, the following can be used according to the invention:

The culture of undifferentiated or dedifferentiated cells: this type of method involves first creating cell lines highly proliferative in agar medium either from meristem cells that are undifferentiated cells, either from dedifferentiated cells (growing as callus after the removal of a fragment of plant, leaf, stem, root or other). These lines are then cultured in liquid medium so as to substantially increase the biomass. At the end of the growth cycle and in environmental conditions to define and optimize (finding the right elicitation medium), the cellular biomass will synthesize the molecules of interest. The culture is then stopped and subjected to an extraction at the optimal time to obtain a maximum quantity of molecules of interest. Existing cell lines already commercially available can also be used initially.

The culture of tissue or organ: this type of culture can concern the root part ("root culture"), the aerial part ("shoot culture") or somatic embryos ("somatic embryo"). In this type of method, distinction is made between cultures having undergone a genomic transformation by Agrobacterium rhizogens bacteria (roots) or by Agrobacterium tumefaciens (stems). Cultures of roots or aerial parts thus transformed have a high growth rate and are genetically very stable. They are used to synthesize the molecules of interest after optimization of the elicitation parameters. These cultures are then subjected to an extraction by the conventional means.

Preferably, the present invention is more particularly directed to products derived from in vitro culture of undifferentiated or dedifferentiated cells, hereinafter called plant cell culture.

Schematically in vitro cell culture methods consist of:

- Where appropriate, initially establishing cell lines from calluses (clusters of undifferentiated or dedifferentiated cells) obtained on cuts of plant parts (leaf, root, stem, buds, etc.)

- Selecting a cell line capable of producing a cell biomass at a large-scale according to pre-established criteria (constant phenotype, and optimum and constant production of selected metabolites, ability to proliferate);

- Then from this selected line, generating said cellular biomass, possibly with an elicitation stage, preferably at the end of the proliferation stage; and

- Thirdly, treating the obtained cellular biomass to recover the whole cells, break the cell aggregates by a high pressure homogenization or lyse these cells and possibly then extracting the content of said cells.

It is these extracts and/or whole or disrupted cells that can then be used in cosmetic compositions as active ingredient with a physiologically acceptable excipient and optionally with other additional active ingredients. The EP2319914 patent application describes this in vitro technique for obtaining undifferentiated cells in culture with a high yield of derivatives of caffeic acid for a theoretical list of 33 plant species including Leontopodium alpinum. The concerned caffeic acid derivatives include phenylpropanoid glycosides as well as caffeoylquinic acids. Inhibitory activity of hyaluronidase is presented for Leontopodium alpinum. Protecting hyaluronic acid digestion by hyaluronidase helps preventively to preserve the integrity of the dermis.

SUMMARY OF THE INVENTION

The present invention aims to provide a new use of Leontopodium alpinum cells obtained by an in vitro cell culture method for a cosmetic treatment.

To this aim, the present invention provides the use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinium obtained by in vitro cell culture method for a non-therapeutic cosmetic treatment for restoring the homeostasis of the cells of aged skin and increase their metabolic and energetic activity.

Homeostasis of the cells of aged dermis results in:

1) A rebalancing between MMPs ("Matrix Metallo Proteases" of the dermal matrix) and TIMPs ("Tissue Inhibitor of Metallo Proteinases" of the dermal matrix), in favour of the TIMPs.

There is a large number of MMPs (over 20), produced in a latent form (pro-MMP) to avoid uncontrolled runaway. Their production increases with age but acute or chronic stress amplify this phenomenon. Their excessive production results in the reduction of the strength of the dermis, the loss of its density and to its thinning. Thus MMPs results in faster and worst skin ageing. While the fibres of the dermis in young skin are long, intact and dense, those in aged skin are to the contrary fragmented and less dense. This lesser quality of the fibres reduces the dynamic tension of the dermis, the interactions between the MEC and fibroblasts being also sometimes reduced up to 80%.

MMPs are naturally controlled in the tissues by the TIMP inhibitors which avoid a blind activity. It is the balance of the TIMPs relative to MMPs that determine the level of activity of the MMPs. TIMPs are small glycoproteins whose production is associated with the reduction of chronic diseases associated with MMPs and with the reduction of photo-damages due to UV. By combining themselves with the MMPs, the TIMPs neutralize them and therefore limit the fragmentation of the dermal matrix, which thus retains its elasticity. Furthermore, it is considered that TIMP-1 is a favourable survival factor for fibroblasts and keratinocytes.

The decrease of TIMP-1 production is observed under the effect of UV irradiation but also during the senescence of fibroblasts. This leads to an increase of the MMPs activity.

2) A stimulation of the production of the thrombospondins (TSPs) which are matrix glycoproteins produced by the epidermis and the dermis, and whose scientific interest is increasing. TSP-1, once experimentally overexpressed in mouse epidermis can effectively reduce the harmful effects of UVB irradiation. Interestingly, a clear improvement in the organization of collagen and elastin fibres of the underlying dermis has been noticed, as well as a reduction of the formation wrinkles on skin. This could be linked to the control of pro-angiogenic and proteasic factors. Its counterpart, the TSP-2, has also a significant potential anti-aging role. Indeed, mice that cannot produce this protein show dermal collagen fibres being less organized disordered and their skin is more fragile to extension. Their fibroblasts also provide artificial dermis that are also less dense and slacker, and that does not contract normally.

The TSP-1 and TSP-2 seem to act on several key points of aging, in particular by inhibiting MMP's activity, including that of MMP-9. Moreover, the increase of TSP-1 production is also known to restore the synthesis of procollagen decreased by UVB irradiation. This recovery requires the activation of TGF-β well known to stimulate the synthesis of collagens. In contrast, fibroblasts that cannot produce TSP-2 overproduce MMP-9. Thus, it is clear that the expression of TSPs, including by keratinocytes, represents a favourable benefit for the quality of the dermis and skin, and aside with the TIMPs, limits their aging.

The increase in metabolic activities of dermal fibroblasts results in an improvement of the contractile capacities and an increase of the production of dermal macromolecules. Regarding the energy activity of fibroblasts, every cell of the organism produces, thanks to the oxygen in the air, its own energy from production units, called mitochondria, in greater or lesser number, according to the energy needs of the tissue. With age the mitochondria becomes less efficient and converted less oxygen than before. Fibroblasts have filament-like mitochondria from 1 to 10 μπι in contrast to other cell types, with a strong multiplicative power, that are rather ovoid. However both forms, isolated ovoid form (fission) or filamentous network form (fusion), coexist in each cell type. The preservation of the dynamic passage from one state to another is crucial to the maintenance of mitochondrial functions. If the fission is essential for cell division to divide the pool of mitochondria in daughter cells, the fusion allows pulling genes and molecules that is needed to sustain cellular respiration.

It was observed that senescent cells are rich in the fused form which here again allows a prolongation of their survival.

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinium can be used after being mixed under high pressure, in order to homogenize the medium and break cell aggregates.

According to the invention, the undifferentiated or dedifferentiated Leontopodium alpinium cells may be used whole or lysed, or in the form of a cellular extract produced from these cells.

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinium can be extracted by any physiologically acceptable solvent or any mixture of these solvents. The extraction may be done according to various known methods that can be combined: heat extraction, maceration, decoction, infusion, pressure leaching, ultrasonic, microwave, lysing the cells by any appropriate chemical or physical method. Phase separation can be done by filtration or centrifugation. Alternatively, the biomass can be extracted with a supercritical or subcritical fluid. According to the invention, a higher purification of the cellular extract can also be considered by all industrially available methods, by liquid-liquid partition or chromatography, for example using an adsorbent resin, in order to concentrate the molecules of interest, such as the two leontopodic acids A and/or B.

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinium may also be used in a dried form, prepared by atomization or preferably by lyophilisation. This allows their long-term storage and preserves their biological activity.

In the following description, "plant cells" encompasses the undifferentiated or dedifferentiated cells of Leontopodium alpinium, prepared by an in vitro cell culture process, whether whole or lysed, whether or not having undergone high pressure homogenization, whether in a fresh or dry form, as well as the extracts derived from these cells.

According to the invention, plant cells can be incorporated (suspended or dissolved) in a physiologically acceptable medium and used to make a cosmetic composition intended to restore homeostasis of aged skin cells and increase their metabolic and energy activity.

Preferably, according to the invention, the physiologically acceptable medium is a hydrophilic matrix. More preferably, the cosmetic treatment of the invention is topical.

Surprisingly, the Applicant has shown by in vivo tests that the cosmetic treatment of aged skin consisting of applying a composition comprising undifferentiated or dedifferentiated cells of Leontopodium alpinium obtained by an in vitro cell culture process induces on these skins a cure or repair effect, firming and/or smoothing effect, in particular on the sagging neck area and the fold of the tear trough.

The results of these tests are presented below in the description in more details.

Finally, the invention provides the use of the plant cells as defined above, for the manufacture of a non-therapeutic cosmetic active ingredient, and the use of a composition comprising said active ingredient suspended and/or solubilized in a physiologically acceptable medium which can be a hydrophilic matrix, to restore homeostasis of the cells of aged skin and increase their metabolic and energy activity.

According to the invention, plant cells rich in leontopodic acids A and/or B, preferably A and B, are used. Proteins, amino acids, phytosterols, lipids and polysaccharides have also been identified as classes of compounds in the plant cells used according to the invention.

Finally, according to other features, the present invention provides a cosmetic active ingredient for use according to the invention, comprising undifferentiated or dedifferentiated plant cells of Leontopodium alpinium obtained by an in vitro cell culture method and comprising at least 0.04% of leontopodic acids A and B in a physiologically acceptable matrix. Preferably, in this ingredient and as explained above, the plant cells are whole and/or lysed cells or are present as a cellular extract of said whole and/or lysed cells. To obtain the dedifferentiated or undifferentiated plant cells that can be used according to the invention, the following method may be implemented:

1) From a selected line of Leontopodium alpinum, produce a critical pre-biomass by successive pre- cultures and of increasing sizes;

2) Producing a biomass of said dedifferentiated or undifferentiated cells in a bioreactor from said pre-biomass and a suitable culture medium; and

3) Separating said biomass enriched in leontopodic acids from said culture medium and thus recovering said dedifferentiated or undifferentiated cells.

According to optional features:

1) The production step in a bioreactor may comprise an elicitation step, advantageously increasing the contents of leontopodic acids A and/or B or varying the relative proportion; and/or

2) The biomass is collected from the reactor by filtration after a cultivation time between 7 and 21 days; preferably between 10 and 14 days, allowing advantageously to produce the biggest quantity of biomass, with a high viability; and/or

3) The biomass can be subjected to a step of high pressure homogenization, in order to break the cell aggregates; and/or

4) An additional step of drying the cellular biomass can be added, for a better long-term preservation; and/or

5) The cells may be extracted, in particular for enrichment in leontopodic acids A and B.

Generally, elicitation of the compounds of interest can be done by adding to the culture of microbial fractions (including saccharomyces yeasts), adding to the culture of biological molecules such as for example chitosan, methyljasmonate, jasmonic acid, salicylic acid, adding to the culture of non- biological molecules such as paclobutrazol, applying to the culture a change in temperature, pH or osmotic stress induced by a non-metabolisable sugar, such as mannitol, recourse to an even more drastic impoverishment of the medium in macro-elements and sugar, adding to the culture of adsorbent resins which, in addition to elicit the production of compounds of interest, may trap them. Preferably according to the invention, elicitation is achieved by modifying the culture medium, in particular nutrient levels.

Preparation of compositions for the implementation of the invention

A cosmetic composition, especially topical, includes plant cells in a physiologically acceptable medium. According to the excipient and dosage in plant cells, said composition will be a concentrated active ingredient or a final composition less concentrated directly intended for an end user.

"Physiologically acceptable medium" means according to the present invention, without limitation, an aqueous or hydro-alcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a micro- emulsion, an aqueous gel, an anhydrous gel, a serum, a dispersion of vesicles, or a powder. "Physiologically acceptable" means that the compositions are suitable for topical or transdermal use, in contact with mucous membranes, appendages (nails, hairs), scalp and skin of mammals, particularly human, compositions which may be ingested, or injected into the skin, without risk of toxicity, incompatibility, instability, allergic response, and others.

This "physiologically acceptable medium" forms what is commonly called the excipient of the composition.

The plant cells of the invention may be combined with other active ingredients at effective concentrations that can act synergistically or additionally for reinforcing and achieving the desired effects described for the invention, such as the following agents: UVA and/or UVB filtering agents, hydrating, moisturizing, humectant, calming, dermo-relaxing, slimming, restructuring, firming, replumping, lifting, smoothing, acting on blood microcirculation, inflammation, free radicals, anti- aging, anti-fine lines and wrinkles, lightening, acting on complexion, anti-glycation, anti- carbonylation, pro-pigmenting, acting on stratum corneum, on dermal-epidermal junction, on HSP protein production, on firmness, elasticity and tone of skin, on hair growth or anti-regrowth (including eyelashes and eyebrows), on eye contours (dark circles and under eye bags), peptides, vitamins, etc. The plant cells may be applied according to the invention to the face, body, neckline, scalp, hair, eyelashes, body hair, in whatever form or carriers known to those skilled in the art, in particular in the form of solution, dispersion, emulsion, paste, or powder, individually or as a premix or in vehicles individually or as a premix in vectors such as macro-, micro-, or nano-capsules, macro-, micro- or , nano-spheres, liposomes, oleosomes or chylomicrons, macro-, micro-, or nanoparticles or macro-, micro- or nano-sponges, micro- or nano-emulsions or adsorbed on organic polymer powders, talcs, bentonites, spores or exines, and other inorganic or organic supports.

In cosmetics in particular, applications can be offered particularly in skincare ranges for the face, body, hair and body hairs, and in make-up ranges, including for eyebrows and eyelashes.

In general, the plant cells according to the present invention may be used in any form, in a form bound to or incorporated in or absorbed in or adsorbed on macro-, micro-, and nanoparticles, or macro-, micro-, and nano-capsules, for the treatment of textiles, natural or synthetic fibres, wools, and any materials that may be used for clothing or underwear for day or night intended to come into contact with the skin, handkerchiefs or cloths, to exert their cosmetic effect via this skin/textile contact and to allow continuous topical delivery.

The CTFA (« International Cosmetic Ingredient Dictionary & Handbook » (15th Ed. 2014) published by « the Personal Care Products council », ex- « the Cosmetic, Toiletry, and Fragrance Association, Inc. », Washington, D.C.), describes a non-limited wide variety of cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which are suitable for use as additional ingredients in the compositions according to the present invention.

Further additional skin care actives that are particularly useful can be found in the commercial literature of Sederma and on the website www.sederma.com. The following commercial actives can also be mentioned, as examples: betaine, glycerol, Actimoist Bio 2™ (Active organics), AquaCacteen™ (Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia), Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (Arch Chemicals, Inc), Hydra'Flow™ (Sochibo), Hydromoist L™ (Symrise), RenovHyal™ (Soliance), Seamoss™ (Biotech Marine), Argireline™ (commercial name for the acetyl hexapeptide-3 of Lipotec), spilanthol or an extract of Acmella oleracea known under the commercial name Gatuline Expression™, an extract of Boswellia serrata known under the commercial name Boswellin™, Deepaline PVB™ (Seppic), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), PhytoCellTec™Argan (Mibelle), Papilactyl D™ (Silab), Preventhelia™ (Lipotec), and from Sederma: Subliskin™, Venuceane™, Moist 24™, Vegesome Moist 24™, Essenskin™, Juvinity™, Revidrat™, Resistem™, Chronodyn™, Kombuchka™, Chromocare™, Calmosensine™, Glycokin factor S™, Biobustyl™, Idealift™, Ceramide 2™, Ceramide A2™ et Ceramide H03™, Legance™, Intenslim™, Prodizia™, Beautifeye™, NG-shea butter unsaponifiables (natural grade), Zingerslim™, Meiritage™, Senestem™, Sebuless™, Majestem™, Apiscalp™, Rubistem™, or mixture thereof.

Among other plant extracts which can be combined with the peptide of the invention, there may more particularly be mentioned extracts of Ivy, in particular English Ivy (Hedera Helix), of Bupleurum chinensis, of Bupleurum Falcatum, of arnica {Arnica Montana L), of rosemary {Rosmarinus officinalis N), of marigold {Calendula officinalis), of sage {Salvia officinalis L), of ginseng {Panax ginseng), of ginko biloba, of St.-John's-Wort {Hyperycum Perforatum), of butcher's-broom {Ruscus aculeatus L), of European meadowsweet {Filipendula ulmaria L), of big- flowered Jarva tea {Orthosiphon Stamincus Benth), of algae {Fucus Vesiculosus), of birch {Betula alba), of green tea, of cola nuts {Cola Nipida), of horse-chestnut, of bamboo, of Centella asiatica, of heather, of fucus, of willow, of mouse- ear, of escine, of cangzhu, of chrysanthellum indicum, of the plants of the Armeniacea genus, Atractylodis Platicodon, Sinnomenum, Pharbitidis, Flemingia, of Coleus such as C. Forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C. xanthantus and C. Barbatus, such as the extract of root of Coleus barbatus, extracts of Ballote, of Guioa, of Davallia, of Terminalia, of Barringtonia, of Trema, of antirobia, cecropia, argania, dioscoreae such as Dioscorea opposita or Mexican, extracts of Ammi visnaga, of Siegesbeckia, in particular Siegesbeckia orientalis, vegetable extracts of the family of Ericaceae, in particular bilberry extracts {Vaccinium angustifollium) or Arctostaphylos uva ursi, aloe vera, plant containing sterols (e.g., phytosterol), Manjistha (extracted from plants of the genus Rubia, particularly Rubia Cordifolia), and Guggal (extracted from plants of the genus Commiphora, particularly Commiphora Mukul), kola extract, chamomile, red clover extract, Piper methysticum extract (Kava Kava™ from Sederma), Bacopa monieri extract (Bacocalmine™ from Sederma) and sea whip extract, extracts of Glycyrrhiza glabra, of mulberry, of melaleuca (tea tree), of Larrea divaricata, of Rabdosia rubescens, of Euglena gracilis, of Fibraurea recisa Hirudinea, of Chaparral Sorghum, of sun flower extract, of Enantia chlorantha, of Mitracarpe of Spermacocea genus, of Buchu barosma, of Lawsonia inermis L. , of Adiantium Capillus -Veneris L. , of Chelidonium majus, of Luffa cylindrica, of Japanese Mandarin (Citrus reticulata Blanco var. unshiu), of Camelia sinensis, of Imperata cylindrica, of Glaucium Flavum, of Cupressus Sempervirens , of Polygonatum multiflorum, of loveyly hemsleya, of Sambucus Nigra, of Phaseolus lunatus, of Centaurium, of Macrocystis Pyrifera, of Turnera Diffusa, of Anemarrhena asphodeloides, of Portulaca pilosa, of Humulus lupulus, of Coffea Arabica, of //ex Paraguariensis, or of Globularia Cordifolia, of Albizzia julibrissin, of Oxydendron arboretum, of Zingimber Zerumbet Smith, of Astragalus membranaceus , of Atractylodes macrocephalae, of Plantago lanceolata, of Leontopodium alpinum, of Mirabilis jalapa or of Apium graveolens.

The compositions of the present invention may include other peptides, including, without limitation, di-, tri-, tetra-, penta-and hexapeptides and their derivatives. According to a particular embodiment, the concentration of the additional peptide, in the composition, ranges from lxl0^"7% and 20%, preferably from lxl0^"6% and 10%, preferably between lxl0^"5% and 5% by weight.

According to the present invention, the term "peptide" refers to peptides containing 10 amino acids or less, their derivatives, isomers and complexes with other species such as a metal ion (e.g. copper, zinc, manganese, magnesium, and others). The term "peptides" refers to both natural peptides and synthetic peptides. It also refers to compositions that contain peptides and which are found in nature, and/or are commercially available.

Suitable dipeptides for use herein include but are not limited to Carnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK or TT. Suitable tripeptides for use herein include, but are not limited to RKR, HGG, GHK, GKH, GGH, GHG, KFK, KPK, KMOK, KM0₂K (M0₂ being a di-oxygenated in sulfoxide methionine) or KAvaK. Suitable tetrapeptides for use herein include but are not limited to RSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2) or KTFK (SEQ ID NO: 3). Suitable pentapeptides include, but are not limited to KTTKS (SEQ ID NO: 4). Suitable hexapeptides include but are not limited to GKTTKS (SEQ ID NO: 5) and VGVAPG (SEQ ID NO: 6).

Other suitable peptides for use herein include, but are not limited to: lipophilic derivatives of peptides, preferably palmitoyl derivatives, and metal complexes as aforementioned (e.g. copper complex of the tripeptide HGG). Preferred dipeptide include for example N-Palmitoyl-beta-Ala-His, N-Acetyl-Tyr- Arg-hexadecylester (Calmosensine™, Idealift™ from Sederma). Preferred tripeptide derivatives include for example N-Palmitoyl-Gly-Lys-His, and Pal-Gly-His-Lys, (Pal-GKH and Pal-GHK from Sederma), the copper derivative of HGG (Lamin™ from Sigma), Lipospondin (N-Elaidoyl-KFK) and its analogs of conservative substitution, N-Acetyl-RKR-NH₂ (Peptide CK+), N-Biot-GHK (from Sederma), Pal-KM0₂K (Matrixyl®synthe'6® from Sederma) and derivatives thereof. Suitable tetrapeptide derivatives for use according to the present invention include, but are not limited to, N- Pal-GQPR (SEQ ID NO: 7) (from Sederma) and Ela-KTFK (SEQ ID NO: 8), suitable pentapeptide derivatives for use herein include, but are not limited to, Pal-KTTKS (SEQ ID NO: 9) (available as Matrixyl® from Sederma), Pal-YGGFL (SEQ ID NO: 10) or Pal-YGGFP (SEQ ID NO: 11) or mixtures thereof. Suitable hexapeptide derivatives for use herein include, but are not limited to, Pal- VGVAPG (SEQ ID NO: 12), Pal-GKTTKS (SEQ ID NO: 13), HLDIIW (SEQ ID NO: 14), HKDIITpi (SEQ ID NO: 15), Tpi being the Tryptoline-3-carboxylic acid residue, or HLDIIF (SEQ ID NO: 16), or Pal-, and derivatives thereof. The mixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 7) (Matrixyl® 3000, Sederma) can also be mentioned.

The preferred compositions commercially available containing a tripeptide or a derivative include Biopeptide-CL™, Maxilip™, Biobustyl™, Procapil™ and Matrixyl®synthe'6® of Sederma. The compositions commercially available preferred sources of tetrapeptides include Rigin™, Eyeliss™, Matrixyl® Reloaded and Matrixyl 3000® which contain between 50 and 500 ppm of Pal-GQPR (SEQ ID NO: 7) and an excipient, proposed by Sederma.

The following marketed peptides can be mentioned as well as additional active ingredients:

Vialox™ (INCI name = Pentapeptide-3 (synthetic peptide comprising alanine, arginine, isoleucine, glycine and proline)), Syn-ake™ (β-Ala-Pro-Dab-NH-Bzl) or Syn-Coll™ (Pal-Lys- Val-Lys-OH) marketed by Pentapharm;

- Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH₂ (INCI name = Acetyl hexapeptide-3) (SEQ ID NO: 17), Leuphasyl™ (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO: 18), Aldenine™ (Gly-His-Lys), Trylagen™ (INCI name = Pseudoalteromonas Ferment Extract, Hydro lyzed Wheat Protein, Hydro lyzed Soy Protein, Tripeptide- 10 Citrulline (reaction product of Citrulline and Tripeptide- 10 (synthetic peptide constituted of aspartic acid, isoleucine and lysine)), Tripeptide-1), Eyeseryl™ (Ac- -Ala-His-Ser-His)(SEQ ID NO: 19), Serilesine™ (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO 20) or Decorinyl™ (INCI name: Tripeptide- 10 Citrulline = reaction product of Citrulline and Tripeptide- 10 (synthetic peptide constituted of aspartic acid, isoleucine and lysine) marketed by Lipotec;

- Collaxyl™ (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO 21)) or Quintescine™ (Cys-Gly) marketed by Vincience;

Cytokinol™LS (casein hydrolysate) marketed by Les Laboratoires Serobiologiques/Cognis;

- Kollaren™ (Gly-His-Lys), IP2000™ (Pal-Val-Tyr-Val) or Meliprene™ (INCI name = Monofluoroheptapeptide-1 : reaction product of acetic acide and a synthetic peptide comprising arginine, glycine, glutamic acid, histidine, norleucine, p-fluorophenylalanine and tryptophan) marketed by l'lnstitut Europeen de Biologie Cellulaire;

Neutrazen™ (Pal-His-D-Phe-Arg-NH₂) marketed by Innovations; or

BONT-L-Peptide™ (INCI name = Palmitoyl Hexapeptide-19: reaction product of palmitic acid and Hexapeptide-19 (synthetic peptide constituted of asparagine, aspartic acid, lysine and methionine), Timp-Peptide™ (INCI name = Acetyl Hexapeptide-20: reaction product obtained by acetylation of Hexapeptide-20 (synthetic peptide constituted of alanine, glycine, lysine, valine and proline) or ECM Moduline™ (INCI name = Palmitoyl Tripeptide-28: reaction product of palmitic acid and Tripeptide-28 (synthetic peptide constituted of arginine, lysine and phenylalanine) marketed by lnfinitec Activos. More specifically, the plant cells according to the invention may be combined with at least one of compounds selected from compounds of the vitamin B3, compounds such as niacinamide or tocopherol, retinoid compounds such as retinol, hexamidine, a-lipoic acid, resveratrol or DHEA, hyaluronic acid, peptides, in particular N-acetyl-Tyr-Arg-O-hexadecyl ester, Pal-VGVAPG (SEQ ID NO: 12), Pal-KTTKS (SEQ ID NO: 9), Pal-GHK, Pal-KM0₂K and Pal-GQPR (SEQ ID NO: 7), which are widely used active ingredients in topical cosmetic or dermopharmaceutical compositions.

The present invention also provides a method of cosmetic or dermatological topical treatment for improving the appearance and condition of the skin and its appendages, comprising the topical application to the skin of a subject in need thereof an effective amount of plant cells or a composition comprising them, in a physiologically acceptable excipient.

"Topical treatment" or "topical use" means according to the invention, an application that is intended to act where it is applied: skin, mucosa and/or appendages.

The composition comprising the plant cells according to the invention may be applied locally to targeted areas.

The "effective" amount depends on various factors, such as the age, the condition of the patient, the seriousness of the disorder or pathology, the administration mode, etc. An effective amount means a nontoxic amount enough to achieve the desired effect.

According to other features of the invention:

- The physiologically acceptable medium is a hydrophilic matrix in which said plant cells are suspended and/or solubilized; and/or

- The active ingredient comprises a thickener; and/or

- The active ingredient comprises at least 0.04% of leontopodic acids A and B relative to the total weight of the ingredient. This ingredient can be used thereafter in cosmetic formulations between 0.1 to 10%, preferably between 1 and 5%, more preferably between 2 and 3% and usually 2% by weight of said formulation; which corresponds to leontopodic acid A and B levels in cosmetic formulations between 0.00004% and 0.004%, preferably between 0.0004% and 0.002%, more preferably between 0.0008% and 0.0012% and generally at least 0.0008% with regard to the total weight of composition.

All percentages and ratios used herein are by weight of the total composition and all measurements are made at 25°C unless it is otherwise specified.

For example, for a cosmetic treatment of the face, the European Cosmetics Directive has set a standard amount for applying a cream of 2.72mg/cm²/day/person and for a body lotion of 0.5mg/cm²/day/person .

According to other specific features, the cosmetic treatment method according to the invention can be combined with one or more other treatment methods targeting the skin such as lumino-therapy, heat or aromatherapy treatments.

According to the invention, devices with several compartments or kits may be proposed to apply the method described above which may include for example and non-restrictively, a first compartment containing a composition comprising active cells according to the invention, and in a second compartment another active ingredient and/or excipient, the compositions contained in the said first and second compartments in this case being considered to be a combination composition for simultaneous, separate or stepwise use in time, particularly in one of the treatment methods recited above.

The method of treatment according to the invention is particularly suitable for a cosmetic treatment for repairing firming and smoothing treatment on an aged skin more particularly at the level of the sagging surface of the neck and the fold of the tear trough.

DETAILED DESCRIPTION

The present invention will be better understood and other advantages will appear from the following detailed description of an example of preparation of the plant cells, and in vitro and in vivo tests realized on these cells.

A) Example of preparation of plant cells

Creation of a cell line

Selected pieces of leaves of the Leontopodium alpinum genus are collected, washed and cut into small pieces of several mm, so as to provide from 200 to 1500 explants. After a series of decontamination treatments and then of sterilization, the pieces are placed on an agar culture medium in the presence of a nutrient medium containing plant growth hormones to induce the call genesis (formation of a callus). After an appropriate period of time, a mass of dedifferentiated cells or callus is formed, then transferred on a larger area and in a fresh culture medium to be able to multiply. A number of subcultures (transfers to fresh culture medium) is performed to stabilize the cell line, that is to say, until the latter has a high and constant speed of proliferation, preservation of phenotype, a constant content of bioactive compounds of interest (primary and secondary metabolites).

The cell line is then subjected to a selection step consisting of culturing the cells for a suitable time, collecting the formed aggregates of cells and inoculating them in a liquid culture medium for a period of time for obtaining the multiplication of the cellular aggregate. The best cell line will be the one to obtain as quickly as possible and reproducibly an important biomass having an optimal content of selected metabolites, the best biological activity and a homogeneous phenotype.

The cell line was also chosen for its ability to produce leontopodic acids A and B in an amount of about 5% by weight measured with respect to the dry weight of cells as, measured by HPLC.

Industrial method for preparing a biomass of undifferentiated or dedifferentiated cells of Leontopodium alpinum and treatment of this biomass

A cell line prepared as described above or an existing line is used to start.

The leontopodium alpinum line is at first multiplied to obtain a sufficient amount of biomass of dedifferentiated cells in order to perform the large-scale production step. The following steps are implemented:

a) Inoculation of the selected line in a liquid medium and cultivation during a time sufficient to obtain an increase in biomass of at least 300%;

b) Optionally, transfer the suspension obtained in a) in a fresh liquid medium and again cultivation during a sufficient time to obtain a biomass increase of 300%;

c) Optionally repeating step b);

d) Transfer of the cellular suspensions obtained at the steps a) to c) in a bioreactor with fresh liquid medium, and conduct the cultivation under such conditions and for a time sufficient to obtain a cellular biomass containing the metabolites of interest that is to say the phenylpropanoid glycosides and leontopodic acids in sufficient quantities, this production step in a bioreactor comprising an elicitation stage achieved by modifying the rates of the culture medium nutrients.

The bioreactor:

Volume: 5 to 50 times greater than the volume of the biomass used as inoculum; internal surface of the bioreactor smooth and uniform (no edges or comers which could cause the rupture of the cell walls). Growing conditions:

Culture medium: medium containing mineral salts (solution of macro-elements and micro-elements), vitamins, plant hormones as well as sucrose. Vegetable agar is added in the solid media.

Temperature: between 15°C and 35°C, preferably between 20°C and 30°C and still more preferably at 25°C.

Duration: between 7 and 21 days, preferably between 10 and 14 days.

Stirring of the biomass: it is important that the biomass be aerated optimally, and at the same time, be kept stirred either by internal means, or by an external means. It is necessary that the agitation, although small, be effective, especially in the final stages when the biomass is in large quantities. For the purposes of the present invention, internally suitable means are stirring helix rotating between 20 and 120 rpm, preferably at 60 rpm, or externally orbitally rotating means preferably rotating between 40 and 200 rpm and preferably at about 120 rpm.

Oxygenation: normally performed using sterile air at a rate of 0.5 to 4 litres per minute, preferably between 2 and 2.5 litres per minute, for a volume of 10 litres of biomass. Alternatively, gas mixtures containing from 10% to 100% v/v oxygen may be used. It is preferable to use means for diffusing air or oxygen with a nozzle having a flow rate comprised between 10 ml/min and 600 ml min and preferably between 50 ml/min and 350 ml/min.

Treatment of the obtained biomass

Filtration to eliminate the culture medium and recover the cell biomass. This biomass may be characterized by its equivalent content of lyophilized cells.

High pressure homogenization of the cellular biomass: allows a reduction in the size of the cellular aggregates; some cells may be broken and then a mixture of whole cells and crushed cells can be obtained, preferably keeping 10% of whole cells. Characterization of the active compounds contained in the cells by analytical determination of primary and secondary metabolites produced by the culture including protein, phenylpropanoid glycosides comprising leontopodic acids A and B.

Optionally:

1) Drying the cells including by lyophilisation or atomization to allow a greater stability of the compounds of interest, improve the long-term storage without the need to add preservatives.

2) Extracting the content of the cells by grinding/lysis/breaking down of the cells and separation of liquid and solid phases (by centrifugation or filtration or other means), to obtain a specific cellular extract.

3) Purification of the cellular extract to increase the content of leontopodic acids.

B) Preparation of an active ingredient for a use according to the invention

The biomass of cells, either as obtained above after filtration, or either in a dried form, or else the resulting extract, can be mixed with a physiologically acceptable medium forming the excipient.

As a preferred example, this physiologically acceptable medium is a hydrophilic matrix in which the cells are suspended, for example glycerol and/or butylene glycol in the case of a cosmetic composition. Additives may also be added if necessary, such as antimicrobial agents, anti-oxidants, stabilizing agents, agents acting on the pH, emulsifiers or thickening agents, including a thickener such as xanthan gum which will promote the maintaining of the cells in suspension.

An active ingredient for a cosmetic use may thus be formed for the implementation of the invention, comprising for example 20% by weight of fresh biomass of whole dedifferentiated cells (corresponding to about 1% of dry cells), in a physiologically acceptable excipient mixture consisting of glycerol (approximately 80%) and xanthan gum (0.3% by weight), said ingredient having a final content of about 0.05% of leontopodic acids A and B (about 15% acid A and 85% acid B).

This ingredient can be used to prepare cosmetic formulations as discussed below in the Galenic paragraph F).

According to the invention plant cells comprising a different content of leontopodic acids could of course be used to, especially higher, either obtained directly by the in vitro method (e.g. using an appropriate elicitation for increasing the rate), or obtained through a purification/concentration step of the obtained cells (e.g. by a concentration step after extraction of the cellular content).

For example, it is possible to produce plant cells in the form of a purified extract comprising a high rate of leontopodic acids, for example a rate of 25% relative to the dry matter, said cells being themselves used to make an active ingredient as described above.

C) in vitro test results

In vitro tests were carried out using an ethanol/water extract (70/30) of freeze-dried cells of the cellular culture of Leontopodium Alpinium (21% of freeze-dried cells obtained according to the above example in the solvent mixture). This solution is the stock solution. If 0.2% of this solution into a test medium is taken, the resulting solution will be 420ppm cell equivalent. The expression "a cellular extract at X cell equivalent" will thereafter be used.

1) Restauration of the homeostasis of dermal cells

The following tests were carried out using, when desired, experimental models of accelerated aging (by UVA or UVB irradiations) to demonstrate the advantages of the product according to the invention for restoring the homeostasis of the dermis cells.

a) Thrombospondins synthesis (TSP)

Principle: normal human keratinocytes (NHK) in culture are contacted with the cellular extract of the invention at 140 and 280 ppm cell equivalent for 48h. TSP-1 and -2 synthesis are evaluated by an ELISA method in the cell culture medium.

Table 1: TSP-2 production by NHK +/- the cellular extract of the invention (n=3).

These results show that the cellular extract according to the invention at 280ppm cell equivalent significantly induces the production of TSP-2 in the NHK (+ 51%, p<0.01).

This TSP increase is limiting MMP activity with the result of improving the quality of the dermis, b) Reduction of the synthesis of stress-induced MMP

Principle: dermal human fibroblasts (DHF) are cultured in the presence of the cellular extract according to the invention (70, 140 or 280ppm cell equivalent) for 24 hours. Then, after rinsing, the layers are exposed to an UVA irradiation to increase the pool production of MMP by these cells and thus to model an accelerated cell aging. The cells are then returned to the culture medium in contact with the extract according to the invention for 24 hours. MMP production in the culture medium is monitored using Multiplex/ELISA methods.

Table 2: Variation in the production of MMP following UVA irradiation of fibroblasts in contact with the cellular extract according to the invention (n = 4).

MMP-1 MMP-7 MMP-9

Variation Variation Variation (pg/mL/10⁶ (pg/mL/10⁶ (pg/mL/10⁶

(%) (%) (%) cells) cells) cells)

Control 1622 ± 47 Reference 657 ± 196 Reference 22.5 ± 3.1 Reference

-42%; -41%; -17%;

70ppm 942 ± 82 388 ± 136 18.8 ± 2.2

p<0.01 p<0.07 dns

-51%; -59%; -25%;

140ppm 794 ± 55 266 ± 134 16.8 ± 1.9

p<0.01 p<0.02 p=0.02 -53%; -56%; -21%;

280ppm 762 ± 40 289 ± 159 17.8 ± 1.8

p<0.01 p<0.03 p<0.04

UVA stress applied on fibroblasts for modelling aging has increased the production of MMP-1 (+ 292%, p<0.01), MMP-7 (48%, p=0.08) and MMP-9 (+ 51%; p<0.01). The contact of the cells with the extract according to the invention at 280ppm cell equivalent has greatly reduced the induction of MMP-1 (-53%, p<0.01), MMP-7 (-59%, p<0.02) and MMP-9 (-25%, p=0.02). Thus, the extract according to the invention can limit the formation of proteases that fragment the dermal extracellular matrix.

c) Increase of the stress induced TIMP synthesis

Principle: In parallel of the previous test, the TIMP production was followed using Multiplex/ELISA methods in the culture medium.

Table 3: Variation of the TIMP production following UVA irradiation on fibroblasts in contact with the cellular extract of the invention (n=4).

The contact of cells with the cellular extract according to the invention at 280ppm cell equivalent can re-activate the induction of TIMP-1 (+ 170%, p<0.01), TIMP-2 (141 %, p<0.01), TIMP-3 (+ 75%, p<0.01) and TIMP-4 (37%, p<0.01). Thus, the product according to the invention can highly induce specific inhibitors of MMPs.

d) Effect according to the invention of the extract on the basal production of dermal macromolecules

Collagen I

Dermal human fibroblasts (DHF) are cultured. After reaching confluence the cells are placed in contact with Vitamin C +/- the extract according to the invention at 140ppm cell equivalent and returned to culture for 72 hours. The supernatants are removed and cell layers extracted to quantify the number of cells. The determination of type I collagen is performed on the supematants by ELISA. No toxicity was observed compared to the control.

Table 4: Collagen I synthesis on DHF (n=5)

Hyaluronic acid

Dermal human fibroblasts (DHF) are cultured. After reaching confluence the cells are placed in contact with the cellular extract according to the invention at 210ppm and 420ppm cell equivalent and returned to culture for 72 hours. The supematants are then removed and cell layers extracted to quantify the number of cells. The dosage of hyaluronic acid is performed on the supematants by ELISA method. No toxicity was observed compared to the control.

Table 6: Synthesis of hyaluronic acid (HA) by HDF (n=5)

For the 3 macromolecules tested, a stimulation of their production by fibroblasts in the presence of the cellular extract according to the invention exists.

2) Improvement of the cellular and energetic dynamism

a) Contraction of modelled dermis

Principle: FHD of culture are aged in an accelerated manner using H₂0₂. The cells are then returned to their culture medium for 24 hours. Then, these cells are included in a model of dermis and, after polymerization, receive the cellular extract according to the invention (140 or 280ppm cell equivalent). The contraction of the dermis model is followed for 4 days. Photos are used to quantify the differences by image analysis. Table 7: Modulation of the contraction of a dermal model by FHD +/- a cellular extract according to the invention (n=6).

These results confirm that contacting the cells with H₂0₂ can reduce the contractile capacity of these cells (-33%; p<0.01). The extract according to the invention at 280ppm cell equivalent reduces, in a dose-dependent manner, the effect of accelerated aging on the contractile ability of the cells (+54% contraction compared to the control, p<0.01).

b) Mitochondrial dynamism

Principle: Aged cells are more retracted than young cells. The preservation of cell shape and of aspect of its mitochondrial network are therefore good indicators of their dynamism. Dermal human fibroblasts (DHF) in culture are stressed with UVB exposure in order to obtain a retracted phenotype and a fragmented mitochondrial network. Immediately after irradiation, cells are then returned to culture medium together with the cellular extract according to the invention (280ppm cell equivalent) and fixed after 18h. The cells are then labelled with Mitotraker specific dye and the network is quantified from photos by extracting beforehand the network connections using a specific computer tool.

The photos show that exposure, even minor of DHF to UVB, causes their shrinking and fragmentation of their mitochondrial network (less mitochondria in filaments, more puncture structures). Contact with the cellular extract according to the invention, contacted just after irradiation, can standardize the aspect of cells by maintaining their non-retracted phenotype and restore a less fragmented aspect to the mitochondrial network.

Table 8: Modulation mitochondrial connections by HDF +/- a cellular extract of the invention (n=28).

Therefore these results show that the mitochondrial network is highly disturbed for several hours after UVB irradiation, even low (22% fewer connections; p<0.02). Contact with the cellular extract according to the invention placed after irradiation can maintain the network and connections to the level of the non-irradiated control (+ 31%, p<0.05 vs. UVB alone). The cellular extract according to the invention can therefore restore the cellular and mitochondrial dynamism.

D) Galenic

Various cosmetic formulations are described below. Additional active ingredients, coming when appropriate in support and/or in addition to the activity of the active ingredient according to the invention containing undifferentiated cells or dedifferentiated of Leontopodium alpinum, can be added in the correct formulation phase according to their hydrophobic or hydrophilic nature. These ingredients can be of any category according to their(s) function(s), site of application (body, face, neck, chest, hands, etc.), the desired end and the targeted consumer, for example specific anti-wrinkle, moisturizing, anti-dark circles, firming, anti-glycation, volumizing, soothing, myo-relaxing, anti-redness, detoxifying, etc.

Active ingredient used in the galenic formulations given below: 20% by weight, based on the total weight of the ingredient composition, of fresh undifferentiated or dedifferentiated cells (biomass) of the invention (corresponding to about 1% of dry cells), the ingredient having a final content of leontopodic A and B acid of about 0.05% in a physiologically acceptable excipient mixture consisting of glycerol, xanthan gum thickener and citric acid to adjust the pH when appropriate.

This ingredient is preconized between 0.1 and 10%, preferably between 1 and 5%, more preferably between 2 and 3% and typically at 2%.

« Cream » form, particularly well adapted to the neck

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water

Optasense G83™ 0.30 Carbomer

Phase B

Brij S2-SS-(RB)™ 0.40 Steareth-2

Brij S IO-SO-(RB)™ 1.20 Steareth-10

Crodafos CES-PA-(RB)™ 4.00 Cetearyl Alcohol & Dicetyl Phosphate & Ceteth-

10 Phosphate

Crodacol CS 90-PA-(RB)™ 1.50 Cetearyl Alcohol

Laurocapram 2.50 Laurocapram

BRB CM 56™ 2.00 Cyclopentasiloxane & Cyclohexasiloxane

Crodamol OSU-LQ-(RB)™ 7.00 Diethylhexyl Succinate

Phase C

Glycerin 4.00 Glycerin

Octanediol 0.50 Caprylyl Glycol

Phase D

Phenoxyethanol qs Phenoxyethanol Phase E

Potassium sorbate qs Potassium Sorbate

Phase F

H₂0 4.00 Water

NaOH 30 % 0.40 Sodium Hydroxide

Phase G

Active ingredient according to 1.00 to 5.00 /

the invention

Phase H

Perfume 0.10 Fragrance

Protocol: weigh phase A and let swell for 30 minutes without stirring. Heat phase A at 75°C in a water bath. Weigh phase B and heat to 75°C in a water bath. Mix well. Weigh and melt phase C at 45°C. Add phase D to phase C, cooled beforehand. Pour phase C+D in phase A, under Staro stirring v=500rpm. Mix well. Pour phase B in the previous phase under Staro stirring v=1000rpm. Mix well. Extemporaneously, add phase E, homogenize. Add phase F, homogenize. Add phase G below 45°C, homogenize, 1 hour. Add phase H, mix well.

Examples of ingredients which may be added to this formulation:

• M A T R I X Y L ' s vn t h c ' 6^® : peptide-based anti-wrinkle ingredient marketed by Sederma (WO2010/082175) which helps repair skin damage caused by aging.

• MATRIXYL^®3000 : peptide-based anti-wrinkle ingredient marketed by Sederma (WO2005/048968) comprising two matrikines Pal-GHK and Pal-GQPR, which in synergy helps repairing skin damages caused by aging.

• PPvODIZIA™: active ingredient marketed by SEDERMA (WO2013/046137), comprising an extract of Albizia julibrissin, fighting the signs cutaneous fatigue: dark circles, under eye bags, dull complexion and drawn features, by repairing and protection the skin against the caused by damages of glycation and glycoxydation.

2) « Serum » form

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water

Potassium sorbate qs Potassium Sorbate

Phase B

Butylene glycol 3.00 Butylene Glycol

Phenoxyethanol qs Phenoxyethanol

Keltrol CG-SFT™ 0.30 Xanthan Gum

Satiagel VPC 614™ 0.20 Chondrus Crispus (Carrageenan) Extract Perfume 0.10 Fragrance

Phase C

H₂0 0.20 Water

Lactic acid 0.02 Lactic Acid

Phase D

Active ingredient of the 1.00 to

/

invention 5.00

Protocol: Weigh phase A under propeller v=300rpm. Weigh and homogenize phase B. Add phase B in phase A under rapid propeller stirring v=600rpm. Let homogenize for 1 hour. Adjust the pH to 6.00±0.10 with phase C. Add phase D in the previous phase and homogenize.

Examples of ingredients which may be added to this formulation:

• KOMBUCHKA™: active ingredient marketed by Sederma (WO2004/012650), acting on skin radiance, glycation, redensifying adipocyte population, and improving the quality of the skin (smoothness, skin tone).

• REVIDRATE™: active marketed by Sederma (WO 2011/086532) in particular improves the cohesion of the epidermis and its hydration.

• IDEALIFT™: active ingredient marketed by Sederma (WO2010/136965 - Butylene Glycol (and) Water (and) Sorbitan Laurate (and) Hydroxyethylcellulose (and) Acetyl Dipeptide-1 Cetyl Ester) who fights flaccidity of the face and improves resistance to gravity.

3) « Lotion » form, well adapted in particular to the neck

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water

Potassium sorbate 0.10 Potassium Sorbate

Phase B

Glycerin 2.00 Glycerin

Phenoxyethanol qs Phenoxyethanol

Keltrol CG-SFT™ 0.60 Xanthan Gum

Vivapur CS 032™ 0.25 Microcrystalline Cellulose (and) Xanthan Gum

Phase C

Span 40-PW-(MV)™ 1.00 Sorbitan Palmitate

Span 60-PW-(MV)™ 1.50 Sorbitan Stearate

Crodamol GTCC-LQ-(MV)™ 4.00 Caprylic/Capric Triglyceride

Prisorine 3505™ 4.00 Isostearic Acid Phase D

H₂0 0.30 Water

Lactic acid 0.03 Lactic Acid

Phase E

Active ingredient of the 1.00 to /

invention 5.00

Phase F

Perfume 0.10 Fragrance

Protocol: Weigh phase A. Weigh and mix phase B. Add phase B into phase A under propeller v=800rpm. Mix well. Heat phase A+B at 75°C in a water bath. Weigh and heat phase C at 75°C in a water bath. Pour phase C into phase A+B under very strong Staro stirring v=1000rpm; mix well. Adjust the pH to 5.60+0.10 with phase D below 35°C. Add phase E; mix well. Add phase F; mix well.

Examples of ingredients which may be added to this formulation:

• PACIFEEL™: active ingredient actif marketed by Sederma, comprising a natural extract of the Mirabilis jalapa plant also known as the Marvel of Peru, which alleviates cutaneous discomfort, fades redness of sensitive and reactive skin and strengthens and hydrates the epidermis.

• RESISTEM™: anti-aging marketed by Sederma (WO 2012/104774), helping the skin to build its own anti-aging defense system, based on an extract obtained by cell culture of Globularia cordifolia plant.

• MEIRITAGE™: anti-ageing active ingredient based on three plant extracts (Astragalus membranaceus (Huang Qi), Bupleurum falcatum (Chai Hu) and Atractylodes macrocephala (Bai Zhu), which improves the uniformity and radiance of skin.

4) « Gel » form for eve contour, particularly adapted to the « tear trough »

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water

Potassium sorbate 0.10 Potassium Sorbate

Sodium disulfite 0.01 Sodium Sulfite

Phase B

Keltrol CG-SFT™ 0.50 Xanthan Gum

N-HANCE HP40™ 0.25 Hydroxypropyl Guar

Zemea™ 1.90 Propanediol

Phenoxyethanol qs Phenoxyethanol Phase C

Crodamol ISIS-LQ-(MV)™ 2.90 Isostearyl Isostearate

Span 20-LQ-(SG)™ 0.70 Sorbitan Laurate

Ethanol 4.80 Ethanol

Perfume 0.10 Fragrance

Phase D

H₂0 2.00 Water

Lactic acid 0.02 Lactic Acid

Phase E

Active ingredient of the 1.00 to /

invention 5.00

Protocol: Weigh phase A and homogenize. Weigh phase B and homogenize. Add phase B to phase A under slow propeller stirring v=600rpm for 30 min. Weigh phase C and mix. Add phase C into phase A+B under vigorous staro stirring v=3000rpm. Adjust the pH to 5.50 ± 0.10 with phase D. Add phase E in the previous phase and homogenize.

Examples of ingredients which may be added to this formulation:

• MATRIXYL^®svnthe ' 6^® : as recited above .

• HALOXYL™: active ingredient for dark circles marketed by Sederma (WO2005/102266).

Haloxyl™ combines Pal-GHK and Pal-GQPR matrikines with N-hydroxysuccinimide (NHS) and the chrysin flavonoid. The Pal-GHK and Pal-GQPR reinforce firmness and tone of the eye contour; chrysin and N-hydroxysuccinimide activate the elimination of blood origin pigments responsible for the color of dark circles but also of the local inflammation.

• EYELISS™: active ingredient marketed by Sederma (WO2003/068141) that helps prevent against the appearance of bags under the eyes. It combines three components: hesperidin methyl chalcone reducing capillary permeability, Valyl-Tryptophan (VW) dipeptide which promotes lymphatic circulation and Pal-GQPR lipopeptide that improves firmness, elasticity and reduces inflammation.

« Complexion base » serum form with solar protection

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water

Potassium Sorbate 0.10 Potassium Sorbate

Phase B

Glycerin 2.00 Glycerin

Phenoxyethanol qs Phenoxyethanol Keltrol CG-SFT™ 0.60 Xanthan Gum

Vivapur CS 032™ 0.25 Microcrystalline Cellulose & Xanthan Gum

Phase C

Eusolex 4360™ 1.80 Benzophenone-3

Eusolex 2292™ 3.50 Ethylhexyl Methoxycinnamate & BHT

Span 40-PW-(MV)™ 1.00 Sorbitan Palmitate

Span 60-PW-(MV)™ 1.50 Sorbitan Stearate

Crodamol GTCC-LQ-(RB)™ 1.35 Caprylic/Capric Triglyceride

Prisorine 3505™ 1.35 Isostearic Acid

Phase D

H₂0 0.20 Water

Lactic acid 0.02 Lactic Acid

Phase E

Active ingredient of the invention 1.00 to

5.00

Phase H

Perfume 0.10 Fragrance

Protocol: Weigh phase A and put under propeller stirring v=500rpm. Weigh phase B and homogenize. Add phase B into phase A under propeller stirring v=800rpm; mix well. Heat phase A+B at 75°C in a water bath. Weigh phase C and heat at 75°C using a water bath. Add phase C into phase A+B under staro stirring v=1000rpm. Homogenize 30 min. Adjust the pH to 5.80+0.1 with phase D, below 35°C. Add phase E, homogenize. Add phase F, homogenize.

Eusolex 4360^1M is a UVA filter and Eusolex 2292^1M a UVB filter.

Examples of ingredients which may be added to this formulation:

• DERMAXYL™: anti-aging active ingredient marketed by SEDERMA (WO2004/101609) which smoothes wrinkles and repair the skin barrier.

• VENUCEANE™: active marketed by Sederma (WO2002/066668) comprising a Thermus thermophiles biotechnological extractthat prevents visible signs of photo-aging (spots, wrinkles, dryness ...), protects cell structures from damages caused by UV and strengthens skin integrity.

• MELASLOW™: active ingredient marketed by Sederma that promotes lightening of skin tone and depigmentation of spots (extract of Japanese Mandarin reticulata Blanco var. unshiu Citrus).

6) Acid pH cream form

PRODUCT % INCI NAME

Phase A

H₂0 QsplOO Water Potassium Sorbate 0.10 Potassium Sorbate

Phase B

Glycerin 5.00 Glycerin

Phenoxyethanol qs Phenoxyethanol

Keltrol CG-SFT™ 0.60 Xanthan Gum

Supercol GF™ 0.25 Cyamopsis Tetragonoloba (Guar) Gum

Phase C

Span 40-PW-(MV)™ 1.00 Sorbitan Stearate

Span 60-PW-(MV)™ 1.50 Sorbitan Palmitate

Crodamol IPIS-LQ-(MV)™ 4.00 Isopropyl Isostearate

Prisorine 3505™ 4.00 Isostearic Acid

Phase D

H₂0 QsplOO Water

Lactic acid 0.04 Lactic Acid

Phase E

Active ingredient of the invention 1.00 to

5.00

Phase F

Perfume 0.10 Fragrance

Phase G

Fruitbio™ 0.90 Water (Aqua) - Lactic acid - Camelia sinensis leaf extract - Glycerin - Citric acid - Malic acid

Protocol: Weigh phase A and put under propeller stirring v=130rpm. Weigh and homogenize phase B. Add phase B into phase A under propeller stirring v=400rpm; mix well for 30 minutes. Heat phase A+B at 75°C in a water bath. Weigh phase C and heat to 75°C in a water bath. Add phase C into phase A+B under staro stirring v=1000rpm; homogenizing 30 min. Adjust the pH to 5.60+0.10 with phase D, below 35°C. Add phase E in the previous phase, mix well. Add phase F, homogenize. Adjust the pH to 3.50+0.10 with phase G.

Fruitbio™ is an active ingredient marketed by Sederma consisting of a complex of a-hydroxyacids associated with a green tea extract.

Examples of ingredients which may be added to this formulation:

• CALMOSENSINE™: soothing active for sensitive skins marketed by Sederma (WO 1998/07744) comprising the Tyr-Arg lipo-dipeptide. It reduces discomfort feelings.

• PHYTOTONINE™: Active marketed by Sederma comprising a synergistic combination of three plant actives, flavonoids of Arnica montana flowers, saponins of rhizomes of Polygonatum multiflorum (Solomon's Seal) and proanthocyanidins of cones of Cupressus sempervirens (Cypress); significantly improves the appearance of "blotchy"skin.

• CHROMOCARE™: antiaging active combining a Rabdosia rubescens extract rich in oridonine and a Siegesbeckia orientalis extract rich in darutoside, marketed by Sederma (WO2010/1 19423) which unify and rejuvenate the complexion.

7) Thick cream form, in particular for a night cream

Protocol: weigh phase A. Weigh phase B and mix Add phase B into phase A and mix well. Heat phase A+B at 75°C in a water bath. Weigh phase C and heat to 75°C in a water bath. Mix well. Heat phase A to 90°C on a hot plate while mixing with a spatula. Add phase D to phase C at 75°C in a water bath. Add phase A to phase A+B at 75°C in a water bath. Slowly add phase A+B+E to phase C+D under strong agitation (v=2500rpm, then 1500rpm outside bath). Add phase F. Ceramide 2™ is an active ingredient marketed by Sederma, consisting of a pure substance identical to the ceramides present in the skin, which constitute 40 to 50% of cutaneous lipids. This ingredient has a restructuring function, maintains the hydration and integrity of the skin barrier.

E) In vivo tests

The evaluation of the in vivo efficacy of the treatment of the invention was conducted on the neck and on the fold of the tear trough.

Principle

The study was conducted on women, 59 years (41 - 71ans) mean age with several visible signs of aging:

On the neck with a measurement of the sagging surface, by image analysis on standardized photos, and an expert evaluation on these photos.

On the fold of the tear trough (the line through which tears move before rolling down the cheeks), with a measurement of its surface by fringe projection.

For the neck, a panel of 31 people of a mean age of 60 years [45 - 71 years] was selected based on the visible importance of the sagging surface.

For the tear trough, 21 people aged over 55 years (mean age 64 years) [56 - 71 years] were included on the basis of the visible extent of this type of fold.

Protocol

Specific inclusion criteria

Volunteers needed to have definite sagging neck skin surface and a clearly visible fold in the area of the so-called tear trough. The women had to have had hormonal consistency in the three months preceding the test as well as during the test. A seven-day washout period during which the volunteer could only use a simple moisturizing cream on the face was required. During the test, subjects were asked to only expose themselves moderately to the sun and only use the provided products.

Study type and duration

The study was a simple blind on the neck and the face. Each volunteer applied a cream according to the invention (formula of example 1 of the Galenic at 2%) and a placebo cream and in a contralateral on his face, except for the neck where, for practical reasons, only the 2% cream according to the invention was applied. Both creams were massaged into the skin twice daily for 6 weeks.

The study synopsis is shown in the diagram below.

TO T 3 weeks T 6 weeks

>

Dermatop, fold of the tear trough Dermatop, fold of the tear trough

Photos (face & neck) Photos (neck) Photos (face & neck)

Statistical testing was performed using the Student's t test or, if needed, a Wilcoxon signed-rank test. The tests were conducted on paired series. For the self-evaluation, a Chi2 test was used. Study of the sagging surface of the neck and smoothing

The efficacy of the product according to the invention on the neck consisted in measuring the sagging part of the neck by an image analysis of standardized photographs, and an expert assessment on these photographs.

Photos were realized at TO and 3 weeks using a photographic bench (From Orion Concept Company, France) equipped with a high definition digital camera, a special lighting as well as a restraint system of the volunteers. The position of the face, photographs and lighting parameters were standardized and controlled to be reproduced over time.

The sagging surface of the neck was measured on side face photographs, by image analysis. Anatomical irremovable landmarks (scars, spots, et.) were used to precisely define the contours of the surface of the neck including the sagging surface.

These results show a reduction from 3 weeks of the sagging surface of the neck; this reduction reached 11% (p < 0.01) of the surface measured at TO. In parallel, a strong responder rate was observed (77%). Regarding the smoothing effect of the skin of the neck, it was appreciated by a panel of seven expert judges who have viewed photographs of 28 volunteers having folds on the neck. The experts found a smoothing effect in 56% of the cases after 6 weeks.

Study of the tear trough fold

The tear trough is the hollow where the tears come flowing before rolling down the cheeks. This suborbital hollow is continuous with the lower lid and will sometimes extend between the cheek and cheekbone. This hollows significantly with age thereby increasing its volume. The factors involved are usually fat melting and flaccidity.

To test the efficacy of the treatment of the invention on this site, a contactless FOITS system ("Fast Optical In vivo Topometry System") was used. This system is based on the analysis of the projection of optical fringes on the studied skin area, here the tear trough. The apparatus used (of the Dermatop company; Eotech, France) consists of a projector and a camera which are joined and form a specific angle, allowing triangulation. The study of the deformation of the fringes by the site relief allows a 3D reconstruction of the relief. The objectification of the effect according to the invention was made using the Aeva™ software (Eotech, France), by analyzing data on the extent of the tear trough measured by its circumference. In parallel, the volume parameter of the trough was obtained by the same procedure.

Table 10: Variation of the extent and volume of the tears trough after application of the invention. (N=21 volunteers; >55years)

-significant difference.

The results show that the treatment according to the invention leads to a 9% reduction of the extent of the tear trough (measured in circumference), this reduction being significant compared to placebo (p <0.01). In contralateral, applying a placebo produces no significant change. Moreover, the volume of the tear trough of the people over 55 years is reduced by 8.8%, this reduction being significant (p <0.01) compared to placebo.

Claims

1. Use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinium obtained by in vitro cell culture method for a non-therapeutic cosmetic treatment for restoring the homeostasis of the cells of aged skin and increase their metabolic and energetic activity.

2. Use according to claim 1 of whole and/or lysed plant cells.

3. Use according to claim 2 of a cellular extract of said whole and/or lysed plant cells.

4. Use according to anyone of claims 1 to 3 of plant cells in dried form.

5. Use according to anyone of claims 1 to 4 of a cosmetic composition comprising said plant cells suspended and/or dissolved in a physiologically acceptable medium.

6. Use according to claim 5, wherein said physiologically acceptable medium is a hydrophilic matrix.

7. Use according to anyone of claims 1 to 6, wherein said treatment is topical.

8. Use according to anyone of claims 1 to 7 for a tensing and smoothing repairing treatment on an aged skin.

9. Use according to claim 8, for a cosmetic treatment of the sagging surface of the neck and/or the fold of the tear trough.

10. Use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinium obtained by in vitro cell culture method for producing a non-therapeutic cosmetic active ingredient to restore homeostasis of the cells of aged skin and increase their metabolic and energetic activity.

11. Use according to claim 10, wherein said active ingredient comprises at least 0.04% of leontopodic A and B acids.

12. Cosmetic active ingredient, for a use according to anyone of claims 1 to 9, comprising undifferentiated or dedifferentiated plant cells of Leontopodium alpinium obtained by in vitro cell culture method and comprising at least 0.04% of leontopodic A and B acids in a physiologically acceptable matrix.

13. Ingredient according to claim 12, wherein the plant cells are whole and/or lysed.

14. Ingredient according to claim 13, comprising a cellular extract of said whole and/or lysed plant cells.