FR3101542A1 - USE OF PLANT LEONTODIUM ALPINUM cells FOR ANTI-GLYCATION cosmetic TREATMENT - Google Patents

Abstract

According to the invention, there is proposed the use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinum obtained by cell culture in vitro, for a non-therapeutic cosmetic treatment to prevent the glycation of macro-molecules of the skin, in particular proteins, nucleic acids and / or lipids.

Description

USE OF LEONTODIUM ALPINUM PLANT CELLS FOR AN ANTIGLYCATION COSMETIC TREATMENT

The present invention relates to the use of the plant Leontopodium nivale subsp. alpinum Cass, hereafter called Leontopodium alpinum , for a cosmetic treatment, in particular by topical route.

The present invention thus relates to the cosmetics industry and to hygiene and personal care products, applying to the skin and its integuments, of animal or human mammals.

Leontopodium alpinum, also known as Edelweiss, is a herbaceous plant of the Asteraceae family. It grows at altitude above 1500 m on the mountain ranges of the Pyrenees, the Alps and the Himalayas, in inhospitable areas such as ravines, rocky areas, cold and very exposed to UV rays. As a result, it is a plant that has an excellent adaptation to these extreme conditions through a panoply of molecules of interest and thanks to the protective hairs on its flower and its leaves. On the other hand, it is relatively little distributed and thus rather rare.

Plant products can be obtained by conventional extraction methods directly from the whole plant or its parts or by in vitro culture methods either by cell culture or by tissue culture from cell lines or tissue from different plant organs.

The present invention relates more particularly to the products obtained by in vitro culture of cells or tissues.

Obtaining extracts of plant origin by in vitro culture has many advantages over the agro-industrial route (cultivation of plants in the open field and subsequent extraction in the factory). Due to the total control of the 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 that may come from plant parasites). In addition, the strict control of in vitro culture conditions reduces the risk of spontaneous variation of the strain and guarantees a reproducible profile of secondary metabolites which correspond to the molecules of interest sought, unlike culture in the open field where the problem arises. variability, linked to climatic, meteorological and geographical conditions and their vagaries. Moreover, this technology overcomes obstacles such as the natural biological cycle of the plant and the seasonality of production of secondary metabolites, allowing better security and speed of supply. In addition, the environmental impact is minimal because it substantially limits water consumption, avoids consumption of arable land, and prevents soil pollution. In addition, biodiversity is preserved since all it takes is a plant or even a seed to initiate a new culture in vitro . Finally, this technology offers the possibility of directing cellular metabolism towards the production of molecules of interest (elicitation of cultures) and of carrying out controlled and relatively rapid protocols with a view to increasing the yields of certain molecules, those produced in particular in low amount in the plant.

Among the techniques existing to date in the field of the in vitro culture of plants, it is possible to use according to the invention:

• culture of undifferentiated or dedifferentiated cells: this type of method first involves the creation of highly proliferative cell lines in agar medium either from meristematic cells which are undifferentiated cells, or from dedifferentiated cells (growing in the form of calluses, following the removal of a plant fragment, leaf, stem, root or other). These lines are then cultured in a liquid medium so as to substantially increase the biomass. At the end of the growth cycle and under medium conditions to be defined and optimized (search for the right elicitation medium), the cell biomass will synthesize the molecules of interest. The culture is then stopped and subjected to an extraction at the optimal moment in order to obtain a maximum quantity of molecules of interest. Initially, existing cell lines that are already commercially available can also be used.
• tissue or organ culture: this type of culture may concern the root part (“root culture”), the aerial part (“shoot culture”) or the somatic embryos (“somatic embryo”). In this type of method, a distinction is made between cultures that have undergone genomic transformation by the bacteria Agrobacterium rhizogens (roots) or Agrobacterium tumefaciens (stems). Cultures thus transformed from roots or aerial parts 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 extracted by conventional means.

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

Schematically, in vitro cell culture methods consist of:

• if necessary initially, to establish cell lines from calluses (clusters of undifferentiated or dedifferentiated cells) obtained from cuts of plant parts (leaf, root, stem, buds, etc.);
• select a cell line capable of large-scale cell biomass production according to pre-established criteria (constant phenotype and optimal and constant production of selected metabolites, ability to proliferate);
• then from this selected line, to generate said cell biomass, optionally with an elicitation step, preferably at the end of the proliferation phase; And
• in a third step, in treating the cell biomass obtained to recover the whole cells, break the cell aggregates by homogenization under high pressure or lyse these cells and, if necessary, then extract the content of said cells.

It is these extracts and/or whole or lysed cells which can then be used in cosmetic compositions as an active ingredient with, where appropriate, a physiologically acceptable excipient and possibly other complementary active ingredients.

Patent application EP2319914 describes this in vitro technique for obtaining undifferentiated cells in culture with a high yield of caffeic acid derivatives for a theoretical list of 33 plant species including Leontopodium alpinum . Relevant caffeic acid derivatives include phenylpropanoid glycosides as well as caffeoylquinic acids. Hyaluronidase inhibitory activity is shown for Leontopodium alpinum . Protecting hyaluronic acid from digestion by hyaluronidase allows preventive maintenance of the integrity of the dermis.

The aim of the present invention is to propose a new use of Leontopodium alpinum cells obtained by an in vitro cell culture process for a cosmetic treatment.

To this end, it proposes the use of undifferentiated or dedifferentiated Leontopodium alpinum cells obtained by an in vitro cell culture process for a non-therapeutic cosmetic treatment to prevent or slow down the glycation of macromolecules in the skin, in particular proteins, nucleic acids and/or lipids.

The treatment according to the invention therefore makes it possible to protect the skin from the damage caused by glycation.

The human body needs sugar to produce its energy. However, the sugar that is consumed is never fully used by the body for this beneficial purpose. A residual part of this absorbed sugar will react in a non-enzymatic way with the amino groups of proteins, nucleic acids or lipids to create advanced glycation products (in English "AGE products" for "Advanced Glycosylated End-products" or AGEs ) that accumulate over time in the tissues.

The formation of many glycated proteins whose functional, enzymatic and structural properties are altered has consequences for the proper functioning of the cell or the organism.

Concerning the skin, AGEs are found not only in the dermis but also in the epidermis, up to the stratum corneum . Organs are all the more affected when their proteins have a long lifespan, such as collagen, elastin, fibronectin and laminins. This has the consequence of altering the mechanical and elastic properties of the extracellular matrix of the dermis, which becomes less flexible, more rigid, but also more flabby and less reactive.

Within the cells of the skin, the mitochondrial proteins are glycated which causes reductions in the yield in the production of ATP and a deficiency in the production of energy.

AGEs also modify the permeability of vessels, by glycation / alteration of their matrix proteins. The micro-inflammatory reactions that then take place lead to a decrease in flow and a leak of blood compounds outside the vessels. This leads to an accumulation of liquids and waste around them which forms dark circles and certain types of puffiness.

AGEs therefore represent a major source of cellular dysfunction and their accumulation tends to alter the properties and performance of tissues and cells: less flexibility, less mobility, less reactivity, less energy production. All functions are impaired.

The impact on the skin of these biochemical processes, the effects of which have just been described at the cellular level, is therefore significant. Glycation results in dull, flabby, lackluster skin, with a tired appearance, for example with dark circles and bags under the eyes, drawn features, skin lacking tone and suppleness.

There is therefore a real need in this field in cosmetics.

The applicant has shown that the undifferentiated or dedifferentiated cells of Leontopodium alpinum according to the invention have a preventive effect against glycation, to slow down or even prevent its implementation. Such an effect is independent of age, the use according to the invention not being limited to the treatment of aged skin.

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be used in particular:

• to prevent skin fatigue; and or
• to prevent loss of skin suppleness; and or
• to maintain the radiance of the complexion of the skin and its appendages; and or
• to prevent the formation of dark circles and bags under the eyes.

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

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be used whole or lysed, or else in the form of a cell extract produced from these cells (with or without the cell walls removed).

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be extracted with any physiologically acceptable solvent, or with any mixture of these solvents. The extraction can be done according to the various known processes that can be combined: hot, by maceration, decoction, infusion, pressure, leaching, ultrasound, microwaves, by lysing the cells by any process appropriate chemical or physical. The separation of the phases can be done by filtration or centrifugation. Alternatively, it is possible to extract the biomass with a supercritical or subcritical fluid.

According to the invention, it is also possible to envisage a thorough purification of the cellular extract by all the methods available industrially, by liquid-liquid partition or chromatography, in particular using an adsorbent resin, in order to concentrate the molecules of interest such as the two leontopod acids A and/or B.

According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum can also be used in a form dried by spraying or lyophilization, preferably. This allows their long-term storage and preserves their biological activity. This also makes it possible to advantageously produce anhydrous formulations, of the powder type for example.

In the rest of the description, the expression "plant cells" will encompass the undifferentiated or dedifferentiated cells of Leontopodium alpinum obtained by an in vitro cell culture process, whether whole or lysed, whether or not they have undergone homogenization. at high pressure, whether fresh or in dry form, as well as extracts from these cells (whether or not they have had their cell walls removed).

According to the invention, the plant cells can be incorporated (for example placed in suspension or dissolved) in a physiologically acceptable medium and used to produce a cosmetic composition.

They can also be used as they are, optionally ground to define and homogenize their particle size, in a dry powder formula, for example for care make-up. The dried plant cells are then included in an anhydrous cosmetic composition.

According to another embodiment, a cosmetic composition comprising said plant cells suspended and/or solubilized in a hydrophilic matrix is used according to the invention.

Preferably again, the cosmetic treatment according to the invention is topical.

Surprisingly, the Applicant has shown by in vivo tests that the cosmetic treatment of skin, by applying a composition containing undifferentiated or dedifferentiated cells of Leontopodium alpinum obtained by an in vitro cell culture process, induced on these skins an anti-glycation effect.

The test results are presented in more detail below in the description.

The invention finally proposes the use of plant cells, as defined above, for the manufacture of a non-therapeutic cosmetic active ingredient, as well as the use of a composition comprising said active ingredient intended to prevent the glycation of skin macro-molecules, including proteins, nucleic acids and lipids. Examples of active ingredients and cosmetic compositions are given later in the description.

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 categories of compounds in the plant cells used according to the invention.

To obtain the dedifferentiated or undifferentiated cells that can be used according to the invention, the following process can be implemented:

1. from a selected line of Leontopodium alpinum , produce a critical pre-biomass by successive pre-cultures of increasing sizes;
2. producing a biomass of said 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 undifferentiated cells.

According to other optional features:

1. the bioreactor production step may include an elicitation step, this advantageously making it possible to increase the levels of leontopodium acids A and/or B or to vary the relative proportion thereof; and or
2. the biomass from the reactor is harvested by filtration after a culture time of between 7 and 21 days; preferably between 10 and 14 days, this advantageously allowing the highest quantity of biomass to be produced, with high viability; and or
3. biomass can be subjected to a homogenization step under high pressure, in order to break up cellular aggregates; and or
4. an additional step of drying the cellular biomass can be added, so as to preserve it in the long term; and or
5. the cells can be extracted in particular for the purpose of enrichment in leontopodic acids A and B.

In general, the elicitation of the compounds of interest can be done by the addition to the culture of microbial fractions (in particular saccharomyces yeasts): the addition to the culture of molecules of biological origin such as for example the chitosan, methyljasmonate, jasmonic acid, salicylic acid; the addition to the culture of molecules of non-biological origin such as paclobutrazol; the application to the culture of a variation in temperature, pH or osmotic stress induced by a non-metabolisable sugar, such as mannitol; resorting to an even more drastic depletion of the environment in macroelements and sugar; the addition to the culture of adsorbent resins which, in addition to eliciting the production of the compounds of interest, can trap them.

Preferably according to the invention, the elicitation is carried out by modifying the culture medium, in particular the levels of nutrients.

Preparation of compositions for the implementation of the invention

A cosmetic composition, in particular topical, comprises plant cells in a physiologically acceptable medium. Depending on the excipient and the plant cell dosage, this composition will constitute a concentrated active ingredient or a less concentrated final composition directly intended for the end user.

By "physiologically acceptable medium" is meant according to the present invention, without being limiting, an aqueous or hydro-alcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a microemulsion, an aqueous gel, an anhydrous gel , a serum, a dispersion of vesicles, a powder.

"Physiologically acceptable" means that the compositions are suitable for topical or oral use, in contact with the mucous membranes, the nails, the scalp, the hair, the hairs and the skin of mammals and more particularly human, compositions being able to be ingested or injected into the skin, without risk of toxicity, incompatibility, instability, allergic response, and others. This "physiologically acceptable medium" forms what is conventionally called the excipient of the composition.

The plant cells can be combined with other active ingredients at effective concentrations that can act synergistically or as reinforcement to achieve the desired effects described for the invention, such as the following agents: screening out radiation, in particular UVA and/or UVB, moisturizing, humectant, calming, muscle relaxant, slimming, restructuring, firming, plumping, tightening, acting on microcirculation, acting on inflammation, on free radicals, , anti-wrinkle, lightening, acting on the radiance of the complexion , anti-glycation, pro-pigmenting, acting on the stratum corneum, on the dermis-epidermis junction, on the production of HSPs protein, on firmness, elasticity, skin tone, hair regrowth peptides, vitamins etc

The plant cells can be applied according to the invention to the face, the body, the neckline, the scalp, the hair, the eyelashes, the body hair, in any form or vehicle known to those skilled in the art. , in particular in the form of a solution, dispersion, emulsion, paste or powder, individually or as a premix or being conveyed individually or as a premix by vectors such as macrocapsules, microcapsules or nanocapsules, macrospheres , microspheres, or nanospheres, liposomes, oleosomes or chylomicrons, macroparticles, microparticles or nanoparticles, macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or adsorbed on powdery organic polymers, talcs , bentonites, spores or exines and other mineral or organic carriers.

In cosmetics in particular, applications can be proposed in particular in skin care ranges for the face, body, hair and body hair and make-up-care ranges, in particular eyelashes and eyebrows.

In general, the plant cells according to the present invention can be used in any form, in a bound, incorporated or adsorbed form on macro-, micro-, and nanoparticles, or on macro-, micro- and nanocapsules, for the treatment of textiles, natural or synthetic fibers, wool, and all materials intended to come into contact with the skin and which can be used in clothing, day or night underwear, handkerchiefs , or tissues, in order to exert its cosmetic effect via this skin/textile contact and allow continuous topical delivery.

The CTFA ("International cosmetic ingredient dictionary & handbook" (16th edition, 2016) published by "the Cosmetic, Toiletry, and Fragrance Association, Inc.", Washington, D.C.) describes a wide variety, without limitation, of cosmetic ingredients and pharmaceuticals commonly used in the skin care industry, which are suitable for use as additional ingredients in the compositions according to the present invention.

Other additional skincare actives that are particularly useful can be found in Sederma's marketing literature and at www.sederma.com.

The following commercial active ingredients can also be cited 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™ (trade name for Lipotec's acetyl hexapeptide-3), spilanthol or an extract of Acmella oleracea known as Gatuline Expression™, an extract of Boswellia serrata known as Boswellin™, Deepaline PVB™ ( Seppic), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), PhytoCellTec™Argan (Mibelle), Papilactyl D™ (Silab), Preventhelia™ (Lipotec), or one or more of the following active ingredients sold by 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™, Ceramide HO3™, Legance™, Intenslim™, Prodizia™, Beautifeye™, Pacifeel TM , Zingerslim ^™ , Meiritage™, Senestem™, Sebuless™, Majestem™, Apiscalp™, Rubistem ™, Citystem ^TM , Neonyca ^TM , Unsaponifiable Shea Butter ^TM NG, Majestem ^TM , Hydronesis ^TM , Poretect ^TM , Crystalide ^TM , Amberstem ^TM or mixtures thereof.

Among the plant extracts (in the form of conventional extracts or prepared by an in vitro method) which can be combined with the cyclic peptide(s) according to the invention, mention may be made, in addition and in in particular, extracts of ivy, for example climbing ivy (Hedera helix ), Bupleurum chinensis , Bupleurum falcatum , arnica (Arnica montana L.) , rosemary ( Rosmarinus officinalis N. ), marigold ( Calendula officinalis ) , sage ( Salvia officinalis L. ), ginseng ( Panax ginseng ), ginko biloba, St. John's wort ( Hyperycum perforatum ), butcher's broom ( Ruscus aculeatus L. ), ulmaria ( Filipendula ulmaria L. ), orthosiphon ( Orthosiphon stamincus Benth.), seaweed ( Fucus vesiculosus ), birch ( Betula alba ), green tea, kola nut ( Cola nipida ), horse chestnut, bamboo, Centella asiatica , heather, fucus, willow, mouse-ear, extracts of escine, extracts of cangzhu, extracts of crysanthellum indicum , of plants of the genus Armeniacea, Atractylodis platicodon , Sinnomenum , pharbitidis , Flemingia , of Coleus such as C. forskohlii , C. blumei , C. esquirolii , C. scutellaroides, C. xanthantus and C. barbatus , as an extract from the roots of Coleus barbatus , extracts from Ballote , Guioa , Davallia , Terminalia , Barringtonia , Trema , Antirobia , Cecropia , Argania , Dioscoreae as Dioscorea opposita or extracts of Ammi visnaga , of Siegesbeckia , in particular Siegesbeckia orientalis , of plant extracts of the family Ericaceae , in particular of blueberry ( Vaccinium angustifollium ) extracts of Arctostaphylos uva ursi , Aloe vera , of plants containing sterols (in particular phytosterols), Manjistha (extract of plants of the genus Rubia , in particular Rubia cordifolia ), Guggal (extract of plants of the genus Commiphora , in particular Commiphora mukul ), an extract of kola, chamomile, red clover, Piper methysticum ( Kava Kava from Sederma), Bacopa monieri (Bacocalmine™, Sederma) and sea whip, Glycyrrhiza glabra , mulberry, melaleuca (tea tree), Larrea divaricata , Rabdosia rubescens , Euglena gracilis , Fibraurea recisa hirudinea , Chaparral sorghum , sunflower flower, Enantia chlorantha , Mitracarpus of the genus Spermacocea, Buchu barosma , Lawsonia inermis L. , Adiantium capillus-veneris L. , Chelidonium majus , Luffa cylindrica , of “Japanese Mandarin” ( Citrus reticulata blanco var. unshiu ), Camelia sinensis , Imperata cylindrical , Glaucium flavum , Cupressus sempervirens , Polygonatum multiflorum , Loveyly hemsleya , Sambucus nigra , Phaseolus lunatus , Centaurium , Macrocystis pyrifera , Turnera diffusa , Anemarrhena asphodeloides , Portulaca pilosa , Humulus lupulus , Arabica coffee, Ilex paraguariensis , Globularia cordifolia , Oxydendron arboreum , Albizzia julibrissin , Zingiber zerumbet smith , Astragalus membranaceus , Atractylodes macrocephalae , Plantago lanceolata , Mirabilis jalapa , Apium graveolens, Marrubium vulgare or orchids.

The compositions according to the present invention may comprise one or more peptides, including, but not limited to, di-, tri-, tetra-, penta- and hexa-peptides and their derivatives. According to a particular embodiment, the concentration of the additional peptide, in the composition, varies between 1x10 ^-7 % and 20%, preferably between 1x10 ^-6 % and 10%, preferentially between 1x10 ^-5 % and 5%, by weight . The term “peptide” designates here peptides containing 10 amino acids or less, their derivatives, isomers and complexes with other species such as a metal ion (eg copper, zinc, manganese, magnesium, and others). The term "peptides" refers to both natural peptides and synthetic peptides. It also refers to compositions which contain peptides and which occur in nature, and/or which are commercially available.

Non-limiting examples of dipeptides which can be used in the context of the present invention include Carnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM or PP.

Non-limiting examples of tripeptides include RKR, HGG, GHK, GGH, GHG, KFK, KAvaK, KβAK, KAbuK, KAcaK, KPK, KMOK, KMO2K, PPL, PPR, SPR, QPA, LPA or SPA.

Non-limiting examples of tetrapeptides are RSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2) or KTFK (SEQ ID NO: 3), KTAK (SEQ ID NO: 4), KAYK (SEQ ID NO: 5 ) or KFYK (SEQ ID NO: 6). A non-limiting example of a pentapeptide is KTTKS (SEQ ID NO: 7) and of hexapeptides GKTTKS (SEQ ID NO: 8) and VGVAPG (SEQ ID NO: 9).

Other peptides that can be used in the context of the present invention can be chosen from the following list without it being limiting: the lipophilic derivatives of peptides, preferably the palmitoyl and myristoyl derivatives, and the complexes with the metal ions mentioned above (eg copper complex of the HGG tripeptide). Preferred dipeptides include, for example, N-Palmitoyl-beta-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (Calmosensine™, Idealift™, Sederma), Pal-KT, Pal-RT (Sederma). The preferred tripeptides include in particular N-Pal-Gly-Lys-His, (Pal-GKH, Sederma), the copper derivative of HGG (Lamin™, Sigma), Pal-GHK, lipospondin (N-Elaidoyl-KFK) and its conservative substitution analogs, N-Acetyl-RKR-NH ₂ (Peptide CK+), N-Biot-GHK (Sederma), Pal-KAvaK, Pal-KβAlaK, Pal-KAbuK, Pal-KAcaK, Pal-KMO2K (Sederma) and their derivatives.

Mention may also be made here of the anti-aging tripeptides of general formula X–Pro*–Pro*–Xaa–Y described in application WO2015181688 with Xaa chosen from Leu, Arg, Lys, Ala, Ser, and Asp, at the N terminal end , X chosen from H, -CO-R1 and -SO2-R1 and at the C terminal end Y chosen from OH, OR1, NH2, NHR1 or NR1R2, R1 and R2 being, independently of each other, chosen from a alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonyl, phosphorylated and/or sulfur-containing, said group possibly possessing in its backbone a heteroatom in particular O, S and/or or N, and Pro* corresponding to Proline, an analogue or a derivative thereof; including, for example, Myr-PPL-OH and Myr-PPR-OH.

Mention may also be made here of the pro-pigmenting and/or pro-Mec dipeptides and tripeptides of general formula X–(Xaa1)n–Pro*–Xaa2–Y described in application WO2014/080376, with n=0, 1 or 2, Xaa1 a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, Pro, and analogs or derivatives thereof; or a polar amino acid selected from Ser, Thr, Tyr, Asp, Glu and derivatives and analogs thereof; and when n=2 the two amino acids Xaa1 may be the same or different; Xaa2 a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, and analogs or derivatives thereof; a basic amino acid selected from Arg, Lys, His, and derivatives and analogs thereof; at the N-terminus of the peptide, X is selected from H, -CO-R1 and -SO2-R1; at the C terminal end of the peptide, Y is chosen from OH, OR1, NH2, NHR1 or NR1R2, R1 and R2 being, independently of one another, chosen from an alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group , which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonyl, phosphorylated and/or sulphurous, said group possibly possessing in its skeleton a heteroatom in particular O, S and/or N; Pro* being Proline, an analogue or derivative thereof; including for example the peptides Pal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPA-OH, Myr-SPA-OH, Pal-PM-OH, Pal-PA-OH and Pal-PP -OH.

Tetrapeptide derivatives that can be used in the context of the present invention include, but are not limited to, Pal-GQPR (Sederma) (SEQ ID NO: 10), Pal-KTFK (SEQ ID NO: 11) or Ela-KTFK (SEQ ID NO: 12), Ela-KTAK (SEQ ID NO: 13), Ela-KAYK (SEQ ID NO: 14) or Ela-KFYK (SEQ ID NO: 15). Usable pentapeptide derivatives are, but are not limited to, Pal-KTTKS (SEQ ID NO: 16) (MATRIXYL™, Sederma), Pal-YGGFX (SEQ ID NO: 17) with X being L or P, Pal- HLDIIX with X being W, F, Y, Tic, 7-hydroxy-Tic or Tpi (SEQ ID NO: 18), or a mixture thereof. Useful hexapeptide derivatives include, but are not limited to: Pal-VGVAPG (SEQ ID NO: 19), Pal-GKTTKS (SEQ ID NO: 20) and derivatives. Mention may also be made of the mixture Pal-GHK and Pal-GQPR (SEQ ID NO: 10) (Matrixyl 3000™, Sederma).

Preferred commercially available compositions containing a tripeptide or derivative include Biopeptide CL™, Maxilip™ or Biobustyl™ from Sederma. Preferred, commercially available compositions sources of tetrapeptides include: RIGIN™, Eyeliss™, Matrixyl™ Reloaded and Matrixyl 3000™, which contain between 50 and 500 ppm palmitoyl-GQPR (SEQ ID NO: 10) and an excipient, offered by Sederma.

The following commercial peptides may also be mentioned as additional active ingredients:

• Vialox™ (INCI name = Pentapeptide-3 (synthetic peptide comprising alanine, arginine, isoleucine, glycine and proline)), Syn-ake™ (β-APDab-NH-Bzl) or Syn-Coll™ (Pal-KVK -OH) sold by Pentapharm,
• Argireline™ (Ac-EEMQRR-NH₂(INCI name = Acetyl hexapeptide-3) (SEQ ID NO: 21), Leuphasyl™ (YAGFL) (SEQ ID NO: 22), Aldenine™ (GHK), Trylagen^TM(INCI name = Pseudoalteromonas Ferment Extract, Hydro lyzed Wheat Protein, Hydro lyzed Soy Protein, Tripeptide-10 Citrulline (product of the reaction of Citrulline and Tripeptide-10 (synthetic peptide consisting of aspartic acid, isoleucine and lysine) ), Tripeptide-1), Eyeseryl™ (Ac-β-AHSH)(SEQ ID NO: 23), Serilesine™ (SIKVAV) (SEQ ID NO: 24) or Decorinyl™ (INCI name: Tripeptide-10 Citrulline = product of the reaction of Citrulline and Tripeptide-10 (synthetic peptide consisting of aspartic acid, isoleucine and lysine) sold by the company Lipotec,
• Collaxyl™ (GPQGPQ (SEQ ID NO: 25)) or Quintescine™ (CG) sold by Vincience,
• Cytokinol™LS (casein hydrolyzate) sold by Laboratoires Serobiologiques/Cognis,
• Kollaren™ (GHK), IP2000™ (Pal-VYV) or Meliprene™ (INCI name = Monofluoroheptapeptide-1: reaction product of acetic acid and a synthetic peptide containing arginine, glycine, glutamic acid, histidine, norleucine, p-fluorophenylalanine and tryptophan) sold by the European Institute of Cell Biology,
• Neutrazen™ (Pal-HFR-NH ₂ ) sold by the company Innovations, or
• BONT-L-Peptide™ (INCI name = Palmitoyl Hexapeptide-19: reaction product of palmitic acid and Hexapeptide-19 (synthetic peptide consisting of asparagine, aspartic acid, lysine and methionine), Timp-Peptide™ (INCI name = Acetyl Hexapeptide-20: product obtained by acetylation of Hexapeptide-20 (synthetic peptide consisting of alanine, glycine, lysine, valine and proline) or ECM Moduline™ (INCI name = Palmitoyl Tripeptide-28: product of the reaction of palmitic acid and Tripeptide-28 (synthetic peptide consisting of arginine, lysine and phenylalanine) sold by the company Infinitec Activos.

It is also possible to combine the plant cells according to the invention with one or more cyclic peptides, in particular those extracted from linseed oil described in the Applicant's patent application FR1850845.

More specifically, the plant cells according to the invention can also be combined with at least one of the compounds chosen from vitamin B3 compounds, compounds such as niacinamide or tocopherol, retinoid compounds such as retinol, hexamidine , α-Lipoic Acid, Resveratrol or DHEA, Hyaluronic Acid, Peptides including N-Acetyl-Tyr-Arg-O-Hexadecyl, Pal-VGVAPG (SEQ ID NO: 19), Pal -KTTKS (SEQ ID NO: 16), Pal-GHK, Pal-KMO2K, Pal-GQPR (SEQ ID NO: 10) and Pal-K(P)HG, which are classic active ingredients used in the compositions topical cosmetics or dermo-pharmaceuticals.

The present invention also provides a cosmetic or dermatological topical treatment method for improving the appearance and general condition of the skin and its appendages by preventing the glycation of macro-molecules of the skin, in particular the proteins , nucleic acids and lipids, comprising the topical application to the skin of a subject in need thereof of an effective amount of plant cells or of a composition comprising them, in a physiologically acceptable excipient.

By "topical treatment" or "topical use" is meant an application that is intended to act where it is applied: skin, mucous membrane, appendages.

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

The "effective" amount depends on various factors, such as the age, condition of the patient, severity of the disorder or condition, and mode of administration. An effective amount means a non-toxic amount sufficient to achieve the desired effect.

When the plant cells are used in liquid form, a composition forming the active ingredient comprising said plant cells preferably comprises at least 0.04% of leontopodium acids A and B based on the total weight of the ingredient. This ingredient can then be used in final cosmetic formulations between 0.1 and 10%, preferably between 1 and 5%, more preferably between 2 and 3% and generally 2% by weight of said formulation; which corresponds to contents of leontopodic acids A and B in the final cosmetic formulations comprised between 0.00004% and 0.004%, preferably comprised between 0.0004% and 0.002%, more preferably comprised between 0.0008% and 0 0.0012% and generally at least 0.0008% relative to the total weight of composition.

When the plant cells are used in solid form, the composition forming the active ingredient comprising said plant cells preferably comprises approximately 5% of leontopodic acids A and B relative to the total weight of the ingredient. This ingredient can then be used in the final cosmetic formulations in suitable proportions to obtain the preferred content of leontopodic acids A and B mentioned above.

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

By way of example, for a cosmetic treatment of the face, the European Directive on Cosmetics has set a standard application quantity of a cream of 2.72 mg/cm ² /day/person and for a body lotion of 0.5 mg/cm ² /day/person.

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

According to the invention, it is possible to propose devices with several compartments or kits intended for the implementation of the method described above, and which could comprise, by way of example, and without this being limiting, in a first compartment a composition containing active cells according to the invention and in a second compartment an excipient and/or additional active ingredient, the compositions contained in said first and second compartments being considered here as a combination composition for simultaneous use, separate or spread in time in particular in one of the treatments defined above.

The present invention will be better understood and other advantages will appear in the light of the detailed description which will follow of an example of preparation of plant cells, and of in vitro and in vivo tests carried out on these cells.

A) Example of plant cell preparation

Creation of a cell line

Selected pieces of leaves of the genus Leontopodium alpinum are taken, washed and cut into small pieces of a few mm, so as to produce from 200 to 1500 explants. After a series of decontamination and then sterilization treatments, the pieces are placed on an agar culture medium in the presence of a nutrient medium containing plant growth hormones in order to induce callogenesis (formation of a callus).

After an appropriate period of time, a clump of dedifferentiated cells or callus forms, which is transferred to a larger area and into fresh culture medium so that it can multiply. A number of subcultures (transfers to fresh culture medium) are carried out to stabilize the cell line, i.e. until it exhibits a high and constant rate of proliferation, preservation of the phenotype, a constant content of bioactive compounds of interest (primary and secondary metabolites).

The cell line is then subjected to a selection step which consists in cultivating the cells for an appropriate period, in removing the aggregates of cells formed and inoculating them in a liquid culture medium for a period allowing the multiplication of the cell line to be obtained. cellular aggregate. The best cell line will be the one making it possible to obtain as quickly as possible and in a reproducible manner a large biomass with an optimal content of selected metabolites, the best biological activity and a homogeneous phenotype.

This was also chosen for its ability to produce leontopodic acids A and B in an amount of approximately 5% by weight measured relative to the dry weight of the cells, measured by HPLC.

Industrial process for obtaining a biomass of undifferentiated or dedifferentiated cells of Leontopodium alpinum and treatment of this biomass

We will start from a cell line prepared as described above or from an existing line.

The Leontopodium alpinum line is first multiplied to obtain a sufficient quantity of dedifferentiated cell biomass in order to carry out the large-scale production step.

The following steps are implemented:

1. Inoculation of the selected line in a liquid medium and culture for a sufficient time to obtain an increase in biomass of at least 300%;
2. Optionally, transfer the suspension obtained in a) to a fresh liquid medium and culture again for a sufficient time to obtain an increase in biomass of at least 300%;
3. Optionally repeat step b);
4. Transfer of the cell suspensions obtained in stages a) to c) in a bioreactor with fresh liquid medium, and conduct of the culture under such conditions and for a sufficient time to obtain a cell biomass containing the metabolites of interest, i.e. that is to say the phenylpropanoids glycosides and leontopod acids in sufficient quantities, this step of production in a bioreactor comprising an elicitation step carried out by modifying the nutrient levels of the culture medium.

The bioreactor :

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

Cultivation conditions :

Culture medium: medium comprising mineral salts (solution of macroelements and microelements), vitamins, plant hormones and sucrose. Vegetable agar is added to the solid media.

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

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

Biomass agitation: It is important that the biomass is optimally aerated, and at the same time it is kept agitated either by internal or external means. It is necessary that the agitation, although weak, is effective, especially in the final stages, when the biomass is in large quantity. For the purposes of the present invention, suitable means of internal agitation are propellers rotating between 20 and 120 revolutions/min, preferably at 60 revolutions/min, or externally means of orbital agitation preferably rotating between 40 and 200 rpm and preferably at about 120 rpm.

Oxygenation: normally carried out using sterile air, at a flow rate of 0.5 to 4 liters per minute, preferably between 2 and 2.5 liters per minute, for a volume of 10 liters of biomass. Alternatively, gas mixtures containing 10% to 100% v/v oxygen can be used. It will be preferable to use air or oxygen diffusion means with a nozzle having a flow rate between 10 ml/min and 600 ml/min and preferably between 50 ml/min and 350 ml/min .

Treatment of the biomass obtained

Filtration to remove culture medium and recover cell biomass. This biomass can be characterized by its equivalent rate of freeze-dried cells.

High pressure homogenization of cellular biomass: allows a reduction in the size of cellular aggregates; some cells can be burst and we can then be in the presence of a mixture of whole cells and crushed cells, 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 the content of proteins, phenylpropanoids glycosides including leontopodic acids A and B.

Optional:

1) drying of the cells in particular by lyophilization or atomization to allow greater stability of the compounds of interest, to improve long-term storage without having to add preservatives.

2) extraction of the contents of the cells by crushing/lysis/bursting of the cells and separation of the liquid and solid phases (by centrifugation or filtration or other), in order to obtain a specific cell extract.

3) Purification of cell extract to increase leontopodic acid content.

B) Preparation of active ingredients for use according to the invention

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

As a preferred example, this physiologically acceptable medium is a hydrophilic matrix in which a biomass of whole cells is suspended, for example in the case of a cosmetic composition of glycerol and/or butylene glycol. Additives can also be added if necessary, such as antimicrobial agents, antioxidants, stabilizing agents, agents acting on the pH, emulsifying agents or thickening agents, in particular a thickener such as xanthan gum which will promote the keeping the cells in suspension.

An active ingredient for cosmetic use can 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 consisting of glycerol (approximately 80%) and xanthan gum (0.3% by weight), said ingredient having a final level of approximately 0.05% of leontopodic acids A and B (approximately 15% of acid A and 85% of B).

It goes without saying that according to the invention one could use plant cells comprising a different rate of leontopodic acids, in particular higher, or obtained directly thanks to the in vitro process (for example thanks to an appropriate elicitation making it possible to increase the rate ), or obtained by a phase of purification/concentration of the cells obtained (for example by a concentration step after extraction of the cell content).

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

Thus, according to another example, an active ingredient, suitable for anhydrous formulations such as compact powder formulations for make-up-care, can be formed from freeze-dried cells then ground, for example to obtain a particle size between 5 and 30 μm suitable for this type formulation, and having a higher rate of leontopodic acids (A+B), in particular from 1 to 15%, in particular from 3 to 8%, preferably from approximately 5% (always with the same proportion of approximately 15 % of acid A and 85% of B), for a more pronounced and more immediate effect, particularly localized.

Examples of cosmetic formulations using these ingredients for an anti-glycation effect according to the invention are presented below in Galenic point F).

C) Results of in vitro tests

The in vitro tests were carried out using an Ethanol/Water extract (70/30) of freeze-dried cells from the cell culture of Leontopodium alpinum (21% of freeze-dried cells obtained according to the example above in the mixture of solvents ). This solution is the stock solution and for example 0.1% of this solution in a test medium corresponds to a solution at 210ppm of cell equivalent.

Principle

This test uses a model protein, serum albumin, which serves as a target and an edible reducing sugar from fruits. The protein is progressively glycated (linked to sugar) in an irreversible way, during a heat incubation step, this modification is followed by fluorescence. The extract was brought into contact with the protein during this phase and the glycation modifications were monitored.

Results

Variation in glycation of protein albumin Glycation / UF* Variation Positive control (Aminoguanidine)
0.03% 4818.5 -61% 100 ppm cell-equivalent 3566.5 -70% 210 ppm cell-equivalent 2216.8 -81% 420 ppm cell-equivalent 1546.5 -87%

These results show the strong anti-glycant potential of the active ingredient according to the invention, which is capable of reducing the formation of AGEs and potentially controlling their effects on dull skin and protein denaturation.

D) Galenic

Different cosmetic formulations are described below. Additional active ingredients, coming if necessary in support and / or in addition to the activity of the active ingredient according to the invention based on undifferentiated or dedifferentiated cells of Leontopodium alpinum , can be added in the appropriate phase according to their hydrophobic or hydrophilic nature. These ingredients can be of any category depending on their function(s), the place of application (body, face, neck, chest, hands, etc.), the desired final effect and the targeted consumer, for example specific anti-wrinkle, moisturizing, concealer, firming, anti-glycation, volumizing, soothing, myo-relaxing, anti-redness, detoxifying, etc.

Active ingredient No. 1 used in the galenic formulations given below:

20% by weight, relative to the total weight of the composition of the ingredient, of fresh undifferentiated or dedifferentiated cells (biomass) according to the invention (corresponding to approximately 1% of dry cells), the ingredient having a final content of leontopod acids A and B of approximately 0.05% in a physiologically acceptable excipient mixture consisting of glycerol, thickener xanthan gum and citric acid to adjust the pH if necessary.

This ingredient is recommended between 0.1 and 10%, preferably between 1 and 5%, more preferably between 2 and 3% and generally 2%.

Active ingredient No. 2 used in the galenic formulations given below:

Freeze-dried then ground cells having a particle size of approximately 15 μm (± 10 μm) and having a level of leontopodic acids (A+B) of at least 3.5%, preferably of approximately 6%.

This ingredient is recommended between 0.0025 and 2%, preferably between 0.005 and 0.1%, more preferably between 0.01 and 0.05% and generally 0.03%.

1) "Cream" shape

Product % INCI name Part A _H2O Qsp100 Water Optasense G83 ^TM 0.30 Carbomer
Part B Brij S2-SS-(RB) ^TM 0.40 Steareth-2 Brij S10-SO-(RB) ^TM 1.20 Steareth-10 Crodafos CES-PA-(RB) ^TM 4.00 Cetearyl Alcohol & Dicetyl Phosphate & Ceteth-10 Phosphate Crodacol CS 90-PA-(RB) ^TM 1.50 Cetearyl Alcohol Laurocapram 2.50 Laurocapram BRB CM 56 ^TM 2.00 Cyclopentasiloxane & Cyclohexasiloxane Crodamol OSU-LQ-(RB) ^TM 7.00 Diethylhexyl Succinate Part C Glycerin 4.00 Glycerine Octanediol 0.50 Caprylyl Glycol Part D Phenoxyethanol qs Phenoxyethanol Part E Potassium sorbate qs Potassium Sorbate Part F _H2O 4.00 Water NaOH 30% 0.40 Sodium Hydroxide Part G Active ingredient No. 1 according to the invention 1.00 to 5.00 /

Protocol : Swell part A without stirring and heat it to 75°C in a water bath. Heat part B to 75°C in a bain-marie. Melt part C at 45°C. Add part D to part C, previously cooled. Pour part C+D into part A, stirring staro v= 500 rpm. Pour part B into the previous part, stirring staro v= 1000 rpm. Extemporaneously, add part E, then part F, mix well. Add part G, below 45°C, mix well.

Example of ingredient that can be added to this formulation :

• AQUALANCE ^TM : osmoprotective moisturizing active ingredient marketed by Sederma (WO2009/104118) composed of homarin and erythritol.
• CITYSTEM ^TM : active ingredient based on plant cells obtained in vitro from Marrubium vulgare with a high concentration of Forsythoside B; used against the attacks of pollution: makes the skin soft and smooth, refines the skin texture, reduces the visibility of comedones, leaving the skin radiant and purified.
• EYELISS ^TM : is an active marketed by SEDERMA (WO2003/068141), which helps to prevent and fight against the appearance of bags under the eyes. 3% of this ingredient can for example be added to phase E of the formulation.

2) Serum form "foundation base" with sun protection

Product % INCI name Part A _H2O Qsp100 Water Potassium sorbate 0.10 Potassium Sorbate Part B Glycerin 2.00 Glycerine Phenoxyethanol qs Phenoxyethanol Keltrol CG- ^SFTTM 0.60 Xanthan Gum Vivapur CS 032 ^TM 0.25 Microcrystalline Cellulose & Xanthan Gum Part C Eusolex ^4360TM 1.80 Benzophenone-3 Eusolex ^2292TM 3.50 Ethylhexyl Methoxycinnamate & BHT Span 40-PW-(MV) ^TM 1.00 Sorbitan Palmitate Span 60-PW-(MV) ^TM 1.50 Sorbitan Stearate Crodamol GTCC-LQ-(RB) ^TM 1.35 Caprylic/Capric Triglyceride Prisorin 3505 ^TM 1.35 Isostearic Acid Part D _H2O 0.20 Water Lactic acid 0.02 Lactic acid Part E Active ingredient No. 1 according to the invention 1.00 to 5.00

Protocol : Place part A under propeller stirring v=500rpm. Add part B to part A with propeller stirring v=800rpm; homogenize well. Heat part A+B to 75°C in a bain-marie. Heat part C in a water bath at 75°C, add it to part A+B with staro stirring v=1000rpm. Homogenize. Adjust the pH to 5.80±0.1 with part D, below 35°C. Add part E and mix well.

Eusolex 4360 ^TM is a UVA filter and Eusolex 2292 ^TM a UVB filter.

Examples of ingredients that can be added to this formulation :

• VENUCEANE ^TM : active ingredient marketed by Sederma (WO2002/066668) which prevents visible signs of photoaging (spots, wrinkles, dryness, etc.), protects cellular structures from damage caused by UV rays and strengthens the integrity of the skin.
• PACIFEEL ^TM : active ingredient marketed by Sederma, comprising an extract of natural origin from the Mirabilis jalapa (Night Beauty) plant which relieves feelings of discomfort (itching, tingling), reduces the redness of sensitive and reactive skin.
• PRODIZIA ™: active ingredient marketed by Sederma comprising an extract of Albizia julibrissin , which promotes the visible reduction of signs of fatigue: dark circles, puffiness, dullness and drawn features by repairing and protecting the skin from damage caused by glycation.

3) Powder form for make-up

Product % inci name Part A Talc
Kaolin
Potassium sorbate
Phenoxyethanol Qsp100
2.50
qs
qs Talc
Kaolin
Potassium Sorbate
Phenoxyethanol Part B Active ingredient No. 2 according to the invention 0.03 /

Examples of ingredients that can be added to this formulation :

• VEGESOME MOIST 24 ^TM : active ingredient marketed by Sederma specially designed for the formulation of moisturizing make-up powders; it is a powder composed of 25 μm hollow particles ( Lycopodium clavatum exines) loaded with an extract of Imperata cylindrica with moisturizing properties.

Claims (11)

Use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinum obtained by an in vitro cell culture process comprising leontopodium acid A and/or B for a non-therapeutic cosmetic treatment to prevent the glycation of macro-molecules of the skin. Use according to claim 1 for preventing glycation of proteins, nucleic acids and/or lipids. Use according to Claim 1 or 2, characterized in that the plant cells are whole and/or lysed. Use according to claim 1 or 2, characterized in that a cell extract of said plant cells is used. Use according to one of Claims 1 to 4, characterized in that the plant cells are used in dried form. Use according to Claim 5, characterized in that the dried plant cells are ground so as to form a powder. Use according to Claim 5 or 6, characterized in that the dried plant cells are included in an anhydrous cosmetic composition. Use according to one of Claims 1 to 6, characterized in that a cosmetic composition is used comprising the said plant cells incorporated into a physiologically acceptable medium. Use according to Claim 8, characterized in that the physiologically acceptable medium is a hydrophilic matrix. Use according to Claim 9, characterized in that the said plant cells are suspended and/or solubilized in the physiologically acceptable medium. Use according to one of Claims 1 to 10, characterized in that the treatment is topical.