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WO2021064173A1 - Use of leontodium alpinum plant cells for an anti-glycation anti-ageing skin treatment - Google Patents

Use of leontodium alpinum plant cells for an anti-glycation anti-ageing skin treatment Download PDF

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Publication number
WO2021064173A1
WO2021064173A1 PCT/EP2020/077659 EP2020077659W WO2021064173A1 WO 2021064173 A1 WO2021064173 A1 WO 2021064173A1 EP 2020077659 W EP2020077659 W EP 2020077659W WO 2021064173 A1 WO2021064173 A1 WO 2021064173A1
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WO
WIPO (PCT)
Prior art keywords
cells
plant cells
use according
pal
glycation
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Ceased
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PCT/EP2020/077659
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French (fr)
Inventor
Philippe Mondon
Nathalie ROUYER
Laure BERNARD
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Sederma SA
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Sederma SA
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Publication date
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Priority to CN202080069753.1A priority Critical patent/CN114502140A/en
Priority to EP20785974.5A priority patent/EP4037656A1/en
Publication of WO2021064173A1 publication Critical patent/WO2021064173A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to the use of the Leontopodium nivale subsp. alpinum Cass plant, hereinafter referred to Leontopodium alpinum, for a cosmetic treatment, in particular a topical treatment.
  • 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.
  • Leontopodium alpinum also called Edelweiss
  • This plant is an herbaceous plant of the Asteraceae family. It grows at high altitude, above 1500m, in the Pyrenees, Alps, and Himalayas mountains, in 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 flowers 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 by 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.
  • 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.
  • the environmental impact is minimal because substantially limiting water consumption, avoiding consumption of arable land, and preventing pollution of the soil.
  • biodiversity is preserved since one plant or even one seed is enough to initiate a new in vitro culture.
  • 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.
  • the culture of undifferentiated or dedifferentiated cells involves first the creation of highly proliferative cell lines in agar medium either from meristem cells that are undifferentiated cells, or from dedifferentiated cells (growing as callus after the removal of a fragment of plant, leaf, stem, root or other). Thereafter, these lines are cultured in liquid medium 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 can concern the root part ("root culture”), the aerial part ("shoot culture”) or somatic embryos ("somatic embryo”).
  • root culture the root part
  • shoot culture the aerial part
  • somatic embryo somatic embryos
  • 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 conventional means.
  • 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:
  • 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.
  • 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.
  • the present invention provides the use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinum obtained by in vitro cell culture method for a non-therapeutic cosmetic treatment for preventing or slowing down the glycation of macro-molecules in the skin, in particular proteins, nucleic acids and/or lipids.
  • the treatment according to the invention is adapted to protect the skin from damages caused by glycation.
  • the human body needs sugar to produce its energy. However, the sugar that is consumed is never used entirely by the body for this beneficial purpose. A residual part of this absorbed sugar will react non- enzymatically on the amino groups of proteins, nucleic acids or lipids to create advanced glycation products (“AGE products” for “Advanced Glycosylated End-products”) which accumulate over time in the tissues.
  • AGE products for “Advanced Glycosylated End-products”
  • AGEs are found not only in the dermis but also in the epidermis, up to the stratum corneum. Organs are more affected because their proteins have a long lifespan, such as collagen, elastin, fibronectin, and laminins. The consequence is the altering of the mechanical and elastic properties of the extracellular matrix of the dermis, which becomes less flexible, more rigid, but also more flaccid and less reactive.
  • mitochondrial proteins are glycated causing decreased efficiency in ATP production and a deficiency in energy production.
  • AGEs also modify the permeability of vessels, by glycation/alteration of their matrix proteins.
  • the micro-inflammatory reactions which then take place lead to a decrease in the flow and leakage of blood compounds outside the vessels. This leads to a build-up of fluids and wastes around them which forms dark circles and certain types of under eye bags.
  • 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 altered.
  • 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.
  • the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be used in particular:
  • the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be used after being mixed under high pressure, to homogenize the medium and break cell aggregates.
  • the undifferentiated or dedifferentiated Leontopodium alpinum cells may be used whole or lysed, or in the form of a cellular extract produced from these cells (cleared or not of the cell walls).
  • the undifferentiated or dedifferentiated cells of Leontopodium alpinum 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.
  • the biomass can be extracted with a supercritical or subcritical fluid.
  • 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.
  • the undifferentiated or dedifferentiated cells of Leontopodium alpinum 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.
  • Anhydrous formulations, of powder type for example, can also thus advantageously be produced.
  • plant cells encompasses the undifferentiated or dedifferentiated cells of Leontopodium alpinum, 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 (cleared or not of the cell walls).
  • the plant cells can be incorporated (for example suspended or solubilized) in a physiologically acceptable medium and used to prepare a cosmetic composition.
  • a cosmetic composition according to the invention comprising said plant cells suspended and/or solubilized in a hydrophilic matrix is used.
  • the cosmetic treatment according to the invention is topical.
  • the applicant has shown by in vivo tests that the cosmetic treatment of skins consisting of applying a composition comprising undifferentiated or dedifferentiated cells of Leontopodium alpinum obtained by an in vitro cell culture process induces on these skins an anti-glycation effect.
  • 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 prevent the gly cation of macro-molecules in the skin, in particular proteins, nucleic acids and lipids.
  • a physiologically acceptable medium which can be a hydrophilic matrix
  • active ingredients and cosmetic compositions are given below in the description.
  • 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.
  • the production step in a bioreactor may comprise an elicitation step, advantageously for increasing the contents of leontopodic acids A and/or B or for varying the relative proportion thereof;
  • 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 possible quantity of biomass, with a high viability; and/or
  • the biomass can be subjected to a step of high-pressure homogenization, to break the cell aggregates; and/or
  • An additional step of drying the cellular biomass can be added, for a better long-term preservation;
  • the cells may be extracted, in particular for enrichment in leontopodic acids A and B.
  • 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.
  • microbial fractions including saccharomyces yeasts
  • biological molecules such as for example chitosan, methyljasmonate, jasmonic acid, salicylic acid
  • non- biological molecules such as paclobutrazol
  • elicitation is achieved by modifying the culture medium, in particular nutrient levels.
  • a cosmetic composition especially topical, includes plant cells in a physiologically acceptable medium. According to the excipient and dosage of the 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.
  • 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, re plumping, 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.
  • UVA and/or UVB filtering agents hydrating, moisturizing, hume
  • 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.
  • 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.
  • CTFA « International Cosmetic Ingredient Dictionary & Handbook » (19th Ed. 2019) published by « the Personal Care Products council » ex- « the Cosmetic, Toiletry, and Fragrance Association, Inc.» Washington, D.C.
  • CTFA 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.
  • the following commercial actives can also be mentioned, as examples: betaine, glycerol, Actimoist Bio 2TM (Active organics), AquaCacteenTM (Mibelle AG Cosmetics), AquaphylineTM (Silab), AquaregulKTM (Solabia), CarcilineTM (Greentech), CodiavelaneTM (Biotech Marine), DermafluxTM (Arch Chemicals, Inc), Hydra'FlowTM (Sochibo), Hydromoist LTM (Symrise), RenovHyalTM (Soliance), SeamossTM (Biotech Marine), ArgirelineTM (commercial name for the acetyl hexapeptide-3 of Lipotec), spilanthol or an extract of Acmella oleracea known under the commercial name Gatuline ExpressionTM, an extract of Boswellia serrata known under the commercial name BoswellinTM, Deepaline PVBTM (Seppic), Syn-AKETM (Pentapharm), AmelioxTM, BioxiliftTM (Silab
  • 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
  • compositions of the present invention may include one or more additional peptides, including, without limitation, di-, tri-, tetra-, penta-and hexapeptides and their derivatives.
  • 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.
  • peptide refers here 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).
  • 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 Camosine (bAH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM or PP.
  • Suitable tripeptides for use herein include, but are not limited to RKR, HGG, GKH, GHK, GGH, GHG, KGH, KHG, KFK, KAvaK, KbAK, KAbuK, KAcaK, KPK, KMOK, KM0 2 K (M0 2 being a di- oxygenated sulfoxide methionine), KVK, PPL, PPR, SPR, QPA, LPA, SPA, K(Ac)HG or K(Ac)GH, K(Ac) being a lysine with the amine function of the lateral chain acetylated, as disclosed in WO2017/216177, K(P)HG or K(P)GH, K(P) being a lysine with its lateral chain grafted with a proline, K(Pyr)HG or K(Pyr)GH, K(Pyr) being a lysine with its lateral chain grafted with a py
  • Suitable tetrapeptides for use as additional peptides herein include but are not limited to RSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2), KTFK (SEQ ID NO: 3), KTAK (SEQ ID NO: 4), KAYK (SEQ ID NO: 5) or KFYK (SEQ ID NO: 6).
  • pentapeptide is the KTTKS (SEQ ID NO: 7) and the KTSKS (SEQ ID NO: 8), and a suitable examples of hexapeptides are the GKTTKS (SEQ ID NO: 9), GKTSKS (SEQ ID NO: 10) and VGVAPG (SEQ ID NO: 11).
  • Suitable peptides for use according to the present invention can be selected, this list being not limitative, from: lipophilic derivatives of peptides, preferably palmitoyl (Pal) derivatives or myristoyl (Myr), and metal complexes as aforementioned (e.g. copper complex of the tripeptide HGG or GHK).
  • Preferred dipeptides include for example N-Palmitoyl- -Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (CalmosensineTM, IdealiftTM from Sederma), Pal-RT or Pal-KT (from Sederma).
  • Preferred tripeptide derivatives include for example Pal-GKH and Pal -GHK (from Sederma), the copper derivative of HGG (LaminTM from Sigma), Lipospondin (N-Elaidoyl-KFK) and its analogs of conservative substitution, N- Acetyl-RKR-NH2 (Peptide CK+), N-Biot-GHK (from Sederma), Pal-KAvaK, Pal-K AlaK, Pal- KAbuK, Pal-KAcaK, or Pal-KMCEK (Matrixyl®synthe’6® from Sederma), Pal-KVK (Syn-CollTM of DSM), and derivatives thereof.
  • R 1 and R 2 being, independently of one another, chosen from a alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfurized, said group possibly possessing in its backbone a heteroatom particularly O, S and/or or N, and Pro* corresponding to Proline, an analogue or derivative thereof; comprising, for example, Myr-PPL-OH and Myr-PPR-OH.
  • Suitable tetrapeptide derivatives for use as additional peptides according to the present invention include, but are not limited to, Pal-GQPR (SEQ ID NO: 12) (from Sederma), Pal-KTFK (SEQ ID NO: 13) or Ela-KTFK (SEQ ID NO: 14), Ela-KTAK (SEQ ID NO: 15), Ela-KAYK (SEQ ID NO: 16) or Ela- KFYK (SEQ ID NO: 17).
  • Suitable pentapeptide derivatives for use as additional peptides herein include, but are not limited to, Pal-KTTKS (SEQ ID NO: 18) (available as Matrixyl® from Sederma), Pal- KTSKS (SEQ ID NO: 19), Pal-YGGFXaa (SEQ ID NO: 20) with Xaa being Leu or Pro, or mixtures thereof.
  • Suitable hexapeptide derivatives for use herein include, but are not limited to, Pal-VGVAPG (SEQ ID NO: 21), Pal-GKTTKS (SEQ ID NO: 22), Pal-GKTSKS (SEQ ID NO: 23), Pal-HLDIIXaa with Xaa being Trp, Phe, Tyr, Tic, 7-hydroxy-Tic ou Tpi (SEQ ID NO: 24) and derivatives thereof.
  • the mixture of Pal-GHK and Pal-GQPR SEQ ID NO: 12) (Matrixyl® 3000, Sederma) can also be mentioned.
  • CollaxylTM Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO 29)
  • QuintescineTM Cys-Gly marketed by Vincience
  • 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: 21), Pal- KTTKS (SEQ ID NO: 18), Pal-KTSKS (SEQ ID NO: 19), Pal-GHK, Pal-KM0 2 K and Pal-GQPR (SEQ ID NO: 12), and Pal-K(P)HG (with a proline grafted on the lysine), which are widely used active ingredients in topical cosmetic or dermopharmaceutical compositions.
  • compounds of the vitamin B3 compounds such as niacinamide or tocopherol, retinoid compounds
  • 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, by preventing the glycation of macro-molecules in the skin, in particular proteins, nucleic acids and lipids, 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.
  • 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 non toxic amount enough to achieve the desired effect.
  • a composition forming the active ingredient comprising said plant cells preferably comprises at least 0.04% of leontopodic 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 of 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% relative to the total weight of the composition.
  • the composition forming the active ingredient comprising said plant cells preferably comprises about 5% leontopodic acids A and B based on the total weight of the ingredient. This ingredient can then be used in the final cosmetic formulations in proportions suitable for obtaining the preferred content of leontopodic acids A and B mentioned above.
  • the European Cosmetics Directive has set a standard amount for applying a cream of 2.72mg/cm 2 /day/person and for a body lotion of 0.5mg/cm 2 /day/person.
  • 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.
  • 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.
  • Selected pieces of leaves of the Leontopodium alpinum genus are collected, washed, and cut into small pieces of several mm, 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).
  • 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 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.
  • 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.
  • step 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 The bioreactor:
  • 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.
  • 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.
  • 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.
  • This biomass may be characterized by its equivalent content of lyophibzed 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.
  • the biomass of cells can be mixed with a physiologically acceptable medium forming the excipient.
  • 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).
  • 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 content), or obtained through a purification/concentration step of the obtained cells (e.g. by a concentration step after extraction of the cellular content).
  • an active ingredient suitable for anhydrous formulations such as compact powder formulations for makeup-skincare, can be formed from lyophilized cells then grounded, for example to obtain a particle size of between 5 and 30 pm suitable for this type of formulation, and having a higher leontopodic acid content (A+B), in particular from 1 to 15%, in particular from 3 to 8%, preferably around 5% (always with the same proportion of around 15% of acid A and 85% of B), for a more pronounced and more immediate effect, especially localized.
  • A+B leontopodic acid content
  • This test uses a model protein, the serum albumin, which serves as a target, and an edible reducing sugar from fruits.
  • the protein is gradually glycated (bound to sugar) in an irreversible manner, during a heat incubation step, this change is followed by fluorescence.
  • the extract is contacted with the protein during this phase and the glycation changes are monitored.
  • 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 N°1 used in the galenic formulations given below.
  • the ingredient composition 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%.
  • Lyophilized and then grounded cells having a particle size of approximately 15 pm ( ⁇ 10 pm) and having a leontopodic acid content (A ⁇ B) of at least 3.5%, preferably of around 6%.
  • This ingredient is recommended between 0.025 and 2%, preferably between 0.005 and 0.1%, more preferably between 0.01 and 0.05% and generally 0.03%.
  • Protocol weigh part A and let swell for 30 minutes without stirring. Heat part A at 75°C in a water bath.
  • AQUALANCETM osmoprotective active moisturizing ingredient marketed by Sederma (W02009/104118) composed of homarin and erythritol.
  • CITYSTEMTM active ingredient based on plant cells obtained in vitro from Marrubium vulgare with a high concentration of Forsythoside B; used against pollution attacks: makes the skin soft and smooth, refines the skin texture, reduces the visibility of comedones, leaving the skin radiant and purified.
  • EYELISSTM is an active marketed by SEDERMA (W02003/068141), which helps prevent and fight against the appearance of bags under the eyes. 3% of this ingredient can for example be added to part E of the formulation.
  • Eusolex 4360TM is a UVA filter and Eusolex 2292TM a UVB filter.
  • VENUCEANETM active ingredient marketed by Sederma (W02002/066668) which prevents visible signs of photoaging (spots, wrinkles, dryness, etc.), protects cell structures from UV damages and strengthens skin integrity.
  • PACIFEELTM active ingredient marketed by Sederma, comprising an extract of natural origin from the plant Mirabilis jalapa ( « Belle de touch ») which relieves feelings of discomfort (itching, tingling), reduces redness in sensitive and reactive skins.
  • PRODIZIATM 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.
  • VEGESOME MOIST 24TM active ingredient marketed by Sederma specially designed for the formulation of moisturizing makeup powders; it is a powder composed of 25 pm hollow particles (exins of Lycopodium clavatum) loaded with an Imperata cylindrica extract having moisturizing properties.

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Abstract

According to the invention, there is proposed the use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinum obtained by in vitro cell culture, 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 ANTI-GLYCATION ANTI-AGEING SKIN
TREATMENT
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, in particular a topical treatment. 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 alpinum, also called Edelweiss, is an herbaceous plant of the Asteraceae family. It grows at high altitude, above 1500m, in the Pyrenees, Alps, and Himalayas mountains, in 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 flowers 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 by 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 the creation of highly proliferative cell lines in agar medium either from meristem cells that are undifferentiated cells, or from dedifferentiated cells (growing as callus after the removal of a fragment of plant, leaf, stem, root or other). Thereafter, these lines are cultured in liquid medium 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 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 alpinum obtained by in vitro cell culture method for a non-therapeutic cosmetic treatment for preventing or slowing down the glycation of macro-molecules in the skin, in particular proteins, nucleic acids and/or lipids.
The treatment according to the invention is adapted to protect the skin from damages caused by glycation.
The human body needs sugar to produce its energy. However, the sugar that is consumed is never used entirely by the body for this beneficial purpose. A residual part of this absorbed sugar will react non- enzymatically on the amino groups of proteins, nucleic acids or lipids to create advanced glycation products (“AGE products” for “Advanced Glycosylated End-products”) which accumulate over time in the tissues.
The formation of many glycated proteins whose functional, enzymatic, and structural properties are altered, has consequences on 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 more affected because their proteins have a long lifespan, such as collagen, elastin, fibronectin, and laminins. The consequence is the altering of the mechanical and elastic properties of the extracellular matrix of the dermis, which becomes less flexible, more rigid, but also more flaccid and less reactive.
Within skin cells, mitochondrial proteins are glycated causing decreased efficiency in ATP production and a deficiency in energy production.
AGEs also modify the permeability of vessels, by glycation/alteration of their matrix proteins. The micro-inflammatory reactions which then take place lead to a decrease in the flow and leakage of blood compounds outside the vessels. This leads to a build-up of fluids and wastes around them which forms dark circles and certain types of under eye bags.
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 altered.
The impact on the skin of these biochemical processes, whose effects have just been described at the cellular level, is therefore significant. Glycation results in dull, flaccid, without radiance, tired-looking skin, for example with dark circles and bags under the eyes, drawn features, and skin lacking in 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 a loss of flexibility of the skin; and/or
- to preserve the radiance of the complexion of the skin and its appendages; and/or
- to prevent the formation of dark circles and under eyes bags.
According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum can be used after being mixed under high pressure, to homogenize the medium and break cell aggregates. According to the invention, the undifferentiated or dedifferentiated Leontopodium alpinum cells may be used whole or lysed, or in the form of a cellular extract produced from these cells (cleared or not of the cell walls).
According to the invention, the undifferentiated or dedifferentiated cells of Leontopodium alpinum 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 alpinum 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. Anhydrous formulations, of powder type for example, can also thus advantageously be produced.
In the following description, "plant cells" encompasses the undifferentiated or dedifferentiated cells of Leontopodium alpinum, 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 (cleared or not of the cell walls).
According to the invention, the plant cells can be incorporated (for example suspended or solubilized) in a physiologically acceptable medium and used to prepare a cosmetic composition.
They can also be used as they are, optionally crushed to define and homogenize their particle size, in a dry powder formula, for example for make-up care. The dried plant cells are then included in an anhydrous cosmetic composition. According to another embodiment, a cosmetic composition according to the invention comprising said plant cells suspended and/or solubilized in a hydrophilic matrix is used.
More preferably, the cosmetic treatment according to the invention is topical.
Surprisingly, the applicant has shown by in vivo tests that the cosmetic treatment of skins consisting of applying a composition comprising undifferentiated or dedifferentiated cells of Leontopodium alpinum obtained by an in vitro cell culture process induces on these skins an anti-glycation effect.
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 prevent the gly cation of macro-molecules in the skin, in particular proteins, nucleic acids and lipids. Examples of active ingredients and cosmetic compositions are given below 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 classes of compounds in the plant cells used according to the invention.
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) Produce a biomass of said dedifferentiated or undifferentiated cells in a bioreactor from said pre biomass and a suitable culture medium; and
3) Separate said biomass enriched in leontopodic acids from said culture medium and thus recover said dedifferentiated or undifferentiated cells.
According to optional features:
1) The production step in a bioreactor may comprise an elicitation step, advantageously for increasing the contents of leontopodic acids A and/or B or for varying the relative proportion thereof; 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 possible quantity of biomass, with a high viability; and/or
3) The biomass can be subjected to a step of high-pressure homogenization, 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 of the 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, re plumping, 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 for example, 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 » (19th Ed. 2019) 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™, Citystem™, Neonyca™, NG Insaponifiables de Beurre de Karite™, Majestem™, Hydronesis™, Poretect™, Crystalide™, Amberstem™, Synchrolife™, Feminage™, 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 Law sonia 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 Ilex 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 Mirabilis jalapa, of Apium graveolens, of Marrubium vulgare, Buddleja davidii Franch, Engelhardia chrysolepis, Syringa vulgaris or orchids.
The compositions of the present invention may include one or more additional 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 lxl07% and 20%, preferably from lxl06% and 10%, preferably between lxl05% and 5% by weight. The term “peptide” refers here 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 Camosine (bAH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM or PP.
Suitable tripeptides for use herein include, but are not limited to RKR, HGG, GKH, GHK, GGH, GHG, KGH, KHG, KFK, KAvaK, KbAK, KAbuK, KAcaK, KPK, KMOK, KM02K (M02 being a di- oxygenated sulfoxide methionine), KVK, PPL, PPR, SPR, QPA, LPA, SPA, K(Ac)HG or K(Ac)GH, K(Ac) being a lysine with the amine function of the lateral chain acetylated, as disclosed in WO2017/216177, K(P)HG or K(P)GH, K(P) being a lysine with its lateral chain grafted with a proline, K(Pyr)HG or K(Pyr)GH, K(Pyr) being a lysine with its lateral chain grafted with a pyroglutamic acid, K(Hyp)HG or K(Hyp)GH, K(Hyp) being a lysine with its lateral chain grafted with a hydroxyproline, as disclosed in WO2016/097965.
Suitable tetrapeptides for use as additional peptides herein include but are not limited to RSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2), KTFK (SEQ ID NO: 3), KTAK (SEQ ID NO: 4), KAYK (SEQ ID NO: 5) or KFYK (SEQ ID NO: 6).
A suitable non limitative example of pentapeptide is the KTTKS (SEQ ID NO: 7) and the KTSKS (SEQ ID NO: 8), and a suitable examples of hexapeptides are the GKTTKS (SEQ ID NO: 9), GKTSKS (SEQ ID NO: 10) and VGVAPG (SEQ ID NO: 11).
Other suitable peptides for use according to the present invention can be selected, this list being not limitative, from: lipophilic derivatives of peptides, preferably palmitoyl (Pal) derivatives or myristoyl (Myr), and metal complexes as aforementioned (e.g. copper complex of the tripeptide HGG or GHK). Preferred dipeptides include for example N-Palmitoyl- -Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (Calmosensine™, Idealift™ from Sederma), Pal-RT or Pal-KT (from Sederma). Preferred tripeptide derivatives include for example 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-KAvaK, Pal-K AlaK, Pal- KAbuK, Pal-KAcaK, or Pal-KMCEK (Matrixyl®synthe’6® from Sederma), Pal-KVK (Syn-Coll™ of DSM), and derivatives thereof.
Mention may also be made here of the anti -aging tripeptides of general Formula X-Pro*-Pro*-Xaa-Y described in WO2015181688 application with Xaa selected from Leu, Arg, Lys, Ala, Ser, and Asp, at the N-terminus , X chosen from H, -CO-R1 and -SO2-R1 and at the C-terminal end Y chosen from OH, OR1, NH2, NHR1 or NR1 R2. R1 and R2 being, independently of one another, chosen from a alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy group, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfurized, said group possibly possessing in its backbone a heteroatom particularly O, S and/or or N, and Pro* corresponding to Proline, an analogue or derivative thereof; comprising, for example, Myr-PPL-OH and Myr-PPR-OH. Here can further be cited also the propigmenting and/or pro-mec dipeptides and tripeptides of general Formula X-(Xaai)n-Pro*-Xaa2-Y disclosed in WO2014/080376, with n=0, 1 or 2, Xaai an hydrophobic aminoacid selected from Ala, Val, Met, Leu, Iso, Phe, Pro, and analogs and derivatives thereof; or a polar aminoacid selected from Ser, Thr, Tyr, Asp, Glu and analogs and derivatives thereof; and when n=2 the two aminoacids Xaai being the same or different; Xaa2 being an hydrophobic aminoacid selected from Ala, Val, Met, Leu, Iso, Phe, and analogs and derivatives thereof, or a basic aminoacid selected from Arg, Lys, His, and analogs and derivatives thereof; at the N terminal end X being selected from H, -CO-Ri and -SO2-R1; at the C terminal end Y being selected from OH, ORi, N¾, NHRi or NR1R2; Ri and R2 being, independently from each other, selected from an alkyl, aryl, aralkyl, alkylaryl, alkoxy et aryloxy group, that can be linear, branched, cyclic polycyclic, saturated, unsaturated, hydroxylated, carbonylated, phosphorylated and/or sulfured, said group having or not an O, S and/or N heteroatom in its skeleton and Pro* corresponding to a Proline, analog or derivative thereof; comprising for example the following peptides Pal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPAOH, Myr-SPA-OH, Pal-PM- OH, Pal-PA-OH and Pal-PP-OH.
Suitable tetrapeptide derivatives for use as additional peptides according to the present invention include, but are not limited to, Pal-GQPR (SEQ ID NO: 12) (from Sederma), Pal-KTFK (SEQ ID NO: 13) or Ela-KTFK (SEQ ID NO: 14), Ela-KTAK (SEQ ID NO: 15), Ela-KAYK (SEQ ID NO: 16) or Ela- KFYK (SEQ ID NO: 17). Suitable pentapeptide derivatives for use as additional peptides herein include, but are not limited to, Pal-KTTKS (SEQ ID NO: 18) (available as Matrixyl® from Sederma), Pal- KTSKS (SEQ ID NO: 19), Pal-YGGFXaa (SEQ ID NO: 20) with Xaa being Leu or Pro, or mixtures thereof.
Suitable hexapeptide derivatives for use herein include, but are not limited to, Pal-VGVAPG (SEQ ID NO: 21), Pal-GKTTKS (SEQ ID NO: 22), Pal-GKTSKS (SEQ ID NO: 23), Pal-HLDIIXaa with Xaa being Trp, Phe, Tyr, Tic, 7-hydroxy-Tic ou Tpi (SEQ ID NO: 24) and derivatives thereof. The mixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 12) (Matrixyl® 3000, Sederma) can also be mentioned.
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™ (b-Ala-Pro-Dab-NH-Bzl) or Syn-Coll™ (Pal-Lys-Val-Lys-OH) marketed by Pentapharm;
Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NEE (INCI name = Acetyl hexapeptide-3) (SEQ ID NO: 25), Leuphasyl™ (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO: 26), 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: 27), Serilesine™ (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO 28) 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 29)) 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 Tlnstitut Europeen de Biologie Cellulaire;
Neutrazen™ (Pal-His-D-Phe-Arg-FMT) 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 lnfmitec Activos.
It is also possible to envisage combining 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 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: 21), Pal- KTTKS (SEQ ID NO: 18), Pal-KTSKS (SEQ ID NO: 19), Pal-GHK, Pal-KM02K and Pal-GQPR (SEQ ID NO: 12), and Pal-K(P)HG (with a proline grafted on the lysine), 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, by preventing the glycation of macro-molecules in the skin, in particular proteins, nucleic acids and lipids, 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 non toxic amount enough 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 leontopodic 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 of 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% relative to the total weight of the composition. When the plant cells are used in solid form, the composition forming the active ingredient comprising said plant cells preferably comprises about 5% leontopodic acids A and B based on the total weight of the ingredient. This ingredient can then be used in the final cosmetic formulations in proportions suitable for obtaining 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 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. 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, 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, 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 Leontoyodium 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 lyophibzed 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 active ingredients 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).
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 content), 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 content of leontopodic acids, for example a content of 25% relative to the dry matter, said cells being themselves used to make an active ingredient as described above. Thus, according to another example, an active ingredient, suitable for anhydrous formulations such as compact powder formulations for makeup-skincare, can be formed from lyophilized cells then grounded, for example to obtain a particle size of between 5 and 30 pm suitable for this type of formulation, and having a higher leontopodic acid content (A+B), in particular from 1 to 15%, in particular from 3 to 8%, preferably around 5% (always with the same proportion of around 15% of acid A and 85% of B), for a more pronounced and more immediate effect, especially localized.
Examples of cosmetic formulations using these ingredients for an anti-glycation effect according to the invention are presented below under Galenic point F).
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 alpinum (21% of freeze-dried cells obtained according to the above example in the solvent mixture). This solution is the stock solution and for example 0.1% of this solution into a test medium corresponds to a solution at 210ppm cell equivalent.
Principle
This test uses a model protein, the serum albumin, which serves as a target, and an edible reducing sugar from fruits. The protein is gradually glycated (bound to sugar) in an irreversible manner, during a heat incubation step, this change is followed by fluorescence. The extract is contacted with the protein during this phase and the glycation changes are monitored.
Results [Table 1]
Figure imgf000016_0001
* FU: fluorescence unit
These results show the strong anti-glycant potential of the active agent according to the invention which can reduce the formation of AGEs and potentially can control their effects on dull skin and protein denaturation.
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 N°1 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%. Active ingredient N°2 used in the galenic formulations given below.
Lyophilized and then grounded cells having a particle size of approximately 15 pm (±10 pm) and having a leontopodic acid content (A±B) of at least 3.5%, preferably of around 6%.
This ingredient is recommended between 0.025 and 2%, preferably between 0.005 and 0.1%, more preferably between 0.01 and 0.05% and generally 0.03%. 1) Cream form
[Table 2]
Figure imgf000017_0001
Figure imgf000018_0001
Protocol: weigh part A and let swell for 30 minutes without stirring. Heat part A at 75°C in a water bath.
Weigh part B and heat to 75°C in a water bath. Mix well. Weigh and melt part C at 45°C. Add part D to part C, cooled beforehand. Pour part C+D in part A, under Staro stirring v=500rpm. Mix well. Pour part B in the previous part under Staro stirring v=1000rpm. Mix well. Extemporaneously, add part E, homogenize. Add part F, homogenize. Add part G below 45 °C, homogenize, 1 hour.
Examples of ingredients which may be added to this formulation:
• AQUALANCE™: osmoprotective active moisturizing ingredient marketed by Sederma (W02009/104118) composed of homarin and erythritol.
• CITYSTEM™: active ingredient based on plant cells obtained in vitro from Marrubium vulgare with a high concentration of Forsythoside B; used against pollution attacks: makes the skin soft and smooth, refines the skin texture, reduces the visibility of comedones, leaving the skin radiant and purified.
• EYELISS™: is an active marketed by SEDERMA (W02003/068141), which helps prevent and fight against the appearance of bags under the eyes. 3% of this ingredient can for example be added to part E of the formulation.
2) Serum form, foundation with sun protection
[Table 3]
Figure imgf000018_0002
Figure imgf000019_0001
Protocol: Put 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 water bath. Heat part C in a water bath at 75°C, add it to part A+B with stirring staro 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™ is a UVA filter and Eusolex 2292™ a UVB filter.
Examples of ingredients which may be added to this formulation:
• VENUCEANE™: active ingredient marketed by Sederma (W02002/066668) which prevents visible signs of photoaging (spots, wrinkles, dryness, etc.), protects cell structures from UV damages and strengthens skin integrity. · PACIFEEL™: active ingredient marketed by Sederma, comprising an extract of natural origin from the plant Mirabilis jalapa (« Belle de nuit ») which relieves feelings of discomfort (itching, tingling), reduces redness in sensitive and reactive skins.
• PRODIZIA™: 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.
3) Powder form for makeup
[Table 4]
Figure imgf000019_0002
Examples of ingredients which may be added to this formulation:
• VEGESOME MOIST 24™: active ingredient marketed by Sederma specially designed for the formulation of moisturizing makeup powders; it is a powder composed of 25 pm hollow particles (exins of Lycopodium clavatum) loaded with an Imperata cylindrica extract having moisturizing properties.

Claims

1. Use of undifferentiated or dedifferentiated plant cells of Leontopodium alpinum obtained by in vitro cell culture method for a non-therapeutic cosmetic treatment to prevent glycation of macro molecules in the skin.
2. Use according to claim 1 for preventing glycation of proteins, nucleic acids and/or lipids.
3. Use according to claim 1 or 2, wherein the plant cells are whole and/or lysed.
4. Use according to claim 1 or 2, wherein a cellular extract of said plant cells is used.
5. Use according to anyone of claims 1 to 4, wherein plant cells in a dried form are used.
6. Use according to claim 5, wherein the dried plant cells are grounded to form a powder.
7. Use according to claim 5 or 6, wherein the dried plant cells are included in an anhydrous cosmetic composition.
8. Use according to anyone of claims 1 to 6, wherein a cosmetic composition comprising said plant cells incorporated into a physiologically acceptable medium is used.
9. Use according to claim 8, wherein the physiologically acceptable medium is a hydrophilic matrix.
10. Use according to claim 9, wherein said plant cells are suspended and/or dissolved in the physiologically acceptable medium.
11. Use according to anyone of claims 1 to 10, wherein the treatment is topical.
PCT/EP2020/077659 2019-10-04 2020-10-02 Use of leontodium alpinum plant cells for an anti-glycation anti-ageing skin treatment Ceased WO2021064173A1 (en)

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047538A1 (en) * 1999-12-23 2001-07-05 L'oreal Genus leontopodium plant extract and compositions containing same
WO2002066668A2 (en) 2001-02-21 2002-08-29 Sederma Method for producing proteins by fermentation of microorganisms from the thermus family, the protein mixture thus obtained and cosmetic compositions containing same
WO2003068141A2 (en) 2002-02-15 2003-08-21 Sederma Cosmetic or dermopharmaceutical compositions which are used to reduce bags and circles under the eyes
WO2009104118A1 (en) 2008-02-22 2009-08-27 Sederma Moisturizing cosmetic composition comprising a combination of homarine and erythritol
EP2319914A1 (en) 2009-11-09 2011-05-11 I.R.B. Istituto Di Ricerche Biotecnologiche S.r.l. Preparation and use of plant meristem cells with a high content of caffeic acid derivatives
WO2013046137A2 (en) 2011-09-27 2013-04-04 Sederma New cosmetic use of an albizia julibrissin extract and corresponding topical composition
EP2623094A1 (en) * 2012-02-02 2013-08-07 DSM IP Assets B.V. Use of an edelweiss extract
WO2014080376A2 (en) 2012-11-26 2014-05-30 Sederma Pro-pigmenting peptides
WO2015181688A1 (en) 2014-05-22 2015-12-03 Sederma Peptides, compositions comprising them and uses in particular cosmetic uses
KR20160043551A (en) * 2014-10-13 2016-04-22 주식회사 바이오에프디엔씨 Anti-inflammation and Anti-aging composition for skin external application comprising Leontopodium alpinum Cell Culture Extract and Methods for preparing the Same
WO2016097965A1 (en) 2014-12-16 2016-06-23 Sederma Peptidic compounds, compositions comprising them and uses of said compounds, in particular cosmetic uses
FR3031454A1 (en) * 2015-01-13 2016-07-15 Sederma Sa USE OF LEONTOPODIUM ALPINUM VEGETABLE CELLS FOR COSMETIC TREATMENT AND CORRESPONDING ACTIVE COSMETIC INGREDIENT
WO2017216177A1 (en) 2016-06-14 2017-12-21 Sederma Peptide, composition comprising said peptide and uses thereof, in particular cosmetic uses

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047538A1 (en) * 1999-12-23 2001-07-05 L'oreal Genus leontopodium plant extract and compositions containing same
WO2002066668A2 (en) 2001-02-21 2002-08-29 Sederma Method for producing proteins by fermentation of microorganisms from the thermus family, the protein mixture thus obtained and cosmetic compositions containing same
WO2003068141A2 (en) 2002-02-15 2003-08-21 Sederma Cosmetic or dermopharmaceutical compositions which are used to reduce bags and circles under the eyes
WO2009104118A1 (en) 2008-02-22 2009-08-27 Sederma Moisturizing cosmetic composition comprising a combination of homarine and erythritol
EP2319914A1 (en) 2009-11-09 2011-05-11 I.R.B. Istituto Di Ricerche Biotecnologiche S.r.l. Preparation and use of plant meristem cells with a high content of caffeic acid derivatives
WO2013046137A2 (en) 2011-09-27 2013-04-04 Sederma New cosmetic use of an albizia julibrissin extract and corresponding topical composition
EP2623094A1 (en) * 2012-02-02 2013-08-07 DSM IP Assets B.V. Use of an edelweiss extract
WO2014080376A2 (en) 2012-11-26 2014-05-30 Sederma Pro-pigmenting peptides
WO2015181688A1 (en) 2014-05-22 2015-12-03 Sederma Peptides, compositions comprising them and uses in particular cosmetic uses
KR20160043551A (en) * 2014-10-13 2016-04-22 주식회사 바이오에프디엔씨 Anti-inflammation and Anti-aging composition for skin external application comprising Leontopodium alpinum Cell Culture Extract and Methods for preparing the Same
WO2016097965A1 (en) 2014-12-16 2016-06-23 Sederma Peptidic compounds, compositions comprising them and uses of said compounds, in particular cosmetic uses
FR3031454A1 (en) * 2015-01-13 2016-07-15 Sederma Sa USE OF LEONTOPODIUM ALPINUM VEGETABLE CELLS FOR COSMETIC TREATMENT AND CORRESPONDING ACTIVE COSMETIC INGREDIENT
WO2017216177A1 (en) 2016-06-14 2017-12-21 Sederma Peptide, composition comprising said peptide and uses thereof, in particular cosmetic uses

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"International Cosmetic Ingredient Dictionary & Handbook", 2019, THE PERSONAL CARE PRODUCTS COUNCIL
ANONYMOUS: "DSM ALPAFLOR RANGE", DSM, 1 January 2013 (2013-01-01), XP055359178, Retrieved from the Internet <URL:http://www.sellcare.ch/download.php?file_id=273&download=true> [retrieved on 20170327] *
ANONYMOUS: "Edelweiss stem cell mask", GNPD, 31 December 2013 (2013-12-31), XP002744324 *
KEISS H-P ET AL: "A LIGNAN FROM LEONTOPODIUM ALPINUM INHIBITS THE TNF-ALPHA-INDUCED EXPRESSION OF ICAM-1 AND E-SELECTIN IN A NF-KAPPAB INDEPENDENT MANNER", NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY, SPRINGER, DE, vol. 367, no. SUPPL. 01, 1 March 2003 (2003-03-01), pages R16, XP009051975, ISSN: 0028-1298 *

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