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EP2983464A1 - Procédé de production de cellules méristématiques de plantago lanceolata, composition comprenant lesdites cellules ou leur extrait cellulaire et ses utilisations cosmétiques, nutraceutiques et dermatologiques - Google Patents

Procédé de production de cellules méristématiques de plantago lanceolata, composition comprenant lesdites cellules ou leur extrait cellulaire et ses utilisations cosmétiques, nutraceutiques et dermatologiques

Info

Publication number
EP2983464A1
EP2983464A1 EP14719110.0A EP14719110A EP2983464A1 EP 2983464 A1 EP2983464 A1 EP 2983464A1 EP 14719110 A EP14719110 A EP 14719110A EP 2983464 A1 EP2983464 A1 EP 2983464A1
Authority
EP
European Patent Office
Prior art keywords
cells
phase
skin
cellular extract
biomass
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP14719110.0A
Other languages
German (de)
English (en)
Inventor
Giovanna Pressi
Roberto Dal Toso
Philippe Mondon
Emmanuel DORIDOT
Caroline RINGENBACH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sederma SA
Croda Italiana SpA
Original Assignee
Sederma SA
IRB Istituto di Ricerche Biotecnologiche SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from IT000529A external-priority patent/ITMI20130529A1/it
Priority claimed from FR1353103A external-priority patent/FR3004193B1/fr
Application filed by Sederma SA, IRB Istituto di Ricerche Biotecnologiche SpA filed Critical Sederma SA
Publication of EP2983464A1 publication Critical patent/EP2983464A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/68—Plantaginaceae (Plantain Family)
    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • 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 is directed to meristematic cells from a new cell line of Plantago lanceolata, cells thus obtained, an extract of said cells, compositions comprising them and uses in the fields of cosmetics, nutraceutics and dermatology.
  • the present invention is directed to the cosmetics and personal care product industry applied to skin and appendages (such as body hair, eyelashes, eyebrows, nails, hair) of mammals, human or animal.
  • skin and appendages such as body hair, eyelashes, eyebrows, nails, hair
  • Plantago lanceolata is an herbaceous perennial plant of the Plantaginaceae family that grows in all temperate climates of Europe and north and central Asia. It is also called plantain, ribwort plantain, ribgrass, ripple-grass, English plantain, buckhorn plantain, narrowleaf plantain, lanceleaf plantain, ribleaf and lamb's tongue.
  • the extracts can be obtained by usual extraction methods directly from plant parts or by in vitro methods of plant culture of meristematic cells (or undifferentiated cells).
  • In vitro methods have advantages including to overcome the fluctuations related to culture conditions and to reproducibly stimulate production by plant cells of secondary metabolites of particular interests, which otherwise have low yields, as it is the case for phenylpropanoid glycosides for example.
  • Profile and quantities of bioactive compounds present in the cells obtained by in vitro methods will be different from those obtained by other extraction techniques, the dedifferentiated cells being not capable of producing all secondary metabolites and the cultures being advantageously oriented towards the production of selected secondary metabolites.
  • a cell extract or meristematic cell culture will therefore not necessarily have at the end the same activities that another type of extract, in particular compared to a plant extract not realized by cell culture.
  • a first step where appropriate, the establishment of cell lines from callus (cluster of undifferentiated cells) obtained on cuts of plant parts (leaf, root, stem...); - Selection of a cell line capable of large-scale producing a biomass of meristematic cells according to pre-established criteria (constant phenotype and optimal and consistent production of selected metabolites, ability to proliferate);
  • Patent application EP2319914 discloses this technique for obtaining meristematic cells with a high yield in caffeic acid derivatives for a theoretical list of 33 plant species including Plantago lanceolata.
  • the referred caffeic acid derivatives include phenylpropanoid glycosides and caffeoylquinic acids.
  • Phenylpropanoid glycoside properties have been widely disclosed, mainly antioxidant and antiinflammatory properties that can be efficiently be efficiently handledd in the areas of cosmetics, dermatology and nutraceuticals.
  • the aim of the present invention is to propose a vegetable biological material from a plant of the Plantago lanceolata specie for industrially obtaining, that is to say in a reproducible manner, by an in vitro cell culture a composition having remarkable activities, in particular in relation to the presence of phenylpropanoid glycosides.
  • the present invention proposes a method of in vitro producing of Plantago lanceolata meristematic cells comprising phenylpropanoid glycosides from a stabilized cell line, characterized in that the cell line is the selected IRB PL3 cell line.
  • Meristematic cells thus obtained are rich in phenylpropanoid glycosides, mainly in plantamajoside.
  • the proportion by weight of total glycosides phenylpropanoid in the lyophilized biomass, evaluated by UV spectrometry at 330 nm, expressed as plantamajoside equivalent, is about 10%.
  • Proteins, amino acids, lipids, and polysaccharides have also been identified as classes of compounds in the cells of the invention.
  • the selection and use of the IRB PL3 Plantago lanceolata cell line provides meristematic cells producing advantageously unexpected and remarkable results at all levels of skin: dermis, dermal/epidermal junction (DEJ) and epidermis, as well as remarkable anti-inflammatory, anti-oxidant and anti-glycation results, thereby contemplating applications in the areas covered by the invention. It was also demonstrated that the products obtained with cells according to the invention advantageously act on a number of micro-RNAs. Micro-RNAs are produced by the cell from DNA, such as mRNA, and play a crucial role in controlling many physiological processes by inhibiting the synthesis of specific proteins. The results on micro-RNAs clearly reinforce the intended applications and open to other perspectives.
  • the product can advantageously be positioned as a global anti-age.
  • the present invention also provides the use of Plantago lanceolata cells to densify dermis, strengthen dermal-epidermal junction, thicken epidermis and/or provide anti-glycation defense means to skin.
  • the method according to the invention can be achieved according to the following steps:
  • the bioreactor production stage comprises an elicitation step, thus advantageously allowing increasing the content of plantamajoside and more generally in phenylpropanoid glycosides (PPG); and/or
  • the biomass from the reactor is collected by filtration after a culture period of 7 to 21 days, preferably between 10 and 14 days, thus advantageously producing the greatest quantity of biomass, with high viability; and/or
  • Elicitation of compounds of interest can generally be done by adding microbial fractions to the culture (for example sacchoromyces yeasts), adding biological molecules to the culture such as chitosan, methyljasmonate, jasmonic acid or salicylic acid, adding non-biological molecules to the culture such as paclobutrazol, application to the culture of a variation of temperature, pH or an osmotic stress induced by a non-metabolizable sugar, such as mannitol, the use of a more drastic depletion of macroelements and sugar in the medium, and/or adding to the culture adsorbent resins which, besides eliciting production of interest compounds, can trap them.
  • microbial fractions for example sacchoromyces yeasts
  • biological molecules such as chitosan, methyljasmonate, jasmonic acid or salicylic acid
  • non-biological molecules such as paclobutrazol
  • elicitation is performed by changing the culture medium, including the nutrient levels.
  • the subject matter of the present invention is also the selected IRB PL3 Plantago lanceolata line, meristematic cells of Plantago lanceolata obtained through the use of this line, the invention method disclosed above and a cellular extract obtained from said cells, the extract being for example made by cell lysis, then a centrifugation step followed by filtration, so as to recover the inside of the cells and to eliminate cell walls.
  • the present invention also provides a composition
  • a composition comprising Plantago lanceolata meristematic cells or a cell extract according to the invention in a physiologically acceptable medium and cosmetic, nutraceutical or dermatological uses of said composition, of said cells or said cellular extract.
  • MicroRNAs (or miRNAs) were discovered in 1993. Since then, the role of microRNAs has been pinpointed and seems to have expanded over the years. MicroRNAs are numbered in the order in which they were discovered, forming a different class of very small (20 to 25 nucleotides long), non- coding RNAs. MicroRNAs are produced by cellular DNA, just like mRNA, and play a key role in controlling numerous physiological processes by inhibiting specifically the synthesis of various proteins. Once produced, the microRNA specifically binds to the beginning of its own mRNA, thereby rendering the mRNA information impossible for the ribosome to read or turning it into the target of proteins that will destroy them. These post-transcriptional control phenomena are real natural on-off switches. They may affect at least 30% of gene production. Some miRNAs, repressor of collagen I, collagen IV and elastin synthesis were identified. Some other miRNAs determine the appearance of senescent phenotypes, in particular in fibroblasts.
  • the loss of density and thinning of the dermis are related to a reduction in the synthesis of macromolecules (in particular collagen I, collagen IV, elastin and hyaluronic acid) by dermal fibroblasts, the cells in charge of producing them.
  • macromolecules in particular collagen I, collagen IV, elastin and hyaluronic acid
  • TIMP tissue Inhibitor of Metalloproteinase
  • the increase in MMP degradation activity and the reduction of protein synthesis also affects the dermal-epidermal junction (DEJ), where key skin proteins are found.
  • Collagen IV is one of these key proteins. It is organized into a thin layer to which other structures essential for skin homeostasis, like laminins, attach. With age, collagen IV becomes more fragmented and its production slows, just like for laminins. In certain areas, this leads to changes in the relationships between melanocytes, keratinocytes and the DEJ. The result is an increase in skin pigmentation, including the appearance of age spots or senile lentigo in these areas.
  • Epidermis also undergoes the effects of aging. Cell renewal is less important and therefore the skin becomes thinner. Furthermore, the connections between the cells are less strong. Filaggrin and hyaluronic acid are less synthesized, causing a lesser intercellular cohesion and higher water loss in the stratum corneum.
  • pre-inflammatory mediators prostaglandins and interleukins types
  • pro-oxidant compounds that will increase all the deleterious effects already described by oxidizing and thus degrading/inactivating the molecules that contribute to cellular homeostasis (lipids, proteins, sugars, nucleic acids)
  • lipids, proteins, sugars, nucleic acids lipids, proteins, sugars, nucleic acids
  • glycation of proteins enhances aging by modifying all skin proteins (structural or functional), and in particular dermal proteins such as collagen, disorganizing the extracellular matrix and making it lose its tonicity, its flexibility. This glycation will generate an increase in radical products which in turn will disrupt the metabolisms and degrade cell structures of the skin.
  • the meristematic cells, the cellular extract or a composition comprising them according to the invention act advantageously in five different and complementary directions of skin aging as discussed above.
  • the dermis keeps its density, hydration, will recover elasticity and firmness; the face will gain a smooth relief (reduction of wrinkle depth).
  • meristematic cells of the invention result in superior effects on the synthesis of dermal macromolecules (collagen I and hyaluronic acid), the synthesis of molecules responsible for the DEJ strengthening (laminins) and the strengthening of the epidermal barrier (hyaluronic acid).
  • meristematic cells according to the invention showed a protective effect of cells toward a cytotoxic effect demonstrated contrariwise with verbascoside.
  • Topical application of meristematic cells, of the cellular extract or of a composition containing them, according to the invention (or a composition containing the meristematic cells or their extracts according to the invention), for the treatment pathologies of the skin chosen among:
  • Orally to obtain a beneficial effect to strengthen the intestinal wall.
  • a beneficial effect to strengthen the intestinal wall For example, possible absorption of 10 to 50mg of lyophilized cells per day and per person.
  • the present invention therefore proposes a composition, especially topical, comprising meristematic cells, or an extract of said cells according to the invention in a physiologically acceptable medium.
  • this composition will be for example a concentrated active ingredient or a less concentrated final composition directly intended to the end user.
  • Physiologically acceptable medium means according to the present invention, without limitation, an aqueous or hydroalcoholic 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 oral use, in contact with mucous membranes, appendages (nails, hair and body hair), 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 meristematic cells and/or the cellular extract of these cells may be combined with other active ingredients in effective concentrations to act synergistically or to reinforce and to achieve the desired effects described in the invention, such as the following ingredients: radiation filters, including UVA and/or UVB, moisturizing, calming, muscle relaxant, slimming, restructuring, firming, re-fillling, tensing, acting on microcirculation, acting on inflammation, on free radicals, vitamins, anti-wrinkle lightening agents, etc.
  • active ingredients including UVA and/or UVB, moisturizing, calming, muscle relaxant, slimming, restructuring, firming, re-fillling, tensing, acting on microcirculation, acting on inflammation, on free radicals, vitamins, anti-wrinkle lightening agents, etc.
  • composition according to the invention can be applied to the face, body, neck, neckline, scalp, hair, eyelashes, body hair, in any form or vehicles known from the ones skilled in the art, in particular in the form of solution, dispersion, emulsion, paste or powder, individually or as a premix in vectors such as macrocapsules, microcapsules or nanocapsules, macrospheres, microspheres or nanospheres, liposomes, oleosomes or chylomicrons, macroparticules, microparticules or nanoparticules, macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or adsorbed on organic polymer powders, talcs, bentonites, spores or exines and other inorganic or organic supports.
  • applications can be proposed in particular in the ranges of skin care products for face, body, hair and body hair and makeup-care lines, including eyebrows and eyelashes.
  • the cells or the cellular extract of the present invention may in general be used in any form whatsoever, in a form bound to or incorporated in or absorbed in or adsorbed on macro-, micro-, and nanoparticles, or macro-, micro-, and nanocapsules, for the treatment of textiles, natural or synthetic fibers, 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 or therapeutical effect via this skin/textile contact and to permit continuous topical delivery.
  • CTFA International cosmetic ingredient dictionary & handbook (13th Ed. 2010) (published by the Cosmetic, Toiletry, and Fragrance Combination, Inc., Washington, D.C.) describes a non-limited wide variety of cosmetic and pharmaceutical ingredients conventionally used in the skin care industry that can be used as additional ingredients/compounds in the compositions of the present invention.
  • betain betain, 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 (trade name of the acetyl hexapeptide-3 of Lipotec), spilanthol or an extract of Acmella oleracea known under the name Gatuline ExpressionTM, an extract of Boswellia serrata known under the name BoswellinTM, Deepaline PVBTM (Seppic), Syn-AKETM (Pentapharm), AmelioxTM, BioxiliftTM (Silab), Phy
  • 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 (Col
  • 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, of Globularia Cordifolia, of Albizzia julibrissin, Oxydendron arboretum or of Zingimber Zerumbet Smith.
  • compositions of the present invention may include other peptides, including, without limitation, the 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 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).
  • 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 which are found in nature, and/or are commercially available.
  • Suitable dipeptides for use within the scope of the present invention include but are not limited to carnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK or TT.
  • Non limitative suitable tripeptides for use herein include, but are not limited to RKR, HGG, GHK, GKH, GGH, GHG, KFK, KPK, KMOK, KMO 2 K or KAvaK.
  • Non limitative suitable tetrapeptides for use herein include but are not limited to RSRK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2) or KTFK (SEQ ID NO: 3).
  • Non limitative suitable pentapeptides include, but are not limited to KTTKS (SEQ ID NO: 4) and hexapeptides include but are not limited to GKTTKS (SEQ ID NO: 5) and VGVAPG (SEQ ID NO: 6).
  • Suitable peptides for use in the context of the present invention include, but are not limited to: lipophilic derivatives of peptides, preferably palmitoyl derivatives, and metal complexes as aforementioned (e.g. copper complex of the tripeptide HGG).
  • Preferred dipeptide derivatives include N-Palmitoyl-beta-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (CalmosensineTM, IdealiftTM from Sederma).
  • Preferred tripeptide derivatives include in particular the N-Palmitoyl-Gly-Lys-His, and Pal- Gly-His-Ly, (Pal-GKH and Pal-GHK from Sederma), the copper derivative of HGG (LaminTM from Sigma), Lipospondin (N-Elaidoyl-KFK) and its analogues of conservative substitution, N-Acetyl- RKR-NH 2 (Peptide CK+), N-Biot-GHK (from Sederma), Pal-KM0 2 K (Sederma) and derivatives thereof.
  • Suitable tetrapeptide derivatives for use according to the present invention include, but are not limited to, N-palmitoyl-GQPR (SEQ ID NO: 7) (from Sederma), Ela-KTFK (SEQ ID NO: 8).
  • Suitable pentapeptide derivatives for use herein include, but are not limited to, N-Palmitoyl-KTTKS (SEQ ID NO: 9) (available as MatrixylTM from Sederma), N-Palmitoyl-Tyr-Gly-Gly-Phe-X (SEQ ID NO: 10) with X Met or Leu or mixtures thereof.
  • Suitable hexapeptide derivatives for use herein include, but are not limited to, N-Palmitoyl- VGVAPG (SEQ ID NO: 11), Pal-GKTTKS (SEQ ID NO: 12) and derivatives thereof.
  • the mixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 7) (MatrixylTM 3000, Sederma) can also be mentioned.
  • compositions commercially available containing a tripeptide or a derivative include Biopeptide-CLTM, MaxilipTM, BiobustylTM, ProcapilTM and MatrixylTMsynthe'6TM of Sederma.
  • compositions commercially available preferred sources of tetrapeptides include RiginTM, EyelissTM, MatrixylTM Reloaded and Matrixyl 3000TM which contain between 50 and 500 ppm of Palmitoyl- GQPR (SEQ ID NO: 7) and carrier, proposed by Sederma.
  • peptides can be mentioned as well as additional active ingredients: VialoxTM, Syn-akeTM or Syn-CollTM (Pentapharm), Hydroxyprolisilane CNTM (Exsymol), ArgirelineTM, LeuphasylTM, AldenineTM, TrylgenTM, EyeserylTM, SerilesineTM or DecorinylTM (Lipotec), CollaxylTM or QuintescineTM (Vincience), BONT-L-PeptideTM (lnfinitec Activos), CytokinolTMLS (Laboratoires Serobi GmbH/Cognis), KollarenTM, IP2000TM or MelipreneTM (lnstitut Europeen de Biologie Cellulaire), NeutrazenTM (Innovations), ECM-ProtectTM (Atrium Innovations), Timp-PeptideTM or ECM ModulineTM (lnfinitec Activos).
  • the present invention also provides a topical cosmetic or dermopharmaceutical treatment method to improve the appearance and condition of the skin and its appendages, comprising the topical application to the skin of a subject in need thereof of an effective amount of meristematic cells or of their cellular extract according to the invention or of a composition according to the invention comprising said cells or said cellular extracts as recited above.
  • Topical treatment or “topical use” means an application that is intended to act where it is applied: skin, mucous, appendages.
  • the meristematic cells, the cellular extract or composition according to the invention can be applied locally applied to targeted areas.
  • the “effective” amount depends on various factors, such as the age, the condition of the patient, the severity of the disorder or disease and the administration mode.
  • An effective amount means a nontoxic amount enough to achieve the desired effect.
  • the meristematic cells or cellular extract to be present in an effective amount are generally present in an amount ranging from 0.000001% and 15% based on the total weight of the composition, preferably between 0.0001% and 10% depending on the destination of the composition and the more or less pronounced desired effect.
  • the physiologically acceptable medium is a hydrophilic matrix wherein said cells are in suspension; and/or
  • composition comprises a thickening agent and/or undergoes high pressure homogenization; and/or
  • composition forming a cosmetic ingredient, includes at least 0.1 % of phenylpropanoid glycosides, usually approximately 0.1%. This ingredient is then used for example in an amount of several % to 20% to prepare cosmetic formulations.
  • the SCCS'S Stemific Committee on Consumer Safety
  • Guidance for the testing of cosmetic substances and their safety evaluation (8 th Revision, 11 dec. 2012) has set a standard amount for applying a cream of 2.72 mg/cm 2 /day/person and for a body lotion of 0.5 mg/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 the active cells of the invention, and in a second compartment an excipient and/or an additional active, the compositions contained in the said first and second compartments in this case being considered to be a combination composition for simultaneous, separate or stepwise use in time, particularly in one of the treatment methods recited above.
  • the method of treatment according to the invention is particularly suitable for:
  • cosmetic applications are also possible, for example, for a treatment for slimming, loss of elasticity, detoxification, anti-glycation, tensing, anti-fatigue, for a treatment of under-eyes bags and/or dark circles, soothing, firming, treatment of hair and body hairs, action on skin radiance etc. for a preventive or curative action.
  • Figure 1 shows a skin deformation graph using a CutometerTM illustrating the different measured parameters.
  • a dedifferentiated cell mass or callus is formed and transferred on a larger area and in a fresh culture medium to be able to multiply.
  • a number of subcultures is performed for stabilizing the cell line, i.e. until it has a high and constant speed of proliferation, preservation of phenotype and a constant content of bioactive compounds of interest (primary and secondary metabolites).
  • the cell line is subsequently subjected to a selection step of cultivating the cells for an appropriate period, collecting the formed cell aggregates and inoculating these on a liquid culture medium for a period enough to obtain the multiplication of cellular aggregate.
  • the best cell line will be the one giving as quickly as possible and in a reproductible manner a large biomass having an optimal content in selected metabolites, the best biological activity and a homogeneous phenotype.
  • the inventors have selected the IRB PL3 Plantago lanceolata cell line.
  • This cell line was also selected for its ability to produce phenylpropanoid glycosides in an amount of about 10% as measured by weight of total phenylpropanoid glycosides expressed as plantamajoside relative to the dry cell weight.
  • the Plantago lanceolata IRB PL3 line is initially multiplied to obtain sufficient biomass of meristematic cells to perform the step of large-scale production.
  • step b) optionally repeating step b)
  • this step of bioreactor production comprising an step elicitation achieved by modifying the content of nutrients of the culture medium.
  • the bioreactor The bioreactor:
  • volume 5 to 50 times larger than the volume of biomass used as inoculum; smooth and uniform internal surface bioreactor (no edges or angles which could cause the rupture of cell walls).
  • Culture medium medium comprising mineral salts (macronutrients and micronutrients solution), vitamins, plant hormones and sucrose. Vegetable agar is added to solid media.
  • Temperature between 15°C and 35°C, preferably between 20°C and 30°C, and more preferably at 25°C.
  • Duration between 7 and 21 days, preferably between 10 and 14 days.
  • Biomass agitation it is important that biomass be optimally ventilated 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 steps, when the biomass is in large quantities.
  • suitable agitation by internally means are propellers rotating between 20 and 120 rpm, preferably 60 rpm, or externally by orbital agitation means rotating preferably between 40 and 200 rpm and preferably about 120 rpm.
  • Oxygenation usually carried out using sterile air, at a rate of 0.5 to 4 liters per minute, preferably between 2 and 2.5 liters per minute, for a volume of 10 liters of biomass.
  • gas mixtures containing from 10% to 100% v/v of oxygen may be used. It is preferable to use diffusion means of air or oxygen with a nozzle having a flow rate of between lOml/min and 600ml/min and preferably between 50ml/min and 350ml/min.
  • This biomass may be characterized by an equivalent rate of lyophilized cells.
  • Characterization of the active compounds contained in the cells by analytical determination of primary and secondary metabolites produced by the culture, including protein and PPG.
  • High pressure homogenization of the cellular culture medium reduces the size of cellular aggregates, certain cells can be broken and a mixture of whole cells and crushed cells can be obtained;
  • the cell biomass either as obtained at point B) after filtration, or in a dried form, or the cellular extract, can be mixed in a physiologically acceptable medium forming the excipient.
  • the physiologically acceptable medium is a hydrophilic matrix in which the cells are suspended, for example in the case of a cosmetic composition glycerol and/or butylene glycol.
  • Additives may also be added if necessary, such as antimicrobial agents, antioxidants, stabilizing agents, agents acting on pH, emulsifying agents or thickeners, such as a xanthan gum thickener which will promote the maintaining of the cells in suspension.
  • a supplementary high-pressure homogenization step can be provided to obtain a fine particle suspension.
  • An ingredient for cosmetic purposes, concentrated in active compounds can thus be formed comprising for example 20% by weight of fresh biomass of whole meristematic cells (corresponding to about 1 % of dry cells) relative to the total weight of the composition, having a final content of about 0.1% of phenylpropanoid glycosides, in a physiologically acceptable excipient mixture consisting of glycerol (approximately 80%) and xanthan gum (0.3 wt%).
  • Collagen I is the most abundant protein in the dermis. It is an essential component to have a firm and elastic skin and tends to be less produced with increasing age.
  • NHF in culture were exposed to the invention product for 6 days.
  • Collagen I synthesis was measured using photography after immune-labelling.
  • Cellular nuclei were counted using a DNA stain (Hoescht 33258) to standardize the data.
  • AFU arbitrary fluorescence unit; no toxicity was noted compared to the control.
  • Elastin is synthesized and secreted in the extracellular space by dermal fibroblasts first in pro-elastin, then in tropo-elastin. Elastin is the major component up to 90% of elastic fibers. Elastin and fibrillin forming the elastic fibers, and collagen, are the main constituents of the extracellular matrix. It is important to stimulate their synthesis which decrease with age.
  • Normal human fibroblasts were grown in contact or not with the test products at various concentrations. Elastin synthesis produced by the cells was then quantified by immuno-labeling and image analysis on the fixed layers. A nuclear counter staining with Hoechst reagent complements the study and can be used to weight the results.
  • the application of the corticosteroid reduces the elastin level by 25% with the placebo.
  • the invention product allows raising it up to a higher level than the level of unstressed placebo skins.
  • Hyaluronic acid is an essential component of the dermis.
  • the interest of hyaluronic acid lies in its viscoelastic properties and its ability to capture water.
  • water fills the spaces between collagen and elastin fibers in the dermis. This contributes to skin flexibility and prevents wrinkle formation. This substance decreasing with age, skin becomes dry and wrinkles.
  • the product according to the invention stimulates the hyaluronic acid synthesis by normal human fibroblasts.
  • NHFs human dermal fibroblasts
  • the product of the invention was contacted with the product of the invention for 3 and 24 hours.
  • the miRNAs were extracted and were studied by transcriptional analysis after checking the quality of RNA by capillary electrophoresis and determining the miRNA percentage. The values obtained at each time point were compared with the control without product.
  • Dermal proteases are induced by various stresses and during ageing. They contribute to fragmentation and increased degradation of dermal macromolecules (collagen I in particular), rendering skin less firm, less dense and less flexible. MMPs are controlled by different mechanisms including TIMPs produced by the cells. These TIMPs are glycoproteins responsible for inhibiting the activity of MMPs by complexation. They are also controlled by miRNAs.
  • Oxidative stress causes on the control an increase in MMP-1 and -2 (+21%; +21%) and a decrease in TIMP-1 and -2 (-30% and -41%).
  • MicroRNA-21 is known to repress TIMP-1 production and to facilitate MMPs action. NHFs were contacted with the invention product for 3 and 24 hours. Each time the miRNAs were extracted and quantified as mentioned above.
  • AFU arbitrary fluorescence unit; AFU variation considered case/control AFU; no toxicity was observed compared to the control.
  • the decrease of miR-21 expression will increase TIMP-1 production and thus will reduce MMPs activity.
  • the product according to the invention thus advantageously protects the dermal macromolecules against degradation by proteases.
  • the product according to the invention protects the dermal extracellular matrix from degradation by proteases. This is particularly sought for anti-aging action.
  • DEJ or dermo-epidermal junction is a thick membrane that ensures the cohesion between the dermis and the epidermis.
  • Collagen IV is a type collagen mainly located at the basal membrane or dermo-epidermal junction (DEJ). It is one of the essential elements of the skin, not by quantity but by its role at the level of the DEJ. With age, collagen IV is more fragmented because attacked by MMPs and at the same time less synthesized, like laminins, resulting in some areas in an impaired JDE, disturbed relationships between melanocytes, keratinocytes and JDE, resulting in increased skin pigmentation.
  • DEJ dermo-epidermal junction
  • AFU arbitrary fluorescence unit
  • the product according to the invention therefore advantageously stimulates the synthesis of collag skin explants.
  • Basal keratinocytes are attached to each other by forming a thin layer bonded to the dermis via the DEJ and its complex network of proteins and fibers.
  • the laminins boat anchor- shaped proteins, whose branches bind together collagen IV, dermal proteoglycans and keratinocytes.
  • Laminins decrease in quantity with age and at the level of senile lentigo. Thus stimulating their synthesis is of great interest.
  • Human keratinocytes were contacted with the invention product during 3 days.
  • a dosage of laminin was carried out on culture supernatants using an ELISA method.
  • An estimate of cell amount by Hoechst assay was used to weight this dosage.
  • Epidermis thinning is one aspect of aging, hence the need to act also in the direction of the reinforcement of epidermis and therefore also to act on the reinforcement of keratinocyte differentiation.
  • a mixture of neutral and polar lipids is dominant in the deep layers and is gradually replaced by a more apolar lipidic content, including ceramides, free sterols and free fatty acids, as well as variable quantities of triglycerides, sterol esters and other non-polar components.
  • the decrease in cell number is classic after contact with a pro-differentiator product and does not reflect cytotoxicity of this product, but rather a lower adhesion to the support of the differentiated cells.
  • the product of the invention stimulates the synthesis of epidermal lipids. This result is consistent because differentiation is accompanied by accumulation of ceramides which are neutral lipids,
  • Tight junctions form a protection system which strongly binds the cells together via the actin network in the upper part of the epidermis. They have a role as guardian of the water homeostasis, preventing water evaporation. ZO (zona occludens) -1 and claudin-1 are involved in this network.
  • Human keratinocytes were contacted with the invention product at 0.12% for 2 days and immunofluorescent labelling of ZO-1 and claudin-1 was carried out.
  • a nuclear counter staining with Hoechst dye associated with a count is used to weight the data.
  • the compound of the invention induces a significant increase of two proteins of tight junctions: ZO-1 and claudin-1 at the level of the corneocytes and therefore contributes to strengthen the skin barrier and the cohesion of the epidermal cells.
  • Loricrin and filaggrin are two specific proteins of keratinocyte differentiation.
  • the effect of the product according to the invention on their synthesis was evaluated. Confluent human keratinocytes were contacted with the product of the invention at 0.12% in a medium containing calcium, and differentiation was followed. After culture, the layers are rinsed, fixed and loricrin and filaggrin synthesis are identified by immunofluorescence and quantified by image analysis. An Hoechst counter staining of nuclei completes the study and weights these results.
  • the decrease in cell number is a classic phenomenon after contact with a pro-differenciator product and does not reflect cytotoxicity of this product, but rather a lower adhesion to the support of the differentiated cells.
  • the product according to the invention strongly stimulates the filaggrin and loricrin synthesis, and therefore differentiation of keratinocytes.
  • Thinning and drying of the epidermis is due to HA deficiency. This happens with aging.
  • Human keratinocytes are cultured for 24hrs.
  • the cells are contacted or not with the product according to the invention at 0.12% for 3 days.
  • the culture supernatants are removed and a hyaluronic acid assay is performed by ELISA, an estimate of the amount of cells by a BCA assay (measuring the amount of protein) is used to weight the results.
  • the product according to the invention significantly stimulates the synthesis of HA in keratinocytes. e) Stimulation of CD44 synthesis
  • CD44 is a surface receptor of HA involved in interactions between cells and cell adhesion.
  • Keratinocytes are cultured. After a growth phase of one week, the confluent cells are contacted with the product according to the invention at 0.12% during 48h. After the contact, the layers are rinsed, fixed and CD44 revealed by immunofluorescence. The labelled layers are then photographed and the specific labelling of each protein of interest is quantified by image analysis using Image JTM software. A counter staining of nuclei with Hoechst dye associated with a count is used to weight these data.
  • the product according to the invention increases the synthesis of CD44. This effect combined with the effect of stimulating the synthesis of hyaluronic acid participates in improving the hydration of the epidermis, and increase its thickness.
  • IL-6 for example, is a cytokine whose serum levels are increased in elderly; IL-6 also induces premature senescence in human fibroblasts.
  • PGE2 form a critical compound for senescence induction and for maintaining an inflammatory condition; they also occur on melanocytes by increasing tyrosinase activity and dendricity which produces hyperpigmentation.
  • aged skin cells produce more IL-6 and IL-8 in response to a stress than young cells.
  • a decrease in production of these inflammatory mediators in the basal state in the absence of stress is advantageous in an anti-aging perspective.
  • Normal human keratinocytes are cultured until a confluent.
  • Invention product 0.15% 10 + 0.5 -91 % ; p ⁇ 0.01
  • Invention product 0.15% 561 + 42 -65% ; p ⁇ 0.01
  • the product according to the invention reduces strongly and significantly pre- inflammatory messengers that promote ageing.
  • DPPH l,l-diphenyl-2-picryl hydrazyl
  • DPPH2 l,l-diphenyl-2-picryl hydrazine
  • This conversion is accompanied by discoloration (violet to yellow), which can be followed in time by spectrophotometry at 517nm. Control having no scavenging activity maintains a constant optical density.
  • Cell membranes are composed of oxidizable phospholipids.
  • a cell-free model unsaturated phospholipid based liposomes
  • a reproducible and physiological oxidative stress UVA radiation
  • UVA radiation oxidative stress
  • the product according to the invention advantageously has a good effect against free radicals and inhibits lipid peroxidation induced by UVA.
  • ROS excess in the cell leads to the damage increase and at long-term to cellular aging.
  • ROS evaluation was performed on human dermal fibroblasts (NHF) with the DCFH-DA probe that, once in the cell, becomes fluorescent in contact with ROS (fluorescence level directly proportional to the ROS amount).
  • the fluorescence obtained in the cells in contact with the invention product was quantified and compared with that of the control without product. Raw results were reduced to the number of cells.
  • AFU arbitrary fluorescence unit; AFU variation considered case/control AFU; no toxicity was observed compared to the control.
  • the product according to the invention thus advantageously protects fibroblast against ROS that may damage its lipid, protein or nucleic structures.
  • SOD is a metalloenzyme which is part of the defense system of the cell against free radicals by performing superoxide anion dismutation into oxygen and hydrogen peroxide.
  • Mitochondria contain a manganese superoxide dismutase (SOD2) which represents the first anti-ROS defense line. Mice skin deficient in this enzyme is very impaired and prematurely aged, in particular epidermis is thinner. The amount of SOD2 was assessed by ELISA in the HDF cell layers after contact with the product according to the invention and compared to the control without product. Raw results were reduced to cell number.
  • the product according to the invention increases basally cell SOD activity and therefore advantageously enhances their potential of defense against oxidation
  • Protein glycation increases with age; it affects all skin proteins (structural or functional) and in particular dermal proteins such as collagen, disrupting the extracellular matrix and making the skin lose its tone and flexibility. This glycation will generate an increase of radical products which in turn will disrupt the metabolism and degrade cell structures of the skin.
  • a model protein the BSA (bovine serum albumin)
  • BSA bovine serum albumin
  • AGEs advanced glycation end products
  • the product according to the invention decreases the glycation of BSA protein model as from a dose of 0.06%.
  • the product of the invention has a protective action with respect to protein glycation.
  • NHFs were contacted with the invention product for 3 and 24 hours. Each time the miRNAs were extracted and were studied by transcriptional analysis after checking the quality of RNA by capillary electrophoresis and determining the percentage of miRNAs. The values obtained at each time point were compared with the control without product.
  • the product according to the invention reduces expression of miR-30a, 34a, 181a and -152 described in the scientific literature as senescence accelerators.
  • the product of the invention has therefore a profile of anti-aging activities very interesting especially for cosmetics.
  • Normal human fibroblasts were cultured for 24 hours. The cells are contacted or not with the test product (product of the invention or verbascoside) for 6 days. Collagen I synthesis produced by the cells is then quantified by immune-labelling and image analysis on the fixed layers. A counter nuclear staining with Hoechst complements the study and is used to weight the results.
  • the product according to the invention strongly stimulates the synthesis of collagen I, whereas verbascoside is inactive.
  • Normal human fibroblasts were cultured for 24 hours. The cells were contacted or not with the test product (product of the invention or verbascoside). The synthesis of hyaluronic acid produced by the cells was then quantified by immune-labelling and image analysis on the fixed layers. A counter nuclear staining with Hoechst complements the study and was used to weight the results.
  • Table 23 Compared effect between verbascoside and the invention product on hyaluronic acid synthesis on human fibroblasts
  • the product according to the invention strongly stimulates the synthesis of hyaluronic acid.
  • Verbascoside also has a stimulating effect but much weaker.
  • Human keratinocytes are cultured for 24hrs. The cells are contacted or not with the test product (product of the invention or verbascoside) for 3 days. Culture supernatants are taken and a dosage of the amount of laminin and hyaluronic acid is conducted by the ELISA method, an estimate of the amount of cells by Hoechst assay (measuring the amount of DNA) or BCA (measure the amount of protein) is used to weight the results,
  • Table 24 Compared effect between verbascoside and the product of the invention on laminin synthesis on human keratinocytes
  • the product according to the invention stimulates strongly and in a dose-dependent manner the laminin synthesis, whereas verbascoside is inactive.
  • fibroblasts it was observed with keratinocytes a beginning of toxicity at 8.8ppm of verbascoside, which does not appear with the product according to the invention,
  • Table 25 Compared effect between verbascoside and the invention product on hyaluronic acid synthesis on human keratinocytes
  • test product product according to the invention or verbascoside
  • a calcium containing medium a calcium containing medium
  • loricrin synthesis is highlighted by immunofluorescence and quantified by image analysis.
  • a counter staining of nuclei with Hoechst completes the study and weights these results.
  • Table 26 Compared effect between verbascoside and the invention product on loricrin synthesis on human keratinocytes
  • the decrease in cell number is classic after a pro-differentiating contact with a product and does not reflect cytotoxicity of this product, but rather a lower adhesion of the differentiated cells to the support.
  • the product according to the invention strongly stimulates the synthesis of loricrin, and therefore the differentiation of the keratinocytes, whereas verbascoside is inactive.
  • Additional cosmetic active ingredients in support and/or in complement of the activity of the active ingredient according to the invention if necessary can be added to the appropriate phase according to their hydrophobic or hydrophilic nature.
  • These ingredients can be of any class according to their(s) function(s), place of application (body, face, neck, chest, hands, hair, eyelashes, eyebrows, body hair, etc.), final desired effect and target consumer, for example anti-aging, anti-wrinkle, moisturizing, anti-wrinkle, firming, anti-glycation, slimming, soothing, myo-relaxing, anti- redness, anti-stretch marks, etc.
  • Active ingredient according to the invention used in the galenic formulas given below 20% in weight with regard to the total weight of the composition of fresh whole meristematic cells (corresponding to 1% of dried cells), the ingredient having a final content of 0.1% of total phenylpropanoid glycosides (expressed in plantamajoside) in an physiologically acceptable excipient mixture of glycerin, xanthan gum thickener and citric acid to regulate pH if necessary.
  • MATRIXYL synthe'6TM peptide-based anti-wrinkle ingredient marketed by Sederma (WO2010/082175) which helps repair skin damage caused by aging. 3% by weight can be added for example at the end of the formulation.
  • MATRIXYLTM3000 peptide-based anti-wrinkle ingredient marketed by Sederma (WO2005/048968) comprising two matrikines Pal-GHK and Pal-GQPR, which in synergy helps repairing skin damages caused by aging. 3% by weight can be added for example at the end of the formulation.
  • RESISTEMTM anti-aging marketed by Sederma (WO2012/104774), helping the skin to build its own anti-aging defense system, based on an extract obtained by cell culture of Globularia cordifolia plant. 2% can be added at the end of the formulation in phase E.
  • RIGINTM active marketed by SEDERMA (WO2000/433417) improving the elasticity and firmness of the skin, increasing hydration and smoothing the skin. 3% by weight of this ingredient may for example be added at the end of the formulation. Night cream form
  • MEIRIT AGETM anti-ageing active ingredient based on three plant extracts (Astragalus membranaceus (Huang Qi), Bupleurum falcatum (Chai Hu) and Atractylodes macrocephala (Bai Zhu), which improves the uniformity and radiance of skin. 4) Eve contour gel form
  • BEAUTIFEYETM active ingredient marketed by Sederma ; an association of an Albizi julibrissin and of darutoside extracted from Siegesbeckia orientalis, demonstrates a lifting action on sagging upper eyelids. 3% can be added at the end of the formulation before phase E.
  • MATRIXYL synthe'6TM mentioned above. 3% in weight can be added at the end of the formulation.
  • HALOXYLTM active ingredient for dark circles marketed by Sederma (WO2005/102266).
  • HaloxylTM combines Pal-GHK and Pal-GQPR matrikines with N-hydroxysuccinimide (NHS) and the chrysin flavonoid.
  • the Pal-GHK and Pal-GQPR reinforce firmness and tone of the eye contour; chrysin and N-hydroxysuccinimide activate the elimination of blood origin pigments responsible for the color of dark circles but also of the local inflammation. 3% by weight of this ingredient may be added at the end of the formulation.
  • EYELISSTM active ingredient marketed by Sederma (WO2003/068141) that helps prevent against the appearance of bags under the eyes. It combines three components: hesperidin methyl chalcone reducing capillary permeability, Valyl-Tryptophan (VW) dipeptide which promotes lymphatic circulation and Pal-GQPR lipopeptide that improves firmness, elasticity and reduces inflammation. 3% by weight of this ingredient may be added at the end of the formulation.
  • ⁇ CALMOSENSINETM soothing active for sensitive skins marketed by Sederma (WO 1998/07744) comprising the Tyr-Arg lipo-dipeptide. It reduces discomfort feelings.
  • PHYTOTONINETM active marketed by Sederma comprising a synergistic combination of three vegetable actives, flavonoids from Arnica montana flowers, saponins from rhizomes of Polygonatum multiflorum (Solomon's Seal) and proanthocyanidins from Cupressus sempervirens cones (Cypress); clearly improves the appearance of "blotchy skin”.
  • CHROMOCARETM anti-age active ingredient marketed by Sederma (WO2010/119423) ; an association of an extract of Rabdosia rubescens rich in oridonine and an extract of Siegesbeckia orientalis rich in darutoside ; evens and rejuvenates the complexion. 6) Alcoholic gel form
  • AQUALANCETM osmo-protector moisturising active ingredient marketed by Sederma (WO2009/104118) comprising homarine and erythritol.
  • NG Birch SapTM raw sap from birch sapwood; skin toning and moisturizing marketed by Sederma.
  • VENUCEANETM active marketed by Sederma (WO2002/066668) comprising a Thermus thermophiles biotechnological extract,that prevents visible signs of photo-aging (spots, wrinkles, dryness %), protects cell structures from damages caused by UV and strengthens skin integrity. 5% can be added for example after phase G.
  • MELASLOWTM active marketed by Sederma, promotes complexion lightening and age spot depigmentation (extract of Japanese Mandarin Citrus reticulata Blanco var. unshiu).
  • RE VIDR ATETM active marketed by Sederma (WO 2011/086532) that in particular improves the cohesion of the epidermis and its hydration. 2% of this ingredient may for example be added to phase C of the formulation.
  • Shea butter is an ingredient with nourishing and protective properties for the treatment of skin damaged by the environment.
  • ⁇ INTENSLIMTM slimming active ingredient marketed by Sederma (WO2013/105048) corresponding to a synergistic combination of extracts obtained by Globularia cordifolia plant cell culture, Zingiber zerumbet Smith titrated in zerumbone and vegetable caffeine obtained by supercritical C0 2 extraction.
  • JUVINITYTM active marketed by Sederma (WO 2011/125039) reducing signs of aging on the face and neckline, smoothing wrinkles, densifying and restructuring the dermis.
  • RENO V AGETM global anti-aging active ingredient marketed by Sederma (WO2006/ 120646).
  • Bio-BustylTM active marketed by Sederma based on peptides (PalGHK and PalVGVAPG) and a bacterial filtrate having a global action on firmness and tone of the bust.
  • the volunteers were required to present visible signs of ageing on the tested body parts, such as senescence spots, sagging hand skin and/or deep crow's feet. Since not all volunteers presented all three parameters in an optimal way at the time (sometimes with differences between two sides), all tests were not done on each volunteer, and subpopulations were created to have consistent panels for each test.
  • the study was carried out as a single-bind study on the back of the hand and the face.
  • the cream according to the invention and the placebo cream, used contralaterally, were applied and massaged in twice daily for a period of 2 months.
  • a Cutometer® MPA580 (Courage & Khazaka, Germany) was used to measure the elasticity and firmness of the skin of the hand.
  • This device which is widely used to assess the effects of cosmetic products, measures the deformation of an area of skin subject to mechanical stress (suction) and the skin's ability to return to its original shape (i.e. recuperate).
  • the 2- mm probe was used with 500mbar of negative pressure.
  • the figure 1 of the drawings show an example of skin deformation graph thus obtained.
  • the Reviscometer® RV 600 (Courage & Khazaka, Germany) measures the propagation time for a sound wave emitted at the surface of the dermis and epidermis. This parameter is called Resonance Running Time (or RRT).
  • RRT Resonance Running Time
  • the skin probe of the instrument contains an acoustic transmitter separated from the receiver by a distance of 2mm. The probe does not penetrate at more than 0.5mm, it is thus possible to study the qualities of the dermis. The speed with which the sound propagated in a material depends on the density and tension of the material.
  • the device measures RRTs at different angles by rotating the probe on the surface of the skin. This provides a mean propagation time (mean RRT), which is described as diminishing with age on the hand, on other sites and when there is tissue breakdown.
  • the mean RTT therefore is reflective of the quality of all the fibers; it diminishes concomitantly with the density of the tissue, contrary to the RRT max.
  • the RRT max increases following this breakdown. The waves move along the strongest fibers, which are those that are least rapidly broken down, and are accentuated.
  • Ultrasonography or echography
  • ultrasound waves encounter tissue in the human body, they are reflected and send back a signal, or an "echo".
  • the intensity of an echo is translated by the ultrasound into digital grayscale, creating a reliable anatomical image of the area being explored.
  • the instrument used was a DermascanTM C (Cortex Technology, Denmark), which has a 20-Mhz frequency probe that provides images that are 12mm in width and 15mm in penetration. A successive sequence of images provides 5 representative images for study.
  • the improvement in elasticity and firmness is correlated with the increase of the dermal thickness observed here and the increase in density measured using the Re viscometer®. This translates into a qualitative improvement in the skin, and in the dermal fibers in particular.
  • the epidermis thins with age, especially on the hand.
  • the epidermis was analyzed using confocal laser scanning microscopy with a Vivascope® 3000 (Mavig, Germany), which has a portable, maneuverable probe.
  • the light emitted by the instrument (830nm wavelength) is reflected differently in the sky depending on the refraction index of the structures it hits (keratin, melanin and micro-cellular structures, as well as collagen, will appear in white on a greyscale digital image).
  • the Vivascope® enables to "lighten” a specific point in the skin (focalization of illumination) and in parallel precisely detects the returning light (focalization of the detection).
  • This instrument is called “Confocal” for "conjugate focal planes”. This enables to perform a real non-invasive biopsy of the skin in real time to study healthy skin, its pigmentation, its ageing, its photoageing and the thickness of its different layers.
  • the depigmentation effect is observable both for areas with spots and for the areas around the spots, leading to an overall lightening effect.

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Abstract

Selon la présente invention, des cellules méristématiques sont obtenues à partir d'une lignée cellulaire sélectionnée et sont principalement constituées de plantamajoside. Ces cellules peuvent être employées avantageusement dans des produits cosmétiques, en particulier des produits nutraceutiques anti-vieillissement ou des applications dermatologiques.
EP14719110.0A 2013-04-08 2014-04-03 Procédé de production de cellules méristématiques de plantago lanceolata, composition comprenant lesdites cellules ou leur extrait cellulaire et ses utilisations cosmétiques, nutraceutiques et dermatologiques Ceased EP2983464A1 (fr)

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IT000529A ITMI20130529A1 (it) 2013-04-08 2013-04-08 Processo di produzione di cellule meristematiche di plantago lanceolata, composizione comprendente dette cellule o il loro estratto cellulare e l'utilizzo cosmetico, nutraceutico e dermatologico
FR1353103A FR3004193B1 (fr) 2013-04-08 2013-04-08 Procede de fabrication de cellules meristematiques de plantago lanceolata, composition comprenant lesdites cellules ou leur extrait cellulaire, et utilisations cosmetiques, nutraceutiques et dermatologiques
PCT/IB2014/060404 WO2014167464A1 (fr) 2013-04-08 2014-04-03 Procédé de production de cellules méristématiques de plantago lanceolata, composition comprenant lesdites cellules ou leur extrait cellulaire et ses utilisations cosmétiques, nutraceutiques et dermatologiques

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CN109156346B (zh) * 2018-08-31 2021-09-07 江苏农林职业技术学院 百可花腋芽微繁方法
CN108904414A (zh) * 2018-10-10 2018-11-30 广州科盈化妆品有限公司 一种快速祛皱眼霜
CN109984043B (zh) * 2019-05-15 2022-03-11 西南林业大学 一种以叶片为外植体的樟叶越桔组织培养方法
CN110622862B (zh) * 2019-10-16 2022-04-19 西南林业大学 一种樟叶越桔悬浮细胞培养体系的建立方法
CN112409310B (zh) * 2020-12-18 2023-04-21 许昌学院 一种具有lsd1抑制活性的化合物、制备方法及应用
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
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