WO2023042826A1 - Cals dérivés d'une plante fagaceae, extrait de cals, procédé et composition cosmétique les utilisant - Google Patents

Cals dérivés d'une plante fagaceae, extrait de cals, procédé et composition cosmétique les utilisant Download PDF

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WO2023042826A1
WO2023042826A1 PCT/JP2022/034255 JP2022034255W WO2023042826A1 WO 2023042826 A1 WO2023042826 A1 WO 2023042826A1 JP 2022034255 W JP2022034255 W JP 2022034255W WO 2023042826 A1 WO2023042826 A1 WO 2023042826A1
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callus
acid
extract
plant
fagaceous
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English (en)
Japanese (ja)
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若奈 村上
祐太 木村
翔多朗 鈴木
史訓 駒井
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株式会社アルビオン
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Priority to JP2023548470A priority Critical patent/JPWO2023042826A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • the present invention provides a callus derived from a fagaceous plant, a method for producing the callus, an extract of the callus derived from the fagaceous plant, a method using the callus extract, and a cosmetic composition containing the callus extract derived from the fagaceae plant. about things.
  • Patent Document 1 discloses a skin cosmetic containing a combination of an extract extracted from parsley and an extract extracted from a beech tree. Plant-derived cosmetic raw materials can be incorporated into cosmetics with the expectation of various useful activities on the skin.
  • the present invention provides callus from seeds and germination of fagaceae plants, methods for producing the callus, extracts of callus from seeds and germinations of fagaceae plants, methods using the callus extracts, and fagaceae plants. It is an object of the present invention to provide a cosmetic composition comprising a callus extract of plant seeds and germination.
  • a callus consisting of cells of a seed of a fagaceous plant or a germination body derived from a seed of a fagaceous plant.
  • the callus of [1] wherein the cells are cells treated with a plant growth regulator.
  • a plant growth regulator is synonymous with a plant growth regulator.
  • auxin is picloram, dicamba, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5 -trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 2-methyl-4-chlorophenoxybutyric acid and naproanilide, or salts thereof.
  • the callus according to any one of [1] to [4], wherein the cell is a seed cell of a fagaceous plant.
  • auxin is picloram, dicamba, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5 -trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 2-methyl-4-chlorophenoxybutyric acid, and naproanilide, or a salt thereof.
  • [12-1] The method for producing callus according to any one of [9] to [12], wherein the cell is a seed cell of a fagaceous plant.
  • [12-2] The method for producing callus according to any one of [9] to [12], wherein the cell is a seed-derived germination cell of a fagaceous plant.
  • [12-3] The method for producing callus according to [12-2], wherein the germinated body is a germinated body obtained by germination of a seed of a fagaceous plant under sterile germination conditions.
  • [13] The method for producing callus according to any one of [9] to [12] and [12-1] to [12-3], wherein the fagaceous plant is Japanese beech.
  • [14] A callus extract derived from the callus of any one of [1] to [8].
  • the callus extract of [14] which is extracted with water or a mixture of water and alcohol.
  • the callus extract of [14] which is an extract obtained with a mixture of water and ethanol.
  • [16] The group consisting of antioxidant, melanin synthesis suppression, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anticancer
  • the callus extract of [14], [15] or [15-1] which has one or more actions selected from: [16-1]
  • a cosmetic composition comprising the callus extract of any one of [14] to [16] and a cosmetic base.
  • Anti-oxidation, suppression of melanin synthesis, anti-aging, whitening, enhancement of cell proliferation, collagen comprising applying the callus extract of any one of [14] to [16] to the skin of a human subject
  • biochemically useful callus of seeds and germinations of fagaceous plants methods for producing the callus, and callus of seeds and germinations of fagaceous plants, which are suitable for blending in cosmetics and the like.
  • An extract, a method using the callus extract thereof, and a cosmetic composition comprising the callus extract of seeds and germinations of fagaceous plants can be provided.
  • FIG. 1 is a photograph showing an example of callus production in Examples 1 and 2.
  • FIG. FIG. 2 is a graph showing cell viability when callus extracts were applied to cells, FIG. 2A showing viability of normal human dermal fibroblasts (NHDF) and FIG. Fig. 3 shows the viability of metamorphic cells (NHEK).
  • FIG. 3 is a graph showing selected representative ones (SOD2, KITLG, SIRT1, FGF7) among the results of gene expression analysis in fibroblasts.
  • FIG. 4 is a graph showing representative results (FGF2, MMP1, HAS2, ADAM10) extracted from the results of gene expression analysis in fibroblasts.
  • FIG. 5 is a graph showing selected representative ones (WNT5A, DKK3, KITLG, DKK1, AQP3) among the results of gene expression analysis in epidermal keratinocytes.
  • Callus of the present invention relates to callus (hereinafter also referred to as “callus of the present invention") consisting of cells of seeds of fagaceous plants or seed-derived germinations of fagaceous plants.
  • callus means a mass of undifferentiated plant cells obtained by culturing part of a plant body.
  • a part of a plant body means an element (for example, seed, root, leaf, stem, flower, etc.) constituting a plant and a material obtained by further cutting or separating the element.
  • a callus is neither a leaf, nor a stem, nor a root, nor any other plant tissue, but a mass of cells that can differentiate into any tissue of its originating plant (i.e., the plant from which the callus originates). could be. Therefore, callus does not include tissues or cells simply removed from plants (mere leaf fragments, stem segments, etc.).
  • a callus can be produced by dedifferentiating the material removed from the plant by stimulating it with a plant hormone or the like.
  • callus is sometimes referred to as "healing cells" because it is formed to cover wounds of plants in the natural world. Callus does not include these natural substances themselves.
  • callus refers to what is artificially produced.
  • a callus is composed of a cell mass, ie, an aggregate of a plurality of cells.
  • the present invention provides callus consisting of seed cells of a fagaceous plant. In another aspect, the present invention provides callus composed of seed-derived germination cells of a fagaceous plant.
  • Fagaceae plants include, for example, those belonging to the subfamily Fagoideae, Quercoideae, Castaneoideae, and further, for example, the genus As, beech genus (Fagus), Antarctic beech genus (Nothofagus), Quercus genus (Quercus), Trigonobalanus (Castanea), Chestnut genus (Castanea), Castanopsis (Castanopsis), Lithocarpus (Lithocarpus), Nisematebashii Examples include plants belonging to the Fagaceae family such as the genus Notholithocarpus and the genus Chrysolepis. Fagaceous plants are plants that become trees, and are usually woody plants, most of which are trees (arbors).
  • Fagaceae plants include beech, oak, konara oak, mizunara oak, oak, sawtooth oak, matebashi, shii, oak, chestnut, white oak, and oak. be able to.
  • Plants of the genus Fagaceae (scientific name: Fagus, English name: beech) are particularly preferable as fagaceous plants, and among them, beech (scientific name: Fagus crenata, English name: Japanese beech) and European beech (scientific name: Fagus sylvatica) , English name: European beech) is more preferred, and beech (Japanese beech) is even more preferred.
  • Japanese beech is sometimes referred to as shiro-buna, kuro-buna, dog-buna, hon-buna, buckwheat-buckwheat, kohabuna, oohabuna, etc., but all of these are included.
  • the scientific name of Japanese beech and Japanese beech is Fagus japonica, in this specification, they are included in beech (Japanese beech).
  • the place of production of the fagaceous plant is not particularly limited, but preferably Japanese or European beech can be used.
  • beech from Shirakami-Sanchi in Japan a mountainous region spanning the northwestern part of Akita Prefecture and the southwestern part of Aomori Prefecture
  • Seeds of fagaceae plants can be used by collecting seeds that naturally fall from fagaceae plants (eg beech trees). Seeds that are generally called acorns can be used. However, an acorn correctly means a fruit (also called a nut or solid), and within the acorn is the seed. Compared to the use of bark, the use of seeds has the advantage that existing plants are not damaged and raw materials can be collected continuously. In one aspect of the present invention, callus is induced from seeds of fagaceous plants.
  • the sprout derived from the seed of the fagaceae plant is a plant body obtained by germinating the seed of the fagaceous plant described above.
  • Germination means that the seed absorbs water and the radicle, which is part of the embryonic tissue, breaks through the seed coat and emerges.
  • Germination can be carried out artificially, or naturally germinated seeds may be used, but it is preferable to carry out the germination artificially.
  • Artificial germination can be performed by a known method, and preferably includes, for example, a method under aseptic germination conditions.
  • the seed germinates to obtain a germination body.
  • Aseptic culture conditions are, for example, using a medium for plant culture such as WPM medium as a medium, room temperature (for example, 20 to 25 ° C.), day length longer than a certain time (for example, 12 hours or more). , over a period of days to months (eg, weeks to a month).
  • a medium for plant culture such as WPM medium as a medium, room temperature (for example, 20 to 25 ° C.), day length longer than a certain time (for example, 12 hours or more).
  • days to months eg, weeks to a month.
  • the germination for example, those at the stage where the radicle has broken the seed coat, at the stage of sprout (seedling), and further at the stage of sprout can be used.
  • a germinant may have a root (such as a radicle), a leaf (such as a cotyledon), and a stem (such as a hypocotyl).
  • the germination body may be used at the cotyledon development stage.
  • callus is induced from germinated seeds of fagaceous plants.
  • seeds of a fagaceous plant or germination cells derived from seeds of a fagaceous plant are cultured and proliferated in a medium in the presence of a plant growth regulator.
  • the seeds are cut into a plurality of pieces (for example, two pieces).
  • the formation of cuts facilitates the formation of callus.
  • the seeds can be cut, for example, so that the major axis of the oval shape is divided into about two parts.
  • it is preferable to sterilize the seeds with an alcohol such as ethanol or a sterilizing liquid such as a sodium hypochlorite solution.
  • Some or all of the seed cells are then placed in culture medium. When using a portion of the seed cells, the seed cells can be appropriately cut into appropriate sizes and used.
  • a part of the germinated bodies preferably the hypocotyl (the part of the stem between the root and the leaf), is cut off from the germinated bodies obtained by the above-described method, and then cut off. are cut into appropriate sizes as necessary and placed in the culture medium.
  • the cells are exposed and the cells of the germination body are ready for culturing.
  • the cells (seed cells or germination cells) placed in the medium are cultured and proliferated by cell culture, and are induced into callus, which is a cell mass. At this time, for example, a part of the seed or the germination is raised by cell proliferation. The portion that swells due to this proliferation becomes a callus.
  • the medium may be a solid medium or a liquid medium, but a solid medium is preferred.
  • seed or germination cells can be cultured by placing them directly on the medium (by contacting the medium).
  • a medium containing additive components such as plant growth regulators can be used.
  • the medium (basal medium) is not particularly limited. ), among which WPM medium is preferred.
  • WPM represents Woody plant medium
  • BTM represents Broadleaf Tree Medium
  • MS represents Murasige Skoog.
  • the composition of media is known. Media are commercially available.
  • the medium can be formed into a solid medium by, for example, heating a solution obtained by dissolving a composition serving as a substrate of the medium in water, adding a support (e.g., agar, etc.), and cooling and solidifying the solution. can be done. At this time, by adding the plant growth regulator to the solution, a medium containing the plant growth regulator can be obtained.
  • a support e.g., agar, etc.
  • a plant growth regulator is a substance that regulates plant growth (preferably growth promotion), and may include, for example, what is called a plant hormone.
  • Plant growth regulators include auxin, cytokinin, gibberellin, abscisic acid, ethylene, and combinations of two or more thereof. Auxins are preferably used as plant growth regulators. When auxin is used, callus can be produced efficiently.
  • a plant growth regulator is synonymous with a plant growth regulator.
  • auxins include picloram, dicamba, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5- from trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 2-methyl-4-chlorophenoxybutyric acid, and naproanilide, or salts thereof (for example inorganic salts such as sodium and calcium salts) It is preferably selected. Thereby, callus can be produced more efficiently.
  • Auxin is more preferably picloram or dicamba, and still more preferably picloram.
  • auxin and cytokinin are also preferable to use as a plant growth regulator. Thereby, callus can be produced more efficiently. In particular, it is preferable to combine them when culturing germinal cells.
  • the cytokinin may be a synthetic cytokinin. Cytokinins include 6-benzylaminopurine, trans-Zeatin and the like, with 6-benzylaminopurine being preferred.
  • a specific combination of auxin and cytokinin is, for example, a combination of picloram and 6-benzylaminopurine.
  • the concentration of auxin (eg, picloram) in the medium is preferably 0.1-20 ⁇ M, more preferably 0.5-10 ⁇ M, and 1-10 ⁇ M. is more preferred, and 1 ⁇ M is even more preferred.
  • the concentration of cytokinin (eg, 6-benzylaminopurine) in the medium is preferably 1-10 ⁇ M, more preferably 2 ⁇ M.
  • a combination of 0.1-20 ⁇ M auxin and 1-10 ⁇ M cytokinin for example, a combination of 0.1-20 ⁇ M picloram and 1-10 ⁇ M 6-benzylaminopurine, specifically is preferably a combination of 1 ⁇ M picloram and 2 ⁇ M 6-benzylaminopurine, or a combination of 10 ⁇ M picloram and 2 ⁇ M 6-benzylaminopurine.
  • the conditions for cell culture are not particularly limited, but it can be carried out in a culture room under conditions of 20-25°C and a day length of 12 hours or more.
  • the cell culture time is preferably 2 weeks to 2 months, more preferably 1 month.
  • cell culture may be continued until it is confirmed by visual observation that a sufficient amount of callus is obtained.
  • Cell culture may or may not be performed under sterile conditions.
  • callus with different morphologies eg, color, hardness
  • white callus can be obtained from beech seeds, brown callus or green callus from beech germlings (see Examples).
  • Such calluses also differ in hardness. It has been confirmed that these calluses differ not only in morphology but also in action.
  • the cells in the medium proliferate by cell culture and produce callus, which is a cell mass.
  • Callus can be formed on the surface of seeds or sprouts in a raised manner. Callus can be used for extraction separately from seeds and germination bodies (so to speak, mother body) used for culture, or together with seeds and germination bodies (for each culture).
  • callus extracts derived from the above callus.
  • callus extracts extracts from callus are simply referred to herein as "callus extracts.”
  • Callus produced by the production method described above can be used. Therefore, the callus extract is an extract from fagaceous plant seed callus or an extract from fagaceous plant seed germinated callus.
  • Extraction from callus can be performed by a method that conforms to the extraction method used when producing known plant extracts.
  • the callus can be freeze-dried to remove moisture, the resulting dried product is cut or pulverized into small pieces or powder, and extracted with an extraction solvent.
  • an organic solvent water, an organic solvent, or a mixture of water and an organic solvent can be used.
  • a hydrophilic organic solvent or a hydrophobic organic solvent can be used, but a hydrophilic organic solvent is preferred, an alcohol (eg, a lower alcohol) is preferred, and ethanol or methanol is preferred.
  • Preferred solvents include water or mixtures of water and alcohols such as ethanol.
  • the ratio of the organic solvent (eg, alcohol) to water (organic solvent/water) is preferably 1/99 to 99/1, preferably 1/9. ⁇ 9/1 is more preferred, for example a proportion of organic solvent (eg alcohol) of 50% is more preferred.
  • the extraction may be performed at room temperature or under heating. For example, extraction can be performed under temperature conditions from room temperature to the boiling point of the organic solvent used (eg, 80°C). The extraction may be performed in a standing state or in a stirred state.
  • the extraction time is not particularly limited, but may be, for example, 1 hour to 1 day, preferably 1 hour.
  • a solution (callus extract) containing the components extracted from the callus can be obtained by removing the residue (solid matter) by a method such as filtration.
  • the extract can be used as an extract as it is or after being concentrated.
  • an extract in the form of solid, oil or paste can be obtained by removing liquid components (water, alcohol, etc.) from the extract.
  • the callus extract is preferably in the form of a solid powder. Callus extracts can be stored at room temperature or refrigerated, for example, in amber glass containers.
  • the callus extract thus obtained has anti-oxidation, suppression of melanin synthesis, anti-aging, whitening, enhancement of cell proliferation, synthesis of collagen, synthesis of hyaluronic acid, anti-inflammatory, gene repair, cell differentiation, cell regeneration, promotion of metabolism, hair growth, and It can have one or more actions selected from the group consisting of anti-cancer.
  • the callus extract is preferably one or more selected from the group consisting of antioxidant, melanin synthesis suppression, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, metabolism promotion, and hair growth. It is an active callus extract.
  • the above effects of the callus extract have been experimentally confirmed as described in Examples below.
  • a cosmetic having the above effects can be obtained. It can also be applied to topical administration type medicines.
  • the above action may be exerted orally, and can be applied to pharmaceuticals (oral administration type), foods, and the like. More preferably, it is used in applications other than medicine, preferably in cosmetics or foods, and even more preferably in cosmetics.
  • cosmetics may include quasi-drugs. However, it may be used in cosmetics other than quasi-drugs.
  • the callus induced from the beech seeds and the germinated body has an improved amount and quality of the active ingredient compared to the active ingredient possessed by the beech seed itself and the germinated body itself.
  • the callus formed by cell proliferation increases the active ingredient along with the proliferation.
  • the callus extract extracted from the callus with increased active components also has an increased active component, and that the above-mentioned useful effects are brought about.
  • the active ingredient can be increased by cell culture, so a useful beech extract can be obtained more efficiently than when the plant itself is used.
  • the present invention is not limited to the above speculation.
  • the present invention relates to cosmetic compositions comprising a callus extract and a cosmetic base.
  • the callus extract those described above can be used.
  • callus extract anti-oxidation, suppression of melanin synthesis, anti-aging, whitening, enhancement of cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, promotion of metabolism, and hair growth.
  • a cosmetic product can be obtained which can have one or more effects selected from the group consisting of: Cosmetics preferably have one or more actions selected from the group consisting of antioxidant, melanin synthesis suppression, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, metabolism promotion, and hair growth.
  • the specific efficacy and effect of the cosmetic produced by the above action are not particularly limited, but include whitening, anti-wrinkle, anti-blemish, improvement of firmness, improvement of skin moisture, improvement of skin barrier effect, Promotion of turnover of skin cells and improvement of resilience from damage such as ultraviolet rays.
  • the cosmetic base may be a component that can be blended as a cosmetic.
  • a cosmetic base means a component that forms the skeleton of a cosmetic product.
  • examples of cosmetic bases include, but are not limited to, water, oils, alcohols (monohydric or polyhydric alcohols), surfactants, emulsifiers, suspending agents, polymers, powders, and the like. be done.
  • oil agents include, but are not limited to, natural or synthetic ester oils, hydrocarbon oils, higher alcohols, fatty acids, silicone oils, and the like, and specifically, are not limited to these. No, but for example liquid paraffin, petroleum jelly, ethyl oleate, stearic acid, palmitic acid, squalane, cetanol, cholesterol, beeswax, shea butter, behenyl alcohol, cetostearyl alcohol, batyl alcohol, jojoba oil, macadamia nut oil, meadowfoam oil, hydrogenated coconut oil, hydrogenated palm oil, hydrogenated castor oil stearate, olive oil, hydrogenated polyisobutene, polyethylene glycol, dimethylpolysiloxane, dimethicone, and the like.
  • alcohols include, but are not limited to, ethanol, isopropanol, butanol, glycerin, 1,3-butylene glycol, propylene glycol, dipropylene glycol, and the like. be done.
  • surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. Specific examples include, but are not limited to, sodium laurate, Sodium lauryl sulfate, polysorbate 80, glyceryl oleate, polyglyceryl laurate, sorbitan stearate and the like.
  • polymers include naturally occurring polymers and synthetic polymers, and specifically, but not limited to, gums such as xanthan gum, gellan gum, gum arabic, guar gum, hydroxypropyl Cellulose such as cellulose, hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol and the like.
  • gums such as xanthan gum, gellan gum, gum arabic, guar gum, hydroxypropyl Cellulose such as cellulose, hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol and the like.
  • powders include inorganic powders and organic powders, but are not limited to these, but examples include titanium oxide, zinc oxide, talc, mica, silica, polyethylene terephthalate (PET) powder, and the like. mentioned.
  • the cosmetic composition may contain ingredients other than the cosmetic base.
  • ingredients other than the cosmetic base examples include water-soluble components, oil-soluble components, moisturizers, thickeners, pigments, ultraviolet absorbers, film-forming agents, pH adjusters, anti-fading agents, antioxidants, antifoaming agents, Cosmetic ingredients, preservatives, fragrances, and the like.
  • the cosmetic composition may contain an oil agent, a water-soluble polymer and a powder that do not constitute a cosmetic base.
  • the cosmetic composition may contain a physiologically active substance (so-called active ingredient) other than the callus extract.
  • the cosmetic composition may be a composition in an appropriate form that can be used as cosmetics.
  • Forms of cosmetic compositions include, for example, aqueous solutions, oils, emulsions (O/W type, W/O type, W/O/W type, etc.), pastes, powders, solids, and the like.
  • a spray agent, a mist agent, or the like may be used.
  • the cosmetic composition itself may be used as a cosmetic product.
  • the cosmetic composition may be used as a raw material (work-in-progress) for manufacturing the final cosmetic product.
  • the cosmetic composition can be obtained by appropriately mixing the above callus extract with the raw materials to be blended into the cosmetic composition as described above. Accordingly, the present invention provides the use of the above callus extracts for the manufacture of cosmetic compositions.
  • the cosmetic composition may be a composition for skin cosmetics.
  • the skin may be the skin of the face and the skin of parts of the body other than the face (head, neck, shoulders, hands, feet, etc.), with facial skin and scalp being preferred.
  • Skin cosmetics are not particularly limited, but specific examples include emulsions, creams, serums, lotions, hand creams, eye creams, body creams, makeup cosmetics, concealers, cheeks, eye creams, Cosmetics such as shadows (eye colors), makeup bases, foundations (eg, liquid foundations, solid foundations), and sunscreens are exemplified.
  • examples of cosmetics for the scalp include cosmetics such as hair growth agents, hair growth agents, and hair tonics.
  • the cosmetic composition may be a composition for cosmetics for hair.
  • hair cosmetics include cosmetics such as hair cream, hair wax, hair rinse, hair mask, and hair treatment.
  • the present invention provides antioxidant, melanin synthesis suppression, anti-aging, whitening, and cell proliferation enhancement comprising applying the above callus extract to the skin of a human subject.
  • the callus extract is, as described above, obtained from the seed of the fagaceous plant or from the germinated callus derived from the seed of the fagaceous plant.
  • the application of the callus extract to the skin of human subjects is preferably non-medical application.
  • the callus extract is applied to human skin for cosmetic use, preferably by means of a cosmetic composition.
  • the callus extract can be applied to the skin of a human subject by applying the cosmetic composition to the skin.
  • the callus extract or components therein permeate the skin, and the above effects can be exhibited.
  • application of the callus extract to the skin does not involve pharmaceutical therapeutic use.
  • the present invention provides antioxidation, suppression of melanin synthesis, One or more actions selected from the group consisting of anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anticancer.
  • the callus extract is, as described above, obtained from the seed of the fagaceous plant or from the germinated callus derived from the seed of the fagaceous plant.
  • the callus extract in one aspect, contains normal human fibroblasts (also referred to herein simply as “human fibroblasts” or “fibroblasts”) or normal human epidermal keratinocytes (herein Also referred to herein simply as “human epidermal keratinocytes” or “epidermal keratinocytes”).
  • the callus extract is applied to the skin of a human subject. Such applications are also referred to as so-called in vivo.
  • the callus extract is preferably applied to human skin by means of a cosmetic composition. By applying the cosmetic composition to the skin of a human subject, the callus extract can be provided to human fibroblasts or human epidermal keratinocytes within the skin.
  • the callus extract or components therein permeate the skin and affect normal human fibroblasts or normal human epidermal keratinocytes, resulting in the above effects.
  • the expression level of related genes can be increased.
  • the callus extract can, in other embodiments, be subjected to cultured normal human fibroblasts or human epidermal keratinocytes. Such applications are also referred to as so-called in vitro.
  • components in the callus extract affect normal human fibroblasts or normal human epidermal keratinocytes. , can increase the expression of genes associated with the above effects.
  • callus extracts can also be useful for animals other than humans (particularly mammals such as dogs, cats, rats, and mice). Callus extracts are also applicable to animals other than humans. When applied to animals other than humans (for example, applied to the skin), the same effects as described above (for example, increase in gene expression level, etc.) can be exhibited.
  • FIG. 1 is a photograph showing an example of callus production in Examples 1 and 2, and FIG. 1A shows beech seeds.
  • Example 1 Preparation of beech seed callus The pericarp and seed coat were removed from beech seeds, cut into two, and sterilized by soaking in 70% ethanol for 30 seconds and 10% sodium hypochlorite solution for 5 minutes (Fig. 1B). The seeds were cut, for example, so that the long axis of the oval shape was divided into about two parts. On the other hand, picloram was added to the raw material of WPM medium to prepare a solution, and this mixed solution was placed in a cell culture dish to prepare a WPM medium containing 10 ⁇ M picloram (referred to as WPM medium 1). Seeds from which the pericarp and seed coat were removed were added to this picloram-containing WPM medium, and cultured at 25° C.
  • Example 1 The callus obtained in Example 1 is hereinafter referred to as callus 1.
  • the culture was removed and a portion of the callus was excised and used for the next extraction step.
  • Example 2 Production of callus from beech seed germination by removing pericarp and seed coat from beech seeds, placing the seeds in WPM medium (phytohormone-free medium), and aseptically Within one month of culturing, germinated bodies (seedlings) were obtained (Fig. 1C). The hypocotyl (the part between the leaf and the root) of the germination was cut and collected, and cut into pieces of a size similar to a cube with a side of 3 to 10 mm.
  • WPM medium phytohormone-free medium
  • WPM medium 2 a WPM medium containing 10 ⁇ M picloram and 2 ⁇ M 6-benzylaminopurine
  • WPM medium 3 a WPM medium containing 1 ⁇ M picloram and 2 ⁇ M 6-benzylaminopurine
  • callus 2 The callus obtained from WPM medium 2 (hereinafter referred to as callus 2) was brown.
  • the callus obtained from WPM medium 3 (hereinafter referred to as callus 3) was green.
  • the culture was removed and a portion of the callus was excised and used for the next extraction step.
  • Example 3 Preparation of callus extract
  • callus extract 1 the callus extract obtained from callus 1
  • callus extract 2 the callus extract obtained from callus 3
  • callus extract 3 the callus extract obtained from callus 3
  • Example 4 Gene Expression Biochemical Assay of Human Skin Cells with Callus Extracts1. Preparation of sample for assay In order to clarify the functionality of the beech callus extract on human skin, the beech callus extract was applied to human skin cells and the gene expression level was analyzed using real-time PCR. investigated the effect of The total amount of each callus extract obtained above was dissolved in 1 to 1.5 mL of dimethyl sulfoxide (DMSO) to prepare assay samples.
  • DMSO dimethyl sulfoxide
  • a European beech bud extract (hereinafter referred to as "comparative extract 1"), which is generally commercially available as a raw material for cosmetics, was used.
  • the European beech bud extract is an extract (not derived from callus) obtained by concentrating the extract obtained by extracting the seedlings of European beech (scientific name: Fagus sylvatica, English: European beech) with water.
  • FIG. 2 is a graph showing cell viability when callus extract is applied to cells.
  • the addition concentration the highest concentration within the range that does not adversely affect the cells (decrease in viability) was adopted. For example, when callus extract 2 is applied to fibroblasts, it is 0.012% (w/v) (Fig. 2A), and when callus extract 3 is applied to epidermal keratinocytes, it is 0.112% (w/v). ) (Fig. 2B).
  • Cells normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in a cell culture dish, and assay samples were added when an appropriate cell amount was reached.
  • fibroblasts experiments were conducted with callus extracts 1 to 3 and comparative extract 1.
  • epidermal keratinocytes experiments were conducted with callus extracts 2 and 3 and comparative extract 1.
  • Cells were cultured for 24 hours after sample addition and cells were analyzed by real-time PCR. QuantStudio 12K Flex (ThermoFisher) was used as a real-time PCR system.
  • ADAM10 hair growth
  • ADAM12 hair growth
  • PPARG hair growth, antioxidant
  • CCND1 gene repair
  • GLO1 gene repair
  • PARK7 gene repair
  • RBMX gene repair
  • TP53BP1 gene repair
  • CCL2 anti-inflammatory
  • IL1A anti-inflammatory
  • IL6 anti-inflammatory
  • NFKB1 anti-inflammatory
  • PTGS2 anti-inflammatory
  • STAT3 anti-inflammatory
  • TGFB1 anti-inflammatory
  • EXT1 anti-cancer
  • NF1 anti-inflammatory
  • ADAM10 hair growth
  • CEBPA hair growth
  • CCND1 gene repair
  • GLO1 gene repair
  • HAGH gene repair
  • PARK7 gene repair
  • RBMX gene repair
  • TP53BP1 gene repair
  • COL17A1 cell differentiation regeneration
  • DKK3 cell differentiation/regeneration
  • ITGA6 cell differentiation/regeneration
  • LAMA5 cell differentiation/regeneration
  • WNT5A cell differentiation/regeneration
  • CXCL8 anti-inflammatory
  • IL1A anti-inflammatory
  • NFKB1 anti-inflammatory
  • PTGS2 anti-inflammatory
  • STAT3 anti-inflammatory
  • TGFB1 anti-inflammatory
  • GABPA anti-inflammatory, antioxidant, anti-aging
  • CDKN2A anti-cancer
  • EXT1 anti-cancer
  • PTEN PTEN
  • Results Fibroblast Gene Expression Table 1 shows the results of gene expression levels in normal human dermal fibroblasts (NHDF).
  • NHDF normal human dermal fibroblasts
  • the mRNA expression level was evaluated as a relative value with respect to the control (control value is set to 1).
  • usefulness was indicated as "high” when the callus extract (at least one of callus extracts 1 to 3) showed particularly high usefulness.
  • upregulation increase in expression level
  • downregulation decrease in expression level
  • FIGS. 3 and 4 show graphs showing representative results extracted from the results of gene expression analysis in fibroblasts. "High” and “Low” on the vertical axis of the graph mean “highly effective” and “lowly effective”, respectively.
  • FIG. 3A is a graph of SOD2, which encodes a reactive oxygen scavenging enzyme, so an increase in the expression level suggests an improvement in antioxidant activity.
  • FIG. 3B is a graph of KITLG, which is a gene that promotes melanin synthesis, so a decrease in the expression level suggests an improvement in melanin synthesis inhibitory action.
  • FIG. 3C is a graph of SIRT1, and increased expression suggests improved anti-aging effects.
  • FIG. 3D is a graph of FGF7, and decreased expression suggests improved whitening effect.
  • FIG. 3A is a graph of SOD2, which encodes a reactive oxygen scavenging enzyme, so an increase in the expression level suggests an improvement in antioxidant activity.
  • FIG. 3B is a graph of KITLG,
  • FIG. 4A is a graph of FGF2, and an increase in the expression level suggests an improvement in cell proliferation-enhancing action.
  • FIG. 4B is a graph of MMP1, in which decreased expression suggests enhanced collagen synthesis.
  • FIG. 4C is a graph of HAS2, and an increase in the expression level suggests an improvement in hyaluronic acid synthesis.
  • FIG. 4D is a graph of ADAM10, and decreased expression suggests improved hair growth.
  • the above table shows the superiority of the beech callus-derived extract.
  • callus extract 2 showed the gene DKK3, whose expression level leads to increased cell proliferation, and FGF2 showed a higher expression level in callus extracts 1 and 2 than comparative extract 1.
  • the expression level of gene HAS2, which promotes hyaluronic acid synthesis was higher in callus extracts 1 and 2 than in comparative extract 1.
  • the gene FGF7 whose expression level decreases leads to whitening in all callus extracts 1 to 3
  • the gene KITLG in callus extract 1 has a lower expression level than the comparative extract 1
  • the gene DKK1 whose expression level increases leads to whitening in callus extract 1.
  • the expression level increased from the comparative extract 1.
  • the beech callus-derived extract contained a plurality of genes suggesting higher efficacy than the comparative extract, suggesting that these callus extracts are more effective than the comparative extract for the living body including the skin.
  • the test on biological utility there may be differences in the test results due to the target biological factors and / or test conditions, but from the above results, as a whole, the beech callus-derived It can be said that the effectiveness of the extract was demonstrated.
  • Table 2 shows the results of gene expression levels in normal human epidermal keratinocytes (NHEK).
  • NHEK normal human epidermal keratinocytes
  • the mRNA expression level was evaluated as a relative value with respect to the control (control value is set to 1).
  • usefulness was indicated as "high” when the callus extract (at least one of callus extracts 2 and 3) showed particularly high usefulness.
  • upregulation increase in expression level
  • downregulation decrease in expression level
  • FIG. 5 shows a graph showing representative results extracted from gene expression analysis results in epidermal keratinocytes. "High” and “Low” on the vertical axis of the graph mean “highly effective” and “lowly effective”, respectively.
  • FIG. 5A is a graph of WNT5A, and a decrease in the expression level suggests an improvement in cell differentiation/regeneration action.
  • FIG. 5B is a graph of DKK3, and a decrease in the expression level suggests an improvement in cell differentiation/regeneration action.
  • FIG. 5C is a graph of KITLG, and a decrease in the expression level suggests an improvement in melanin synthesis inhibitory action.
  • FIG. 5D is a graph of DKK1, and an increase in the expression level suggests an improvement in melanocyte growth inhibitory activity.
  • FIG. 5E is a graph of AQP3, with increased expression suggesting improved metabolism.
  • the above table shows the superiority of the beech callus-derived extract.
  • the expression level of AQP3, a gene whose expression level promotes metabolism, is greater in callus extract 2 than in comparative extract 1.
  • the expression levels of genes DKK1 and LAMC2, whose expression level increases lead to skin whitening, are increased in callus extracts 2 and 3 compared to comparative extract 1.
  • the callus extract showed a higher gene expression level than the comparative extract for some genes, suggesting that the callus extract is more effective than the comparative extract for living organisms including the skin.
  • the beech callus extract of the example has anti-aging, whitening and melanin synthesis-related effects, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, And it was confirmed that it can have an action against cancer. In particular, excellent effects were confirmed in suppressing melanin synthesis, whitening, enhancing cell proliferation, and synthesizing hyaluronic acid. Therefore, by using the beech callus extract, highly functional cosmetics can be obtained. It should be noted that the above actions (eg, antioxidant action, gene repair action, etc.) may be exhibited not only when directly applied to the skin but also when orally ingested. Therefore, by using the beech callus extract, it is possible to obtain orally ingested medicines and functional foods (for example, beauty foods, health foods, etc.).
  • orally ingested medicines and functional foods for example, beauty foods, health foods, etc.

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Abstract

La présente invention concerne des cals constitués de cellules de graines d'une plante Fagaceae ou de corps germinatifs dérivés de graines d'une plante Fagaceae. La présente invention concerne également un procédé de production des cals, un extrait des cals, une composition cosmétique contenant l'extrait de cals, etc. La présente invention permet de procurer : des cals de graines et de corps en germination d'une plante Fagaceae, lesdits cals étant utiles comme matière première de cosmétiques et comme cosmétique ; un procédé de production des cals ; un extrait de cals de graines et de corps en germination d'une plante Fagaceae ; un procédé utilisant l'extrait de cals ; et une composition cosmétique contenant un extrait de cals de graines et de corps en germination d'une plante Fagaceae.
PCT/JP2022/034255 2021-09-14 2022-09-13 Cals dérivés d'une plante fagaceae, extrait de cals, procédé et composition cosmétique les utilisant WO2023042826A1 (fr)

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WO2002022838A2 (fr) * 2000-09-18 2002-03-21 The Scotts Company Paturins transgeniques
JP2004018471A (ja) * 2002-06-18 2004-01-22 Shiseido Co Ltd Ｉｖ型、ｖｉｉ型コラーゲン産生促進剤および皮膚基底膜ケア用組成物
KR20140090896A (ko) * 2013-01-10 2014-07-18 홍소영 방울 양배추 추출물을 유효성분으로 하는 화장료 조성물
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