WO2023042826A1 - Calluses derived from fagaceae plant, callus extract, and method and cosmetic composition using same - Google Patents

Calluses derived from fagaceae plant, callus extract, and method and cosmetic composition using same 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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PCT/JP2022/034255
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French (fr)
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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    • 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.

Abstract

The present invention pertains to calluses consisting of cells of seeds of a Fagaceae plant or seed-derived germinating bodies of a Fagaceae plant. The present invention also pertains to a method for producing the calluses, an extract of the calluses, a cosmetic composition containing the callus extract, etc. The present invention makes it possible to provide: calluses of seeds and germinating bodies of a Fagaceae plant, said calluses being useful as a starting material of cosmetics and as a cometic; a method for producing the calluses; an extract of calluses of seeds and germinating bodies of a Fagaceae plant; a method using the callus extract; and a cosmetic composition containing a callus extract of seeds and germinating bodies of a Fagaceae plant.

Description

ブナ科植物由来カルス、カルス抽出物、それを用いた方法および化粧品組成物Fagaceous plant-derived callus, callus extract, method using the same, and cosmetic composition
　本発明は、ブナ科植物由来のカルス、そのカルスの製造方法、ブナ科植物由来のカルスの抽出物、そのカルス抽出物を用いた方法、および、ブナ科植物由来のカルス抽出物を含む化粧品組成物に関する。 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.
　従来、化粧品の原料として、植物の抽出物が用いられている。その中で、植物としてブナを由来とする化粧品原料も知られている。例えば、特許文献１では、パセリから抽出されるエキスとブナの木から抽出されるエキスを組み合わせて含有する皮膚化粧料が開示されている。植物由来の化粧品原料は、皮膚へのさまざまな有用な活性を期待して、化粧品に配合され得る。 Traditionally, plant extracts have been used as raw materials for cosmetics. Among them, cosmetic raw materials derived from beech as a plant are also known. For example, 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.
特開平１１－３３５２５７号公報JP-A-11-335257
　本発明者らは、鋭意検討を行った結果、ブナ科植物の種子および発芽体から誘導されたカルスの抽出物が、生化学的に有用な活性を有することを見出し、本発明を完成させるに至った。
　したがって、本発明は、ブナ科植物の種子および発芽体のカルス、そのカルスの製造方法、ブナ科植物の種子および発芽体のカルスの抽出物、そのカルス抽出物を用いた方法、および、ブナ科植物の種子および発芽体のカルス抽出物を含む化粧品組成物、を提供することを目的とする。 As a result of intensive studies, the present inventors found that extracts of callus induced from seeds and germinations of fagaceous plants have biochemically useful activities. Arrived.
Therefore, 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.
　本発明は、下記に挙げられる実施態様を含むが、これらに限定されるものではない。
［１］　ブナ科植物の種子またはブナ科植物の種子由来の発芽体の、細胞からなる、カルス。
［２］　前記細胞が、植物生長調節物質で処理した細胞である、［１］に記載のカルス。なお、本明細書において、植物生長調節物質は、植物生長調整物質と同義である。
［３］　植物生長調節物質が、オーキシン（ａｕｘｉｎ）である、［２］に記載のカルス。
［４］　オーキシンが、ピクロラム（ｐｉｃｌｏｒａｍ）、ジカンバ（ｄｉｃａｍｂａ）、インドール－３－酢酸、インドール－３－酪酸、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、２，４－ジクロロフェノキシ酢酸、２，４，５－トリクロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酪酸、および、ナプロアニリド、またはそれらの塩、から選択される、［３］に記載のカルス。
［５］　前記細胞が、ブナ科植物の種子の細胞である、［１］～［４］のいずれか１つに記載のカルス。
［６］　前記細胞が、ブナ科植物の種子由来の発芽体の細胞である、［１］～［４］のいずれか１つに記載のカルス。
［７］　前記発芽体が、ブナ科植物の種子を無菌発芽の条件下で発芽させた発芽体である、［６］に記載のカルス。
［８］　ブナ科植物が、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）である、［１］～［７］のいずれか１つに記載のカルス。
［９］　ブナ科植物の種子またはブナ科植物の種子由来の発芽体の細胞を、培地中、植物生長調節物質の存在下で培養して増殖させることを含む、カルスの製造方法。
［１０］　植物生長調節物質が、オーキシン（ａｕｘｉｎ）である、［９］に記載のカルスの製造方法。
［１１］　オーキシンが、ピクロラム（ｐｉｃｌｏｒａｍ）、ジカンバ（ｄｉｃａｍｂａ）、インドール－３－酢酸、インドール－３－酪酸、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、２，４－ジクロロフェノキシ酢酸、２，４，５－トリクロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酪酸、および、ナプロアニリド、またはそれらの塩、から選択される、［１０］に記載のカルスの製造方法。
［１２］　前記培地が、ＷＰＭ培地である、［９］～［１１］のいずれか１つに記載のカルスの製造方法。
［１２－１］　前記細胞が、ブナ科植物の種子の細胞である、［９］～［１２］のいずれか１つに記載のカルスの製造方法。
［１２－２］　前記細胞が、ブナ科植物の種子由来の発芽体の細胞である、［９］～［１２］のいずれか１つに記載のカルスの製造方法。
［１２－３］　前記発芽体が、ブナ科植物の種子を無菌発芽の条件下で発芽させた発芽体である、［１２－２］に記載のカルスの製造方法。
［１３］　ブナ科植物が、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）である、［９］～［１２］、［１２－１］～［１２－３］のいずれか１つに記載のカルスの製造方法。
［１４］　［１］～［８］のいずれか１つに記載のカルス由来の、カルス抽出物。
［１５］　水、または水とアルコールの混合液による抽出物である、［１４］に記載のカルス抽出物。
［１５－１］　水とエタノールの混合液による抽出物である、［１４］に記載のカルス抽出物。
［１６］　抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を有する、［１４］、［１５］または［１５－１］に記載のカルス抽出物。
［１６－１］　化粧品組成物に使用するための、［１４］～［１６］のいずれか１つに記載のカルス抽出物。
［１７］　［１４］～［１６］のいずれか１つに記載のカルス抽出物、および化粧品基剤を含む、化粧品組成物。
［１７－１］　抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、新陳代謝促進、または育毛のための、［１７］に記載の化粧品組成物。
［１８］　［１４］～［１６］のいずれか１つに記載のカルス抽出物をヒト被験者の皮膚に適用することを含む、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を供するための方法。
［１８－１］　医薬以外の用途で、例えば、化粧品用途、食品用途など、好ましくは化粧品用途で、カルス抽出物を適用するものである、［１８］に記載の方法。
［１９］　［１４］～［１６］いずれか１つに記載のカルス抽出物を供することを含む、ヒト繊維芽細胞またはヒト表皮角化細胞における、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用に関連する遺伝子の発現量を増加するための方法。［１９－１］　医薬以外の用途で、例えば、化粧品用途、食品用途、好ましくは化粧品用途で、カルス抽出物を供するものである、［１９］に記載の方法。
［２０］　化粧品組成物の製造のための、［１４］～［１６］のいずれか１つに記載のカルス抽出物の使用。
［２０－１］　化粧品組成物が、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、新陳代謝促進、または育毛のためのものである、［２０］に記載の使用。 The present invention includes, but is not limited to, the embodiments listed below.
[1] A callus consisting of cells of a seed of a fagaceous plant or a germination body derived from a seed of a fagaceous plant.
[2] The callus of [1], wherein the cells are cells treated with a plant growth regulator. In addition, in this specification, a plant growth regulator is synonymous with a plant growth regulator.
[3] The callus of [2], wherein the plant growth regulator is auxin.
[4] 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.
[5] The callus according to any one of [1] to [4], wherein the cell is a seed cell of a fagaceous plant.
[6] The callus according to any one of [1] to [4], wherein the cell is a seed-derived germination cell of a fagaceous plant.
[7] The callus of [6], wherein the germinating body is a germinating body obtained by germinating a seed of a fagaceous plant under aseptic germination conditions.
[8] The callus of any one of [1] to [7], wherein the fagaceous plant is Japanese beech.
[9] A method for producing callus, which comprises culturing and proliferating the seed of a fagaceous plant or the germination cell derived from the seed of a fagaceous plant in a medium in the presence of a plant growth regulator.
[10] The method for producing callus according to [9], wherein the plant growth regulator is auxin.
[11] 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] The method for producing callus according to any one of [9] to [11], wherein the medium is WPM medium.
[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].
[15] The callus extract of [14], which is extracted with water or a mixture of water and alcohol.
[15-1] 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] The callus extract of any one of [14] to [16], for use in a cosmetic composition.
[17] A cosmetic composition comprising the callus extract of any one of [14] to [16] and a cosmetic base.
[17-1] The cosmetic composition of [17], which is for anti-oxidation, suppression of melanin synthesis, anti-aging, whitening, enhancement of cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, promotion of metabolism, or hair growth.
[18] 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 A method for providing one or more effects selected from the group consisting of synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism enhancement, hair growth, and anti-cancer.
[18-1] The method according to [18], wherein the callus extract is applied for uses other than medicine, such as cosmetic use, food use, and the like, preferably cosmetic use.
[19] antioxidation, suppression of melanin synthesis, anti-aging, whitening, and anti-aging in human fibroblasts or human epidermal keratinocytes, comprising providing the callus extract of any one of [14] to [16]. Expression of genes associated with one or more actions selected from the group consisting of cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anticancer Method for increasing quantity. [19-1] The method according to [19], wherein the callus extract is provided for non-medical uses, such as cosmetic uses, food uses, preferably cosmetic uses.
[20] Use of the callus extract of any one of [14] to [16] for the production of a cosmetic composition.
[20-1] The cosmetic composition is for antioxidant, suppression of melanin synthesis, anti-aging, whitening, enhancement of cell proliferation, synthesis of collagen, synthesis of hyaluronic acid, anti-inflammatory, promotion of metabolism, or hair growth, [20 ].
　本発明によれば、化粧品などへの配合に適した、生化学的に有用な、ブナ科植物の種子および発芽体のカルス、そのカルスの製造方法、ブナ科植物の種子および発芽体のカルスの抽出物、そのカルス抽出物を用いた方法、および、ブナ科植物の種子および発芽体のカルス抽出物を含む化粧品組成物、を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, 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.
図１は、実施例１および２のカルスの製造の一例の様子を示す写真である。FIG. 1 is a photograph showing an example of callus production in Examples 1 and 2. FIG. 図２は、カルス抽出物を細胞に適用したときの細胞生存率を示すグラフであり、図２Ａは、正常ヒト皮膚線維芽細胞（ＮＨＤＦ）の生存率を示し、図２Ｂは、正常ヒト表皮角化細胞（ＮＨＥＫ）の生存率を示す。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). 図３は、線維芽細胞での遺伝子発現解析結果のうち、代表的なもの（ＳＯＤ２、ＫＩＴＬＧ、ＳＩＲＴ１、ＦＧＦ７）を抜粋して表したグラフである。FIG. 3 is a graph showing selected representative ones (SOD2, KITLG, SIRT1, FGF7) among the results of gene expression analysis in fibroblasts. 図４は、線維芽細胞での遺伝子発現解析結果のうち、代表的なもの（ＦＧＦ２、ＭＭＰ１、ＨＡＳ２、ＡＤＡＭ１０）を抜粋して表したグラフである。FIG. 4 is a graph showing representative results (FGF2, MMP1, HAS2, ADAM10) extracted from the results of gene expression analysis in fibroblasts. 図５は、表皮角化細胞での遺伝子発現解析結果のうち、代表的なもの（ＷＮＴ５Ａ、ＤＫＫ３、ＫＩＴＬＧ、ＤＫＫ１、ＡＱＰ３）を抜粋して表したグラフである。FIG. 5 is a graph showing selected representative ones (WNT5A, DKK3, KITLG, DKK1, AQP3) among the results of gene expression analysis in epidermal keratinocytes.
　以下、本発明の好ましい実施態様について説明する。 Preferred embodiments of the present invention will be described below.
カルス
　本発明は、ブナ科植物の種子またはブナ科植物の種子由来の発芽体の、細胞からなる、カルス（以下「本発明カルス」とも称する）、に関する。 Callus 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.
　本明細書において、「カルス」とは、植物体の一部を培養して得られる、未分化の植物細胞の塊を意味する。植物体の一部とは、植物を構成する一要素（例えば、種、根、葉、茎、花など）およびその要素をさらに切断または分離して得たものを意味する。カルスは、葉でも茎でも根でもなく、またその他植物組織でもなく、そして、分化してその元の植物（すなわち、カルスの供給源となる植物）の組織の何にでもなり得る細胞の塊であり得る。したがって、単に植物から取り出しただけの組織や細胞（単なる葉の断片や茎の切片など）は、カルスには含まれない。植物から取り出したものを植物ホルモン等による刺激で脱分化させることにより、カルスが生成され得る。ここで、カルスは、広義には、別名として、自然界では植物の傷を覆うように形成されることから「癒傷細胞（ゆしょうさいぼう）」と称される場合もあるが、本発明のカルスとはこれら天然物そのものの物質は含まない。本明細書において、カルスとは人工的に作製したものを意味する。カルスは、細胞塊、すなわち、複数の細胞の集合体、により構成される。 As used herein, "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. Here, in a broad sense, 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. As used herein, callus refers to what is artificially produced. A callus is composed of a cell mass, ie, an aggregate of a plurality of cells.
　本発明では、一の態様において、ブナ科植物の種子の細胞からなるカルス、を提供する。また、本発明では、別の態様において、ブナ科植物の種子由来の発芽体の細胞からなるカルス、を提供する。 In one aspect, 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.
　ブナ科（Ｆａｇａｃｅａｅ）植物としては、例えば、亜科（Ｓｕｂｆａｍｉｌｙ）として、ブナ亜科（Ｆａｇｏｉｄｅａｅ）、コナラ亜科（Ｑｕｅｒｃｏｉｄｅａｅ）、クリ亜科（Ｃａｓｔａｎｅｏｉｄｅａｅ）に属するものが挙げられ、さらに、例えば、属として、ブナ属（Ｆａｇｕｓ）、ナンキョクブナ属（Ｎｏｔｈｏｆａｇｕｓ）、コナラ属（Ｑｕｅｒｃｕｓ）、カクミガシ属（Ｔｒｉｇｏｎｏｂａｌａｎｕｓ）、クリ属（Ｃａｓｔａｎｅａ）、シイ属（クリガシ属）（Ｃａｓｔａｎｏｐｓｉｓ）、マテバシイ属（Ｌｉｔｈｏｃａｒｐｕｓ）、ニセマテバシイ属（Ｎｏｔｈｏｌｉｔｈｏｃａｒｐｕｓ）、トゲガシ属（Ｃｈｒｙｓｏｌｅｐｉｓ）などのブナ科に属する植物が挙げられる。ブナ科植物は、樹木となる植物であり、通常、木本（ｗｏｏｄｙ　ｐｌａｎｔ）であり、多くは、高木（ｔｒｅｅ、ａｒｂｏｒ）である。 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).
　ブナ科植物は、具体的には、例えば、ブナ（ｂｅｅｃｈ）、ナラ、コナラ、ミズナラ、オーク、クヌギ、マテバシイ、椎（シイ）、樫（カシ）、栗（クリ）、シラカシ、カシワなどを挙げることができる。ブナ科植物としては、特に、ブナ属（学名：Ｆａｇｕｓ、英語名：ｂｅｅｃｈ）の植物が好ましく、その中でも、ブナ（学名：Ｆａｇｕｓ　ｃｒｅｎａｔａ、英語名：Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）、およびヨーロッパブナ（学名：Ｆａｇｕｓ　ｓｙｌｖａｔｉｃａ、英語名：Ｅｕｒｏｐｅａｎ　ｂｅｅｃｈ）がより好ましく、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）がさらに好ましい。ブナは、シロブナ、クロブナ、イヌブナ、ホンブナ、ソバグリ、コハブナ、オオハブナなどと称されることもあるが、これらは全て含まれる。なお、クロブナおよびイヌブナは、学名がＦａｇｕｓ　ｊａｐｏｎｉｃａであるが、本明細書では、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）に含まれるものとする。また、ブナ科植物の産地は、特に限定されるものではないが、好ましくは、日本産またはヨーロッパ産のブナを用いることができる。特に好ましくは、例えば、日本の白神山地（秋田県北西部と青森県南西部にまたがる山地）のブナを用いることができる。 Specific examples of 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. Although the scientific name of Japanese beech and Japanese beech is Fagus japonica, in this specification, they are included in beech (Japanese beech). In addition, the place of production of the fagaceous plant is not particularly limited, but preferably Japanese or European beech can be used. Particularly preferably, for example, beech from Shirakami-Sanchi in Japan (a mountainous region spanning the northwestern part of Akita Prefecture and the southwestern part of Aomori Prefecture) can be used.
　ブナ科植物の種子は、ブナ科植物（例えばブナの木）から自然に落下した種子を採取して用いることができる。種子は、一般にドングリと称されるものを用いることができる。ただし、ドングリは正確には果実（堅果または堅実とも称される）を意味し、ドングリ内に種子が含まれる。種子を用いることにより、樹皮などを利用する場合に比べて、現存する植物を傷つけず、また、持続的に原料を採取できるというメリットがある。本発明の一態様では、ブナ科植物の種子から、カルスが誘導される。 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.
　また、ブナ科植物の種子由来の発芽体は、上記で述べたブナ科植物の種子を発芽させることにより得られる植物体である。発芽とは、種子が吸水して、胚組織の一部である幼根（のちに根となる器官）が種皮を破って現れることを意味する。発芽は、人工的に行うことができ、あるいは、自然に発芽したものを用いてもよいが、人工的に行う方が好ましい。人工的な発芽は、公知の方法で行うことができ、例えば、無菌発芽の条件下での方法が好ましくは挙げることができる。例えば、種子（正確には種子を含む堅果）の果皮と種皮を除去し、種子内部を露出させ、無菌培養を行うことにより、種子が発芽して、発芽体を得ることができる。無菌培養の条件は、例えば、培地としてＷＰＭ培地などの植物培養用の培地を用い、室温（例えば２０～２５℃）、一定時間より長い日長時間（例えば１２時間以上）の日長の条件で、数日から数カ月の期間（例えば、数週間から１カ月）で行うことができる。発芽体としては、例えば、幼根が種皮を破った段階のものから、芽生え（実生）（ｓｅｅｄｌｉｎｇ）の段階、さらにはスプラウト（Ｓｐｒｏｕｔ）の段階のものまでを用いることができる。発芽体としては、芽生え（実生）（ｓｅｅｄｌｉｎｇ）が好ましい。発芽体は、根（幼根など）、葉（子葉など）、および茎（胚軸など）を有するものであってもよい。発芽体は、子葉展開期のものを用いてもよい。本発明の一態様では、ブナ科植物の種子の発芽体から、カルスが誘導される。 In addition, 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. For example, by removing the pericarp and seed coat of a seed (exactly a nut containing a seed), exposing the inside of the seed, and performing sterile culture, 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). As 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. As a germination body, a seedling is preferable. 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. In one aspect of the present invention, callus is induced from germinated seeds of fagaceous plants.
カルスの製造
　本発明の一態様によるカルスの製造方法は、ブナ科植物の種子またはブナ科植物の種子由来の発芽体の細胞を、培地中、植物生長調節物質の存在下で培養して増殖させることを含む。 Production of Callus In a method for producing callus according to an aspect of the present invention, 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. Including.
　カルスの製造にあたって、種子からカルスを誘導する場合、まず、種子の果皮と種皮を除去する。これにより、種子の細胞が露出し、種子の細胞が培養しやすい状態となる。このとき、好ましくは、種子を、複数個（例えば２つ）に切り分けることが好ましい。切れ目ができることにより、カルスが形成されやすくなる。種子は、例えば、卵型形状の長径を約２分するように切り分けることができる。また、種子を、エタノールなどのアルコールや次亜塩素酸ナトリウム溶液などの殺菌液で、殺菌することが好ましい。次いで、種子の細胞の一部または全部を、培地に入れる。種子の細胞の一部を用いるときは、適宜に種子の細胞を適度の大きさに切断して用いることができる。 In the production of callus, when inducing callus from seeds, first remove the pericarp and seed coat of the seeds. As a result, the seed cells are exposed and the seed cells can be easily cultured. At this time, preferably, 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. Moreover, 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.
　また、発芽体からカルスを誘導する場合、上記で述べた方法により得た発芽体について、まず、発芽体の一部、好ましくは胚軸（根と葉の間の茎の部分）を切り取り、これを必要に応じて適宜の大きさに切断し、培地に入れる。発芽体から切り取ることにより、細胞が露出し、発芽体の細胞が培養しやすい状態となる。 In the case of inducing callus from germinated bodies, first, 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. By cutting the germination body, the cells are exposed and the cells of the germination body are ready for culturing.
　培地に入れられた細胞（種子の細胞または発芽体の細胞）は、細胞培養によって、細胞が培養されて増殖し、細胞塊であるカルスへと誘導される。このとき、例えば、種子または発芽体の一部が、細胞の増殖によって盛り上がる。この増殖で盛り上がった部分がカルスとなる。 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.
　培地は、固体培地でも液体培地でもよいが、固体培地が好ましい。固体培地の場合、種子または発芽体の細胞を、培地の上に直接載せることによって（培地に接触させることによって）、それらの細胞を培養することができる。培地は、植物生長調節物質などの添加成分を含む培地を使用することができる。 The medium may be a solid medium or a liquid medium, but a solid medium is preferred. In the case of solid media, seed or germination cells can be cultured by placing them directly on the medium (by contacting the medium). As the medium, a medium containing additive components such as plant growth regulators can be used.
　培地（基本培地）としては、特に限定されるものではないが、例えば、ＷＰＭ培地、ＢＴＭ培地、ＭＳ培地、およびそれらの希釈物（例えば、１／２ＷＰＭ培地、１／２ＢＴＭ培地、１／２ＭＳ培地）などが挙げられ、このうち、ＷＰＭ培地が好ましい。なお、培地において、ＷＰＭは、Ｗｏｏｄｙ　ｐｌａｎｔ　ｍｅｄｉｕｍを表し、ＢＴＭは、Ｂｒｏａｄｌｅａｆ　Ｔｒｅｅ　Ｍｅｄｉｕｍを表し、ＭＳは、Ｍｕｒａｓｉｇｅ　Ｓｋｏｏｇを表す。培地の組成は、公知である。培地は、商業的に入手可能である。 The medium (basal medium) is not particularly limited. ), among which WPM medium is preferred. In the medium, WPM represents Woody plant medium, BTM represents Broadleaf Tree Medium, and MS represents Murasige Skoog. The composition of media is known. Media are commercially available.
　培地は、例えば、培地の基質となる組成物を水に溶解して得た溶液を加熱し、支持体（例えば寒天など）を入れた後、冷却して固めることによって、固体培地を形成することができる。このとき、溶液に、植物生長調節物質を添加することにより、植物生長調節物質を含有する培地を得ることができる。 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.
　植物生長調節物質は、植物の生長を調整する物質（好ましくは生長促進）であり、例えば、植物ホルモンと称せられるものが含まれ得る。植物生長調節物質としては、オーキシン（ａｕｘｉｎ）、サイトカイニン（ｃｙｔｏｋｉｎｉｎ）、ジベレリン（ｇｉｂｂｅｒｅｌｌｉｎ）、アブシジン酸（ａｂｓｃｉｓｉｃ　ａｃｉｄ）、エチレン（ｅｔｈｙｌｅｎｅ）、およびそれらの２種以上の組み合わせなどを用いることができる。植物生長調節物質として、オーキシンを用いることが好ましい。オーキシンを用いた場合、カルスを効率よく製造することができる。なお、本明細書において、植物生長調節物質は植物生長調整物質と同義である。 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. In addition, in this specification, a plant growth regulator is synonymous with a plant growth regulator.
　さらに、オーキシンは、ピクロラム（ｐｉｃｌｏｒａｍ）、ジカンバ（ｄｉｃａｍｂａ）、インドール－３－酢酸、インドール－３－酪酸、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、２，４－ジクロロフェノキシ酢酸、２，４，５－トリクロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酪酸、および、ナプロアニリド（ｎａｐｒｏａｎｉｌｉｄｅ）、またはこれらの塩（例えば、ナトリウム塩およびカルシウム塩などの無機物の塩）から選択されるものであることが好ましい。それにより、カルスをさらに効率よく製造することができる。オーキシンは、ピクロラム、ジカンバがより好ましく、ピクロラムがさらに好ましい。 In addition, 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.
　また、植物生長調節物質として、オーキシンとサイトカイニンの組み合わせを用いることも好ましい。それにより、カルスをさらに効率よく製造することができる。特に、発芽体の細胞を培養する場合に、これらを組み合わせることが好ましい。サイトカイニンは、合成サイトカイニンであってもよい。サイトカイニンとしては、６－ベンジルアミノプリン（６－ｂｅｎｚｙｌａｍｉｎｏｐｕｒｉｎｅ）、ゼアチン（ｔｒａｎｓ－Ｚｅａｔｉｎ）などを挙げることができ、６－ベンジルアミノプリンが好ましい。オーキシンとサイトカイニンの具体的な組み合わせとして、例えば、ピクロラムと６－ベンジルアミノプリンの組み合わせが好ましい。 It is also preferable to use a combination of auxin and cytokinin 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.
　植物生長調節物質としてオーキシンを用いる場合、培地におけるオーキシン（例えばピクロラム）の濃度は、０．１～２０μＭであることが好ましく、０．５～１０μＭであることがより好ましく、１～１０μＭであることがさらに好ましく、１μＭであることがよりさらに好ましい。また、植物生長調節物質としてサイトカイニンを用いる場合、培地におけるサイトカイニン（例えば６－ベンジルアミノプリン）の濃度は、１～１０μＭであることが好ましく、２μＭであることがさらに好ましい。また、オーキシンとサイトカイニンを組み合わせる場合、０．１～２０μＭのオーキシンと１～１０μＭのサイトカイニンの組み合わせ、例えば、０．１～２０μＭのピクロラムと１～１０μＭの６－ベンジルアミノプリンの組み合わせ、具体的には、１μＭのピクロラムと２μＭの６－ベンジルアミノプリンの組み合わせ、または１０μＭのピクロラムと２μＭの６－ベンジルアミノプリンの組み合わせ、が好ましい。 When auxin is used as a plant growth regulator, the concentration of auxin (eg, picloram) in the medium is preferably 0.1-20 μM, more preferably 0.5-10 μM, and 1-10 μM. is more preferred, and 1 μM is even more preferred. When cytokinin is used as a plant growth regulator, the concentration of cytokinin (eg, 6-benzylaminopurine) in the medium is preferably 1-10 μM, more preferably 2 μM. Further, when combining auxin and cytokinin, a combination of 0.1-20 μM auxin and 1-10 μM cytokinin, for example, a combination of 0.1-20 μM picloram and 1-10 μM 6-benzylaminopurine, specifically is preferably a combination of 1 μM picloram and 2 μM 6-benzylaminopurine, or a combination of 10 μM picloram and 2 μM 6-benzylaminopurine.
　細胞培養の条件としては、特に限定されるものではないが、２０～２５℃、１２時間以上の日長条件下の培養室内で行うことができる。細胞培養の時間は２週間～２ヶ月が好ましく、１ヶ月がより好ましい。あるいは、細胞培養は、目視観察で十分な量のカルスが得られるのが確認されるまで行うようにしてもよい。細胞培養は、無菌条件で行ってもよいし、そうでなくてもよい。 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. Alternatively, 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.
　カルス製造においては、使用する植物生長調節物質の種類およびその使用する量または濃度の違いにより、形態（例えば、色、堅さ）の異なるカルスが得られることが確認されている。例えば、一実施態様では、ブナ種子から白色のカルス、ブナ発芽体から褐色のカルスまたは緑色のカルスが得られ得る（実施例参照）。また、このようなカルスは堅さをも異なる。これらのカルスは、形態だけでなく、作用にも違いがあることが確認されている。 In callus production, it has been confirmed that callus with different morphologies (eg, color, hardness) can be obtained depending on the type of plant growth regulator used and the amount or concentration used. For example, in one embodiment, 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.
　上記のようにして、培地中の細胞（種子の細胞または発芽体の細胞）は、細胞培養によって増殖し、細胞塊であるカルスが産生する。カルスは、種子または発芽体の表面に、盛り上がるようにして形成され得る。カルスは、培養に用いた種子および発芽体（いわば母体）から切り離して、または、種子および発芽体とともに（培養物ごと）、抽出に用いることができる。 As described above, the cells in the medium (seed cells or germination cells) 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).
　本発明の一態様は、上記のカルス由来のカルス抽出物に関する。ここで、カルスからの抽出物は、本明細書中では単に「カルス抽出物」と呼称する。カルスは、上記で述べた製造方法によって製造されたものを用いることができる。したがって、カルス抽出物は、ブナ科植物種子カルスからの抽出物、または、ブナ科植物種子発芽体カルスからの抽出物である。 One aspect of the present invention relates to a callus extract derived from the above callus. Here, 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.
　カルスからの抽出は、公知の植物エキスを製造するときの抽出方法に準じた方法で行うことができる。例えば、カルスを凍結乾燥して水分を除去し、得られた乾燥物を切断または粉砕するなどして、小片あるいは粉末にし、これを抽出用の溶媒によって抽出することにより、抽出を行うことができる。溶媒としては、水、有機溶媒、または水と有機溶媒の混合液を用いることができる。有機溶媒としては、親水性有機溶媒または疎水性有機溶媒を用いることができるが、親水性の有機溶媒が好ましい、アルコール（例えば、低級アルコール）が好ましく、エタノールまたはメタノールが好ましい。好ましい溶媒としては、水、または、水とエタノールなどのアルコールとの混合液が挙げられる。水と有機溶媒（例えばアルコール）の混合液を用いる場合、水に対する有機溶媒（例えばアルコール）の比率（有機溶媒／水）は、体積比で、１／９９～９９／１が好ましく、１／９～９／１がより好ましく、例えば、有機溶媒（例えばアルコール）の割合５０％がより好ましい。抽出は、室温で行ってもよいし、加温下で行ってもよい。例えば、室温から使用する有機溶媒の沸点（例えば８０℃）の温度条件で抽出を行うことができる。抽出は、放置状態で行ってもよいし、撹拌された状態で行ってもよい。抽出時間は、特に限定されるものではないが、例えば、１時間～１日であってよく、好ましくは、１時間であってよい。 Extraction from callus can be performed by a method that conforms to the extraction method used when producing known plant extracts. For example, 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. . As the solvent, water, an organic solvent, or a mixture of water and an organic solvent can be used. As the organic solvent, 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. When a mixture of water and an organic solvent (eg, alcohol) is used, 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.
　抽出後、濾過などの方法によって、残渣（固形物）を取り除くことにより、カルスからの抽出成分が含まれる溶液（カルス抽出液）を得ることができる。抽出液は、そのまま、あるいは濃縮して、抽出物として使用することができる。好ましくは、抽出液から液体成分（水、アルコールなど）を除去することにより、固体、オイルまたはペーストの形態の抽出物を得ることができる。カルス抽出物は、好ましくは、固体粉末の形態である。カルス抽出物は、例えば、褐色のガラス容器に入れて、室温または冷蔵で保管することができる。 After extraction, 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. Preferably, 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.
　こうして得られたカルス抽出物は、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を有し得るものである。カルス抽出物は、好ましくは、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、新陳代謝促進、および育毛からなる群から選択される１つ以上の作用を有するカルス抽出物である。カルス抽出物の上記の作用は、後述の実施例で述べるように実験的に確かめられている。これらの作用は、化粧品的に、医薬的に、およびその他の生理学的に、利用することができる。例えば、カルス抽出物を化粧品組成物に配合することにより、上記の作用を有する化粧品を得ることができる。また、局所投与型の医薬品に応用することもできる。また、上記の作用は、経口によって発揮される可能性があり、医薬品（経口投与型）、および食品などにも応用可能である。より好ましくは、医薬以外の用途での使用であり、化粧品または食品の用途が好ましく、化粧品の用途がさらに好ましい。なお、本明細書において、化粧品には、医薬部外品が含まれ得る。ただし、医薬部外品を除いた化粧品において使用されてもよい。 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. These effects can be exploited cosmetically, medicinally, and otherwise physiologically. For example, by blending a callus extract into a cosmetic composition, a cosmetic having the above effects can be obtained. It can also be applied to topical administration type medicines. In addition, 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. In this specification, cosmetics may include quasi-drugs. However, it may be used in cosmetics other than quasi-drugs.
　ここで、仮説に縛られるものではないが、ブナの種子および発芽体から誘導されたカルスは、ブナの種子自体および発芽体自体が有する活性成分よりも、活性成分の量や質が向上している可能性がある。これは、細胞が増殖して形成されるカルスによって、その増殖とともに、活性成分が増加するからではないかと推測される。そして、活性成分が増加したカルスから抽出されたカルス抽出物においても活性成分が増加し、上記のような有用な作用をもたらすのではないかと推測される。そして、カルスを用いる場合、細胞培養によって活性成分を増やすことができるため、植物自体を用いる場合よりも、効率よく有用なブナの抽出物を得ることができるものである。ただし、本発明は、上記の推測に限定されるものではない。 Here, although not bound by any hypothesis, 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. there may be. It is presumed that this is because the callus formed by cell proliferation increases the active ingredient along with the proliferation. It is also presumed that 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. When callus is used, 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. However, the present invention is not limited to the above speculation.
化粧品組成物
　本発明は、一態様において、カルス抽出物および化粧品基剤を含む化粧品組成物に関する。カルス抽出物は、上記で述べたものを用いることができる。カルス抽出物を配合することにより、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、および育毛からなる群から選択される１つ以上の作用を有し得る化粧品を得ることができる。化粧品は、好ましくは、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、新陳代謝促進、および育毛からなる群から選択される１つ以上の作用を有する化粧品である。上記の作用によって奏せられる化粧品の具体的な効能および効果としては、特に限定されるものではないが、美白、シワ抑制、シミ抑制、ハリ改善、肌の潤い改善、肌のバリア効果の向上、肌細胞のターンオーバーの促進、および紫外線等のダメージからの回復力向上、などが挙げられる。 Cosmetic Compositions In one aspect, the present invention relates to cosmetic compositions comprising a callus extract and a cosmetic base. As the callus extract, those described above can be used. By blending 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. It is a cosmetic that has 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.
　化粧品基剤は、化粧品として配合することが可能な成分であってよい。本明細書において、化粧品基剤とは、化粧品の形態の骨格を形成する成分を意味する。化粧品基剤としては、これに限定されるものではないが、水、油剤、アルコール類（一価または多価のアルコール）、界面活性剤、乳化剤、懸濁剤、高分子、粉体などが挙げられる。 The cosmetic base may be a component that can be blended as a cosmetic. As used herein, 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.
　油剤としては、これに限定されるものではないが、天然または合成の、エステル油、炭化水素油、高級アルコール、脂肪酸、シリコーン油などが挙げられ、具体的には、これに限定されるものではないが、例えば、流動パラフィン、ワセリン、オレイン酸エチル、ステアリン酸、パルミチン酸、スクワラン、セタノール、コレステロール、ミツロウ、シア脂、ベヘニルアルコール、セトステアリルアルコール、バチルアルコール、ホホバ油、マカデミアンナッツ油、メドウフォーム油、水添ヤシ油、水添パーム油、ステアリン酸水添ヒマシ油、オリーブ油、水添ポリイソブテン、ポリエチレングリコール、ジメチルポリシロキサン、ジメチコン、などが挙げられる。 Examples of 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.
　アルコール類（一価または多価のアルコール）としては、これに限定されるものではないが、例えば、エタノール、イソプロパノール、ブタノール、グリセリン、１，３－ブチレングリコール、プロピレングリコール、ジプロピレングリコールなどが挙げられる。 Examples of alcohols (monohydric or polyhydric alcohols) include, but are not limited to, ethanol, isopropanol, butanol, glycerin, 1,3-butylene glycol, propylene glycol, dipropylene glycol, and the like. be done.
　界面活性剤としては、アニオン界面活性剤、カチオン界面活性剤、ノニオン界面活性剤、両性界面活性剤が挙げられ、具体的には、これに限定されるものではないが、例えば、ラウリン酸ナトリウム、ラウリル硫酸ナトリウム、ポリソルベート８０、オレイン酸グリセリル、ラウリン酸ポリグリセリル、ステアリン酸ソルビタンなどが挙げられる。 Examples of 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.
　高分子としては、天然由来の高分子、合成高分子が挙げられ、具体的には、これに限定されるものではないが、例えば、キサンタンガム、ジェランガム、アラビアガム、グアガムなどのガム類、ヒドロキシプロピルセルロース、ヒドロキシエチルセルロース、メチルセルロース、エチルセルロース、カルボキシメチルセルロースなどのセルロース類、ヒアルロン酸ナトリウム、カルボキシビニルポリマー、ポリビニルアルコールなどが挙げられる。 Examples of 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.
　粉体としては、無機の粉体、有機の粉体が挙げられ、これに限定されるものではないが、例えば、酸化チタン、酸化亜鉛、タルク、マイカ、シリカ、ポリエチレンテレフタレート（ＰＥＴ）粉末などが挙げられる。 Examples of 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.
　化粧品組成物は、化粧品基剤以外の成分を含有してもよい。そのような成分として、例えば、水溶性成分、油溶性成分、保湿剤、増粘剤、色素、紫外線吸収剤、被膜形成性剤、ｐＨ調整剤、褪色防止剤、酸化防止剤、消泡剤、美容成分、防腐剤、香料などが挙げられる。また、化粧品組成物は、化粧品基剤を構成しない油剤、水溶性高分子および粉体を含んでもよい。また、化粧品組成物は、上記のカルス抽出物以外の生理活性を有する物質（いわゆる有効成分）を含んでもよい。 The cosmetic composition may contain ingredients other than the cosmetic base. Examples of such components 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. In addition, the cosmetic composition may contain an oil agent, a water-soluble polymer and a powder that do not constitute a cosmetic base. In addition, the cosmetic composition may contain a physiologically active substance (so-called active ingredient) other than the callus extract.
　化粧品組成物は、化粧品として使用することが可能な適宜の形態の組成物であってよい。化粧品組成物の形態としては、例えば、水溶液、オイル、エマルジョン（Ｏ／Ｗ型、Ｗ／Ｏ型、Ｗ／Ｏ／Ｗ型など）、ペースト、粉末、固形状物、などが挙げられる。また、スプレー剤、ミスト剤などであってもよい。 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. Alternatively, a spray agent, a mist agent, or the like may be used.
　化粧品組成物は、それ自体で化粧品として使用してもよい。あるいは、化粧品組成物は、最終の化粧品を製造するための原料（仕掛品）として使用してもよい。 The cosmetic composition itself may be used as a cosmetic product. Alternatively, the cosmetic composition may be used as a raw material (work-in-progress) for manufacturing the final cosmetic product.
　化粧品組成物は、上記のカルス抽出物を、上記で述べたような化粧品組成物に配合される原料と、適宜に混合することにより得ることができる。したがって、本発明は、化粧品組成物の製造のための、上記カルス抽出物の使用、を提供する。 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.
　化粧品組成物は、皮膚用の化粧品のための組成物であり得る。皮膚は、顔の皮膚、および顔以外の身体の部位（頭、首、肩、手、足など）の皮膚であってよく、顔皮膚および頭皮が好ましい。皮膚用化粧品としては、特に限定されるものではないが、具体的には、例えば、乳液、クリーム、美容液、化粧水、ハンドクリーム、アイクリーム、ボディクリーム、メーキャップ化粧料、コンシーラー、チーク、アイシャドウ（アイカラー）、化粧用下地、ファンデーション（例えば、リキッドファンデーション、固形ファンデーション）、日焼け止め、などの化粧品が例示される。また、特に、頭皮用の化粧品としては、育毛剤、発毛剤、養毛剤などの化粧品が例示される。 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. In particular, examples of cosmetics for the scalp include cosmetics such as hair growth agents, hair growth agents, and hair tonics.
　化粧品組成物は、毛髪用の化粧品のための組成物であってもよい。毛髪用化粧品としては、例えば、ヘアクリーム、ヘアワックス、ヘアリンス、ヘアマスク、ヘアトリートメントなどの化粧品が例示される。 The cosmetic composition may be a composition for cosmetics for hair. Examples of hair cosmetics include cosmetics such as hair cream, hair wax, hair rinse, hair mask, and hair treatment.
カルス抽出物のヒト被験者皮膚への適用
　本発明は、一態様において、上記のカルス抽出物をヒト被験者の皮膚に適用することを含む、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を供するための方法、に関する。カルス抽出物は、上記で述べたとおり、ブナ科植物の種子またはブナ科植物の種子由来の発芽体のカルスから得られたものである。 Application of Callus Extract to Human Subject Skin In one aspect, 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. , a method for providing one or more effects selected from the group consisting of collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism enhancement, 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.
　カルス抽出物のヒト被験者の皮膚への適用は、医薬的な適用以外の適用であることが好ましい。例えば、カルス抽出物は、化粧品用途として、好ましくは、化粧品組成物によって、ヒトの皮膚に適用される。化粧品組成物をヒト被験者の皮膚に塗布することにより、カルス抽出物を皮膚に適用することができる。カルス抽出物が皮膚に適用されることにより、例えば、カルス抽出物またはその中の成分が皮膚に浸透して、上記の作用が奏され得る。好ましくは、カルス抽出物の皮膚への適用は、医薬的な治療用途を含まない。 The application of the callus extract to the skin of human subjects is preferably non-medical application. For example, 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. By applying the callus extract to the skin, for example, the callus extract or components therein permeate the skin, and the above effects can be exhibited. Preferably, application of the callus extract to the skin does not involve pharmaceutical therapeutic use.
カルス抽出物による遺伝子発現量の増加方法
　本発明は、一態様において、上記のカルス抽出物を供することを含む、正常ヒト繊維芽細胞または正常ヒト表皮角化細胞における、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用に関連する遺伝子の発現量を増加するための方法、に関する。カルス抽出物は、上記で述べたとおり、ブナ科植物の種子またはブナ科植物の種子由来の発芽体のカルスから得られたものである。 Method for Increasing Gene Expression Level by Callus Extract In one aspect, 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. A method for increasing the expression level of related genes. 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.
　カルス抽出物は、一態様において、ヒト皮膚内の正常ヒト繊維芽細胞（本明細書中、単に「ヒト繊維芽細胞」または「繊維芽細胞」とも称する）または正常ヒト表皮角化細胞（本明細書中、単に「ヒト表皮角化細胞」または「表皮角化細胞」とも称する）に供され得る。この場合、カルス抽出物は、ヒト被験者の皮膚に適用される。このような適用は、いわゆるインビボ（ｉｎ　ｖｉｖｏ）とも称せられる。カルス抽出物は、好ましくは、化粧品組成物によって、ヒトの皮膚に適用される。化粧品組成物をヒト被験者の皮膚に塗布することにより、カルス抽出物を皮膚内のヒト繊維芽細胞またはヒト表皮角化細胞に供することができる。カルス抽出物が皮膚に適用されることにより、例えば、カルス抽出物またはその中の成分が皮膚に浸透し、正常ヒト繊維芽細胞または正常ヒト表皮角化細胞に影響を与えて、上記の作用に関連する遺伝子の発現量を増加させることができる。 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"). In this case, 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. When the callus extract is applied to the skin, for example, 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.
　カルス抽出物は、他の態様において、培養された正常ヒト繊維芽細胞またはヒト表皮角化細胞に供され得る。このような適用は、いわゆるインビトロ（ｉｎ　ｖｉｔｒｏ）とも称せられる。カルス抽出物が正常ヒト繊維芽細胞または正常ヒト表皮角化細胞に適用されることにより、例えば、カルス抽出物中の成分が、正常ヒト繊維芽細胞または正常ヒト表皮角化細胞に影響を与えて、上記の作用に関連する遺伝子の発現量を増加させることができる。 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. By applying the callus extract to normal human fibroblasts or normal human epidermal keratinocytes, for example, 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.
　ここで、カルス抽出物は、ヒト以外の動物（特に哺乳動物、例えば、イヌ、ネコ、ラット、マウスなど）にも有用であり得る。カルス抽出物は、ヒト以外の動物にも適用可能である。ヒト以外の動物に適用（例えば皮膚に塗布）した場合も、上記で述べたのと同様の効果（例えば遺伝子発現量の増加など）が奏され得る。 Here, 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.
　以下、本発明を実施例により説明するが、本発明は実施例に限定されるものではない。 Although the present invention will be described below with reference to examples, the present invention is not limited to the examples.
材料
ブナ種子
　日本の白神山地（秋田県）で自生するブナ（学名：Ｆａｇｕｓ　ｃｒｅｎａｔａ、英語名：Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）から地上に自然に落下した種子を採取した。以下の実験では、このように採取したブナ種子を用いた。図１は、実施例１および２のカルスの製造の一例の様子を示す写真であり、図１Ａは、ブナ種子を示す。 Materials Beech Seeds Seeds that naturally fell to the ground were collected from beech (scientific name: Fagus crenata, English name: Japanese beech) growing wild in Shirakami Mountains (Akita Prefecture) in Japan. The beech seeds collected in this manner were used in the following experiments. FIG. 1 is a photograph showing an example of callus production in Examples 1 and 2, and FIG. 1A shows beech seeds.
実施例１．ブナ種子のカルスの製造
　ブナ種子から果皮と種皮を除去し、２つに切り分け、７０％エタノールに３０秒、１０％次亜塩素酸ナトリウム溶液に５分間浸漬することによりこの種子を殺菌した（図１Ｂ）。種子は、例えば、卵型形状の長径を約２分するように切り分けた。一方、ＷＰＭ培地の原料にピクロラムを加えて溶液を調製し、この混合液を細胞培養ディッシュに入れて、１０μＭのピクロラムを含むＷＰＭ培地（ＷＰＭ培地１と称する）を調製した。このピクロラム含有ＷＰＭ培地に、果皮と種皮を除去した種子を加え、２５℃、１６時間日長の条件で、培養した。培養の３０日後、培養物の細胞が増殖し、細胞塊であるカルスの形成が確認された（図１Ｄ）。カルスは白色であった。以下、実施例１で得られたカルスをカルス１と称する。培養物を取り出し、カルスの部分を切り取り、次の抽出工程に用いた。 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 μM 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. under the conditions of 16-hour photoperiod. After 30 days of culture, the cells in the culture proliferated and the formation of callus, which is a cell mass, was confirmed (Fig. 1D). The callus was white. 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.
実施例２．ブナ種子発芽体のカルスの製造
　ブナ種子から果皮と種皮を除去し、この種子を、ＷＰＭ培地（植物ホルモンフリー培地）に入れ、２５℃、１６時間日長の条件で、無菌発芽させることにより、培養１ヶ月以内で、発芽体（芽生え）を得た（図１Ｃ）。発芽体の胚軸（葉と根の間の部分）を切断して採取し、１辺３～１０ｍｍの立方体と同程度の大きさの断片にした。一方、ＷＰＭ培地の原料にピクロラムおよび６－ベンジルアミノプリン（以下「６－ＢＰ」とも称する）を加えて溶液を調製し、この混合液を細胞培養ディッシュに入れて、ピクロラムと６－ベンジルアミノプリンを含むＷＰＭ培地を調製した。ここで、培地は、１０μＭのピクロラムと２μＭの６－ベンジルアミノプリンを含むＷＰＭ培地（ＷＰＭ培地２と称する）と、１μＭのピクロラムと２μＭの６－ベンジルアミノプリンを含むＷＰＭ培地（ＷＰＭ培地３と称する）の２種類の培地を調製した。このＷＰＭ培地に、発芽体の胚軸の断片を加え、２５℃、１６時間日長の条件で、培養した。培養の３０日後、培養物の細胞が増殖し、細胞塊であるカルスの形成が確認された（図１Ｅおよび図１Ｆ）。ＷＰＭ培地２で得たカルス（以下、カルス２と称する）は褐色であった。ＷＰＭ培地３で得たカルス（以下、カルス３と称する）は緑色であった。培養物を取り出し、カルスの部分を切り取り、次の抽出工程に用いた。 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. On the other hand, picloram and 6-benzylaminopurine (hereinafter also referred to as “6-BP”) were added to the raw materials of WPM medium to prepare a solution, and the mixture was placed in a cell culture dish to obtain picloram and 6-benzylaminopurine. A WPM medium containing was prepared. Here, the medium was a WPM medium containing 10 μM picloram and 2 μM 6-benzylaminopurine (referred to as WPM medium 2) and a WPM medium containing 1 μM picloram and 2 μM 6-benzylaminopurine (referred to as WPM medium 3). ) were prepared. Fragments of germination hypocotyls were added to this WPM medium and cultured at 25° C. with a day length of 16 hours. After 30 days of culture, the cells in the culture proliferated and the formation of callus, which is a cell mass, was confirmed (FIGS. 1E and 1F). 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.
実施例３．カルス抽出物の製造
　実施例１および２で得た各カルス（カルス１～３）を、凍結乾燥し、得られた乾燥物を粉砕することにより、粉末にした。カルス粉末０．５～１ｇに、抽出溶媒として５０％エタノール水４０ｍＬを加え、室温で振とうさせ抽出を行った。抽出開始から１時間後、濾過により、固形分を除去し、エバポレートにより濾液を濃縮した。濃縮物は、固体となった。これにより、カルス抽出物を得た。以下、カルス１から得たカルス抽出物を「カルスエキス１」と称し、カルス２から得たカルス抽出物を「カルスエキス２」と称し、カルス３から得たカルス抽出物を「カルスエキス３」と称する。 Example 3. Preparation of callus extract Each callus obtained in Examples 1 and 2 (callus 1 to 3) was freeze-dried and the resulting dried material was pulverized into powder. To 0.5 to 1 g of callus powder, 40 mL of 50% ethanol water was added as an extraction solvent, and extracted by shaking at room temperature. After 1 hour from the start of extraction, solid content was removed by filtration, and the filtrate was concentrated by evaporation. The concentrate became a solid. A callus extract was thus obtained. Hereinafter, the callus extract obtained from callus 1 is referred to as "callus extract 1", the callus extract obtained from callus 2 is referred to as "callus extract 2", and the callus extract obtained from callus 3 is referred to as "callus extract 3". called.
実施例４．カルス抽出物によるヒト皮膚細胞の遺伝子発現
生化学的アッセイ
１．アッセイ用サンプルの調製
　ブナカルス抽出物のヒト皮膚に対する機能性を明らかにするために、ブナカルス抽出物をヒト皮膚細胞に適用し、リアルタイムＰＣＲを用いた遺伝子発現量の解析をすることによって、ブナカルス抽出物の作用を調べた。
　上記で得た各カルス抽出物全量を、１～１．５ｍＬのジメチルスルホキシド（Dimethyl sulfoxide）（ＤＭＳＯ）に溶解し、アッセイ用サンプルを調製した。
　比較用のサンプルとして、化粧品原料として一般に市販されているヨーロッパブナ芽エキス（以下、「比較エキス１」と称する）を用いた。ヨーロッパブナ芽エキスは、ヨーロッパブナ（学名：Ｆａｇｕｓ　ｓｙｌｖａｔｉｃａ、英名：Ｅｕｒｏｐｅａｎ　ｂｅｅｃｈ）の幼芽を水で抽出した抽出物を濃縮して得たエキスである（カルス由来のものではない）。 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.
As a sample for comparison, 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.
２．細胞培養
　ヒト皮膚由来細胞として、正常ヒト皮膚線維芽細胞（ＮＨＤＦ）および正常ヒト表皮角化細胞（ＮＨＥＫ）の２種類の細胞を用いた。細胞培養は、培地として、ＮＨＤＦにはＤＭＥＭ（Ｎａｃａｌａｉ　ｔｅｓｑｕｅ）＋１０％ウシ血清アルブミン（ＢＳＡ）を用い、ＮＨＥＫにはＣｎＴ－Ｐｒｉｍｅ（ＣＥＬＬｎＴＥＣ）を用い、３７℃で、ＣＯ２インキュベーターでインキュベートすることにより行った。
　ここで、添加濃度の検討のため、事前に、カルス抽出物を１／３倍希釈で９段階まで希釈して得たサンプルを、培養した細胞に添加し、サンプル添加２４時間後の細胞生存率を調査した。繊維芽細胞の調査には、カルス２の抽出物を用い、および、表皮角化細胞の調査には、カルス３の抽出物を用いた。図２は、カルス抽出物を細胞に適用したときの細胞生存率を示すグラフである。添加濃度は、細胞に悪影響（生存率の低下）を及ぼさない範囲での最も高い濃度を採用することにした。例えば、カルスエキス２を繊維芽細胞に適用する場合、０．０１２％（ｗ／ｖ）となり（図２Ａ）、カルスエキス３を表皮角化細胞に適用する場合、０．１１２％（ｗ／ｖ）となった（図２Ｂ）。
　細胞（正常ヒト皮膚線維芽細胞および正常ヒト表皮角化細胞）を、細胞培養ディッシュで培養し、適宜の細胞量となったときに、アッセイ用サンプルを添加した。なお、線維芽細胞については、カルスエキス１～３および比較エキス１について実験を実施し、表皮角化細胞については、カルスエキス２、３および比較エキス１について実験を実施した。
　サンプル添加後、２４時間、細胞培養し、細胞をリアルタイムＰＣＲで分析した。リアルタイムＰＣＲシステムとしては、ＱｕａｎｔＳｔｕｄｉｏ　１２Ｋ　Ｆｌｅｘ（ＴｈｅｒｍｏＦｉｓｈｅｒ）を用いた。細胞からのＲＮＡ抽出にはＳｕｐｅｒＰｒｅｐ（商標）　ＩＩ　Ｃｅｌｌ　Ｌｙｓｉｓ　＆　ＲＴ　Ｋｉｔ（ＴＯＹＯＢＯ）を用い、逆転差反応にはＲｅｖｅｒＴｒａ　Ａｃｅ　ｑＰＣＲ　ＲＴ　Ｍａｓｔｅｒ　Ｍｉｘ（ＴＯＹＯＢＯ）を用いた。リアルタイムＰＣＲの反応はＴａｑＭａｎ　Ｆａｓｔ　Ａｄｖａｎｃｅｄ　Ｍａｓｔｅｒ　ＭｉｘとＴａｑＭａｎ　Ａｒｒａｙカード（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ）を用いて行った。これにより、抗炎症、抗がん性、遺伝子修復などの機能に関与する４６種類の遺伝子についての発現量の変化を解析することができる。各遺伝子の発現量は、サンプル無添加をコントロールとし、１８Ｓ　ｒＲＮＡをベースにした相対値（ΔΔＣｔ法に基づく相対値）で評価した。 2. Cell Culture As human skin-derived cells, two types of cells, normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK), were used. Cell culture was carried out using DMEM (Nacalai tesque) + 10% bovine serum albumin (BSA) for NHDF and CnT-Prime (CELLnTEC) for NHEK as media, and incubating at 37°C in a CO 2 incubator. gone.
Here, in order to examine the addition concentration, a sample obtained by diluting the callus extract up to 9 steps by 1/3 dilution in advance was added to the cultured cells, and the cell viability 24 hours after addition of the sample was investigated. Extracts of callus 2 were used for fibroblast studies and extracts of callus 3 were used for epidermal keratinocyte studies. FIG. 2 is a graph showing cell viability when callus extract is applied to cells. As for 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. As for fibroblasts, experiments were conducted with callus extracts 1 to 3 and comparative extract 1. As for 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. SuperPrep™ II Cell Lysis & RT Kit (TOYOBO) was used for RNA extraction from cells, and ReverTra Ace qPCR RT Master Mix (TOYOBO) was used for reverse differential reaction. The real-time PCR reaction was performed using TaqMan Fast Advanced Master Mix and TaqMan Array card (Thermo Fisher). As a result, it is possible to analyze changes in the expression levels of 46 types of genes involved in functions such as anti-inflammatory, anti-cancer, and gene repair. The expression level of each gene was evaluated as a relative value based on 18S rRNA (relative value based on the ΔΔCt method) using no sample as a control.
　正常ヒト皮膚線維芽細胞（ＮＨＤＦ）については、次の４２種類の遺伝子を解析した。なお、括弧内は、その遺伝子に関連する作用（効能および効果）を表す。
　ＡＤＡＭ１０（育毛）、ＡＤＡＭ１２（育毛）、ＰＰＡＲＧ（育毛・抗酸化）、ＣＣＮＤ１（遺伝子修復）、ＧＬＯ１（遺伝子修復）、ＰＡＲＫ７（遺伝子修復）、ＲＢＭＸ（遺伝子修復）、ＴＰ５３ＢＰ１（遺伝子修復）、ＣＣＬ２（抗炎症）、ＩＬ１Ａ（抗炎症）、ＩＬ６（抗炎症）、ＮＦＫＢ１（抗炎症）、ＰＴＧＳ２（抗炎症）、ＳＴＡＴ３（抗炎症）、ＴＧＦＢ１（抗炎症）、ＥＸＴ１（抗がん）、ＮＦ１（抗がん）、ＰＴＥＮ（抗がん）、ＲＢ１（抗がん）、ＳＭＡＤ４（抗がん）、ＴＰ５３（抗がん）、ＳＯＤ１（抗酸化）、ＳＯＤ２（抗酸化）、ＳＯＤ３（抗酸化）、ＳＩＲＴ１（抗老化）、ＳＩＲＴ２（抗老化）、ＣＯＬ１Ａ１（コラーゲン合成）、ＣＯＬ３Ａ１（コラーゲン合成）、ＣＯＬ７Ａ１（コラーゲン合成）、ＭＭＰ１（コラーゲン合成）、ＤＫＫ３（増殖亢進）、ＦＧＦ２（増殖亢進）、ＰＴＧＥＳ３（増殖亢進）、ＶＥＧＦＡ（増殖亢進）、ＣＤ４４（ヒアルロン酸合成）、ＨＡＳ２（ヒアルロン酸合成）、ＶＣＡＮ（ヒアルロン酸合成）、ＣＬＵ（美白）、ＦＧＦ７（美白）、ＫＩＴＬＧ（メラニン合成関連）、ＮＲＧ１（メラニン合成関連）、ＤＫＫ１（メラニン合成関連）。 For normal human dermal fibroblasts (NHDF), the following 42 types of genes were analyzed. The numbers in parentheses indicate the action (efficacy and effect) related to the gene.
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) cancer), PTEN (anti-cancer), RB1 (anti-cancer), SMAD4 (anti-cancer), TP53 (anti-cancer), SOD1 (antioxidant), SOD2 (antioxidant), SOD3 (antioxidant), SIRT1 (anti-aging), SIRT2 (anti-aging), COL1A1 (collagen synthesis), COL3A1 (collagen synthesis), COL7A1 (collagen synthesis), MMP1 (collagen synthesis), DKK3 (proliferation), FGF2 (proliferation), PTGES3 ( proliferation), VEGFA (proliferation enhancement), CD44 (hyaluronic acid synthesis), HAS2 (hyaluronic acid synthesis), VCAN (hyaluronic acid synthesis), CLU (whitening), FGF7 (whitening), KITLG (related to melanin synthesis), NRG1 ( related to melanin synthesis), DKK1 (related to melanin synthesis).
　正常ヒト表皮角化細胞（ＮＨＥＫ）については、次の４１種類の遺伝子を解析した。なお、括弧内は、その遺伝子に関連する作用（効能および効果）を表す。
　ＡＤＡＭ１０（育毛）、ＣＥＢＰＡ（育毛）、ＣＣＮＤ１（遺伝子修復）、ＧＬＯ１（遺伝子修復）、ＨＡＧＨ（遺伝子修復）、ＰＡＲＫ７（遺伝子修復）、ＲＢＭＸ（遺伝子修復）、ＴＰ５３ＢＰ１（遺伝子修復）、ＣＯＬ１７Ａ１（細胞分化・再生）、ＤＫＫ３（細胞分化・再生）、ＩＴＧＡ６（細胞分化・再生）、ＬＡＭＡ５（細胞分化・再生）、ＷＮＴ５Ａ（細胞分化・再生）、ＣＸＣＬ８（抗炎症）、ＩＬ１Ａ（抗炎症）、ＮＦＫＢ１（抗炎症）、ＰＴＧＳ２（抗炎症）、ＳＴＡＴ３（抗炎症）、ＴＧＦＢ１（抗炎症）、ＧＡＢＰＡ（抗炎症・抗酸化・抗老化）、ＣＤＫＮ２Ａ（抗がん）、ＥＸＴ１（抗がん）、ＰＴＥＮ（抗がん）、ＳＭＡＤ４（抗がん）、ＴＰ５３（抗がん）、ＳＯＤ１（抗酸化）、ＳＯＤ２（抗酸化）、ＰＰＡＲＧ（抗酸化・育毛）、ＳＩＲＴ１（抗老化）、ＳＩＲＴ２（抗老化）、ＣＥＲＳ３（セラミド合成）、ＥＬＯＶＬ１（セラミド合成）、ＥＬＯＶＬ４（セラミド合成）、ＡＱＰ３（新陳代謝）、ＦＬＧ（新陳代謝）、ＬＯＲ（新陳代謝）、ＣＤ４４（ヒアルロン酸合成）、ＨＡＳ３（ヒアルロン酸合成）、ＤＫＫ１（メラニン合成関連）、ＫＩＴＬＧ（メラニン合成関連）、ＬＡＭＣ２（美白）。 For normal human epidermal keratinocytes (NHEK), the following 41 types of genes were analyzed. The numbers in parentheses indicate the action (efficacy and effect) related to the gene.
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 ( anticancer), SMAD4 (anticancer), TP53 (anticancer), SOD1 (antioxidant), SOD2 (antioxidant), PPARG (antioxidant/hair growth), SIRT1 (antiaging), SIRT2 (antiaging) , CERS3 (ceramide synthesis), ELOVL1 (ceramide synthesis), ELOVL4 (ceramide synthesis), AQP3 (metabolism), FLG (metabolism), LOR (metabolism), CD44 (hyaluronic acid synthesis), HAS3 (hyaluronic acid synthesis), DKK1 ( related to melanin synthesis), KITLG (related to melanin synthesis), LAMC2 (whitening).
３．結果
繊維芽細胞の遺伝子発現
　表１は、正常ヒト皮膚繊維芽細胞（ＮＨＤＦ）の遺伝子発現量の結果を示している。各遺伝子の発現量は、ｍＲＮＡ発現量を、コントロールとの相対値として評価した（コントロールの値を１とする）。ここで、各遺伝子について、カルスエキス（カルスエキス１～３の少なくとも１つ）が特に高い有用性を示したものには有用性を「ｈｉｇｈ」として表した。また、好ましい発現の方向性について、アップレギュレーション（発現量増加）が好ましい場合は、「ｕｐ」と記載し、ダウンレギュレーション（発現量減少）が好ましい場合は、「ｄｏｗｎ」と記載した。発現量評価として、発現量の増加が大きいものを「＋」、発現量の増加が著しく大きいものを「＋＋」で表し、また、発現量の減少が大きいものを「－」、発現量の増加が著しく大きいものを「－－」で表した。 3. Results Fibroblast Gene Expression Table 1 shows the results of gene expression levels in normal human dermal fibroblasts (NHDF). For the expression level of each gene, the mRNA expression level was evaluated as a relative value with respect to the control (control value is set to 1). Here, for each gene, usefulness was indicated as "high" when the callus extract (at least one of callus extracts 1 to 3) showed particularly high usefulness. In addition, with regard to the preferred direction of expression, when upregulation (increase in expression level) is preferred, it is indicated as "up", and when downregulation (decrease in expression level) is preferred, it is indicated as "down". As the expression level evaluation, "+" indicates a large increase in the expression level, "++" indicates a significant increase in the expression level, "-" indicates a large decrease in the expression level, and "-" indicates an increase in the expression level. A significantly large value is indicated by "--".
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　図３～図４は、線維芽細胞での遺伝子発現解析結果のうち、代表的なものを抜粋して表したグラフを示す。グラフ縦軸の「高」および「低」は、それぞれ、「効果が高い」および「効果が低い」ことを意味する。
　図３Ａは、ＳＯＤ２のグラフであり、活性酸素消去酵素をコードすることから、発現量増加は、抗酸化作用の向上を示唆する。図３Ｂは、ＫＩＴＬＧのグラフであり、メラニン合成の促進を示す遺伝子であることから、発現量減少は、メラニン合成抑制作用の向上を示唆する。図３Ｃは、ＳＩＲＴ１のグラフであり、発現量増加は、抗老化作用の向上を示唆する。図３Ｄは、ＦＧＦ７のグラフであり、発現量減少は、美白作用の向上を示唆する。図４Ａは、ＦＧＦ２のグラフであり、発現量増加は、細胞増殖亢進作用の向上を示唆する。図４Ｂは、ＭＭＰ１のグラフであり、発現量減少は、コラーゲン合成作用の向上を示唆する。図４Ｃは、ＨＡＳ２のグラフであり、発現量増加は、ヒアルロン酸合成作用の向上を示唆する。図４Ｄは、ＡＤＡＭ１０のグラフであり、発現量減少は、育毛作用の向上を示唆する。 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. 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.
　上記の表から、ブナカルス由来のエキスの優位性が示される。例えば、発現量増加が細胞増殖亢進に繋がる遺伝子ＤＫＫ３はカルスエキス２で、ＦＧＦ２はカルスエキス１、２で比較エキス１より大きい発現量となった。同じくヒアルロン酸合成を促進する遺伝子ＨＡＳ２の発現量はカルスエキス１、２で比較エキス１より大きくなった。発現量低下が美白に繋がる遺伝子ＦＧＦ７はカルスエキス１～３全てで、遺伝子ＫＩＴＬＧはカルスエキス１で比較エキス１より発現量が減り、逆に発現量増加が美白に繋がる遺伝子ＤＫＫ１はカルスエキス１で比較エキス１より発現量が増加した。このように、ブナカルス由来のエキスにおいて、比較エキスより高い効能を示唆する遺伝子が複数個あったことから、これらのカルスエキスが比較エキスより皮膚を含む生体に有効であることが示唆された。なお、生物的有用性に関する試験においては、対象となる生物的な要素および／または試験条件に起因して試験結果に差異が生じる場合があり得るが、上記の結果から、全体として、ブナカルス由来のエキスの有効性が示されたと言える。 The above table shows the superiority of the beech callus-derived extract. For example, 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. Similarly, 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, and the gene DKK1 whose expression level increases leads to whitening in callus extract 1. The expression level increased from the comparative extract 1. As described above, 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. In addition, in 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.
表皮角化細胞の遺伝子発現
　表２は、正常ヒト表皮角化細胞（ＮＨＥＫ）の遺伝子発現量の結果を示している。各遺伝子の発現量は、ｍＲＮＡ発現量を、コントロールとの相対値として評価した（コントロールの値を１とする）。ここで、各遺伝子について、カルスエキス（カルスエキス２～３の少なくとも１つ）が特に高い有用性を示したものには有用性を「ｈｉｇｈ」として表した。また、好ましい発現の方向性について、アップレギュレーション（発現量増加）が好ましい場合は、「ｕｐ」と記載し、ダウンレギュレーション（発現量減少）が好ましい場合は、「ｄｏｗｎ」と記載した。発現量評価として、発現量の増加が大きいものを「＋」、発現量の増加が著しく大きいものを「＋＋」で表し、また、発現量の減少が大きいものを「－」、発現量の増加が著しく大きいものを「－－」で表した。 Gene Expression in Epidermal Keratinocytes Table 2 shows the results of gene expression levels in normal human epidermal keratinocytes (NHEK). For the expression level of each gene, the mRNA expression level was evaluated as a relative value with respect to the control (control value is set to 1). Here, for each gene, usefulness was indicated as "high" when the callus extract (at least one of callus extracts 2 and 3) showed particularly high usefulness. In addition, with regard to the preferred direction of expression, when upregulation (increase in expression level) is preferred, it is indicated as "up", and when downregulation (decrease in expression level) is preferred, it is indicated as "down". As the expression level evaluation, "+" indicates a large increase in the expression level, "++" indicates a significant increase in the expression level, "-" indicates a large decrease in the expression level, and "-" indicates an increase in the expression level. A significantly large value is indicated by "--".
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図５は、表皮角化細胞での遺伝子発現解析結果のうち、代表的なものを抜粋して表したグラフを示す。グラフ縦軸の「高」および「低」は、それぞれ、「効果が高い」および「効果が低い」ことを意味する。
　図５Ａは、ＷＮＴ５Ａのグラフであり、発現量減少は、細胞分化・再生作用の向上を示唆する。図５Ｂは、ＤＫＫ３のグラフであり、発現量減少は、細胞分化・再生作用の向上を示唆する。図５Ｃは、ＫＩＴＬＧのグラフであり、発現量減少は、メラニン合成抑制作用の向上を示唆する。図５Ｄは、ＤＫＫ１のグラフであり、発現量増加は、メラノサイト増殖抑制作用の向上を示唆する。図５Ｅは、ＡＱＰ３のグラフであり、発現量増加は、新陳代謝作用の向上を示唆する。 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.
　上記の表から、ブナカルス由来のエキスの優位性が示される。発現量増加が新陳代謝を促進する遺伝子ＡＱＰ３の発現量はカルスエキス２で比較エキス１より大きい。また、発現量増加が美白に繋がる遺伝子ＤＫＫ１とＬＡＭＣ２はカルスエキス２、３で比較エキス１より発現量が増加する。このように、一部の遺伝子においてはカルスエキスが比較エキスより優位な遺伝子発現量となったことから、カルスエキスは比較エキスより皮膚を含む生体に有効である点があることが示唆された。 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. In addition, 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. As described above, 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.
評価
　以上から、実施例のブナカルスエキスは、抗老化、美白およびメラニン合成関連作用、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんに対して、作用を有し得ることが確認された。特に、メラニン合成抑制、美白、細胞増殖亢進、ヒアルロン酸合成などにおいて、優れた効果が確認された。したがって、ブナカルスエキスを用いることにより、機能性の高い化粧品を得ることができる。なお、上記の作用（例えば、抗酸化作用、遺伝子修復作用など）は、皮膚に直接塗布したときだけではなく、経口で摂取した際にも機能性を発揮する可能性がある。そのため、ブナカルスエキスを用いることにより、経口摂取の医薬品や、機能性を有する食品（例えば、美容食品、健康食品など）を得ることができる。 Evaluation From the above, 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.).

Claims (20)

  1. 　ブナ科植物の種子またはブナ科植物の種子由来の発芽体の、細胞からなる、カルス。 A callus consisting of cells of seeds of fagaceous plants or germination bodies derived from seeds of fagaceous plants.
  2. 　前記細胞が、植物生長調節物質で処理した細胞である、請求項１に記載のカルス。 The callus according to claim 1, wherein the cells are cells treated with a plant growth regulator.
  3. 　植物生長調節物質が、オーキシン（ａｕｘｉｎ）である、請求項２に記載のカルス。 The callus according to claim 2, wherein the plant growth regulator is auxin.
  4. 　オーキシンが、ピクロラム（ｐｉｃｌｏｒａｍ）、ジカンバ（ｄｉｃａｍｂａ）、インドール－３－酢酸、インドール－３－酪酸、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、２，４－ジクロロフェノキシ酢酸、２，４，５－トリクロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酪酸、および、ナプロアニリド、またはそれらの塩、から選択される、請求項３に記載のカルス。 Auxins are picloram, dicamba, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxy 4. The callus of claim 3, selected from acetic acid, 2-methyl-4-chlorophenoxyacetic acid, 2-methyl-4-chlorophenoxybutyric acid and naproanilide, or salts thereof.
  5. 　前記細胞が、ブナ科植物の種子の細胞である、請求項１～４のいずれか１項に記載のカルス。 The callus according to any one of claims 1 to 4, wherein the cells are seed cells of a fagaceous plant.
  6. 　前記細胞が、ブナ科植物の種子由来の発芽体の細胞である、請求項１～４のいずれか１項に記載のカルス。 The callus according to any one of claims 1 to 4, wherein the cell is a seed-derived germination cell of a fagaceous plant.
  7. 　前記発芽体が、ブナ科植物の種子を無菌発芽の条件下で発芽させた発芽体である、請求項６に記載のカルス。 The callus according to claim 6, wherein the germinated body is a germinated body obtained by germination of a seed of a fagaceous plant under sterile germination conditions.
  8. 　ブナ科植物が、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）である、請求項１～７のいずれか１項に記載のカルス。 The callus according to any one of claims 1 to 7, wherein the fagaceous plant is Japanese beech.
  9. 　ブナ科植物の種子またはブナ科植物の種子由来の発芽体の細胞を、培地中、植物生長調節物質の存在下で培養して増殖させることを含む、カルスの製造方法。 A method for producing callus, which comprises culturing and proliferating seeds of a fagaceous plant or seed-derived germination cells in a medium in the presence of a plant growth regulator.
  10. 　植物生長調節物質が、オーキシン（ａｕｘｉｎ）である、請求項９に記載のカルスの製造方法。 The method for producing callus according to claim 9, wherein the plant growth regulator is auxin.
  11. 　オーキシンが、ピクロラム（ｐｉｃｌｏｒａｍ）、ジカンバ（ｄｉｃａｍｂａ）、インドール－３－酢酸、インドール－３－酪酸、ナフタレン酢酸、ナフトキシ酢酸、フェニル酢酸、２，４－ジクロロフェノキシ酢酸、２，４，５－トリクロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酢酸、２－メチル－４－クロロフェノキシ酪酸、および、ナプロアニリド、またはそれらの塩、から選択される、請求項１０に記載のカルスの製造方法。 Auxins are picloram, dicamba, indole-3-acetic acid, indole-3-butyric acid, naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxy The method for producing callus according to claim 10, wherein the callus is selected from acetic acid, 2-methyl-4-chlorophenoxyacetic acid, 2-methyl-4-chlorophenoxybutyric acid, and naproanilide, or salts thereof.
  12. 　前記培地が、ＷＰＭ培地である、請求項９～１１のいずれか１項に記載のカルスの製造方法。 The method for producing callus according to any one of claims 9 to 11, wherein the medium is WPM medium.
  13. 　ブナ科植物が、ブナ（Ｊａｐａｎｅｓｅ　ｂｅｅｃｈ）である、請求項９～１２のいずれか１項に記載のカルスの製造方法。 The method for producing callus according to any one of claims 9 to 12, wherein the fagaceous plant is Japanese beech.
  14. 　請求項１～８のいずれか１項に記載のカルス由来の、カルス抽出物。 A callus extract derived from the callus according to any one of claims 1 to 8.
  15. 　水、または水とアルコールの混合液による抽出物である、請求項１４に記載のカルス抽出物。 The callus extract according to claim 14, which is an extract with water or a mixture of water and alcohol.
  16. 　抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を有する、請求項１４または１５に記載のカルス抽出物。 selected from the group consisting of anti-oxidation, suppression of melanin synthesis, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer. 16. The callus extract of claim 14 or 15, which has one or more effects of
  17. 　請求項１４～１６のいずれか１項に記載のカルス抽出物、および化粧品基剤を含む、化粧品組成物。 A cosmetic composition comprising the callus extract according to any one of claims 14 to 16 and a cosmetic base.
  18. 　請求項１４～１６のいずれか１項に記載のカルス抽出物をヒト被験者の皮膚に適用することを含む、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用を供するための方法。 Antioxidation, melanin synthesis suppression, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, comprising applying the callus extract according to any one of claims 14 to 16 to the skin of a human subject , anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism enhancement, hair growth, and anti-cancer.
  19. 　請求項１４～１６のいずれか１項に記載のカルス抽出物を供することを含む、ヒト繊維芽細胞またはヒト表皮角化細胞における、抗酸化、メラニン合成抑制、抗老化、美白、細胞増殖亢進、コラーゲン合成、ヒアルロン酸合成、抗炎症、遺伝子修復、細胞分化、細胞再生、新陳代謝促進、育毛、および抗がんからなる群から選択される１つ以上の作用に関連する遺伝子の発現量を増加するための方法。 Antioxidation, melanin synthesis suppression, anti-aging, whitening, cell proliferation enhancement in human fibroblasts or human epidermal keratinocytes, comprising providing the callus extract according to any one of claims 14 to 16, increasing expression of genes associated with one or more actions selected from the group consisting of collagen synthesis, hyaluronic acid synthesis, anti-inflammatory, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anticancer way for.
  20. 　化粧品組成物の製造のための、請求項１４～１６のいずれか１項に記載のカルス抽出物の使用。 Use of the callus extract according to any one of claims 14-16 for the production of a cosmetic composition.
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