Callus derived from Fagaceae plants, callus extract, method using same and cosmetic composition

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

[Technical Field]

[0001] The present invention relates to callus derived from a plant of the Fagaceae family, a method for producing the callus, an extract of callus derived from a plant of the Fagaceae family, a method using the callus extract, and a cosmetic composition containing the extract of callus derived from a plant of the Fagaceae family. [Background technology]

[0002] Plant extracts have traditionally been used as raw materials for cosmetics. Among these, cosmetic raw materials derived from beech trees are also known. For example, Patent Document 1 discloses a skin cosmetic containing a combination of extracts extracted from parsley and extracts extracted from beech trees. Plant-derived cosmetic raw materials can be incorporated into cosmetics in the hope of providing various beneficial effects on the skin. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 11-335257 Summary of the Invention [Problem to be solved by the invention]

[0004] As a result of extensive research, the present inventors have discovered that extracts from callus induced from seeds and germinated bodies of Fagaceae plants have biochemically useful activities, leading to the completion of the present invention. Therefore, the present invention aims to provide callus of seeds and germinated bodies of Fagaceae plants, a method for producing the callus, an extract of callus of seeds and germinated bodies of Fagaceae plants, a method using the callus extract, and a cosmetic composition containing an extract of callus of seeds and germinated bodies of Fagaceae plants. [Means for solving the problem]

[0005] The present invention includes, but is not limited to, the embodiments listed below. [1] A callus consisting of cells from the seeds of Fagaceae plants or from germinants derived from the seeds of Fagaceae plants. [2] The callus according to [1], wherein the cells are cells treated with a plant growth regulator. In this specification, the plant growth regulator is synonymous with the plant growth regulator. [3] The callus described in [2], wherein the plant growth regulator is an auxin. [4] The callus described in [3], wherein the auxin is selected from 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. [5] The callus according to any one of [1] to [4], wherein the cells are seed cells of a plant of the Fagaceae family. [6] A callus according to any one of [1] to [4], wherein the cells are cells of a germinant derived from the seeds of a plant of the Fagaceae family. [7] The callus described in [6], wherein the germinated body is a germinated body obtained by germinating seeds of a Fagaceae plant under sterile germination conditions. [8] A callus according to any one of [1] to [7], wherein the Fagaceae plant is Japanese beech. [9] A method for producing callus, comprising culturing and growing seeds of a Fagaceae plant or cells of germinated bodies derived from seeds of a Fagaceae plant in a medium in the presence of a plant growth regulator.

[10] A method for producing callus according to [9], wherein the plant growth regulator is an auxin.

[11] A method for producing callus described in

[10] , wherein the auxin is selected from 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 a WPM medium. [12-1] The method for producing a callus according to any one of [9] to

[12] , wherein the cells are seed cells of a Fagaceae plant. [12-2] A method for producing a callus according to any one of [9] to

[12] , wherein the cells are cells of germinants derived from seeds of a plant of the Fagaceae family. [12-3] A method for producing callus described in [12-2], wherein the germinated body is a germinated body obtained by germinating seeds of a Fagaceae 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 Fagaceae plant is Japanese beech.

[14] A callus extract derived from the callus described in any one of [1] to [8].

[15] A callus extract according to

[14] , which is an extract using water or a mixture of water and alcohol. [15-1] A callus extract described in

[14] , which is an extract using a mixture of water and ethanol.

[16] A callus extract described in

[14] ,

[15] or [15-1], having one or more effects selected from the group consisting of antioxidant, inhibition of melanin synthesis, anti-aging, whitening, enhanced cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer. [16-1] A callus extract according to any one of

[14] to

[16] for use in a cosmetic composition.

[17] A cosmetic composition comprising the callus extract described in any one of

[14] to

[16] and a cosmetic base. [17-1] A cosmetic composition according to

[17] for antioxidant, melanin synthesis inhibition, anti-aging, whitening, cell proliferation promotion, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, metabolism promotion, or hair growth.

[18] A method for providing one or more effects selected from the group consisting of antioxidant, inhibition of melanin synthesis, anti-aging, whitening, increased cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer, comprising applying a callus extract described in any one of

[14] to

[16] to the skin of a human subject. [18-1] The method described in

[18] , in which the callus extract is applied for purposes other than medicine, such as cosmetic purposes or food purposes, preferably cosmetic purposes.

[19] A method for increasing the expression level of genes associated with one or more effects selected from the group consisting of antioxidation, inhibition of melanin synthesis, anti-aging, whitening, enhanced cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolic promotion, hair growth, and anti-cancer in human fibroblasts or human epidermal keratinocytes, comprising providing the callus extract described in any one of

[14] to

[16] . [19-1] The method described in

[19] , in which the callus extract is provided for use other than pharmaceuticals, for example, for cosmetic or food applications, preferably for cosmetic applications.

[20] Use of a callus extract according to any one of

[14] to

[16] for the manufacture of a cosmetic composition. [20-1] The use described in

[20] , wherein the cosmetic composition is for antioxidant, melanin synthesis inhibition, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, metabolism promotion, or hair growth. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide biochemically useful callus of seeds and germinated bodies of Fagaceae plants that is suitable for incorporation into cosmetics, etc., a method for producing said callus, an extract of callus of seeds and germinated bodies of Fagaceae plants, a method using said callus extract, and a cosmetic composition containing an extract of callus of seeds and germinated bodies of Fagaceae plants. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a photograph showing an example of callus production in Examples 1 and 2. [Figure 2] Figure 2 is a graph showing cell viability when callus extract is applied to cells, where Figure 2A shows the viability of normal human dermal fibroblasts (NHDFs) and Figure 2B shows the viability of normal human epidermal keratinocytes (NHEKs). [Figure 3] FIG. 3 is a graph showing representative gene expression analysis results in fibroblasts (SOD2, KITLG, SIRT1, FGF7). [Figure 4] FIG. 4 is a graph showing representative gene expression analysis results in fibroblasts (FGF2, MMP1, HAS2, ADAM10). [Figure 5] FIG. 5 is a graph showing representative gene expression analysis results in epidermal keratinocytes (WNT5A, DKK3, KITLG, DKK1, AQP3). DETAILED DESCRIPTION OF THE INVENTION

[0008] Preferred embodiments of the present invention will now be described.

[0009] callus The present invention relates to a callus (hereinafter also referred to as "the callus of the present invention") consisting of cells of the seeds of Fagaceous plants or germinants derived from the seeds of Fagaceous plants.

[0010] As used herein, "callus" refers to a mass of undifferentiated plant cells obtained by culturing a part of a plant. A part of a plant refers to a component of a plant (e.g., a seed, root, leaf, stem, flower, etc.) and any other component obtained by further cutting or separating the component. Callus is not a leaf, stem, root, or other plant tissue, but rather a mass of cells that can differentiate into any tissue of the original plant (i.e., the plant from which the callus is derived). Therefore, callus does not include tissues or cells simply extracted from a plant (e.g., a leaf fragment or a stem segment). Callus can be generated by dedifferentiating tissue extracted from a plant using stimulation with plant hormones or other factors. In a broad sense, callus is also sometimes referred to as "wound-healing cells" because they form in nature to cover wounds in plants. However, the callus of the present invention does not include these natural substances. As used herein, callus refers to an artificially produced material. Callus is composed of a cell mass, i.e., an aggregate of multiple cells.

[0011] In one aspect, the present invention provides a callus consisting of cells from the seed of a Fagaceae plant. In another aspect, the present invention provides a callus consisting of cells from germinants derived from the seed of a Fagaceae plant.

[0012] Examples of plants of the Fagaceae family include those belonging to the subfamilies Fagoideae, Quercoideae, and Castaneoideae, and further include plants of the Fagaceae family such as the genera Fagus, Nothofagus, Quercus, Trigonobalanus, Castanea, Castanopsis, Lithocarpus, Notholithocarpus, and Chrysolepis. Fagaceae plants are arboreal plants, typically woody plants, and many are tall trees or arbors.

[0013] Specific examples of Fagaceae plants include beech, oak, konara oak, mizunara oak, oak, sawtooth oak, edulis oak, chinquapin, oak, chestnut, white oak, and oak. Particularly preferred Fagaceae plants are plants of the genus Fagus (scientific name: Fagus, English name: beech), and among them, beech (scientific name: Fagus crenata, English name: Japanese beech) and European beech (scientific name: Fagus sylvatica, English name: European beech) are more preferred, and Japanese beech is even more preferred. Beech is also called white beech, black beech, dog beech, hon beech, buckwheat, small beech, and large beech, and all of these are included. The scientific name for black beech and Japanese beech is Fagus japonica, but in this specification they are considered to be included in the Japanese beech. The origin of the Fagaceae plants is not particularly limited, but beech from Japan or Europe can be used. It is particularly preferable to use beech from the Shirakami Mountains in Japan (a mountain range spanning northwestern Akita Prefecture and southwestern Aomori Prefecture), for example.

[0014] Seeds of Fagaceae plants can be collected and used after naturally falling from Fagaceae plants (e.g., beech trees). Seeds that are generally called acorns can be used. However, acorns technically refer to fruits (also called nuts or nut), and the seeds are contained within the acorns. Using seeds has the advantage of not damaging existing plants and allowing for sustained harvesting of raw materials, compared to using bark, etc. In one aspect of the present invention, callus is induced from Fagaceae plant seeds.

[0015] Furthermore, germinated plants derived from the seeds of Fagaceae plants are plants obtained by germinating the seeds of the Fagaceae plants described above. Germination refers to the process in which a seed absorbs water and the radicle (an organ that will later become a root), a part of the embryonic tissue, breaks through the seed coat to emerge. Germination can be performed artificially, or naturally germinated seeds can be used, but artificial germination is preferred. Artificial germination can be performed using known methods, and a preferred example is a method under aseptic germination conditions. For example, the pericarp and seed coat of a seed (or more precisely, a nut containing a seed) can be removed to expose the seed interior, and then the seed can be aseptically cultured to germinate the seed and obtain germinated plants. Aseptic culture conditions include, for example, using a plant culture medium such as WPM medium, at room temperature (e.g., 20 to 25°C), under a photoperiod longer than a certain period (e.g., 12 hours or more), for a period of several days to several months (e.g., several weeks to one month). The germinants that can be used range from those at the stage where the radicle has broken through the seed coat to those at the seedling stage and even those at the sprout stage. The germinants are preferably seedlings. The germinants may have roots (radicles, etc.), leaves (cotyledons, etc.), and stems (hypocotyls, etc.). The germinants used may be those at the cotyledon expansion stage. In one embodiment of the present invention, callus is induced from germinants of seeds of Fagaceae plants.

[0016] Callus production A method for producing callus according to one embodiment of the present invention comprises culturing and growing cells of seeds of a Fagaceous plant or germinants derived from seeds of a Fagaceous plant in a medium in the presence of a plant growth regulator.

[0017] When inducing callus from seeds for the production of callus, first, the pericarp and seed coat of the seed are removed. This exposes the seed cells, making them easier to culture. At this stage, it is preferable to cut the seeds into multiple pieces (for example, two pieces). The cuts facilitate callus formation. For example, the seeds can be cut so that the major axis of the oval shape is divided into approximately two parts. It is also preferable to sterilize the seeds with an alcohol such as ethanol or a sterilizing solution such as a sodium hypochlorite solution. Next, some or all of the seed cells are placed in a culture medium. When using some of the seed cells, the seed cells can be cut into appropriate sizes as needed.

[0018] When inducing callus from germinated bodies, first, a portion of the germinated body obtained by the method described above, preferably the hypocotyl (the stem portion between the roots and the leaves), is cut into appropriate sizes as needed, and placed in a culture medium. By cutting from the germinated body, the cells are exposed, making it easier to culture the germinated cells.

[0019] Cells (seed cells or germinated cells) placed in a medium are cultivated and multiplied by cell culture, and are induced to form a callus, a mass of cells. At this time, for example, a part of the seed or germinated cell will swell due to cell proliferation. This proliferated part becomes a callus.

[0020] The medium may be a solid medium or a liquid medium, but a solid medium is preferred. In the case of a solid medium, the cells of the seed or germinated plant can be cultured by placing them directly on the medium (by contacting the medium). The medium may contain additional components such as plant growth regulators.

[0021] The medium (basal medium) is not particularly limited, but examples include WPM medium, BTM medium, MS medium, and dilutions thereof (e.g., 1 / 2 WPM medium, 1 / 2 BTM medium, 1 / 2 MS medium), among which WPM medium is preferred. In the media, WPM stands for Woody Plant Medium, BTM stands for Broadleaf Tree Medium, and MS stands for Murasige Skoog. The composition of the medium is publicly known. The medium is commercially available.

[0022] A solid medium can be formed by, for example, dissolving a composition that serves as the substrate of the medium in water, heating the resulting solution, adding a support (e.g., agar), and then cooling and solidifying the solution. At this time, a plant growth regulator can be added to the solution to obtain a medium containing the plant growth regulator.

[0023] Plant growth regulators are substances that regulate plant growth (preferably growth promoters), and may include, for example, substances known as plant hormones. Examples of plant growth regulators that can be used include auxin, cytokinin, gibberellin, abscisic acid, ethylene, and combinations of two or more of these. Auxin is preferably used as the plant growth regulator. When auxin is used, callus can be produced efficiently. In this specification, the term "plant growth regulator" is synonymous with "plant growth regulator."

[0024] Furthermore, the auxin is preferably selected from 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 (e.g., inorganic salts such as sodium salts and calcium salts). This allows for more efficient callus production. The auxin is more preferably picloram or dicamba, with picloram being even more preferred.

[0025] It is also preferable to use a combination of auxin and cytokinin as a plant growth regulator. This allows for more efficient callus production. This combination is particularly preferable when culturing germinated cells. The cytokinin may be a synthetic cytokinin. Examples of cytokinins include 6-benzylaminopurine and trans-zeatin, with 6-benzylaminopurine being preferred. A specific example of a combination of auxin and cytokinin is the combination of picloram and 6-benzylaminopurine.

[0026] When auxin is used as the plant growth regulator, the concentration of auxin (e.g., picloram) in the medium is preferably 0.1 to 20 μM, more preferably 0.5 to 10 μM, even more preferably 1 to 10 μM, and even more preferably 1 μM. When cytokinin is used as the plant growth regulator, the concentration of cytokinin (e.g., 6-benzylaminopurine) in the medium is preferably 1 to 10 μM, and even more preferably 2 μM. When auxin and cytokinin are combined, a combination of 0.1 to 20 μM auxin and 1 to 10 μM cytokinin is preferred, such as a combination of 0.1 to 20 μM picloram and 1 to 10 μM 6-benzylaminopurine, for example, a combination of 0.1 to 20 μM picloram and 1 to 10 μM 6-benzylaminopurine, specifically, a combination of 1 μM picloram and 2 μM 6-benzylaminopurine, or a combination of 10 μM picloram and 2 μM 6-benzylaminopurine.

[0027] The conditions for cell culture are not particularly limited, but can be carried out in a culture room at 20-25°C with 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 a sufficient amount of callus is visually confirmed to have been obtained. Cell culture may or may not be carried out under sterile conditions.

[0028] It has been confirmed that callus with different morphologies (e.g., color, hardness) can be obtained by varying the type of plant growth regulator used and the amount or concentration of the plant growth regulator used. For example, in one embodiment, white callus can be obtained from beech seeds, and brown or green callus can be obtained from beech germinating plants (see Examples). Such callus also differs in hardness. It has been confirmed that these calluses differ not only in morphology but also in their functions.

[0029] As described above, cells in the medium (seed cells or germinant cells) are grown by cell culture, producing callus, a mass of cells. Callus can form as a raised mass on the surface of the seed or germinant. Callus can be used for extraction either separately from the seeds and germinants (the mother body, so to speak) used for culture, or together with the seeds and germinants (the entire culture).

[0030] One aspect of the present invention relates to a callus extract derived from the above-mentioned callus. Herein, the extract from the callus is simply referred to as "callus extract." The callus may be produced by the production method described above. Therefore, the callus extract is an extract from the seed callus of a Fagaceae plant or an extract from the callus of a germinated seed of a Fagaceae plant.

[0031] Extraction from callus can be performed using methods similar to those used to produce known plant extracts. For example, callus can be freeze-dried to remove moisture, then the resulting dried material can be cut or crushed into small pieces or powder, and then extracted with an extraction solvent. The solvent can be water, an organic solvent, or a mixture of water and an organic solvent. While either hydrophilic or hydrophobic organic solvents can be used, hydrophilic organic solvents are preferred. Alcohols (e.g., lower alcohols) are preferred, with ethanol or methanol being preferred. Preferred solvents include water or a mixture of water and an alcohol such as ethanol. When using a mixture of water and an organic solvent (e.g., alcohol), the ratio of organic solvent (e.g., alcohol) to water (organic solvent / water) is preferably 1 / 99 to 99 / 1 by volume, more preferably 1 / 9 to 9 / 1, with a ratio of 50% organic solvent (e.g., alcohol) being more preferred. Extraction can be performed at room temperature or under heating. For example, extraction can be performed at temperatures ranging from room temperature to the boiling point of the organic solvent (e.g., 80°C). The extraction may be carried out while the mixture is left standing or while being stirred. The extraction time is not particularly limited, but may be, for example, 1 hour to 1 day, and preferably 1 hour.

[0032] After extraction, residue (solid matter) can be removed by filtration or other methods to obtain a solution (callus extract) containing components extracted from the callus. The extract can be used as is or after concentration. Preferably, liquid components (water, alcohol, etc.) can be removed from the extract to obtain an extract in the form of a solid, oil, or paste. The callus extract is preferably in the form of a solid powder. The callus extract can be stored, for example, in a brown glass container at room temperature or in a refrigerator.

[0033] The callus extract thus obtained may have one or more effects selected from the group consisting of antioxidant, melanin synthesis inhibition, anti-aging, skin whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer. The callus extract preferably has one or more effects selected from the group consisting of antioxidant, melanin synthesis inhibition, anti-aging, skin whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, metabolism promotion, and hair growth. The above effects of the callus extract have been experimentally confirmed, as described in the Examples below. These effects can be utilized in cosmetic, pharmaceutical, and other physiological applications. For example, by incorporating the callus extract into a cosmetic composition, cosmetics having the above effects can be obtained. It can also be used in topical pharmaceuticals. Furthermore, the above effects may be exerted orally, making it applicable to pharmaceuticals (orally administered), foods, and the like. More preferably, it is used in applications other than medicine, and more preferably in cosmetics or foods, and even more preferably in cosmetics. In this specification, cosmetics may include quasi-drugs. However, it may also be used in cosmetics other than quasi-drugs.

[0034] Without being bound by any hypothesis, it is possible that callus induced from beech seeds and germinated tissues may contain higher quantities and quality of active ingredients than the active ingredients contained in the beech seeds and germinated tissues themselves. It is speculated that this is because the active ingredients increase as cells proliferate to form callus. It is also speculated that the active ingredients in callus extracts extracted from callus with increased active ingredients also increase, resulting in the above-mentioned beneficial effects. Furthermore, when callus is used, the active ingredients can be increased by cell culture, making it possible to obtain useful beech extracts more efficiently than when the plant itself is used. However, the present invention is not limited to the above speculation.

[0035] Cosmetic composition In one aspect, the present invention relates to a cosmetic composition comprising a callus extract and a cosmetic base. The callus extract may be any of those described above. By incorporating the callus extract, cosmetics can be obtained that have one or more effects selected from the group consisting of antioxidant, melanin synthesis inhibition, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, and hair growth. The cosmetics preferably have one or more effects selected from the group consisting of antioxidant, melanin synthesis inhibition, anti-aging, whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, metabolism promotion, and hair growth. Specific efficacy and effects of the cosmetics achieved by the above effects include, but are not limited to, whitening, wrinkle prevention, blemish prevention, firmness improvement, improved skin moisture, improved skin barrier function, promotion of skin cell turnover, and improved recovery from damage caused by ultraviolet rays and the like.

[0036] A cosmetic base may be a component that can be incorporated into cosmetics. In this specification, the term "cosmetic base" refers to a component that forms the framework of the 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, etc.

[0037] Examples of oils include, but are not limited to, natural or synthetic ester oils, hydrocarbon oils, higher alcohols, fatty acids, and silicone oils. Specific examples include, but are not limited to, liquid paraffin, petrolatum, 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, and dimethicone.

[0038] Examples of alcohols (monohydric or polyhydric alcohols) include, but are not limited to, ethanol, isopropanol, butanol, glycerin, 1,3-butylene glycol, propylene glycol, and dipropylene glycol.

[0039] 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, and sorbitan stearate.

[0040] Examples of polymers include naturally occurring polymers and synthetic polymers, and specific examples include, but are not limited to, gums such as xanthan gum, gellan gum, gum arabic, and guar gum, celluloses such as hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, and carboxymethyl cellulose, sodium hyaluronate, carboxyvinyl polymer, and polyvinyl alcohol.

[0041] Examples of powders include inorganic powders and organic powders, including, but not limited to, titanium oxide, zinc oxide, talc, mica, silica, and polyethylene terephthalate (PET) powder.

[0042] The cosmetic composition may contain ingredients other than the cosmetic base. Examples of such ingredients include water-soluble ingredients, oil-soluble ingredients, moisturizers, thickeners, pigments, UV absorbers, film-forming agents, pH adjusters, anti-fading agents, antioxidants, antifoaming agents, cosmetic ingredients, preservatives, and fragrances. The cosmetic composition may also contain oils, water-soluble polymers, and powders that do not constitute the cosmetic base. The cosmetic composition may also contain physiologically active substances (so-called active ingredients) other than the callus extract.

[0043] The cosmetic composition may be in any suitable form that can be used as a cosmetic. Examples of the cosmetic composition include aqueous solutions, oils, emulsions (O / W type, W / O type, W / O / W type, etc.), pastes, powders, solids, etc. The cosmetic composition may also be in the form of a spray, mist, etc.

[0044] The cosmetic composition may be used as a cosmetic product itself, or may be used as a raw material (work-in-progress) for producing a final cosmetic product.

[0045] A cosmetic composition can be obtained by appropriately mixing the above-mentioned callus extract with the raw materials to be incorporated into the cosmetic composition as described above. Thus, the present invention provides use of the above-mentioned callus extract for the production of a cosmetic composition.

[0046] The cosmetic composition may be a composition for skin cosmetics. The skin may be facial skin or skin on parts of the body other than the face (such as the head, neck, shoulders, hands, and feet), 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, blushers, eye shadows (eye colors), makeup bases, foundations (such as liquid foundations and solid foundations), and sunscreens. In particular, scalp cosmetics include hair growth agents, hair growth promoters, and hair care products.

[0047] The cosmetic composition may be a composition for hair cosmetics. Examples of hair cosmetics include hair cream, hair wax, hair rinse, hair mask, and hair treatment.

[0048] Application of callus extract to the skin of human subjects In one aspect, the present invention relates to a method for providing one or more effects selected from the group consisting of antioxidation, inhibition of melanin synthesis, anti-aging, skin whitening, promotion of cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, promotion of metabolism, hair growth, and anti-cancer, comprising applying the above-mentioned callus extract to the skin of a human subject. The callus extract is obtained from the seeds of Fagaceae plants or calluses of germinated bodies derived from the seeds of Fagaceae plants, as described above.

[0049] The application of the callus extract to the skin of a human subject is preferably an application other than a pharmaceutical application. For example, the callus extract is applied to human skin for cosmetic use, preferably by a cosmetic composition. The callus extract can be applied to the skin by applying the cosmetic composition to the skin of a human subject. By applying the callus extract to the skin, for example, the callus extract or components therein can penetrate the skin and exert the above-mentioned effects. Preferably, the application of the callus extract to the skin does not include pharmaceutical therapeutic use.

[0050] Method for increasing gene expression using callus extract In one aspect, the present invention relates to a method for increasing the expression level of genes associated with one or more functions selected from the group consisting of antioxidation, melanin synthesis inhibition, anti-aging, skin whitening, cell proliferation enhancement, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer in normal human fibroblasts or normal human epidermal keratinocytes, comprising providing the above-mentioned callus extract. The callus extract is obtained from the seeds of Fagaceae plants or callus of germinated bodies derived from the seeds of Fagaceae plants, as described above.

[0051] In one aspect, the callus extract can be applied to normal human fibroblasts (also referred to herein simply as "human fibroblasts" or "fibroblasts") or normal human epidermal keratinocytes (also referred to herein simply as "human epidermal keratinocytes" or "epidermal keratinocytes") in human skin. In this case, the callus extract is applied to the skin of a human subject. Such application is also referred to as in vivo. The callus extract is preferably applied to human skin using a cosmetic composition. By applying the cosmetic composition to the skin of a human subject, the callus extract can be applied to human fibroblasts or human epidermal keratinocytes in the skin. By applying the callus extract to the skin, for example, the callus extract or components therein can penetrate the skin and affect normal human fibroblasts or normal human epidermal keratinocytes, thereby increasing the expression level of genes related to the above-mentioned effects.

[0052] In another embodiment, the callus extract can be applied to cultured normal human fibroblasts or human epidermal keratinocytes. This application is also referred to as in vitro. By applying the callus extract to normal human fibroblasts or normal human epidermal keratinocytes, for example, the components in the callus extract can affect the normal human fibroblasts or normal human epidermal keratinocytes, thereby increasing the expression levels of genes related to the above-mentioned effects.

[0053] Here, the callus extract may also be useful for animals other than humans (particularly mammals, such as dogs, cats, rats, and mice). The callus extract can also be applied to animals other than humans. When applied to animals other than humans (for example, by applying it to the skin), the same effects as those described above (for example, increased gene expression levels) can be achieved. [Example]

[0054] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0055] material beech seeds Seeds that had naturally fallen to the ground from beech (scientific name: Fagus crenata, English name: Japanese beech) growing wild in the Shirakami Mountains (Akita Prefecture) of Japan were collected. Beech seeds collected in this way were used in the following experiments. Figure 1 is a photograph showing an example of callus production in Examples 1 and 2, and Figure 1A shows a beech seed.

[0056] Example 1. Production of beech seed callus The pericarp and seed coat were removed from beech seeds, cut into two pieces, and sterilized by immersion in 70% ethanol for 30 seconds and 10% sodium hypochlorite solution for 5 minutes (Figure 1B). For example, the seeds were cut into pieces approximately halfway along the major axis of their ovoid shape. Separately, picloram was added to the WPM medium ingredients to prepare a solution. This mixture was placed in a cell culture dish to prepare WPM medium containing 10 μM picloram (referred to as WPM medium 1). The seeds from which the pericarp and seed coat had been removed were added to this picloram-containing WPM medium and cultured at 25°C under a 16-hour photoperiod. After 30 days of culture, the cells in the culture proliferated, and callus formation, a cell mass, was confirmed (Figure 1D). The callus was white. Hereinafter, the callus obtained in Example 1 is referred to as callus 1. The culture was removed, and the callus portion was excised and used in the subsequent extraction process.

[0057] Example 2. Production of callus from germinated beech seeds The pericarp and seed coat were removed from beech seeds, which were then placed in WPM medium (a plant hormone-free medium) and germinated under conditions of 25°C and a 16-hour photoperiod. Germinated plants (sprouts) were obtained within one month of incubation (Figure 1C). The hypocotyls (the area between the leaves and roots) of the germinated plants were cut and harvested into cube-sized pieces measuring 3–10 mm on each side. A solution containing picloram and 6-benzylaminopurine (hereinafter also referred to as "6-BP") was prepared by adding picloram and 6-benzylaminopurine (hereinafter referred to as "6-BP") to the WPM medium ingredients. This mixture was then placed in a cell culture dish to prepare WPM medium containing picloram and 6-benzylaminopurine. Two types of WPM medium were prepared: WPM medium containing 10 μM picloram and 2 μM 6-benzylaminopurine (referred to as WPM medium 2) and WPM medium containing 1 μM picloram and 2 μM 6-benzylaminopurine (referred to as WPM medium 3). Fragments of the hypocotyls from the germinated plants were added to this WPM medium and cultured at 25°C with a 16-hour photoperiod. After 30 days of culture, the cells in the cultures proliferated, and the formation of callus, a cell mass, was confirmed (Figures 1E and 1F). The callus obtained on WPM medium 2 (hereafter referred to as callus 2) was brown. The callus obtained on WPM medium 3 (hereafter referred to as callus 3) was green. The cultures were removed, and the callus portions were cut out and used in the subsequent extraction process.

[0058] 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 to powder. 40 mL of 50% ethanol water was added as an extraction solvent to 0.5 to 1 g of callus powder, and the mixture was shaken at room temperature to perform extraction. One hour after the start of extraction, the solids were removed by filtration, and the filtrate was concentrated by evaporation. The concentrate became a solid. This resulted in a callus extract. Hereinafter, the callus extract obtained from callus 1 will be referred to as "callus extract 1," the callus extract obtained from callus 2 will be referred to as "callus extract 2," and the callus extract obtained from callus 3 will be referred to as "callus extract 3."

[0059] Example 4. Gene expression in human skin cells by callus extract Biochemical assays 1. Preparation of Samples for Assay To clarify the functionality of beech callus extract on human skin, the extract was applied to human skin cells and the effects of the beech callus extract were investigated by analyzing gene expression levels using real-time PCR. The entire amount of each callus extract obtained above was dissolved in 1 to 1.5 mL of dimethyl sulfoxide (DMSO) to prepare a sample for assay. As a comparative sample, we used a commercially available European beech bud extract (hereafter referred to as "Comparative Extract 1"), which is a cosmetic ingredient. European beech bud extract is an extract obtained by concentrating an extract obtained by extracting the young buds of European beech (scientific name: Fagus sylvatica, English name: European beech) with water (it is not derived from callus).

[0060] 2.Cell culture Two types of human skin-derived cells were used: normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK). Cell culture was performed in DMEM (Nacalai tesque) + 10% bovine serum albumin (BSA) medium for NHDF and CnT-Prime (CELLnTEC) medium for NHEK, at 37°C in a CO2 incubator. To determine the appropriate concentration, the callus extract was diluted 1 / 3 times to nine different concentrations and then added to the cultured cells. Cell viability was assessed 24 hours after addition. Extract from callus 2 was used for the fibroblast study, and extract from callus 3 was used for the epidermal keratinocyte study. Figure 2 shows the cell viability of the callus extracts applied to the cells. The highest concentration that did not adversely affect the cells (reduced viability) was used. For example, when callus extract 2 was applied to fibroblasts, the concentration was 0.012% (w / v) (Figure 2A), and when callus extract 3 was applied to epidermal keratinocytes, the concentration was 0.112% (w / v) (Figure 2B). Cells (normal human skin fibroblasts and normal human epidermal keratinocytes) were cultured in cell culture dishes, and when the appropriate cell mass was reached, the assay sample was added. For fibroblasts, experiments were conducted using callus extracts 1 to 3 and comparative extract 1, and for epidermal keratinocytes, experiments were conducted using callus extracts 2, 3, and comparative extract 1. After adding the sample, the cells were cultured for 24 hours and then analyzed by real-time PCR. The real-time PCR system used was the QuantStudio 12K Flex (ThermoFisher). RNA was extracted from the cells using the SuperPrep™ II Cell Lysis & RT Kit (TOYOBO), and the reverse transcription reaction was performed using ReverTra Ace qPCR RT Master Mix (TOYOBO). Real-time PCR reactions were performed using TaqMan Fast Advanced Master Mix and TaqMan Array cards (ThermoFisher). This allowed for the analysis of changes in the expression levels of 46 genes involved in functions such as anti-inflammation, anti-cancer, and gene repair. The expression level of each gene was evaluated using a control without sample addition as a relative value based on 18S rRNA (relative value based on the ΔΔCt method).

[0061] The following 42 genes were analyzed in normal human dermal fibroblasts (NHDF). Note that the parentheses indicate the effects (efficacy and efficacy) associated with each gene. ADAM10 (hair growth), ADAM12 (hair growth), PPARG (hair growth / antioxidation), 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-cancer), PTEN (anti-cancer), RB1 (anti-cancer), SMAD4 (anti-cancer), TP53 (anti-cancer), SOD1 (anti-oxidation), SOD2 (anti-oxidation) ), SOD3 (antioxidant), SIRT1 (anti-aging), SIRT2 (anti-aging), COL1A1 (collagen synthesis), COL3A1 (collagen synthesis), COL7A1 (collagen synthesis), MMP1 (collagen synthesis), DKK3 (proliferation enhancement), FGF2 (proliferation enhancement), PTGES3 (proliferation enhancement), VEGFA (proliferation enhancement), CD44 (hyaluronic acid synthesis), HAS2 (hyaluronic acid synthesis), VCAN (hyaluronic acid synthesis), CLU (skin whitening), FGF7 (skin whitening), KITLG (melanin synthesis related), NRG1 (melanin synthesis related), DKK1 (melanin synthesis related).

[0062] The following 41 genes were analyzed in normal human epidermal keratinocytes (NHEK). Note that the numbers in parentheses indicate the effects (efficacy and effectiveness) associated with each 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 and regeneration), DKK3 (cell differentiation and regeneration), ITGA6 (cell differentiation and regeneration), LAMA5 (cell differentiation and regeneration), WNT5A (cell differentiation and 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 (anti-cancer), SMAD4 (anti-cancer), TP53 (anti-cancer), SOD1 (anti-oxidation), SOD2 (anti-oxidation), PPARG (anti-oxidation / hair growth), SIRT1 (anti-aging), SIRT2 (anti-aging), CERS3 (ceramide synthesis), ELOVL1 (ceramide synthesis), ELOVL4 (ceramide synthesis), AQP3 (metabolism), FLG (metabolism), LOR (metabolism), CD44 (hyaluronic acid synthesis), HAS3 (hyaluronic acid synthesis), DKK1 (melanin synthesis related), KITLG (melanin synthesis related), LAMC2 (whitening).

[0063] 3.Results Fibroblast gene expression Table 1 shows the results of gene expression in normal human dermal fibroblasts (NHDF). The expression level of each gene was evaluated relative to the control mRNA expression level (control value was set to 1). For each gene, the callus extract (at least one of callus extracts 1-3) showed particularly high usefulness, which was indicated as "high." Regarding the preferred direction of expression, "up" was used when upregulation (increased expression level) was preferred, and "down" was used when downregulation (decreased expression level) was preferred. Regarding the expression level evaluation, a large increase in expression level was indicated by "+," a significantly large increase in expression level was indicated by "++," a large decrease in expression level was indicated by "-," and a significantly large increase in expression level was indicated by "--."

[0064] [Table 1]

[0065] Figures 3 and 4 show graphs illustrating representative results of gene expression analysis in fibroblasts. "High" and "Low" on the vertical axis of the graphs represent "high effect" and "low effect," respectively. Figure 3A is a graph of SOD2, which encodes an enzyme that scavenges reactive oxygen species, so increased expression suggests improved antioxidant activity. Figure 3B is a graph of KITLG, which is a gene that promotes melanin synthesis, so decreased expression suggests improved melanin synthesis inhibitory activity. Figure 3C is a graph of SIRT1, whose increased expression suggests improved anti-aging activity. Figure 3D is a graph of FGF7, whose decreased expression suggests improved whitening activity. Figure 4A is a graph of FGF2, whose increased expression suggests improved cell proliferation promotion activity. Figure 4B is a graph of MMP1, whose decreased expression suggests improved collagen synthesis activity. Figure 4C is a graph of HAS2, whose increased expression suggests improved hyaluronic acid synthesis activity. Figure 4D is a graph of ADAM10, whose decreased expression suggests improved hair growth activity.

[0066] The table above demonstrates the superiority of beech callus-derived extracts. For example, the gene DKK3, whose increased expression leads to enhanced cell proliferation, was expressed at a higher level in callus extract 2, while FGF2 was expressed at a higher level in callus extracts 1 and 2 than in comparison extract 1. Similarly, the expression level of the gene HAS2, which promotes hyaluronic acid synthesis, was higher in callus extracts 1 and 2 than in comparison extract 1. The gene FGF7, whose decreased expression leads to skin whitening, was expressed at a lower level in all callus extracts 1 through 3, while the gene KITLG was expressed at a lower level in callus extract 1 than comparison extract 1. Conversely, the gene DKK1, whose increased expression leads to skin whitening, was expressed at a higher level in callus extract 1 than comparison extract 1. Thus, the presence of several genes in the beech callus-derived extracts suggesting greater efficacy than the comparison extracts, suggesting that these callus extracts may be more effective for the body, including the skin, than the comparison extracts. Although there may be differences in the results of tests on biological usefulness due to the biological factors and / or test conditions, the above results indicate that the beech callus-derived extract is generally effective.

[0067] Gene expression in epidermal keratinocytes Table 2 shows the results of gene expression in normal human epidermal keratinocytes (NHEK). The expression level of each gene was evaluated relative to the control mRNA expression level (control value was set to 1). Here, for each gene, the usefulness of callus extracts (at least one of callus extracts 2-3) was indicated as "high." Regarding the preferred direction of expression, upregulation (increased expression level) was indicated as "up," and downregulation (decreased expression level) was indicated as "down." Regarding the expression level evaluation, a large increase in expression level was indicated as "+," a significantly large increase in expression level was indicated as "++," a large decrease in expression level was indicated as "-," and a significantly large increase in expression level was indicated as "--."

[0068] [Table 2]

[0069] Figure 5 shows a graph depicting representative results of gene expression analysis in epidermal keratinocytes. "High" and "Low" on the vertical axis of the graph represent "high effect" and "low effect," respectively. Figure 5A is a graph of WNT5A, and a decrease in expression level suggests improved cell differentiation and regeneration. Figure 5B is a graph of DKK3, and a decrease in expression level suggests improved cell differentiation and regeneration. Figure 5C is a graph of KITLG, and a decrease in expression level suggests improved melanin synthesis inhibitory activity. Figure 5D is a graph of DKK1, and an increase in expression level suggests improved melanocyte proliferation inhibitory activity. Figure 5E is a graph of AQP3, and an increase in expression level suggests improved metabolic activity.

[0070] The table above shows the superiority of the extract derived from beech callus. The expression level of the gene AQP3, whose increased expression promotes metabolism, was higher in callus extract 2 than in comparison extract 1. Additionally, the expression levels of the genes DKK1 and LAMC2, whose increased expression leads to skin whitening, were higher in callus extracts 2 and 3 than in comparison extract 1. As such, the callus extract had a higher gene expression level than the comparison extract for some genes, suggesting that the callus extract may be more effective for the living body, including the skin, than the comparison extract.

[0071] evaluation From the above, it was confirmed that the beech callus extract of the examples may have effects on anti-aging, whitening, melanin synthesis-related effects, cell proliferation promotion, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer. In particular, excellent effects were confirmed in melanin synthesis inhibition, whitening, cell proliferation promotion, and hyaluronic acid synthesis. Therefore, the use of beech callus extract can make highly functional cosmetics. The above effects (e.g., antioxidant effect, gene repair effect, etc.) may be functional not only when applied directly to the skin but also when taken orally. Therefore, the use of beech callus extract can make oral medicines and functional foods (e.g., beauty foods, health foods, etc.).

Claims

1. A beech callus extract, which is an extract from callus consisting of cells of seeds of beech plants or germinants derived from seeds of beech plants, The Fagaceae plant is the Japanese beech. The beech callus extract.

2. 2. The beech callus extract of claim 1, wherein the cells are cells treated with a plant growth regulator.

3. 3. The beech callus extract according to claim 2, wherein the plant growth regulator is an auxin.

4. 4. The beech callus extract according to claim 3, wherein the auxin is selected from 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.

5. 2. The beech callus extract according to claim 1, wherein the cells are seed cells of a beech plant.

6. 2. The beech callus extract according to claim 1, wherein the cells are cells of germinants derived from the seeds of a beech plant.

7. 7. The beech callus extract according to claim 6, wherein the germinants are germinants obtained by germinating seeds of a beech plant under sterile germination conditions.

8. 2. The beech callus extract according to claim 1, which is an extract with water or a mixture of water and alcohol.

9. The beech callus extract of claim 1 has one or more effects selected from the group consisting of antioxidant, inhibition of melanin synthesis, anti-aging, whitening, enhanced cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, metabolism promotion, hair growth, and anti-cancer.

10. Cultivating and growing cells of seeds of a plant of the family Fagaceae or germinated cells derived from seeds of a plant of the family Fagaceae in a medium in the presence of a plant growth regulator to obtain callus; obtaining an extract from said callus; A method for producing a beech callus extract, comprising: The Fagaceae plant is the Japanese beech. The manufacturing method.

11. 11. The method for producing beech callus extract according to claim 10, wherein the plant growth regulator is an auxin.

12. 12. The method for producing beech callus extract according to claim 11, wherein the auxin is selected from 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.

13. A method for producing beech callus extract according to claim 10, wherein the medium is WPM medium.

14. The method for producing a beech callus extract according to any one of claims 10 to 13, wherein the Fagaceae plant is Japanese beech.

15. A cosmetic composition comprising the beech callus extract according to any one of claims 1 to 9 and a cosmetic base.

16. A method for increasing the expression level of genes associated with one or more functions selected from the group consisting of antioxidation, inhibition of melanin synthesis, anti-aging, whitening, enhanced cell proliferation, collagen synthesis, hyaluronic acid synthesis, anti-inflammation, gene repair, cell differentiation, cell regeneration, promotion of metabolism, hair growth, and anti-cancer in human fibroblasts or human epidermal keratinocytes, comprising providing a beech callus extract described in any one of claims 1 to 9.

17. Use of the beech callus extract according to any one of claims 1 to 9 for the preparation of a cosmetic composition.

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