Cosmetic composition

Abstract

An object of the present invention is to provide a composition containing an active ingredient that is a component contained in a safe natural product and that can suppress or improve skin aging such as wrinkles and sagging. The above object is achieved by a cosmetic composition containing at least one active ingredient selected from the group consisting of 5,7,3',4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone, and 5-OH-3,7,3',4'-tetramethoxyflavone, and a product containing at least one of these ingredients.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition, particularly a cosmetic composition containing components derived from natural products.

Background Art

[0002] The skin, the largest organ of the human body, is the outermost layer of the living body and plays a role in the forefront of biological defense, and it is required to be kept healthy and aesthetically normal. The skin (stratum corneum) is subjected to many stresses due to ultraviolet rays, aging, stress, diseases, eating habits, etc., and various troubles such as deterioration of skin function and skin aging occur.

[0003] Wrinkles are formed by the action of proteolytic enzymes (such as elastase and collagenase) induced by skin aging (photoaging) due to aging or sunlight. Although the mechanism of skin aging is not clear, ultraviolet rays are considered to be the largest environmental factor involved in wrinkle formation. Reactive oxygen species produced by ultraviolet rays not only cause acute inflammation such as sunburn, but also are known to induce photoaging when their production is repeatedly chronic. There is a high demand for anti-wrinkle agents that suppress skin aging and improve wrinkles for the health and beauty of the skin.

[0004] On the other hand, an important factor that determines the color of the skin and hair is a pigment substance called melanin, which is produced by melanocytes present in the basal layer of the epidermis and hair follicles. The control of melanin has an aesthetic impact as well as protection against ultraviolet rays. Congenital or acquired loss or decrease in melanin production ability results in vitiligo and leukoderma. Gray hair that occurs with aging is also thought to be due to a decrease in melanin production ability in the hair follicles.

[0005] In addition, cultures that consider brown skin to be healthy and beautiful exist not only in Europe and the Americas but also widely. There is a great demand for making the skin and hair black or dark brown. Since sunburn caused by sunlight exposes people to ultraviolet rays and involves significant risks such as the occurrence of skin cancer, sunburn accelerators that do not rely on sunlight are considered useful. These skin problems due to skin aging and beauty needs are common worldwide. Even in Japan alone, the market size of cosmetics exceeds one trillion yen.

[0006] To date, as things for improving wrinkle formation and skin aging, the use of antioxidants such as vitamin E and regulators of the extracellular matrix such as collagen and elastin have been proposed. Also, for gray hair that occurs with aging, it has been dyed with dyes as a symptomatic treatment.

[0007] Components derived from natural products are expected from the viewpoint of safety as things that can show an anti-wrinkle effect. For example, the anti-wrinkle effect of black ginger extract is described in Non-Patent Document 1 below (the entire description of this document is incorporated herein by reference). Also, Patent Document 1 below (the entire description of this document is incorporated herein by reference) describes that black ginger extract has an antioxidant effect. Furthermore, Non-Patent Document 2 below (the entire description of this document is incorporated herein by reference) describes that among the components contained in black ginger, 5,7-dimethoxyflavone has a melanin formation promoting effect.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, since the black ginger extract described in Patent Document 1 and Non-Patent Document 1 contains various components, the antioxidant effect of the black ginger extract is small, and the anti-wrinkle effect achieved thereby is weak.

[0011] In addition, according to the investigation by the present inventors, the melanin formation promoting effect of 5,7-dimethoxyflavone described in Non-Patent Document 2 is very small.

[0012] Therefore, a first problem to be solved by the present invention is to provide a composition containing an active ingredient that is a component derived from a natural product with excellent safety and can suppress or improve skin aging such as wrinkles and sagging.

[0013] Another problem to be solved by the present invention is to provide a composition containing an active ingredient that is a component derived from a natural product with excellent safety and can promote melanin synthesis to suppress or improve whitening of hair and skin.

Means for Solving the Problems

[0014] In order to solve the above problems, the present inventors have conducted intensive research and development, and examined the physiological activities of various natural products and components derived from natural products. As a result, they succeeded in isolating 11 polymethoxyflavonoids contained in black ginger. Then, as a result of repeatedly testing the physiological activities of these polymethoxyflavonoids through trial and error, surprisingly, 5,7,3’,4’-tetramethoxyflavone, 5,7,4’-trimethoxyflavone, and 5-OH-3,7,3’,4’-tetramethoxyflavone were found to have an effect of suppressing the activity of proteolytic enzymes involved in wrinkle formation and inflammatory factors in the skin.

[0015] Even more surprisingly, the present inventors found that 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, and 5-OH-3,7,4’-trimethoxyflavone have an effect of promoting the activation of enzymes involved in the melanin synthesis pathway.

[0016] Finally, the present inventors have completed a composition containing the above polymethoxyflavonoids and a composition containing these components as active ingredients. The present invention has been completed based on these findings and successful examples.

[0017] Therefore, according to one aspect of the present invention, the following compositions and methods [1] to

[10] are provided. [1] A cosmetic composition containing at least one active ingredient selected from the group consisting of 5,7,3’,4’-tetramethoxyflavone, 5,7,4’-trimethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, and a composition containing at least one of these. [2] The composition according to [1], which has at least one action selected from the group consisting of an elastase activation inhibitory action, a collagenase activation inhibitory action, and an inflammatory cytokine activation inhibitory action. [3] The composition according to any one of [1] to [2], which is at least one composition selected from the group consisting of an anti-wrinkle composition, an anti-callus composition, an anti-skin inflammation composition, an anti-aging composition, and a skin-improving composition. [4] A beauty composition containing at least one active ingredient selected from the group consisting of 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, 5-OH-3,7,4’-trimethoxyflavone, and at least one inclusion thereof. [5] The composition according to [4], which has a tyrosinase activation promoting action. [6] The composition according to any one of [4] to [5], which is at least one composition selected from the group consisting of an anti-vitiligo composition, an anti-gray hair composition, an anti-white hair composition, an anti-aging composition, a sunburn promoting composition, and a skin darkening composition. [7] The composition according to any one of [1] to [6], which is a cosmetic composition. Use method of at least one active ingredient selected from the group consisting of 5,7,3’,4’-tetramethoxyflavone, 5,7,4’-trimethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, 5-OH-3,7,4’-trimethoxyflavone, and a composition containing at least one of these components for producing the composition according to any one of [1] to [7]. A method for treating, preventing, suppressing or improving a symptom or disease selected from the group consisting of wrinkles, sagging skin, skin inflammation, aging, skin abnormalities, leukotrichia, white hair and vitiligo, which comprises applying the composition according to any one of [1] to [7] to an individual to be used. A method for promoting sunburn or blackening the skin, which comprises applying the composition according to any one of [4] to [7] to an individual to be used.

Advantages of the Invention

[0018] The composition according to one embodiment of the present invention contains, as an active ingredient, a component contained in black ginger, which is a natural product whose safety has been demonstrated, such as being originally used in herbal medicines and is hardly known to exhibit side effects. Therefore, it can be safely used for the individual to be used. In addition, black ginger grows wild in Southeast Asia and can be obtained in large quantities. Furthermore, since the above components contained in black ginger are commercially available, it can be expected to be manufactured on an industrial scale with high economic efficiency.

[0019] The composition according to one embodiment of the present invention can be expected to treat, prevent, suppress or improve various skin-related diseases, symptoms and abnormalities in addition to wrinkles, sagging skin, skin inflammation, aging, skin abnormalities, etc. through the action of suppressing the activity of proteolytic enzymes involved in wrinkle formation and the action of suppressing the production of inflammatory factors involved in wrinkle formation.

[0020] Furthermore, the composition of one embodiment of the present invention can be expected to treat, prevent, suppress, or improve vitiligo, gray hair, leukoderma, aging, etc. through the action of activating enzymes involved in the melanin synthesis pathway, and can also be expected to be used for cosmetic purposes such as promoting sunburn and skin darkening.

[0021] The composition of one embodiment of the present invention not only can be used topically, but also contains inner beauty components (inner cosmeceuticals) with few side effects and can be orally ingested, so it can be expected to be used as a health food.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Hereinafter, the details of the composition and method which are one aspect of the present invention will be described. However, the technical scope of the present invention is not limited only by the matters in this section, and the present invention can take various aspects as long as its object is achieved.

[0024] Unless otherwise defined, each term in this specification is used in the meaning usually used by those skilled in the art and should not be construed as having an unduly restrictive meaning. Also, the speculations and inferences made in this specification are based on the inventors' knowledge and experience so far, and thus the technical scope of the present invention is not limited only by such speculations and inferences.

[0025] The "composition" is not particularly limited as having the commonly used meaning. For example, it is a substance formed by combining two or more substances. Specifically, examples include those formed by combining an active ingredient with another substance, those formed by combining two or more active ingredients, etc. More specifically, examples include solid compositions and liquid compositions formed by combining one or more active ingredients with one or more solid components or solvent components.

[0026] The term "and / or" means any one of the listed multiple related items, or any combination or all combinations of two or more of them.

[0027] "Content" is synonymous with "concentration" and means the ratio of the amount of a component to the total amount of the composition. However, the total content of the components shall not exceed 100%.

[0028] In this specification, "~" in a numerical range means a range including the numerical values before and after it. For example, "0 mass% ~ 100 mass%" means a range that is 0 mass% or more and 100 mass% or less.

[0029] The term "activation inhibitory action" of a substance refers to at least any one of the actions of reducing the degree of the catalytic action or physiological action inherent in the substance, reducing the expression level of the gene of the substance, reducing the translation amount of the gene product (protein) of the substance, and inhibiting the production of the gene product of the substance.

[0030] The term "activation promoting action" of a substance refers to at least any one of the actions of enhancing the degree of the catalytic action or physiological action inherent in the substance, increasing the expression level of the gene of the substance, increasing the translation amount of the gene product (protein) of the substance, and promoting the production of the gene product of the substance.

[0031] The term "production promoting action" of a substance refers to at least any one of the actions of increasing the expression level of the gene of the substance, increasing the translation amount of the gene product (protein) of the substance, and promoting the production of the gene product of the substance.

[0032] [Summary of the Invention] The composition of the first aspect of the present invention contains at least one active ingredient selected from the group consisting of 5,7,3',4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone, and 5-OH-3,7,3',4'-tetramethoxyflavone and a substance containing at least one of these components.

[0033] The composition of the first aspect of the present invention may have at least one action selected from the group consisting of an action of suppressing the activation of proteolytic enzymes involved in wrinkle formation and an action of suppressing the activation of inflammatory factors in the skin, by the active ingredient contained therein. By having these actions, the composition of the first aspect of the present invention can typically take forms such as a composition for anti-wrinkle, a composition for anti-dry skin, a composition for anti-skin inflammation, a composition for anti-aging, and a composition for improving skin abnormalities. Another aspect of the present invention is a method of applying an active ingredient to a subject to treat, prevent, suppress, or improve wrinkles, dry skin, skin inflammation, aging, and skin abnormalities.

[0034] The composition of the second aspect of the present invention contains at least one active ingredient selected from the group consisting of 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, 5-OH-3,7,4’-trimethoxyflavone, and a substance containing at least one of these components.

[0035] The composition of the second aspect of the present invention may have an action of promoting the activation of enzymes involved in the melanin synthesis pathway, by the active ingredient contained therein. By having these actions, the composition of the second aspect of the present invention can typically take forms such as a composition for anti-vitiligo, a composition for anti-gray hair, a composition for anti-white spot, a composition for anti-aging, a composition for promoting sunburn, and a composition for skin darkening. Another aspect of the present invention is a method of applying an active ingredient to a subject to treat, prevent, suppress, or improve vitiligo, gray hair, and white spots. Another aspect of the present invention is a method of applying an active ingredient to a subject to promote sunburn or darken the skin.

[0036] In this specification, the compositions of the first aspect of the present invention and the compositions of the second aspect of the present invention may be collectively referred to as "the compositions of one aspect of the present invention". Further, the active ingredients contained in the compositions of the first aspect of the present invention and the active ingredients contained in the compositions of the second aspect of the present invention may be collectively referred to as "active ingredients", and the effects that the compositions of the first aspect of the present invention may have and the effects that the compositions of the second aspect of the present invention may have may be collectively referred to as "effective effects".

[0037] [Active ingredient] The active ingredient is classified as a polymethoxyflavonoid and has the following general formula (I) [Chemical formula] (wherein R l , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 each independently represents hydrogen, a hydroxyl group or a methoxy group.) is included in the compounds shown in

[0038] The active ingredient in the composition of the first aspect of the present invention may be any one, two or all three of 5,7,3',4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3',4'-tetramethoxyflavone. The active ingredient in the composition of the second aspect of the present invention may be any one, two, three, four, five, six or all seven of 3,5,7,3',4'-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetramethoxyflavone, 5-OH-3,7,3',4'-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4'-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone and 5-OH-3,7,4'-trimethoxyflavone.

[0039] 5,7,3’,4’-Tetramethoxyflavone, 5,7,4’-trimethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, and 5-OH-3,7,4’-trimethoxyflavone (hereinafter, these may be collectively referred to as "polymethoxyflavonoids") are not particularly limited as long as they are as commonly known, and may be commercially available, synthetic, or extracted and / or separated from natural products. The positions of the methoxy groups and hydroxy groups in the polymethoxyflavonoids can be confirmed from the general formula (I).

[0040] The active ingredient may be a composition containing any one or more of the polymethoxyflavonoids. The composition is not particularly limited as long as it contains the polymethoxyflavonoids, and examples thereof include plant extracts, preferably black ginger extracts.

[0041] Black ginger ( Kaempferia parviflora ) is a plant of the genus Zingiber of the Zingiberaceae family, which is known to grow wild in Southeast Asia and other places. Black ginger is known to have effects such as enhancing energy, tonifying the body, lowering blood sugar levels, restoring physical strength, improving the digestive system, treating leukorrhea, hemorrhoids, hemorrhoid diseases, heartburn, stomatitis, joint pain, and stomach pain. Since black ginger is a natural plant that has been ingested by humans for a long time and has high safety, the composition of one aspect of the present invention can be applied in a parenteral or oral form.

[0042] The part of black ginger used is not particularly limited as long as it contains the active ingredient that contributes to the desired effective action, and examples thereof include roots, stems, leaves, flowers, branches, etc., and preferably the rhizome containing a large amount of polymethoxyflavonoids.

[0043] The method for producing a black ginger extract containing polymethoxyflavonoids from black ginger is not particularly limited, and examples include a method that at least includes contacting the black ginger extract with a polymethoxyflavonoid-soluble solvent.

[0044] The black ginger used as the raw material for the black ginger extract may be in the as-collected state or in a state subjected to processing such as drying after collection. When time is required until the processing after collection, it is preferably stored by storage means commonly used by those skilled in the art such as low-temperature storage to prevent deterioration of the raw material of black ginger.

[0045] Since the raw material of the black ginger extract may contain a large amount of moisture depending on the part of the black ginger used, it is preferably in a dried state. The drying treatment is not particularly limited, and examples include a treatment of drying such that the mass after drying (dry mass) of the raw material of the black ginger extract is about 1 / 2 to 1 / 10, preferably about 1 / 3 to 1 / 6, of the mass before drying (wet mass). The drying treatment can be performed by any method known to those skilled in the art such as hot air drying, high-pressure steam drying, electromagnetic wave drying, and freeze drying. Drying by heating can be performed, for example, at a temperature and for a time at which the raw material of the black ginger extract does not change color when heated at 30°C to 100°C for 24 hours to 72 hours.

[0046] From the viewpoint of efficiently performing the extraction operation, the raw material of the black ginger extract is preferably in a crushed, pulverized, or mashed state. The method for crushing, pulverizing, or mashing the raw material is not particularly limited, and examples include methods using a cutter, a cutting machine, a crusher, a blender, a mixer, a mill, a grinder, a kneader, a mortar, and the like.

[0047] As the extraction solvent for extracting the black ginger extract, it is preferably an extraction solvent containing at least an organic solvent. Examples of the extraction solvent include a mixed solvent of water and an organic solvent, lower alcohols such as methanol, ethanol, propanol, isopropanol, and butanol; lower esters such as ethyl acetate and methyl acetate; and organic solvents such as dimethyl sulfoxide, acetonitrile, acetone, hexane, glycerin, and propylene glycol, but are not limited thereto.

[0048] As the organic solvent, a single species may be used, or a mixed solvent of a plurality of species may be used. When a mixed solvent of water and an organic solvent is used as the extraction solvent, the content of the organic solvent in the mixed solvent is preferably 50% by volume or more, more preferably 80% by volume or more. When the content of the organic solvent in the mixed solvent is less than 50% by volume, it tends to be difficult to efficiently extract polymethoxyflavonoids. Note that as the organic solvent, lower alcohols are preferred, and ethanol and methanol are more preferred.

[0049] As the extraction operation, the raw material of the black ginger extract is immersed in the extraction solvent for a predetermined time. In such an extraction operation, in order to increase the extraction efficiency, a stirring operation, heating, etc. may be performed as necessary. Further, in order to reduce the impurities extracted from the raw material, a water extraction operation or a hot water extraction operation may be performed separately prior to the extraction operation. Since polymethoxyflavonoids are components insoluble in water, by boiling black ginger in hot water, for example, unnecessary impurities can be removed prior to the extraction operation.

[0050] By performing a solid-liquid separation operation after the extraction operation, the black ginger extract and the residue of the raw material can be separated. The method of the solid-liquid separation operation is not particularly limited, and for example, known separation methods such as filtration and centrifugation can be used.

[0051] The black ginger extract may be concentrated as necessary. By removing the extraction solvent contained in the black ginger extract as necessary, an oily or solid black ginger extract having viscosity can be obtained. The method of concentration is not particularly limited, and for example, it may be carried out by placing it at room temperature under reduced pressure or by heating, or it may be carried out by freeze-drying.

[0052] Black ginger contains flavonoids other than polymethoxyflavonoids listed as active ingredients. For example, by fractionating the black ginger extract using a column, an extract containing a desired polymethoxyflavonoid at a high concentration can be obtained. The packing material used for the separation operation using the column is not particularly limited, and examples thereof include silica gel bonded with an octadecyl group.

[0053] The method for confirming and quantifying the polymethoxyflavonoids contained in the black ginger extract is not particularly limited, and for example, it can be carried out by high performance liquid chromatography (HPLC) as described in the examples below.

[0054] The content of polymethoxyflavonoids in the black ginger extract is not particularly limited. For example, in terms of the total amount per mass of the black ginger extract, it is 0.1% by mass or more, preferably 1 to 99% by mass, and more preferably 5 to 90% by mass. Also, the content of 5,7,3’,4’-tetramethoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 5,7,4’-trimethoxyflavone in the black ginger extract is, for example, 1.0% by mass or more, preferably 5.0% by mass or more, per mass of the black ginger extract. The content of 5-OH-3,7,3’,4’-tetramethoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 3,5,7,3’,4’-pentamethoxyflavone in the black ginger extract is, for example, 1.0% by mass or more, preferably 5.0% by mass or more, per mass of the black ginger extract. The content of 3,5,7-trimethoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 3,5,7,4’-tetramethoxyflavone in the black ginger extract is, for example, 0.5% by mass or more, preferably 2.5% by mass or more, per mass of the black ginger extract. The content of 5-OH-3,7,3’,4’-tetramethoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 5-OH-7-methoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 5-OH-7,4’-dimethoxyflavone in the black ginger extract is, for example, 0.1% by mass or more, preferably 0.5% by mass or more, per mass of the black ginger extract. The content of 5-OH-3,7-dimethoxyflavone in the black ginger extract is, for example, 0.2% by mass or more, preferably 1.0% by mass or more, per mass of the black ginger extract.The content of 5-OH-3,7,4'-trimethoxyflavone in the black ginger extract is, for example, 0.3% by mass or more, preferably 1.5% by mass or more, per mass of the black ginger extract. The upper limit of the content of polymethoxyflavonoids in the black ginger extract is not particularly limited and is typically 99% by mass or less in total.

[0055] [Composition] By containing an active ingredient, the composition of the first aspect of the present invention exhibits either or both of the actions of suppressing the activation of proteolytic enzymes and inflammatory factors involved in wrinkle formation.

[0056] As the action of suppressing the activation of proteolytic enzymes involved in wrinkle formation that the composition of the first aspect of the present invention has, it is preferably either one of the actions of suppressing elastase activation and suppressing collagenase activation, and more preferably both of these actions. As the action of suppressing the activation of inflammatory factors involved in wrinkle formation that the composition of the first aspect of the present invention has, it is preferably the action of suppressing the activation of inflammatory cytokines, and more preferably the action of suppressing the activation of interleukin-1α (IL-1α).

[0057] The effective action that the composition of the first aspect of the present invention has can be confirmed by measuring the titer, gene expression level, and / or protein production amount of the activity related to enzymes and inflammatory factors induced by external stimuli of skin fibroblasts or epidermal keratinocytes as described in the examples below. At this time, using the system without adding the composition of the first aspect of the present invention as a control, if the titer, gene expression level, and / or protein production amount of the activity related to enzymes and inflammatory factors induced by external stimuli of cells in the system added with the composition of the first aspect of the present invention are smaller than those of the control, it can be evaluated that the composition of the first aspect of the present invention has an effective action.

[0058] The composition of the second aspect of the present invention shows an effect of promoting the activation of enzymes involved in the melanin synthesis pathway by containing an active ingredient. As the effect of promoting the activation of enzymes involved in the melanin synthesis pathway that the composition of the second aspect of the present invention has, it is preferably an effect of promoting tyrosinase activation.

[0059] The effective effect that the composition of the second aspect of the present invention has can be confirmed by measuring the titer of the activity, gene expression level, and / or protein production amount related to the enzyme produced by melanoma cells as described in the examples below. At this time, using the system without adding the composition of the second aspect of the present invention as a control, if the titer of the activity, gene expression level, and / or protein production amount related to the enzyme of the cells in the system to which the composition of the second aspect of the present invention is added is smaller than that of the control, it can be evaluated that the composition of the second aspect of the present invention has an effective effect.

[0060] The composition of one aspect of the present invention is not particularly limited as long as it is a composition used for cosmetic purposes, and can take various forms such as, for example, a composition for cosmetics, a composition for foods and drinks, a composition for pharmaceuticals, etc. Therefore, a specific embodiment of the present invention is a parenteral or oral composition for cosmetics, a composition for foods and drinks, a composition for pharmaceuticals, a composition for quasi-drugs, a composition for animal feeds, etc. containing an active ingredient.

[0061] The content of the active ingredient contained in the composition of one aspect of the present invention is not particularly limited as long as the desired effective effect can be recognized. For example, as a parenteral cosmetic composition, it is 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more; as an oral cosmetic composition, it is 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more. The upper limit of the content is not particularly limited, but is typically 1% by mass.

[0062] The individual and method of using the composition according to one aspect of the present invention are not particularly limited. For example, the individual to be used may be an animal, especially a mammal. Examples of mammals include humans, dogs, cats, cows, horses, etc., and among these, humans are preferably used.

[0063] The dosage form and dosage, such as the amount used per application, the amount used per day, the period of use, and the interval between uses, of the composition according to one aspect of the present invention are not particularly limited and can be appropriately set according to the usage mode, the state of the individual to be used, etc. The interval between uses can be, for example, once, twice, three times, or several times a day, continuously applied for a certain period, that is, two days or more, preferably one week or more, more preferably two weeks or more, still more preferably one month or more, and even more preferably six months or one year or more. It is preferable to apply the composition according to one aspect of the present invention every day, but as long as it is continuously applied during the period, it is not necessary to apply the composition according to one aspect of the present invention every day.

[0064] The composition according to one aspect of the present invention may be applied parenterally or orally as described above. However, since it can be directly applied to the affected part where the effective action is exerted, it is preferably a parenteral cosmetic composition, and more preferably a cosmetic composition and a parenteral pharmaceutical composition.

[0065] The composition according to one aspect of the present invention may contain other components in addition to the active ingredient as long as it can solve the problems of the present invention.

[0066] Examples of other components in the case of a parenteral cosmetic composition include components usually contained in ordinary cosmetics and pharmaceuticals. More specifically, bases such as water, oily components, moisturizers, cooling agents, preservatives, chelating agents, pH adjusters, antioxidants, ultraviolet absorbers, ultraviolet scattering agents, thickeners, whitening agents, vitamins, various other medicinal components, powders, fragrances, colorants, etc. can be mentioned. The content of other components can be appropriately set by those skilled in the art as long as it does not prevent the solution of the problems of the present invention. Some of the other components are listed below, but these are merely examples and are not limiting.

[0067] The oily component includes, for example, an oily emollient component, etc., and preferably is an oily emollient component that is liquid or semi-solid at room temperature (25 °C). Specific examples of the oily emollient component include ester oils such as neopentyl glycol dicaprate, triethylhexanoin, isopropyl palmitate, cetyl 2-ethylhexanoate, isononyl isononanoate, isotridecyl isononanoate, diisopropyl sebacate, pentaerythrityl tetra(2-ethylhexanoate), diethylhexyl succinate, and alkyl benzoates having 12 to 15 carbon atoms; silicone oils such as dimethylpolysiloxane, dimethylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, and silicone oils modified with higher alcohols; glyceride oils such as acetoglyceryl, glyceryl triisooctanoate, glyceryl triisostearate, glyceryl triisopalmitate, glyceryl monostearate, glyceryl di(2-heptylundecanoate), and glyceryl trimyristate; animal oils such as liquid lanolin; fluorine oils such as fluoropolyethers and perfluoroalkyl ether silicones; and natural oily components such as olive oil, jojoba oil, lavender oil, evening primrose oil, avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, southern magnolia oil, castor oil, linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, sinagiri oil, Japanese cedar oil, and germ oil, but are not limited thereto. The oily emollient component can be used alone or in combination of two or more of the above-mentioned ones. The content of the oily emollient component is preferably 0% by mass to 10% by mass.

[0068] Examples of the humectant include glycerin, diglycerin, trehalose, butylene glycol (BG), xylitol, maltitol, maltose, sorbitol, glucose, fructose, hydrolyzed elastin, sodium lactate, cyclodextrin, pyrrolidone carboxylic acid, etc., and one of these can be used alone or in combination of two or more. The content of the humectant is preferably 0% by mass to 15% by mass.

[0069] Examples of the preservative include benzoic acid, salicylic acid, phenol, sorbic acid, methyl paraoxybenzoate, parachlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichlorocarbanilide, photosensitizer, phenoxyethanol, etc., and one of these can be used alone or in combination of two or more. The content of the preservative is preferably 0% by mass to 1% by mass.

[0070] Examples of the pH adjuster include lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, malic acid, etidronic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, sodium hydroxide, potassium hydroxide, triethanolamine, monoethanolamine, etc., and one of these can be used alone or in combination of two or more. The content of the pH adjuster is preferably 0% by mass to 1% by mass.

[0071] Examples of the antioxidant include vitamin E such as dibutylhydroxytoluene, butylhydroxyanisole, δ-tocopherol and its derivatives, thiotaurine, Portulaca oleracea extract, β-carotene, catechin compounds, flavonoid compounds, polyphenol compounds, etc., and one of these can be used alone or in combination of two or more. The content of the antioxidant is preferably 0% by mass to 1% by mass.

[0072] Examples of the thickener include water-soluble polysaccharides such as hyaluronic acid, sodium hyaluronate, acetylated hyaluronic acid, sodium acetylated hyaluronate, chondroitin sulfate, sodium chondroitin sulfate, fucoidan, tuberose polysaccharide, xanthan gum; natural polymers such as carrageenan and alginic acid; semi-synthetic polymers such as sodium carboxymethyl cellulose; and synthetic polymers such as carbomer, polyacrylic acid, sodium polyacrylate, and (meth)acrylic acid / (meth)acrylic acid alkyl copolymer. Carbomer is commercially available, for example, as "Carbopol 980", "Carbopol 910", "Carbopol 934", "Carbopol 940", "Carbopol 941", "Carbopol 981" (each manufactured by Lubrizol Advanced Materials, Inc.). The (meth)acrylic acid / (meth)acrylic acid alkyl copolymer is commercially available, for example, as "Carbopol SC-500", "Carbopol 1382", "Carbopol ETD2020", "PEMULEN TR-1", "PEMULEN TR-2" (each manufactured by Lubrizol Advanced Materials, Inc.). Sodium carboxymethyl cellulose is commercially available, for example, as "CMC Daicel" (manufactured by Daicel Chemical Industries, Ltd.). The thickener can be used alone or in combination of two or more of these. The content of the thickener is preferably 0% by mass to 1% by mass.

[0073] The usage modes and dosage forms as a cosmetic composition are not particularly limited. For example, skin care cosmetics, makeup cosmetics, fragrance cosmetics, body care cosmetics, etc. can be mentioned. More specifically, creams, emulsions, foundations, lotions, beauty essences, hair tonics, hair creams, shampoos, hair rinses, treatments, facial cleansers, foundations, hair growth agents, aqueous ointments, sprays, gels, lotions, packs, facial cleansing creams, hand creams, makeup cleansing, makeup bases, concealers, blush, eyeshadows, eyeliners, eyebrows, lipsticks, hair removal / epilation creams, all-in-one cosmetics, etc. can be mentioned. The composition of one aspect of the present invention is preferably in the form of an oil-in-water emulsion cosmetic in the form of a dosage form having a certain viscosity such as a gel, emulsion, lotion, cream, etc., so that it can have a good feel in use on the skin and hair.

[0074] The usage modes and dosage forms as a composition for parenteral pharmaceuticals are not particularly limited. For example, injections, patches, ointments, creams, etc. can be mentioned.

[0075] The composition of one aspect of the present invention may be an oral cosmetic composition. In this case, it is preferably a composition for foods and drinks and a composition for oral pharmaceuticals.

[0076] A specific aspect of the composition for foods and drinks is, for example, a functional food and drink that has a certain functionality for the living body. Functional food and drinks include, for example, so-called health food and drinks such as foods for specified health uses, foods with functional claims, nutritional functional foods, health functional foods, special-purpose foods, nutritional supplements, health supplements, supplements, beauty foods, etc., as well as foods for specific persons such as foods for infants, foods for pregnant and lactating women, and foods for the elderly. Furthermore, functional food and drinks include health food and drinks to which a health claim based on the food standards of the Codex (Joint FAO / WHO Food Standards Programme) is applied.

[0077] The composition for oral pharmaceuticals can take any form of oral pharmaceuticals as long as it can exhibit an effective action after application. Further, the composition for oral pharmaceuticals may contain other physiologically active substances having an effective action other than the active ingredient as long as the object of the present invention can be achieved, and may be used in combination with other pharmaceuticals containing the physiologically active substance as an active ingredient.

[0078] The usage modes and dosage forms of the oral cosmetic composition are not particularly limited. For example, it can take forms such as tablets, powders, granules, powders, capsules, pills, lozenges, liquids, etc., but is not limited thereto. Tablets include sugar-coated tablets, coated tablets, buccal tablets, etc. Capsules include both hard capsules and soft capsules. Granules include coated granules. Further, liquids include suspensions, emulsions, syrups, elixirs, etc. Syrups include dry syrups, etc.

[0079] When it is used as a composition for foods and drinks, for example, it can be added to bread, cookies, biscuits, wheat for adding to cooked rice, miscellaneous grains, udon, soba, pasta and other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, miso, soy sauce and other soy products, tea, coffee, cocoa, soft drinks, fruit drinks and other non-alcoholic beverages, medicinal liquors, other alcoholic beverages, candies, chocolate and other snack foods, chewing gum, senbei, yokan and other confectionery made from soybeans to form a general form of foods and drinks.

[0080] The oral cosmetic composition only needs to contain an active ingredient, but it may also be a combination with other ingredients. The other ingredients are not particularly limited. For example, additives for foods and drinks or oral pharmaceuticals can be used as other ingredients. For example, various excipients, binders, lubricants, stabilizers, diluents, extenders, emulsifiers, colorants, fragrances, essential oils, thickeners, brighteners, buffers, etc. can be used as other ingredients. The content of the other ingredients can be appropriately selected according to the form of the composition of one aspect of the present invention as long as it does not prevent the solution of the problems of the present invention.

[0081] Examples of excipients include lactose, glucose, sucrose, mannitol, potato starch, dextrin, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder, and the like. Examples of binders include starch, tragacanth gum, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. Examples of disintegrants include starch, agar, gelatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. Examples of lubricants include talc, magnesium stearate, and the like. As the coloring agent, those that are permitted to be added to foods, beverages, and oral pharmaceuticals can be used, and there is no particular limitation.

[0082] When making tablets or granules, if desired, they can be coated with sucrose, gelatin, purified shellac, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, methyl methacrylate, methacrylic acid polymer, etc., and can also be coated in multiple layers. Furthermore, granules or powders can be filled into capsules such as ethyl cellulose or gelatin to make capsule preparations.

[0083] The composition of one aspect of the present invention is not particularly limited with respect to its production method. For example, depending on the usage modes such as cosmetics, foods, beverages, and pharmaceuticals, it can be produced by mixing the active ingredient and optionally other ingredients by methods known to those skilled in the art.

[0084] The composition of one aspect of the present invention is not particularly limited with respect to its method of use, and for example, according to the usage modes such as cosmetics, food and drink products, pharmaceuticals, etc., the commonly used methods can be adopted.

[0085] The composition of one aspect of the present invention can be made into a container-packed composition packed in a container and sealed. The container is not particularly limited, and examples include packaging containers such as metals such as aluminum, paper, plastics such as PET and PTP, single-layer or laminated film bags, retort pouches, vacuum packs, aluminum containers, plastic containers, glass containers, bottles, and cans. In order to avoid deterioration over time, the composition of one aspect of the present invention is preferably sterilized by pressurization and / or heating after being packed in a container and sealed. The container-packed composition is preferably obtained by dispensing, filling and / or individual packaging the produced composition, and can be independently distributed and commercially available by itself.

[0086] [Specific embodiments of the composition] The composition of the first aspect of the present invention can be in the form of a composition for suppressing, improving, alleviating, treating and / or preventing the formation of wrinkles occurring in the skin through the action of suppressing the activation of proteolytic enzymes related to wrinkle formation, that is, an anti-wrinkle composition. Similarly, the composition of the first aspect of the present invention can be in the form of an anti-chapping composition, an anti-aging composition, a composition for improving skin abnormalities, etc. through the action of suppressing the activation of proteolytic enzymes related to wrinkle formation.

[0087] The composition of the first aspect of the present invention can be in the form of a composition for suppressing, improving, alleviating, treating and / or preventing inflammation in the skin through the action of suppressing the activation of inflammatory factors in the skin, that is, an anti-skin inflammation composition, etc.

[0088] The composition of the second aspect of the present invention promotes melanin synthesis in the skin and hair through the action of promoting the activation of enzymes involved in the melanin synthesis pathway, thereby suppressing, improving, alleviating, treating, and / or preventing leukotrichia and vitiligo, that is, it can take the form of a composition for anti-leukotrichia and a composition for anti-vitiligo. Similarly, the composition of the second aspect of the present invention can take the form of a composition for anti-gray hair, a composition for anti-aging, a composition for promoting sunburn, a composition for skin darkening, etc. through the action of promoting the activation of enzymes involved in the melanin synthesis pathway.

[0089] Thus, the composition of one aspect of the present invention is preferably a composition for treating, preventing, suppressing, alleviating, and / or improving symptoms or diseases of the skin and hair, particularly those associated with aging, through the corresponding effective action. Furthermore, the composition of one aspect of the present invention can be used for subsequent treatment. The individual using the composition of one aspect of the present invention may be a healthy individual, but preferably an individual with wrinkles, dullness, inflammation, etc. on the skin, or an individual with whitening of the skin or hair, and an individual at risk of developing these.

[0090] As described in the examples to be described later, the active ingredient in the composition of the first aspect of the present invention has an elastase activation inhibitory action, a collagenase activation inhibitory action, and an inflammatory cytokine activation inhibitory action. Therefore, another aspect of the present invention is a composition for inhibiting elastase activation or an elastase activation inhibitor, a composition for inhibiting collagenase activation or a collagenase activation inhibitor, a composition for inhibiting inflammatory cytokine activation or an inflammatory cytokine activation inhibitor, and a composition for inhibiting interleukin-1α activation in skin cells or an interleukin-1α activation inhibitor in skin cells, which contain the active ingredient.

[0091] As described in the examples to be described later, the active ingredient in the composition of the second aspect of the present invention has a tyrosinase activation promoting action. Therefore, another aspect of the present invention is a composition for promoting tyrosinase activation or a tyrosinase activation promoter, which contains the active ingredient.

[0092] [Method] The manufacturing method and the usage method of the composition of one aspect of the present invention are included in the present invention as another aspect.

[0093] The method of one aspect of the present invention is a method of using at least one active ingredient selected from the group consisting of 5,7,3’,4’-tetramethoxyflavone, 5,7,4’-trimethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 3,5,7,3’,4’-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5-OH-3,7,3’,4’-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4’-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, 5-OH-3,7,4’-trimethoxyflavone, and a composition containing at least one of these components for manufacturing the composition of one aspect of the present invention.

[0094] Another method of the present invention is a method for treating, preventing, suppressing, or improving symptoms or diseases selected from the group consisting of wrinkles, sagging skin, skin inflammation, aging, and skin abnormalities, including applying the composition of the first aspect of the present invention to an individual to be used.

[0095] Another method of the present invention is a method for treating, preventing, suppressing, or improving symptoms or diseases selected from the group consisting of vitiligo, white hair, leukoderma, and aging, including applying the composition of the second aspect of the present invention to an individual to be used.

[0096] Another method of the present invention is a method for promoting sunburn or darkening the skin, including applying the composition of the second aspect of the present invention to an individual to be used.

[0097] The method of one aspect of the present invention can incorporate various steps and operations before, after, or during the above-described steps as long as the problems of the present invention can be solved.

[0098] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the problems of the present invention can be solved.

Example

[0099] [Example 1. Evaluation of the inhibitory effect of methoxyflavonoid on elastase activation] (1-1) Test samples and materials Normal human dermal fibroblasts (normal human skin fibroblasts) were purchased from TOYOBO Co., Ltd. (Catalog number: GCA10605F). DMEM was purchased from SIGMA Co., Ltd. (Catalog number: D6429-500ML). Other reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. or SIGMA Co., Ltd. The 96-well microplate was purchased from Nunc Co., Ltd. (Catalog number: 161093).

[0100] The compounds to be used were isolated from black ginger by liquid-liquid partitioning with an organic solvent, chromatography using a reverse-phase resin (Diaion HP20 manufactured by Mitsubishi Chemical Corporation), and preparative HPLC using a reverse-phase ODS column (Wakopak Wakosil-II 5C18RS Prep 28×250mm manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and each compound was identified by NMR and other structure analyses. In addition, the method for confirming and quantifying polymethoxyflavonoids contained in the black ginger extract was performed by high-performance liquid chromatography (HPLC) using a reverse-phase ODS column (Wakopak Wakosil-II 5C18RS Prep 4.6×250mm manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The test samples were prepared by diluting the isolated compounds with DMSO.

[0101] (1-2) Pre-culture The elastase activity of normal human dermal fibroblasts increases upon stimulation with IL-1α. Therefore, normal human dermal fibroblasts were cultured in a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)) at 5.0×10 4Prepared to be cells / ml, and dispensed 100 μl / well of this into a 96-well microplate. Incubated for 24 hours at 37 °C in 5% CO 2 in the presence.

[0102] (1-3) Elastase enzyme activity test To each well of the pre-cultured culture plate, IL-1α (final concentration 0.1 nM) and the test sample were added so that the final concentration of the isolated compound was 15 or 30 μM. After culturing the plate with the test sample added for 72 hours, the culture supernatant was removed. Then, after adding 0.5 vol% TritonX-100 solution to the cells remaining in the well to lyse the cells, the enzyme activity of elastase was measured with a plate reader by further adding substrate buffer. As the substrate, Suc-(Ala) 3 -pNA (STANA) was used, and the amount of p-nitroaniline produced by the action of elastase was measured by the absorbance at 405 nm. The results obtained by testing with DMSO added instead of the test sample were used as the control (CN). The results obtained by testing without adding the test sample and DMSO using normal human skin fibroblasts not stimulated with IL-1α were used as the blank (BL). Also, as a positive control, the results obtained by testing with 30 μM resveratrol instead of the test sample were obtained. Dunnett's test was performed on the obtained values against CN. The significance levels were set at a significance level of 5% and 1%.

[0103] (1-4) Elastase gene expression test To each well of the pre-cultured culture plate, the test sample was added so that the final concentration of the isolated compound was 30 μM. After culturing the plate with the test sample added for 24 hours, total RNA was extracted and cDNA was prepared from the cells using TRIZOL Reagent (Catalog No.: 15596-018) manufactured by Thermo Fisher Scientific, and the expression level of the elastase gene was measured by real-time PCR.

[0104] (1-5) Elastase protein expression test To each well of the pre-cultured culture plate, the test sample was added such that the final concentrations of the isolated compounds were 10, 15, and 30 μM. After culturing the plate with the test sample added for 24 hours, it was washed with PBS, and then the cells were lysed using RIPA Buffer (Catalog number: 182-02451) manufactured by Fujifilm Wako Pure Chemical Corporation to prepare a cell extract, and the expression level of elastase protein (NEP) was measured by Western blotting (Control: β-actin).

[0105] (1-6) Test Results and Evaluation As shown in Figure 1, 11 types of polymethoxyflavonoids, MF1 to MF11, were isolated from black ginger extract.

[0106] Among the methoxyflavonoids shown in Figure 1, 5,7,3’,4’-tetramethoxyflavone (MF1), 5,7,4’-trimethoxyflavone (MF4), and 5-OH-3,7,3’,4’-tetramethoxyflavone (MF7) significantly suppressed the elastase enzyme activity that increased upon IL-1α stimulation (Figure 2). Similarly, MF1, MF4, and MF7 suppressed the elastase gene expression that increased upon IL-1α stimulation (Figure 3). Furthermore, MF4 suppressed the elastase protein expression that increased upon IL-1α stimulation (Figure 4).

[0107] As described above, MF1, MF4, and MF7 suppress elastase, a protease involved in wrinkle formation, at the enzyme level, gene level, and protein level, thereby suppressing elastase activity, and it was found to be useful for the prevention and improvement of wrinkles.

[0108] [Example 2. Evaluation of the IL-1α Gene Expression Suppressing Effect of Methoxyflavonoids] (2-1) Test Samples and Materials As a human epidermal keratinocyte cell line, human keratinocyte HaCaT (Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. J Cell Biol. 1988 Mar;106(3):761-71.) was used. The 24-well microplates were purchased from TPP (Catalog number: 92024).

[0109] (2-2) Pre-culture In the human epidermal keratinocyte cell line (human keratinocyte HaCaT), the expression of inflammatory genes such as IL-1α increases upon UV-B irradiation stimulation. Therefore, the human epidermal keratinocyte cell line was prepared to a concentration of 5.0×10 4 cells / ml using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)), and this was dispensed into a 24-well multiplate at 500 μl / well. The culture plate containing the human epidermal keratinocyte cell line was cultured at 37°C and 5% CO 2 for 24 hours in the presence. Then, after removing the culture supernatant, it was further cultured for 24 hours using a new medium (DMEM medium containing 0.1 vol% FBS (fetal bovine serum)).

[0110] (2-3) IL-1α gene expression test To each well of the pre-cultured culture plate, the test sample was added such that the final concentrations of the isolated compounds were 10, 20, and 30 μM. The culture plate to which the test sample was added was irradiated with UV-B at 50 mJ / cm 2 and cultured for 24 hours. Then, Total RNA was recovered from the cultured cells, and the expression level of the IL-1α gene was measured by real-time PCR. Dunnett's test was performed on the obtained values against the control (CN). The significance levels were set at a 5% and 1% risk rate.

[0111] (2-4) Test results and evaluation Among the methoxyflavonoids, 5,7,4’-trimethoxyflavone (MF4) significantly suppressed the expression of IL-1α that was increased by UV-B stimulation (Figure 5).

[0112] From these results, it was found that MF4 is useful for suppressing the inflammatory reaction by suppressing the production of inflammatory cytokines in keratinocytes at the gene level.

[0113] [Example 3. Evaluation of the inhibitory effect of methoxyflavonoid on elastase activation and the inhibitory effect on collagenase (MMP-1) activation] (3-1) Test samples and materials The 12-well co-culture system plate was purchased from Falcon (Catalog number: 353043).

[0114] (3-2) Pre-culture Normal human skin fibroblasts increase elastase enzyme activity and collagenase (MMP-1) gene expression by co-culture with UV-B-stimulated human epidermal keratinocyte cell line (human keratinocyte HaCaT). Therefore, the human epidermal keratinocyte cell line was adjusted to 5.0×10 4 cells / ml using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)), and 500 μl / well of this was dispensed into the transwell of a 12-well co-culture system plate.

[0115] Next, normal human skin fibroblasts were adjusted to 5.0×10 4 cells / ml using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)), and 1,500 μl / well of this was dispensed into the plate well of a 12-well co-culture system plate.

[0116] The culture plate containing normal human skin fibroblasts and the human epidermal keratinocyte cell line was cultured at 37 °C in 5% CO 2 for 24 hours in the presence. Then, after removing the culture supernatant, it was further cultured for 24 hours using a new medium (DMEM medium containing 0.1 vol% FBS (fetal bovine serum)).

[0117] (3-3) Collagenase (MMP-1) Gene Expression Test UV-B was irradiated only on the human epidermal keratinocyte cell line (human keratinocyte HaCaT) of the Transwell at 50 mJ / cm 2 and the test sample was added to each well of the Transwell plate so that the final concentration of the isolated compound became 15 and 30 μM.

[0118] After culturing the culture plate for 24 hours, Total RNA was recovered from the cells after culture, and the expression level of the collagenase (MMP-1) gene was measured by real-time PCR method.

[0119] (3-4) Elastase Enzyme Activity Test After culturing the culture plate for 48 hours, the enzyme activity of elastase was measured in the same manner as in the above (1-3).

[0120] (3-5) Test Results and Evaluation Among the methoxyflavonoids, 5,7,4’-trimethoxyflavone (MF4) suppressed the increase in elastase activity (Figure 6). In addition, MF4 also suppressed the increase in the expression of the collagenase (MMP-1) gene (Figure 7).

[0121] From these results, it was found that MF4 is useful for the prevention and improvement of wrinkles because it suppresses the increase in the elastase enzyme activity and the increase in the expression of the collagenase (MMP-1) gene of skin fibroblasts induced by UV-B-exposed keratinocytes.

[0122] [Example 4. Evaluation of the Promoting Effect of Methoxyflavonoid on Tyrosinase Activation] (4-1) Test Samples and Materials The human HMV-II melanoma cell line was purchased from RIKEN BRC (Catalog number: RCB07777). Ham’s F-12 medium was purchased from Fujifilm Wako Pure Chemical Corporation (Catalog number: 087-08335). Other reagents were purchased from Fujifilm Wako Pure Chemical Corporation or SIGMA.

[0123] (4-2) Pre-culture The human HMV-II melanoma cell line was seeded at 1.0×10 4 cells / well in a 96-well microplate using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)) and cultured at 37 °C in 5% CO 2 for 24 hours in the presence.

[0124] (4-3) Tyrosinase enzyme activity test The test sample was added to each well of the pre-cultured culture plate so that the final concentration of the isolated compound was 40 μM. After culturing the plate with the test sample added for 72 hours, the culture supernatant was removed.

[0125] Next, 20 μL of T.A Lysis Buffer (1% sodium deoxycholate + 0.5% TritonX-100 + 100 mM PMSF (Phenylmethylsulfonyl fluoride)) was added to the cells remaining in the well, and the cells were lysed by shaking for 30 minutes.

[0126] After shaking, 5 μL of the cell lysate was taken out from each well, and the protein amount was determined by measuring the absorbance at 570 nm using a BCA Protein Assay Kit.

[0127] 200 μL of Mixed Buffer (N,N-dimethylformamide + L-DOPA (3,4-dihydroxy-L-phenylalanine) + MBTH (3-methyl-2-benzo-thiazolinone hydrazine hydrochloride)) was added to the remaining cell lysate. The absorbance at 505 nm was measured every 10 minutes while incubating at 37 °C.

[0128] The difference in absorbance per hour was determined, and the enzyme activity per 1 mg of protein (Δ absorbance / h / mg) was determined.

[0129] (4-4) Test results and evaluation Among the methoxyflavonoids, 3,5,7,3’,4’-pentamethoxyflavone (MF2), 3,5,7-trimethoxyflavone (MF5), 3,5,7,4’-tetramethoxyflavone (MF6), 5-OH-3,7,3’,4’-tetramethoxyflavone (MF7), 5-OH-7-methoxyflavone (MF8), 5-OH-7,4’-dimethoxyflavone (MF9), 5-OH-3,7-dimethoxyflavone (MF10), and 5-OH-3,7,4’-trimethoxyflavone (MF11) increased the activity of tyrosinase, which is the rate-limiting enzyme for melanin synthesis, in the human HMV-II melanoma cell line (Figure 8).

[0130] From these results, it was found that MF2, MF5, MF6, MF7, MF8, MF9, MF10, and MF11 are useful for darkening hair and skin because they promote tyrosinase activity and thus melanin synthesis in human cells.

Industrial Applicability

[0131] The composition according to one aspect of the present invention contains a methoxyflavonoid derived from black ginger as an active ingredient, and thus exhibits an anti-inflammatory effect based on an inflammatory factor inhibitory action, an anti-wrinkle effect based on an elastase activation inhibitory action and a collagenase activation inhibitory action, and a hair and skin darkening promotion effect based on a tyrosinase activation promotion action and a melanin production promotion action. It can be used as a composition for drinking, food, and cosmetics, particularly as a composition for inhibiting skin aging, improving skin metabolism, and for beauty purposes. Cross-reference to related applications

[0132] This application claims the priority of Japanese Patent Application No. 2019-010756 filed on January 25, 2019, the entire description of which is incorporated herein by reference.

Claims

1. An elastase activation inhibitor selected from the group consisting of 5,7,3',4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone, and 5-OH-3,7,3',4'-tetramethoxyflavone.

2. A collagenase MMP-1 activation inhibitor or an interleukin-1α activation inhibitor, which is 5,7,4'-trimethoxyflavone.

3. A tyrosinase activation promoter selected from the group consisting of 3,5,7,3',4'-pentamethoxyflavone, 3,5,7,4'-tetramethoxyflavone, 5-OH-3,7,3',4'-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4'-dimethoxyflavone, 5-OH-3,7-dimethoxyflavone, and 5-OH-3,7,4'-trimethoxyflavone.

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