JPWO2020153466A1 - Cosmetological composition - Google Patents

Abstract

An object of the present invention is to provide a composition containing an active ingredient capable of suppressing or improving skin aging such as wrinkles and tarmi, while being a component contained in a natural product having excellent safety. It is in. The above objectives are 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3', 4'-tetramethoxyflavone and at least these. It is solved by a cosmetological composition or the like containing at least one active ingredient selected from the group consisting of the inclusions of one ingredient.

Description

The present invention relates to cosmetological compositions, in particular cosmetological compositions containing components derived from natural products.

The skin, which is the largest organ of the human body, plays the role of the front line of biological defense in the outermost layer of the living body, and it is required to keep it healthy and aesthetically normal. The skin (stratum corneum) receives a lot of stress due to ultraviolet rays, aging, stress, illness, eating habits, etc., and causes various troubles such as deterioration of skin function and skin aging.

Wrinkles are formed by the action of proteolytic enzymes (elastase, collagenase, etc.) induced by skin aging (photoaging) caused by aging and sunlight. Although the mechanism of skin aging is not clear, UV light is considered to be the largest environmental factor involved in wrinkle formation. It is known that active oxygen produced by ultraviolet rays not only causes acute inflammation such as sunburn, but also induces photoaging by chronically repeating its production. For skin health and beauty, there is a high demand for anti-wrinkle agents that suppress skin aging and improve wrinkles.

On the other hand, an important factor that determines the color of skin and hair is a pigment substance called melanin, which is produced by melanocytes present in the basal layer of the epidermis and hair roots. Control of melanin has aesthetic implications as well as UV protection. Congenital or acquired deficiency or decreased melanin-producing ability results in poliosis and vitiligo. The white hair that occurs with aging is also thought to be due to the decline in melanin-producing ability in the hair roots.

In addition, not only in Europe and the United States, there is a widespread culture that brown skin is healthy and beautiful, and there is a great demand for making skin and hair black or dark brown. Since sunburn caused by sunlight is exposed to ultraviolet rays, there is a great risk of developing skin cancer, and therefore, a sunburn accelerator that does not depend on sunlight is considered to be useful. These skin problems and cosmetology needs due to skin aging are common throughout the world, and the cosmetics market size exceeds 1 trillion yen in Japan alone.

So far, it has been proposed to use antioxidants such as vitamin E and extracellular matrix regulators such as collagen and elastin to improve wrinkle formation and skin aging. In addition, the gray hair that occurs with aging is dyed with a dye as a symptomatic treatment.

A component derived from a natural product is expected from the viewpoint of safety as a substance capable of exhibiting an anti-wrinkle effect. For example, the following Non-Patent Document 1 (the entire description of the document is incorporated herein by reference) describes the anti-wrinkle effect of the black ginger extract. Further, Patent Document 1 below (the entire description of the document is incorporated herein by reference) describes that the black ginger extract has an antioxidant effect. Further, in Non-Patent Document 2 below (the entire description of the document is incorporated herein by reference), it is stated that 5,7-dimethoxyflavone among the components contained in black ginger has a melanin formation promoting action. There is.

Japanese Unexamined Patent Publication No. 2012-176976

Photodermatology, Photoimmunology & Photomedicine 30: 237-245, (2014) Experimental Dermatology 20 (5): 445-447 (2011)

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. be.

Further, according to the investigation by the present inventors, the melanin formation promoting action of 5,7-dimethoxyflavone described in Non-Patent Document 2 is very small.

Therefore, the present invention provides a composition containing an active ingredient capable of suppressing or improving skin aging such as wrinkles and tarmi, while being a component derived from a natural product having excellent safety. Is the first problem to be solved by the invention.

Further, the present invention is a composition containing an active ingredient capable of promoting melanin synthesis and suppressing or improving whitening of hair or skin, while being a component derived from a natural product having excellent safety. Providing goods is the second problem to be solved by the invention.

The present inventors have diligently pursued research and development in order to solve the above problems, and investigated the bioactive effects of a wide variety of natural products and components derived from natural products. As a result, 11 kinds of polymethoxyflavonoids contained in black ginger Was successfully isolated. As a result of repeated trial and error tests on the physiologically active effects of these polymethoxyflavones, surprisingly, 5,7,3', 4'-tetramethoxyflavones, 5,7,4'-trimethoxyflavones and It was found that 5-OH-3,7,3', 4'-tetramethoxyflavone has an action of suppressing the activity of proteolytic enzymes involved in wrinkle formation and inflammatory factors in the skin.

Even more surprising, we found that 3,5,7,3', 4'-pentamethoxyflavones, 3,5,7-trimethoxyflavones, 3,5,7,4'-tetramethoxyflavones. , 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'-trimethoxyflavones have been found to have the effect of promoting the activation of enzymes involved in the melanin synthesis pathway.

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

Therefore, according to one aspect of the present invention, the compositions and methods according to the following [1] to [10] are provided.
[1] 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3', 4'-tetramethoxyflavone and at least one of them. A cosmetological composition comprising at least one active ingredient selected from the group consisting of seed inclusions.
[2] The composition according to [1], wherein the composition 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 is at least one composition selected from the group consisting of an anti-wrinkle composition, an anti-tarmi composition, an anti-skin inflammation composition, an anti-aging composition and a skin improving composition. The composition according to any one of [1] to [2].
[4] 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, A cosmetic composition comprising 4'-trimethoxyflavone and at least one active ingredient selected from the group consisting of at least one of these.
[5] The composition according to [4], wherein the composition has a tyrosinase activation promoting action.
[6] The composition is a group consisting of an anti-politicia composition, an anti-white hair composition, an anti-white spot disease composition, an anti-aging composition, a sunburn promoting composition, and a skin blackening composition. The composition according to any one of [4] to [5], which is at least one composition selected from the above.
[7] The composition according to any one of [1] to [6], wherein the composition is a cosmetic composition.
[8] 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone for producing the composition according to any one of [1] to [7]. , 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 How to use at least one active ingredient selected from the group consisting of -3,7-dimethoxyflavones and 5-OH-3,7,4'-trimethoxyflavones and inclusions of at least one of these ingredients.
[9] Wrinkles, tarmi, skin inflammation, aging, skin abnormalities, poliosis, canities and vitiligo, which include applying the composition according to any one of [1] to [7] to the individual used. A method of treating, preventing, suppressing or ameliorating a symptom or disease selected from the group consisting of illnesses.
[10] A method for promoting sunburn or a method for blackening the skin, which comprises applying the composition according to any one of [4] to [7] to the individual to be used.

The composition of one aspect of the present invention contains components contained in black ginger, which is a natural product whose safety has been demonstrated by being used in crude drugs and the like, and little is known to exhibit side effects. Since it is contained as an active ingredient, it can be safely used for the individual used. In addition, black ginger grows naturally in Southeast Asia and is available in large quantities, and since the above ingredients contained in black ginger are commercially available, it can be manufactured on an industrial scale with high economic efficiency. You can expect it.

The composition of one aspect of the present invention has wrinkles, tarmi, skin inflammation, aging, and skin abnormalities 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. In addition to the above, it can be expected to treat, prevent, suppress or ameliorate various skin diseases, symptoms and abnormalities.

Furthermore, the composition of one aspect of the present invention can be expected to treat, prevent, suppress or improve poliosis, white hair, vitiligo, aging, etc. through the action of activating enzymes involved in the melanin synthesis pathway. Furthermore, it can be expected to be used for cosmetic purposes such as promoting sunburn and darkening the skin.

The composition of one aspect of the present invention is not only used for external use, but also contains internal and external cosmetological ingredients (inner cosmetics) that have few side effects and can be taken orally, so that it can be used as a health food. You can expect it.

FIG. 1 shows the structural formula of methoxyflavonoids isolated from black ginger extract as described in Examples described later. FIG. 2 is a diagram showing the results of measuring the enzymatic activity of elastase in IL-1α-stimulated normal human skin fibroblasts, as described in Examples described later. FIG. 3 is a diagram showing the results of measuring the gene expression level of elastase in IL-1α-stimulated normal human skin fibroblasts as described in Examples described later. FIG. 4 is a diagram showing the results of measuring the expression level of elastase protein in IL-1α-stimulated normal human skin fibroblasts, as described in Examples described later. FIG. 5 is a diagram showing the results of measuring the expression level of the IL-1α gene in a human epidermal keratinized cell line irradiated with UVB, as described in Examples described later. FIG. 6 is a diagram showing the results of measuring the enzymatic activity of elastase in the co-culture method of normal human skin fibroblasts and UV-B-irradiated keratinocytes (HaCaT) as described in Examples described later. .. FIG. 7 measures the gene expression level of collagenase (MMP-1) in the co-culture method of normal human skin fibroblasts and UV-B-irradiated keratinocytes (HaCaT) as described in Examples described later. It is a figure which shows the result. FIG. 8 is a diagram showing the results of measuring the enzymatic activity of tyrosinase in human HMV-II melanoma cells as described in Examples described later.

Hereinafter, the details of the composition and the method which are one aspect of the present invention will be described, but the technical scope of the present invention is not limited to the matters of this item, and the present invention is as long as the object is achieved. Can take various aspects in.

Unless otherwise specified, each term herein is used in the sense commonly used by one of ordinary skill in the art and should not be construed as having an unreasonably limited meaning. Further, since the speculations and inferences made in the present specification are based on the knowledge and experience of the present inventors, the technical scope of the present invention is limited only by such speculations and inferences. It is not something that will be done.

The "composition" is not particularly limited as having a meaning usually used, but is, for example, a combination of two or more kinds of substances, specifically, a combination of an active ingredient and another substance. , A combination of two or more active ingredients, and more specifically, a combination of one or more active ingredients and one or more solid or solvent components. Examples thereof include solid compositions and liquid compositions.

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

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

In the present specification, "~" in the numerical range is a range including the numerical values before and after it, and for example, "0% by mass to 100% by mass" is 0% by mass or more and 100% by mass or less. It means a certain range.

The term "inhibition of activation" of a substance means to diminish the degree of catalytic action or physiological action inherent in the substance, to reduce the expression level of the gene of the substance, and to translate the gene product (protein) of the substance. It refers to at least one of the actions of reducing the amount of the substance and inhibiting the production of the gene product of the substance.

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

The term "promoting action" of a substance means 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. It refers to at least one of the actions.

[Outline of the present invention]
The composition of the first aspect of the present invention is 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3', 4'-. It contains at least one active ingredient selected from the group consisting of tetramethoxyflavones and the inclusions of at least one of these ingredients.

The composition of the first aspect of the present invention is at least one 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, depending on the active ingredient contained therein. May have action. By having these actions, the composition of the first aspect of the present invention is typically an anti-wrinkle composition, an anti-tarmi composition, an anti-skin inflammation composition, an anti-aging composition and a skin abnormality. It may take an aspect such as an improvement composition. Another aspect of the present invention is a method of applying an active ingredient to a used individual to treat, prevent, suppress or ameliorate wrinkles, tarmi, skin inflammation, aging and skin abnormalities.

The composition of the second aspect of the present invention is 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 It contains at least one active ingredient selected from the group consisting of 5-OH-3,7,4'-trimethoxyflavones and inclusions of at least one of these ingredients.

The composition of the second aspect of the present invention may have an action of promoting activation of an enzyme involved in the melanin synthesis pathway depending on the active ingredient contained therein. By having these actions, the composition of the second aspect of the present invention is typically a composition for anti-paliosis, a composition for anti-white hair, a composition for anti-white spot disease, a composition for anti-aging, and the like. It may take aspects such as a sunburn promoting composition and a skin blackening composition. Another aspect of the present invention is a method of treating, preventing, suppressing or ameliorating poliosis, canities and vitiligo by applying the active ingredient to the individual used. Another aspect of the present invention is a method of applying an active ingredient to an individual to be used to promote sunburn or a method of blackening the skin.

In the present specification, the composition of the first aspect of the present invention and the composition of the second aspect of the present invention may be collectively referred to as "the composition of one aspect of the present invention". In addition, the active ingredient contained in the composition of the first aspect of the present invention and the active ingredient contained in the composition of the second aspect of the present invention may be collectively referred to as an "active ingredient", and the first aspect of the present invention. The action that the composition may have and the action that the composition of the second aspect of the present invention may have may be collectively referred to as "effective action".

（式中、Ｒ_ｌ、Ｒ_２、Ｒ_３、Ｒ_４、Ｒ_５、Ｒ_６、Ｒ_７、Ｒ_８、Ｒ_９及びＲ_１０は、それぞれ独立して、水素、水酸基又はメトキシ基を表す。）
に示される化合物に包含される。[Active ingredient]
The active ingredient is classified into polymethoxyflavonoids, and has the following general formula (I).

(In the formula, R _l , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ and R ₁₀ independently represent hydrogen, hydroxyl group or methoxy group, respectively.)
Included in the compounds shown in.

The active ingredients in the composition of the first aspect of the present invention are 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3', It can be any one or two or all three of the 4'-tetramethoxyflavones. The active ingredient in the composition of the second aspect of the present invention is 3,5,7,3', 4'-pentamethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetra. Methoxyflavone, 5-OH-3,7,3', 4'-tetramethoxyflavone, 5-OH-7-methoxyflavone, 5-OH-7,4'-dimethoxyflavone, 5-OH-3,7- Any one of dimethoxyflavones and 5-OH-3,7,4'-trimethoxyflavones, 2 types, 3 types, 4 types, 5 types or 6 types or all 7 types can be used.

5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 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-methoxyflavones, 5-OH-7,4'-dimethoxyflavones, 5-OH-3,7-dimethoxyflavones and 5-OH-3,7,4'-trimethoxyflavones (hereinafter referred to as these). (Sometimes collectively referred to as "polymethoxyflavonoid") is not particularly limited as long as it is generally known, and even if it is commercially available, it is synthesized. Alternatively, it may be extracted and / or separated from a natural product. The positions of the methoxy group and the hydroxy group in the polymethoxyflavonoid can be confirmed from the general formula (I).

The active ingredient may be an inclusion containing any one or more of polymethoxyflavonoids. The content is not particularly limited as long as it contains the polymethoxyflavonoid, but is, for example, a plant extract, preferably a black ginger extract.

Black ginger ( Kaempferia parviflora ) is a plant belonging to the genus Kaempferia of the family Zingiberaceae, which is known to grow naturally in Southeast Asia and the like. Black ginger is known to have effects such as energizing, nourishing tonic, lowering blood sugar level, recovering physical strength, improving digestive system, subvaginal band, hemorrhoids, hemorrhoids, nausea, stomatitis, arthralgia, and stomach pain. Has been done. Since black ginger is a natural plant that has been ingested by humans for a long period of time and has high safety, the composition of one aspect of the present invention can be applied in a parenteral or oral form. ..

The site of use of black ginger is not particularly limited as long as it contains an active ingredient that contributes to a desired effective action, and examples thereof include roots, stems, leaves, flowers, branches, etc., but polymethoxyflavonoids are preferable. It is a rhizome containing a lot.

The method for producing a black ginger extract containing polymethoxyflavonoid from black ginger is not particularly limited, and examples thereof include a method including at least contacting the black ginger extract with a polymethoxyflavonoid-soluble solvent.

The black ginger used as a raw material for the black ginger extract may be in a as-collected state or may be in a state of being subjected to processing such as drying after being collected. If it takes time to process the ginger after collection, it is preferable to store it by a storage means usually used by those skilled in the art such as low temperature storage in order to prevent deterioration of the raw material of black ginger.

The raw material of the black ginger extract is preferably in a dry state because it contains a large amount of water depending on the part of the black ginger used. The drying treatment is not particularly limited, and for example, the mass (dry mass) of the raw material of the black ginger extract after drying is about 1/2 to 1/10, preferably 1/10 of the mass before drying (wet mass). Examples thereof include a treatment of drying to about 1/3 to 1/6. 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 time at which the raw material of the black ginger extract is not discolored by heating at 30 ° C. to 100 ° C. for 24 hours to 72 hours.

The raw material of the black ginger extract is preferably in a crushed, crushed or ground state from the viewpoint of efficiently performing the extraction operation. The method of crushing, crushing or grinding the raw material is not particularly limited, and examples thereof include a method using a cutter, a cutting machine, a crusher, a blender, a mixer, a mill, a grinder, a kneader, a mortar and the like.

The extraction solvent for extracting the black ginger extract 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; dimethylsulfoxide, acetonitrile, acetone, hexane and glycerin. , Organic solvents such as propylene glycol and the like, but are not limited thereto.

As the organic solvent, a single species may be used, or a mixed solvent in which a plurality of species are mixed 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. As the organic solvent, lower alcohol is preferable, and ethanol and methanol are more preferable.

As an 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, a stirring operation, heating, or the like may be performed as necessary in order to improve the extraction efficiency. 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 insoluble in water, unnecessary impurities can be removed prior to the extraction operation by boiling black ginger in boiling water, for example.

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, a known separation method such as filtration or centrifugation can be used.

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

Black ginger contains flavonoids other than the 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 filler used for the separation operation using a column is not particularly limited, and examples thereof include silica gel to which an octadecyl group is bonded.

The method for confirming and quantifying the polymethoxyflavonoid contained in the black ginger extract is not particularly limited, and for example, it can be performed by high performance liquid chromatography (HPLC) as described in Examples described later.

The content of polymethoxyflavonoid in the black ginger extract is not particularly limited, but for example, the total amount per mass of the black ginger extract is 0.1% by mass or more, preferably 1 to 99% by mass, and more. It is preferably 5 to 90% by mass. 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. Is. 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, per mass of the black ginger extract. % Or more. 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. Is. 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, based on the 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, per mass of the black ginger extract. % Or more. 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, based on the 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. be. 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.

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

As the activation inhibitory action of the proteolytic enzyme involved in wrinkle formation contained in the composition of the first aspect of the present invention, it is preferable that the action is any one of an elastase activation inhibitory action and a collagenase activation inhibitory action. Both of these actions are more preferred. The composition of the first aspect of the present invention preferably has an inflammatory cytokine activation inhibitory action as an inflammatory factor activation inhibitory action involved in wrinkle formation, and interleukin-1α (IL-1α) activation. It is more preferable that it has an inhibitory effect.

The effective action of the composition of the first aspect of the present invention is the activity related to enzymes and inflammatory factors induced by external stimulation of skin fibroblasts or epidermal keratinocytes, as described in Examples described later. It can be confirmed by measuring the titer, gene expression level and / or protein production amount of. At this time, the system to which the composition of the first aspect of the present invention is added is used as a control, and the system to which the composition of the first aspect of the present invention is added is an enzyme or an inflammatory factor induced by an external stimulus of cells rather than the control. If the titer of the activity, the gene expression level and / or the protein production amount is small, the composition of the first aspect of the present invention can be evaluated to have an effective action.

The composition of the second aspect of the present invention exhibits an activation promoting action of an enzyme involved in the melanin synthesis pathway by containing an active ingredient. The tyrosinase activation-promoting action is preferable as the activation-promoting action of the enzyme involved in the melanin synthesis pathway of the composition of the second aspect of the present invention.

The effective action of the composition of the second aspect of the present invention is to measure the activity titer, gene expression level and / or protein production amount of the enzyme produced by melanoma cells, as described in Examples described later. It can be confirmed by doing. At this time, the system to which the composition of the second aspect of the present invention is added is used as a control, and the system to which the composition of the second aspect of the present invention is added is more active than the control and the gene expression level is higher than that of the control. And / or if the amount of protein produced is small, the composition of the second aspect of the present invention can be evaluated to have an effective effect.

The composition of one aspect of the present invention is not particularly limited as long as it is a composition used for cosmetic purposes, but may take various forms such as a cosmetic composition, a food and drink composition, and a pharmaceutical composition. .. Therefore, a specific embodiment of the present invention is a composition for parenteral or oral cosmetics, a composition for food and drink, a composition for pharmaceuticals, a composition for quasi-drugs, which contains an active ingredient. It is a composition for animal feed and the like.

The content of the active ingredient contained in the composition of one aspect of the present invention is not particularly limited as long as it has a desired effective effect, but is 0.00001, for example, as a parenteral cosmetic composition. By mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more; 0.0001% by mass or more, preferably 0.001% by mass as an oral cosmetic composition. As mentioned above, it is more preferably 0.01% by mass or more. The upper limit of the content is not particularly limited, but is typically 1% by mass.

The individual used and the method of using the composition of one aspect of the present invention are not particularly limited, and examples thereof include animals, particularly mammals, and mammals include humans, dogs, cats, cows, and horses. Of these, humans are preferred.

The usage and dosage of the composition of one aspect of the present invention, such as the amount of one-time use, the amount of daily use, the period of use, the interval of use, etc., are not particularly limited, and are appropriately set according to the mode of use, the condition of the individual used, and the like. Can be done. The interval of use is, for example, once, twice, three times or several times a day 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. Even more preferably, it may be continuously applied for 6 months or 1 year or more. The composition of one aspect of the invention is preferably applied daily, but the composition of one aspect of the invention may not be applied daily as long as it is continuously applied during the period.

As described above, the composition of one aspect of the present invention may be applied parenterally or orally, but it may be applied directly to the affected area where the effective action is exerted. It is preferably a parenteral beauty composition, and more preferably a cosmetic composition and a parenteral pharmaceutical composition.

The composition of one aspect of the present invention may contain other components in addition to the active ingredient as long as the problems of the present invention can be solved.

Other ingredients in the case of a parenteral cosmetic composition include, for example, ingredients contained in ordinary cosmetics and pharmaceuticals, and more specifically, bases such as water, oily ingredients, and moisturizers. Agents, refreshing agents, preservatives, chelating agents, pH regulators, antioxidants, UV absorbers, UV scatterers, thickeners, whitening agents, vitamins, various other medicinal ingredients, powders, fragrances, coloring materials, etc. Can be mentioned. The contents of other components may be appropriately set by those skilled in the art as long as they do not interfere with solving the problems of the present invention. Some of the other components are listed below, but these are examples only and are not limited.

Examples of the oily component include an oily emollient component, which is preferably an oily emollient component that is liquid or semi-solid at room temperature (25 ° C.). Specific examples of the oily emollient component include neopentyl glycol dicaprate, triethylhexanoin, isopropyl palmitate, cetyl 2-ethylhexanoate, isononyl isononanoate, isotridecyl isononanoate, diisopropyl sebacate, and pentaeryth tetra2-ethylhexanoate. Ester oils such as lithyl, diethylhexyl succinate, alkylbenzoate having 12 to 15 carbon atoms; silicone oils such as dimethylpolysiloxane, dimethylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, higher alcohol modified silicone oils; Glyceride oils such as acetoglyceryl, glyceryl triisooctanoate, glyceryl triisostearate, glyceryl triisopalmitate, glyceryl monostearate, glyceryl di-2-heptylundecanoate, glyceryl trimyristinate; animal oils such as liquid lanolin; fluoropoly Fluorine oils such as ether, perfluoroalkyl ether silicone; 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 ka oil, castor oil, flaxseed oil, saflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, brown seed oil, kaya oil, rice bran oil, cinnamon oil, Japanese millet oil, germ oil, etc. Natural oily ingredients, etc., but are not limited to these. As the oily emollient component, one of the above-mentioned ones can be used alone or in combination of two or more. The content of the oily emollient component is preferably 0% by mass to 10% by mass.

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

Examples of the preservative include benzoic acid, salicylic acid, phenolic acid, sorbic acid, methyl paraoxybenzoate, parachlormethacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichlorocarbanilide, photosensitive element, phenoxyethanol and the like. 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.

Examples of the pH adjuster include lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, malic acid, ethidronic acid, potassium carbonate, sodium hydrogencarbonate, ammonium hydrogencarbonate, sodium hydroxide, potassium hydroxide, and triethanolamine. Examples thereof include monoethanolamine, 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.

Examples of the antioxidant include vitamin E and its derivatives such as dibutylhydroxytoluene, butylhydroxyanisole, and δ-tocopherol, thiotaurine, mematsuyoigusa extract, β-carotene, catechin compounds, flavonoid compounds, polyphenol compounds, and the like. One type can be used alone, or two or more types can be used in combination. The content of the antioxidant is preferably 0% by mass to 1% by mass.

Examples of the thickener include water-soluble polysaccharides such as hyaluronic acid, sodium hyaluronate, acetylated hyaluronic acid, acetylated sodium hyaluronate, chondroitin sulfate, sodium chondroitin sulfate, fucoidan, tuberose polysaccharide, and xanthan gum; Natural polymers such as; semi-synthetic polymers such as sodium carboxymethyl cellulose; carbomer, polyacrylic acid, sodium polyacrylate, acrylic acid-based polymers such as (meth) acrylic acid / (meth) alkyl acrylate copolymers, etc. Examples include synthetic polymers. Carbo-mer is, for example, as a commercial product, "Carbo-mer 980", "Carbo-mer 910", "Carbo-mer 934", "Carbo-mer 940", "Carbo-mer 941", "Carbo-mer 981" (each of Lubrizol Advanced Materials, Inc. It is commercially available as a product). The (meth) acrylic acid / (meth) acrylic acid alkyl copolymer is, for example, "Carbo-mer SC-500", "Carbo-mer 1382", "Carbo-mer ETD2020", "PEMULEN TR-1", "PEMULEN TR-". 2 ”(Made by Lubrizol Advanced Materials, Inc., respectively) and the like. Sodium carboxymethyl cellulose is commercially available, for example, as a commercial product such as "CMC Daicel" (manufactured by Daicel Chemical Industries, Ltd.). The thickener may be used alone or in combination of two or more. The content of the thickener is preferably 0% by mass to 1% by mass.

The mode of use and dosage form as a cosmetic composition are not particularly limited, and examples thereof include skin care cosmetics, makeup cosmetics, fragrance cosmetics, and body care cosmetics, and more specifically, creams, milky lotions, foundations, and facial cleansers. , Beauty liquid, hair tonic, hair cream, shampoo, hair rinse, treatment, face wash, foundation, hair restorer, aqueous ointment, spray, gel, lotion, pack, face wash cream, hand cream, makeup cleansing, makeup base, concealer, cheek Examples include red, eye shadow, eye liner, eyebrow, lipstick, hair removal / hair removal cream, and all-in-one cosmetics. The composition of one aspect of the present invention is used as an oil-in-water emulsified cosmetic for skin and hair by taking a dosage form having a certain viscosity such as a gel, a milky lotion, a lotion, and a cream. It is preferable because it can have a feel.

The mode of use and dosage form as a composition for parenteral pharmaceuticals are not particularly limited, and examples thereof include injections, patches, ointments, and creams.

The composition of one aspect of the present invention may be a cosmetic composition for oral use, and in this case, a composition for food and drink and a composition for oral pharmaceutical products are preferable.

A specific embodiment of the composition for food and drink is, for example, a functional food and drink which is a food and drink having a certain functionality with respect to a living body. Functional foods and drinks include, for example, foods and drinks for specified health use, foods and drinks with functional claims, foods and drinks with nutritional functions, foods and drinks with health functions, foods and drinks for special purposes, foods and drinks for nutritional supplements, foods and drinks for health supplements, supplements, beauty foods, etc. In addition to the so-called healthy foods and drinks, it includes foods and drinks for specific persons such as foods and drinks for infants, foods and drinks for pregnant women, and foods and drinks for the elderly. Further, functional foods and drinks include healthy foods and drinks to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied.

Oral pharmaceutical compositions can take the form of any oral pharmaceutical as long as they can exhibit effective action after application. In addition, the composition for oral pharmaceutical products may contain other bioactive substances having an active action other than the active ingredient as long as the object of the present invention can be achieved, and the bioactive substance is contained as the active ingredient. It may be used in combination with other drugs.

The mode of use and dosage form of the cosmetological composition for oral use are not particularly limited, and may be in the form of tablets, powders, granules, powders, capsules, pills, troches, liquids and the like. Not limited. The tablets include sugar-coated tablets, coated tablets, buccal agents and the like. Capsules include both hard and soft capsules. Granules also include coated granules. Further, the liquid agent includes a suspension agent, an emulsion, a syrup agent, an elixir agent and the like. The syrup agent includes dry syrup and the like.

When used as a composition for food and drink, for example, bread, cookies, biscuits, wheat for adding rice, miscellaneous grains, udon, buckwheat, pasta and other noodles, cheese, yogurt and other dairy products, jam, mayonnaise, miso, soy sauce and others. Made from soybean products, tea, coffee, cocoa, soft drinks, fruit drinks and other non-alcoholic drinks, medicinal liquors, other alcoholic drinks, candies, chocolate and other snacks, chewing gum, senbei, sheep and other soybeans. It can be added to confectionery or the like to form a general food or drink.

The cosmetological composition for oral use may be one containing an active ingredient, but may be a combination with other ingredients. The other ingredients are not particularly limited, but for example, additives for foods and drinks or oral pharmaceuticals can be used as other ingredients. For example, various excipients, binders, lubricants, stabilizers, diluents, bulking agents, emulsifiers, colorants, fragrances, perfume oils, thickeners, brighteners, buffers, etc. may be used as other components. Can be done. The content of other components can be appropriately selected depending on the form of the composition of one aspect of the present invention as long as it does not interfere with the solution of the problem of the present invention.

Examples of the excipient include lactose, glucose, sucrose, mannitol, horse belly starch, dextrin, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, citrus powder, gentian powder and the like. Examples of the binder include starch, tragant rubber, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxylmethyl cellulose and the like. Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, methyl cellulose, ethyl cellulose, carboxyl methyl cellulose and the like. Examples of the lubricant include talc and magnesium stearate. As the colorant, those that are permitted to be added to foods and drinks and oral medicines can be used, and are not particularly limited.

In the case of tablets and granules, if desired, sucrose, gelatin, purified cellulose, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, phthalate cellulose acetate, hydroxypropylmethyl cellulose phthalate, methyl methacrylate, etc. It may be coated with a methacrylic acid polymer or the like, or may be coated with a plurality of layers. Further, granules and powders can be packed into capsules such as ethyl cellulose and gelatin to form capsules.

The composition of one aspect of the present invention is not particularly limited in terms of the production method thereof, and for example, depending on the mode of use of cosmetics, foods and drinks, pharmaceuticals, etc., the active ingredient and optionally other ingredients may be obtained by a method known to those skilled in the art. It can be manufactured by mixing with an ingredient or the like.

The composition of one aspect of the present invention is not particularly limited in terms of the method of use thereof, and for example, a method usually used can be adopted depending on the mode of use of cosmetics, foods and drinks, pharmaceuticals and the like.

The composition of one aspect of the present invention can be a packaged composition packed in a container and sealed. The container is not particularly limited, but for example, a metal such as aluminum, paper, a plastic such as PET or PTP, a single-layer or laminated film bag, a retort pouch, a vacuum pack, an aluminum container, a plastic container, a glass container, a bottle, etc. Examples include packaging containers such as cans. In order to avoid deterioration over time, the composition of one aspect of the present invention is preferably packed in a container, sealed, and then sterilized by pressurization and / or heating. It is preferable that the packaged composition is one in which the produced composition can be dispensed, filled and / or individually packaged and independently put into distribution and marketed by itself.

[Specific aspects of the composition]
The composition of the first aspect of the present invention is a composition for suppressing, improving, alleviating, treating and / or preventing the formation of wrinkles generated in the skin through the activation inhibitory action of proteolytic enzymes involved in wrinkle formation. That is, it may take the form of an anti-wrinkle composition. Similarly, the composition of the first aspect of the present invention has aspects such as an anti-talmi composition, an anti-aging composition, and a skin abnormality improving composition through the action of suppressing the activation of proteolytic enzymes involved in wrinkle formation. It can be taken.

The composition of the first aspect of the present invention is 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, for anti-skin inflammation. It may take an aspect such as a composition.

The composition of the second aspect of the present invention suppresses, improves, alleviates poliosis and vitiligo by promoting melanin synthesis in skin and hair through the activation promoting action of enzymes involved in the melanin synthesis pathway. It may take the form of a composition for treatment and / or prevention, i.e., an anti-politic acid composition and an anti-white spot disease composition. Similarly, the composition of the second aspect of the present invention is used for anti-white hair composition, anti-aging composition, sunburn promoting composition, and skin blackening through the activation promoting action of enzymes involved in the melanin synthesis pathway. It may take an aspect such as a composition.

As described above, the composition of one aspect of the present invention is used for treating, preventing, suppressing, alleviating and / or ameliorating a skin or hair symptom or disease caused in the skin or hair, particularly associated with aging, through a corresponding effective action. It is preferably a composition. Moreover, the composition of one aspect of the invention can be used for prognostic treatment. The individual using the composition of one aspect of the present invention may be a healthy individual, but an individual having wrinkles, tarmi, inflammation, etc. on the skin, or whitening of the skin or hair. , And it is preferable that they are individuals at risk of manifestation.

As described in Examples 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 an elastase activation inhibitory composition or an elastase activation inhibitor, a collagenase activation inhibitory composition or a collagenase activation inhibitor, an inflammatory cytokine activation, which contains an active ingredient. An inhibitory composition or an inflammatory cytokine activation inhibitor and an interleukin-1α activation inhibitor in skin cells or an interleukin-1α activation inhibitor in skin cells.

As described in Examples 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 tyrosinase activation-promoting composition or a tyrosinase activation-promoting agent containing an active ingredient.

[Method]
The method for producing and using the composition of one aspect of the present invention is included in the present invention as another aspect.

The method of one aspect of the present invention is 5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone, 5-for producing the composition of one aspect of the present invention. 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, A method of using at least one active ingredient selected from the group consisting of 7-dimethoxyflavones and 5-OH-3,7,4'-trimethoxyflavones and inclusions of at least one of these ingredients.

A method of another aspect of the invention comprises applying the composition of the first aspect of the invention to a user, a symptom selected from the group consisting of wrinkles, tarmi, skin inflammation, aging and skin abnormalities. A method of treating, preventing, suppressing or ameliorating a disease.

A method of another aspect of the invention comprises applying the composition of the second aspect of the invention to a user, a symptom or disease selected from the group consisting of poliosis, canities, vitiligo and aging. Is a method of treating, preventing, suppressing or ameliorating.

Another method of the present invention is a method of promoting sunburn or a method of blackening the skin, which comprises applying the composition of the second aspect of the present invention to a used individual.

As long as the problem of the present invention can be solved, the method of one aspect of the present invention can incorporate various steps and operations in the pre-stage, the post-stage, or the process of the above-mentioned process.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. ..

[Example 1. Evaluation of elastase activation inhibitory effect of methoxyflavonoids]
(1-1) Test Samples and Materials Normal human skin fibroblasts (normal human skin fibroblasts) were purchased from TOYOBO (catalog number: GCA10605F). DMEM was purchased from SIGMA (catalog number: D6429-500ML). Other reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. or SIGMA. The 96-well microplate was purchased from Nunc (catalog number: 161093).

Liquid-liquid distribution with an organic solvent from black ginger, chromatography using a reverse-phase resin (Diaion HP20 manufactured by Mitsubishi Chemical Co., Ltd.) and reverse-phase ODS column (Wakopak Wakosil-II 5C18RS Prep 28 manufactured by Fujifilm Wako Junyaku Co., Ltd.) The compounds to be used were isolated by preparative HPLC using (× 250 mm), and each compound was identified by structural analysis such as NMR. In addition, the method for confirming and quantifying the polymethoxyflavonoids contained in the black ginger extract is a high-performance liquid using a reverse-phase ODS column (Wakopak Wakosil-II 5C18RS Rep 4.6 × 250 mm manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). It was performed by chromatography (HPLC). Test samples were prepared by diluting the isolated compound with DMSO.

(1-2) Preculture In normal human skin fibroblasts, elastase activity is increased by IL-1α stimulation. ^{Therefore, normal human skin fibroblasts were prepared at 5.0 × 10 4} cells / ml using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)), and 100 μl of this was placed in a 96-well microplate. Dispensed at / well. The cells were cultured for 24 hours at 37 ° C. in the _{presence of 5% CO 2.}

(1-3) Elastase enzyme activity test To each well of the pre-cultured culture plate, IL-1α (final concentration 0.1 nM) and a test sample were added so that the final concentration of the isolated compound was 15 or 30 μM. .. After culturing the plate containing the test sample for 72 hours, the culture supernatant was withdrawn. Then, 0.5 vol% Triton X-100 solution was added to the cells remaining in the well to dissolve the cells, and then a substrate buffer was further added to measure the enzymatic activity of elastase with a plate reader. Using Suc- (Ala) _3- pNA (STANA) as a substrate, the amount of p-nitroaniline produced by the action of elastase was measured by the absorbance at 405 nm. The result obtained by adding DMSO instead of the test sample was used as control (CN). The results obtained by testing using normal human skin fibroblasts not stimulated with IL-1α without adding the test sample and DMSO were designated as blank (BL). In addition, as a positive control, 30 μM resveratrol was used instead of the test sample, and the results obtained were obtained. The obtained values were subjected to Dunnett's test against CN. The significance level was set to risk rates of 5% and 1%.

(1-4) Elastase gene expression test A test sample was added to each well of the pre-cultured culture plate so that the final concentration of the isolated compound was 30 μM. After culturing the plate containing the test sample for 24 hours, total RNA was extracted and cDNA was prepared from the cells using TRIZOL Reagent (catalog number: 15596-018) manufactured by Thermo Fisher Scientific, and the elastase gene was prepared by real-time PCR. The expression level was measured.

(1-5) Elastase Protein 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 10, 15 and 30 μM. After culturing the plate containing the test sample for 24 hours, washing with PBS, the cells were lysed using RIPA Buffer (catalog number: 182-02451) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. to prepare a cell extract. , The expression level of elastase protein (NEP) was measured by Western blotting (control: β-actin).

(1-6) Test Results and Evaluation As shown in FIG. 1, 11 kinds of polymethoxyflavonoids of MF1 to MF11 were isolated from the black ginger extract.

Among the methoxyflavonoids shown in FIG. 1, 5,7,3', 4'-ttramethoxyflavonoid (MF1), 5,7,4'-trimethothoxyflavonoid (MF4) and 5-OH-3,7,3', 4'- Methoxythyflavonoid (MF7) significantly suppressed the elastase enzyme activity increased by IL-1α stimulation (Fig. 2). Similarly, MF1, MF4 and MF7 suppressed IL-1α-stimulated elastase gene expression (FIG. 3). Furthermore, MF4 suppressed the expression of elastase protein that was increased by IL-1α stimulation (Fig. 4).

As described above, MF1, MF4 and MF7 suppress elastase, which is a proteolytic enzyme involved in wrinkle formation, at the enzyme level, gene level and protein level, and thus suppress elastase activity, which is useful for the prevention and improvement of wrinkles. It turned out to be.

[Example 2. Evaluation of IL-1α gene expression inhibitory effect of methoxyflavonoids]
(2-1) Test Samples and Materials As human epidermal keratinized cell lines, human keratinocyte HaCaT (Normal keratinization in a pontaneusly immortalized aneuploid human human keratinocyte Cell line Cell line. . J Cell Biol. 1988 Mar; 106 (3): 761-71.) Was used. The 24-well microplate was purchased from TPP (catalog number: 92024).

(2-2) In the pre-cultured human epidermal keratinized cell line (human keratinocyte HaCaT), the expression of inflammatory genes such as IL-1α is increased by UV-B irradiation stimulation. Therefore, a human epidermal keratinized cell line was prepared using a medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)) so as to be 5.0 × 10 ⁴ cells / ml, and 500 μl of this was prepared in a 24-well multiplate. Dispensed at / well. Culture plates containing human epidermal keratinized cell lines were cultured for 24 hours at 37 ° C. in the presence of _{5% CO 2.} Then, after extracting the culture supernatant, the cells were further cultured for 24 hours using a new medium (DMEM medium containing 0.1 vol% FBS (fetal bovine serum)).

(2-3) IL-1α 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 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 cells after culturing, and the expression level of the IL-1α gene was measured by a real-time PCR method. The obtained values were subjected to Dunnett's test against CN. The significance level was set to risk rates of 5% and 1%.

(2-4) Test Results and Evaluation Of the methoxyflavonoids, 5,7,4'-trimethoxyflavonoids (MF4) significantly suppressed the expression of IL-1α, which was increased by UV-B stimulation (FIG. 5).

From this result, 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.

[Example 3. Evaluation of elastase activation inhibitory effect and collagenase (MMP-1) activation inhibitory effect of methoxyflavonoids]
(3-1) Test sample and material The 12-well co-culture system plate was purchased from Falcon (catalog number: 353043).

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

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

Culture plates containing normal human skin fibroblasts and human epidermal keratinized cell lines were cultured for 24 hours at 37 ° C. in the presence of _{5% CO 2.} Then, after extracting the culture supernatant, the cells were further cultured for 24 hours using a new medium (DMEM medium containing 0.1 vol% FBS (fetal bovine serum)).

(3-3) Collagenase (MMP-1) gene expression test Only the human epidermal keratinized cell line (human keratinocyte HaCaT) of the ^{transwell was irradiated with UV-B at 50 mJ / cm 2} , and each well of the trans plate was subjected to. The test sample was added so that the final concentration of the isolated compound was 15 and 30 μM.

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

(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 (1-3) above.

(3-5) Test Results and Evaluation Of the methoxyflavonoids, 5,7,4'-trimethoxyflavonoids (MF4) suppressed the increase in elastase activity (Fig. 6). MF4 also suppressed the increased expression of the collagenase (MMP-1) gene (Fig. 7).

From these results, MF4 suppresses the increase in elastase enzyme activity and collagenase (MMP-1) gene expression in skin fibroblasts induced by UV-B-exposed keratinocytes, thus preventing or improving wrinkles. It turned out to be useful.

[Example 4. Evaluation of tyrosinase activation promoting action of methoxyflavonoids]
(4-1) Test Samples and Materials Human HMV-II melanoma cell lines were purchased from RIKEN BRC (catalog number: RCB07777). Ham's F-12 medium was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (catalog number: 087-08335). Other reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. or SIGMA.

^{(4-2) Precultured human HMV-II melanoma cell line is seeded in 96-well microplate at 1.0 × 10 4} cells / well using medium (DMEM medium containing 10 vol% FBS (fetal bovine serum)). Then, the cells were cultured for 24 hours at 37 ° C. in the _{presence of 5% CO 2.}

(4-3) Tyrosinase enzyme activity test A 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 containing the test sample for 72 hours, the culture supernatant was withdrawn.

Then, to the cells remaining in the well, T.I. 20 μL of A Lysis Buffer (1% sodium deoxycholate + 0.5% TritonX-100 + 100 mM PMSF (Phenylmethylsulphonyl fluid)) was added, and the cells were lysed by shaking for 30 minutes.

After shaking, 5 μL of cytolytic solution was taken out from each well, and the absorbance at 570 nm was measured using a BCA Protein Assay Kit to determine the amount of protein.

Mixed Buffer (N, N-dimethylformamide + L-DOPA (3,4-dihydroxy-L-phenylalanine) + MBTH (3-methyl-2-benzo-thiazolinone) lydopa) was added to the remaining cytolysate. Absorbance 505 nm was measured every 10 minutes while incubating at 37 ° C.

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

(4-4) Test Results and Evaluation Among the methoxyflavonoids, 3,5,7,3', 4'-pentamethoxyflavone (MF2), 3,5,7-trimethoxoflavonoid (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-dimethoxyflavonoids (MF10) and 5-OH-3,7,4'-trimethoxyflavonoids (MF11) increase the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, in human HMV-II melanoma cell lines. (Fig. 8).

From these results, MF2, MF5, MF6, MF7, MF8, MF9, MF10 and MF11 are useful for blackening hair and skin because they promote tyrosinase activity and promote melanin synthesis in human cells. It turned out that there was.

By containing methoxyflavonoids derived from black ginger as an active ingredient, the composition of one aspect of the present invention has an anti-inflammatory effect based on an inflammatory factor inhibitory effect, an anti-inflammatory effect based on an elastase activation inhibitory effect, and an anti-collagenase activation inhibitory effect. As a composition for drinking, edible and cosmetics that exerts an effect of promoting blackening of hair and skin based on the wrinkle effect, tyrosinase activation promoting action and melanin production promoting action, especially for skin aging suppressing composition and skin metabolism improving. It can be used as a composition and a cosmetic composition.

Cross-reference of related applications

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 (7)

5,7,3', 4'-tetramethoxyflavone, 5,7,4'-trimethoxyflavone and 5-OH-3,7,3', 4'-tetramethoxyflavone and at least one component thereof. A cosmetological composition containing at least one active ingredient selected from the group consisting of the inclusions of. The composition according to claim 1, wherein the composition 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. The composition is at least one composition selected from the group consisting of an anti-wrinkle composition, an anti-tarmi composition, an anti-skin inflammation composition, an anti-aging composition and a skin abnormality improving composition. , The composition according to any one of claims 1 and 2. 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'- A cosmetic composition comprising at least one active ingredient selected from the group consisting of trimethoxyflavones and the inclusions of at least one of these ingredients. The composition according to claim 4, wherein the composition has a tyrosinase activation promoting action. The composition is selected from the group consisting of an anti-politicia composition, an anti-white hair composition, an anti-white spot disease composition, an anti-aging composition, a sun-promoting composition, and a skin blackening composition. The composition according to any one of claims 4 to 5, which is at least one composition. The composition according to any one of claims 1 to 6, wherein the composition is a cosmetic composition.

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