ESTROGEN RECEPTOR β mRNA EXPRESSION PROMOTER, TYPE III COLLAGEN mRNA EXPRESSION PROMOTER, ELASTIN mRNA EXPRESSION PROMOTER, MATRIX METALLOPROTEINASE-1 mRNA EXPRESSION INHIBITOR, AND MATRIX METALLOPROTEINASE-3 mRNA EXPRESSION INHIBITOR

Abstract

To discover a substance having estrogen receptor β mRNA expression-promoting activity, type III collagen mRNA expression-promoting activity, elastin mRNA expression-promoting activity, matrix metalloproteinase-1 mRNA expression-inhibiting activity, or matrix metalloproteinase-3 mRNA expression-inhibiting activity, and to provide an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor, comprising such a substance as an active ingredient.SOLUTION: An estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor, comprise a Citrus junos extract as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor. [Background technology]

[0002] Estrogen, also known as follicle hormone, regulates the reproductive function as a female hormone together with progesterone, a luteinizing hormone.In addition, estrogen is known to act on skin fibroblasts to promote the biosynthesis of collagen and hyaluronic acid, to show antagonistic action with male hormones to suppress the hyperactivity of sebaceous glands activated by male hormones, and to act on epidermal keratinocytes to improve the barrier function against UV exposure, etc.Therefore, if the secretion ability of estrogen decreases, the frequency of skin aging symptoms such as decreased skin firmness and increased wrinkles will increase, and the occurrence of acne due to excessive sebum secretion and skin diseases such as rough skin due to decreased skin barrier function will increase.

[0003] For example, in postmenopausal women, a significant decrease in blood estrogen concentration leads to changes in the overall function of the skin. In particular, collagen fibers in the dermis are significantly reduced, resulting in skin aging phenomena such as decreased skin firmness, increased wrinkles, and sagging skin. In addition, a decrease in blood estrogen concentration can cause menopausal disorders, including sudden hot flashes, such as a sudden feeling of heat in the face and body, an increased heart rate, and excessive sweating.

[0004] On the other hand, it is known that female hormone replacement therapy (HRT) for postmenopausal women improves wrinkles and blood circulation, and the application of estrogen is known to improve wrinkles, elasticity, and moisture content of the skin and suppress sebum secretion from the sebaceous glands. It is also known that the intake of isoflavones, a type of phytoestrogen found in soybeans, significantly improves the symptoms of hot flashes.

[0005] Estrogen binds to two types of estrogen receptors (Estrogen Receptor α (ERα) and Estrogen Receptor β (ERβ)), and the effects of estrogen are exerted by the cooperative action of these receptors (ERα and ERβ). After estrogen binds to the Ligand Binding Domain (LBD) of ERα and ERβ, causing a conformational change, dimers (three types of combinations: ERα / α, ERβ / β, and ERα / β) are formed. The dimeric estrogen receptor (ER) binds to estrogen response elements present on the genome in the nucleus, where it recruits various coactivators and then controls the transcriptional activity of target genes.

[0006] Due to the different combinations of target genes, ERα and ERβ exert different physiological functions. For example, ERα can activate breast cancer cell proliferation, while ERβ can suppress its proliferation, which are opposite roles. ERβ also functions to protect the skin from photoaging.

[0007] Therefore, it is believed that selective activation of ERβ can prevent and improve skin aging symptoms caused by estrogen deficiency, menopausal disorders such as hot flashes, etc. 3,9-dihydrochypterocarpan and the like have been known to activate estrogen receptor β (see Patent Document 1).

[0008] One of the factors that cause wrinkles and sagging skin due to aging is a decrease in skin elasticity (flexibility). Human skin is composed of the stratum corneum, epidermis, basement membrane, and dermis, with the dermis occupying the largest area. Type I collagen and type III collagen, which are abundant in the dermis, decrease with age due to a decline in fibroblast function, etc., and this decrease is thought to cause a loss of skin elasticity (flexibility) and contribute to the occurrence of wrinkles and sagging. In particular, type III collagen is said to have the function of imparting flexibility, but it is known that the proportion of type III collagen in the dermis of adult tissue is extremely low compared to infant tissue, and tends to decrease rapidly with age.

[0009] Furthermore, Ehlers-Danlos syndrome type IV, which is caused by an abnormality in the collagen fibril formation mechanism, is characterized by noticeable wrinkles on the skin, bleeding is easily triggered by even the slightest external force, and there is a risk of symptoms such as arterial rupture and intestinal perforation. These diseases are thought to be caused by insufficient synthesis of type III collagen. Thus, type III collagen is an essential component for building normal, soft skin and suppressing skin aging, and is also an important component that can improve various diseases caused by insufficient synthesis of type III collagen.

[0010] Therefore, there is a need for the development of a type III collagen production promoter that can effectively promote the production of type III collagen and contains a naturally derived, highly safe active ingredient that can be taken orally on a continuous basis. Conventionally, isostearyl ascorbyl phosphate and the like have been known to have the effect of promoting the production of type III collagen (see Patent Document 2).

[0011] The dermis and epidermis of the skin are composed of epidermal cells, fibroblasts, and the extracellular matrix, such as elastin and collagen, which is located outside these cells and supports the skin structure. In young skin, these skin tissues maintain homeostasis, ensuring moisture retention, flexibility, elasticity, etc., and the skin is maintained in a firm, glossy, and moist appearance.

[0012] However, due to the influence of certain external factors such as ultraviolet radiation, extremely dry air, and excessive skin washing, as well as the progression of aging, the production of elastin, collagen, and other major components of the dermal extracellular matrix decreases, leading to their degeneration and degradation. As a result, the keratin begins to peel abnormally, the skin loses its firmness and luster, and symptoms of aging such as rough skin and wrinkles appear.

[0013] Thus, changes associated with skin aging, such as wrinkle formation, loss of firmness, and reduced elasticity, are associated with the decrease and degeneration of dermal extracellular matrix components such as elastin and collagen.

[0014] Among these matrix components, elastin is a fiber that provides elasticity to skin tissue. Its production decreases with age, and its degradation and denaturation are accelerated by ultraviolet rays. A decrease in normal elastin reduces skin elasticity, which can lead to wrinkles and sagging. Therefore, if we can promote elastin production, it is thought that wrinkles and sagging will be less likely to occur, and that symptoms of skin aging such as loss of firmness and loss of elasticity can be prevented and improved.

[0015] In addition to skin tissue, elastin is widely expressed in tissues in the body that require elasticity, such as the lungs and blood vessels. It is known that a decrease in normal elastin from these tissues with aging leads to a decrease in elasticity in the lungs, blood vessels, etc., which can cause pulmonary diseases such as emphysema, hypertension, and vascular diseases such as aneurysms. Therefore, if elastin production can be promoted, it is thought that a decrease in elasticity in the lungs, blood vessels, etc. will be less likely to occur, and pulmonary diseases such as emphysema, hypertension, and vascular diseases such as aneurysms will be prevented and treated. Extracts from plants of the Brachytheciaceae family have been known to have the effect of promoting elastin production (see Patent Document 3).

[0016] In recent years, degradation and reconstruction of proteolytic enzymes called matrix metalloproteinases (hereinafter sometimes referred to as "MMPs") have been identified as factors that induce the reduction and degeneration of dermal extracellular matrix components. MMPs are broadly classified into the following five groups based on differences in their primary structure and substrate specificity (see Patent Document 4). (1) Collagenase group (MMP-1, MMP-8, MMP-13, MMP-18) (2) Gelatinase group (MMP-2, MMP-9) (3) Stromelysin group (MMP-3, MMP-10, MMP-11) (4) Membrane-bound matrix metalloproteinases (MMP-14, MMP-15, MMP-16, MMP-17) (5) Matrilysin group (MMP-7)

[0017] Among MMPs, MMP-1 is known to be an enzyme that degrades type I, type II, and type III collagen, which are major components of the extracellular matrix of the skin. Furthermore, MMP-1 expression is significantly increased by UV irradiation, contributing to UV-induced collagen loss and degeneration, and is thought to be a major factor in the formation of wrinkles and loss of skin elasticity. Furthermore, increased MMP-1 activity leads to the destruction of the extracellular matrix. Extracellular matrix destruction is known to be associated with various diseases, including cancer invasion and metastasis, rheumatoid arthritis, osteoarthritis, periodontal disease, and age-related macular degeneration.

[0018] Therefore, it is believed that by inhibiting the activity of MMP-1, it is possible to prevent or improve skin aging symptoms such as wrinkle formation and loss of elasticity, as well as to prevent, treat, or improve diseases associated with the destruction of the extracellular matrix. For example, extracts of mikkhu alba (see Patent Document 5) and albizia julibrissin (see Patent Document 6) have been known to have an inhibitory effect on MMP-1 activity.

[0019] Changes in the amount of dermal extracellular matrix components are known to be related to fibroblasts, which produce the dermal extracellular matrix, and matrix metalloproteinase (MMP-3). MMP-3 and MMP-10, which belong to the stromelysin group, are known to be enzymes that degrade proteoglycans, type IV collagen, laminin, fibronectin, and other proteins. Therefore, MMP-3 activity can cause skin aging symptoms, such as wrinkle formation. MMP-3 has also been suggested to be involved in cancer invasion and metastasis, angiogenesis in cancer cells, and other factors (see Non-Patent Document 1). Therefore, inhibiting MMP-3 activity is thought to prevent or improve skin aging symptoms, as well as inhibit cancer invasion and metastasis, angiogenesis in cancer cells, and other factors. Examples of substances known to inhibit MMP-3 activity include tormentilla extract and black tea extract (see Patent Document 7). [Prior art documents] [Patent documents]

[0020] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-79152 [Patent Document 2] Japanese Patent Publication No. 2022-86491 [Patent Document 3] Japanese Patent Application Laid-Open No. 2005-60341 [Patent Document 4] Japanese Patent Application Laid-Open No. 2000-344672 [Patent Document 5] Japanese Patent Application Laid-Open No. 2013-177439 [Patent Document 6] Japanese Patent Application Laid-Open No. 2010-235548 [Patent Document 7] Japanese Patent Application Laid-Open No. 2001-192316 [Non-patent literature]

[0021] [Non-Patent Document 1] Cancer and Metastasis Reviews,2004,Vol.23,pp.101-117 Summary of the Invention [Problem to be solved by the invention]

[0022] The present invention aims to discover highly safe compositions derived from natural products that have the effects of promoting estrogen receptor β mRNA expression, promoting type III collagen mRNA expression, promoting elastin mRNA expression, suppressing matrix metalloproteinase-1 mRNA expression, and suppressing matrix metalloproteinase-3 mRNA expression, and to provide an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor that contain these as active ingredients. [Means for solving the problem]

[0023] In order to solve these problems, the present invention provides an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor, each of which contains yuzu extract as an active ingredient. [Effects of the Invention]

[0024] According to the present invention, it is possible to provide an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor, each of which contains as an active ingredient a yuzu extract, which is a highly safe composition derived from natural products. DETAILED DESCRIPTION OF THE INVENTION

[0025] An embodiment of the present invention will be described. The estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor of this embodiment contain yuzu extract as an active ingredient.

[0026] The raw material used for extraction to obtain the active ingredient in this embodiment is yuzu (scientific name: Citrus junos). Yuzu (Citrus junos) is an evergreen tree of the genus Rutaceae that has been cultivated in China and Japan for a long time and is readily available in these regions. The parts of the yuzu tree that can be used as the raw material for extraction can be appropriately selected depending on the purpose, and examples include leaves, trunks, branches, flowers, buds, fruit, peel, pit, seeds, roots, and mixtures thereof, with the peel being preferred.

[0027] Although the details of the substances contained in the extract from the above-mentioned extraction raw material that have the effects of promoting estrogen receptor β mRNA expression, promoting type III collagen mRNA expression, promoting elastin mRNA expression, and suppressing matrix metalloproteinase-1 mRNA expression and matrix metalloproteinase-3 mRNA expression are unknown, an extract having the effects of promoting estrogen receptor β mRNA expression, promoting type III collagen mRNA expression, promoting elastin mRNA expression, suppressing matrix metalloproteinase-1 mRNA expression and matrix metalloproteinase-3 mRNA expression can be obtained from the above-mentioned extraction raw material by an extraction method commonly used for plant extraction, etc. Note that the extract in this embodiment includes an extract obtained from the extraction raw material by extraction treatment, a diluted or concentrated extract, a dried product obtained by drying the extract, or any of these roughly purified or purified products.

[0028] The extract can be obtained by drying the raw material, either directly or after crushing using a crusher, and then subjecting it to extraction with an extraction solvent. Drying can be performed in the sun or using a commonly used dryer. The raw material can also be used after pretreatment, such as degreasing, using a nonpolar solvent such as hexane. Pretreatment, such as degreasing, allows for efficient extraction of plants with a polar solvent.

[0029] The solvent used for extraction may be water, a hydrophilic organic solvent, or a mixture thereof, and is preferably used at room temperature or a temperature below the boiling point of the solvent. Components contained in the extraction material that have the effects of promoting estrogen receptor β mRNA expression, type III collagen mRNA expression, elastin mRNA expression, matrix metalloproteinase-1 mRNA expression, and matrix metalloproteinase-3 mRNA expression can be easily extracted by extraction using a polar solvent as the extraction solvent.

[0030] Examples of water that can be used as an extraction solvent include pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, and water that has undergone various treatments. Treatments that can be applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, etc. Therefore, water that can be used as an extraction solvent in the present invention also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0031] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0032] When a mixture of two or more polar solvents is used as the extraction solvent, the mixing ratio can be adjusted appropriately. For example, when using a mixture of water and a lower aliphatic alcohol, it is preferable to mix 10 parts by volume of water with 1 to 90 parts by volume of the lower aliphatic alcohol. When using a mixture of water and a lower aliphatic ketone, it is preferable to mix 10 parts by volume of water with 1 to 40 parts by volume of the lower aliphatic ketone. When using a mixture of water and a polyhydric alcohol, it is preferable to mix 10 parts by volume of water with 1 to 90 parts by volume of the polyhydric alcohol.

[0033] The extraction process does not require the use of a special extraction method as long as the soluble components contained in the extraction material can be dissolved into the extraction solvent. Extraction can be performed at room temperature or under reflux heating. For example, the extraction material is placed in a treatment tank filled with the extraction solvent, stirred as needed, and left to stand for 30 minutes to 4 hours to dissolve the soluble components, followed by filtration to remove solids, to obtain an extract. Distilling the extraction solvent from the resulting extract yields a paste-like concentrate, which can then be further dried to obtain a dried product. When water is used as the extraction solvent, the extraction conditions are 50 to 95°C for approximately 1 to 4 hours. When a mixture of water and ethanol is used as the extraction solvent, the extraction conditions are 40 to 80°C for approximately 30 minutes to 4 hours.

[0034] The extract obtained as described above may be subjected to treatments such as dilution, concentration, drying, purification, etc. according to conventional methods to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude or purified product thereof.

[0035] The obtained extract can be used as an active ingredient in an estrogen receptor β mRNA expression promoter, a type III collagen mRNA expression promoter, an elastin mRNA expression promoter, a matrix metalloproteinase-1 mRNA expression inhibitor, and a matrix metalloproteinase-3 mRNA expression inhibitor, but a concentrated solution or a dried product is preferred. When obtaining a dried product, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.

[0036] Furthermore, since the extract has a unique odor and taste, it may be purified for the purpose of decolorization, deodorization, etc., to the extent that its physiological activity is not reduced, but since it is not used in large quantities when added to cosmetics, there is no practical problem in using it unpurified. Purification can be carried out by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, etc.

[0037] The yuzu extract obtained in the manner described above has the effects of promoting estrogen receptor β mRNA expression, type III collagen mRNA expression, elastin mRNA expression, and inhibiting matrix metalloproteinase-1 mRNA expression and matrix metalloproteinase-3 mRNA expression, and therefore can be used as the active ingredient of an estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor by utilizing these effects.

[0038] The estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to this embodiment may consist of yuzu extract alone, or may be a formulation of yuzu extract.

[0039] The extract can be formulated into any dosage form, such as powder, granules, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to a conventional method. In this case, examples of auxiliary agents that can be used include excipients, stabilizers, and odor masking agents. Examples of the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor formulated from the extract include ointments and topical liquids.

[0040] The type III collagen mRNA expression promoter and elastin mRNA expression promoter according to this embodiment may contain, as an active ingredient, estrogen (17β-estradiol) or an estrogen preparation formulated from estrogen (17β-estradiol) together with yuzu extract. When the type III collagen mRNA expression promoter and elastin mRNA expression promoter each contain, as an active ingredient, estrogen or an estrogen preparation together with yuzu extract, the expression of type III collagen mRNA and elastin mRNA can be more effectively promoted. When the type III collagen mRNA expression promoter and elastin mRNA expression promoter according to this embodiment use a combination of yuzu extract and the estrogen or estrogen preparation as active ingredients, the content ratio (by mass) of the yuzu extract to the estrogen or estrogen preparation in the type III collagen mRNA expression promoter and elastin mRNA expression promoter can be appropriately determined depending on the strength of the type III collagen mRNA expression promoting effect and elastin mRNA expression promoting effect of each of the two.

[0041] The estrogen receptor β mRNA expression promoter of this embodiment can promote the expression of estrogen receptor β mRNA through the estrogen receptor β mRNA expression promoting effect of yuzu extract. This can prevent and improve skin aging symptoms such as loss of skin firmness and increased wrinkles, skin diseases such as acne caused by excessive sebum secretion, rough skin caused by a decrease in the skin's barrier function, and menopausal disorders including symptoms known as hot flashes. However, in addition to these uses, the estrogen receptor β mRNA expression promoter of this embodiment can also be used for any other use where it is meaningful to exert an estrogen receptor β mRNA expression promoting effect.

[0042] The type III collagen mRNA expression promoter according to this embodiment can promote the expression of type III collagen mRNA through the type III collagen mRNA expression-promoting activity of yuzu extract. This can prevent, treat, or improve various diseases caused by defective synthesis of type III collagen, such as skin aging symptoms due to aging and type IV clinical symptoms of Ehlers-Danlos syndrome, which is caused by an abnormality in the collagen fibril formation mechanism. However, in addition to these uses, the type III collagen mRNA expression promoter according to this embodiment can also be used for any other use where it is meaningful to exert the type III collagen mRNA expression-promoting activity.

[0043] The elastin mRNA expression promoter of this embodiment can promote elastin mRNA expression through the elastin mRNA expression-promoting activity of yuzu extract. This can prevent, treat, or improve skin aging symptoms such as wrinkles, sagging, loss of firmness, and reduced elasticity, as well as lung diseases such as emphysema, high blood pressure, and vascular diseases such as aneurysms. However, in addition to these uses, the elastin mRNA expression promoter of this embodiment can also be used for any other use where it is meaningful to exert an elastin mRNA expression-promoting activity.

[0044] The matrix metalloproteinase-1 mRNA expression inhibitor according to the present embodiment can inhibit matrix metalloproteinase-1 mRNA expression through the matrix metalloproteinase-1 mRNA expression inhibitory activity of the yuzu extract. This inhibits collagen degradation, preventing or ameliorating changes associated with skin aging, such as wrinkles, dullness, loss of texture, and loss of elasticity, and also preventing, treating, or ameliorating diseases associated with the breakdown of the extracellular matrix (e.g., cancer invasion and metastasis, rheumatoid arthritis, osteoarthritis, periodontal disease, age-related macular degeneration, etc.). However, the matrix metalloproteinase-1 mRNA expression inhibitor according to the present embodiment can be used in all applications where the inhibitory activity against matrix metalloproteinase-1 mRNA expression is significant.

[0045] The matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiment can inhibit matrix metalloproteinase-3 mRNA expression through the matrix metalloproteinase-3 mRNA expression inhibitory effect of the yuzu extract. This can prevent or improve changes associated with skin aging, such as wrinkles, dullness, loss of texture, and loss of elasticity, as well as inhibit cancer invasion and metastasis, angiogenesis in cancer cells, etc. However, in addition to these uses, the matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiment can also be used for any other uses where the inhibitory effect on matrix metalloproteinase-3 mRNA expression is significant.

[0046] The estrogen receptor β mRNA expression enhancer, type III collagen mRNA expression enhancer, elastin mRNA expression enhancer, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiments can be administered to a patient by subcutaneous administration, intramuscular administration, intravenous administration, oral administration, transdermal administration, etc., and a method suitable for the prevention, treatment, etc. of the disease can be appropriately selected depending on the type of disease. Furthermore, the dosage of the estrogen receptor β mRNA expression enhancer, type III collagen mRNA expression enhancer, elastin mRNA expression enhancer, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiments can be increased or decreased as appropriate depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0047] Furthermore, the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiment have excellent estrogen receptor β mRNA expression promoting effects, type III collagen mRNA expression promoting effects, elastin mRNA expression promoting effects, matrix metalloproteinase-1 mRNA expression inhibiting effects, and matrix metalloproteinase-3 mRNA expression inhibiting effects, and are therefore suitable for incorporation into cosmetics such as skin cosmetics, scalp cosmetics, and hair cosmetics, as well as foods and beverages.

[0048] Cosmetics that can contain the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor include, for example, ointments, creams, emulsions, lotions, packs, foundations, hair tonics, hair lotions, shampoos, rinses, soaps, etc. When the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor are contained in cosmetics, the amount of each of the promoters can be adjusted appropriately depending on the type of cosmetic. A preferred blending ratio is about 0.0001 to 10% by mass, calculated as the standard extract, and a particularly preferred blending ratio is about 0.001 to 1% by mass, calculated as the standard extract. The cosmetic composition may contain main ingredients, auxiliary ingredients, or other ingredients typically used in the manufacture of cosmetics, such as astringents, disinfectants / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / antiallergic agents, antioxidants / active oxygen scavengers, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, and fragrances, as long as they do not interfere with the extract's effects of promoting estrogen receptor β mRNA expression, promoting type III collagen mRNA expression, promoting elastin mRNA expression, and inhibiting matrix metalloproteinase-1 or matrix metalloproteinase-3 mRNA expression. Such combinations can result in more versatile products, and the synergistic effects of the combined active ingredients can sometimes produce superior effects beyond those normally expected.

[0049] Foods and beverages refer to those that pose little risk to human health and are ingested orally or by gastrointestinal administration in normal social life, and are not limited to administrative classifications such as foods, drugs, or quasi-drugs. Therefore, the "foods and beverages" of this embodiment broadly include compositions that constitute orally ingested general foods, health foods (functional foods and beverages), health functional foods (foods for specified health uses, foods with nutrient claims, foods with functional claims), quasi-drugs, drugs, etc. The foods and beverages of this embodiment may be those that, or their packaging, display the effects of the extracts promoting estrogen receptor β mRNA expression, promoting type III collagen mRNA expression, promoting elastin mRNA expression, inhibiting matrix metalloproteinase-1 mRNA expression, or inhibiting matrix metalloproteinase-3 mRNA expression, or may be health functional foods (foods for specified health uses, foods with functional claims, foods with nutrient claims), quasi-drugs, or drugs.

[0050] When the above extracts or the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor formulated from the above extracts are incorporated into foods and beverages, the amount of active ingredient can be appropriately adjusted taking into account the intended use, symptoms, gender, etc., but it is preferable to adjust the extract intake to approximately 1 to 1,000 mg per adult per day, taking into account the general intake of the food and beverage to be added. When the food and beverage to be added is in the form of granules, tablets, or capsules, the amount of the above extracts or the estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor formulated from the above extracts is typically 0.1 to 100% by mass, preferably 5 to 100% by mass, of the food and beverage to be added.

[0051] The estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to the present embodiment have excellent estrogen receptor β mRNA expression promoting effects, type III collagen mRNA expression promoting effects, elastin mRNA expression promoting effects, matrix metalloproteinase-1 mRNA expression inhibiting effects, and matrix metalloproteinase-3 mRNA expression inhibiting effects, and can therefore be suitably used as reagents for research into the mechanisms of these actions.

[0052] The estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor according to this embodiment are preferably applied to humans, but can also be applied to animals other than humans as long as their respective functional effects are achieved. [Example]

[0053] The present invention will be explained in more detail below by showing production examples, test examples, etc., but the present invention is not limited to the following production examples, test examples, etc.

[0054] [Production Example 1] Production of yuzu extract 500 mL of 50% by volume ethanol solution was added to 50 g of yuzu peel, and reflux extraction was carried out at 80-90°C for 2 hours using a reflux extractor, followed by hot filtration. The resulting extract was dried to obtain yuzu extract (18.65 g).

[0055] [Test Example 1] Test of estrogen receptor β mRNA expression promoting effect in epidermal keratinocytes The yuzu extract obtained in the above production example was used as a test sample and tested for its promoting effect on estrogen receptor β (ERβ) mRNA expression by the following method.

[0056] Human epidermal keratinocyte cell line (HaCaT) was cultured in a 24-well culture plate at 2 × 10 5 Cells / mL were seeded and cultured for 24 hours. After culture, a test sample, yuzu extract (Production Example 1), was added to each well to final concentrations of 10 μg / mL, 100 μg / mL, and 1000 μg / mL, and cultured for 6 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0057] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse-transcribed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers specific to the target molecule to measure the expression levels of ERβ mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of ERβ mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0058] From the corrected values ​​obtained, the ERβ mRNA expression promotion rate (%) was calculated according to the following formula. ERβ mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when the sample is added" and B represents the "corrected value when no sample is added." The results are shown in Table 1.

[0059] [Test Example 2] Test of estrogen receptor β mRNA expression promoting effect in skin fibroblasts The yuzu extract obtained in the above production example was used as a test sample and tested for its promoting effect on estrogen receptor β (ERβ) mRNA expression by the following method.

[0060] Human normal skin fibroblast cell line (TIG-113) was cultured in a 24-well culture plate at 2 × 10 5 Cells / mL were seeded and cultured for 24 hours. After culture, a test sample, yuzu extract (Production Example 1), was added to each well to final concentrations of 10 μg / mL, 100 μg / mL, and 1000 μg / mL, and cultured for 6 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0061] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse-transcribed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers specific to the target molecule to measure the expression levels of ERβ mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of ERβ mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0062] From the corrected values ​​obtained, the ERβ mRNA expression promotion rate (%) was calculated according to the following formula. ERβ mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when the sample is added" and B represents the "corrected value when no sample is added." The results are shown in Table 1.

[0063] [Test Example 3] Type III collagen mRNA expression promoting effect test The yuzu extract obtained in the above Production Example was used as a test sample and tested for its type III collagen (COL3A1) mRNA expression promoting effect by the following method.

[0064] Human epidermal keratinocyte cell line (HaCaT) was cultured in a 24-well culture plate at 2 × 10 5 Cells / mL were seeded and cultured for 24 hours. After culture, a test sample, yuzu extract (Production Example 1), was added to each well to a final concentration of 100 μg / mL, and cultured for 6 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0065] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse-transcribed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers specific to the target molecule to measure the expression levels of COL3A1 mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of COL3A1 mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0066] From the corrected values ​​obtained, the COL3A1 mRNA expression promotion rate (%) was calculated using the following formula. COL3A1 mRNA expression promotion rate (%) = A / B x 100 In the formula, A represents the "corrected value when the sample is added" and B represents the "corrected value when no sample is added." The results are shown in Table 1.

[0067] [Test Example 4] Elastin mRNA expression promoting effect test The yuzu extract obtained in the above production example was used as a test sample, and the elastin (ELN) mRNA expression promoting effect was tested by the following method.

[0068] Human epidermal keratinocyte cell line (HaCaT) was cultured in a 24-well culture plate at 2 × 10 5Cells / mL were seeded and cultured for 24 hours. After culture, a test sample, yuzu extract (Production Example 1), was added to each well to a final concentration of 100 μg / mL or 1000 μg / mL, and cultured for 6 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0069] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse transcription was performed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers for the target molecule to measure the expression levels of ELN mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of ELN mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0070] From the corrected values ​​obtained, the ELN mRNA expression promotion rate (%) was calculated using the following formula. ELN mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when the sample is added" and B represents the "corrected value when no sample is added." The results are shown in Table 1.

[0071] [Table 1]

[0072] As shown in Table 1, it was confirmed that yuzu extract has excellent effects of promoting the expression of estrogen receptor β (ERβ) mRNA, type III collagen (COL3A1) mRNA, and elastin (ELN) mRNA. In particular, it was confirmed that yuzu extract has excellent effects of promoting the expression of estrogen receptor β (ERβ) mRNA in both epidermal keratinocytes (HaCaT) and skin fibroblasts (TIG-113).

[0073] [Test Example 5] Matrix metalloproteinase-1 mRNA expression inhibitory effect test The yuzu extract obtained in the above Production Example was used as a test sample and tested for its inhibitory effect on matrix metalloproteinase-1 (MMP-1) mRNA expression by the following method.

[0074] Human normal skin fibroblast cell line (TIG-110) was cultured in a 24-well culture plate at 2 × 10 5 After culturing, yuzu extract (Production Example 1) was added to each well as a test sample to a final concentration of 1 μg / mL, 10 μg / mL, or 100 μg / mL, and the cells were cultured for 24 hours. After culturing, the culture medium was replaced with HBSS(+) and the cells were cultured at 7.5 J / cm 2 After UV irradiation, the HBSS(+) was immediately removed, and 1 mL of 10% FBS-containing DMEM was added to each well and cultured for 24 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0075] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse-transcribed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers specific to the target molecule to measure the expression levels of MMP-1 mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of MMP-1 mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0076] From the corrected values ​​obtained, the MMP-1 mRNA expression inhibition rate (%) was calculated according to the following formula. MMP-1 mRNA expression suppression rate (%)={1-(AC) / (BC)}×100 In the formula, A represents the "corrected value when sample is added and UV light is irradiated," B represents the "corrected value when no sample is added and UV light is irradiated," and C represents the "corrected value when no sample is added and UV light is not irradiated." The results are shown in Table 2.

[0077] [Test Example 6] Matrix metalloproteinase-3 mRNA expression inhibitory effect test The yuzu extract obtained in the above Production Example was used as a test sample and tested for its inhibitory effect on matrix metalloproteinase-3 (MMP-3) mRNA expression by the following method.

[0078] Human normal skin fibroblast cell line (TIG-110) was cultured in a 24-well culture plate at 2 × 10 5 After culturing, a test sample, yuzu extract (Production Example 1), was added to each well to a final concentration of 10 μg / mL or 100 μg / mL, and the cells were cultured for 24 hours. After culturing, the culture medium was replaced with HBSS(+) and 7.5 J / cm2 After UV irradiation, the HBSS(+) was immediately removed, and 1 mL of 10% FBS-containing DMEM was added to each well and cultured for 24 hours. As a control, the same amount of 50 wt% ethanol was added and cultured in the same manner.

[0079] After culturing, 1 μg of total RNA was extracted from the cells using TRI Reagent (registered trademark, Sigma-Aldrich). 5× First Strand Buffer, 0.1 M DTT, 10 mM dNTP Mix, 10 pmol / μL Oligo(dt)18, and M-MLV Reverse Transcriptase were added and reverse-transcribed for 50 minutes at 42°C using a PTC-200 thermal cycler. The resulting cDNA was subjected to gene expression analysis by quantitative PCR using primers specific to the target molecule to measure the expression levels of MMP-3 mRNA and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression level of MMP-3 mRNA was corrected for the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and the corrected value was calculated.

[0080] From the corrected values ​​obtained, the MMP-3 mRNA expression inhibition rate (%) was calculated according to the following formula. MMP-3 mRNA expression suppression rate (%)={1-(AC) / (BC)}×100 In the formula, A represents the "corrected value when sample is added and UV light is irradiated," B represents the "corrected value when no sample is added and UV light is irradiated," and C represents the "corrected value when no sample is added and UV light is not irradiated." The results are shown in Table 2.

[0081] [Table 2]

[0082] As shown in Table 2, it was confirmed that yuzu extract has excellent inhibitory effects on matrix metalloproteinase-1 (MMP-1) mRNA expression and matrix metalloproteinase-3 (MMP-3) mRNA expression. [Industrial Applicability]

[0083] The estrogen receptor β mRNA expression promoter, type III collagen mRNA expression promoter, elastin mRNA expression promoter, matrix metalloproteinase-1 mRNA expression inhibitor, and matrix metalloproteinase-3 mRNA expression inhibitor of the present invention can significantly contribute to the prevention or improvement of skin aging symptoms, etc.

Claims

1. An estrogen receptor β mRNA expression promoter comprising a yuzu extract as an active ingredient.

2. A type III collagen mRNA expression promoter characterized by containing yuzu extract as an active ingredient.

3. An elastin mRNA expression promoter characterized by containing yuzu extract as an active ingredient.

4. A matrix metalloproteinase-1 mRNA expression inhibitor characterized by containing a yuzu extract as an active ingredient.

5. A matrix metalloproteinase-3 mRNA expression inhibitor characterized by containing a yuzu extract as an active ingredient.

Images