JP7642607B2 - Composition for inhibiting or improving decline in skin barrier function, composition for inhibiting or improving decline in expression of type 4 collagen, and method for screening for substance having effect of inhibiting or improving decline in skin barrier function - Google Patents

Description

The present invention relates to a composition for inhibiting or improving the decline in skin barrier function and a composition for inhibiting or improving the decline in expression of type 4 collagen. The present invention also relates to a method for inhibiting the decline in skin barrier function or improving skin barrier function and a method for inhibiting the decline in expression of type 4 collagen or improving said expression. The present invention also relates to a screening method for a substance that has the effect of inhibiting or improving the decline in skin barrier function, etc.

The skin is broadly composed of two layers, the epidermis and the dermis, from the surface. The epidermis is composed of four layers, from the deepest layer to the most deep layer: the basal layer, the spinous layer, the granular layer, and the stratum corneum. The basal layer is attached to the dermis via a basement membrane. The basement membrane is composed of components such as type 4 collagen and type 7 collagen, and serves to bind the basal layer of the epidermis to the dermis, but its function decreases with age.

The skin has a barrier function that prevents foreign substances from entering the body from the outside and maintains moisture in the skin by preventing excessive evaporation of water from the skin. When the skin barrier function is weakened due to aging or other reasons, it can cause dry skin. Patent Document 1 describes a skin barrier function improver that contains an extract of Aspalathus linearis.

JP 2009-256244 A

The present invention aims to provide a composition for inhibiting or improving a decline in skin barrier function, which can inhibit the decline in skin barrier function or improve said function. The present invention also aims to provide a composition for inhibiting or improving a decline in the expression of type 4 collagen. The present invention also aims to provide a method for inhibiting a decline in skin barrier function or improving said function, and a method for inhibiting a decline in the expression of type 4 collagen or improving said expression. Furthermore, the present invention aims to provide a method for screening a substance that has the effect of inhibiting or improving the decline in skin barrier function.

As a result of research conducted to solve the above problems, the inventors found that by suppressing the decrease in expression of type 4 collagen in skin cells or improving said expression, the decrease in skin barrier function can be suppressed or said function can be improved. Based on this finding, the inventors conducted further research and completed the present invention.

The present invention includes, but is not limited to, the following compositions for inhibiting or improving a decline in skin barrier function, compositions for inhibiting or improving a decline in the expression of type 4 collagen, and the like.
[1] A composition for inhibiting or improving a decline in skin barrier function through inhibiting or improving the decline in type IV collagen expression, comprising as an active ingredient one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia, extracts of plants of the genus Camellia of the family Theaceae, extracts of plants of the genus Spiraea of the family Rosaceae, and extracts of plants of the genus Mentha of the family Lamiaceae.
[2] The composition for inhibiting or improving a decline in skin barrier function according to the above-mentioned [1], wherein the plant of the genus Rosaceae is rose, the yeast of the genus Yarrowia is Yarrowia lipolytica, the plant of the genus Theaceae is tea plant, the plant of the genus Spiraea of the Rosaceae is Meadowsweet, and the plant of the genus Mentha of the Lamiaceae is Mentha piperita.
[3] A composition for inhibiting or improving a decline in skin barrier function according to [1] or [2] above, wherein the plant of the genus Rosaceae is rose petals containing one or more compounds selected from the group consisting of delphinidin-type anthocyanins, rosacyanin compounds, and rosadelphin compounds, the plant of the genus Camellia of the family Theaceae is tea leaves, the plant of the genus Spiraea of the family Rosaceae is meadowsweet flowers, and the plant of the genus Mentha of the family Lamiaceae is the whole plant of Mentha piperita.
[4] A composition for inhibiting or improving a decline in skin barrier function, comprising an extract of a plant of the genus Hamamelid in the family Hamamelidaceae and/or an extract of a plant of the genus Plantago in the family Plantaginaceae as an active ingredient.
[5] A composition for inhibiting or improving a decline in skin barrier function according to the above [4], wherein the Hamamelidaceae family Hamamelidaceae plant is Hamamelis, and the Plantago major family plant is Plantago major.
[6] A composition for inhibiting or improving a decline in skin barrier function according to the above [4] or [5], wherein the Hamamelidaceae family Hamamelidaceae plant is a Hamamelis leaf, and the Plantago major family Plantago seed is Plantago major.
[7] A composition for inhibiting or improving a decline in skin barrier function according to any one of [4] to [6] above, which inhibits or improves the decline in skin barrier function through the inhibition or improvement of the decline in basement membrane function.
[8] The composition for inhibiting or improving a decline in skin barrier function described in [7] above, wherein the inhibition or improvement of decline in basement membrane function is mediated by inhibition or improvement of decline in expression of type 4 collagen.
[9] A composition for inhibiting or improving a decline in skin barrier function, comprising an agent for inhibiting or improving a decline in basement membrane function as an active ingredient.
[10] The composition for inhibiting or improving the decline of skin barrier function described in [9] above, wherein the agent for inhibiting or improving the decline of basement membrane function inhibits the decline of basement membrane function or improves the basement membrane function by inhibiting or improving the decline of expression of type 4 collagen.
[11] A composition for inhibiting or improving the decrease in expression of type 4 collagen, comprising as an active ingredient one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia, extracts of plants of the genus Camellia of the family Theaceae, extracts of plants of the genus Hamamelidaceae, extracts of plants of the genus Spiraea of the family Rosaceae, extracts of plants of the genus Plantago major of the family Plantaginaceae, and extracts of plants of the genus Mentha of the family Lamiaceae.
[12] A composition for inhibiting or improving the decrease in expression of type 4 collagen according to [11] above, wherein the plant of the genus Rosaceae is rose, the yeast of the genus Yarrowia is Yarrowia lipolytica, the plant of the genus Theaceae is tea plant, the plant of the genus Hamamelidaceae is Hamamelis, the plant of the genus Spiraea of the Rosaceae is Meadowsweet, the plant of the genus Plantago major of the Plantaginaceae family is Plantago major, and the plant of the genus Mentha of the Lamiaceae family is Mentha piperita.
[13] A composition for inhibiting or improving the decrease in expression of type 4 collagen according to [11] or [12] above, wherein the plant of the genus Rosaceae is rose petals containing one or more compounds selected from the group consisting of delphinidin-type anthocyanins, rosacyanin compounds, and rosadelphin compounds, the plant of the genus Camellia of the family Theaceae is tea leaves, the plant of the genus Hamamelidaceae is witch hazel leaves, the plant of the genus Spiraea of the family Rosaceae is meadowsweet flowers, the plant of the genus Plantago major of the family Plantaginaceae is plantago major seeds, and the plant of the genus Mentha of the family Lamiaceae is peppermint whole plant.
[14] The composition according to any one of [1] to [13] above, which is an external preparation for the skin.
[15] The composition according to any one of [1] to [14] above, which is a cosmetic preparation.
[16] Use of one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia, extracts of plants of the genus Camellia, extracts of plants of the genus Spiraea, and extracts of plants of the genus Mentha in the family Lamiaceae, for inhibiting or improving the decrease in expression of type 4 collagen, thereby inhibiting or improving the decrease in skin barrier function or improving skin barrier function.
[17] Use of an extract of a plant of the genus Hamamelid in the family Hamamelidaceae and/or an extract of a plant of the genus Plantago in the family Plantaginaceae for suppressing a decline in skin barrier function or improving skin barrier function.
[18] Use of one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae (Rosaceae), extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia (Yarrowia), extracts of plants of the genus Camellia (Theaceae), extracts of plants of the genus Hamamelidaceae (Witch Hazel), extracts of plants of the genus Spiraea (Rosaceae), extracts of plants of the genus Plantago (Plantaginaceae), and extracts of plants of the genus Mentha (Lamiaceae), for suppressing a decrease in the expression of type 4 collagen or improving the expression.
[19] A method for inhibiting a decline in skin barrier function or improving skin barrier function by administering one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skin, extracts of yeast of the genus Yarrowia, extracts of plants of the genus Camellia of the family Theaceae, extracts of plants of the genus Spiraea of the family Rosaceae, and extracts of plants of the genus Mentha of the family Lamiaceae.
[20] A method for suppressing a decline in skin barrier function or improving skin barrier function, comprising administering an extract of a plant of the genus Hamamelid in the family Hamamelidaceae and/or an extract of a plant of the genus Plantago in the family Plantaginaceae.
[21] A method for suppressing a decrease in the expression of type 4 collagen or improving the expression, comprising administering one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia, extracts of plants of the genus Camellia of the family Theaceae, extracts of plants of the genus Hamamelidaceae, extracts of plants of the genus Spiraea of the family Rosaceae, extracts of plants of the genus Plantago major of the family Plantaginaceae, and extracts of plants of the genus Mentha of the family Lamiaceae.
[22] A method for screening for a substance having an effect of inhibiting or improving a decline in skin barrier function, comprising the steps of culturing human epidermal keratinocytes in vitro in a medium containing a test substance or a control medium not containing the test substance, and comparing the expression levels of type 4 collagen in the skin cells cultured in the medium containing the test substance and the control skin cells cultured in the control medium, wherein if the expression level of type 4 collagen in the skin cells cultured in the medium containing the test substance is greater than the expression level of type 4 collagen in the control skin cells, the test substance is selected as a substance having an effect of inhibiting or improving a decline in skin barrier function.

The present invention can provide a composition for inhibiting or improving a decline in skin barrier function, which can inhibit the decline in skin barrier function or improve said function. Furthermore, the present invention can provide a composition for inhibiting or improving the decline in expression of type 4 collagen. Furthermore, the present invention can provide a method for inhibiting a decline in skin barrier function or improving said function, and a method for inhibiting a decline in expression of type 4 collagen or improving said expression. The present invention can provide a method for screening for a substance that has the effect of inhibiting or improving the decline in skin barrier function.

A first aspect of the present invention is a composition for inhibiting or improving the decrease in expression of type 4 collagen, which comprises as an active ingredient one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae (Rosaceae), extracts of grape seeds and/or skins, extracts of yeast of the genus Yarrowia, extracts of plants of the genus Camellia (Theaceae), extracts of plants of the genus Hamamelidaceae (Witch hazel), extracts of plants of the genus Spiraea (Rosaceae), extracts of plants of the genus Plantago (Plantaginaceae), and extracts of plants of the genus Mentha (Lamiaceae). The composition for inhibiting or improving the decrease in expression of type 4 collagen according to the first aspect of the present invention is also referred to as the composition according to the first aspect of the present invention.
The above extract has an effect of suppressing the decrease in expression of type 4 collagen or improving said expression. Suppressing the decrease in expression of type 4 collagen includes suppressing the progress of the decrease in expression of type 4 collagen, maintaining the expression of type 4 collagen, reducing (alleviating) the degree of the decrease in expression of type 4 collagen, etc. Improving the expression of type 4 collagen includes restoring the expression of type 4 collagen, etc. Restoration includes at least partial restoration. Expression of type 4 collagen includes expression of a gene encoding type 4 collagen and expression at the protein level of type 4 collagen, and may be either or both of these. Type 4 collagen is preferably human type 4 collagen, more preferably human type 4 collagen α1 chain. Human type 4 collagen α1 chain is a protein encoded by gene type 4 collagen A1 (COL4A1). In one aspect, the composition for suppressing or improving the decrease in expression of type 4 collagen of the present invention can be used to suppress the decrease in expression of human type 4 collagen α1 chain or improve said expression, thereby suppressing the decrease in expression of human type 4 collagen α1 chain or improving said expression.

The expression of type 4 collagen decreases (the amount of expression decreases) due to external factors such as ultraviolet rays and internal factors such as aging.
As shown in the examples below, when the decrease in the expression of type 4 collagen in epidermal keratinocytes was suppressed or improved, the decrease in the transepithelial electrical resistance (TEER) of the skin was suppressed or improved. This indicates that the decrease in the skin barrier function was suppressed or the skin barrier function was improved. Although the detailed mechanism by which the decrease in the expression of type 4 collagen was suppressed or improved by suppressing or improving the decrease in the expression of type 4 collagen is not clear, it is presumed that the decrease in the function of the basement membrane was suppressed or improved by suppressing or improving the decrease in the expression of type 4 collagen, and the decrease in the skin barrier function was suppressed or improved as a result of maintaining the homeostasis of the skin. Therefore, it is expected that the decrease in the skin barrier function can be suppressed or improved by suppressing or improving the decrease in the basement membrane function. Since the above-mentioned plant or yeast extract has an excellent effect of suppressing or improving the decrease in the expression of type 4 collagen, it can be used to suppress the decrease in the basement membrane function or improve the function.
From the viewpoint of the effect of inhibiting the decrease or improving the expression of type 4 collagen, in the composition of the first aspect of the present invention, the above-mentioned extract is preferably one or more selected from the group consisting of extracts of plants of the genus Rosaceae in the Rosaceae family, extracts of grape seeds and/or skin, and extracts of plants of the genus Spiraea in the Rosaceae family.

The skin barrier function refers to the barrier function of the skin that prevents the intrusion of foreign substances into the living body from the outside and maintains moisture in the skin by preventing excessive evaporation of water inside the body from the skin. Inhibiting the decline of the skin barrier function includes inhibiting the progression of the decline of the skin barrier function, maintaining the skin barrier function, reducing (alleviating) the degree of the decline of the skin barrier function, etc. Improving the skin barrier function includes restoring the skin barrier function, etc. The decline of the skin barrier function may be a decline of the skin barrier function due to aging, a decline of the skin barrier function due to exposure to ultraviolet rays, etc.

As described above, by suppressing or improving the decline in basement membrane function, it is possible to suppress the decline in skin barrier function or improve said function.
A second aspect of the present invention is a composition for inhibiting or improving a decline in skin barrier function, comprising an agent for inhibiting or improving the decline in basement membrane function as an active ingredient. The composition for inhibiting or improving a decline in skin barrier function according to the second aspect of the present invention is hereinafter also referred to as the composition according to the second aspect of the present invention.

Known functions of the basement membrane include mechanical support connecting the epidermis and dermis, and selective filtering for material exchange between the epidermis and dermis. Inhibiting the decrease in expression of basement membrane components such as type 4 collagen or improving said expression is effective in inhibiting the decrease in or improving the function of the basement membrane.
The agent for inhibiting or improving the decline of basement membrane function is not particularly limited as long as it has an effect of inhibiting or improving the decline of basement membrane function, for example, an effect of inhibiting or improving the decline of expression of a component of the basement membrane. The agent for inhibiting or improving the decline of basement membrane function is preferably an agent for inhibiting or improving the decline of type IV collagen through the decline or improvement of the expression of type IV collagen. For example, the above-mentioned plant or yeast extract having an effect of inhibiting or improving the decline of type IV collagen expression can be used as the agent for inhibiting or improving the decline of basement membrane function. In one aspect, the agent for inhibiting or improving the decline of basement membrane function can be one or more extracts selected from the group consisting of an extract of a plant of the genus Rosaceae (Rosaceae), an extract of grape seeds and/or skin, an extract of yeast of the genus Yarrowia (Yarrowia), an extract of a plant of the genus Camellia (Yarrowia), an extract of a plant of the genus Hamamelidaceae (Witch hazel), an extract of a plant of the genus Spiraea (Rosaceae), an extract of a plant of the genus Plantago major (Plantaginaceae), and an extract of a plant of the genus Mentha (Mintha) (Lamiaceae).
Among the above extracts, one or more selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skin, and extracts of plants of the genus Spiraea, are preferred as agents for inhibiting or improving the decline in basement membrane function.

A third aspect of the present invention is a composition for inhibiting or improving a decline in skin barrier function through inhibiting or improving the decline in type IV collagen, comprising as an active ingredient one or more extracts selected from the group consisting of an extract of a plant of the genus Rosaceae (Rosaceae), an extract of grape seeds and/or skins, an extract of a yeast of the genus Yarrowia (Yarrowia), an extract of a plant of the genus Camellia (Theaceae), an extract of a plant of the genus Spiraea (Rosaceae), and an extract of a plant of the genus Mentha (Lamiaceae). The composition for inhibiting or improving a decline in skin barrier function according to the third aspect of the present invention is hereinafter also referred to as the composition according to the third aspect of the present invention.
In the composition according to the third aspect of the present invention, the extract is preferably one or more selected from the group consisting of an extract of grape seeds and/or skin, an extract of a yeast of the genus Yarrowia, an extract of a plant of the genus Camellia in the family Theaceae, and an extract of a plant of the genus Spiraea in the family Rosaceae.

A fourth aspect of the present invention is a composition for inhibiting or improving a decline in skin barrier function, which comprises an extract of a Hamamelidaceae plant of the genus Hamamelid and/or an extract of a Plantago genus plant of the family Plantaginaceae as an active ingredient. The composition for inhibiting or improving a decline in skin barrier function according to the fourth aspect of the present invention is hereinafter also referred to as the composition according to the fourth aspect of the present invention. The composition according to the fourth aspect of the present invention preferably comprises an extract of a Hamamelidaceae plant of the genus Hamamelid as an active ingredient.

The above extracts used in the composition of the third aspect and the composition of the fourth aspect of the present invention have an effect of suppressing or improving the decline in type 4 collagen expression. This makes it possible to suppress or improve the decline in basement membrane function. In one aspect, the composition for suppressing or improving the decline in skin barrier function of the third aspect and the fourth aspect of the present invention can be used to suppress the decline in skin barrier function or improve the skin barrier function through the suppression or improvement of the decline in basement membrane function. In addition, the composition for suppressing or improving the decline in skin barrier function of the fourth aspect of the present invention can be used to suppress the decline in skin barrier function or improve the skin barrier function through the suppression or improvement of the decline in basement membrane function through the suppression or improvement of the decline in type 4 collagen expression. In one aspect, the composition for suppressing or improving the decline in skin barrier function of the fourth aspect of the present invention can be used to suppress or improve the decline in skin barrier function by suppressing or improving the decline in type 4 collagen expression.

Hereinafter, the composition for inhibiting or improving the decline in type 4 collagen expression according to the first aspect of the present invention, and the compositions for inhibiting or improving the decline in skin barrier function according to the second, third, and fourth aspects of the present invention will be collectively referred to as the composition of the present invention. In the composition of the present invention, two or more of the above extracts may be used in combination as active ingredients.

In the present invention, the plant of the genus Rosaceae (Rosa) of the family Rosaceae is a rose of the genus Rosaceae, represented by Rosa hybrida (scientific name).
As the plant of the genus Rosaceae, a rose containing one or more compounds selected from the group consisting of delphinidin-type anthocyanins, rosacyanin compounds, and rosadelphin compounds is preferred, and a rose containing one or more compounds selected from the group consisting of delphinidin-type anthocyanins, rosacyanin compounds, and rosadelphin compounds in its petals is more preferred. Among these, a rose containing delphinidin-type anthocyanins in its petals is preferred.

Delphinidin-type anthocyanins are pigments that are found in large quantities in purple or blue petals. Examples of delphinidin-type anthocyanins include delphinidin, malvidin, petunidin, and compounds obtained by modifying these with sugars, acyl groups, etc. Examples of roses that contain delphinidin-type anthocyanins include roses that are blue or purple in color, such as rose varieties such as Suntory Blue Rose Applause (registered trademark). The presence of delphinidin-type anthocyanins in roses can be confirmed by HPLC, for example, using the method described in Patent Publication No. 5697040.

The rosacyanin compound is one or more compounds selected from the group consisting of rosacyanin A1, rosacyanin A2, and rosacyanin B. The rose containing a rosacyanin compound may be any rose containing one or more of the above compounds, but is preferably a rose containing rosa rosacyanin A1, rosadelphin A2, and rosadelphin B. Rosacyanin A1 is a compound represented by the following general formula (I) in which R ¹ is a hydrogen atom, rosacyanin A2 is a compound represented by the following general formula (II) in which R ² is a hydrogen atom, and rosacyanin B is a compound represented by the following general formula (III) in which R ³ is a hydrogen atom.

(In the above formula, the coordination of the hydroxyl group at the 1-position of glucose (wavy line) indicates tautomerism between α-form and β-form. ^R1 represents a hydrogen atom or a hydroxyl group.)

(In the above formula, ^R2 represents a hydrogen atom or a hydroxyl group.)

(In the above formula, ^R3 represents a hydrogen atom or a hydroxyl group.)

Roses containing rosacyanin compounds include roses that exhibit a blue or purple color tone, such as Madame Biole, Purple Rain, Lavande, Manhattan Blue, Chantilly Lace, Blue Moon, Tassogale, Charles de Gaulle, Violet Dolly, Blue Ribbon, Aozora, Lady X, Blue Bayou, and Sterling Silver. These roses are preferred because they contain rosacyanin compounds in their petals.

The rosadelphin compound is one or more compounds selected from the group consisting of rosadelphin A1, rosadelphin A2, and rosadelphin B. The rose containing the rosadelphin compound may be any rose containing one or more of the above compounds, but is preferably a rose containing rosadelphin A1, rosadelphin A2, and rosadelphin B. Rosadelphin A1 is a compound represented by the above general formula (I) in which R ¹ is a hydroxyl group, rosadelphin A2 is a compound represented by the above general formula (II) in which R ² is a hydroxyl group, and rosadelphin B is a compound represented by the above general formula (III) in which R ³ is a hydroxyl group.

The above-mentioned rosadelphin compound is not present in wild roses, but is produced in roses into which a gene encoding flavonoid 3',5'-hydroxylase (hereinafter also referred to as the flavonoid 3',5'-hydroxylase gene) has been introduced by techniques such as genetic recombination, or in roses that have the flavonoid 3',5'-hydroxylase gene obtained from a genetically modified rose as a parent. The blue pigment delphinidin is synthesized, and this compound is then combined with gallates and tellimagrandins to produce the compound.

As roses containing the above-mentioned rosadelphin compound, roses into which a flavonoid 3',5'-hydroxylase gene has been introduced by a technique such as genetic recombination, and roses having a flavonoid 3',5'-hydroxylase gene obtained from such a genetically modified rose as a parent can be used. As such roses, for example, rose varieties such as Suntory Blue Rose Applause (registered trademark) can be used. It is known that the petals of Suntory Blue Rose Applause (registered trademark) contain rosacyanin A1, rosacyanin A2, and rosacyanin B, as well as rosadelphin A1, rosadelphin A2, and rosadelphin B (for example, WO2010/110382). Suntory Blue Rose Applause (registered trademark) can be obtained from Suntory Flowers Ltd. (Tokyo, Japan). Roses having a flavonoid 3',5'-hydroxylase gene can also be produced by the method described in WO2010/110382, etc. Examples of the flavonoid 3',5'-hydroxylase gene include the plant-derived genes described in WO2004/020637, and for example, the flavonoid 3',5'-hydroxylase gene derived from a pansy of the genus Viola (such as black pansy (Viola spp. cv Black Pansy)) is preferred. Roses having a flavonoid 3',5'-hydroxylase gene are preferred because they contain rosadelphin compounds in their petals. All academic and patent documents described in this specification are incorporated herein by reference.

The presence of rosacyanin compounds and/or rosadelphin compounds in roses can be confirmed by HPLC-TOF-MS, for example, using the methods described in WO2010/110382, WO2015/178385, etc.

The grape seeds and skins are the seeds and skins of grapes (scientific name: Vitis spp.) of the Vitaceae Vitis plant. The grape varieties are not particularly limited, but examples include black grapes such as Cabernet Sauvignon, Concord, Merlot, and Muscat Bailey A; and white grapes such as Chardonnay and Koshu. Among these, Chardonnay or black grapes are preferred, black grapes are more preferred, and Cabernet Sauvignon, Merlot, and the like are even more preferred. The grapes in the grape seed and/or skin extract may be one type or two or more types. The grape seed and/or skin extract is preferably an extract of grape seeds and skin.

As the yeast of the genus Yarrowia, Yarrowia lipolytica is preferred. The extract of the yeast of the genus Yarrowia is preferably an extract of Yarrowia lipolytica.

As a plant of the genus Theaceae Camellia, tea plant (scientific name: Camellia sinensis) is preferred.
As a plant of the Hamamelidaceae Hamamelis genus, Hamamelidaceae, Hamamelis (scientific name: Hamamelis virginiana) is preferred.
As a plant of the genus Rosaceae Filipendula of the family Rosaceae, meadowsweet (scientific name: Filipendula ulmaria) is preferred.

As a plant of the genus Plantaginaceae of the family Plantaginaceae, Plantago major (scientific name: Plantago major) is preferable.
As a plant of the genus Mentha of the family Lamiaceae, peppermint (scientific name: Mentha x piperita) is preferred.

For the preparation of the plant extract, any part of the above-mentioned plant can be used, for example, roots, rhizomes, stems, leaves, branches, bark, flowers, seeds (including seed coat, germ, endosperm, hypocotyl, cotyledons, etc.), fruits (including pulp and pericarp), or a combination of two or more of these. The plant extract can be produced by extracting any part of the plant with a solvent.
Preferred parts of the plant for preparing the plant extract include, for example, the following parts: As the plant extract of the present invention, the extract of the following parts of the plant is preferred.
For plants of the genus Rosaceae, flowers, especially petals, are preferred. For grapes, seeds and/or skins are used, preferably seeds and skins. For plants of the genus Theaceae, Camellia, and for plants of the genus Hamamelidaceae, leaves are preferred. For plants of the genus Spiraea, Rosaceae, flowers are preferred. For plants of the genus Plantago, Plantaginaceae, seeds are preferred. For plants of the genus Mentha, Lamiaceae, the whole plant is preferred.
The above-mentioned plant parts may be subjected to the extraction step as they are, or may be subjected to the extraction step after being crushed, cut or dried. In the present invention, the plant extract may be an extract obtained by extracting the above-mentioned plant as it is, or may be an extract obtained by extracting the above-mentioned plant after crushing, cutting or drying. Preferably, it is an extract obtained by extracting the above-mentioned plant after crushing, cutting or drying.

The method of extracting plants to obtain a plant extract is not particularly limited, and can be obtained by a normal extraction method used for extracting plant components. The extraction method can be set appropriately, and the extraction conditions are not particularly limited. For example, it is preferable to obtain a plant extract by extracting the above-mentioned plant with a solvent (extraction solvent) at room temperature or under heating. In preparing a plant extract, the above-mentioned plant as a raw material may be subjected to the extraction process as it is, or may be subjected to the extraction process after being crushed, cut or dried.

The extraction solvent used in the preparation of the plant extract can be appropriately selected, and those commonly used for the extraction of plant components can be used. Examples of the extraction solvent include water; monohydric alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propanol, and butanol; polyhydric alcohols having 2 to 5 carbon atoms, such as ethylene glycol, propylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, and 2,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; and squalane. These may be used alone, or a mixed solvent (mixture) of two or more types may be used. As the monohydric alcohol having 1 to 5 carbon atoms, those having 1 to 4 carbon atoms are preferable, those having 2 to 4 carbon atoms are more preferable, and ethanol is even more preferable. As the polyhydric alcohol having 2 to 5 carbon atoms, a dihydric or trihydric alcohol having 2 to 4 carbon atoms is preferred, a dihydric alcohol is more preferred, and 1,3-butylene glycol is even more preferred. Among them, as the extraction solvent, water, a monohydric alcohol having 1 to 5 carbon atoms, a polyhydric alcohol having 2 to 5 carbon atoms, or a mixed solvent of two or more of these is preferred, water, a monohydric alcohol having 2 to 4 carbon atoms or an aqueous solution thereof, a dihydric alcohol having 2 to 4 carbon atoms or an aqueous solution thereof is more preferred, water, ethanol, an aqueous ethanol solution, 1,3-butylene glycol, and an aqueous 1,3-butylene glycol solution are even more preferred, and water, an aqueous ethanol solution, or an aqueous 1,3-butylene glycol solution is particularly preferred. In one aspect, the aqueous ethanol solution and the aqueous 1,3-butylene glycol solution have an ethanol or 1,3-butylene glycol concentration of 10 to 98% by volume, more preferably 30 to 90% by volume, and even more preferably 30 to 70% by volume. The above-mentioned solvent extract can be suitably used as the plant extract in the present invention.

During extraction, an acid or alkali may be added to adjust the pH of the extraction solvent. After extraction, it is preferable to remove the plant residue (the plant body or parts thereof after extraction) from the extract. There are no particular limitations on the method for removing the plant residue from the extract, and examples include known separation means such as filtration and centrifugation.

As an example of a plant extraction method, for example, the following method can be used. The plant is crushed as is or after drying, and 0.1 to 30 times the weight of the extraction solvent is added, and the plant is extracted at room temperature under normal pressure for preferably 10 minutes to 15 days, more preferably 30 minutes to 10 days, and even more preferably 1 hour to 7 days, or extracted near the boiling point of the extraction solvent for preferably 10 minutes to 1 day (more preferably 10 minutes to 2 hours), and then filtered to obtain a filtrate. During extraction, the mixture may be left to stand or may be stirred as appropriate. The obtained filtrate (plant extract) may be used as a plant extract as is, or may be diluted, concentrated, dried, etc. as necessary.

In the present invention, the plant extract obtained by extraction can be used as it is as a plant extract. In addition, as long as the effect of the present invention is not impaired, it may be diluted, concentrated or dried by a known method to form a diluted solution, concentrate or powder, or prepared in a paste form for use. Examples of drying methods include freeze-drying and spray-drying. Furthermore, the above-mentioned plant extract, its concentrate or dried powder, etc. may be further purified, such as by deodorization and decolorization, as necessary, within a range that does not impair the effect of the present invention. Such a purification method may be performed by any ordinary means selected at will.
The plant extract of the present invention includes various solvent extracts obtained by the above-mentioned extraction methods, their dilutions, their concentrates, their dry powders, and their purified products. The extract can also be used after diluting or dissolving in a solvent different from the extraction solvent.
In addition, the above-mentioned plant extracts are commercially available, and commercially available products can also be used.

The extract of the yeast of the genus Yarrowia is preferably an extract of the yeast cells or the cell culture of the yeast of the genus Yarrowia. Examples of the extract of the yeast cells or the cell culture include an extract of the yeast cells or the cell culture containing the yeast cells (preferably a cell culture solution) which is subjected to a cell disruption treatment such as autolysis or enzymatic decomposition, and the yeast cell contents are dissolved in the culture solution or the like (cell disruption product), and a product from which the cell residue is removed from the cell disruption treatment or the cell culture (cell disruption product). Preferably, the extract is an extract of the yeast cells or the cell culture containing the yeast cells (preferably a cell culture solution) which is subjected to autolysis treatment. The cell disruption product or the product from which the cell residue is removed may be subjected to a sterilization treatment such as a heat treatment, if necessary. The above yeast extract may be one which has been subjected to such a sterilization treatment. The form of the extract of yeast of the genus Yarrowia is not particularly limited, but examples thereof include a paste, suspension, extract, liquid, powder, and the like.

In one embodiment, the extract of a plant of the genus Rosaceae is preferably an ethanol extract or an aqueous ethanol extract of rose (preferably petals).
As the extract of grape seeds and/or skin, an ethanol extract or an aqueous ethanol extract of grape seeds and/or skin is preferred.
As the extract of a plant of the genus Camellia in the family Theaceae, an ethanol extract or an aqueous ethanol extract of the tea plant (preferably the leaves) is preferred.
As the extract of a plant of the genus Hamamelis in the family Hamamelidaceae, a 1,3-butylene glycol extract or an aqueous 1,3-butylene glycol extract of Hamamelis virginiana (preferably the leaves) is preferred.
As an extract of a plant of the genus Spiraea in the family Rosaceae, a 1,3-butylene glycol extract or an aqueous 1,3-butylene glycol extract of Meadowsweet (preferably flowers) is preferred.As an extract of a plant of the genus Plantago in the family Plantaginaceae, an ethanol extract or an aqueous ethanol extract of Plantago major (preferably seeds) is preferred.
As the extract of a plant of the genus Mentha in the family Lamiaceae, an ethanol extract, an aqueous ethanol extract, or a hot water extract of peppermint (preferably the whole plant) is preferred.

The form of the composition of the present invention is not particularly limited, and may be powder, granule, paste, solid, or liquid, and may be appropriately selected. In addition to the above extract, other components may be blended into the composition of the present invention, so long as the effect of the present invention is not impaired. Examples of other components include components that can be used in cosmetics, foods and beverages, medicines, quasi-drugs, etc., as described below.
The composition for suppressing or improving the decrease in type 4 collagen expression and the composition for suppressing or improving the decrease in skin barrier function of the present invention can be provided in the form of an agent, for example, but is not limited to this form. The agent can be provided as a composition as it is, or as a composition containing the agent. In one aspect, the composition of the present invention can also be called an agent for suppressing or improving the decrease in type 4 collagen expression and an agent for suppressing or improving the decrease in skin barrier function.

The composition for inhibiting or improving a decrease in type 4 collagen expression according to the first aspect of the present invention is used to inhibit a decrease in or improve the expression of type 4 collagen in a subject, preferably to inhibit a decrease in or improve the expression of type 4 collagen in the skin of a subject. The compositions for inhibiting or improving a decrease in skin barrier function according to the second, third, and fourth aspects of the present invention are used to inhibit a decrease in or improve the skin barrier function of a subject.
The composition of the present invention can be used to prevent or improve problems caused by, for example, a decrease in skin barrier function, a decrease in basement membrane function, or a decrease in type 4 collagen expression. Examples of such problems include dry skin, itching, dryness, sensitive skin, rough skin, etc. In one embodiment, the composition of the present invention can be used to prevent or improve dry skin, itching, dryness, sensitive skin, rough skin, etc. by suppressing or improving the decrease in skin barrier function, or suppressing or improving the decrease in type 4 collagen expression in the skin. Prevention of problems includes prevention of onset, delay of onset, reduction in onset rate, etc. Improvement of problems includes alleviation of symptoms, improvement of symptoms, suppression of progression of symptoms, etc.

The composition of the present invention can be used for various applications such as cosmetics, food and beverages, medicines, and quasi-drugs. The composition of the present invention may itself be a cosmetic, food and beverage, medicine, or quasi-drug, etc. for inhibiting or improving the decline in expression of type 4 collagen, or for inhibiting or improving the decline in skin barrier function, or may be a material or preparation, etc., used in combination with the cosmetic, food and beverage, medicine, or quasi-drug, etc.

The composition of the present invention is preferably used, for example, as an external preparation for the skin. External preparations for the skin include cosmetics, medicines, and quasi-drugs, and are preferably cosmetics.
The composition of the present invention can also be used as a drug or quasi-drug other than an external skin preparation.
In another embodiment, the composition of the present invention is preferably formulated as a food or drink. The case where the composition of the present invention is formulated as a cosmetic, a medicine, an external preparation for skin such as a quasi-drug, a food or drink, etc. will be described below.

When the composition of the present invention is used as a skin topical preparation, the dosage form is not particularly limited, and can be any form such as a solution, emulsion, cream, gel, powder, aerosol, mist, capsule, and sheet. The skin topical preparation is preferably a cosmetic. The product form of the cosmetic is also not particularly limited, and examples thereof include skin care cosmetics such as face wash, makeup remover, lotion, serum, pack, emulsion, cream, and sunscreen; makeup cosmetics such as foundation, makeup base, lipstick, eye shadow, eyeliner, mascara, eyebrow, blusher, and nail enamel; hair cosmetics such as hair shampoo, hair rinse, hair styling product, hair dye, and hair growth agent; cleansing products such as soap and body soap; and bath additives.

The above-mentioned cosmetic preparation may appropriately contain one or more of the following components, such as carriers and additives acceptable for cosmetic preparations, within the scope of the present invention, such as water, alcohols, oils, surfactants, thickeners, metal soaps, gelling agents, powders, chelating agents, water-soluble polymers, film-forming agents, resins, inclusion compounds, antibacterial agents, deodorants, salts, pH adjusters, UV absorbers, extracts derived from animals, plants, and microorganisms other than those mentioned above, keratolytic agents, enzymes, hormones, other vitamins, moisturizers, bactericides, anti-inflammatory agents, and fragrances, within the scope of the present invention. The cosmetic preparation may be produced by a general production method. For example, the cosmetic preparation may be obtained by mixing the above-mentioned extract used as the active ingredient with one or more components that can be used in the above-mentioned cosmetic preparation, and then processing the mixture into the desired form.

When the topical skin preparation is a drug or quasi-drug, components such as carriers and additives acceptable for drugs or quasi-drugs may be used within the scope that does not impair the effects of the present invention. Examples of such components include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, etc., which can be used as needed.

The composition of the present invention can also be a medicine or quasi-drug other than the above-mentioned skin topical preparation. Such medicine or quasi-drug may be administered orally or parenterally. The medicine or quasi-drug can be in the form of an oral administration preparation (internal preparation) or a parenteral administration preparation. Examples of the dosage form of an oral administration preparation include liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewable preparations, etc. Examples of the dosage form of a parenteral administration preparation include injections, infusions, etc. Such medicines or quasi-drugs may contain components such as carriers and additives that are acceptable for medicines or quasi-drugs. Examples of such components include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents, etc., which can be used as needed. Medicines and quasi-drugs can be manufactured by general manufacturing methods. For example, the extract can be mixed with one or more ingredients that can be used in pharmaceuticals or quasi-drugs, and then processed into the desired form.

When the composition of the present invention is used as a food or beverage, the food or beverage is not particularly limited, and examples thereof include general food or beverage, health food, functional food, and food for specified health uses. The form of the food or beverage is also not particularly limited. The above-mentioned health food, functional food, and food for specified health uses can also be in various formulation forms, such as liquids, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewable agents, and liquid diets.

The above-mentioned food and beverage products may contain components acceptable for food and beverage products, such as other food and beverage ingredients, additives that are added to food and beverage products, etc., within the scope of not impairing the effects of the present invention. Such food and beverage products can be manufactured by general manufacturing methods. For example, in the manufacture of food and beverage products, the above-mentioned extract used as an active ingredient in the present invention may be added to food and beverage ingredients, etc.

When the composition of the present invention is in the form of a skin topical preparation, a medicine or quasi-drug other than a skin topical preparation, a food or beverage, etc., the content of the above extract used as an active ingredient in the composition is, for example, preferably 0.0000001 to 100% by weight, more preferably 0.000001 to 100% by weight, and even more preferably 0.00001 to 10% by weight, calculated as the weight of the dry matter of the extract. When two or more types of extracts are contained, the above content is the total content.

The amount of the composition of the present invention to be used is not particularly limited, and may be an amount (effective amount) that can suppress or improve the decline of skin barrier function and suppress or improve the decline of type 4 collagen expression, and can be appropriately set according to the condition, weight, sex, age, or other factors of the subject. The amount of the composition of the present invention to be used is, for example, 0.01 to 500 mg of the above extract (dry matter equivalent) used as the active ingredient per adult (60 kg) per day in the case of a skin external preparation such as a cosmetic. The dosage when a pharmaceutical or quasi-drug is orally administered is, for example, 0.01 to 500 mg of the above extract (dry matter equivalent) per adult (60 kg) per day. In the case of a food or drink, the intake amount is, for example, 0.01 to 500 mg of the above extract (dry matter equivalent) per adult (60 kg) per day. The above amount of extract can be ingested or administered in a single dose or in multiple doses. When two or more types of extracts are used, the amount of the above extract is the total amount. There are no particular limitations on the timing of applying an external skin preparation containing the above extract to the skin, or the timing of ingesting or administering a food or drink, medicine or quasi-drug.

The subject to which the composition of the present invention is applied (which can also be referred to as the subject to which it is administered) is not particularly limited, and can be applied to humans or non-human mammals, etc., but humans are preferred. Subjects to which the composition of the present invention is applied include, for example, subjects who wish or need to inhibit or improve the decline in skin barrier function, subjects who wish or need to inhibit or improve the decline in expression of type 4 collagen, etc. Examples of such subjects include people who experience dry skin, itchiness, dryness, sensitive skin, and rough skin. The composition of the present invention can be ingested or administered by various methods known per se depending on its form, dosage form, etc. In one aspect, the composition of the present invention is preferably applied to the skin.

The above extract used as an active ingredient of the composition of the present invention can be used to suppress the decrease in the expression of type 4 collagen in a subject or to improve said expression. The above extract can be used to suppress the decrease in the skin barrier function in a subject or to improve the skin barrier function, preferably to suppress the decrease in the skin barrier function or to improve the skin barrier function by suppressing or improving the decrease in the expression of type 4 collagen.

The present invention also encompasses the following uses and methods.
Use of one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia, extracts of plants of the genus Camellia, extracts of plants of the genus Spiraea, and extracts of plants of the genus Mentha in the family Lamiaceae, for inhibiting or improving the decrease in expression of type 4 collagen, thereby inhibiting or improving the decrease in skin barrier function or improving skin barrier function.
A method for inhibiting a decline in skin barrier function or improving skin barrier function by administering one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae, extracts of grape seeds and/or skin, extracts of yeast of the genus Yarrowia, extracts of plants of the genus Camellia of the family Theaceae, extracts of plants of the genus Spiraea of the family Rosaceae, and extracts of plants of the genus Mentha of the family Lamiaceae.

Use of an extract of a plant of the genus Hamamelis of the family Hamamelidaceae and/or an extract of a plant of the genus Plantago of the family Plantaginaceae for inhibiting a decline in skin barrier function or improving skin barrier function.
A method for inhibiting a decline in skin barrier function or improving skin barrier function, comprising administering an extract of a plant of the genus Hamamelid in the family Hamamelidaceae and/or an extract of a plant of the genus Plantago in the family Plantaginaceae.

2. Use of one or more extracts selected from the group consisting of extracts of plants of the genus Rosaceae (Rosaceae), extracts of grape seeds and/or skins, extracts of yeasts of the genus Yarrowia (Yarrowia), extracts of plants of the genus Camellia (Theaceae), extracts of plants of the genus Hamamelidaceae (Witch hazel), extracts of plants of the genus Spiraea (Rosaceae), extracts of plants of the genus Plantago (Plantaginaceae), and extracts of plants of the genus Mentha (Lamiaceae), for suppressing a decrease in the expression of type 4 collagen or improving said expression.
A method for suppressing a decrease in expression of type 4 collagen or improving said expression, comprising administering one or more of the above extracts.

The above extract is preferably administered (applied) to the skin and can be used to suppress or improve a decrease in skin barrier function, to suppress or improve a decrease in type 4 collagen expression in the skin, or to suppress or improve a decrease in basement membrane function. In one aspect, the above extract can be used to prevent or improve problems caused by a decrease in skin barrier function, a decrease in basement membrane function, or a decrease in type 4 collagen expression. The above extract can be used, for example, to prevent or improve dry skin, itchiness, dryness, sensitive skin, rough skin, etc. by suppressing or improving a decrease in skin barrier function, or suppressing or improving a decrease in type 4 collagen expression in the skin.

In the above-mentioned uses and methods, the above-mentioned extract, the subject to which it is administered, the dosage of the extract, and the preferred aspects thereof are the same as those in the above-mentioned composition of the present invention.The above-mentioned extract may be used as it is, or may be used in combination with other ingredients.The composition of the present invention may be used.The above-mentioned extract may be used in the form of the above-mentioned skin external preparation, food, drink, etc.
The use may be a therapeutic use or a non-therapeutic use. The method may be a therapeutic method or a non-therapeutic method, the latter being a concept that does not include medical procedures, i.e., surgery, treatment or diagnosis of humans.

Another aspect of the present invention is the use of the above extract for producing a composition for inhibiting or improving the decline of skin barrier function, or the use of the above extract for producing a composition for inhibiting or improving the decline of expression of type 4 collagen. The extract and preferred aspects thereof are the same as those in the composition of the present invention described above.
Further aspects of the present invention are use of an agent for inhibiting or improving a decline in basement membrane function to inhibit the decline in skin barrier function or improve the skin barrier function; and a method for inhibiting the decline in skin barrier function or improving the skin barrier function by administering an agent for inhibiting or improving a decline in basement membrane function.
The agent for inhibiting or ameliorating the decline in basement membrane function and preferred embodiments thereof are the same as those described above.

The present invention also encompasses a method for screening for a substance that has an effect of inhibiting or improving the decline in skin barrier function. The screening method of the present invention will now be described.
The screening method of the present invention for a substance having an effect of inhibiting or improving the decline of skin barrier function comprises the steps of culturing human epidermal keratinocytes in vitro in a medium containing a test substance or a control medium not containing the test substance, and comparing the expression level of type 4 collagen in the skin cells cultured in the medium containing the test substance and the control skin cells cultured in the control medium. If the expression level of type 4 collagen in the skin cells cultured in the medium containing the test substance is greater than the expression level of type 4 collagen in the control skin cells, the test substance is selected as a substance having an effect of inhibiting or improving the decline of skin barrier function.

In the screening method of the present invention, human epidermal keratinocytes are cultured in vitro in a medium containing a test substance or a control medium not containing the test substance. Human epidermal keratinocytes are commercially available, and commercially available products can be used.
The test substance is not particularly limited, and examples thereof include plant extracts, cell extracts, cell culture supernatants, fermentation products, proteins, peptides, vitamins, synthetic compounds, etc. These may be known substances or novel substances.

The process of culturing human epidermal keratinocytes in vitro in a medium containing a test substance or a control medium not containing the test substance is also referred to as a culturing process. The process of comparing the expression levels of type 4 collagen in skin cells cultured in a medium containing the test substance (also referred to as test skin cells) and control skin cells cultured in a control medium is also referred to as a type 4 collagen expression comparison process.

In the culturing step, the conditions for culturing human epidermal keratinocytes are not particularly limited, and known conditions may be adopted. The medium containing the test substance can be prepared by mixing the test substance with the medium. The medium may be one used for culturing human epidermal keratinocytes, or a commercially available product may be used. Examples of the medium include HuMedia-KB2 medium (manufactured by Kurabo Industries, Ltd.) and DMEM medium. The medium may contain insulin, human EGF, hydrocortisone, serum, antibacterial agents, etc. The culture time is not particularly limited, and may be appropriately set depending on the culture conditions, etc. The control medium may be the same as the medium containing the test substance, except that it does not contain the test substance. For example, human epidermal keratinocytes may be cultured in the medium containing the test substance or the control medium for preferably 1 to 72 hours, more preferably 3 to 48 hours.
In the culture step, stress may be applied, such as ultraviolet irradiation, oxidative stress, etc. For example, when culture is performed under stressful conditions, such as ultraviolet irradiation, oxidative stress, etc., it is possible to screen for ingredients that are effective in suppressing a decrease in skin barrier function caused by the stress or improving skin barrier function under stress.

After the culturing step, a step of comparing the expression levels of type 4 collagen is performed. After the above culturing, the expression levels of type 4 collagen in the skin cells cultured in a medium containing the test substance and in the control skin cells cultured in a control medium are compared. If the expression level of type 4 collagen in the skin cells cultured in a medium containing the test substance is greater than the expression level of type 4 collagen in the control skin cells, the test substance is selected as a substance having an effect of inhibiting or improving the decline of skin barrier function.

The expression level of type 4 collagen can be measured by measuring the amount of mRNA encoding type 4 collagen or the amount of protein of type 4 collagen. The amount of mRNA or protein can be measured by a known method.
The amount of mRNA encoding type 4 collagen can be measured by quantitative PCR, Northern blotting, microarray, live imaging using a fluorescent probe, etc. The amount of protein of type 4 collagen can be measured by immunochemical assay, mass spectrometry, etc. Examples of immunochemical assays include Enzyme Linked Immunosorbent Assay (ELISA) and Western blotting. A commercially available ELISA kit (manufactured by MyBioSource) can also be used to measure the amount of protein. The base sequence and amino acid sequence (ORF) of human type 4 collagen are known, and the base sequence (mRNA) is registered in Genbank, a public database, under Accession No. AH002741. Those skilled in the art can easily design and synthesize primers for measuring the amount of mRNA encoding type 4 collagen, and amplify DNA, etc.

As described above, by suppressing the decrease in the expression of type 4 collagen or improving said expression, it is possible to suppress or improve the decrease in skin barrier function. Therefore, when the expression level of type 4 collagen in the test skin cells is greater than the expression level of type 4 collagen in the control skin cells, the test substance can be determined to be a substance that has an effect of suppressing or improving the decrease in skin barrier function. According to the above screening method, it is possible to screen for substances that have an effect of suppressing or improving the decrease in skin barrier function.

The present invention will now be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.

<Preparation Example 1>
Extracts were prepared from the following plants and their parts:
(1) Rose (Rosa hybrida, variety "Suntory Blue Rose Applause" (registered trademark), Suntory Flowers Ltd.): petals (2) Grape (Vitis spp.) (Cabernet Sauvignon): seeds and skin (3) Tea plant (Camellia sinensis): leaves (4) Hamamelis virginiana: leaves (5) Summer snowflake (Filipendula ulmaria): flowers (6) Plantain (Plantago major): seeds (7) Mentha x The above rose (cultivar "Suntory Blue Rose Applause") is a blue rose containing delphinidin-type anthocyanins, rosacyanin A1, rosacyanin A2, and rosacyanin B, as well as rosadelphin A1, rosadelphin A2, and rosadelphin B in the petals.

The crushed plants of (1) to (7) above were soaked in a solvent and extracted. More specifically, for (1), (2), (3), (6), and (7) above, the crushed plants were soaked in 10 times the plant's weight in 30 to 90 vol% ethanol aqueous solution, and extracted for 10 minutes to 7 days at room temperature with stirring once a day to obtain an extract. For (4) and (5) above, 1,3-butylene glycol aqueous solution was used instead of ethanol aqueous solution, and extraction was performed using the above method.

From the obtained extract, plant residues were removed by filtration, and the filtrate was obtained as a plant extract.
The obtained plant extracts were concentrated in an evaporator and then freeze-dried to obtain powdered extracts of each plant. The powdered plant extracts (rose petal extract, grape seed and skin extract, tea leaf extract, witch hazel leaf extract, meadowsweet flower extract, plantago major seed extract, and peppermint extract) were used as extract samples for the evaluation described below.

<Preparation Example 2>
The powdered Yarrowia lipolytica extract was obtained by culturing Yarrowia lipolytica, allowing it to autolyze, and then subjecting it to heat sterilization and sterilization.
In the examples described below, for the Yarrowia lipolytica extract, the above powder was used as the extract sample.

Example 1: Effect of overexpression of type 4 collagen on skin barrier under inflammatory stimulation A plasmid (hereinafter, COL4A1 plasmid) was prepared by incorporating COL4A1 (RefSeq ID: NM_001845.6) downstream of the CMV promoter. According to the recommended protocol, the COL4A1 plasmid and Lipofectamine 3000 Reagent (manufactured by Thermo Fisher Scientific) were diluted with Opti-MEM (registered trademark) I Reduced Serum Media (manufactured by Thermo Fisher Scientific) (hereinafter, Opti-MEM) to prepare two solutions with different plasmid concentrations (hereinafter, Opti-MEM solutions) (plasmid concentration: 5 μg/mL or 20 μg/mL). As a control, a solution was also prepared to which Opti-MEM was added instead of the plasmid.

Using HuMedia-KG2 (recommended medium) (Kurabo Industries, Ltd.), normal human adult epidermal keratinocytes (Kurabo Industries, Ltd.) were seeded at a concentration of 3 x ¹⁰⁴ cells/200 μL/well in 24-well Cell Culture Inserts (Millipore Industries, Ltd.), and 10 μL of any of the Opti-MEM solutions was added immediately. Note that 900 μL of the recommended medium was added to the 24-well plate (AGC Technoglass, Ltd.). Then, 30 minutes later, _hydrogen peroxide solution (Nacalai Tesque, Inc.) was added to a final concentration of 100 μM only for some groups ( _H2O2 treatment). Four hours after seeding, the medium was replaced with the recommended medium. 24 hours after seeding, the medium was replaced with the recommended medium in which the Ca2 ⁺ concentration in the medium was increased to 1.41 mM by adding _CaCl2 (hereinafter, referred to as the irregular recommended medium). The medium was replaced with the irregular recommended medium every 2 to 3 days.

96 hours, 120 hours, and 144 hours after seeding, transepithelial electrical resistance (TEER (Ω·cm ² )) was evaluated using Millicell ERS-2 (Merck). After 144 hours after seeding, cells were lysed using isogen (Nippon Gene Co., Ltd.), and mRNA was extracted. Then, cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit and RNase Inhibitor, and quantitative PCR was performed using TaqMan Fast Universal PCR Master Mix (both manufactured by Thermo Fisher Scientific) and primers to evaluate the expression level of human type 4 collagen A1 (COL4A1). Eukaryotic 18S rRNA was used as an endogenous control.

The primers used to measure COL4A1 expression levels (COL4A1) and to quantify endogenous controls (Eukaryotic 18S rRNA) were #Hs00266237_m1 and #Hs99999901_s1 (both manufactured by Thermo Fisher Scientific), respectively.

The results are shown in Tables 1 and 2. Table 1 shows the gene expression level of COL4A1 when the COL4A1 expression level in the group without plasmid addition and without H ₂ O ₂ treatment (H ₂ O ₂ (-), plasmid (-)) is set to 1. In Tables 1 and 2, H ₂ O ₂ (+) is the group treated with H ₂ O _2. Plasmid (-) indicates the group without COL4A1 plasmid addition, and plasmid (+) indicates the group with COL4A1 plasmid addition. * indicates that there was a significant difference (p<0.05) (Dunnett's test (vs. group without plasmid addition and treated with H ₂ O ₂ )). A decrease in the COL4A1 expression level was observed with H ₂ O ₂ stimulation, but it was confirmed that the introduction of the COL4A1 plasmid significantly and dose-dependently suppressed or improved the decrease in expression compared to the H ₂ O ₂ stimulation group (*: p<0.05).

Table 2 shows the TEER values 144 hours after seeding. * indicates that there was a significant difference (p _{<0.05) (Dunnett's test (vs. group without plasmid addition and H2O2 treatment)). A significant decrease in TEER was observed with H2O2 stimulation , but} the introduction of the COL4A1 plasmid was confirmed to improve TEER _dose -dependently and significantly in the high-dose group compared to the _H2O2 stimulation group (*: p<0.05).

Examples 2 to 9: Evaluation of the inhibitory or ameliorating effect of extract samples on the decrease in type IV collagen gene expression Normal human adult epidermal keratinocytes (Kurabo) were seeded in a 12-well plate (AGC Technoglass) at a concentration of 5 x ¹⁰⁴ cells/mL/well using the recommended medium, and cultured for 48 hours. After the medium was replaced with the recommended medium containing the extract sample to start sample treatment, hydrogen peroxide solution (Nacalai Tesque) was added to a final concentration of 100 μM 30 minutes after the start of sample treatment. After 3 hours from the start of hydrogen peroxide solution treatment, the medium was replaced with the recommended medium not containing the extract sample. After 48 hours from the start of sample treatment, the cells were lysed and collected using RLT Buffer (containing 1% 2-mercaptoethanol), and mRNA was extracted using RNeasy Mini Kit and RNase-Free DNase Set (both manufactured by Qiagen). Thereafter, cDNA synthesis and quantitative PCR were carried out in the same manner as in Example 1 to evaluate the expression level of COL4A1.

As a control, cells were cultured in the same manner as above, except that instead of the extract sample, dimethyl sulfoxide (DMSO) was added to a final concentration of 0.1%, and hydrogen peroxide was added and the expression level of COL4A1 was evaluated.

The extract samples used in Examples 2 to 9 were rose petal extract, grape seed and skin extract, tea leaf extract, witch hazel leaf extract, meadowsweet flower extract, plantago major seed extract, Yarrowia lipolytica extract, or peppermint extract prepared in Preparation Examples 1 and 2. The extract samples were dissolved in ultrapure water or DMSO and added to the medium. The concentration of the extract sample in the medium was 5 to 20 μg/mL. When dissolved in ultrapure water, DMSO was added separately so that the final DMSO concentration of the recommended medium containing any of the extract samples was 0.1%.

The results of Examples 2 to 9 (average values of N=3 to 4) are shown in Table 3. The results shown in Table 3 are the relative values (%) of the expression level of COL4A1 in the cells treated with the extract samples, when the expression level of COL4A1 in the control is taken as 100%. * indicates that there was a significant difference (p<0.05) from the control (Student's t-test). Table 3 shows the results when the expression of COL4A1 was highest in the extract sample (test substance) concentration range of 5 to 20 μg/mL. Rose petal extract, grape seed and skin extract, tea leaf extract, witch hazel leaf extract, meadowsweet flower extract, psyllium seed extract, Yarrowia lipolytica extract, and peppermint extract were confirmed to have a significant effect of suppressing the decrease in COL4A1 expression or improving the expression compared to the control (*: p<0.05).

Examples 10 to 15: Evaluation of the effect of extract samples in inhibiting or improving the decline of skin barrier function Normal human adult epidermal keratinocytes were seeded in Millicell Cell Culture Inserts at a concentration of 3 x ¹⁰⁴ cells/200 μL/well using the recommended medium containing the extract sample. 30 minutes after seeding, hydrogen peroxide water was added to a final concentration of 100 μM. 4 hours after seeding, the medium was replaced with the recommended medium not containing the extract sample. 24 hours after seeding, the medium was replaced with the irregular recommended medium. Thereafter, the transepithelial electrical resistance (TEER) was evaluated using the same method as in Example 1.

For the control, cells were cultured in the same manner as above, except that the recommended medium containing DMSO at a final concentration of 0.1% was used instead of the recommended medium containing the extract sample, and hydrogen peroxide was added and the transepithelial electrical resistance value was evaluated.

The extract samples used in Examples 10 to 15 were the extracts prepared in Preparation Examples 1 and 2 and shown in Table 4. The extract samples were dissolved in ultrapure water or DMSO and added to the culture medium. The concentration of the extract sample in the culture medium was 5 to 20 μg/mL. When dissolved in ultrapure water, DMSO was added separately so that the final DMSO concentration of the recommended culture medium containing any of the extract samples was 0.1%.

The results (average of N=4-6) are shown in Table 4. The results shown in Table 4 are the relative values (%) of TEER in cells treated with the extract samples, with the control TEER taken as 100%. Table 4 shows the results when the extract sample (test substance) concentration was 5-20 μg/mL and the measurement time was in the range of 96-144 hours after seeding, and the TEER value was the highest. Six types of extracts were confirmed to be highly effective in inhibiting or improving the decrease in TEER, and furthermore, significant changes were confirmed compared to the control for grape seed and skin extract, tea leaf extract, witch hazel leaf extract, meadowsweet flower extract, and Yarrowia lipolytica extract (*: p<0.05) (Student's t-test).

It has been revealed that the extracts of the above plants and the extract of Yarrowia lipolytica have the effect of suppressing the decline in skin barrier function or improving said function.

Claims (6)

A composition for inhibiting or improving the decrease in expression of type 4 collagen, comprising as active ingredients one or more extracts selected from the group consisting of a rose petal extract containing one or more compounds selected from the group consisting of a delphinidin-type anthocyanin, a rosacyanin compound, and a rosadelphin compound, an extract of a yeast of the genus Yarrowia, and an extract of a plant of the genus Plantago of the family Plantaginaceae. 2. The composition for inhibiting or improving a decrease in expression of type 4 collagen according to claim 1 , wherein the Yarrowia yeast is Yarrowia lipolytica , and the Plantago genus plant of the Plantaginaceae family is Plantago major. 3. The composition for inhibiting or improving the decrease in expression of type 4 collagen according to claim 1 or 2 , wherein the plant of the genus Plantago in the family Plantaginaceae is Plantago major seed. The composition according to any one of claims 1 to 3 , which is an external preparation for the skin. The composition according to any one of claims 1 to 4 , which is a cosmetic. Use of one or more extracts selected from the group consisting of a rose petal extract containing one or more compounds selected from the group consisting of a delphinidin-type anthocyanin, a rosacyanin compound, and a rosadelphin compound, an extract of a yeast of the genus Yarrowia, and an extract of a plant of the genus Plantago in the family Plantaginaceae, for suppressing a decrease in the expression of type 4 collagen or improving said expression (however, medical procedures for humans are excluded).