Expression prompter for glucose transporter type 1

Abstract

This expression promoter for glucose transporter type 1 contains a water-soluble plant-derived polysaccharide, wherein the water-soluble plant-derived polysaccharide contains, as constituent sugars, galactose, arabinose, and galacturonic acid at a specified ratio.

Description

Glucose transporter 1 expression promoter

[0001] The present invention relates to an agent for promoting the expression of glucose transporter 1.

[0002] Glucose transporter 1 is present in the cell membrane of many cells and plays a role in taking up glucose into the cell. In recent years, the physiological significance of promoting the expression of glucose transporter 1 has attracted attention. For example, Patent Document 1 discloses a blood-brain barrier protector containing, as an active ingredient, tricaffeoylquinic acid or a salt thereof, which promotes the expression of glucose transporter 1.

[0003] Japanese Patent Application Laid-Open No. 2021-19532

[0004] An object of the present invention is to provide an agent for promoting the expression of glucose transporter 1.

[0005] The present disclosure provides, for example, the inventions described in the following [1] to

[30] : [1] A glucose transporter 1 expression promoter comprising a water-soluble plant polysaccharide, wherein the proportion of galactose in all constituent sugars of the water-soluble plant polysaccharide is 15 to 60 mol %, the proportion of arabinose in all constituent sugars of the water-soluble plant polysaccharide is 10 to 50 mol %, and the proportion of galactose in all constituent sugars of the water-soluble plant polysaccharide is 15 to 60 mol %, the proportion of arabinose in all constituent sugars of the water-soluble plant polysaccharide is 10 to 50 mol %, and the proportion of galactose in all constituent sugars of the water-soluble plant polysaccharide is 50 to 98 mol %. [1a] An expression promoter for glucose transporter 1, comprising water-soluble black tea leaf polysaccharides, wherein the water-soluble black tea leaf polysaccharides contain galactose, arabinose, and galacturonic acid as constituent sugars, wherein the proportion of galactose in the total constituent sugars of the water-soluble black tea leaf polysaccharides is 20 to 30 mol %, and the proportion of arabinose is 30 to 40 mol %, and the total proportion of galactose and arabinose in the total constituent sugars of the water-soluble black tea leaf polysaccharides is 60 to 90 mol %. [2] The expression promoter according to [1] or [1a], which is at least one selected from the group consisting of an expression inhibitor of galectin-7, a claudin-1 expression promoter, and a filaggrin expression promoter. [3] The expression promoter according to any of [1] to [2], which is at least one therapeutic or preventive agent selected from the group consisting of dry skin, a decrease in epidermal barrier function, and rough skin. [4] The expression promoter according to any one of [1], [2], and [3], wherein the water-soluble plant polysaccharide is black tea leaf polysaccharide. [5] The expression promoter according to [4], wherein the black tea leaf polysaccharide contains 20 to 30 mol% of galactose, 30 to 40 mol% of arabinose, and 15 to 25 mol% of galacturonic acid in all sugar constituents, and the total proportions of galactose, arabinose, and galacturonic acid in all sugar constituents of the black tea leaf polysaccharide is 70 to 90 mol%. [6] The expression promoter according to any one of [1a], [4], and [5], wherein the black tea leaf polysaccharide contains 5 to 20% by mass of black tea leaf polysaccharides having a molecular weight of 100 to 1,000 and 10 to 30% by mass of black tea leaf polysaccharides having a molecular weight of 3,000 to 50,000.[7] The expression promoter according to any one of [1], [2], and [3], wherein the water-soluble plant polysaccharide is soybean polysaccharide. [8] The expression promoter according to [7], wherein the soybean polysaccharide contains 30 to 50 mol% of galactose, 15 to 35 mol% of arabinose, and 5 to 20 mol% of galacturonic acid in all sugar constituents, and the total proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the soybean polysaccharide is 60 to 90 mol%. [9] The expression promoter according to [7] or [8], wherein the soybean polysaccharide contains soybean polysaccharides having a molecular weight of 30,000 to 1,000,000 in an amount of 20 mass% or more.

[10] The expression promoter according to any one of [1] to [9], wherein the expression promoter is for use in the epidermis.

[11] The expression promoter according to any one of [1] to

[10] , wherein the expression promoter is an external preparation.

[12] The expression promoter according to any one of [1] to

[11] , which is a cosmetic.

[13] A method for promoting expression of glucose transporter 1, comprising administering a water-soluble plant polysaccharide to a subject in need thereof, wherein the water-soluble plant polysaccharide contains 15 to 60 mol% of galactose, 10 to 50 mol% of arabinose, and 4 to 35 mol% of galacturonic acid in all sugar constituents, and the total proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the water-soluble plant polysaccharide is 50 to 98 mol%. [13a] A method for promoting expression of glucose transporter 1, comprising administering water-soluble black tea leaf polysaccharides to a subject in need thereof, wherein the water-soluble black tea leaf polysaccharides contain galactose, arabinose, and galacturonic acid as constituent sugars, the proportion of galactose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 20 to 30 mol %, and the proportion of arabinose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 30 to 40 mol %, and the total proportion of galactose and arabinose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 60 to 90 mol %.

[14] A water-soluble plant polysaccharide for use in a therapeutic method for promoting expression of glucose transporter 1, wherein, based on all sugar constituents of the water-soluble plant polysaccharide, the proportion of galactose is 15 to 60 mol%, the proportion of arabinose is 10 to 50 mol%, and the proportion of galacturonic acid is 4 to 35 mol%, and the total proportion of galactose, arabinose, and galacturonic acid based on all sugar constituents of the water-soluble plant polysaccharide is 50 to 98 mol%. [14a] A water-soluble black tea leaf polysaccharide for use in a therapeutic method for promoting expression of glucose transporter 1, the water-soluble black tea leaf polysaccharide comprising galactose, arabinose, and galacturonic acid as constituent sugars, the proportion of galactose in the total constituent sugars of the water-soluble black tea leaf polysaccharide being 20 to 30 mol % and the proportion of arabinose being 30 to 40 mol %, and the total proportion of galactose and arabinose in the total constituent sugars of the water-soluble black tea leaf polysaccharide being 60 to 90 mol %.

[15] Use of a water-soluble plant polysaccharide in a non-therapeutic method for promoting expression of glucose transporter 1, wherein the proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the water-soluble plant polysaccharide is 15 to 60 mol%, 10 to 50 mol%, and 4 to 35 mol%, respectively, and the total proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the water-soluble plant polysaccharide is 50 to 98 mol%. [15a] Use of water-soluble black tea leaf polysaccharides in a non-therapeutic method for promoting expression of glucose transporter 1, wherein the water-soluble black tea leaf polysaccharides contain galactose, arabinose, and galacturonic acid as constituent sugars, the proportion of galactose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 20 to 30 mol %, and the proportion of arabinose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 30 to 40 mol %, and the total proportion of galactose and arabinose in all constituent sugars of the water-soluble black tea leaf polysaccharides is 60 to 90 mol %.

[16] The method, water-soluble plant polysaccharide, or use according to any one of

[13] to [15a], wherein the method is at least one method selected from the group consisting of a method for suppressing expression of galectin-7, a method for promoting expression of claudin-1, and a method for promoting expression of filaggrin.

[17] The method, water-soluble plant polysaccharide, or use according to any one of

[13] to

[16] , wherein the method is a method for treating or preventing at least one condition selected from the group consisting of dry skin, a decrease in the epidermal barrier function, and rough skin.

[18] The method, water-soluble plant polysaccharide, or use according to any one of

[13] to

[17] , wherein the water-soluble plant polysaccharide is administered transdermally.

[19] Use of a water-soluble plant polysaccharide for the production of an expression promoter for glucose transporter 1, wherein the proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the water-soluble plant polysaccharide is 15 to 60 mol%, 10 to 50 mol%, and 4 to 35 mol%, respectively, and the total proportion of galactose, arabinose, and galacturonic acid in all sugar constituents of the water-soluble plant polysaccharide is 50 to 98 mol%. [19a] Use of water-soluble black tea leaf polysaccharides for the manufacture of an agent for promoting the expression of glucose transporter 1, wherein the water-soluble black tea leaf polysaccharides contain galactose, arabinose, and galacturonic acid as constituent sugars, the proportion of galactose in the total constituent sugars of the water-soluble black tea leaf polysaccharides being 20 to 30 mol % and the proportion of arabinose being 30 to 40 mol %, and the total proportion of galactose and arabinose in the total constituent sugars of the water-soluble black tea leaf polysaccharides being 60 to 90 mol %.

[20] The use according to

[19] or [19a], wherein the agent for promoting the expression of glucose transporter 1 is at least one selected from the group consisting of an inhibitor of galectin-7 expression, a claudin-1 expression promoter, and a filaggrin expression promoter.

[21] The use according to any of

[19] to

[20] , wherein the agent for promoting the expression of glucose transporter 1 is at least one therapeutic or preventive agent selected from the group consisting of dry skin, a decrease in epidermal barrier function, and rough skin.

[22] The use according to any one of

[19] to

[21] , wherein the expression promoter is for use in the epidermis.

[23] The use according to any one of

[19] to

[22] , wherein the expression promoter is an external preparation.

[24] The use according to any one of

[19] to

[23] , wherein the expression promoter is a cosmetic.

[25] The method, water-soluble plant polysaccharide, or use according to any one of

[13] ,

[14] ,

[15] ,

[16] to

[19] , and

[20] to

[24] , wherein the water-soluble plant polysaccharide is black tea leaf polysaccharide.

[26] The method, water-soluble plant polysaccharide, or use according to

[25] , wherein, of all sugar constituents of the black tea leaf polysaccharide, galactose accounts for 20 to 30 mol%, arabinose for 30 to 40 mol%, and galacturonic acid for 15 to 25 mol%, and the sum of the proportions of galactose, arabinose, and galacturonic acid for 70 to 90 mol%.

[27] The method, water-soluble plant polysaccharide, or use according to any one of [13a], [14a], [15a], [19a],

[25] , and

[26] , wherein the proportion of the black tea leaf polysaccharides having a molecular weight of 100 to 1000 is 5 to 20% by mass, and the proportion of the black tea leaf polysaccharides having a molecular weight of 3000 to 50000 is 10 to 30% by mass.

[28] The method, water-soluble plant polysaccharide, or use according to any one of

[13] ,

[14] ,

[15] ,

[16] to

[19] , and

[20] to

[24] , wherein the water-soluble plant polysaccharide is soybean polysaccharide.

[29] The method, water-soluble plant polysaccharide, or use according to

[28] , wherein the soybean polysaccharide contains 30 to 50 mol% of galactose, 15 to 35 mol% of arabinose, and 5 to 20 mol% of galacturonic acid in total sugar components, and the total proportions of galactose, arabinose, and galacturonic acid in total sugar components is 60 to 90 mol%.

[30] The method, water-soluble plant polysaccharide, or use according to any one of [13a], [14a], [15a], [19a],

[28] , and

[29] , wherein the soybean polysaccharide contains soybean polysaccharides having a molecular weight of 30,000 to 1,000,000 in an amount of 20 mass% or more.

[0006] According to the present invention, an agent for promoting the expression of glucose transporter 1 can be provided.

[0007] 1 shows the integral and differential molecular weight distributions of black tea leaf polysaccharides. 2 shows the integral and differential molecular weight distributions of soybean polysaccharides. 3 shows the expression level of glucose transporter 1 in a skin model when black tea leaf extract is added. 4 shows the moisture content of the stratum corneum of an epidermal model when black tea leaf extract is added. 5 shows the amount of galectin-7 in the medium when black tea leaf extract is added to an epidermal model. 6 shows the expression level of claudin-1 in a skin model when black tea leaf extract is added. 7 shows the expression level of filaggrin in a skin model when black tea leaf extract is added. 8 shows the expression level of glucose transporter 1 in a skin model when soybean extract is added. 9 shows the moisture content of the stratum corneum of an epidermal model when soybean extract is added. 10 shows the expression level of galectin-7 in the medium when soybean extract is added to an epidermal model. 11 shows the expression level of claudin-1 in a skin model when soybean extract is added. 12 shows the expression level of filaggrin in a skin model when soybean extract is added.

[0008] Hereinafter, embodiments of the present disclosure will be described in detail.

[0009] The glucose transporter 1 expression promoter according to this embodiment (hereinafter, the "glucose transporter 1 expression promoter according to this embodiment" may also be referred to as the "GLUT1 expression promoter") contains a water-soluble plant polysaccharide. The GLUT1 expression promoter can more effectively promote the expression of glucose transporter 1 in cells that constitute the epidermis. The cells that constitute the epidermis may be, for example, epidermal keratinocytes.

[0010] The water-soluble plant polysaccharides are not particularly limited, and examples thereof include black tea leaf polysaccharides and soybean polysaccharides. Examples of black tea leaf polysaccharides include polysaccharides derived from Darjeeling tea leaves, polysaccharides derived from Dimbula tea leaves, polysaccharides derived from Assam tea leaves, and polysaccharides derived from Uva tea leaves. Examples of soybean polysaccharides include polysaccharides derived from soybean pulp. The GLUT1 expression promoter may contain one type of water-soluble plant polysaccharide, or may contain two or more types of water-soluble plant polysaccharides. In this specification, "plant-based" means a component derived from a plant. In this specification, black tea leaf polysaccharides, which are water-soluble plant polysaccharides, are also referred to as water-soluble black tea leaf polysaccharides.

[0011] The water-soluble plant polysaccharides contain, as constituent sugars, galactose, arabinose, and galacturonic acid, and may further contain monosaccharides such as xylose, fucose, rhamnose, glucose, and mannose.

[0012] The proportion of galactose in all the constituent sugars of the water-soluble plant polysaccharide is 15 to 60 mol %, may be 18 to 55 mol %, or may be 22 to 50 mol %.

[0013] The proportion of arabinose in the total sugar components of the water-soluble plant polysaccharide is 10 to 50 mol %, may be 15 to 45 mol %, or may be 20 to 40 mol %.

[0014] The proportion of galacturonic acid in all constituent sugars of the water-soluble plant polysaccharide may be 4 to 35 mol %, 5 to 30 mol %, or 6 to 25 mol %.

[0015] The total proportion of galactose, arabinose, and galacturonic acid in all constituent sugars of the water-soluble plant polysaccharide may be 50 to 98 mol%, 57 to 90 mol%, or 65 to 85 mol%.

[0016] The proportion of xylose in all constituent sugars of the water-soluble plant polysaccharide may be 0.5 to 10 mol% or 1 to 15 mol%, the proportion of fucose may be 1 to 8 mol% or 2 to 6 mol%, the proportion of rhamnose may be 0.5 to 10 mol% or 1.5 to 7 mol%, the proportion of glucose may be 0.5 to 25 mol% or 1 to 10 mol%, and the proportion of mannose may be 0.1 to 4 mol% or 0.2 to 3 mol%.

[0017] The proportion of galactose in the total sugar components of the black tea leaf polysaccharides may be 20 to 30 mol %, 23 to 30 mol %, or 25 to 29 mol %.

[0018] The proportion of arabinose in the total sugar components of the black tea leaf polysaccharides may be 30 to 40 mol %, 30 to 37 mol %, or 30 to 35 mol %.

[0019] The proportion of galacturonic acid in the total sugar components of the black tea leaf polysaccharides may be more than 0 mol% and not more than 10 mol%, 2 mol% to 6 mol%, 15 to 25 mol%, 15 to 24 mol%, or 16 to 22 mol%.

[0020] The total proportion of galactose and arabinose in all constituent sugars of the black tea leaf polysaccharides may be 50 to 90 mol%, 60 to 90 mol%, 70 to 90 mol%, 58 to 78 mol%, 60 to 75 mol%, or 63 to 73 mol%.

[0021] The total proportion of galactose, arabinose, and galacturonic acid in the total sugar constituents of the black tea leaf polysaccharides may be 70 to 90 mol %, 72 to 85 mol %, or 75 to 82 mol %.

[0022] The proportion of xylose in all constituent sugars of the black tea leaf polysaccharides may be 1 to 8 mol%, or may be 2 to 7 mol%, the proportion of fucose may be 0.1 to 10 mol%, or may be 0.5 to 5 mol%, the proportion of rhamnose may be 1 to 10 mol%, or may be 2 to 8 mol%, the proportion of glucose may be 1 to 10 mol%, or may be 2 to 8 mol%, and the proportion of mannose may be 0.1 to 5 mol%, or may be 0.1 to 3 mol%.

[0023] The proportion of galactose in all the constituent sugars of the soybean polysaccharide may be 30 to 50 mol %, 33 to 48 mol %, or 37 to 46 mol %.

[0024] The proportion of arabinose in all constituent sugars of the soybean polysaccharide may be 15 to 35 mol %, 20 to 34 mol %, or 23 to 33 mol %.

[0025] The proportion of galacturonic acid in all constituent sugars of the soybean polysaccharide may be 5 to 20 mol %, 6 to 15 mol %, or 6.5 to 10 mol %.

[0026] The total proportion of galactose, arabinose, and galacturonic acid in all the constituent sugars of the soybean polysaccharide may be 60 to 90 mol %, 70 to 88 mol %, or 75 to 85 mol %.

[0027] The proportion of xylose in all constituent sugars of the soybean polysaccharide may be 2 to 10 mol% or 3 to 8 mol%, the proportion of fucose may be 1 to 8 mol% or 2 to 6 mol%, the proportion of rhamnose may be 0.5 to 7 mol% or 1.5 to 5 mol%, the proportion of glucose may be 1 to 30 mol% or 2 to 10 mol%, and the proportion of mannose may be 0.05 to 5 mol% or 0.1 to 3 mol%.

[0028] The proportion of each constituent sugar in the total sugar content of the water-soluble plant polysaccharide is measured by the following method. First, the water-soluble plant polysaccharide is converted into monosaccharides by acid hydrolysis, and each monosaccharide is then labeled with 4-aminobenzoic acid ethyl ester by reductive amination. The labeled sample is subjected to reverse-phase chromatography to separate the monosaccharides, and the absorbance of each monosaccharide at 305 nm in ultraviolet light is measured. The concentration of each monosaccharide in the sample is then determined based on the absorbance using an absolute calibration curve method. The acid hydrolysis may be performed, for example, by storing the water-soluble plant polysaccharide in a 3 to 5 mol / L aqueous trifluoroacetic acid solution at 100 to 120°C for 2 to 4 hours. The acid hydrolysis may also be performed by adding a 3 to 5 mol / L aqueous trifluoroacetic acid solution to a plant extract containing the water-soluble plant polysaccharide. Specifically, the measurement may be performed using the method described in the Examples.

[0029] The black tea leaf polysaccharides may contain black tea leaf polysaccharides having a molecular weight of 100 to 1000 and / or black tea leaf polysaccharides having a molecular weight of 3000 to 50000. The proportion of the black tea leaf polysaccharides having a molecular weight of 100 to 1000 in the black tea leaf polysaccharides may be 5 to 20% by mass, or may be 10 to 15% by mass. The proportion of the black tea leaf polysaccharides having a molecular weight of 3000 to 50000 in the black tea leaf polysaccharides may be 10 to 30% by mass, or may be 15 to 25% by mass.

[0030] The soybean polysaccharides may contain the soybean polysaccharides having a molecular weight of 30,000 to 1,000,000. The proportion of the soybean polysaccharides having a molecular weight of 30,000 to 1,000,000 in the soybean polysaccharides may be 20% by mass or more, 40% by mass or more, or 40 to 90% by mass.

[0031] The proportion of water-soluble plant polysaccharides having a molecular weight within a predetermined range is calculated from an integrated molecular weight distribution curve obtained by size exclusion chromatography. Specifically, size exclusion chromatography is performed using a high-performance GPC apparatus (manufactured by Tosoh Corporation, model number: HLC 8420GPC), a "TSKgel guard column α, 6.0 mm ID x 4 cm" (manufactured by Tosoh Corporation) as a guard column, "TSKgel α-M, 7.8 mm ID x 30 cm, particle size 13 μm" (manufactured by Tosoh Corporation) and "TSKgel α-3000, 7.8 mm ID x 30 cm, particle size 7 μm" (manufactured by Tosoh Corporation) as columns, and a 0.5 mmol / L sodium acetate buffer solution as the mobile phase. The measurement conditions are a column temperature of 40°C and a flow rate of 0.6 mL / min. A calibration curve is obtained using standard polyethylene oxide (Tosoh SE Kit, manufactured by Tosoh). Samples subjected to size exclusion chromatography may be ones from which impurities have been removed, if necessary. For example, when using a plant extract as described below as a sample, impurities may be removed by adding the plant extract to a sodium acetate buffer, stirring, allowing to stand for 18 hours or more, and then centrifuging the mixture at 12,000 g for 5 minutes to obtain a supernatant, and then removing unnecessary precipitates from the supernatant using a chromatodisc with a filter pore size of 0.2 μm.

[0032] The GLUT1 expression promoter may contain the water-soluble plant polysaccharide by containing a plant extract containing the water-soluble plant polysaccharide. The plant extract is not particularly limited as long as it contains the water-soluble plant polysaccharide, and examples thereof include black tea leaf extract, soybean extract, etc. Examples of black tea leaf extract include Darjeeling tea leaf extract, Dimbula tea leaf extract, Assam tea leaf extract, Uva tea leaf extract, etc. Examples of soybean extract include soybean pulp extract, etc. The GLUT1 expression promoter may contain one type of plant extract, or may contain two or more types of plant extracts.

[0033] The content of the water-soluble plant polysaccharides in the plant extract may be 30 to 98% by mass, or 40 to 95% by mass, based on the total dry weight of the plant extract.

[0034] The content of black tea leaf polysaccharides in the black tea leaf extract may be 30 to 70% by mass, or 40 to 60% by mass, based on the total dry weight of the black tea leaf extract.

[0035] The soybean polysaccharide content in the soybean extract may be 50 to 98% by mass, or 60 to 95% by mass, based on the total dry weight of the soybean extract.

[0036] The content of the water-soluble plant polysaccharides in the plant extract is calculated by calculating the total content of each monosaccharide in the plant extract from the concentration of each monosaccharide determined in the measurement of the proportion of each constituent sugar in the total sugar content of the water-soluble plant polysaccharide. Specifically, the content of the water-soluble plant polysaccharides in the plant extract can be determined by the method described in the Examples.

[0037] In addition to the water-soluble plant polysaccharides, the plant extract may contain, for example, proteins and polyphenols such as theaflavins and thearubigins. For example, the black tea leaf extract may contain polyphenols, and the content of polyphenols in the black tea leaf extract may be 1 to 20% by mass, or 3 to 15% by mass, based on the total dry weight of the black tea leaf extract.

[0038] The content of theaflavins and thearubigins in the plant extract is measured by measuring the absorbance at 375 nm of an aqueous solution containing the plant extract, determining the concentrations of theaflavins and thearubigins in the aqueous solution based on the absorbance using an absolute calibration curve method, and calculating the content. Specifically, the measurement can be performed using the method described in the Examples.

[0039] The method for producing the plant extract may be, for example, as follows. First, purified water, an aqueous acetic acid solution, or the like is added to a plant-derived raw material such as black tea leaves (e.g., Darjeeling tea leaves) or dried soybean pulp, and the water-soluble plant polysaccharides are extracted by heating and pressurizing. Next, solids are removed from the resulting extract using a 100-mesh sieve or the like, and the extract is then dried by freeze-drying or the like. From the viewpoint of efficient extraction of the water-soluble plant polysaccharides, the heating and pressurizing is preferably carried out for 1 to 3 hours at a temperature of 105 to 135°C and a pressure of 0.12 to 0.31 MPa. From the viewpoint of extracting the water-soluble plant polysaccharides with a more excellent GLUT1 expression-promoting effect, the heating and pressurizing is preferably carried out at a temperature of 115 to 130°C and a pressure of 0.15 to 0.3 MPa. Furthermore, in the method for producing the water-soluble plant polysaccharides, a plant-derived raw material from which unnecessary components have been removed in advance may be used as the plant-derived raw material. A method for removing unnecessary components may be, for example, a method in which purified water is added to a plant-derived raw material, followed by heat treatment at 70 to 100°C to extract the unnecessary components, and the remaining raw material is recovered.

[0040] The black tea leaf extract can be obtained, for example, by using black tea leaves such as Darjeeling tea leaves as a raw material in the above-mentioned method for producing a plant extract. It is preferable to use black tea leaves such as Darjeeling tea leaves from which unnecessary components have been removed. A preferred method for removing unnecessary components involves first adding hot water at 90 to 100°C to black tea leaves such as Darjeeling tea leaves, stirring for 2 to 4 minutes, and then recovering the tea leaves through a 100-mesh sieve or the like. Purified water is then added to the tea leaves, followed by heating and stirring at 70 to 90°C for 3 hours or more, and then recovering the tea leaves from which unnecessary components have been removed through a 100-mesh sieve or the like. In the method for producing the black tea leaf extract, the heating and pressurizing is preferably carried out by adding purified water to the raw material. Known black tea leaves such as Darjeeling tea leaves may be used.

[0041] The soybean extract can be obtained, for example, by using dried soybean lees or other okara as a raw material in the above-mentioned plant extract production method. The heating and pressurizing in the soybean extract production method is preferably carried out by adding a 2.5 to 10 mmol / L aqueous acetic acid solution to the raw material. The dried soybean lees or other okara may be any known okara.

[0042] The GLUT1 expression promoter can exhibit at least one effect selected from the group consisting of suppressing the expression of galectin-7, promoting the expression of claudin-1, and promoting the expression of filaggrin. The above effect is exhibited more effectively in cells constituting the epidermis, and can be exhibited even more effectively in cells constituting the epidermis with reduced barrier function and / or reduced moisture content in the stratum corneum. That is, the GLUT1 expression promoter may be at least one selected from the group consisting of a galectin-7 expression inhibitor, a claudin-1 expression promoter, and a filaggrin expression promoter, or may be at least one selected from the group consisting of a galectin-7 expression inhibitor, a claudin-1 expression promoter, and a filaggrin expression promoter in cells constituting the epidermis, or may be at least one selected from the group consisting of a galectin-7 expression inhibitor, a claudin-1 expression promoter, and a filaggrin expression promoter in cells constituting the epidermis with reduced barrier function and / or reduced moisture content in the stratum corneum. In cells constituting epidermis with reduced barrier function, increased expression of galectin-7, decreased expression of claudin-1, decreased expression of filaggrin, etc. may occur due to the reduced barrier function. Furthermore, a reduced barrier function of the epidermis is likely to cause dry skin, rough skin, etc. Here, epidermis with reduced barrier function refers to epidermis in which the normal barrier function has been impaired due to a disruption of the sebum film and / or stratum corneum, and examples include epidermis treated with a surfactant such as sodium dodecyl sulfate. The cells constituting the epidermis may be, for example, epidermal keratinocytes.

[0043] When the GLUT1 expression promoter contains the above-mentioned black tea leaf polysaccharides, the above-mentioned effect can be particularly effectively achieved by using the agent on the epidermis after the barrier function has been weakened and / or the epidermis after the moisture content of the stratum corneum has decreased.

[0044] When the GLUT1 expression promoter contains the above-mentioned soybean polysaccharide, the above-mentioned effect can be particularly effectively achieved by using the agent on the epidermis before the barrier function is impaired and / or on the epidermis before the moisture content of the stratum corneum is reduced.

[0045] It is known that the expression level of galectin-7 decreases as rough skin improves (for example, JP 2015-042970 A). Therefore, the GLUT1 expression promoter can be a therapeutic or preventive agent for rough skin. When the GLUT1 expression promoter contains the black tea leaf polysaccharide, the GLUT1 expression promoter can be more effective as a preventive agent for rough skin. When the GLUT1 expression promoter contains the soybean polysaccharide, the GLUT1 expression promoter can be more effective as a therapeutic agent for rough skin.

[0046] Claudin 1 is a component of tight junctions. Therefore, the GLUT1 expression promoter can be a therapeutic or preventive agent for a decrease in the barrier function of the epidermis. When the GLUT1 expression promoter contains the black tea leaf polysaccharide, the GLUT1 expression promoter can be more effective as a preventive agent for a decrease in the barrier function of the epidermis. When the GLUT1 expression promoter contains the soybean polysaccharide, the GLUT1 expression promoter can be more effective as a therapeutic agent for a decrease in the barrier function of the epidermis.

[0047] The GLUT1 expression promoter can increase the moisture content of the stratum corneum. This effect can be more effectively achieved in epidermis with reduced barrier function. Furthermore, filaggrin is a raw material for natural moisturizing factors. From the above, the GLUT1 expression promoter can be a therapeutic or preventive agent for dry skin. When the GLUT1 expression promoter contains the black tea leaf polysaccharide, the GLUT1 expression promoter can be more effective as a preventive agent for dry skin. When the GLUT1 expression promoter contains the soybean polysaccharide, the GLUT1 expression promoter can be more effective as a therapeutic agent for dry skin.

[0048] The GLUT1 expression promoter can be applied to humans and non-human mammals, preferably humans, including mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys.

[0049] The GLUT1 expression promoter is preferably applied to the epidermis, i.e., is for use on the epidermis, including any epidermis of the body, such as the scalp, face (forehead, cheeks, lips, nose, ears, etc.), neck, shoulders, back, chest, abdomen, genitals, arms, hands, lower limbs, feet, nails, hair, etc.

[0050] The GLUT1 expression promoter may contain the water-soluble plant polysaccharide in an amount of 0.001 to 5% by mass, or 0.01 to 0.5% by mass, based on the total amount of the agent.

[0051] When the GLUT1 expression promoter contains the above-mentioned plant extract, it may contain 0.01 to 30% by mass, or 0.1 to 10% by mass, of the above-mentioned plant extract based on the total amount of the agent.

[0052] The dosage of the GLUT1 expression promoter may be 0.001 to 60 g, or 0.01 to 40 g per day for a subject weighing 60 kg.

[0053] The GLUT1 expression promoter may be administered 1 to 3 times per day, or 1 to 2 times per day.

[0054] The administration method of the GLUT1 expression promoter may be transdermal administration, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, or ophthalmic administration, and transdermal administration is preferred. That is, the GLUT1 expression promoter may be an external preparation. Examples of transdermal administration methods include a method of applying the agent to a body surface such as the epidermis, or a method of applying the agent to the epidermis.

[0055] The GLUT1 expression promoter may be in the form of, for example, a solid; a liquid (solution or suspension); an emulsion such as a lotion or cream; a paste; a gel; or a mousse.

[0056] The GLUT1 expression promoter can be used for both therapeutic and non-therapeutic purposes (e.g., cosmetic purposes). Specifically, the GLUT1 expression promoter may be a drug, quasi-drug, cosmetic, or food, and is preferably a cosmetic. Drugs, quasi-drugs, cosmetics, and foods containing the GLUT1 expression promoter can each be manufactured according to conventional methods. The content of the GLUT1 expression promoter in the drug, quasi-drug, cosmetic, or food is not particularly limited and can be freely set depending on the purpose.

[0057] The GLUT1 expression promoter may contain ingredients other than the water-soluble plant polysaccharides or plant extracts, provided that the ingredients do not impair the effect of the promoter as a preventive or ameliorating agent for cellular aging. Specific examples of such ingredients include moisturizing ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell activating ingredients, anti-aging ingredients, blood circulation promoting ingredients, UV protection ingredients, whitening ingredients, vitamins, proteins, peptides, amino acids, alcohols, etc. These active ingredients may be used singly or in combination as needed.

[0058] When the GLUT1 expression promoter is a drug, quasi-drug, cosmetic, or food, the drug, quasi-drug, cosmetic, or food may contain, in addition to the GLUT1 expression promoter, ingredients that are typically used in drugs, quasi-drugs, cosmetics, or foods. The drug, quasi-drug, cosmetic, or food according to one embodiment may contain bases, carriers, additives, etc. that are typically used in drugs, quasi-drugs, cosmetics, or foods. Examples of additives include excipients, oils, powders, buffers, solubilizers, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, antiseptics, pigments, colorants, fragrances, gloss-imparting agents, gelling agents, alcohols, water-soluble polymers, film-forming agents, resins, keratolytic agents, etc. The bases, carriers, and various additives described above may be used singly or in combination, as needed.

[0059] When the GLUT1 expression promoter is a pharmaceutical product, the pharmaceutical product may be in the form of, for example, an aerosol, a liquid, a suspension, an emulsion, a cream, an ointment, a gel, a liniment, a lotion, a cataplasm, a tape, an eye drop, a nose drop, an ear drop, a suppository, an elixir, a capsule, a granule, a pill, a powder, a tablet, a syrup, an injection, or a troche. When the GLUT1 expression promoter is used as a pharmaceutical product for the epidermis, it is preferably an external preparation. By using it as an external preparation, the water-soluble plant polysaccharide acts directly on epidermal cells, thereby achieving a more effective effect. The external preparation preferably has the form of an aerosol, a liquid, a suspension, an emulsion, a cream, an ointment, a gel, a liniment, a lotion, a cataplasm, or a tape.

[0060] When the GLUT1 expression promoter is a quasi-drug or cosmetic, the quasi-drug or cosmetic may take the form of, for example, skin lotion, emulsion, cream, gel, serum, sunscreen cosmetic, pack, hand cream, foot cream, body lotion, body cream, and other basic cosmetics; cleansing cosmetics such as face wash, makeup remover, soap, body shampoo, shampoo, rinse, conditioner, and nail polish remover; makeup cosmetics such as foundation, makeup base, lip balm, lipstick, cheek color, eye color, eyebrow pencil, nail polish, and hair color; antiperspirant; bath additives; perfume; foods and beverages such as nutritional functional foods, functional foods, foods for specified health uses, and foods for special dietary uses. When used as a quasi-drug or cosmetic for the epidermis, it is preferably an external preparation, just like a pharmaceutical. By using it as an external preparation, for example, the water-soluble plant polysaccharide acts directly on epidermal cells, thereby more effectively exerting its effects. The quasi-drugs or cosmetics as external preparations are preferably in the form of quasi-drugs and cosmetics other than foods and drinks.

[0061] The present invention will be described in more detail below with reference to examples, but is not limited to these examples. In the tables and figures, black tea A to D and soybean A to D represent black tea leaf extracts A to D and soybean extracts A to D, respectively.

[0062] Example 1: Method for preparing plant extract (test sample) (Black tea leaf extract A) 500 mL of boiling water (tap water) at approximately 100°C was added to 10 g of dried Darjeeling tea leaves (manufactured by Lipton), and after stirring for 3 minutes, the solids were removed using a 100 mesh sieve and then filtered through a sterilized filter with a pore size of 0.45 μm to obtain black tea liquid A. Black tea liquid A was freeze-dried to obtain black tea extract A.

[0063] (Black Tea Leaf Extracts B to D) 60 g of dried Darjeeling tea leaves (manufactured by Lipton) were added to 500 mL of approximately 100°C hot water (tap water), stirred for 3 minutes, and the tea leaves were filtered through a 100-mesh sieve. 1500 mL of purified water was added to the tea leaves, and the mixture was heated and stirred in an 80°C hot bath for at least 3 hours. The tea leaves were then filtered to obtain black tea leaf residue. The black tea leaf residue was divided into three portions (approximately 20 g each), and 200 mL of purified water was added to each portion. The mixture was then pressurized and heated in an autoclave (under conditions of 106°C and 0.02 MPa, 121°C and 0.1 MPa, or 130°C and 0.2 MPa for 3 hours, respectively). Note that the pressurization referred to here refers to pressure applied relative to atmospheric pressure (approximately 0.101 MPa). Solids were removed from the obtained extract using a 100-mesh sieve, and the extract was then filtered through a sterilized filter with a pore size of 0.45 μm. The obtained clarified filtrate was freeze-dried to obtain black tea leaf extracts B to D (black tea leaf extract B: 106°C, black tea leaf extract C: 121°C, black tea leaf extract D: 130°C).

[0064] (Soybean extract A) 200 mL of hot water (tap water) at about 100°C was added to 10 g of dried soybean refuse (manufactured by AEON Corporation), stirred for 3 minutes, and then the solid content was removed using a 100-mesh sieve, followed by filtration through a sterilized filter with a pore size of 0.45 µm to obtain soybean liquid A. Soybean liquid A was freeze-dried to obtain soybean extract A.

[0065] (Soybean Extracts B to D) 400 mL of 5 mmol / L aqueous acetic acid solution (pH 3.5) was added to three 20 g portions of dried soybean pulp (manufactured by AEON Corporation), and the mixture was pressurized and heated in an autoclave (for 3 hours under conditions of 106°C and 0.02 MPa, 121°C and 0.1 MPa, and 130°C and 0.2 MPa, respectively). Note that the above pressurization refers to pressurization against atmospheric pressure (approximately 0.101 MPa). Solids were removed from the obtained extract using a 100-mesh sieve, and then the extract was filtered through a sterilized filter with a pore size of 0.45 μm. The obtained clear filtrate was freeze-dried to obtain Soybean Extracts B to D (Soybean Extract B: 106°C, Soybean Extract C: 121°C, Soybean Extract D: 130°C).

[0066] Example 2: Molecular weight distribution measurement The integral and differential molecular weight distributions of the black tea leaf polysaccharides A to D and the soybean polysaccharides A to D contained in the black tea leaf extracts A to D and the soybean extracts A to D, respectively, were determined by size exclusion chromatography (hereinafter also referred to as "GPC"). From the integral molecular weight distributions, the proportion (mass %) of each polysaccharide having a molecular weight within a predetermined range was calculated. An "HLC 8420GPC" (manufactured by Tosoh Corporation) was used as the high-speed GPC apparatus, a "TSKgel guard column α, 6.0 mm I.D. × 4 cm" (manufactured by Tosoh Corporation) was used as the guard column, and "TSKgel α-M, 7.8 mm I.D. × 30 cm, particle size 13 μm" (manufactured by Tosoh Corporation) and "TSKgel α-3000, 7.8 mm I.D. × 30 cm, particle size 7 μm" (manufactured by Tosoh Corporation) were used as the columns. Ten milligrams of each test sample (black tea leaf extracts A-D and soybean extracts A-D) obtained in Example 1 was added to 1 mL of sodium acetate buffer, gently stirred, and then allowed to stand at room temperature for 18 hours. The resulting mixture was then centrifuged (12,000 g, 5 minutes), and the supernatant was passed through a chromatodisc (0.2 μm) to obtain the black tea leaf polysaccharides or soybean polysaccharides for analysis. Using the above-described equipment and samples, analysis was performed under conditions of a flow rate of 0.6 mL / min, a column temperature of 40°C, and a sample injection volume of 10 μL, using a mobile phase (0.5 mmol / L sodium acetate buffer (pH 5)). The integral and differential molecular weight distributions were determined using standard polyethylene oxide (Tosoh SE Kit, manufactured by Tosoh) as a standard marker with a known molecular weight. The results for the proportion of each polysaccharide within the specified molecular weight range are shown in Tables 1 and 2, and the integral and differential molecular weight distributions are shown in Figures 1 and 2. 1 and 2, the integral molecular weight distribution is a plot of the integral value of the weight fraction, and the differential molecular weight distribution is a plot of the differential value of the curve of the integral molecular weight distribution. Also, [Log M] means the common logarithm of the molecular weight.

[0067]

[0068]

[0069] Example 3: Sugar composition analysis High performance liquid chromatography (hereinafter also referred to as "HPLC") was used to determine the contents (mass%) of black tea leaf polysaccharides A to D and soybean polysaccharides A to D based on the dry weight of black tea leaf extracts A to D and soybean extracts A to D, and the proportion (mol%) of each monosaccharide in the total sugar constituents of each polysaccharide, based on the dry weight of each black tea leaf extract A to D and soybean extract A to D. An "LC-2010" (manufactured by Shimadzu Corporation) was used as the HPLC apparatus, and a "Honenpak C18" (manufactured by Seikagaku Corporation) was used as the column. To 100 μL of a 1 mg / mL aqueous solution of each test sample (black tea leaf extracts A-D and soybean extracts A-D) obtained in Example 1 or a standard sugar mixture solution containing 12.5 mmol / L each of galacturonic acid, galactose, mannose, glucose, arabinose, xylose, fucose, and rhamnose, 2.0 mL of 100 μL of a 4 mol / L aqueous trifluoroacetic acid solution (final concentration: 2 mol / L) was added and incubated at 110°C for 2 hours. The mixture was then allowed to cool, and the trifluoroacetic acid was removed using a centrifugal concentrator (25°C, 2 hours), followed by lyophilization. The dried sample was dissolved in 50 μL of sterile purified water. 40 μL of ABEE labeling reagent (Seikagaku Corporation) was added to 10 μL of the solution, and the mixture was incubated at 80°C for 1 hour. Then, 200 μL of sterile purified water and 200 μL of chloroform were added, followed by centrifugation, and the supernatant was subjected to HPLC analysis. Using the above equipment and various supernatants, HPLC analysis was performed using a mobile phase (0.2 mol / L potassium borate buffer (pH 9.0) / acetonitrile (93 / 7, v / v)) under conditions of a flow rate of 1 mL / min, a column temperature of 30°C, and a UV wavelength of 305 nm. The concentrations of eight sugars (galacturonic acid, galactose, mannose, glucose, arabinose, xylose, fucose, and rhamnose) were determined using the absolute calibration curve method. Each of the above supernatants was subjected to HPLC analysis twice, and the average of the two analyses was calculated as the concentration. The percentage of each polysaccharide (sugars (mass%)) based on the dry weight of each extract and the percentage of each monosaccharide in the total sugar constituents of each polysaccharide (sugar composition (mol%)) are shown in Table 3 for black tea leaf extracts A to D, and in Table 4 for soybean extracts A to D.In Tables 3 and 4, "GalA," "Gal," "Man," "Glu," "Ara," "Xyl," "Fuc," and "Rha" represent galacturonic acid, galactose, mannose, glucose, arabinose, xylose, fucose, and rhamnose, respectively.

[0070]

[0071]

[0072] Example 4: Absorbance Measurement 100 μL of each of the 0.1 to 2 mg / mL aqueous solutions of black tea leaf extracts A to D obtained in Example 1 and a 0.01 to 2 mg / mL aqueous solution of theaflavin (biochemical grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was dispensed into a 96-well microplate. After dispensing, the absorbance (375 nm) of theaflavins and thearubigins was measured using a microplate reader. For the quantification of theaflavins and thearubigins in black tea leaf extracts A to D, the concentrations were calculated using the absolute calibration curve method. The results are shown in Table 3.

[0073] Example 5: Epidermal model A human 3D cultured epidermal model was used as the epidermal model. A LabCyte EPI-KIT (manufactured by Japan Tissue Engineering Co., Ltd.) was used to analyze the expression levels of proteins in the cells and culture medium of the human 3D cultured epidermal model. For the human 3D cultured epidermal model, 1.5 mL of assay medium was dispensed into a 12-well assay plate in advance, and 0.5 mL of a suspension of epidermal keratinocytes diluted in assay medium was seeded into each well of a cell culture insert. The model was incubated overnight at 37°C in 5% CO ２ The cells were cultured in an incubator. The assay medium in the cell culture inserts was removed the next day. The medium in the 12-well assay plate was then replaced with the same assay medium every 2-3 days and cultured for 14 days. The human 3D cultured epidermal model after 14 days of culture was used in the tests described below. The 3D cultured epidermal model has a basal layer, spinous layer, granular layer, and stratum corneum, and is used as an in vitro model that physiologically and functionally reproduces the state of human epidermis.

[0074] Example 6 The effects of the black tea leaf extracts A to D obtained in Example 1 on the expression level of glucose transporter 1 (hereinafter also referred to as "GLUT1") were evaluated.

[0075] Example 6-1: Preparation of sample for GLUT1 expression analysis 50 μL of phosphate buffer solution (hereinafter, "phosphate buffer solution" may also be referred to as "PBS(-)") containing 0.1% by mass of each of the test samples (black tea leaf extracts A to D) obtained in Example 1 was added to a cell culture insert on which a human three-dimensional cultured epidermal model was mounted, and the cells were incubated at 37°C and 5% CO for 24 hours. ２ The tissue was cultured in an incubator. After washing with PBS(-), the tissue from the human 3D cultured epidermal model was transferred to a 1.5 mL sample tube, and 200 μL of RIPA buffer (manufactured by Nacalai Tesque) was added. The tissue was then homogenized using an ultrasonic disrupter (Bioruptor UCD-250, manufactured by Sonic Bio) at a high output level (250 W) for 2 minutes (1 cycle [ON 10 seconds, OFF 10 seconds] x 6), followed by centrifugation to separate the polycarbonate membrane and insoluble protein as a precipitate, and the supernatant was collected as a protein extract. The protein content in each protein extract was quantified using the Bradford method.

[0076] Example 6-2 Preparation of Control Samples Protein extracts were collected in the same manner as in Example 6-1, except that PBS(-) not containing the test sample was used instead of PBS(-) containing the test sample, and the protein in each protein extract was quantified by the Bradford method.

[0077] Example 6-3: GLUT1 expression analysis Western blot analysis was performed on each protein extract using anti-GLUT1 antibody and anti-β-actin antibody. Specifically, each protein extract was loaded onto a 5-20% acrylamide gel so that the protein amounts were equal. The anti-GLUT1 antibody (Proteintech) and HRP-labeled anti-β-actin antibody (Proteintech) were used according to the manufacturer's instructions. For GLUT1, an HRP-labeled antibody (Proteintech) was used as the secondary antibody. The HRP was developed with 3,3'-diaminobenzidine (DBA). The intensity of the developed color was quantified using the open-source software "Image J," and the results are shown in Figure 3.

[0078] As shown in Figure 3, black tea leaf extract A did not increase the amount of GLUT1 expression in the skin model, but black tea leaf extracts B to D increased the amount of GLUT1 expression in the skin model. Black tea leaf extract D had the strongest effect of increasing GLUT1 expression, followed by black tea leaf extract C.

[0079] Example 7 The effects of black tea leaf extracts A to D obtained in Example 1 on the stratum corneum moisture content, galectin-7 expression level, claudin-1 (CLDN-1) expression level, and filaggrin (FLG) expression level after treatment with sodium dodecyl sulfate (SDS) were evaluated.

[0080] Example 7-1: Preparation of sample for expression analysis and measurement of moisture content of stratum corneum To a cell culture insert equipped with a human three-dimensional cultured epidermal model, 50 μL of PBS(−) containing 0.1% by mass of each of the test samples (black tea leaf extracts A to D) obtained in Example 1 was added, and the cells were incubated at 37°C and 5% CO for 24 hours. ２The skin models were cultured in an incubator. After washing with PBS(-), 50 μL of PBS containing 0.03% by mass of the surfactant SDS (biochemical grade, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the cell culture insert in order to reduce the barrier function of the epidermal model, and the cells were incubated for 4 hours. The assay medium from each well was then collected for the analysis of galectin-7 expression levels described below, and the skin models were washed with PBS(-) and then detached from the cell culture insert. The cells were then incubated at 37°C, 5% CO ２ The epidermal models were allowed to stand in an incubator for 30 minutes. The moisture content of the stratum corneum of each epidermal model was then measured three times for each model using an epidermal stratum corneum moisture content meter (SKICON-200EX, Yayoi Co., Ltd.). The mean values ​​± standard deviations are shown in Figure 4. After measuring the moisture content of the epidermal stratum corneum, the epidermal models were transferred to 1.5 mL sample tubes, and 200 μL of RIPA buffer (Nacalai Tesque) was added. The samples were homogenized using an ultrasonic homogenizer (Bioruptor UCD-250, Sonic Bio) at a high output level (250 W) for 2 minutes (1 cycle [ON 10 seconds, OFF 10 seconds] × 6). The samples were then centrifuged to separate the polycarbonate membrane and insoluble proteins as precipitates, and the supernatant was collected as a protein extract. Protein content in each protein extract was quantified using the Bradford method.

[0081] Example 7-2: Preparation of a control sample and measurement of the moisture content of the stratum corneum The moisture content of the stratum corneum was measured, and an assay medium and a protein extract were collected in the same manner as in Example 7-1, except that PBS(-) containing no test sample was used as a control instead of PBS(-) containing a test sample. The protein extract was quantified by the Bradford method. Furthermore, the moisture content of the stratum corneum was measured, and an assay medium and a protein extract were collected in the same manner as in Example 7-1, except that PBS(-) containing no test sample was used as a control (SDS-) instead of PBS(-) containing a test sample, and PBS(-) containing no SDS was used instead of PBS(-) containing 0.03% by mass of SDS. The protein extract was quantified by the Bradford method.

[0082] As shown in Figure 4, black tea leaf extract A did not increase the moisture content of the stratum corneum of the skin model, but black tea leaf extracts B to D increased the moisture content of the stratum corneum of the skin model. This effect of increasing the moisture content of the stratum corneum was equally strong for black tea leaf extracts D and C, followed by black tea leaf extract B.

[0083] Example 7-3: Galectin-7 expression analysis Because most of galectin-7 is released extracellularly, the expression level of galectin-7 in the assay medium was evaluated by measuring the galectin-7 in the assay medium using ELISA (Human Galectin-7 DuoSet Kit, manufactured by R&D Systems). The measurement wavelength was 450 nm, and the reference wavelength was 620 nm. Galectin-7 was measured three times, and the mean value ± standard deviation is shown in Figure 5.

[0084] As shown in Figure 5, black tea leaf extract A hardly reduced the expression level of galectin-7, but black tea leaf extracts B to D reduced the expression level of galectin-7. Black tea leaf extract D had the strongest effect of reducing the expression level of galectin-7, followed by black tea leaf extract C.

[0085] Example 7-4: CLDN-1 expression analysis Western blot analysis was performed using anti-CLDN-1 antibody and anti-β-actin antibody. Each protein extract was loaded onto a 5-20% acrylamide gel so that the protein amounts were equal. Anti-CLDN-1 antibody (Proteintech) and HRP-labeled anti-β-actin antibody (Proteintech) were used according to the manufacturer's instructions. For CLDN-1, an HRP-labeled antibody (Proteintech) was used as the secondary antibody. HRP was developed with 3,3'-diaminobenzidine. The intensity of the developed color was quantified using the open source software "Image J," and the CLDN-1 expression levels are shown in Figure 6.

[0086] As shown in Figure 6, black tea leaf extracts B to D significantly increased the CLDN-1 expression level compared to black tea leaf extract A. This CLDN-1 expression level increasing effect was strongest for black tea leaf extract C, followed by black tea leaf extract D.

[0087] Example 7-5: FLG expression analysis FLG in each protein extract was measured by ELISA (Enzyme-linked Immunosorbent Assay Kit for Filaggrin (FLG), manufactured by CLOUD-CLONE CORP. WUHAN). The measurement wavelength was 450 nm, and the reference wavelength was 620 nm. The amount of FLG when each test sample was added was calculated relative to the amount of filaggrin in the control (SDS-) as 1. FLG was measured three times, and the average value ± standard deviation is shown in Figure 7.

[0088] As shown in Figure 7, black tea leaf extract A did not increase the amount of FLG expression, but black tea leaf extracts B to D increased the amount of FLG expression. Black tea leaf extracts D and C had similarly strong effects on increasing FLG expression, followed by black tea leaf extract B.

[0089] Example 8 The effects of soybean extracts A to D on the expression level of GLUT1 were evaluated.

[0090] Example 8-1: Preparation of sample for GLUT1 expression analysis 50 μL of PBS(−) containing 0.1% by mass of each of the test samples (soybean extracts A to D) obtained in Example 1 was added to a culture cup containing a human 3D cultured epidermal model, and the culture was incubated at 37°C and 5% CO for 24 hours. ２ The tissue was cultured in an incubator. After washing with PBS(-), the tissue from the human 3D cultured epidermal model was transferred to a 1.5 mL sample tube, and 200 μL of RIPA buffer (manufactured by Nacalai Tesque) was added. The tissue was then homogenized using an ultrasonic disrupter (Bioruptor UCD-250, manufactured by Sonic Bio) at a high output level (250 W) for 2 minutes (1 cycle [ON 10 seconds, OFF 10 seconds] x 6), followed by centrifugation to separate the polycarbonate membrane and insoluble protein as a precipitate, and the supernatant was collected as a protein extract. The protein content in each protein extract was quantified using the Bradford method.

[0091] Example 8-2 Preparation of Control Samples Protein extracts were collected in the same manner as in Example 8-1, except that PBS(-) not containing the test sample was used instead of PBS(-) containing the test sample, and the protein in each protein extract was quantified by the Bradford method.

[0092] Example 8-3: GLUT1 expression analysis Western blot analysis was performed on each protein extract using an anti-GLUT1 antibody and an anti-β-actin antibody. Specifically, each protein extract was loaded onto a 5-20% acrylamide gel so that the protein amounts were equal. The anti-GLUT1 antibody (Proteintech) and the HRP-labeled anti-β-actin antibody (Proteintech) were used according to the manufacturer's instructions. For GLUT1, an HRP-labeled antibody (Proteintech) was used as the secondary antibody. The HRP was developed with 3,3'-diaminobenzidine (DBA). The intensity of the developed color was quantified using the open-source software "Image J," and the results are shown in Figure 8.

[0093] As shown in Figure 8, soybean extract A did not increase the amount of GLUT1 expression in the skin model, but soybean extracts B to D increased the amount of GLUT1 expression in the skin model. Soybean extract C had the strongest effect of increasing GLUT1 expression, followed by soybean extract D.

[0094] Example 9 The effects of soybean extracts A to D obtained in Example 1 on the stratum corneum moisture content, galectin-7 expression level, CLDN-1 expression level, and FLG expression level after SDS treatment were evaluated.

[0095] Example 9-1: Preparation of sample for expression analysis and measurement of moisture content of stratum corneum To a cell culture insert on which a human 3D cultured epidermal model was mounted, 50 μL of PBS(−) containing 0.0025% by mass of SDS (biochemical grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added in order to reduce the barrier function of the skin model, and the sample was incubated at 37°C and 5% CO for 24 hours. ２After washing with PBS(-), 50 μL of phosphate buffer solution (PBS(-)) containing 0.1% of each of the test samples (soybean extracts A to D) obtained in Example 1 was added to the cell culture insert, and the cells were cultured at 37°C and 5% CO for 24 hours. ２ The skin models were then washed with PBS(-), detached from the cell culture insert, and incubated at 37°C, 5% CO ２ The samples were allowed to stand in an incubator for 30 minutes. The moisture content of the stratum corneum of each epidermal model was then measured three times for each model using an epidermal stratum corneum moisture content meter (SKICON-200EX, Yayoi Co., Ltd.). The mean values ​​± standard deviations are shown in Figure 9 . After measuring the moisture content of the epidermal stratum corneum, the tissue samples were transferred to 1.5 mL sample tubes, and 200 μL of RIPA buffer (Nacalai Tesque) was added. The samples were homogenized using an ultrasonic homogenizer (Bioruptor UCD-250, Sonic Bio) at a high output level (250 W) for 2 minutes (1 cycle [ON 10 seconds, OFF 10 seconds] × 6). The samples were then centrifuged to separate the polycarbonate membrane and insoluble proteins as precipitates, and the supernatant was collected as a protein extract. Protein content in each protein extract was quantified using the Bradford method.

[0096] Example 9-2: Preparation of a control sample and measurement of the moisture content of the stratum corneum The moisture content of the stratum corneum was measured, and an assay medium and a protein extract were collected in the same manner as in Example 9-1, except that PBS(-) containing no test sample was used as a control instead of PBS(-) containing a test sample. The protein content of the protein extract was quantified by the Bradford method. Furthermore, the moisture content of the stratum corneum was measured, and an assay medium and a protein extract were collected in the same manner as in Example 9-1, except that PBS(-) containing no test sample was used as a control (SDS-) instead of PBS(-) containing a test sample, and PBS(-) containing no SDS was used instead of PBS(-) containing 0.0025% by mass of SDS. The protein content of the protein extract was quantified by the Bradford method.

[0097] As shown in FIG. 9, soybean extracts C and D significantly increased the moisture content of the stratum corneum of the skin model compared to soybean extract A.

[0098] Example 9-3: Analysis of Galectin-7 Expression Galectin-7 in the assay medium was measured by ELISA (Human Galectin-7 DuoSet Kit, manufactured by R&D Systems) to evaluate the expression level of galectin-7. The measurement wavelength was 450 nm, and the reference wavelength was 620 nm. Galectin-7 was measured three times, and the mean value ± standard deviation is shown in Figure 10.

[0099] 10 , soybean extracts B to D significantly reduced the expression level of galectin-7 compared to soybean extract A. This effect of reducing the expression level of galectin-7 was equally strong for soybean extracts C and D, followed by soybean extract B.

[0100] <Example 9-4: CLDN-1 expression analysis> Western blot analysis was performed using anti-CLDN-1 antibody and anti-β-actin antibody. Each protein extract was loaded onto a 5-20% acrylamide gel so that the protein amounts were equal. Anti-CLDN-1 antibody (Proteintech) and HRP-labeled anti-β-actin antibody (Proteintech) were used according to the manufacturer's instructions. For CLDN-1, an HRP-labeled antibody (Proteintech) was used as the secondary antibody. HRP was developed with 3,3'-diaminobenzidine. The intensity of the developed color was quantified using the open source software "Image J," and the CLDN-1 expression levels are shown in Figure 11.

[0101] 11, soybean extract A did not increase the amount of CLDN-1 expression, but soybean extracts B to D increased the amount of CLDN-1 expression in the skin model. This effect of increasing CLDN-1 expression was equally strong for soybean extracts C and D, followed by soybean extract B.

[0102] Example 9-5: FLG expression analysis FLG in each protein extract was measured by ELISA (Enzyme-linked Immunosorbent Assay Kit for Filaggrin (FLG), manufactured by CLOUD-CLONE CORP. WUHAN). The measurement wavelength was 450 nm, and the reference wavelength was 620 nm. The amount of FLG in each test sample was calculated relative to the amount of FLG in the control (SDS-) as 1. FLG was measured three times, and the average value ± standard deviation is shown in FIG. 12.

[0103] As shown in Figure 12, soybean extract A did not increase the amount of FLG expression, but soybean extracts C and D increased the amount of FLG expression in the skin model. Soybean extract D had the strongest effect of increasing FLG expression, followed by soybean extract C.

Claims

1. An expression promoter for glucose transporter 1 comprising a water-soluble plant polysaccharide, wherein the proportion of galactose in the total sugar constituents of the water-soluble plant polysaccharide is 15 to 60 mol%, the proportion of arabinose in the total sugar constituents of the water-soluble plant polysaccharide is 10 to 50 mol%, and the proportion of galacturonic acid in the total sugar constituents of the water-soluble plant polysaccharide is 4 to 35 mol%, and the total proportion of galactose, arabinose, and galacturonic acid in the total sugar constituents of the water-soluble plant polysaccharide is 50 to 98 mol%.

2. The expression promoter described in claim 1, which is at least one selected from the group consisting of an expression inhibitor of galectin-7, an expression promoter of claudin-1, and an expression promoter of filaggrin.

3. The expression promoter according to claim 1, which is at least one therapeutic or preventive agent selected from the group consisting of dry skin, impaired epidermal barrier function, and rough skin.

4. The expression promoter according to claim 1, wherein the water-soluble plant polysaccharide is black tea leaf polysaccharide.

5. An agent for promoting expression of glucose transporter 1 comprising water-soluble black tea leaf polysaccharides, the water-soluble black tea leaf polysaccharides containing galactose, arabinose, and galacturonic acid as constituent sugars, the proportion of galactose in the total constituent sugars of the water-soluble black tea leaf polysaccharides being 20 to 30 mol%, and the proportion of arabinose in the total constituent sugars of the water-soluble black tea leaf polysaccharides being 30 to 40 mol%, and the sum of the proportions of galactose and arabinose in the total constituent sugars of the water-soluble black tea leaf polysaccharides being 60 to 90 mol%.

6. The expression promoter described in claim 5, wherein the proportion of the black tea leaf polysaccharides having a molecular weight of 100 to 1000 in the black tea leaf polysaccharides is 5 to 20 mass %, and the proportion of the black tea leaf polysaccharides having a molecular weight of 3000 to 50,000 is 10 to 30 mass %.

7. The expression promoter according to claim 1, wherein the water-soluble plant polysaccharide is soybean polysaccharide.

8. The expression promoter according to claim 7, wherein the proportion of galactose in the total sugar constituents of the soybean polysaccharide is 30 to 50 mol%, the proportion of arabinose is 15 to 35 mol%, and the proportion of galacturonic acid is 5 to 20 mol%, and the total proportion of galactose, arabinose, and galacturonic acid in the total sugar constituents of the soybean polysaccharide is 60 to 90 mol%.

9. The expression promoter according to claim 8, wherein the proportion of soybean polysaccharides having a molecular weight of 30,000 to 1,000,000 in the soybean polysaccharides is 20 mass% or more.

10. The expression promoter according to any one of claims 1 to 9, which is for use in the epidermis.

11. The expression promoter according to claim 10, which is an external preparation.

12. The expression promoter according to claim 11, which is a cosmetic product.

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