Agent for amplifying expression of transporter-related gene in epidermal cells

Abstract

To utilize the effect of a diacylglycerol PEG adduct in amplifying the expression of transporter-related genes in epidermal cells.SOLUTION: A agent for amplifying the expression of transporter-related genes in epidermal cells comprises a diacylglycerol PEG adduct as an active ingredient, wherein the diacylglycerol PEG adduct has a structural formula specified herein, where the carbon number of R in the long-chain fatty acid falls within the range of 11-23 and n in the polyethylene glycol chain is within the range of 11-46.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent and a method for amplifying the expression of an epidermal cell transporter-related gene. [Background technology]

[0002] Patent Document 1 discloses a preparation method in which a diacylglycerol polyethylene glycol adduct (hereinafter referred to as a "diacylglycerol PEG adduct") is used as a lipid molecule and mixed with water or a surfactant to spontaneously form a vesicle. Such vesicles are used in drug delivery systems that encapsulate or bind target substances such as proteins and drugs inside or on their surfaces and deliver them to cells in the body. Vesicles made of diacylglycerol PEG adducts have a morphology in which the surface is covered with hydrophilic PEG chains, and have good permeability into the body and stability in blood.

[0003] Patent Document 2 describes that by binding a charged element to the surface of a vesicle made of a diacylglycerol PEG adduct to make it positively charged, the permeability and retention in the stratum corneum of the epidermis can be improved.

[0004] Vesicles in drug delivery systems have been recognized simply as carriers of target drugs. Recently, it has been discovered that the diacylglycerol PEG adducts derived from decomposed vesicles in vivo also exert useful functions in vivo. Patent Document 3 discloses that diacylglycerol PEG adducts contribute to enhanced expression of moisturizing-related substances such as profilaggrin, filaggrin, and natural moisturizing factor NMF in the epidermis. Patent Document 4 discloses that diacylglycerol PEG adducts contribute to enhanced expression of antioxidant-related substances such as oxidative stress response genes Nrf2 and PPARG in the epidermis, and antioxidant enzymes NQO-1, CAT, and HMOX1.

[0005] On the other hand, various transport proteins (so-called transporters) present in the cell membrane function in the uptake and excretion of various substances into epidermal cells. For example, sodium-dependent vitamin C transporters (SVCTs) that transport vitamin C into cells have been discovered, and type 1 (SVCT1) and type 2 (SVCT2) are known (Patent Document 5). For example, xCT proteins are known to be involved in the xCT mechanism that takes up cystine, an amino acid, into cells and releases intracellular glutamic acid to the outside of the cells. Cystine taken up into cells is involved in the production of glutathione via cysteine ​​(Patent Document 6). For example, the ABCC1 protein is known as one of the ABC transporters that has the function of excreting various substances from inside to outside of cells (Patent Document 7). For example, aquaporin proteins are known that function as water channels that regulate the amount of water in cells, and aquaporin 3 (AQP3) in particular is abundant in the epidermis (Patent Document 8). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4497765 (Self-forming thermodynamically stable liposomes and their applications) [Patent Document 2] Patent No. 6297737 (Method for preparing positively charged niosomes and charged niosomes) [Patent Document 3] Patent No. 6805385 (Expression enhancer for moisturizing substances in the epidermis) [Patent Document 4] Patent No. 6860739 (Agent for enhancing expression of antioxidants in the epidermis) [Patent Document 5] International Publication No. 2007 / 094312 (Vitamin C transporter production promoter) [Patent Document 6] JP 2010-280675 A (Composition for enhancing glutathione) [Patent Document 7] JP2022-514669A (Composition containing Lactobacillus rhamnosus extract) [Patent Document 8] JP 2011-32191 A (Aquaporin 3 expression regulator) Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, various epidermal cell transporters are known, but there are few reports on substances that enhance the expression of these transport proteins. As for diacylglycerol PEG adducts, little is known about their relationship with various mechanisms of uptake or excretion of drugs into or from epidermal cells.

[0008] The objective of the present invention is to take advantage of the newly discovered properties of diacylglycerol PEG adducts that relate to epidermal cell transporters, and in particular to amplify the expression of epidermal cell transporter-related genes. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides the following configuration. An embodiment of the present invention is an agent for amplifying epidermal cell transporter-related gene expression, comprising a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct having the following structural formula, in which the number of carbon atoms in R in the long-chain fatty acid is within the range of 11 to 23, and n in the polyethylene glycol chain is within the range of 11 to 46.

[0010] Another aspect of the present invention is a method for amplifying expression of a transporter-related gene in epidermal cells, which uses a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct having the following structural formula, in which the number of carbon atoms in R in the long-chain fatty acid is within the range of 11 to 23, and n in the polyethylene glycol chain is within the range of 11 to 46.

[0011] [ka]

[0012] Preferably, the epidermal cell transporter-related gene is at least one of the group consisting of SVCT2, SLC7A11, glutathione reductase, ABCC1, and aquaporin 3. Preferably, the diacylglycerol PEG adduct is at least one of the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12). Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a solution or vesicle state.

[0013] Yet another aspect of the present invention is a cosmetic preparation comprising the above-mentioned agent for amplifying the expression of an epidermal cell transporter-related gene. Yet another aspect of the present invention is an external skin preparation comprising the above-mentioned agent for amplifying the expression of a transporter-associated gene in epidermal cells. The cosmetic or topical skin preparation may further contain at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, glutathione, and cysteine, in which case it may further contain hydroquinone. Effect of the Invention

[0014] According to the present invention, an agent for amplifying the expression of an epidermal cell transporter-associated gene, which comprises a diacylglycerol PEG adduct as an active ingredient, is provided. Also, according to the present invention, a method for amplifying the expression of an epidermal cell transporter-associated gene, which uses a diacylglycerol PEG adduct as an active ingredient, is provided. [Brief description of the drawings]

[0015] [Figure 1]FIG. 1 is a graph showing the relative expression amplification amount of the SVCT2 gene with respect to GDS23. [Diagram 2] FIG. 2 is a graph showing the relative expression amplification level of the SVCT2 gene in relation to GDM12. [Diagram 3] FIG. 3 is a graph showing the relative expression amplification level of the SLC7A11 gene in GDS23. [Figure 4] FIG. 4 is a graph showing the relative expression amplification level of the SLC7A11 gene in relation to GDM12. [Diagram 5] FIG. 5 is a graph showing the relative expression amplification amount of the GSR gene with respect to GDS23. [Figure 6] FIG. 6 is a graph showing the relative expression amplification amount of the GSR gene in relation to GDM12. [Figure 7] FIG. 7 is a graph showing the relative expression amplification level of the ABCC1 gene in GDS23. [Figure 8] FIG. 8 is a graph showing the relative expression amplification level of the ABCC1 gene in relation to GDM12. [Figure 9] FIG. 9 is a graph showing the relative expression amplification level of the AQP3 gene in relation to GDS23. [Figure 10] FIG. 10 is a graph showing the relative expression amplification level of the AQP3 gene in relation to GDM12. [Figure 11] FIG. 11 is a Western blot band image of SVCT2 protein in relation to GDS23. [Figure 12] FIG. 12 is a graph showing the expression level of SVCT2 protein. [Figure 13] FIG. 13 is a graph showing the results of an evaluation test of the cellular uptake of vitamin C. [Figure 14] FIG. 14 is a graph showing the results of an evaluation test of the cellular uptake of cystine in GDS23. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The present invention has been created by utilizing a newly discovered property of diacylglycerol polyethylene glycol adducts (diacylglycerol PEG adducts), which is the ability to amplify the expression of human epidermal cell transporter genes and genes related thereto.

[0017] 1 is a schematic diagram showing the structural formula of a diacylglycerol PEG adduct, which is a lipid molecule related to the present invention.

[0018] [ka]

[0019] Diacylglycerol PEG adducts consist of a three-carbon glycerol backbone (CH2CHCH2), a linear polyethylene glycol (PEG) chain attached to one of the terminal carbons of the backbone, and the same kind of long-chain fatty acid (COOR) attached to the other two of the three carbons. The PEG chain is hydrophilic, and the long-chain fatty acid is hydrophobic.

[0020] In the following description, when a specific diacylglycerol PEG adduct is referred to, it is referred to as "[di] + [name of long-chain fatty acid] + [glycerol] + [PEG-n]" based on the type of long-chain fatty acid and the number n in the PEG chain. For example, when the long-chain fatty acid is myristic acid and the number n in the PEG chain is 12, it is called "dimyristic acid PEG-12." In addition, a specific diacylglycerol PEG adduct may be further abbreviated.

[0021] The carbon number of R in the long-chain fatty acid can be within the range of 11 to 23. Examples of long-chain fatty acids within this range include myristic acid, palmitic acid, stearic acid, and oleic acid. The number n of the PEG chain can be within the range of 11 to 46. Examples of diacylglycerol PEG adducts related to the present invention include the following. The melting point and abbreviation are shown in parentheses. Glycerol dimyristate PEG-12 (25.0℃: GDM12) Glycerol distearate PEG-12 (40.0℃: GDS12) Glycerol distearate PEG-23 (39.8℃: GDS23) Glycerol dipalmitate PEG-23 (31.2℃:GDP23) Glycerol dioleate PEG-12 (25.0℃:GDO12)

[0022] Various transport proteins (so-called transporters) present in the cell membrane of human epidermal cells are responsible for the uptake and excretion of various substances into epidermal cells. In the present invention, the epidermal cell transporters and their associated genes whose expression was confirmed to be amplified by diacylglycerol PEG adducts are SVCT2, SLC7A11, GSR, ABCC1, and aquaporin 3. These are collectively referred to herein as "epidermal cell transporter-related genes." Each of these genes encodes a corresponding protein or enzyme.

[0023] The SVCT2 protein encoded by the SVCT (sodium-dependent vitamin C transporter) 2 gene forms a pathway necessary for vitamin C (ascorbic acid) to pass into cells. Vitamin C inhibits melanin production in epidermal cells and contributes to whitening. Ascorbic acid can be administered to the skin, for example, as a component of cosmetics. Ascorbic acid has insufficient stability and skin permeability, ascorbic acid derivatives with improved properties are used instead of or in combination with ascorbic acid. Ascorbic acid derivatives are converted to ascorbic acid in the skin or act in the same manner as ascorbic acid.

[0024] The xCT (cystine / glutamate transporter) protein encoded by the SLC7A11 gene takes in the amino acid cystine into cells. Once inside the cells, cystine is reduced to cysteine, which, together with glutamic acid and glycine, produces the tripeptide glutathione (reduced glutathione). Reduced glutathione is one of the antioxidants that protects cells from oxidation by being oxidized (oxidized glutathione). Cystine can be synthesized in the body, but it can also be administered to the skin as an ingredient in cosmetics, etc. Cysteine ​​administered from the outside becomes a dimer of cystine in the skin and is taken up into cells by the xCT transporter.

[0025] Glutathione reductase, encoded by the GSR (glutathione-disulfide reductase) gene, is an enzyme that reduces oxidized glutathione back to reduced glutathione. Although GSR is not a transporter, it is an enzyme that acts in association with the xCT and ABCC1 transporters, and therefore is considered to be a transporter-associated gene in the present invention. Glutathione is an amino acid that can be synthesized in the body, but can also be administered to the skin as an ingredient in cosmetics, etc. Glutathione administered externally (reduced glutathione) is also reduced by glutathione reductase when it is oxidized in the skin.

[0026] The ABCC1 protein, encoded by the ABC (ATP binding cassette) C1 gene, is a member of the ABC transporter family and is also called MRP1. The ABCC1 protein secretes glutathione containing harmful substances such as toxins from within the cells.

[0027] The aquaporin 3 protein encoded by the aquaporin 3 gene functions as a water channel that regulates the amount of water in cells in the epidermis. The aquaporin 3 protein distributes water throughout the epidermis, helping to maintain skin moisture and elasticity.

[0028] The inventors have found that application of diacylglycerol PEG adducts to human epidermis amplifies the expression of epidermal cell transporter-related genes. Amplification of the expression of these epidermal cell transporter-related genes enhances the production of the proteins and enzymes they encode. This is a new effect of diacylglycerol PEG adducts on human epidermis, particularly membrane transport in epidermal cell membranes, and is a new property of diacylglycerol PEG adducts. This property can provide effects such as whitening, antioxidant, detoxification, and moisturizing to the epidermis. This is not simply a physical protective effect on the surface of the epidermis, but an effect obtained in epidermal cells.

[0029] The present invention utilizes the newly discovered properties of the diacylglycerol PEG adduct to provide an agent for amplifying the expression of an epidermal cell transporter-related gene, which contains the diacylglycerol PEG adduct as an active ingredient, and also provides a method for amplifying the expression of an epidermal cell transporter-related gene, which uses the diacylglycerol PEG adduct as an active ingredient.

[0030] In the present invention, when the diacylglycerol PEG adduct is applied to human epidermis, it may be used alone or in combination of two or more kinds.

[0031] According to the present invention, the diacylglycerol PEG adduct that has reached the epidermis can amplify the expression of epidermal cell transporter-related genes and increase the production of the corresponding proteins and enzymes compared to the absence of the diacylglycerol PEG adduct. As a result, not only the state of the epidermis but also the state of the epidermis surface is improved. Therefore, the present invention can provide a cosmetic or skin topical preparation containing a diacylglycerol PEG adduct as an active ingredient as an expression amplifier for epidermal cell transporter-related genes. Both the cosmetic and the skin topical preparation are applied to the epidermal surface. Here, anything other than cosmetics is generally referred to as a skin topical preparation, such as an ointment. Such a cosmetic or skin topical preparation can be provided in various forms, such as an aqueous solution, emulsion, gel, cream, etc. These cosmetic or skin topical preparations can contain other active ingredients and / or various ingredients that are generally contained in addition to the diacylglycerol PEG adduct.

[0032] As one method for delivering the diacylglycerol PEG adduct into the human epidermis, the diacylglycerol PEG adduct can be delivered into the epidermis in the form of a solution in which it is dissolved in water or a specific solvent. For example, a diacylglycerol PEG adduct solution of a specific concentration is prepared in phosphate buffered saline (PBS(-)) as a solvent, and applied to the surface of the epidermis to allow it to penetrate into the epidermis. The applied solution penetrates, for example, into the stratum corneum, the top layer, and then into the granular layer below the stratum corneum, and then into the layer below that. The diacylglycerol PEG adduct then amplifies the expression of epidermal cell transporter-related genes in each layer of the epidermis into which it has penetrated.

[0033] As another method for delivering the diacylglycerol PEG adduct into the epidermis, the diacylglycerol PEG adduct can be delivered into the epidermis in the form of a vesicle. Such a vesicle is formed as a closed spherical shell consisting of a bilayer or a multilayer of bilayers of the diacylglycerol PEG adduct, with hydrophilic PEG chains arranged on the surface of the outermost layer. A vesicle of the diacylglycerol PEG adduct can be prepared and applied to the epidermal surface to penetrate the epidermis. After reaching the epidermis, the vesicle is decomposed and separated into individual molecules, allowing the diacylglycerol PEG adduct itself to exert its effect.

[0034] In conventional drug delivery systems, the diacylglycerol PEG adduct, which is the material of the vesicle, has been considered to be merely a carrier of the target substance, but in the present invention, the diacylglycerol PEG adduct itself is used as an active ingredient. Therefore, in the present invention, the target substance incorporated into the vesicle in a normal drug delivery system is basically unnecessary. In the present invention, by penetrating a vesicle formed by mixing only water and the diacylglycerol PEG adduct into the epidermis, the diacylglycerol PEG adduct itself can function as an expression amplifier for the epidermal cell transporter-related gene.

[0035] Some diacylglycerol PEG adducts spontaneously form vesicles when mixed with water at a certain temperature (see Patent Documents 1 and 2). For example, a suspension of vesicles of GDM12 or GDO12 is obtained by mixing 2% by mass of GDM12 or GDO12 with 98% by mass of deionized water at room temperature and stirring. As another example, a suspension of vesicles of GDS12 or GDS23 is obtained by dissolving 2% by mass of GDS12 or GDS23 at 45 to 55°C, mixing with 98% by mass of deionized water at 45 to 55°C and stirring. As yet another example, a suspension of vesicles of GDP23 is obtained by dissolving 2% by mass of GDP23 at 37°C, mixing with 98% by mass of deionized water at 37°C and stirring. The vesicles are stable even when the suspension obtained at a temperature higher than room temperature is cooled to room temperature.

[0036] As another example, the present invention also includes a case where a vesicle formed by mixing and stirring an aqueous solution of various substances and a diacylglycerol PEG adduct is used instead of the above water. In this case, the substance contained in the aqueous solution can have another function.

[0037] As another example, the present invention also includes a case where the surface of a vesicle formed by mixing and stirring water or an aqueous solution with a diacylglycerol PEG adduct is modified with a charged element such as a cationic surfactant. Patent Document 2 describes that positively charged vesicles are particularly excellent in permeability and retention in the epidermis.

[0038] Below, the relationship between application of diacylglycerol PEG adducts to epidermal cells and epidermal cell transporter-related genes is shown by test data.

[0039] (1) Amplification of epidermal cell transporter-related gene expression A test was carried out to confirm the amplification of mRNA expression of each epidermal cell transporter-related gene.

[0040] (1-1) Test method Normal human epidermal keratinocytes (NHEKs) were 2.0 × 10 4 The cells were seeded onto a 96-well plate at a cell density of 100 cells / well using HuMedia-KG2 medium (Kurabo Industries, Ltd.) and cultured at 37°C and 5% CO2 for 24 hours. Thereafter, using HuMedia-KB2 medium (Kurabo), diacylglycerol PEG adducts were added and cultured for the specified time under conditions of 37°C and 5% CO2. Table 1 shows the target gene, the type of diacylglycerol PEG adduct added, the amount added, and the culture time. The control (no addition) was also cultured under the same conditions.

[0041] [Table 1]

[0042] After culturing for the specified time, RNA was extracted from each sample and control cell. The extracted RNA was reverse transcribed to prepare cDNA, and the mRNA of the target gene was quantified by quantitative real-time PCR expression analysis. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as an internal standard.

[0043] For the analysis, the mRNA expression level of each target gene was corrected by the expression level of GAPDH, an internal standard, in the same sample, and then the corrected value of the sample was calculated as the relative expression level when the correction value of the control was set to 1.

[0044] (1-2) Test results Figure 1 is a graph showing the relative expression level of the SVCT2 gene in GDS23, and Figure 2 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.3 to 1.7 times that of the control. In GDM12, the expression level was confirmed to be about 1.4 times that of the control.

[0045] Figure 3 is a graph showing the relative expression level of the SLC7A11 gene in GDS23, and Figure 4 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 4 to 12 times higher than the control. In GDM12, the expression level was confirmed to be about 1.4 times higher than the control.

[0046] Figure 5 is a graph showing the relative expression level of the GSR gene in GDS23, and Figure 6 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.8 times that of the control, and in GDM12, the expression level was confirmed to be about 1.5 times that of the control.

[0047] Figure 7 is a graph showing the relative expression level of the ABCC1 gene in GDS23, and Figure 8 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 2.1 times that of the control, and in GDM12, the expression level was confirmed to be about 1.3 times that of the control.

[0048] Figure 9 is a graph showing the relative expression level of aquaporin 3 gene in GDS23, and Figure 10 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.6 to 3.5 times that of the control. In GDM12, the expression level was confirmed to be about 1.6 to 2.2 times that of the control.

[0049] (2) Test for enhanced SVCT2 protein production A test to confirm the enhanced production of the protein encoded by the SVCT2 gene was performed using the Western blot method.

[0050] (2-1) Test method Normal human epidermal keratinocytes (NHEKs) were added at 1.0 × 10 5 The cells were seeded onto a 24-well plate at a cell density of 100 cells / well using HuMedia-KG2 medium and cultured at 37°C and 5% CO2 for 24 hours. Then, 50 μM glycerol distearate PEG-23 (GDS23) was added to HuMedia-KB2 medium, and the cells were cultured for 24 hours at 37°C and 5% CO2. The control (no addition) was cultured in HuMedia-KB2 medium alone.

[0051] After the culture, the Western blot method was applied. First, proteins were extracted from the cells using SDS-PAGE containing 10% mercaptoethanol. The protein extract was electrophoresed using a polyacrylamide gel, and the proteins were transferred to a membrane by the semi-dry method. SVCT2 protein was quantified using the specified primary and secondary antibodies shown in Table 2. The amount of SVCT2 protein was corrected by the amount of GAPDH, which is an internal standard in the same sample, and the correction value of the sample was calculated when the correction value of the control was set to 1.

[0052] [Table 2]

[0053] (2-2) Test results Figure 11 is a band image of a Western blot of SVCT2 protein for GDS23. Figure 12 is a graph showing the expression level of SVCT2 protein.

[0054] As shown in Figure 11, an increase in SVCT2 protein was confirmed in the GDS23-added sample. As shown in Figure 12, the amount of SVCT2 protein in the GDS23-added sample was confirmed to be about 1.4-fold higher than that of the control.

[0055] (3) Evaluation of cellular uptake of vitamin C (ascorbic acid) by increasing SVCT2 From the test results of (1) and (2) above, it was confirmed that application of diacylglycerol PEG adduct to epidermal cells enhances the expression of SVCT2 gene, and therefore enhances the production of the vitamin C transporter protein encoded by it. Therefore, the cellular uptake of vitamin C was actually evaluated. This evaluation was performed by quantifying the cytotoxicity caused by hydroquinone (HQ) when diacylglycerol PEG adduct, vitamin C, and hydroquinone (HQ) were added to epidermal cells. Hydroquinone itself has no cytotoxicity and has a whitening effect, but when it is oxidized to benzoquinone, it exhibits cytotoxicity (see Patent Document 4). On the other hand, if hydroquinone is added to the epidermis together with an oxidation inhibitor, it should not be oxidized and its cytotoxicity should be suppressed (the same applies to the evaluation test in (4) below).

[0056] (3-1) Test method Normal human epidermal keratinocytes (NHEKs) were 2.0 × 10 4 The cells were seeded onto a 96-well plate at a cell density of 100 cells / well using HuMedia-KG2 medium and cultured at 37°C and 5% CO2 for 24 hours. Then, using HuMedia-KB2 medium, a sample with 50 μM glycerol distearate PEG-23 (GDS23) added and a sample without GDS23 added were cultured at 37° C. and 5% CO 2 for 24 hours.

[0057] Subsequently, the cells were washed with PBS(-), and the GDS23-added sample and the GDS23-free sample were cultured for 24 hours in the following six types of media. HuMedia-KB2 medium containing 0 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 1 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 5 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 10 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 50 μM ascorbic acid (AsA) HuMedia-KB2 medium containing 100 μM ascorbic acid (AsA)

[0058] The cells were then washed with PBS(-) and cultured in HuMedia-KB2 medium containing 400 μM hydroquinone (HQ) for 24 hours.

[0059] The control (no addition of GDS23, AsA, or HQ) was cultured in HuMedia-KB2 medium alone.

[0060] The cell viability was then measured by neutral red assay. The cell viability of the control was set to 100%, and the cell viability of each of the GDS23-added sample and the GDS23-free sample was calculated.

[0061] (3-2) Test results 13 is a graph showing the results of an evaluation test of the cellular uptake of vitamin C in GDS23. Except for the control on the far left, the six on the left are samples without GDS23 added, and the six on the right are samples with GDS23 added.

[0062] Of the six samples without GDS23, the one on the far left was without ascorbic acid, while the other five were with ascorbic acid. Comparing these, it can be seen that when treated with low concentrations of ascorbic acid (5-100 μM), there was no difference in cell viability compared to the sample without ascorbic acid, indicating that ascorbic acid has almost no effect on cell damage caused by hydroquinone.

[0063] In contrast, the GDS23-added sample had a higher cell viability than the GDS23-free sample, and cell damage caused by hydroquinone was significantly reduced. Among the six GDS23-added samples, the one treated with ascorbic acid had a higher cell viability than the one without ascorbic acid (shown on the far left). This is thought to be because the increase in SVCT2 caused by GDS23 increased the uptake of ascorbic acid into the cells, raising the intracellular ascorbic acid concentration and suppressing the oxidation of hydroquinone.

[0064] Incidentally, among the samples with added GDS23, the cell viability was increased even in the samples without added ascorbic acid compared to the samples without added GDS23. This is thought to be due to the effect of GDS23 itself in reducing HQ cytotoxicity as disclosed in Patent Document 4, and is thought to be due to a mechanism different from the increasing effect of vitamin C transporter of the present invention.

[0065] (4) Evaluation of the effect of increased SLC7A11 on cellular uptake of cystine From the results of the above (1) and (2), it was confirmed that application of diacylglycerol PEG adduct to epidermal cells enhances the expression of SLC7A11 gene, and therefore enhances the production of the xCT transporter protein encoded by it. Therefore, the cellular uptake of cystine was actually evaluated. This evaluation was performed by quantifying the cytotoxicity caused by hydroquinone (HQ) when diacylglycerol PEG adduct, cystine, and hydroquinone (HQ) were added to epidermal cells.

[0066] (4-1) Test method Normal human epidermal keratinocytes (NHEKs) were 2.0 × 10 4 The cells were seeded onto a 96-well plate at a cell density of 100 cells / well using HuMedia-KG2 medium and cultured at 37°C and 5% CO2 for 24 hours. Then, using HuMedia-KB2 medium, a sample with 50 μM glycerol distearate PEG-23 (GDS23) added and a sample without GDS23 added were cultured at 37° C. and 5% CO 2 for 24 hours.

[0067] After washing with PBS(-), the GDS23-added sample and the GDS23-free sample were cultured for 30 hours in the following three media: Cysteine ​​added to the medium is oxidized in the medium or between cells and converted to the dimer cystine, which is then taken up into the cells as cystine. HuMedia-KB2 medium containing 0 μM cysteine ​​(Cys) HuMedia-KB2 medium containing 10 μM cysteine ​​(Cys) HuMedia-KB2 medium containing 50 μM cysteine ​​(Cys)

[0068] The cells were then washed with PBS(-) and cultured in HuMedia-KB2 medium containing 400 μM hydroquinone (HQ) for 24 hours.

[0069] The control (no addition of GDS23, Cys, or HQ) was cultured in HuMedia-KB2 medium alone.

[0070] The cell viability was then measured by neutral red assay. The cell viability of the control was set to 100%, and the cell viability of each of the GDS23-added sample and the GDS23-free sample was calculated.

[0071] (4-2) Test results 14 is a graph showing the results of an evaluation test of the cellular uptake of cystine with respect to GDS23. Except for the control on the far left, the three on the left are samples to which GDS23 was not added, and the three on the right are samples to which GDS23 was added.

[0072] Of the three GDS23-free samples, the one on the far left has no cysteine ​​added, while the other two have cysteine ​​added. Comparing these, the samples treated with 10 μM and 50 μM cysteine ​​have no difference in cell viability compared to the sample without cysteine ​​added, indicating that cysteine ​​(cystine) has almost no effect on cell damage caused by hydroquinone. In other words, it can be seen that cystine is not taken up by cells in the sample without GDS23 added.

[0073] In contrast, the GDS23-added sample had a higher cell viability than the GDS23-free sample, and cytotoxicity caused by hydroquinone was significantly reduced. Among the three GDS23-added samples, the other two cysteine-treated samples had a higher cell viability than the cysteine-free sample on the far left. This is thought to be because GDS23 increased SLC7A11, which increased the uptake of cystine into the cells, increasing the intracellular cystine concentration and enhancing the production of glutathione, thereby suppressing the oxidation of hydroquinone.

[0074] Among the GDS23-added samples, the cysteine-free sample on the far left also showed an increased cell viability compared to the GDS23-free sample. This is thought to be due to the HQ cytotoxicity-reducing effect of GDS23 itself, as disclosed in Patent Document 4, and is thought to be due to a mechanism different from the xCT transporter-increasing effect of the present invention.

[0075] (5) Preparation of cosmetics and topical skin preparations The following are examples of preparations of cosmetics and skin external preparations containing an epidermal cell transporter-related gene expression amplifier containing a diacylglycerol PEG adduct as an active ingredient. Each of the following preparation examples contains an ascorbic acid derivative. The ascorbic acid derivative has improved stability and skin permeability compared to ascorbic acid. After permeation into the skin, the ascorbic acid derivative acts by being converted to ascorbic acid through decomposition or enzyme action, or acts equivalently to ascorbic acid as it is. Examples of ascorbic acid derivatives include 3-O-ethyl ascorbic acid, 3-lauryl glyceryl ascorbic acid, and myristyl 3-glyceryl ascorbic acid. The cosmetics of the present invention may contain either ascorbic acid or an ascorbic acid derivative, or both.

[0076] (5-1) Example of preparing lotion The lotion of Preparation Example 1 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione.

[0077] [Table 3]

[0078] (5-2) Example of emulsion preparation The emulsion of Preparation 2 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, cysteine, and glutathione.

[0079] [Table 4]

[0080] (5-3) Preparation of water-based gel The aqueous gel of Preparation Example 3 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione.

[0081] [Table 5]

[0082] (5-4) Preparation example of hydroquinone-containing cream The hydroquinone-containing cream of Preparation Example 4 contains, in addition to hydroquinone, glycerol dimyristate PEG-12, glycerol distearate PEG-23, ascorbic acid and its derivatives, and glutathione. The antioxidant effects of these substances suppress the cytotoxicity of hydroquinone. As a result, the cosmetic effects of hydroquinone are exerted.

[0083] [Table 6]

[0084] (5-5) Preparation example of hydroquinone-containing ointment The hydroquinone-containing ointment of Preparation Example 5 contains, in addition to hydroquinone, glycerol distearate PEG-23, ascorbic acid, glutathione, and cysteine.

[0085] [Table 7]

[0086] Although not exemplified, there are various combinations of components other than the diacylglycerol PEG adduct. One or more components selected from the group consisting of ascorbic acid, ascorbic acid derivatives, glutathione, and cysteine ​​are selected as necessary. In this case, hydroquinone may be further included. The same applies to skin external preparations other than cosmetics.

[0087] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to these embodiments, and obvious modifications thereto are also included in the present invention.

Claims

1. An agent for amplifying an epidermal cell transporter-related gene expression, comprising a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct having the following structural formula, in which the number of carbon atoms of R in the long-chain fatty acid is within the range of 11 to 23, and n in the polyethylene glycol chain is within the range of 11 to 46. 【Chemistry 1】

2. The agent for amplifying epidermal cell transporter-related gene expression according to claim 1 , wherein the epidermal cell transporter-related gene is at least one selected from the group consisting of SVCT2, SLC7A11, glutathione reductase, ABCC1, and aquaporin 3.

3. 3. The epidermal cell transporter-related gene expression amplifier according to claim 1 or 2, wherein the diacylglycerol PEG adduct is at least one selected from the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12).

4. The agent for amplifying epidermal cell transporter-related gene expression according to claim 1 or 2, wherein the diacylglycerol PEG adduct penetrates into the epidermis in a solution state or a vesicle state.

5. A cosmetic preparation comprising the epidermal cell transporter-related gene expression amplifier according to claim 1 or 2.

6. The cosmetic preparation according to claim 5, further comprising at least one selected from the group consisting of ascorbic acid, an ascorbic acid derivative, glutathione, and cysteine.

7. The cosmetic preparation according to claim 6, further comprising hydroquinone.

8. An external skin preparation comprising the epidermal cell transporter-related gene expression amplifier according to claim 1 or 2.

9. The external skin preparation according to claim 8 , further comprising at least one member selected from the group consisting of ascorbic acid, an ascorbic acid derivative, glutathione, and cysteine.

10. The topical skin preparation according to claim 9, further comprising hydroquinone.

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