Agent for amplifying expression of epidermal barrier function-related gene

Abstract

To utilize action of amplifying expression of an epidermal barrier function-related gene by a diacylglycerol polyethylene glycol adduct.SOLUTION: An agent for amplifying expression of epidermal barrier function-related gene comprises a diacylglycerol polyethylene glycol adduct as an active ingredient, the diacylglycerol PEG adduct having a following structural formula, in which the number of carbon atoms of R in a long-chain fatty acid is in the range from 11 to 23 and n in a polyethylene glycol chain is in the range from 11 to 46.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent and a method for amplifying the expression of epidermal barrier function-related genes. [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, in the epidermis, various proteins or enzymes that constitute the stratum corneum or participate in epidermal cell proliferation are responsible for the epidermal barrier function, which prevents the invasion of bacteria, inflammatory substances, allergens, etc. from the outside into the epidermis, retains moisture to prevent the skin from drying out, and maintains skin homeostasis.

[0006] For example, PPARα, a nuclear receptor, is a protein that controls gene expression related to lipid metabolism and inflammatory response regulation in the skin (Patent Document 5). A large number of synthetic or natural substances are known as ligands that activate PPARα. For example, RARγ, a nuclear receptor for retinoic acid (vitamin A), is a protein that controls gene expression associated with normal differentiation and regeneration of epidermal cells (Patent Document 6). For example, loricrin and involucrin are precursor proteins of the cornified envelope that surrounds keratinocytes, are produced in accordance with the differentiation of epidermal cells, and promote the cornification of the epidermis and stabilize the stratum corneum (Patent Document 7). For example, hyaluronic acid synthase 3 maintains epidermal homeostasis by synthesizing hyaluronic acid in the epidermis and regulating moisture (Patent Document 8). For example, ceramide synthases 2 and 3 produce ceramide, an intercellular lipid that plays an important role in maintaining the epidermal barrier function (Patent Document 9). For example, cholesterol sulfotransferase regulates cholesterol sulfate metabolism in the skin and enhances the epidermal barrier function and moisturization (Patent Document 10). [Prior art documents] [Patent documents]

[0007] [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] JP 2018-48103 A (skin quality improving agent) [Patent Document 6] JP2014-528472A (Anti-aging composition through activation of retinoic acid receptors) [Patent Document 7] JP 2020-160028 A (.. Screening method for skin barrier function improving agents) [Patent Document 8] JP 2011-98983 A (Hyaluronic acid production enhancer) [Patent Document 9] JP 2016-23155 A (Ceramide production promoter and topical skin preparation) [Patent Document 10] Special Publication No. 2019-529515 Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, the diacylglycerol PEG adduct has an effect of enhancing the expression of moisturizing-related substances described in Patent Document 3 and an effect of enhancing the expression of antioxidant substances described in Patent Document 4. These effects contribute to an increase in moisturizing factors in the epidermis and to the alleviation of oxidative stress occurring in the epidermis. However, even if the expression of moisturizing substances and antioxidants is enhanced, if the epidermal barrier function is weakened, the increased moisturizing factors cannot be retained and the occurrence of oxidative stress cannot be prevented. Little is known about how diacylglycerol PEG adducts are related to such epidermal barrier function.

[0009] An object of the present invention is to utilize the newly discovered properties of diacylglycerol PEG adducts that are related to epidermal barrier function, and in particular to amplify the expression of genes related to epidermal barrier function. [Means for solving the problem]

[0010] 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 the expression of genes related to epidermal barrier function, 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.

[0011] Another aspect of the present invention is a method for amplifying expression of an epidermal barrier function-related gene, which uses a diacylglycerol PEG adduct as an active ingredient, and the diacylglycerol PEG adduct has 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.

[0012] [ka]

[0013] Preferably, the diacylglycerol PEG adduct is 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). Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a solution or vesicle state.

[0014] Yet another aspect of the present invention is a cosmetic preparation comprising the above-mentioned epidermal barrier function-related gene expression amplifier. Yet another aspect of the present invention is an external skin preparation comprising the above-mentioned agent for amplifying the expression of genes related to epidermal barrier function. It is preferable that the cosmetic or topical skin preparation further contains one or more members selected from the group consisting of retinoic acid, retinol, retinol derivatives, and retinoic acid derivatives. Effect of the Invention

[0015] According to the present invention, an agent for amplifying the expression of epidermal barrier function-related genes, 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 epidermal barrier function-related genes, which uses a diacylglycerol PEG adduct as an active ingredient, is provided. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a graph showing the relative expression amplification level of the PPARA gene in GDS23. [Diagram 2] FIG. 2 is a graph showing the relative expression amplification level of the PPARA gene in GDM12. [Diagram 3] FIG. 3 is a graph showing the relative expression amplification level of the RARG gene with respect to GDS23. [Figure 4] FIG. 4 is a graph showing the relative expression amplification level of the RARG gene in relation to GDM12. [Diagram 5] FIG. 5 is a graph showing the relative expression amplification amount of the LOR gene with respect to GDS23. [Figure 6] FIG. 6 is a graph showing the relative expression amplification levels of the LOR gene in GDM12. [Figure 7] FIG. 7 is a graph showing the relative expression amplification amount of the IVL gene in relation to GDM12. [Figure 8] FIG. 8 is a graph showing the relative expression amplification level of the HAS3 gene in GDS23. [Figure 9] FIG. 9 is a graph showing the relative expression amplification level of the HAS3 gene in GDM12. [Figure 10] FIG. 10 is a graph showing the relative expression amplification amount of the CERS2 gene with respect to GDS23. [Figure 11] FIG. 11 is a graph showing the relative expression amplification level of the CERS2 gene in relation to GDM12. [Figure 12] FIG. 12 is a graph showing the relative expression amplification amount of the CERS3 gene with respect to GDS23. [Figure 13] FIG. 13 is a graph showing the relative expression amplification level of the SULT2B1 gene in relation to GDS23. [Figure 14] FIG. 14 is a graph showing the relative expression amplification level of the SULT2B1 gene in relation to GDM12. [Figure 15] FIG. 15 shows images showing the results of detection of loricrin (LOR) by a loricrin (LOR) immunostaining method (upper panel) and a hematoxylin-eosin (HE) staining method (lower panel) for GDS23. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 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 genes related to human epidermal barrier function.

[0018] 1 is a schematic diagram showing the structural formula of a diacylglycerol PEG adduct, which is a lipid molecule related to the present invention.

[0019] [ka]

[0020] 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.

[0021] 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.

[0022] 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)

[0023] The human epidermal barrier function is responsible for preventing the intrusion of bacteria, inflammatory substances, allergens, etc. from the outside and avoiding various skin troubles. In the present invention, the epidermal barrier function-related genes whose expression was confirmed to be amplified by diacylglycerol PEG adducts are PPARA, RARG, LOR, IVL, HAS3, CERS2, CERS3, and SULT2B1. In this specification, these are collectively referred to as "epidermal barrier function-related genes". Each of these genes encodes a corresponding protein or enzyme. Substances that activate these genes are expected to be useful in controlling skin functions.

[0024] The PPARα protein encoded by the PPARA gene is one of the homologues of the peroxisome proliferator-activated receptor (PPAR), a nuclear receptor. PPARα has a lipid decomposition function, and PPARα present in the epidermis in particular induces differentiation of keratinocytes and promotes lipid production in the skin, thereby maintaining the epidermal barrier function and suppressing skin inflammation.

[0025] The RARγ protein encoded by the RARG gene is one of the homologues of the nuclear receptor RAR, and its ligand is fat-soluble vitamins, especially retinoic acid. RARγ has a function of maintaining homeostasis, and RARγ present in the epidermis in particular has the function of controlling epidermal cell differentiation and regeneration. Although retinoic acid can be administered externally to the skin, it has poor stability to light and heat and poor skin permeability. Therefore, retinol, retinol derivatives, or retinoic acid derivatives, which have good properties, may be administered to the skin as, for example, a component of a cosmetic product, so that they are converted to retinoic acid in the body and function as a ligand. Retinol derivatives are compounds that are based on retinol and are bonded with ester bonds, etc., and when hydrolyzed, retinol is produced. Retinol is oxidized by enzyme action to become retinoic acid. An example of a retinol derivative is retinol palmitate. Retinoic acid derivatives are compounds based on retinoic acid that are bonded via ester bonds, etc., and become retinoic acid when hydrolyzed, etc. Examples of retinoic acid derivatives include tocopheryl retinoate and hydroxypinacolone retinoate.

[0026] The proteins loricrin and involucrin, encoded by the LOR and IVL genes, respectively, are expressed in the spinous to granular layers as epidermal cells differentiate, and as epidermal cells differentiate, they are cross-linked by enzymes to form the cornified envelope (CE) that covers the keratinocytes, promoting epidermal keratinization and strengthening the skin barrier function.

[0027] Hyaluronan synthase, encoded by the HAS3 gene, synthesizes hyaluronan in the epidermis and regulates the moisture content of the skin, thereby maintaining epidermal homeostasis.

[0028] Ceramide synthase 2 and ceramide synthase 3, encoded by the CERS2 and CERS3 genes, respectively, synthesize ceramide, an intercellular lipid. Ceramide is produced in the stratum granulosum and ultimately forms a lamellar structure between stratum corneum cells, providing a barrier function for the epidermis.

[0029] Cholesterol sulfotransferase encoded by the SULT2B1 gene regulates cholesterol sulfate metabolism in the skin and enhances the skin barrier function and moisturization through differentiation into keratinocytes.

[0030] The inventors have found that application of diacylglycerol PEG adducts to human epidermis amplifies the expression of epidermal barrier function-related genes. Amplification of the expression of these epidermal barrier function-related genes enhances the production of the proteins and enzymes they code for. This is a new action of diacylglycerol PEG adducts related to human epidermis, particularly related to epidermal barrier function, and is a new property of diacylglycerol PEG adducts. This property can provide effects such as preventing the intrusion of harmful substances and allergens from the outside, preventing the occurrence of oxidative stress in the skin due to external factors, and maintaining moisture retention ability to the epidermis. By exerting these effects, dryness of the skin can be avoided, skin homeostasis can be maintained, and inflammatory reactions can be suppressed. This also leads to improvement of symptoms of chronic skin diseases such as atopic dermatitis and infection. These epidermal barrier functions are not simply physical protective effects on the surface of the epidermis, but are effects obtained within epidermal cells.

[0031] The present invention utilizes the newly discovered properties of the diacylglycerol PEG adduct to provide an agent for amplifying the expression of epidermal barrier function-related genes, which contains the diacylglycerol PEG adduct as an active ingredient, and also provides a method for amplifying the expression of epidermal barrier function-related genes, which uses the diacylglycerol PEG adduct as an active ingredient.

[0032] 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.

[0033] According to the present invention, the diacylglycerol PEG adduct that has reached the epidermis can amplify the expression of epidermal barrier function-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 barrier function-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.

[0034] 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 skin surface 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 stratum granulosum below the stratum corneum, and then into the stratum spinosum and stratum basale. The diacylglycerol PEG adduct then amplifies the expression of epidermal barrier function-related genes in each layer of the epidermis into which it has penetrated.

[0035] 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 skin surface to penetrate into 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.

[0036] 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 epidermal barrier function-related genes.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Below, the relationship between application of diacylglycerol PEG adducts to epidermal cells and genes related to epidermal barrier function is shown by test data.

[0041] (1) Test for the amplification of epidermal barrier function-related genes A test was carried out to confirm the amplification of mRNA expression of each epidermal barrier function-related gene.

[0042] (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 each was cultured for a 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.

[0043] [Table 1]

[0044] 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.

[0045] 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.

[0046] (1-2) Test results Figure 1 is a graph showing the relative expression level of the PPARA gene in GDS23, and Figure 2 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was about 2.2 times higher than the control, and in GDM12, the expression level was about 1.7 to 1.8 times higher than the control.

[0047] Figure 3 is a graph showing the relative expression level of the RARG 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 1.3 to 1.7 times that of the control. In GDM12, the expression level was confirmed to be about 1.5 to 1.8 times that of the control.

[0048] Figure 5 is a graph showing the relative expression level of the LOR 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 2.0 to 3.9 times that of the control, and in GDM12, the expression level was confirmed to be about 1.3 times that of the control.

[0049] 7 is a graph showing the relative expression levels of the IVL gene in GDM12. The expression level was confirmed to be about 1.5 times that of the control.

[0050] Figure 8 is a graph showing the relative expression level of the HAS3 gene in GDS23, and Figure 9 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.7 to 2.8 times that of the control, and in GDM12, the expression level was confirmed to be about 1.6 to 2.2 times that of the control.

[0051] Figure 10 is a graph showing the relative expression level of the CERS2 gene in GDS23, and Figure 11 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.1 to 1.4 times that of the control. In GDM12, the expression level was confirmed to be about 1.6 to 1.8 times that of the control.

[0052] 12 is a graph showing the relative expression level of the CERS3 gene in GDS23. The expression level was confirmed to be about twice that of the control.

[0053] Figure 13 is a graph showing the relative expression level of the SULT2B1 gene in GDS23, and Figure 14 is a graph showing the relative expression level in GDM12. In GDS23, the expression level was confirmed to be about 1.2 times that of the control, and in GDM12, the expression level was confirmed to be about 1.6 times that of the control.

[0054] (2) Loricrin production detection test Tests to detect the production of loricrin encoded by the LOR gene were performed using two staining methods: immunostaining with anti-loricrin antibody and hematoxylin-eosin staining.

[0055] (2-1) Test method Processing of the epidermis model A human three-dimensional cultured epidermis model (LabCyte EPI-MODEL24 6D: manufactured by Japan Tissue Engineering) was cultured in medium (assay medium: manufactured by Japan Tissue Engineering) at 37°C for 24 hours. After that, the medium was replaced, and 50 μL of a sample (2% adjusted solution of GDS23) prepared using purified water was applied to the surface of the stratum corneum, and the culture was continued for 24 hours. For comparison, only purified water was applied to the control.

[0056] After that, excess sample was removed from the surface of the stratum corneum of the epidermis model by sucking it up with a sterilized cotton swab, and then the epidermis model was continued to be cultured without applying any new sample to the surface of the stratum corneum. The epidermis model was harvested 9 days after application of the sample.

[0057] Preparation of frozen sections of epidermis model and loricrin immunostaining The collected epidermis model was embedded in a frozen tissue embedding medium (OCT compound: Sakura Finetech Japan) to prepare frozen sections from the three-dimensional cultured skin tissue. The sections were then fixed in a 4% formaldehyde solution and blocked for 1 hour with a 3% BSA / PBS(-) solution containing 10% normal goat serum. After aspirating the blocking agent, anti-loricrin antibody (Proteintech Japan) (diluted 50-fold with 3% BSA / PBS solution) was added to the sections, and the primary antibody reaction was carried out overnight at 4°C. Then, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488 antibody (Abcam) (diluted 250-fold with 3% BSA / PBS solution) was added, and the secondary antibody reaction was carried out at room temperature in the dark for 1 hour. Then, nuclear staining was carried out with DAPI diluted 200-fold with 3% BSA / PBS solution. After that, green and blue fluorescence were observed using a fluorescence microscope (BZ-X810, KEYENCE). Green fluorescence indicates the presence of loricrin. Blue fluorescence stains the nuclei of the epidermis model to confirm that there are no abnormalities in the cells.

[0058] Preparation of frozen sections of the epidermis model and hematoxylin-eosin staining The collected epidermal model was embedded in a frozen tissue embedding medium (OCT compound: Sakura Finetech Japan), and frozen sections were prepared. These were then fixed in a 4% formaldehyde solution and washed with purified water. They were then immersed in hematoxylin solution for staining, washed with running water, and then immersed in eosin solution. The sections were then dehydrated with 90% ethanol and 100% ethanol, cleared with xylene, and mounted. They were then observed. Hematoxylin-eosin staining was performed to confirm that there were no abnormalities in the cells of the epidermal model.

[0059] (2-2) Test results FIG. 15 shows the test results of the loricrin immunostaining method (upper panel) and the hematoxylin-eosin staining method (lower panel).

[0060] The green fluorescence in the fluorescence microscope images in the top row (the upper part of the band extending from the upper left to the lower right) indicates that the amount of loricrin produced in the stratum corneum of the epidermis model to which GDS23 solution was applied was greater than that of the control epidermis model.

[0061] The blue fluorescence in the fluorescence microscope image (top row) (lower part of the band extending from the upper left to the lower right) and the hematoxylin-eosin stained image (bottom row) show that there are no abnormalities in the cells of the epidermis model.

[0062] (3) Preparation of cosmetics and topical skin preparations Below are examples of preparations of cosmetics and topical skin preparations containing an epidermal barrier function-related gene expression amplifier that contains a diacylglycerol PEG adduct as an active ingredient. Each of the following preparation examples contains retinoic acid, retinol, a retinol derivative, and / or a retinoic acid derivative. Retinoic acid can bind to and activate the nuclear receptor RARγ. Each substance other than retinoic acid can act on RARγ by being converted to retinoic acid through hydrolysis, enzymatic action, etc. in the body. As described above, the expression of RARγ is enhanced by the diacylglycerol PEG adduct of the present invention. Therefore, it is expected that the activation of RARγ will be further promoted by enriching the retinoic acid in the body with the above-mentioned substances such as retinoic acid administered from the outside.

[0063] (3-1) Example of preparing lotion The lotion of Preparation Example 1 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, tocopheryl retinoate, retinol, retinol palmitate, and hydroxypinacolone retinoate. [Table 2]

[0064] (3-2) Example of emulsion preparation The emulsion of Preparation Example 2 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, tocopheryl retinoate, retinol, and hydroxypinacolone retinoate. [Table 3]

[0065] (3-3) Preparation of water-based gel The aqueous gel of Preparation Example 3 contains glycerol distearate PEG-23, glycerol dimyristate PEG-12, tocopheryl retinoate, retinol, and hydroxypinacolone retinoate. [Table 4]

[0066] (3-4) Cream preparation example The cream of Preparation Example 4 contains PEG-23 glycerol distearate, PEG-12 glycerol dimyristate, tocopheryl retinoate, hydroxypinacolone retinoate, and retinol. [Table 5]

[0067] (3-5) Preparation of ointment The ointment of Preparation Example 5 contains glycerol distearate PEG-23, glycerol dimyristate PEG-12, tocopheryl retinoate, hydroxypinacolone retinoate, and retinoic acid. [Table 6]

[0068] (3-6) Preparation of a lotion containing retinoic acid The retinoic acid-containing lotion of Preparation Example 6 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, tocopheryl retinoate, and retinoic acid. [Table 7]

[0069] (3-7) Preparation of cream containing retinoic acid The retinoic acid-containing cream of Preparation Example 7 contains glycerol distearate PEG-23, glycerol dimyristate PEG-12, retinoic acid, tocopheryl retinoate, and retinol. [Table 8]

[0070] Although not exemplified, there are various combinations of diacylglycerol PEG adducts and other ingredients in cosmetics or topical skin preparations other than those mentioned above. In addition, when retinoic acid, retinol, a retinol derivative, or a retinoic acid derivative is contained as an ingredient, only one of these or a combination of multiple kinds is selected as necessary.

[0071] 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 the expression of an epidermal barrier function-related gene, 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 epidermal barrier function-related gene expression amplifier according to claim 1, wherein the epidermal barrier function-related gene is at least one selected from the group consisting of PPARA, RARG, LOR, IVL, HAS3, CER2, CER3, and SULT2B1.

3. 3. The agent for enhancing expression of epidermal barrier function-related genes according to claim 1 or 2, wherein the diacylglycerol PEG adduct is 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 expression amplifier for genes related to epidermal barrier function 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 barrier function-related gene expression amplifier according to claim 1 or 2.

6. The cosmetic preparation according to claim 5 , further comprising one or more selected from the group consisting of retinoic acid, retinol, a retinol derivative, and a retinoic acid derivative.

7. An external skin preparation comprising the epidermal barrier function-related gene expression amplifier according to claim 1 or 2.

8. The skin topical preparation according to claim 7, further comprising one or more selected from the group consisting of retinoic acid, retinol, retinol derivatives, and retinoic acid derivatives.

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