Gene expression amplification agent related to epidermal barrier function

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 epidermal barrier function-related gene expression amplifying agent and an expression amplifying method.

Background Art

[0002] Patent Document 1 discloses a preparation method in which a diacylglycerol polyethylene glycol adduct (hereinafter referred to as "diacylglycerol PEG adduct") is used as a lipid molecule and vesicles are spontaneously formed by mixing with water or a surfactant. Such vesicles are used in a drug delivery system for encapsulating or binding target substances such as proteins and drugs inside or on their surface and delivering them to cells in vivo. Vesicles composed of diacylglycerol PEG adducts have a form in which their surface is covered with hydrophilic PEG chains, and have good permeability in vivo and stability in blood.

[0003] Patent Document 2 describes that by binding a charged element to the surface of a vesicle composed 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 a drug delivery system have been simply recognized as carriers of drugs as target substances. Recently, it has been known that the molecules of the diacylglycerol PEG adducts derived from vesicles decomposed in vivo also exert useful effects in vivo. Patent Document 3 discloses that diacylglycerol PEG adducts contribute to enhancing the expression of moisture-related substances such as profilaggrin, filaggrin, and natural moisturizing factor NMF in the epidermis. Further, Patent Document 4 discloses that diacylglycerol PEG adducts contribute to enhancing the expression of antioxidant-related substances such as Nrf2 and PPARG, which are oxidative stress response genes in the epidermis, and antioxidant enzymes such as NQO-1, CAT, and HMOX1.

[0005] On the other hand, in the epidermis, various proteins or enzymes that constitute the stratum corneum of the epidermis or are involved in epidermal cell proliferation play a role in the epidermal barrier function. The epidermal barrier function prevents the invasion of bacteria, inflammatory substances, allergens, etc., from the outside into the epidermis, and retains moisture to prevent skin dryness and maintain skin homeostasis.

[0006] For example, PPARα, a nuclear receptor, is a protein that regulates gene expression related to lipid metabolism and inflammatory response in the skin (Patent Document 5). A great many synthetic and natural substances are known as ligands that activate PPARα. For example, RARγ, the nuclear receptor for retinoic acid (vitamin A), is a protein that regulates gene expression related to the 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. They are produced in accordance with the differentiation of epidermal cells, promoting keratinization of the epidermis and stabilizing 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 levels (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 the metabolism of cholesterol sulfate in the skin, thereby enhancing the epidermal barrier function and moisturizing properties (Patent Document 10). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 4497765 (Self-forming thermodynamically stable liposome and its application) [Patent Document 2] Japanese Patent Publication No. 6297737 (Method for preparing positively charged niosomes and charged niosomes) [Patent Document 3] Japanese Patent Publication No. 6805385 (An agent for enhancing the expression of moisturizing substances in the epidermis) [Patent Document 4] Japanese Patent Publication No. 6860739 (Antioxidant expression enhancer in the epidermis) [Patent Document 5] Japanese Patent Publication No. 2018-48103 (Skin Texture Improving Agent) [Patent Document 6] Japanese Patent Publication No. 2014-528472 (Anti-aging composition through activation of retinoic acid receptors) [Patent Document 7] Japanese Patent Publication No. 2020-160028 (Screening method for skin barrier function improving agents) [Patent Document 8] Japanese Patent Publication No. 2011-98983 (Hyaluronic Acid Production Enhancer) [Patent Document 9] Japanese Patent Publication No. 2016-23155 (Ceramide production promoter and topical skin preparation) [Patent Document 10] Special Publication No. 2019-529515 [Overview of the project] [Problems that the invention aims to solve]

[0008] As described above, diacylglycerol PEG adducts have the effect of enhancing the expression of moisturizing substances described in Patent Document 3 and the effect of enhancing the expression of antioxidant substances described in Patent Document 4. These effects contribute to increasing moisturizing factors in the epidermis and alleviating oxidative stress that occurs in the epidermis. However, even if the expression of moisturizing and antioxidant substances is enhanced, if the epidermal barrier function is impaired, the increased moisturizing factors cannot be retained, nor can the occurrence of oxidative stress be prevented. Little is known about how diacylglycerol PEG adducts are related to this epidermal barrier function.

[0009] The objective of the present invention is to utilize newly discovered properties of diacylglycerol PEG adducts related to epidermal barrier function, and in particular to amplify the expression of epidermal barrier function-related genes.

Means for Solving the Problem

[0010] In order to achieve the above object, the present invention provides the following configuration. An aspect of the present invention is An agent for amplifying the expression of genes related to epidermal barrier function, containing a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct having the following structural formula, the number of carbon atoms of R in the long-chain fatty acid being within the range of 11 to 23, and n in the polyethylene glycol chain being within the range of 11 to 46 Furthermore, the epidermal barrier function-related gene is at least one of the group consisting of PPARA, RARG, and HAS3. .

[0012]

Chemical formula

[0013] Preferably, the diacylglycerol PEG adduct is selected from the group consisting of glyceryl dimyristate PEG-12 (GDM12), glyceryl distearate PEG-12 (GDS12), glyceryl distearate PEG-23 (GDS23), glyceryl dipalmitate PEG-23 (GDP23), and glyceryl dioleate PEG-12 (GDO12). Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a solution state or a vesicle state.

[0014] Another aspect of the present invention is a cosmetic containing the above epidermal barrier function-related gene expression amplifying agent. Another aspect of the present invention is an external preparation for skin containing the above epidermal barrier function-related gene expression amplifying agent. The cosmetic or the external preparation for skin preferably further contains one or more of the group consisting of retinoic acid, retinol, retinol derivatives, and retinoic acid derivatives.

Effect of the Invention

[0015] According to the present invention, an expression amplifier for genes related to epidermal barrier function, which contains a diacylglycerol PEG adduct as an active ingredient, is realized. Further, according to the present invention, a method for amplifying the expression of genes related to epidermal barrier function, which uses a diacylglycerol PEG adduct as an active ingredient, is realized.

Brief Description of Drawings

[0016] [Figure 1] Figure 1 is a graph showing the relative expression amplification amount of the PPARA gene related to GDS23. [Figure 2] Figure 2 is a graph showing the relative expression amplification amount of the PPARA gene related to GDM12. [Figure 3] Figure 3 is a graph showing the relative expression amplification amount of the RARG gene related to GDS23. [Figure 4] Figure 4 is a graph showing the relative expression amplification amount of the RARG gene related to GDM12. [Figure 5] Figure 5 is a graph showing the relative expression amplification amount of the LOR gene related to GDS23. [Figure 6] Figure 6 is a graph showing the relative expression amplification amount of the LOR gene related to GDM12. [Figure 7] Figure 7 is a graph showing the relative expression amplification amount of the IVL gene related to GDM12. [Figure 8] Figure 8 is a graph showing the relative expression amplification amount of the HAS3 gene related to GDS23. [Figure 9] Figure 9 is a graph showing the relative expression amplification amount of the HAS3 gene related to GDM12. [Figure 10] Figure 10 is a graph showing the relative expression amplification amount of the CERS2 gene related to GDS23. [Figure 11] Figure 11 is a graph showing the relative expression amplification amount of the CERS2 gene related to GDM12. [Figure 12] Figure 12 is a graph showing the relative expression amplification amount of the CERS3 gene related to GDS23. [Figure 13] Figure 13 is a graph showing the relative expression amplification levels of the SULT2B1 gene related to GDS23. [Figure 14] Figure 14 is a graph showing the relative expression amplification levels of the SULT2B1 gene related to GDM12. [Figure 15] Figure 15 shows images illustrating the detection results of loricrin (LOR) using the loricrin (LOR) immunostaining method (top panel) and the hematoxylin-eosin (HE) staining method (bottom panel) for GDS23. [Modes for carrying out the invention]

[0017] Embodiments of the present invention will be described below with reference to the drawings. This invention was created by utilizing a newly discovered property of diacylglycerol polyethylene glycol adduct (diacylglycerol PEG adduct). The newly discovered property is the ability to amplify the expression of human epidermal barrier function-related genes.

[0018] The structural formula of the diacylglycerol PEG adduct, a lipid molecule related to the present invention, is schematically shown.

[0019] [ka]

[0020] Diacylglycerol PEG adducts consist of a glycerol skeleton (CH2CHCH2) with three carbon atoms, a PEG chain which is a linear polyethylene glycol bonded to one of the terminal carbon atoms of the skeleton, and identical long-chain fatty acids (COOR) bonded to the other two carbon atoms of the skeleton. The PEG chain portion is hydrophilic, while the long-chain fatty acid portion is hydrophobic.

[0021] In the following explanation, when referring to a specific diacylglycerol PEG adduct, it will be denoted as "[di] + [name of long-chain fatty acid] + [glycerol] + [PEG-n]" based on the type of long-chain fatty acid and the number of n in the PEG chain. For example, if the long-chain fatty acid is myristic acid and the number of n in the PEG chain is 12, it will be referred to as "dimrylistate PEG-12". In addition, specific diacylglycerol PEG adducts may also be referred to by abbreviations.

[0022] The number of carbon atoms in R in long-chain fatty acids can be in the range of 11 to 23. Examples of long-chain fatty acids in this range include myristic acid, palmitic acid, stearic acid, or oleic acid. The number of n atoms in the PEG chain can be in the range of 11 to 46. The following are examples of diacylglycerol PEG adducts related to the present invention. 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 invasion of bacteria, inflammatory substances, allergens, and other substances from the outside, thereby avoiding various skin problems. In this invention, the epidermal barrier function-related genes whose expression amplification has been confirmed 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 codes for a corresponding protein or enzyme. Substances that activate these genes are expected to play a role in controlling skin function.

[0024] The PPARα protein encoded by the PPARA gene is a homolog of the peroxisome proliferator-responsive receptor PPAR, a nuclear receptor. PPARα has a lipid-degrading function, and in particular, PPARα present in the epidermis induces keratinocyte differentiation 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 a homolog of the nuclear receptor RAR and uses fat-soluble vitamins, particularly retinoic acid, as its ligand. RARγ plays a role in maintaining homeostasis, and RARγ present in the epidermis, in particular, regulates epidermal cell differentiation and regeneration. Retinoic acid can be administered externally to the skin, but it has poor stability to light and heat, and poor skin penetration. Therefore, retinol, retinol derivatives, or retinoic acid derivatives, which have good properties in these areas, may be administered to the skin, for example, as ingredients in cosmetics, so that they are converted to retinoic acid in the body and function as ligands. Retinol derivatives are compounds formed by linking retinol with ester bonds or other mechanisms, and when hydrolyzed, retinol is produced. Retinol can be oxidized by enzymatic action to retinoic acid. Retinol derivatives include, for example, retinyl palmitate. Retinoic acid derivatives are compounds formed by linking retinoic acid with ester bonds or other means, and they revert to retinoic acid when hydrolyzed. Examples of retinoic acid derivatives include retinoic acid tocopheryl and retinoic acid hydroxypinacolin.

[0026] The loricrin and involucrin proteins encoded by the LOR and IVL genes, respectively, are expressed in the stratum spinosum and stratum granulosum during epidermal cell differentiation. As epidermal cells differentiate, they are enzymatically cross-linked to form a cornified envelope (CE) that covers the keratinocytes. This promotes keratinization of the epidermis and strengthens the skin's barrier function.

[0027] The hyaluronic acid synthase encoded by the HAS3 gene synthesizes hyaluronic acid in the epidermis, maintaining and regulating the skin's moisture content. This helps maintain epidermal homeostasis.

[0028] The 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 granular layer and ultimately forms a lamellar structure between stratum corneum cells, playing a role in the barrier function of the epidermis.

[0029] The cholesterol sulfotransferase encoded by the SULT2B1 gene regulates the metabolism of cholesterol sulfate in the skin, enhancing the skin's barrier function and moisturizing properties through differentiation into keratinocytes.

[0030] The inventors discovered that applying diacylglycerol PEG adducts to human epidermis amplifies the expression of genes related to epidermal barrier function. This amplification of the expression of these epidermal barrier function-related genes enhances the production of the proteins and enzymes they encode. This is a novel effect of diacylglycerol PEG adducts on human epidermis, particularly related to epidermal barrier function, and represents a new property of diacylglycerol PEG adducts. This property can provide the epidermis with 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 capacity. By exerting these effects, skin dryness can be avoided, skin homeostasis can be maintained, and inflammatory responses can be suppressed. Ultimately, this can lead to improvement in the symptoms of chronic skin diseases such as atopic dermatitis and infections. These epidermal barrier functions are not merely physical protective effects on the surface of the epidermis, but effects obtained within epidermal cells.

[0031] This invention provides an expression amplification agent for epidermal barrier function-related genes, utilizing newly discovered properties of the diacylglycerol PEG adduct, with the diacylglycerol PEG adduct as an active ingredient. Furthermore, this invention provides a method for amplifying the expression of epidermal barrier function-related genes using the diacylglycerol PEG adduct as an active ingredient.

[0032] In this invention, when applying diacylglycerol PEG adducts to human epidermis, one type may be used alone, or multiple types may be used in combination.

[0033] According to the present invention, diacylglycerol PEG adducts that reach the epidermis can amplify the expression of epidermal barrier function-related genes and increase the production of corresponding proteins and enzymes compared to the absence of diacylglycerol PEG adducts. As a result, not only the condition within the epidermis but also the condition of the epidermal surface is improved. Therefore, the present invention can provide cosmetics or topical skin preparations containing diacylglycerol PEG adducts as an active ingredient, acting as an expression amplifier for epidermal barrier function-related genes. Both cosmetics and topical skin preparations are applied to the surface of the epidermis. Here, all substances other than cosmetics are referred to as topical skin preparations, such as ointments. Such cosmetics or topical skin preparations can be provided in various forms, such as aqueous solutions, emulsions, gels, and creams. In addition to diacylglycerol PEG adducts, these cosmetics or topical skin preparations may contain other active ingredients and / or various commonly included components.

[0034] One method for delivering diacylglycerol PEG adducts into the human epidermis is to deliver them in a solution form dissolved in water or a predetermined solvent. For example, a solution of diacylglycerol PEG adducts at a predetermined concentration using phosphate-buffered saline (PBS(-)) as a solvent can be prepared and applied to the skin surface to allow penetration into the epidermis. The applied solution penetrates, for example, into the stratum corneum (the uppermost layer), then further into the granular layer below the stratum corneum, and then into the spinous and basal layers. The diacylglycerol PEG adducts then amplify the expression of epidermal barrier function-related genes in each layer of the epidermis into which they penetrate.

[0035] Another method for delivering diacylglycerol PEG adducts to the epidermis is to deliver them in vesicle form. Such vesicles are formed as closed spherical shells consisting of a bilayer of diacylglycerol PEG adducts, or multiple layers of bilayers, with hydrophilic PEG chains positioned on the outermost surface. By preparing diacylglycerol PEG adduct vesicles and applying them to the skin surface, they can penetrate into the epidermis. After reaching the epidermis, the vesicles decompose and separate into individual molecules, allowing the diacylglycerol PEG adducts themselves to exert their effects.

[0036] In conventional drug delivery systems, diacylglycerol PEG adducts, which are the material for vesicles, have been considered merely carriers of the target substance. However, in the present invention, the diacylglycerol PEG adduct itself is used as the active ingredient. Therefore, in the present invention, the target substance incorporated into the vesicle, which is typically required in conventional drug delivery systems, is essentially unnecessary. In the present invention, by permeating vesicles formed by mixing only water and diacylglycerol PEG adducts 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 predetermined temperature (see Patent Documents 1 and 2). For example, a suspension of GDM12 or GDO12 vesicles can be obtained by mixing 2% by mass of GDM12 or GDO12 with 98% by mass of deionized water at room temperature and stirring. Another example is to dissolve 2% by mass of GDS12 or GDS23 at 45-55°C, then mix with 98% by mass of deionized water at 45-55°C and stir to obtain a suspension of GDS12 or GDS23 vesicles. Yet another example is to dissolve 2% by mass of GDP23 at 37°C, then mix with 98% by mass of deionized water at 37°C and stir to obtain a suspension of GDP23 vesicles. The vesicles remain 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 the use of vesicles formed by mixing and stirring aqueous solutions of various substances with diacylglycerol PEG adducts, instead of the water described above. In this case, the substances contained in the aqueous solution may be given other functions.

[0039] As yet another example, the present invention also includes cases where the surface of vesicles 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 have particularly excellent permeability and retention properties in the epidermis.

[0040] The following study presents experimental data illustrating the relationship between the application of diacylglycerol PEG adducts to epidermal cells and genes related to epidermal barrier function.

[0041] (1) Tests on the amplification of expression of genes related to epidermal barrier function We conducted tests to confirm the amplification of mRNA expression in each gene related to epidermal barrier function.

[0042] (1-1) Test Method Normal human epidermal keratinocytes (NHEKs) 2.0 × 10⁻⁶ 4 Cells were seeded in HuMedia-KG2 medium (Kurabo Industries Ltd.) at a cell density of cells / well into 96-well plates and cultured for 24 hours at 37°C under 5% CO2 conditions. Subsequently, diacylglycerol PEG adducts were added to HuMedia-KB2 medium (Kurabo Industries Ltd.), and each cell was cultured at 37°C under 5% CO2 conditions for a predetermined time. Table 1 shows the target gene, the type of diacylglycerol PEG adduct added, the amount added, and the culture time. The control (no adduct) was cultured under the same conditions.

[0043] [Table 1]

[0044] After culturing each sample for a predetermined 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 the internal standard.

[0045] The analysis involved correcting the mRNA expression level of each target gene using the expression level of GAPDH, an internal standard in the same sample. Furthermore, the corrected value of the sample was calculated as the relative expression level, with the control's corrected value set to 1.

[0046] (1-2) Test results Figure 1 shows the relative expression levels of the PPARA gene in GDS23, and Figure 2 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 2.2 times higher than that of the control. In GDM12, the expression level was approximately 1.7 to 1.8 times higher than that of the control.

[0047] Figure 3 shows the relative expression levels of the RARG gene in GDS23, and Figure 4 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.3 to 1.7 times higher than that of the control. In GDM12, the expression level was approximately 1.5 to 1.8 times higher than that of the control.

[0048] Figure 5 shows the relative expression levels of the LOR gene in GDS23, and Figure 6 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 2.0 to 3.9 times higher than that of the control. In GDM12, the expression level was approximately 1.3 times higher than that of the control.

[0049] Figure 7 shows graphs illustrating the relative expression levels of the IVL gene in GDM12. Expression levels approximately 1.5 times higher than those of the control were observed.

[0050] Figure 8 shows the relative expression levels of the HAS3 gene in GDS23, and Figure 9 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.7 to 2.8 times higher than that of the control. In GDM12, the expression level was approximately 1.6 to 2.2 times higher than that of the control.

[0051] Figure 10 shows the relative expression levels of the CERS2 gene in GDS23, and Figure 11 shows the relative expression levels in GDM12. In GDS23, the expression level was approximately 1.1 to 1.4 times higher than that of the control. In GDM12, the expression level was approximately 1.6 to 1.8 times higher than that of the control.

[0052] Figure 12 shows graphs illustrating the relative expression levels of the CERS3 gene in GDS23. Expression levels approximately twice that of the control were observed.

[0053] Figure 13 and Figure 14 show graphs illustrating the relative expression levels of the SULT2B1 gene in GDS23 and GDM12, respectively. In GDS23, the expression level was approximately 1.2 times higher than that of the control. In GDM12, the expression level was approximately 1.6 times higher than that of the control.

[0054] (2) Test for detection of loricrin production To detect the production of loricrin encoded by the LOR gene, we performed two staining methods: immunohistochemistry using anti-loricrin antibodies and hematoxylin-eosin staining.

[0055] (2-1) Test Method • Processing of the epidermal model A human three-dimensional cultured epidermal model (LabCyte EPI-MODEL24 6D: manufactured by Japan Tissue Engineering Co., Ltd.) was cultured in culture medium (assay medium: manufactured by Japan Tissue Engineering Co., Ltd.) at 37°C for 24 hours. Subsequently, the culture medium was changed, and 50 μL of the sample prepared with purified water (2% GDS23 solution) was applied to the stratum corneum surface, and incubation was continued for 24 hours. The control group was coated with purified water only.

[0056] Subsequently, excess sample was removed from the stratum corneum surface of the epidermal model by aspirating it with a sterile cotton swab. The culture of the epidermal model was then continued without applying any new sample to the stratum corneum surface, and the epidermal model was collected 9 days after the application of the sample.

[0057] • Preparation of frozen sections of an epidermal model and Loricrin immunohistochemistry Frozen sections were prepared from three-dimensional cultured skin tissue, which was embedded in a frozen tissue embedding medium (OCT compound: manufactured by Sakura FineTech Japan) using a recovered epidermal model. After fixing with 4% formaldehyde solution, the sections were 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) (50-fold dilution in 3% BSA / PBS solution) was added to the section, and a primary antibody reaction was carried out overnight at 4°C. Subsequently, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488 antibody (Abcam)) (250-fold dilution in 3% BSA / PBS solution) was added, and a secondary antibody reaction was carried out at room temperature in the dark for 1 hour. Then, nuclear staining was performed with DAPI diluted 200-fold in 3% BSA / PBS solution. After that, green and blue fluorescence was observed using a fluorescence microscope (BZ-X810, KEYENCE). Green fluorescence indicates the presence of loricrin. Blue fluorescence stains the nuclei of the epidermal model and confirms that there are no abnormalities in the cells.

[0058] • Preparation of frozen sections of an epidermal model and hematoxylin-eosin staining method The recovered epidermal model was embedded in a cryogenic tissue embedding medium (OCT compound: manufactured by Sakura FineTech Japan), and frozen sections were prepared. After fixing with 4% formaldehyde solution, the sections were washed with purified water. Subsequently, they were stained by immersion in hematoxylin solution, washed with running water, and then immersed in eosin solution. Furthermore, the sections were dehydrated with 90% ethanol and 100% ethanol, cleared with xylene, and mounted. Observation was then performed. The hematoxylin-eosin staining method was performed to confirm that there were no abnormalities in the cells of the epidermal model.

[0059] (2-2) Test Results Figure 15 shows the results of the Loricrin immunohistochemical staining method (top panel) and the hematoxylin-eosin staining method (bottom panel), respectively.

[0060] The green fluorescence in the upper fluorescence microscope image (the upper part of the band extending from the upper left to the lower right) indicates that the production of loricrin in the stratum corneum of the epidermal model coated with GDS23 solution is higher than that of the control epidermal model.

[0061] The blue fluorescence in the upper fluorescence microscope image (the lower part of the band extending from the upper left to the lower right), and the hematoxylin-eosin stained image in the lower panel, indicate that there are no abnormalities in the cells of the epidermal model.

[0062] (3) Preparation of cosmetics and topical skin preparations The following are examples of preparations for cosmetics and topical skin preparations containing diacylglycerol PEG adducts as active ingredients, which are gene expression amplification agents related to the skin barrier function. Each of the following preparations includes retinoic acid, retinol, retinol derivatives, and / or retinoic acid derivatives. Retinoic acid can bind to and activate the nuclear receptor RARγ. The other substances can act on RARγ by being converted to retinoic acid in the body through hydrolysis or enzymatic action. As described above, the diacylglycerol PEG adduct in the present invention enhances RARγ expression. Therefore, it is expected that the activation of RARγ will be further promoted by enriching the body with retinoic acid through the administration of the above-mentioned retinoic acid from an external source.

[0063] (3-1) Example of lotion preparation The lotion in Adjustment Example 1 contains glycerol dimyristate PEG-12, glycerol distearate PEG-23, tocopheryl retinoate, retinol, retinyl palmitate, and hydroxypinacolin retinoate. [Table 2]

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

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

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

[0067] (3-5) Examples of ointment preparation The ointment in formulation example 5 contains glycerol distearate PEG-23, glycerol dimyristate PEG-12, tocopheryl retinoate, hydroxypinacolin retinoate, and retinoic acid. [Table 6]

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

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

[0070] Although not exemplified, there are many other combinations of diacylglycerol PEG adducts with other ingredients in cosmetics or topical skin preparations. Furthermore, when retinoic acid, retinol, retinol derivatives, or retinoic acid derivatives are included as ingredients, one or more of these may be selected as needed.

[0071] The present invention has been described above with reference to the examples, but the present invention is not limited to these examples, and obvious modifications therefrom are also included in the present invention.

Claims

1. An agent for amplifying the expression of epidermal barrier function-related genes, The active ingredient is a diacylglycerol PEG adduct, wherein the diacylglycerol PEG adduct has the following structural formula, the number of carbon atoms in R in the long-chain fatty acid is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46. An epidermal barrier function-related gene expression amplifier wherein the epidermal barrier function-related gene is at least one from the group consisting of PPARA, RARG, and HAS3. 【Chemistry 1】

2. The epidermal barrier function-related gene expression enhancer according to claim 1, 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).

3. The epidermal barrier function-related gene expression amplification agent according to claim 1, wherein the diacylglycerol PEG adduct penetrates into the epidermis in a solution state or in a vesicle state.

4. A cosmetic composition comprising the epidermal barrier function-related gene expression amplification agent described in claim 1.

5. The cosmetic composition according to claim 4, further comprising one or more from the group consisting of retinoic acid, retinol, retinol derivatives, and retinoic acid derivatives.

6. A topical skin preparation comprising the epidermal barrier function-related gene expression amplification agent described in claim 1.

7. The topical skin preparation according to claim 6, further comprising one or more from the group consisting of retinoic acid, retinol, retinol derivatives, and retinoic acid derivatives.

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