Aquaporin 3 (AQP3) mRNA expression promoter

Abstract

To provide a aquaporin 3 (AQP3) mRNA expression promoter excellent in action effect.SOLUTION: The aquaporin 3 (AQP3) mRNA expression promoter contains nicotinamide as an active ingredient (except a promoter containing a combination of nicotinamide, hexamidine and pentanediol as active ingredients).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a hyaluronan synthase 3 (HAS3) mRNA expression promoter, a serine palmitoyltransferase (SPT) mRNA expression promoter, an aquaporin 3 (AQP3) mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, and an occludin mRNA expression promoter. [Background technology]

[0002] The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrices such as collagen, elastin, and hyaluronic acid that are present outside these cells and support the skin structure. In young skin, fibroblast proliferation is active, and the interaction between fibroblasts, extracellular matrix components, and other skin tissues maintains homeostasis, ensuring moisture retention, flexibility, elasticity, and the like, and maintaining the skin in a firm, glossy, and moist state.

[0003] However, under the influence of certain external factors, such as ultraviolet radiation, extremely dry air, and excessive skin cleansing, or due to aging, the production of collagen, elastin, and hyaluronic acid, the major components of the extracellular matrix, decreases and undergoes degradation and alteration. As a result, the skin's moisturizing function and elasticity decline, and abnormal peeling of the keratin occurs, causing the skin to lose firmness and luster and to exhibit aging symptoms such as rough skin and wrinkles. Thus, changes associated with skin aging, such as wrinkles, dullness, changes in texture, and loss of elasticity, are related to the reduction and denaturation of matrix components such as collagen, elastin, and hyaluronic acid. Therefore, promoting the production of collagen, elastin, and hyaluronic acid is important for preventing, treating, or improving skin aging.

[0004] Among the aforementioned extracellular matrix components, hyaluronic acid is a type of mucopolysaccharide that fills the intercellular spaces to hold cells in place, and also has numerous other functions, such as retaining moisture in the intercellular spaces, providing lubrication and flexibility to tissues, and providing resistance to external forces such as mechanical damage. Promoting hyaluronic acid production is believed to prevent, treat, or improve skin aging symptoms such as rough skin, wrinkles, dullness, changes in texture, loss of elasticity, and loss of moisturizing function. Furthermore, promoting the expression of hyaluronan synthase 3 (HAS3), which is involved in promoting the synthesis of epidermal hyaluronan, is believed to prevent, treat, or improve skin aging. To date, licorice leaf extract (see Patent Document 1) and other substances are known to promote hyaluronan synthase 3 (HAS3) mRNA expression.

[0005] Ceramide is produced from serine and palmitoyl-CoA during the keratinization process of epidermal cells by the action of enzymes including serine palmitoyltransferase (SPT), known as the rate-limiting enzyme in ceramide synthesis. Ceramide is specifically present as the main component of intercellular lipids that cover the outermost layer of the skin, and plays an important role in maintaining the skin's natural barrier membrane function between the body and the outside world.

[0006] The structure of the stratum corneum can be likened to bricks and mortar, with about 15 layers of keratinocytes stacked together by intercellular lipids to form a strong barrier membrane.Keratinocytes retain moisture by containing natural moisturizing factors, primarily amino acids, within the cells, while intercellular lipids are composed primarily of ceramides (about 50%), as well as amphiphilic lipids such as cholesterol and fatty acids, and are characterized by a lamellar structure, in which hydrophobic and hydrophilic regions alternate.

[0007] A decline in skin barrier function due to various internal and external factors increases transepidermal water loss, causing dryness, scaling, itching, and other symptoms, resulting in so-called dry skin. Furthermore, a decline in skin barrier function increases skin inflammation, resulting in a vicious cycle in which the skin's defense function against various external stimuli is impaired. Recent studies have reported a decrease or change in composition of keratinocyte ceramide components (so-called intercellular lipids) due to aging or in patients with atopic dermatitis, known as a barrier disorder (see Non-Patent Document 1), and it has become widely known that ceramides are important for maintaining and improving skin barrier function. Known methods for improving skin barrier function include supplementing ceramides externally (see Non-Patent Document 2) and enhancing ceramide production within the skin (see Non-Patent Document 3).

[0008] In skin cells, aquaporins, known as water channels, are expressed on the cell membrane and are known to take up small molecules such as water from the intercellular space into the cells. Thirteen types of aquaporins (AQP0-AQP12) are known to exist in humans. Epidermal cells primarily contain AQP3, which is thought to take up not only water but also small molecules such as glycerol and urea, which are involved in moisture retention.

[0009] However, AQP3 decreases with age, and it has been suggested that this is one of the reasons for the decline in water retention function. Therefore, it is thought that promoting AQP3 expression may be able to control water retention ability and barrier function due to aging (see Non-Patent Document 4). For example, extracts from star fruit leaves (see Patent Document 2) are known to have the effect of promoting AQP3 expression.

[0010] Epithelial tissue, composed of epithelial cells, is one of the structures that separate the inside and outside of the body. Epithelial tissue has a barrier function that controls the permeation of substances, thereby creating an internal environment in the body that is different from the outside world. This barrier function is mainly formed by intercellular adhesion, one type of adhesion being tight junctions (hereinafter sometimes referred to as "TJs"). TJs are intercellular adhesion structures that not only bring adjacent epithelial cells into close contact with each other, but also control the permeation of substances by sealing the gaps between cells. TJs are composed of cell membrane proteins such as claudins and occludin, and scaffolding proteins such as ZO-1 and ZO-2. These proteins are thought to form the framework of the TJ strand and control the barrier function of TJs (see Non-Patent Document 5).

[0011] To date, more than 20 types of claudin molecules have been reported, forming the claudin family. These are known to show tissue-specific expression patterns, with claudin-1 and claudin-4 being expressed in the epidermis. Claudin-4 is also highly expressed in mucosal epithelia and functions as a barrier to prevent the entry of foreign substances both inside and outside the body.

[0012] Decreased expression of claudins and occludin for some reason leads to impaired TJ function. For example, impaired TJ function in the digestive tract allows food allergens and pathogenic microorganisms to enter the body, which is thought to contribute to inflammatory bowel disease and various infectious diseases. Furthermore, while it was previously believed that the skin's barrier function was solely the responsibility of the stratum corneum, it has recently been found that genetic deletion of TJ components in the granular layer of the epidermis disrupts the skin's barrier function, leading to the belief that TJs also play an important role in skin barrier function (see Non-Patent Document 6). Here, decreased expression of claudins, occludin, and other proteins leads to structural destruction of TJs, which no longer function as a permeable barrier to substances, which is thought to contribute to skin conditions such as dry skin, rough skin, and atopic dermatitis, as well as various infectious diseases.

[0013] Therefore, it is believed that strengthening TJ function through promoting the production of claudins and occludin can strengthen the barrier function in epithelial tissue, and prevent or improve inflammatory bowel disease, food allergies, and various infectious diseases in the digestive tract, while preventing or improving skin conditions such as dry skin, rough skin, atopic dermatitis, and various infectious diseases in the epidermis. Aspalathus linearis extract (Patent Document 3) and the like are known to have the effect of promoting claudin-1 production and occludin production. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-090035 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-191039 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-256244

[0015] [Non-Patent Document 1] J. Dermatol.,1993,Vol.20,No.1,p.1-6 [Non-patent document 2] "Fragrance Journal", 2004, Vol. 32, No. 11, pp. 23-32 [Non-patent document 3] Br. J. Dermatol.,2000,Vol.143,Issue 3,p.524-531 [Non-patent document 4] "Fragrance Journal", 2006, Vol. 34, No. 10, pp. 19-23 [Non-patent document 5] Journal of the Japanese Society of Cosmetic Science, 2007, vol.31, pp.296-301 [Non-patent document 6] J. Cell Biol.,2002,vol.156,pp.1099-1111 Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention aims to provide a hyaluronic acid synthase 3 (HAS3) mRNA expression promoter, a serine palmitoyltransferase (SPT) mRNA expression promoter, aquaporin 3 (AQP3) mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, and an occludin mRNA expression promoter, all of which have excellent effects. [Means for solving the problem]

[0017] In order to solve the above problems, the hyaluronic acid synthase 3 (HAS3) mRNA expression promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide and cytidine.

[0018] In order to solve the above problems, the serine palmitoyltransferase (SPT) mRNA expression promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, and nicotinamide.

[0019] In order to solve the above problems, the aquaporin 3 (AQP3) mRNA expression promoter of the present invention is characterized by containing one or more active ingredients selected from the group consisting of glucuronic acid, citric acid, nicotinamide, cytidine, and uridine.

[0020] In order to solve the above problems, the claudin-1 mRNA expression promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, and 3-hydroxybutyric acid.

[0021] In order to solve the above problems, the claudin-4 mRNA expression promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of cytosine, trimethylglycine, glucuronic acid and citric acid.

[0022] In order to solve the above problems, the occludin mRNA expression promoter of the present invention is characterized by having one or more active ingredients selected from the group consisting of cytosine, trimethylglycine, citric acid, 3-hydroxybutyric acid, and glyceric acid. [Effects of the Invention]

[0023] According to the present invention, it is possible to provide a hyaluronic acid synthase 3 (HAS3) mRNA expression promoter, a serine palmitoyltransferase (SPT) mRNA expression promoter, an aquaporin 3 (AQP3) mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, and an occludin mRNA expression promoter, all of which have excellent effects. DETAILED DESCRIPTION OF THE INVENTION

[0024] Hereinafter, an embodiment of the present invention will be described. [HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, occludin mRNA expression promoter] The HAS3 mRNA expression promoter of this embodiment contains, as an active ingredient, one or more members selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, and cytidine. The SPT mRNA expression promoter of this embodiment contains, as an active ingredient, one or more members selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, and nicotinamide. The AQP3 mRNA expression promoter of this embodiment contains, as an active ingredient, one or more selected from the group consisting of glucuronic acid, citric acid, nicotinamide, cytidine, and uridine. The claudin-1 mRNA expression promoter of this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, and 3-hydroxybutyric acid. The claudin-4 mRNA expression promoter of this embodiment contains, as an active ingredient, one or more selected from the group consisting of cytosine, trimethylglycine, glucuronic acid, and citric acid. The occludin mRNA expression promoter of this embodiment contains, as an active ingredient, one or more selected from the group consisting of cytosine, trimethylglycine, citric acid, 3-hydroxybutyric acid, and glyceric acid.

[0025] The cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, and uridine used in this embodiment are commercially available, and these may be used. Alternatively, they may be produced by isolating and purifying from a plant extract containing cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, or uridine using a known method, or by synthesizing using a known method. The plant extract includes an extract obtained from a plant containing cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, or uridine as an extracting material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof.

[0026] Plants containing cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid or uridine include, but are not limited to, rice.

[0027] The plant extract may be subjected to microbial fermentation, for example, by dissolving the concentrated plant extract in water or by using the plant extract as is without concentrating it to dryness and then inoculating it with a fermenting microorganism.

[0028] The microorganisms that carry out the fermentation are not particularly limited, and examples include lactic acid bacteria, yeast, and koji mold, but yeast is preferred, and Saccharomyces verona is particularly preferred.

[0029] The cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, and uridine used in this embodiment have excellent HAS3 mRNA expression-promoting effects, SPT mRNA expression-promoting effects, AQP3 mRNA expression-promoting effects, claudin-1 mRNA expression-promoting effects, claudin-4 mRNA expression-promoting effects, and occludin mRNA expression-promoting effects, and therefore can be used as active ingredients of HAS3 mRNA expression promoters, SPT mRNA expression promoters, AQP3 mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, and occludin mRNA expression promoters according to this embodiment. The HAS3 mRNA expression promoters, SPT mRNA expression promoters, AQP3 mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, and occludin mRNA expression promoters according to this embodiment can be used in a wide range of applications, such as pharmaceuticals, quasi-drugs, and foods and beverages.

[0030] In this embodiment, any one of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, and uridine may be used as the active ingredient of the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, or occludin mRNA expression promoter, or two or more of these may be mixed and used as the active ingredient. When two or more of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, or uridine are mixed and used as the active ingredient, the mixing ratio may be adjusted appropriately depending on the level of the HAS3 mRNA expression-promoting effect, SPT mRNA expression-promoting effect, AQP3 mRNA expression-promoting effect, claudin-1 mRNA expression-promoting effect, claudin-4 mRNA expression-promoting effect, or occludin mRNA expression-promoting effect of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, and uridine.

[0031] The HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment may consist solely of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, or uridine, or a mixture thereof, or may be a formulation of these active ingredients.

[0032] The HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment can be formulated into any dosage form, such as powder, granules, tablets, or liquid, using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin, or any other auxiliary agent, according to standard methods. In this case, examples of auxiliary agents that can be used include excipients, binders, disintegrants, lubricants, stabilizers, and flavorings / flavoring agents. The HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter can be incorporated into other compositions (e.g., topical skin preparations, oral compositions, etc., as described below) and used as ointments, topical liquids, patches, etc.

[0033] When the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment are formulated, the content of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, uridine, or a mixture thereof is not particularly limited and can be set appropriately depending on the purpose.

[0034] The HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment can be used as an active ingredient, if necessary, by blending other substances (e.g., natural extracts) having an HAS3 mRNA expression-promoting effect, an SPT mRNA expression-promoting effect, an AQP3 mRNA expression-promoting effect, a claudin-1 mRNA expression-promoting effect, a claudin-4 mRNA expression-promoting effect, or an occludin mRNA expression-promoting effect with cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, uridine, or a mixture thereof.

[0035] Methods for administering the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment to patients include transdermal administration, oral administration, etc., and a method suitable for the prevention, treatment, etc. of the disease may be appropriately selected depending on the type of disease. Furthermore, the dosage of the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment may be increased or decreased as appropriate depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0036] The HAS3 mRNA expression promoter of this embodiment can promote the expression of hyaluronan synthase 3 (HAS3) through the HAS3 mRNA expression-promoting effects of cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, and cytidine, thereby promoting the production of hyaluronic acid and preventing, treating, or ameliorating symptoms of skin aging, such as rough skin, wrinkles, dullness, changes in texture, loss of elasticity, and loss of moisturizing function. However, in addition to these uses, the HAS3 mRNA expression promoter of this embodiment can also be used for any other uses in which exerting the above-mentioned effects is meaningful.

[0037] The SPT mRNA expression promoter of this embodiment can promote ceramide synthesis through the SPT mRNA expression-promoting effects of cytosine, trimethylglycine, glucuronic acid, and nicotinamide, thereby strengthening the skin's barrier function and preventing, treating, or ameliorating rough skin, dry skin, and dry skin diseases (e.g., atopic dermatitis). However, in addition to these uses, the SPT mRNA expression promoter of this embodiment can also be used for any other uses where exerting the above-mentioned effects is meaningful.

[0038] The AQP3 mRNA expression promoter of this embodiment can promote the expression of aquaporin 3 (AQP3) through the AQP3 mRNA expression-promoting effects of glucuronic acid, citric acid, nicotinamide, cytidine, and uridine, and can therefore improve age-related moisture retention ability, barrier function, etc. However, in addition to these uses, the AQP3 mRNA expression promoter of this embodiment can also be used for all uses in which exerting the above-mentioned effects is significant.

[0039] The claudin-1 mRNA expression promoter of this embodiment can promote the formation of tight junctions in epithelial tissue through the claudin-1 mRNA expression-promoting effects of cytosine, trimethylglycine, glucuronic acid, and 3-hydroxybutyric acid, thereby enhancing barrier function and moisture retention, and preventing, treating, or ameliorating skin conditions such as dry skin, rough skin, and atopic dermatitis, as well as various infectious diseases. However, in addition to these uses, the claudin-1 mRNA expression promoter of this embodiment can also be used for any other uses in which exerting the above-mentioned effects is meaningful.

[0040] The claudin-4 mRNA expression promoter of this embodiment can promote the formation of tight junctions in epithelial tissue through the claudin-4 mRNA expression-promoting effects of cytosine, trimethylglycine, glucuronic acid, and citric acid, thereby enhancing barrier function and moisture retention function and preventing, treating, or ameliorating skin conditions such as dry skin, rough skin, and atopic dermatitis, as well as various infectious diseases. However, in addition to these uses, the claudin-4 mRNA expression promoter of this embodiment can also be used for any other uses where exerting the above-mentioned effects is significant.

[0041] The occludin mRNA expression promoter of this embodiment can promote the formation of tight junctions in epithelial tissue through the occludin mRNA expression-promoting effects of cytosine, trimethylglycine, citric acid, 3-hydroxybutyric acid, and glyceric acid, thereby enhancing barrier function and moisture retention, and preventing, treating, or ameliorating skin conditions such as dry skin, rough skin, and atopic dermatitis, as well as various infectious diseases. However, in addition to these uses, the occludin mRNA expression promoter of this embodiment can also be used for any other uses in which exerting the above-mentioned effects is meaningful.

[0042] Furthermore, the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment have excellent HAS3 mRNA expression-promoting activity, SPT mRNA expression-promoting activity, AQP3 mRNA expression-promoting activity, claudin-1 mRNA expression-promoting activity, claudin-4 mRNA expression-promoting activity, or occludin mRNA expression-promoting activity, and are therefore suitable for incorporation into, for example, topical skin preparations, oral compositions, and the like. In this case, cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, uridine, or a mixture thereof may be blended directly, or an HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, or occludin mRNA expression promoter formulated from citric acid, uridine, cytidine, trimethylglycine, glyceric acid, 3-hydroxybutyric acid, cytosine, glucuronic acid, nicotinamide, or a mixture thereof may be blended.

[0043] Here, topical skin preparations are not limited to specific categories and include a wide range of skin cosmetics, quasi-drugs, pharmaceuticals, etc. that are used transdermally. Specific examples include ointments, creams, emulsions, skin lotions, beauty serums, lotions, gels, beauty oils, packs, foundations, lip balms, bath additives, hair tonics, hair lotions, shampoos, rinses, soaps, and body shampoos.

[0044] An oral composition refers to a composition that is unlikely to be harmful to human health and that is taken orally or by administration through the digestive tract in normal social life, and is not limited to administrative classifications such as food and drink, medicine, quasi-drug, etc. Therefore, in this embodiment, the "oral composition" broadly includes orally taken general foods, feed, health foods, health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims), quasi-drugs, medicines, etc.

[0045] Furthermore, the HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, and occludin mRNA expression promoter of this embodiment have excellent HAS3 mRNA expression-promoting activity, SPT mRNA expression-promoting activity, AQP3 mRNA expression-promoting activity, claudin-1 mRNA expression-promoting activity, claudin-4 mRNA expression-promoting activity, or occludin mRNA expression-promoting activity, and can therefore be suitably used as reagents for research on the mechanisms of these actions.

[0046] The HAS3 mRNA expression promoter, SPT mRNA expression promoter, AQP3 mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter and occludin mRNA expression promoter of this embodiment are preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as their respective effects are achieved. [Example]

[0047] The present invention will be specifically explained below by showing test examples, but the present invention is not limited to the following examples in any way.

[0048] In the following test examples, the following samples 1 to 9 were used as cytosine, trimethylglycine, glucuronic acid, citric acid, nicotinamide, cytidine, 3-hydroxybutyric acid, glyceric acid, and uridine. Sample 1: Cytosine (Tokyo Chemical Industry Co., Ltd.) Sample 2: Trimethylglycine (Sigma-Aldrich) Sample 3: D-glucuronic acid (Tokyo Chemical Industry Co., Ltd.) Sample 4: Citric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) Sample 5: Nicotinamide (Fujifilm Wako Pure Chemical Industries, Ltd.) Sample 6: Cytidine (Tokyo Chemical Industry Co., Ltd.) Sample 7: DL-3-hydroxybutyric acid (Tokyo Chemical Industry Co., Ltd.) Sample 8: DL-glyceric acid (Tokyo Chemical Industry Co., Ltd.) Sample 9: Uridine (Tokyo Chemical Industry Co., Ltd.)

[0049] [Test Example 1] Hyaluronic acid synthase 3 (HAS3) mRNA expression promoting effect test Cytosine (sample 1), trimethylglycine (sample 2), D-glucuronic acid (sample 3), citric acid (sample 4), nicotinamide (sample 5), and cytidine (sample 6) were tested for their ability to promote HAS3 mRNA expression as follows.

[0050] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0051] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 1 to 6, see Table 1 below for sample concentrations) dissolved in KBM medium was added to each dish and incubated at 37°C and 5% CO2 for 24 hours. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene, Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and total RNA was adjusted to 200 ng / μL.

[0052] Using this total RNA as a template, the mRNA expression levels of HAS3 and GAPDH (an internal standard) were measured. Detection was performed using a real-time PCR system, Thermal Cycler Dice. (R) Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR Prime Script RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of HAS3 mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and normalized with the GAPDH value. The HAS3 mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0053] HAS3 mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 1.

[0054] [Table 1]

[0055] As shown in Table 1, all of Samples 1 to 6 had an excellent effect of promoting HAS3 mRNA expression.

[0056] [Test Example 2] Serine palmitoyltransferase (SPT) mRNA expression promoting effect test Cytosine (sample 1), trimethylglycine (sample 2), D-glucuronic acid (sample 3), and nicotinamide (sample 5) were tested for their SPT mRNA expression promoting activity as follows.

[0057] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 104 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0058] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 1 to 5; see Table 2 below for sample concentrations) dissolved in KBM medium was added to each dish. The cultures were then incubated at 37°C and 5% CO2 for 24 hours. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd., Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0059] Using this total RNA as a template, the mRNA expression levels of SPT and GAPDH as an internal standard were measured. Detection was performed using a real-time PCR device, Thermal Cycler Dice. (R) Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR Prime Script RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of SPT mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The SPT mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0060] SPT mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 2.

[0061] [Table 2]

[0062] As shown in Table 2, it was confirmed that Samples 1 to 3 and 5 all had an excellent effect of promoting SPT mRNA expression.

[0063] [Test Example 3] Aquaporin 3 (AQP3) mRNA expression promoting effect test D-glucuronic acid (sample 3), citric acid (sample 4), nicotinamide (sample 5), cytidine (sample 6), and uridine (sample 9) were tested for their AQP3 mRNA expression promoting activity as follows.

[0064] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0065] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 3-6 and 9; see Table 3 below for sample concentrations) dissolved in KBM medium was added to each dish. The samples were then incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene, Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0066] Using this total RNA as a template, the mRNA expression levels of AQP3 and GAPDH (an internal standard) were measured. Detection was performed using a real-time PCR system, Thermal Cycler Dice. (R) Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of AQP3 mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The AQP3 mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0067] AQP3 mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 3.

[0068] [Table 3]

[0069] As shown in Table 3, it was confirmed that Samples 3 to 6 and 9 all had an excellent effect of promoting AQP3 mRNA expression.

[0070] [Test Example 4] Claudin-1 mRNA expression promoting effect test Cytosine (sample 1), trimethylglycine (sample 2), D-glucuronic acid (sample 3), and DL-3-hydroxybutyric acid (sample 7) were tested for their promoting effect on claudin-1 mRNA expression as follows.

[0071] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0072] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 1-3 and 7; see Table 4 below for sample concentrations) dissolved in KBM medium was added to each dish. The samples were then incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene, Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0073] Using this total RNA as a template, the mRNA expression levels of claudin-1 and GAPDH (an internal standard) were measured. Detection was performed using a real-time PCR system, Thermal Cycler Dice. (R) Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of claudin-1 mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and normalized with the GAPDH value. The claudin-1 mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0074] Claudin-1 mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 4.

[0075] [Table 4]

[0076] As shown in Table 4, it was confirmed that Samples 1 to 3 and 7 all had an excellent effect of promoting the expression of claudin-1 mRNA.

[0077] [Test Example 5] Claudin-4 mRNA expression promoting effect test Cytosine (sample 1), trimethylglycine (sample 2), D-glucuronic acid (sample 3), and citric acid (sample 4) were tested for their promoting effect on claudin-4 mRNA expression as follows.

[0078] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0079] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 1 to 4, see Table 5 below for sample concentrations) dissolved in KBM medium was added to each dish. The cultures were then incubated for 24 hours at 37°C in 5% CO2. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene, Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0080] Using this total RNA as a template, the mRNA expression levels of claudin-4 and GAPDH (an internal standard) were measured. Detection was performed using a real-time PCR system, Thermal Cycler Dice. (R) Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of claudin-4 mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The claudin-4 mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0081] Claudin-4 mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 5.

[0082] [Table 5]

[0083] As shown in Table 5, it was confirmed that all of Samples 1 to 4 had an excellent effect of promoting the expression of claudin-4 mRNA.

[0084] [Test Example 6] Occludin mRNA expression promoting effect test Cytosine (sample 1), trimethylglycine (sample 2), citric acid (sample 4), DL-3-hydroxybutyric acid (sample 7), and DL-glyceric acid (sample 8) were tested for their ability to promote occludin mRNA expression as follows.

[0085] Normal human neonatal epidermal keratinocytes (NHEK) were pre-cultured in normal human epidermal keratinocyte growth medium (KGM), and the cells were harvested by trypsinization. The harvested cells were collected at a concentration of 20 × 10 4 After diluting with KGM medium to a cell density of 40 × 10 cells / mL, 2 mL of the medium was seeded in 35 mm dishes. 4 The cells were cultured overnight at 37°C in 5% CO2 (cells / dish). After culture, the medium was replaced with normal human epidermal keratinocyte basal medium (KBM, the above KGM medium without growth factors (hEGF, BPE, insulin)), and the cells were cultured for an additional 24 hours.

[0086] After 24 hours of incubation, the medium was removed, and 2 mL of test samples (samples 1, 2, 4, 7, and 8; see Table 6 below for sample concentrations) dissolved in KBM medium was added to each dish. The cultures were then incubated at 37°C in 5% CO2 for 24 hours. As a control, KBM medium without sample was used for incubation in the same manner. After incubation, the medium was removed, and total RNA was extracted using ISOGEN II (Nippon Gene, Cat. No. 311-07361). The amount of RNA was measured using a spectrophotometer, and the total RNA was adjusted to 200 ng / μL.

[0087] Using this total RNA as a template, the mRNA expression levels of occludin and GAPDH (an internal standard) were measured. Detection was performed using a real-time PCR system, Thermal Cycler Dice. (R)Real-time 2-step RT-PCR was performed using the Real Time System III (Takara Bio) with the TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (Takara Bio, code No. RR063A). The expression level of occludin mRNA was calculated based on total RNA samples prepared from cells cultured with and without the test sample, and the normalized value was calculated using the GAPDH value. The occludin mRNA expression promotion rate (%) was calculated from the obtained values ​​using the following formula:

[0088] Occludin mRNA expression promotion rate (%) = A / B × 100 The terms in the formula represent the following: A: Correction value when test sample is added B: Corrected value without sample addition The results are shown in Table 6.

[0089] [Table 6]

[0090] As shown in Table 6, it was confirmed that Samples 1, 2, 4, 7 and 8 all had an excellent effect of promoting occludin mRNA expression.

[0091] [Formulation example 1] A cream having the following composition was prepared by a conventional method. Cytosine 0.02g Trimethylglycine 0.02g Glucuronic acid 0.01g Sophora root extract 0.1g Scutellaria root extract 0.1g Liquid paraffin 5.0g White beeswax 4.0g Squalane 10.0g Cetanol 3.0g Lanolin 2.0g Stearic acid 1.0g Polyoxyethylene sorbitan oleate (20E.O.) 1.5g Glyceryl monostearate 3.0g Oil-soluble licorice extract 0.1g 1,3-butylene glycol 6.0g Methyl parahydroxybenzoate 1.5g Fragrance 0.1g Purified water Rest (total amount is 100g)

[0092] [Formulation example 2] An emulsion was prepared in a conventional manner according to the following composition. Citric acid 0.01g Nicotinamide 0.01g Cytidine 0.01g Jojoba oil 4.00g 1,3-butylene glycol 3.00g Arbutin 3.00g Polyoxyethylene cetyl ether (20E.O.) 2.50g 2.00g olive oil Squalane 2.00g Cetyl alcohol 2.00g Glyceryl monostearate 2.00g Polyoxyethylene sorbitan oleate (20E.O.) 2.00g Methyl parahydroxybenzoate 0.15g Stearyl glycyrrhetinate 0.10g Phellodendron bark extract 0.10g Dipotassium glycyrrhizinate 0.10g Ginkgo biloba extract 0.10g Conchiolin 0.10g Phellodendron bark extract 0.10g Chamomile extract 0.10g Fragrance 0.05g Purified water Remainder (total amount is 100g)

[0093] [Formulation example 3] A cosmetic essence having the following composition was prepared by a conventional method. 3-hydroxybutyric acid 0.01g Glyceric acid 0.01g Uridine 0.01g Chamomile extract 0.1g Carrot extract 0.1g Xanthan gum 0.3g Hydroxyethyl cellulose 0.1g Carboxyvinyl polymer 0.1g 1,3-butylene glycol 4.0g Dipotassium glycyrrhizinate 0.1g Glycerin 2.0g Potassium hydroxide 0.25g Fragrance 0.01g Preservative (methyl parahydroxybenzoate) 0.15g Ethanol 2.0g Purified water Remainder (total amount is 100g)

[0094] [Formulation example 4] Tablets having the following composition were prepared by a conventional method. Cytosine 1.0mg Trimethylglycine 1.0mg Glucuronic acid 1.0mg Citric acid 1.0mg Nicotinamide 1.0mg Dolomite (contains 20% calcium and 10% magnesium) 83.4mg Casein phosphopeptide 16.7mg Vitamin C 33.4mg Maltitol 136.8mg Collagen 12.7mg Sucrose fatty acid ester 12.0mg

[0095] [Formulation example 5] An oral liquid preparation having the following composition was prepared by a conventional method. <Composition in 1 ampoule (100 mL)> Cytidine 0.1% by mass 3-hydroxybutyric acid 0.1% by mass Glyceric acid 0.1% by mass Uridine 0.1% by mass Sorbitol 12.0% by mass Sodium benzoate 0.1% by mass Fragrance 1.0% by mass Calcium sulfate 0.5% by mass Purified water remainder (100% by mass) [Industrial Applicability]

[0096] The hyaluronic acid synthase 3 (HAS3) mRNA expression promoter, serine palmitoyltransferase (SPT) mRNA expression promoter, aquaporin 3 (AQP3) mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter and occludin mRNA expression promoter of the present invention can significantly contribute to the prevention, treatment or improvement of skin aging symptoms; improvement or strengthening of skin barrier function and moisture retention ability; prevention, treatment or improvement of dry skin diseases and the like; prevention, treatment or improvement of various infectious diseases; etc.

Claims

[Claim 1] An aquaporin 3 (AQP3) mRNA expression promoter characterized by having nicotinamide as an active ingredient (excluding those having a combination of nicotinamide, hexamidine and pentanediol as active ingredients).

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