Barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter

Abstract

This barrier function enhancer, this occludin mRNA expression promoter, this claudin-1 mRNA expression promoter, this claudin-4 mRNA expression promoter, this tight junction protein-1 mRNA expression promoter, and this tight junction protein-2 mRNA expression promoter contain ganoderic acid as an active ingredient.

Description

Barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter

[0001] The present disclosure relates to barrier function enhancers, occludin mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, tight junction protein-1 mRNA expression promoters, and tight junction protein-2 mRNA expression promoters.

[0002] One of the structures that separates the inside and outside of a living body is epithelial tissue, which is composed of epithelial cells. 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 adhesion between cells, one of which is tight junctions (hereinafter sometimes referred to as "TJ"). 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 (CLDN) and occludin (OCLN), and lining proteins such as tight junction protein-1 (TJP-1, ZO-1 (Zonulaoccludens-1)) and tight junction protein-2 (TJP-2, ZO-2 (Zonulaoccludens-2)). These proteins are thought to form the skeleton of the TJ strand and control the barrier function of the TJ (see Non-Patent Document 1).

[0003] To date, more than 20 types of claudin molecules have been reported, forming the claudin family. These claudins are known to exhibit tissue-specific expression patterns, with claudin-1 (CLDN-1) and claudin-4 (CLDN-4) being expressed in the epidermis. Claudin-4 is also highly expressed in mucosal epithelium and functions as a barrier to prevent the entry of foreign substances both inside and outside the body.

[0004] Decreased expression of claudins, occludin, tight junction protein-1, tight junction protein-2, and the like for some reason leads to impaired TJ function. For example, impaired TJ function in the digestive tract allows food allergens, pathogenic microorganisms, and the like to enter the body, and impaired TJ function is thought to contribute to inflammatory bowel disease and various infectious diseases. Furthermore, while it was previously believed that only the stratum corneum was responsible for the skin's barrier function, it has recently been found that genetic deletion of TJ components present 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 the skin's barrier function (see Non-Patent Document 2). Decreased expression of claudins, occludin, tight junction protein-1, tight junction protein-2, and the like for some reason leads to structural destruction of TJs, which no longer function as a permeable barrier to substances, possibly resulting in skin symptoms such as dry skin, rough skin, atopic dermatitis, and various infectious diseases.

[0005] Therefore, strengthening TJ function through promoting the production of claudins, occludin, tight junction protein-1, and tight junction protein-2 is believed to 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 (see Patent Document 1) is known as a substance that promotes claudin production and occludin production. Furthermore, astilbin (see Patent Document 2) is known as a substance that promotes tight junction protein-1 production and tight junction protein-2 production.

[0006] JP 2009-256244 A JP 2017-75117 A

[0007] Journal of the Japan Cosmetic Science Society, 2007, vol.31, pp.296-301J. Cell Biol., 2002, vol.156, pp.1099-1111

[0008] The present disclosure aims to find, from among highly safe natural products, substances that have the effect of enhancing barrier function, promoting occludin mRNA expression, promoting claudin-1 mRNA expression, promoting claudin-4 mRNA expression, promoting tight junction protein-1 mRNA expression, and promoting tight junction protein-2 mRNA expression, and to provide barrier function enhancers, occludin mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, tight junction protein-1 mRNA expression promoters, and tight junction protein-2 mRNA expression promoters that contain these as active ingredients.

[0009] In order to solve the above problems, the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of the present disclosure contain ganoderic acid as an active ingredient.

[0010] According to the present disclosure, it is possible to provide a highly safe barrier function enhancer, an occludin mRNA expression promoter, a claudin-1 mRNA expression promoter, a claudin-4 mRNA expression promoter, a tight junction protein-1 mRNA expression promoter, and a tight junction protein-2 mRNA expression promoter which have excellent barrier function enhancement effects, occludin mRNA expression promoting effects, claudin-1 mRNA expression promoting effects, claudin-4 mRNA expression promoting effects, tight junction protein-1 mRNA expression promoting effects, and tight junction protein-2 mRNA expression promoting effects.

[0011] Embodiments of the present disclosure are described in detail below. The embodiments of the present disclosure may include a method for treating, preventing, or ameliorating a skin infection by transdermally administering to a patient ganoderic acid or a drug containing ganoderic acid as an active ingredient; use of ganoderic acid for treating, preventing, or ameliorating a skin infection; or use of ganoderic acid for producing an agent for treating, preventing, or ameliorating a skin infection.

[0012] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter according to this embodiment all contain ganoderic acid as an active ingredient.

[0013] Ganoderic acid, the active ingredient in this embodiment, is a terpenoid compound having a carbon number of 5n (n is an integer of 2 or greater), where n isoprene units or isopentane units are structural units. Examples of ganoderic acid in this embodiment include ganoderic acid A shown by the following formula (1), ganoderic acid C2 shown by the following formula (2), and ganoderic acid G shown by the following formula (3). Therefore, the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter according to this embodiment can each contain at least one of ganoderic acid A, ganoderic acid C2, and ganoderic acid G as an active ingredient.

[0014]

[0015]

[0016]

[0017] Ganoderic acid may be obtained by synthesis or by isolation and purification from extracts of plants, fungi, algae, etc. containing ganoderic acid (hereinafter sometimes simply referred to as "plants"). Plant extracts containing ganoderic acid include extracts obtained using plants containing ganoderic acid as the extraction raw material, diluted or concentrated extracts, dried products obtained by drying the extracts, and crude or purified products thereof.

[0018] Plant extracts containing ganoderic acid can be obtained by methods commonly used for plant extraction, including, for example, Ganoderma lucidum (Fr.) Karst.

[0019] Ganoderma lucidum (Fr.) Karst. is a basidiomycete fungus belonging to the Polyporaceae family. There are no particular restrictions on the constituent parts that can be used as the extraction material, and they can be selected appropriately depending on the purpose. However, it is particularly preferable to use the fruiting body.

[0020] A plant extract containing ganoderic acid can be obtained by drying the extraction raw material, pulverizing it directly or using a crusher, and then subjecting it to extraction with an extraction solvent. Drying can be carried out in the sun or using a commonly used dryer. The raw material may also be pretreated, such as by degreasing with a nonpolar solvent such as hexane, before use. Pretreatment, such as degreasing, allows for efficient extraction of the plant with a polar solvent.

[0021] As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent, which is preferably used alone or in combination of two or more at room temperature or at a temperature below the boiling point of the solvent.

[0022] Water that can be used as an extraction solvent includes pure water, tap water, well water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has undergone various treatments. Treatments that can be applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, water that can be used as an extraction solvent in this embodiment also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0023] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0024] When a mixture of two or more polar solvents is used as the extraction solvent, the mixing ratio can be appropriately adjusted. For example, when a mixture of water and a lower aliphatic alcohol is used, it is preferable to mix 10 parts by volume of water with 1 to 90 parts by volume of the lower aliphatic alcohol, when a mixture of water and a lower aliphatic ketone is used, it is preferable to mix 10 parts by volume of water with 1 to 40 parts by volume of the lower aliphatic ketone, and when a mixture of water and a polyhydric alcohol is used, it is preferable to mix 10 parts by volume of water with 10 to 90 parts by volume of the polyhydric alcohol.

[0025] The extraction process is not particularly limited as long as it allows the soluble components contained in the extraction raw material to be dissolved in the extraction solvent, and can be carried out according to conventional methods. For example, an extract can be obtained by immersing the extraction raw material in an extraction solvent in an amount (mass ratio) 5 to 15 times the amount of the extraction raw material, extracting the soluble components at room temperature or under reflux, and then filtering to remove the extraction residue. Distilling the solvent from the obtained extract yields a paste-like concentrate, which can then be further dried to obtain a dried product.

[0026] The method for isolating and purifying ganoderic acid from the extract, concentrate, or dried extract obtained as described above is not particularly limited, and can be carried out by conventional methods. For example, the plant extract can be subjected to column chromatography using a porous substance such as silica gel or alumina, or a porous resin such as a styrene-divinylbenzene copolymer or polymethacrylate, and eluted with water and then alcohol, to obtain ganoderic acid as a fraction eluted with alcohol.

[0027] Examples of alcohols that can be used as an eluent in column chromatography include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol, and aqueous solutions thereof, but are not particularly limited to these.

[0028] Furthermore, the alcohol fraction obtained by column chromatography may be purified using any organic compound purification means, such as reverse-phase silica gel chromatography using ODS, recrystallization, liquid-liquid countercurrent extraction, or column chromatography using an ion exchange resin.

[0029] As will be apparent from the examples described below, the ganoderic acids obtained in this manner, particularly ganoderic acid A, ganoderic acid C2 and ganoderic acid G, have the effects of enhancing barrier function, promoting occludin mRNA expression, claudin-1 mRNA expression, claudin-4 mRNA expression, tight junction protein-1 mRNA expression and tight junction protein-2 mRNA expression, and therefore can be used by utilizing these effects as the active ingredients of barrier function enhancers, occludin mRNA expression promoters, claudin-1 mRNA expression promoters, claudin-4 mRNA expression promoters, tight junction protein-1 mRNA expression promoters and tight junction protein-2 mRNA expression promoters.

[0030] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of this embodiment may consist of ganoderic acid alone, or may be a formulation of ganoderic acid.

[0031] Ganoderic acid can be formulated into any dosage form, such as powder, granules, tablets, or liquid, using pharmaceutically acceptable carriers such as dextrin, cyclodextrin, or other optional auxiliary agents according to conventional methods. In this case, auxiliary agents that can be used include, for example, excipients, binders, disintegrants, lubricants, stabilizers, and flavoring / flavoring agents. Ganoderic acid can be incorporated into other compositions (e.g., skin cosmetics) and used as ointments, topical liquids, patches, and the like.

[0032] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of this embodiment can each be used as an active ingredient in combination with other natural extracts having a barrier function enhancing effect, an occludin mRNA expression promoting effect, a claudin-1 mRNA expression promoting effect, a claudin-4 mRNA expression promoting effect, a tight junction protein-1 mRNA expression promoting effect, or a tight junction protein-2 mRNA expression promoting effect, as needed.

[0033] The administration method of the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, or tight junction protein-2 mRNA expression promoter of this embodiment generally includes transdermal administration, oral administration, etc., but a method suitable for the prevention, amelioration, treatment, etc. of the disease may be appropriately selected depending on the type of disease. Furthermore, the dose of the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, or tight junction protein-2 mRNA expression promoter of this embodiment may also be increased or decreased as appropriate depending on the type and severity of the disease, individual patient differences, administration method, administration period, etc.

[0034] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of this embodiment enhance the barrier function in epithelial tissue through the barrier function enhancing effect, occludin mRNA expression promoting effect, claudin-1 mRNA expression promoting effect, claudin-4 mRNA expression promoting effect, tight junction protein-1 mRNA expression promoting effect, or tight junction protein-2 mRNA expression promoting effect of the active ingredient ganoderic acid, and are effective in treating inflammatory bowel diseases, food allergies, The following skin conditions and skin infections can be prevented, treated, or improved: various infectious diseases transmitted through the digestive tract; dry skin, rough skin, atopic dermatitis, sensitive skin, skin aging symptoms, psoriasis, acne (including hidradenitis suppurativa), burns, diaper rash, Netherton syndrome, actinic keratosis, dermatomycosis, dermatosis, ectodermal dysplasia, contact dermatitis, dermatitis, seborrheic dermatitis, ichthyosis vulgaris, and infections transmitted through the oral cavity and respiratory tract; allergic rhinitis, asthma, irritable bowel syndrome, systemic autoimmune diseases (rheumatoid arthritis, lupus erythematosus, etc.), allergies (food allergies, hay fever, etc.), lifestyle-related diseases (obesity, type 1 or type 2 diabetes, high blood pressure, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), arteriosclerosis, etc.). In other words, the present disclosure may be a method for preventing, ameliorating, or treating inflammatory bowel disease, food allergies, various infectious diseases transmitted through the digestive tract, and the like; skin conditions and various skin infections such as dry skin, rough skin, atopic dermatitis, sensitive skin, skin aging symptoms, psoriasis, acne (including hidradenitis suppurativa), burns, diaper rash, Netherton syndrome, actinic keratosis, dermatomycosis, dermatosis, ectodermal dysplasia, contact dermatitis, dermatitis, seborrheic dermatitis, ichthyosis vulgaris, and infections of the oral cavity or respiratory tract; allergic rhinitis, asthma, irritable bowel syndrome, systemic autoimmune diseases (rheumatoid arthritis, lupus erythematosus, etc.), allergies (food allergies, hay fever, etc.), lifestyle-related diseases (obesity, type 1 or type 2 diabetes, hypertension, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), arteriosclerosis, etc.); and the like, by transdermal or oral administration to a patient of ganoderic acid or a drug containing ganoderic acid as an active ingredient.The present disclosure also relates to ganoderic acid for use in the prevention, amelioration, or treatment of skin conditions and various skin infections, such as inflammatory bowel disease, food allergies, and various infectious diseases transmitted via the digestive tract; dry skin, rough skin, atopic dermatitis, sensitive skin, skin aging symptoms, psoriasis, acne (including hidradenitis suppurativa), burns, diaper rash, Netherton syndrome, actinic keratosis, dermatomycosis, dermatosis, ectodermal dysplasia, contact dermatitis, dermatitis, seborrheic dermatitis, ichthyosis vulgaris, and infections originating from the oral cavity or respiratory tract; allergic rhinitis, asthma, irritable bowel syndrome, systemic autoimmune diseases (such as rheumatoid arthritis and lupus erythematosus), allergies (such as food allergies and hay fever), and lifestyle-related diseases (such as obesity, type 1 or type 2 diabetes, high blood pressure, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD) and arteriosclerosis). Furthermore, the present disclosure relates to the use of ganoderic acid for the manufacture of an agent for the prevention, amelioration, or treatment of inflammatory bowel disease, food allergies, various infectious diseases transmitted via the digestive tract, and the like; skin conditions and various skin infections such as dry skin, rough skin, atopic dermatitis, sensitive skin, skin aging symptoms, psoriasis, acne (including hidradenitis suppurativa), burns, diaper rash, Netherton syndrome, actinic keratosis, dermatomycosis, dermatosis, ectodermal dysplasia, contact dermatitis, dermatitis, seborrheic dermatitis, ichthyosis vulgaris, and infections originating from the oral cavity or respiratory tract; allergic rhinitis, asthma, irritable bowel syndrome, systemic autoimmune diseases (rheumatoid arthritis, lupus erythematosus, etc.), allergies (food allergies, hay fever, etc.), lifestyle-related diseases (obesity, type 1 or type 2 diabetes, hypertension, hyperlipidemia, non-alcoholic fatty liver disease (NAFLD), arteriosclerosis, etc.), and the like. However, in addition to these uses, the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of the present embodiment can also be used for all uses in which it is significant to exert a barrier function enhancing effect, an occludin mRNA expression promoting effect, a claudin-1 mRNA expression promoting effect, a claudin-4 mRNA expression promoting effect, a tight junction protein-1 mRNA expression promoting effect, or a tight junction protein-2 mRNA expression promoting effect.

[0035] Furthermore, the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, or tight junction protein-2 mRNA expression promoter of the present embodiment has excellent barrier function enhancement, occludin mRNA expression promotion, claudin-1 mRNA expression promotion, claudin-4 mRNA expression promotion, tight junction protein-1 mRNA expression promotion, or tight junction protein-2 mRNA expression promotion effects, and is therefore suitable for incorporation into, for example, topical skin preparations or oral compositions. In this case, ganoderic acid may be incorporated as is, or a barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, or tight junction protein-2 mRNA expression promoter formulated from ganoderic acid may be incorporated.

[0036] 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, beauty serums, lotions, packs, foundations, lip balms, bath additives, hair tonics, hair lotions, soaps, body shampoos, etc.

[0037] The amount of ganoderic acid in topical skin preparations can be adjusted appropriately depending on the type of topical skin preparation, but a preferred blending rate is 0.0001 to 10% by mass, and a particularly preferred blending rate is 0.001 to 1% by mass.

[0038] An oral composition refers to a composition that is resistant to harm to human health and is taken orally or by gastrointestinal administration in normal social life, and is not limited to administrative classifications such as food, medicine, or quasi-drug. Therefore, the "oral composition" in this embodiment broadly encompasses orally taken general foods, feeds, health foods, health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims), quasi-drugs, medicines, etc. The oral composition in this embodiment is preferably an oral composition that can display the favorable effects of ganoderic acid on the oral composition or its packaging, and is particularly preferably a health functional food (foods for specified health uses, foods with nutrient functions, foods with functional claims), quasi-drugs, or medicines.

[0039] The amount of ganoderic acid in the oral composition can be varied as appropriate, taking into account the intended use, symptoms, gender, etc., but is preferably adjusted so that the daily intake of ganoderic acid for an adult is approximately 1 to 1,000 mg, taking into account the general intake of the oral composition to which it is added. When the oral composition to which it is added is in the form of granules, tablets, or capsules, the amount of ganoderic acid added is typically 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, of the oral composition to which it is added.

[0040] Furthermore, the barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of the present embodiment have excellent barrier function enhancing effects, occludin mRNA expression promoting effects, claudin-1 mRNA expression promoting effects, claudin-4 mRNA expression promoting effects, tight junction protein-1 mRNA expression promoting effects, and tight junction protein-2 mRNA expression promoting effects, and can therefore be suitably used as reagents for research on the mechanisms of these actions.

[0041] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter of the present embodiment are preferably applied to humans, but can also be applied to animals other than humans as long as their respective action effects are exerted.

[0042] The present disclosure will be described in more detail below with reference to test examples, but the present disclosure is not limited to these examples. In the test examples, ganoderic acid A (manufactured by Cayman Chemical Company), ganoderic acid C2, and ganoderic acid G (all manufactured by TargetMOI Chemical Inc.) were used as test samples.

[0043] Test Example 1: Skin barrier function enhancement test (electrical resistance value TER measurement and permeability evaluation using FITC-Dex) Normal human neonatal skin epidermal keratinocytes (NHEK) were cultured for 3 days using normal human neonatal epidermal keratinocyte medium (KGM). Thereafter, the medium was replaced every 24 hours with hydrocortisone-free KGM containing 20 μM cortisol (CS) (KGM+CS). After 3 days of culture, the cells were harvested by trypsinization. The harvested cells were collected in a volume of 30 × 10 5 The cells were diluted with KGM+CS to a cell density of 1000 cells / mL, and then seeded in the upper layer of a 12-well transwell (Corning, 12 mm diameter, 0.4 μm pores) at 0.5 mL per well. 1.5 mL of KGM+CS was added to the lower layer, and the medium was changed every 24 hours, followed by culturing for 3 days.

[0044] In addition, cells (chronic stress model) treated with cortisol (CS) were prepared by the above-mentioned culture (preculture), and in order to confirm the decline in barrier function due to the influence of cortisol (CS) during the above-mentioned preculture, cells (CS-untreated cells) were prepared by culturing in the same manner as the above-mentioned preculture except that KGM containing no cortisol (CS) and no hydrocortisone was used.

[0045] After pre-incubation, the culture medium was discarded, and 0.5 mL of test samples (ganoderic acid A, ganoderic acid C2, and ganoderic acid G; see Tables 1-3 for sample concentrations) dissolved in 1.5 mmol / L CaCl2-containing KGM + CS was added to the upper layer of each well, and 1.5 mL of 1.5 mmol / L CaCl2-containing KGM + CS was added to the lower layer of each well for incubation. For CS-untreated cells, 1.5 mmol / L CaCl2-containing KGM + CS was added to the upper and lower layers of each well for incubation. Furthermore, as a control, a chronic stress model was cultured with 1.5 mmol / L CaCl2-containing KGM + CS added to the upper and lower layers of each well. 72 hours after the addition of KGM+CS, the electrical resistance (TER) was measured using a Millicell-ERS resistance measurement system (Millipore), and the barrier formation promotion rate (%) of the test sample compared to the control was calculated using the following formula. The results are shown in Tables 1 to 3.

[0046] Barrier formation promotion rate (%) = A / B × 100 In the formula, A represents "TER in the chronic stress model to which CS-untreated cells or the test sample was added during pre-culture," and B represents "TER in the chronic stress model to which the test sample was not added."

[0047] After the TER measurement, the upper and lower layers were washed with PBS(-), and 0.5 mL of FITC-Dextran 4000-conjugate (FITC-Dex, Fluka) dissolved in P buffer (10 mM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl, 145 mM NaCl) to a concentration of 1 mg / mL was added to the upper layer, and 1 mL of P buffer was added to the lower layer, followed by incubation at 37°C for 90 minutes. After incubation, 200 μL of each sample was taken from the lower layer, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 545 nm. The amount of FITC-Dex that had permeated from the upper layer to the lower layer was determined based on the calibration curve, and the FITC-Dex permeation inhibition rate (%) of the test sample compared with the control was calculated using the following formula. The results are shown in Tables 1 to 3.

[0048] FITC-Dex permeation inhibition rate (%) = (1 - A / B) × 100 In the formula, A represents the "amount of FITC-Dex permeated in the chronic stress model to which the test sample was added," and B represents the "amount of FITC-Dex permeated in the chronic stress model to which the test sample was not added."

[0049]

[0050]

[0051]

[0052] As shown in Tables 1 to 3, it was confirmed that ganoderic acid (ganoderic acid A, ganoderic acid C2, and ganoderic acid G) can inhibit the permeation of FITC-Dex and has excellent skin barrier function enhancing effects.

[0053] Test Example 2: Test for promoting occludin (OCLN) mRNA expression Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were collected in a volume of 1.5 x 10 5 The cells were diluted with KGM to a cell density of 1000 cells / mL, and then seeded in 500 μL aliquots per well of a 24-well plate. The culture medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM") and cultured for 72 hours, replacing the medium with fresh cortisol-containing KGM every 24 hours. After 72 hours of culture, the culture medium was discarded, and 250 μL of sample solution (ganoderic acid A, ganoderic acid C2, ganoderic acid G; see Tables 4 to 6 below for sample concentrations) dissolved in KGM was added to each well, resulting in a final concentration of 1.5 mM Ca. 2+ 250 μL of KGM adjusted to 20 μM cortisol was added to each well, and the cells were cultured for 72 hours.

[0054] After the incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 150 ng / μL.

[0055] Using total RNA as a template, the expression levels of occludin mRNA and the internal standard GAPDH mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.) with PrimeScript™ RT Master Mix (Perfect Real Time, Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of occludin mRNA was corrected by the expression level of GAPDH mRNA, and the corrected value was calculated.

[0056] The occludin mRNA expression promotion rate (%) was calculated from the obtained corrected values ​​using the following formula: Occludin mRNA expression promotion rate (%) = A / B × 100, where A represents the "corrected value when sample was added" and B represents the "corrected value when no sample was added." The results are shown in Tables 4 to 6.

[0057] Test Example 3: Claudin-1 (CLDN-1) mRNA Expression Promoting Effect Test Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were collected in a volume of 1.5 × 10 5 The cells were diluted with KGM to a cell density of 1000 cells / mL, and then seeded in 500 μL aliquots per well of a 24-well plate. The culture medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM") and cultured for 72 hours, replacing the medium with fresh cortisol-containing KGM every 24 hours. After 72 hours of culture, the culture medium was discarded, and 250 μL of sample solution (ganoderic acid A, ganoderic acid C2, ganoderic acid G; see Tables 4 to 6 below for sample concentrations) dissolved in KGM was added to each well, resulting in a final concentration of 1.5 mM Ca. 2+ 250 μL of KGM adjusted to 20 μM cortisol was added to each well, and the cells were cultured for 72 hours.

[0058] After the incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 150 ng / μL.

[0059] Using total RNA as a template, the expression levels of claudin-1 mRNA and the internal standard GAPDH mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.) with PrimeScript™ RT Master Mix (Perfect Real Time, Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of claudin-1 mRNA was corrected by the expression level of GAPDH mRNA, and the corrected value was calculated.

[0060] From the obtained corrected values, the claudin-1 mRNA expression promotion rate (%) was calculated using the following formula: Claudin-1 mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when sample was added" and B represents the "corrected value when no sample was added." The results are shown in Tables 4 to 6.

[0061] Test Example 4: Claudin-4 (CLDN-4) mRNA Expression Promoting Effect Test Normal human neonatal epidermal keratinocytes (NHEK) were cultured using normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were collected in a volume of 1.5 × 10 5The cells were diluted with KGM to a cell density of 1000 cells / mL, and then seeded in 500 μL aliquots per well of a 24-well plate. The culture medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM") and cultured for 72 hours, replacing the medium with fresh cortisol-containing KGM every 24 hours. After 72 hours of culture, the culture medium was discarded, and 250 μL of sample solution (ganoderic acid A, ganoderic acid C2, ganoderic acid G; see Tables 4 to 6 below for sample concentrations) dissolved in KGM was added to each well, resulting in a final concentration of 1.5 mM Ca. 2+ 250 μL of KGM adjusted to 20 μM cortisol was added to each well, and the cells were cultured for 72 hours.

[0062] After the incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 150 ng / μL.

[0063] Using total RNA as a template, the expression levels of claudin-4 mRNA and the internal standard GAPDH mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.) with PrimeScript™ RT Master Mix (Perfect Real Time, Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of claudin-4 mRNA was corrected by the expression level of GAPDH mRNA, and the corrected value was calculated.

[0064] From the obtained corrected values, the claudin-4 mRNA expression promotion rate (%) was calculated using the following formula: Claudin-4 mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when sample was added" and B represents the "corrected value when no sample was added." The results are shown in Tables 4 to 6.

[0065] Test Example 5: Test for promoting expression of tight junction protein-1 (TJP-1) mRNA Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were collected at a concentration of 1.5 x 10 5 The cells were diluted with KGM to a cell density of 1000 cells / mL, and then seeded in 500 μL aliquots per well of a 24-well plate. The culture medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM") and cultured for 72 hours, replacing the medium with fresh cortisol-containing KGM every 24 hours. After 72 hours of culture, the culture medium was discarded, and 250 μL of sample solution (ganoderic acid A, ganoderic acid C2, ganoderic acid G; see Tables 4 to 6 below for sample concentrations) dissolved in KGM was added to each well, resulting in a final concentration of 1.5 mM Ca. 2+ 250 μL of KGM adjusted to 20 μM cortisol was added to each well, and the cells were cultured for 72 hours.

[0066] After the incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 150 ng / μL.

[0067] Using total RNA as a template, the expression levels of tight junction protein-1 mRNA and the internal standard GAPDH mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.) with PrimeScript™ RT Master Mix (Perfect Real Time, Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of tight junction protein-1 mRNA was corrected by the expression level of GAPDH mRNA, and the corrected value was calculated.

[0068] The tight junction protein-1 mRNA expression promotion rate (%) was calculated from the obtained corrected value using the following formula: Tight junction protein-1 mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when sample was added" and B represents the "corrected value when no sample was added." The results are shown in Tables 4 to 6.

[0069] Test Example 6: Test for promoting expression of tight junction protein-2 (TJP-2) mRNA Normal human neonatal epidermal keratinocytes (NHEK) were cultured in normal human epidermal keratinocyte growth medium (KGM) and then harvested by trypsin treatment. The harvested cells were collected in a volume of 1.5 x 10 5 The cells were diluted with KGM to a cell density of 1000 cells / mL, and then seeded in 500 μL aliquots per well of a 24-well plate. The culture medium was replaced with KGM containing 20 μM cortisol (hereinafter referred to as "cortisol-containing KGM") and cultured for 72 hours, replacing the medium with fresh cortisol-containing KGM every 24 hours. After 72 hours of culture, the culture medium was discarded, and 250 μL of sample solution (ganoderic acid A, ganoderic acid C2, ganoderic acid G; see Tables 4 to 6 below for sample concentrations) dissolved in KGM was added to each well, resulting in a final concentration of 1.5 mM Ca. 2+ 250 μL of KGM adjusted to 20 μM cortisol was added to each well, and the cells were cultured for 72 hours.

[0070] After the incubation, the culture medium was discarded, and total RNA was extracted using ISOGEN II (NIPPON GENE). The amount of RNA was calculated from the absorbance at a wavelength of 260 nm, and the total RNA was adjusted to 150 ng / μL.

[0071] Using total RNA as a template, the expression levels of tight junction protein-2 mRNA and the internal standard GAPDH mRNA were measured. Detection was performed by a two-step real-time RT-PCR reaction using a real-time PCR device (Thermal Cycler Dice® Real Time System III, Takara Bio Inc.) with PrimeScript™ RT Master Mix (Perfect Real Time, Takara Bio Inc.) and TB Green® Fast qPCR Mix (Takara Bio Inc.). The expression level of tight junction protein-2 mRNA was corrected by the expression level of GAPDH mRNA, and the corrected value was calculated.

[0072] The tight junction protein-2 mRNA expression promotion rate (%) was calculated from the obtained corrected value using the following formula: Tight junction protein-2 mRNA expression promotion rate (%) = A / B × 100 In the formula, A represents the "corrected value when sample was added" and B represents the "corrected value when no sample was added." The results are shown in Tables 4 to 6.

[0073]

[0074]

[0075]

[0076] As shown in Tables 4 to 6, it was confirmed that ganoderic acid A, ganoderic acid C2, and ganoderic acid G all have excellent occludin mRNA expression-promoting effects, claudin-1 mRNA expression-promoting effects, claudin-4 mRNA expression-promoting effects, tight junction protein-1 mRNA expression-promoting effects, and tight junction protein-2 mRNA expression-promoting effects.

[0077] The barrier function enhancer, occludin mRNA expression promoter, claudin-1 mRNA expression promoter, claudin-4 mRNA expression promoter, tight junction protein-1 mRNA expression promoter, and tight junction protein-2 mRNA expression promoter according to the present embodiment can greatly contribute to enhancing the barrier function in epithelial tissue (prevention, treatment, or amelioration of inflammatory bowel disease, food allergies, various infectious diseases transmitted through the digestive tract, etc.; prevention, treatment, or amelioration of skin conditions such as dry skin, rough skin, and atopic dermatitis, various infectious diseases, etc.).

Claims

1. A barrier function enhancer comprising ganoderic acid as an active ingredient.

2. An occludin mRNA expression promoter comprising ganoderic acid as an active ingredient.

3. A claudin-1 mRNA expression promoter comprising ganoderic acid as an active ingredient.

4. A claudin-4 mRNA expression promoter comprising ganoderic acid as an active ingredient.

5. A tight junction protein-1 mRNA expression promoter comprising ganoderic acid as an active ingredient.

6. A tight junction protein-2 mRNA expression promoter comprising ganoderic acid as an active ingredient.