Claudin reduction agent, tight junction relaxant, cosmetic composition, pharmaceutical composition, quasi-drug composition and food composition

Abstract

The present invention provides a claudin-reducing agent, a tight junction relaxant, and a cosmetic composition that contribute to the penetration of active ingredients into the skin. [Solution] The claudin-reducing agent of the present invention contains Swertia japonica extract. In this claudin-reducing agent, Swertia japonica extract is an extract obtained by extracting the whole plant of the Gentianaceae plant Swertia japonica with 1,3-butylene glycol (1,3-BG).

Description

Technical Field

[0001] The present invention relates to a claudin amount reducing agent, a tight junction relaxing agent, a cosmetic composition, a pharmaceutical composition, a quasi-drug composition, and a food composition. Specifically, it relates to a claudin amount reducing agent, a tight junction relaxing agent, a cosmetic composition, a pharmaceutical composition, a quasi-drug composition, and a food composition that contribute to the penetration of active ingredients into the skin.

Background Art

[0002] Epithelial cells cover the surfaces of the body, body cavities, organs, etc., and play a role in maintaining the homeostasis of the body. In epithelial cells, adjacent cells are strongly bound to each other by a cell adhesion device, separating the body and organs from the outside and preventing the invasion of pathogenic bacteria and the leakage of substances.

[0003] In addition, epithelial cells have several cell adhesion devices, and the outermost one is the tight junction. Through this tight junction, the barrier function of the skin can be enhanced, preventing the invasion of external stimulants and the evaporation of internal moisture and moisturizing components.

[0004] Moreover, the main component of the tight junction is a protein called claudin, and the tight junction is formed by the binding of claudins like a zipper.

[0005] Since the function of such tight junctions is related to skin moisturization and component penetration, it has been attracting attention as a target for pharmaceuticals and cosmetics to exert their effects.

[0006] Among these, as a method for enhancing the permeability of the active ingredients of pharmaceuticals and cosmetics into the skin, a method of temporarily relaxing the tight junction, which is a skin barrier, is known. For example, Patent Document 1 discloses a tight junction relaxing agent containing a specific compound.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] Patent No. 7304049 [Overview of the project] [Problems that the invention aims to solve]

[0008] While some of the compounds described in Patent Document 1 are plant-derived, considering their use in cosmetics and the like, an increase in the variety of plant-derived ingredients that can provide tight junction relaxation effects is desirable in terms of increasing the freedom of formulation and making it easier to appeal to the product's image.

[0009] The present invention was conceived in view of the above points, and aims to provide a claudin-reducing agent that contributes to the penetration of active ingredients into the skin, a tight junction relaxant, a cosmetic composition, a pharmaceutical composition, a quasi-drug composition, and a food composition. [Means for solving the problem]

[0010] To achieve the above objective, the claudin-reducing agent of the present invention is configured to contain at least one of Swertia japonica extract and ferulic acid.

[0011] Here, by including Swertia japonica extract, the amount of claudin that forms tight junctions can be reduced. The Swertia japonica extract referred to here is an extract obtained by solvent extraction from the whole plant of the gentian family plant Swertia japonica.

[0012] Furthermore, the inclusion of ferulic acid can reduce the amount of claudin that forms tight junctions.

[0013] Furthermore, reducing the amount of claudin 1 (CLD1) can reduce the amount of claudins within the claudin family that are major claudins constituting tight junctions and that play a barrier function in regulating material permeability. Note that there are multiple families of claudins (e.g., claudins 1-27).

[0014] Furthermore, reducing the amount of claudin 4 (CLD4) can reduce the amount of claudins within the claudin family, which are the main claudins that make up tight junctions and play a barrier role in regulating material permeability.

[0015] Furthermore, in order to achieve the above objectives, the tight junction relaxant of the present invention is configured to contain at least one of Swertia japonica extract and ferulic acid.

[0016] Here, by including Swertia japonica extract, the amount of claudin that forms tight junctions can be reduced, thereby alleviating tight junctions.

[0017] Furthermore, the inclusion of ferulic acid can reduce the amount of claudin that forms tight junctions, thereby easing the tight junctions.

[0018] Furthermore, in order to achieve the above objectives, the cosmetic composition of the present invention is configured to contain the claudin-reducing agent described in claims 1 to 3, and / or the tight junction relaxant described in claim 4.

[0019] Herein, by including the claudin-reducing agent described in claims 1 to 3 and / or the tight junction-relaxing agent described in claim 4, the cosmetic composition can be given the effect of reducing the amount of claudin that forms tight junctions and relaxing tight junctions.

[0020] Also, in order to achieve the above object, the pharmaceutical composition of the present invention is configured to contain an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4.

[0021] Here, by containing an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4, the amount of claudin forming tight junctions can be reduced, and the effect of relaxing tight junctions can be imparted to the pharmaceutical composition.

[0022] Also, in order to achieve the above object, the quasi-drug composition of the present invention is configured to contain an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4.

[0023] Here, by containing an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4, the amount of claudin forming tight junctions can be reduced, and the effect of relaxing tight junctions can be imparted to the quasi-drug composition.

[0024] Also, in order to achieve the above object, the food composition of the present invention is configured to contain an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4.

[0025] Here, by containing an agent for reducing the amount of claudin according to claims 1 to 3 and / or an agent for relaxing tight junctions according to claim 4, the amount of claudin forming tight junctions can be reduced, and the effect of relaxing tight junctions can be imparted to the food composition.

Effects of the Invention

[0026] The agent for reducing the amount of claudin, the tight junction relaxant, the cosmetic composition, the pharmaceutical composition, the quasi-drug composition, and the food composition according to the present invention contribute to the penetration of the active ingredient into the skin.

Brief Description of the Drawings

[0027] [Figure 1] Regarding Examples 1 to 3, (a) is a diagram showing the results of the expression level of claudin 1 (CLD1), and (b) is a diagram showing the results of the expression level of claudin 3 (CLD3). [Figure 2] Regarding Examples 1 to 3, (a) is a diagram showing the results of the expression level of claudin 4 (CLD4), and (b) is a diagram showing the results of the expression level of claudin 7 (CLD7). [Figure 3] Regarding Example 4, (a) is a diagram showing the results of the expression level of claudin 1 (CLD1), and (b) is a diagram showing the results of the expression level of claudin 3 (CLD3). [Figure 4] Regarding Example 4, (a) is a diagram showing the results of the expression level of claudin 4 (CLD4), and (b) is a diagram showing the results of the expression level of claudin 7 (CLD7). [Figure 5] Regarding Example 5, (a) is a diagram showing the results of the expression level of claudin 1 (CLD1), and (b) is a diagram showing the results of the expression level of claudin 3 (CLD3).

Modes for Carrying Out the Invention

[0028] [The First Embodiment of the Present Invention] Hereinafter, the agent for reducing the amount of claudin, which is the first embodiment of the present invention, contains an assembly extract. The agent for reducing the amount of claudin according to the first embodiment of the present invention is also a tight junction relaxant.

[0029] In the claudin-reducing agent according to the first embodiment of the present invention, Swertia japonica extract is an extract obtained from the whole plant of the gentian family plant Swertia japonica with 1,3-butylene glycol (1,3-BG). This extract will be hereinafter referred to as "Swertia japonica extract (BG extract)". Swertia japonica is used as a bitter stomachic and intestinal regulator. It is also incorporated into skincare products and hair growth products due to its vasodilating effect, blood circulation promoting effect, hair growth effect, skin roughness improvement effect, and whitening effect.

[0030] Furthermore, Swertia japonica extract (BG extract) is composed of Swertia japonica extract: 12.50% and 1,3-butylene glycol: 87.50%.

[0031] Furthermore, Swertia japonica extract (BG extract) is an ingredient that reduces the amount of claudin. Also, there are multiple families of claudin (for example, claudin 1 to claudin 27). In the claudin-reducing agent according to the first embodiment of the present invention, the amount of claudin that is reduced is not limited, but it is preferably claudin 1 (CLD1) or claudin 4 (CLD4).

[0032] In this specification, the amount of claudin primarily refers to the amount of claudin protein, but is not limited to this; it may also refer to the amount of claudin mRNA.

[0033] Here, the Swertia japonica extract (BG extract) does not necessarily have to consist of Swertia japonica extract: 12.50% and 1,3-butylene glycol: 87.50%; the content of Swertia japonica extract and 1,3-butylene glycol can be set as appropriate.

[0034] Furthermore, in the claudin-reducing agent according to the first embodiment of the present invention, the concentration of Swertia japonica extract (BG extract) is not particularly limited, but it is preferably 0.12% (v / v) or 0.03% (v / v).

[0035] The claudin reduction agent according to the first embodiment of the present invention can reduce the amount of claudin that forms tight junctions. In particular, it can effectively reduce the amount of claudin 1 (CLD1) and / or claudin 4 (CLD4). As a result, tight junctions can be alleviated.

[0036] The claudin-reducing agent according to the first embodiment of the present invention has the advantage of reducing claudin levels without exhibiting toxicity. Furthermore, it can relax tight junctions without causing their collapse, thereby allowing the expression of intercellular spaces.

[0037] [Second embodiment of the present invention] The claudin-reducing agent according to the second embodiment of the present invention contains Swertia japonica extract. The claudin-reducing agent according to the second embodiment of the present invention is also a tight junction relaxant.

[0038] In the claudin-reducing agent according to the second embodiment of the present invention, Swertia japonica extract is an extract obtained by extracting the whole plant of the Gentianaceae plant Swertia japonica with ethanol. This extract will be hereinafter referred to as "Swertia japonica extract (Et-OH extract)".

[0039] Furthermore, Swertia japonica extract (Et-OH extract) is composed of Swertia japonica components: 1.67%, ethanol: 57.00%, and water: 41.33%.

[0040] Furthermore, Swertia japonica extract (Et-OH extract) is an ingredient that reduces the amount of claudin. Also, there are multiple families of claudin (for example, claudin 1 to claudin 27). In the claudin-reducing agent according to the second embodiment of the present invention, the amount of claudin that is reduced is not limited, but it is preferably claudin 1 (CLD1).

[0041] Here, the Swertia japonica extract (Et-OH extract) does not necessarily have to consist of Swertia japonica components: 1.67%, ethanol: 57.00%, and water: 41.33%; the content of Swertia japonica components, ethanol, and water can be set as appropriate.

[0042] Furthermore, in the claudin-reducing agent according to the second embodiment of the present invention, the concentration of Swertia japonica extract (Et-OH extract) is not particularly limited, but is preferably 3% (v / v).

[0043] The claudin-reducing agent according to the second embodiment of the present invention can reduce the amount of claudin that forms tight junctions. In particular, it can effectively reduce the amount of claudin 1 (CLD1). As a result, tight junctions can be alleviated.

[0044] The claudin-reducing agent according to the second embodiment of the present invention has the advantage of reducing claudin levels without exhibiting toxicity. Furthermore, it can relax tight junctions without causing them to collapse, thereby allowing the expression of intercellular spaces.

[0045] [Third Embodiment of the Present Invention] The claudin-reducing agent according to the third embodiment of the present invention contains ferulic acid (trans-ferulic acid). The claudin-reducing agent according to the third embodiment of the present invention is also a tight junction relaxant.

[0046] In the claudin-reducing agent according to the third embodiment of the present invention, ferulic acid is the sodium salt of ferulic acid dissolved by adding water and sodium hydroxide to the powder reagent. Ferulic acid is an organic compound that exists as a phytochemical in the cell walls of plants, etc.

[0047] Furthermore, due to its strong antioxidant properties, ferulic acid is expected to be effective in preventing Alzheimer's disease and treating diabetes and hypercholesterolemia, and is being applied in a wide range of fields. In cosmetic applications, it is an effective ingredient for improving skin health because it inhibits tyrosinase activity, suppresses melanin production, and has anti-inflammatory effects.

[0048] Furthermore, ferulic acid is an ingredient that reduces the amount of claudin. There are also multiple families of claudin (for example, claudin 1 to claudin 27). In the claudin-reducing agent according to the third embodiment of the present invention, the amount of claudin that is reduced is not limited, but it is preferably claudin 1 (CLD1) or claudin 4 (CLD4).

[0049] Furthermore, in the claudin-reducing agent according to the third embodiment of the present invention, the concentration of ferulic acid is not particularly limited, but is preferably 125 μM or 500 μM.

[0050] The claudin reduction agent according to the third embodiment of the present invention can reduce the amount of claudin that forms tight junctions. In particular, it can effectively reduce the amount of claudin 1 (CLD1) and / or claudin 4 (CLD4). As a result, tight junctions can be alleviated.

[0051] The claudin-reducing agent according to the third embodiment of the present invention has the advantage of reducing claudin levels without exhibiting toxicity. Furthermore, it can relax tight junctions without causing them to collapse, thereby allowing the expression of intercellular spaces.

[0052] The claudin-reducing agents according to the first to third embodiments of the present invention may contain components other than the active ingredient described above. These components other than the active ingredient are not particularly limited and may include, for example, buffering agents, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents, antibacterial agents, and the like.

[0053] Furthermore, the dosage form of the claudin-reducing agent according to the first to third embodiments of the present invention is not particularly limited and includes, for example, topical preparations (e.g., liquids, gels, creams, sticks, sheets), tablets, powders, and granules.

[0054] Furthermore, the target recipients of the claudin-reducing agents according to the first to third embodiments of the present invention are not particularly limited, and examples include humans, animals, tissues collected from them, cells collected from them, and established cell lines.

[0055] Furthermore, the administration route of the claudin-reducing agent according to the first to third embodiments of the present invention is not particularly limited, and for example, parenteral administration (e.g., transdermal administration) and oral administration may be employed.

[0056] Furthermore, as another embodiment of the present invention, a cosmetic composition containing a claudin-reducing agent according to the first to third embodiments of the present invention may also be used.

[0057] Furthermore, this cosmetic composition contains Swertia japonica extract (BG extract), Swertia japonica extract (Et-OH extract), or ferulic acid as active ingredients. In addition, this cosmetic composition can be mixed with various raw materials along with these active ingredients to form a cosmetic.

[0058] Here, the various raw materials used in the cosmetic composition include, for example, bases such as purified water, humectants, oils, surfactants, pH adjusters, thickeners, neutralizing agents, plant extracts and additives, preservatives, and characteristic components. This cosmetic composition can be mixed with these various raw materials as appropriate to make a cosmetic.

[0059] Furthermore, the various ingredients listed here are merely examples, and other components and different proportions can be appropriately set. For example, in addition to the above, depending on the type and use of the formulation, chelating agents (metal ion sequestering agents), astringents, disinfectants, skin activators (vitamins, amino acids and amino acid derivatives), anti-inflammatory agents, and whitening agents (arbutin, tranexamic acid, vitamin C derivatives, etc.) can be included. Fragrances and colorants can also be added as needed.

[0060] Furthermore, examples of cosmetic products include lotions, emulsions, facial cleansers, makeup removers, serums, gels, and creams.

[0061] Furthermore, as another embodiment of the present invention, a pharmaceutical composition containing a claudin-reducing agent according to the first to third embodiments of the present invention is also possible.

[0062] Furthermore, the "pharmaceutical composition" disclosed herein may be obtained by adding the "claudin-reducing agent" disclosed herein as an active ingredient to a "pharmaceutical product" such as a transdermal drug, a transmucosal drug such as a transnasal or enteral drug, an injectable drug, an eye drop drug, or an inhaled drug.

[0063] Furthermore, as another embodiment of the present invention, a quasi-drug composition containing a claudin-reducing agent according to the first to third embodiments of the present invention may also be provided.

[0064] Furthermore, the "quasi-drug composition" disclosed in this specification may be, for example, a "quasi-drug" such as a mouth freshener, medicated toothpaste, medicated soap, medicated cosmetic, antiperspirant spray, underarm odor preventative, bath additive, medicated cream, baby powder, hair growth / hair nourishment agent, or hair dye, to which the "claudin-reducing agent" disclosed herein may be added as an active ingredient.

[0065] Furthermore, as another embodiment of the present invention, a food composition containing a claudin-reducing agent according to the first to third embodiments of the present invention may also be used.

[0066] Furthermore, the "food composition" disclosed herein may be obtained by adding the "claudin-reducing agent" disclosed herein as an active ingredient to "foods" such as fresh foods, animal products, plant products, fungal products, processed foods, health foods, beverages, and seasonings.

[0067] As described above, the claudin-reducing agent to which the present invention is applied contributes to the penetration of active ingredients into the skin. Furthermore, the tight junction relaxant to which the present invention is applied contributes to the penetration of active ingredients into the skin. Furthermore, cosmetic compositions to which the present invention is applied contribute to the penetration of active ingredients into the skin. [Examples]

[0068] The following describes embodiments of the present invention.

[0069] Examples of claudin-reducing agents to which the present invention is applied were prepared, and the following evaluations were performed. (1) Evaluation of changes in protein quantity Keratinocytes were cultured using the procedure described below, and protein expression and the increase or decrease in expression levels were evaluated by Western blotting.

[0070] (Keratinocyte culture conditions) Cells: NHEK (manufactured by Lonza) Culture temperature: 37℃ Carbon dioxide concentration: 5% Culture medium change frequency: 1-2 days After seeding the cells into a 6-well plate, The samples were incubated in a special culture medium (containing 100 μM calcium ions) for 1.9 days. The cells were cultured for 2.6 days in a mixed medium (specialized medium: α-MEM = 7:3, 1.3 mM calcium ions). The cells were cultured for 3.8 days in a mixed medium containing 1 ng / mL of recombinant human EGF. 4. The culture medium was replaced with a medium containing the above-mentioned Swertia japonica extract (BG extract), ferulic acid, and Swertia japonica extract (Et-OH extract) at the following dilution ratios, and then incubated for 1 day. Example 1 involved adding Swertia japonica extract (BG extract) to the culture medium at a concentration of 0.12% (v / v). Example 2 involved adding Swertia japonica extract (BG extract) to the culture medium at a concentration of 0.03% (v / v). Example 3 involved adding ferulic acid to the culture medium at a concentration of 125 μM. Example 4 involved adding ferulic acid to the culture medium at a concentration of 500 μM. Example 5 involved adding Swertia japonica extract (Et-OH extract) to the culture medium at a concentration of 3% (v / v). 5. The cells were washed with HEPES buffer, sample buffer for electrophoresis was added, and the cells were collected using a scraper and cryopreserved. Keratinocytes are keratinocytes that make up more than 90% of the cells that constitute the epidermis, and they form tight junctions when cultured under the above conditions.

[0071] (Western blotting) Western blotting was performed on each sample prepared in the above process according to the following procedure. 1. The frozen samples were thawed, and their viscosity was reduced by ultrasonic irradiation. 2. SDS-PAGE was performed to separate proteins contained in the cells based on differences in molecular weight. 3. Western blotting was used to transfer proteins from the gel after SDS-PAGE to a PVDF membrane. 4. The PVDF membrane was blocked with Tris buffer (t-TBS) containing 3% (v / v) skim milk. 5. Furthermore, the PVDF membrane was immersed in skim milk t-TBS containing the following primary antibodies and incubated overnight at 4°C. Claudin 1 (CLD1) antibody: ThermoFisher 71-7800 (anti-rabbit, polyclonal) Claudin 3 (CLD3) antibody: Merck (SIGMA) SAB3500435 (anti-rabbit, polyclonal) Claudin 4 (CLD4) antibody: Merck (SIGMA) SAB4500432 (anti-rabbit, polyclonal) Claudin 7 (CLD7) antibody: ThermoFisher 34-9100 (anti-rabbit, polyclonal) β-actin antibody: Fujifilm Wako Pure Chemical Industries 010-27841 (anti-mouse, monoclonal) 6. The PVDF membrane was washed with t-TBS, immersed in skim milk t-TBS containing the secondary antibody, and incubated at room temperature for 1 hour. 7. The PVDF film was washed with t-TBS and then washed with pure water. 8. The PVDF film was exposed to a chemiluminescent reagent, and the PVDF film was photographed. Claudin 1 (CLD1), claudin 3 (CLD3), claudin 4 (CLD4), and claudin 7 (CLD7) are claudin family proteins that form tight junctions. Claudin 1 (CLD1) and claudin 4 (CLD4) are major claudins in keratinocytes and are proteins primarily responsible for barrier functions that regulate substance permeability. The β-actin antibody is an antibody that specifically binds to β-actin, a globular protein ubiquitously expressed in all eukaryotic cells, and serves as a positive control to demonstrate that protein expression is normal in each sample.

[0072] The results of the Western blotting performed using the procedure described above are shown in Figures 1 to 5.

[0073] In Figures 1(a) and 2(b), the results are shown from left to right in the lanes for "culture medium only, Example 3 (125 μM ferulic acid), 0.1% BG, Example 1 (Swertia japonica extract (BG extracted) 0.12%), and Example 2 (Swertia japonica extract (BG extracted) 0.03%)." The lane for culture medium only compares protein-derived bands with Example 3 (125 μM ferulic acid). The lane for 0.1% BG compares protein-derived bands with Example 1 (Swertia japonica extract (BG extracted) 0.12%) and Example 2 (Swertia japonica extract (BG extracted) 0.03%). Figure 1(a) shows the results using claudin 1 (CLD1) antibody, Figure 1(b) shows the results using claudin 3 (CLD3) antibody, Figure 2(a) shows the results using claudin 4 (CLD4) antibody, and Figure 2(b) shows the results using claudin 7 (CLD7) antibody.

[0074] Furthermore, in Figures 3(a) to 4(b), the results for "culture medium only" and "Example 4 (500 μM ferulic acid)" are shown from left to right in the lane. The lane for culture medium only is used to compare the protein-derived bands with those of Example 4 (500 M ferulic acid). Figure 3(a) shows the results using claudin 1 (CLD1) antibody, Figure 3(b) shows the results using claudin 3 (CLD3) antibody, Figure 4(a) shows the results using claudin 4 (CLD4) antibody, and Figure 4(b) shows the results using claudin 7 (CLD7) antibody.

[0075] Furthermore, in Figures 5(a) and 5(b), the first lane from the left shows the results for "0.1% BG," and the fourth lane from the left shows the results for "Example 5 (Swertia japonica extract (Et-OH extract) 3%)." The 0.1% BG lane is used to compare the protein-derived bands with those of Example 5 (Swertia japonica extract (Et-OH extract) 0.3%). Figure 5(a) shows the results using a claudin 1 (CLD1) antibody, and Figure 5(b) shows the results using a claudin 3 (CLD3) antibody.

[0076] As shown in Figure 1(a), in Example 3, the band corresponding to claudin 1 (CLD1) was fainter compared to the lane with only culture medium, indicating a decrease in claudin 1 (CLD1) expression. Similarly, in Examples 1 and 2, the band corresponding to claudin 1 (CLD1) was fainter compared to the lane with 0.1% BG, indicating a decrease in claudin 1 (CLD1) expression.

[0077] Furthermore, as shown in Figure 1(b), in Example 3, the band corresponding to claudin 3 (CLD3) was slightly fainter compared to the lane with only culture medium, indicating a decrease in claudin 3 (CLD3) expression. Similarly, in Example 2, the band corresponding to claudin 3 (CLD3) was slightly fainter compared to the lane with 0.1% BG, indicating a decrease in claudin 3 (CLD3) expression.

[0078] Furthermore, as shown in Figure 2(a), in Example 3, the band corresponding to claudin 4 (CLD4) was fainter compared to the lane with only culture medium, confirming a decrease in claudin 4 (CLD4) expression. Similarly, in Examples 1 and 2, the band corresponding to claudin 4 (CLD4) was fainter compared to the lane with 0.1% BG, confirming a decrease in claudin 4 (CLD4) expression. As shown in Figure 2(b), in Examples 2 to 4, no change was observed in the band corresponding to Claudin 7 (CLD7) for the lanes containing only culture medium or 0.1% BG.

[0079] As shown in Figure 3(a), in Example 4, the band corresponding to claudin 1 (CLD1) was fainter compared to the lane containing only the culture medium, confirming a decrease in the expression level of claudin 1 (CLD1).

[0080] Furthermore, as shown in Figure 3(b), in Example 4, the band corresponding to claudin 3 (CLD3) was fainter compared to the lane containing only the culture medium, confirming a decrease in the expression level of claudin 3 (CLD3).

[0081] Furthermore, as shown in Figure 4(a), in Example 4, the band corresponding to claudin 4 (CLD4) was fainter compared to the lane containing only the culture medium, confirming a decrease in the expression level of claudin 4 (CLD4). Furthermore, as shown in Figure 4(b), in Example 4, no change was observed in the band corresponding to Claudin 7 (CLD7) in the lane containing only the culture medium.

[0082] As shown in Figure 5(a), in Example 5, the band corresponding to claudin 1 (CLD1) was fainter in the 0.1% BG lane, confirming a decrease in claudin 1 (CLD1) expression.

[0083] Furthermore, as shown in Figure 5(b), in Example 5, the band corresponding to claudin 3 (CLD3) was slightly fainter compared to the 0.1% BG lane, confirming a decrease in claudin 3 (CLD3) expression.

[0084] (2) Evaluation of tight junction permeability Next, the tight junction permeability was evaluated using the claudin-reducing agent to which the present invention is applied.

[0085] (Keratinocyte culture conditions) Cells: NHEK (manufactured by Lonza) Culture temperature: 37℃ Carbon dioxide concentration: 5% Culture medium change frequency: 2 days After seeding the cells into a 24-transwell plate (Falcon), The samples were incubated in a special culture medium (containing 100 μM calcium ions) for 1.9 days. The cells were cultured for 2.4 days in a mixed medium (specialized medium: α-MEM = 7:3, 1.3 mM calcium ions). 3. The culture medium in the upper tank was removed, and differentiation was induced by drying. Additionally, culture medium was added only to the lower tank, and the cells were incubated for two days. 4. With the upper tank left dry, the cells were cultured for 8 days in a mixed medium containing 1 ng / mL of recombinant human EGF.

[0086] (Evaluation of permeability in tight junctions) 5. A keratin permeability enhancer, 2 μMFITC-Dextran 4,000 (FD4K, manufactured by SIGMA), and 50 μL of a HEPES buffer solution containing the aforementioned Swertia japonica extract (BG extract) or ferulic acid were added to the keratinocytes in the upper cell, and the culture was continued in a CO2 incubator. For Example 6, a culture medium was prepared by adding 0.12% (v / v) Swertia japonica extract (BG extract) and 1% (w / w) MG-2070 (keratin permeabilis agent) to the culture medium. For Example 7, a culture medium was prepared by adding 0.03% (v / v) Swertia japonica extract (BG extract) and 0.25% (w / w) MG-2070 (keratin permeabilis agent) to the culture medium. For Example 8, a culture medium was prepared by adding 125 μM ferulic acid and 0.25% (w / w) MG-753D (keratin permeabilis agent) to the culture medium. For Example 9, a culture medium was prepared by adding 500 μM ferulic acid and 5% (w / w) S-753D (keratin permeabilis agent) to the culture medium. 6. After a certain period of time (1h to 61h) following the start of culture as described in 5. above, 100 μL of the culture medium from the lower tank was sampled. In addition, 3 hours after the start of culture as described in 5. above, the HEPEs buffer solution remaining in the upper tank was removed. 7. The sampled culture medium was transferred to a 96-well plate, and the FITC fluorescence intensity in the medium was measured using a microplate reader. The measurement results are shown as relative values, based on the fluorescence intensity of FTTC measured from a sample (control sample) to which a HEPES buffer solution (containing a keratin permeability enhancer, 2 μMFITC-Dextran 4,000) without Swertia japonica extract (BG extraction) and ferulic acid was added in step 5 above. An increase in fluorescence intensity compared to the control sample indicates that FD4K with a molecular weight of 4000 has permeated through the tight junction, i.e., that the tight junction has been relaxed.

[0087] The results of the tight junction permeability evaluation conducted according to the above procedure are shown in Tables 1 to Figures 4 below.

[0088] (Table 1) JPEG2026059854000002.jpg3731

[0089] (Table 2) JPEG2026059854000003.jpg2332

[0090] (Table 3) JPEG2026059854000004.jpg2330

[0091] (Table 4) JPEG2026059854000005.jpg3631

[0092] As shown in Table 1, in Example 6, an increase in fluorescence intensity was observed after culturing with the addition of 0.12% (v / v) Swertia japonica extract (BG extract). In particular, sampling results at 19, 30, and 43 hours after culturing showed an increase in fluorescence intensity of more than 10% compared to the control sample.

[0093] As shown in Table 2, in Example 7, an increase in fluorescence intensity was observed after culturing with the addition of 0.03% (v / v) Swertia japonica extract (BG extract). In particular, sampling results at 25 and 50 hours after culturing showed an increase in fluorescence intensity of more than 10% compared to the control sample.

[0094] As shown in Table 3, in Example 8, an increase in fluorescence intensity was observed after culturing with the addition of 125 μM ferulic acid. A 10% or greater increase in fluorescence intensity was observed compared to the control sample in the samples taken 1 hour, 18 hours, 48 ​​hours, and 61 hours after culturing.

[0095] As shown in Table 4, in Example 9, an increase in fluorescence intensity was observed after culturing with the addition of 500 μM ferulic acid. In particular, an increase of 10% or more in fluorescence intensity was observed compared to the control sample in the sampling results at 1 hour, 3 hours, 6 hours, 19 hours, 43 hours, and 48 hours after culturing.

Claims

1. A claudin-reducing agent containing at least one of Swertia japonica extract and ferulic acid.

2. Reduces the amount of claudin 1 (CLD1) The claudin content reducing agent according to claim 1.

3. Reduces the amount of claudin 4 (CLD4) A claudin-reducing agent according to claim 1 or claim 2.

4. A tight junction reliever containing at least one of Swertia japonica extract and ferulic acid.

5. A cosmetic composition comprising a claudin-reducing agent according to claims 1 to 3, and / or a tight junction relaxant according to claim 4.

6. A pharmaceutical composition comprising a claudin-reducing agent according to claims 1 to 3, and / or a tight junction relaxant according to claim 4.

7. A quasi-drug composition comprising a claudin-reducing agent according to claims 1 to 3, and / or a tight junction relaxant according to claim 4.

8. A food composition comprising a claudin-reducing agent according to claims 1 to 3, and / or a tight junction relaxant according to claim 4.

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