Claudin amount reducing agent, tight junction relaxing agent, cosmetic composition, pharmaceutical composition, quasi-drug composition and food composition

Abstract

Provided are a Claudin amount reducing agent, a tight junction relaxing agent, and a cosmetic composition that contribute to the penetration of an active ingredient into the skin. **Solution**: The Claudin amount reducing agent of the present invention contains an assembly extract. In this Claudin amount reducing agent, the assembly extract is an extract obtained by extracting the whole herb of the Gentianaceae plant Swertia japonica (scientific name: 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 tight junction is the outermost one. Through this tight junction, the barrier function of the skin can be enhanced, preventing the invasion of external irritants 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 to each other 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 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 Japanese Patent No. 7304049 Summary of the Invention Problems to be Solved by the Invention

[0008] Here, although some of the compounds described in Patent Document 1 also include plant-derived compounds, considering their use in cosmetics and the like, an increase in the types of plant-derived ingredients that can provide a tight junction relaxation effect is preferable in terms of increasing the freedom of formulation and making it easier to appeal to the image of the product.

[0009] The present invention was conceived in view of the above points, and an object thereof is to provide a claudin amount reducing agent, a tight junction relaxant, 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. Means for Solving the Problems

[0010] In order to achieve the above object, the claudin amount reducing agent of the present invention is configured to contain at least one of assembly extract and ferulic acid.

[0011] Here, by containing the assembly extract, the amount of claudin forming tight junctions can be reduced. The assembly extract referred to here is an extract obtained by solvent extraction from the whole herb of the Gentianaceae plant Swertia japonica (scientific name: Swertia japonica).

[0012] Also, by containing ferulic acid, the amount of claudin forming tight junctions can be reduced.

[0013] Also, when reducing the amount of Claudin 1 (CLD1), among the Claudin family, it is possible to reduce the Claudin that is a major Claudin constituting the tight junction and is responsible for the barrier function of regulating substance permeability. Note that there are multiple families of Claudin (for example, Claudin 1 to 27).

[0014] Also, when reducing the amount of Claudin 4 (CLD4), among the Claudin family, it is possible to reduce the Claudin that is a major Claudin constituting the tight junction and is responsible for the barrier function of regulating substance permeability.

[0015] Also, in order to achieve the above object, the tight junction relaxant of the present invention is configured to contain at least one of assembly extract and ferulic acid.

[0016] Here, by containing the assembly extract, the amount of Claudin forming the tight junction can be reduced, and the tight junction can be relaxed.

[0017] Also, by containing ferulic acid, the amount of Claudin forming the tight junction can be reduced, and the tight junction can be relaxed.

[0018] Also, in order to achieve the above object, the cosmetic composition of the present invention is configured to contain the Claudin amount reducing agent according to claims 1 to 3 and / or the tight junction relaxant according to claim 4.

[0019] Here, by containing the Claudin amount reducing agent according to claims 1 to 3 and / or the tight junction relaxant according to claim 4, it is possible to endow the cosmetic composition with the effect of reducing the amount of Claudin forming the tight junction and relaxing the tight junction.

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

[0021] Here, by containing a claudin amount reducing agent according to claims 1 to 3 and / or a tight junction relaxing agent according to claim 4, it is possible to endow the pharmaceutical composition with the effect of reducing the amount of claudin forming the tight junction and relaxing the tight junction.

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

[0023] Here, by containing a claudin amount reducing agent according to claims 1 to 3 and / or a tight junction relaxing agent according to claim 4, it is possible to endow the quasi-drug composition with the effect of reducing the amount of claudin forming the tight junction and relaxing the tight junction.

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

[0025] Here, by containing a claudin amount reducing agent according to claims 1 to 3 and / or a tight junction relaxing agent according to claim 4, it is possible to endow the food composition with the effect of reducing the amount of claudin forming the tight junction and relaxing the tight junction.

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

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0028] [First Embodiment of the Present Invention] Hereinafter, the agent for reducing the amount of claudin according to 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 amount reducing agent according to the first embodiment of the present invention, the Swertia extract is an extract extracted from the whole herb of the Swertia plant (scientific name: Swertia japonica) with 1,3-butylene glycol (1,3-BG). Hereinafter, this extract is referred to as "Swertia extract (BG extraction)". Swertia is used as a bitter stomachic and intestinal regulator. It is also formulated in skin care products and hair growth agents because it has a blood circulation promoting effect, a hair growth promoting effect, a rough skin improving effect, and a whitening effect due to vasodilation.

[0030] In addition, the Swertia extract (BG extraction) is composed of 12.50% Swertia extract and 87.50% 1,3-butylene glycol.

[0031] In addition, the Swertia extract (BG extraction) is a component that reduces the amount of Claudin. There are multiple families of Claudin (for example, Claudin 1 to Claudin 27). In the Claudin amount reducing agent according to the first embodiment of the present invention, the Claudin whose amount is reduced is not limited, but it is preferably Claudin 1 (CLD1) or Claudin 4 (CLD4).

[0032] Note that the amount of Claudin in this specification mainly refers to the amount of Claudin protein, but is not limited thereto, and may be the amount of Claudin mRNA.

[0033] Here, the Swertia extract (BG extraction) does not necessarily have to be composed of 12.50% Swertia extract and 87.50% 1,3-butylene glycol, and the contents of the Swertia extract and 1,3-butylene glycol can be set appropriately.

[0034] In addition, in the Claudin amount reducing agent according to the first embodiment of the present invention, the concentration of the Swertia extract (BG extraction) is not particularly limited, but is preferably 0.12% (v / v) or 0.03% (v / v).

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

[0036] The agent for reducing the amount of claudin according to the first embodiment of the present invention has the advantage of reducing the amount of claudin and not showing toxicity. In addition, while relaxing tight junctions, it can express intercellular spaces without collapsing them.

[0037] [Second Embodiment of the Present Invention] Hereinafter, the agent for reducing the amount of claudin, which is the second embodiment of the present invention, contains assembly extract. The agent for reducing the amount of claudin according to the second embodiment of the present invention is also a tight junction relaxant.

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

[0039] In addition, the assembly extract (Et-OH extract) is composed of 1.67% of assembly components, 57.00% of ethanol, and 41.33% of water.

[0040] In addition, the assembly extract (Et-OH extract) is a component that reduces the amount of claudin. There are multiple families of claudin (for example, claudin 1 to claudin 27). In the agent for reducing the amount of claudin according to the second embodiment of the present invention, the claudin whose amount is reduced is not limited, but it is preferably claudin 1 (CLD1).

[0041] Here, the assembly extract (Et-OH extraction) does not necessarily have to be composed of an assembly component: 1.67%, ethanol: 57.00%, and water: 41.33%. The contents of the assembly component, ethanol, and water can be set as appropriate.

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

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

[0044] The claudin amount reducing agent according to the second embodiment of the present invention has the advantage of reducing the amount of claudin and also showing no toxicity. Also, while relaxing the tight junctions, it can express intercellular spaces without collapsing them.

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

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

[0047] In addition, ferulic acid is expected to prevent Alzheimer's disease and have effects on diabetes and hypercholesterolemia due to its high antioxidant activity, and is applied in a wide range of fields. As a cosmetic ingredient, it is effective for beautiful skin because it suppresses melanin production by inhibiting tyrosinase activity and has an anti-inflammatory effect.

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

[0049] In addition, in the claudin amount 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 amount reducing agent according to the third embodiment of the present invention can reduce the amount of claudin that forms tight junctions. In particular, the amount of claudin 1 (CLD1) and / or claudin 4 (CLD4) can be effectively reduced. As a result, tight junctions can be relaxed.

[0051] The claudin amount reducing agent according to the third embodiment of the present invention has the advantage of reducing the amount of claudin without showing toxicity. In addition, while relaxing tight junctions, it can express intercellular spaces without disrupting them.

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

[0053] In addition, the dosage form of the Claudin amount reducing agent according to the first to third embodiments of the present invention is not particularly limited, and examples thereof include external preparations (for example, liquid preparations, gel preparations, cream preparations, stick preparations, sheet preparations), tablets, powders, and granules.

[0054] In addition, the administration target of the Claudin amount reducing agent according to the first to third embodiments of the present invention is not particularly limited, and examples thereof include humans, animals, tissues collected from these, cells collected from these, and immortalized cells.

[0055] In addition, the administration route of the Claudin amount reducing agent according to the first to third embodiments of the present invention is not particularly limited, and for example, parenteral administration (for example, transdermal administration) and oral administration can be employed.

[0056] In addition, as another embodiment of the present invention, a cosmetic composition containing the Claudin amount reducing agent according to the first to third embodiments of the present invention can also be provided.

[0057] In addition, this cosmetic composition contains assembly extract (BG extraction), assembly extract (Et-OH extraction), or ferulic acid as an active ingredient. In addition, in this cosmetic composition, various raw materials can be mixed 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, moisturizers, oils, surfactants, pH adjusters, thickeners, neutralizers, plant extracts and additives, preservatives, characteristic components, and the like. This cosmetic composition can be appropriately mixed with these various raw materials to form a cosmetic.

[0059] In addition, the various raw materials shown here are merely examples, and it is possible to appropriately set other components and different compounding amounts. For example, in addition to the above, according to the type and use of the preparation, chelating agents (metal ion sequestering agents), astringents, bactericides, skin activators (vitamins, amino acids, and amino acid derivatives), anti-inflammatory agents, whitening agents (arbutin, tranexamic acid, vitamin C derivatives, etc.) can be included. Also, if necessary, flavors and coloring agents can also be compounded.

[0060] Examples of the types of cosmetics include, for example, lotions, milky lotions, facial washes, cleansers, beauty essences, gels, creams, and the like.

[0061] In addition, as another embodiment of the present invention, a pharmaceutical composition containing the agent for reducing the amount of claudin according to the first to third embodiments of the present invention can also be used.

[0062] Note that the "pharmaceutical composition" disclosed in this specification may be, for example, a "pharmaceutical product" such as a transdermal administration drug, a transmucosal administration drug such as nasal or enteral administration, an injection administration drug, an eye drop administration drug, an inhalation administration drug, etc., with the "agent for reducing the amount of claudin" disclosed in this specification added as an active ingredient.

[0063] In addition, as another embodiment of the present invention, a quasi-drug composition containing the agent for reducing the amount of claudin according to the first to third embodiments of the present invention can also be used.

[0064] Note that the "quasi-drug composition" disclosed in the specification may be, for example, an "quasi-drug" such as an oral coolant, a medicinal dentifrice, a medicinal soap, a medicinal cosmetic, an antiperspirant spray, an axillary odor inhibitor, a bath agent, a medicinal cream, a baby powder, a hair growth and hair nourishing agent, a hair dye, etc., with the "agent for reducing the amount of claudin" disclosed in this specification added as an active ingredient.

[0065] In addition, as another embodiment of the present invention, a food composition containing the agent for reducing the amount of claudin according to the first to third embodiments of the present invention can also be used.

[0066] In addition, for the "food composition" disclosed in this specification, for example, to "foods" such as fresh foods, animal foods, plant foods, fungal foods, processed foods, health foods, beverages, seasonings, etc., the "claudin amount reducing agent" disclosed in this specification may be added as an active ingredient.

[0067] As described above, the claudin amount reducing agent to which the present invention is applied contributes to the penetration of the active ingredient into the skin. In addition, the tight junction relaxant to which the present invention is applied contributes to the penetration of the active ingredient into the skin. In addition, the cosmetic composition to which the present invention is applied contributes to the penetration of the active ingredient into the skin.

Examples

[0068] Hereinafter, examples of the present invention will be described.

[0069] Examples of the claudin amount reducing agent to which the present invention is applied were prepared and the following evaluations were conducted. (1) Evaluation of the change in protein amount Keratinocytes were cultured according to the procedure shown below, and the expression and increase or decrease in the amount of protein were evaluated by Western blotting.

[0070] (Culture conditions of keratinocytes) Cells: NHEK (manufactured by Lonza) Culture temperature: 37°C Carbon dioxide concentration: 5% Medium exchange frequency: 1 - 2 days After seeding the cells in a 6-well plate, 1. For 9 days, a dedicated medium (containing 100 μM calcium ions) was used. 2. For 6 days, the cells were cultured in a mixed medium (dedicated medium: α-MEM = 7:3, 1.3 mM calcium ions). 3. For 8 days, the cells were cultured in a mixed medium containing 1 ng / mL of recombinant human EGF. 4. Using the above-mentioned assembly extract (BG extraction), ferulic acid, and assembly extract (Et-OH extraction), after replacing the medium added at the following dilution ratios, the cells were cultured for 1 day. For Example 1, the assembly extract (BG extraction) was added to the medium at a concentration of 0.12% (v / v); for Example 2, the assembly extract (BG extraction) was added to the medium at a concentration of 0.03% (v / v); for Example 3, ferulic acid was added to the medium at a concentration of 125 μM; for Example 4, ferulic acid was added to the medium at a concentration of 500 μM; and for Example 5, the assembly extract (Et-OH extraction) was added to the medium at a concentration of 3% (v / v). 5. The cells were washed with HEPES buffer, an electrophoresis sample buffer was added, and the cells were collected using a scraper and cryopreserved. Note that keratinocytes are keratinized cells that account for more than 90% of the cells constituting the epidermis, and tight junctions are formed by culturing under the above conditions.

[0071] (Western blotting) Western blotting was performed on each sample prepared in the above steps according to the following procedure. 1. The cryopreserved sample was thawed and the viscosity was reduced by ultrasonic irradiation. 2. SDS-PAGE was performed to separate the proteins contained in the cells based on the difference in molecular weight. 3. By Western blotting, the proteins in the gel after SDS-PAGE were transferred to a PVDF membrane. 4. The PVDF membrane was blocked with Tris buffer (t-TBS) containing 3% (v / v) skim milk. 5. Further, the PVDF membrane was immersed in skim milk t-TBS containing the following various 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 010-27841 (anti-mouse, monoclonal) 6. The PVDF membrane was washed with t-TBS and immersed in skim milk t-TBS containing the secondary antibody, and incubated at room temperature for 1 hour. 7. The PVDF membrane was washed with t-TBS and rinsed pure. 8. The PVDF membrane was exposed to chemiluminescence reagent and the PVDF membrane was photographed. Note that Claudin 1 (CLD1), Claudin 3 (CLD3), Claudin 4 (CLD4) and Claudin 7 (CLD7) are claudin family proteins that form tight junctions. Also, Claudin 1 (CLD1) and Claudin 4 (CLD4) are the main claudins in keratinocytes and are proteins that mainly undertake the barrier function of regulating substance permeability. Also, the β-actin antibody is an antibody that specifically binds to β-actin, a globular protein ubiquitously expressed in all eukaryotic cells, and is a positive control to show that protein expression is normally carried out in each sample.

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

[0073] In FIGS. 1(a) to 2(b), the results of "medium only, Example 3 (125 μM ferulic acid), 0.1% BG, Example 1 (assembly extract (BG extraction) 0.12%), Example 2 (assembly extract (BG extraction) 0.03%)" are shown from the left side of the lane. Also, the lane of medium only is the lane for comparing protein-derived bands with Example 3 (125 μM ferulic acid). Further, 0.1% BG is the lane for comparing protein-derived bands between Example 1 (assembly extract (BG extraction) 0.12%) and Example 2 (assembly extract (BG extraction) 0.03%). Also, FIG. 1(a) shows the result using a claudin 1 (CLD1) antibody, FIG. 1(b) shows the result using a claudin 3 (CLD3) antibody, FIG. 2(a) shows the result using a claudin 4 (CLD4) antibody, and FIG. 2(b) shows the result using a claudin 7 (CLD7) antibody.

[0074] In FIGS. 3(a) to 4(b), the results of "medium only, Example 4 (500 μM ferulic acid)" are shown from the left side of the lane. Also, the lane of medium only is the lane for comparing protein-derived bands with Example 4 (500 M ferulic acid). Also, FIG. 3(a) shows the result using a claudin 1 (CLD1) antibody, FIG. 3(b) shows the result using a claudin 3 (CLD3) antibody, FIG. 4(a) shows the result using a claudin 4 (CLD4) antibody, and FIG. 4(b) shows the result using a claudin 7 (CLD7) antibody.

[0075] In FIGS. 5(a) and 5(b), the results of "0.1% BG" at the first position from the left in the lane and "Example 5 (assembly extract (Et-OH extraction) 3%)" at the fourth position from the left in the lane are shown. Also, 0.1% BG is the lane for comparing protein-derived bands with Example 5 (assembly extract (Et-OH extraction) 0.3%). Also, FIG. 5(a) shows the result using a claudin 1 (CLD1) antibody and FIG. 5(b) shows the result using a claudin 3 (CLD3) antibody.

[0076] As shown in Fig. 1(a), for the lane with only the medium, in Example 3, the band corresponding to Claudin 1 (CLD1) became thinner, and a decrease in the expression level of Claudin 1 (CLD1) was confirmed. Similarly, for the lane with 0.1% BG, in Examples 1 and 2, the band corresponding to Claudin 1 (CLD1) became thinner, and a decrease in the expression level of Claudin 1 (CLD1) was confirmed.

[0077] Also, as shown in Fig. 1(b), for the lane with only the medium, in Example 3, the band corresponding to Claudin 3 (CLD3) became slightly thinner, and a decrease in the expression level of Claudin 3 (CLD3) was confirmed. Similarly, for the lane with 0.1% BG, in Example 2, the band corresponding to Claudin 3 (CLD3) became slightly thinner, and a decrease in the expression level of Claudin 3 (CLD3) was confirmed.

[0078] Also, as shown in Fig. 2(a), for the lane with only the medium, in Example 3, the band corresponding to Claudin 4 (CLD4) became thinner, and a decrease in the expression level of Claudin 4 (CLD4) was confirmed. Similarly, for the lane with 0.1% BG, in Examples 1 and 2, the band corresponding to Claudin 4 (CLD4) became thinner, and a decrease in the expression level of Claudin 4 (CLD4) was confirmed. Note that as shown in Fig. 2(b), in Examples 2 to 4, no change was observed in the band corresponding to Claudin 7 (CLD7) for the lane with only the medium or the lane with 0.1% BG.

[0079] As shown in Fig. 3(a), for the lane with only the medium, in Example 4, the band corresponding to Claudin 1 (CLD1) became thinner, and a decrease in the expression level of Claudin 1 (CLD1) was confirmed.

[0080] Also, as shown in Fig. 3(b), for the lane with only the medium, in Example 4, the band corresponding to Claudin 3 (CLD3) became thinner, and a decrease in the expression level of Claudin 3 (CLD3) was confirmed.

[0081] Also, as shown in Fig. 4(a), in Example 4, for the lane of the medium only, the band corresponding to Claudin 4 (CLD4) was thinner, and a decrease in the expression level of Claudin 4 (CLD4) was confirmed. Note that, as shown in Fig. 4(b), in Example 4, no change was observed in the band corresponding to Claudin 7 (CLD7) for the lane of the medium only.

[0082] As shown in Fig. 5(a), in Example 5, for the lane of 0.1% BG, the band corresponding to Claudin 1 (CLD1) was thinner, and a decrease in the expression level of Claudin 1 (CLD1) was confirmed.

[0083] Also, as shown in Fig. 5(b), in Example 5, for the lane of 0.1% BG, the band corresponding to Claudin 3 (CLD3) was slightly thinner, and a decrease in the expression level of Claudin 3 (CLD3) was confirmed.

[0084] (2) Evaluation of tight junction permeability Next, the following evaluation was performed on the tight junction permeability using the agent for reducing the amount of claudin to which the present invention was applied.

[0085] (Culture conditions of keratinocytes) Cells: NHEK (manufactured by Lonza) Culture temperature: 37 °C Carbon dioxide concentration: 5% Medium exchange frequency: every 2 days After seeding the cells in a 24-well transwell plate (manufactured by falcon), 1. They were cultured in a dedicated medium (containing 100 μM calcium ions) for 1.9 days. 2. They were cultured in a mixed medium (dedicated medium: α-MEM = 7:3, 1.3 mM calcium ions) for 4 days. 3. Except for the medium in the upper chamber, differentiation was induced by drying. Also, medium was added only to the lower chamber and cultured for 2 days. 4. With the upper chamber kept dry, it was cultured in a medium containing 1 ng / mL of recombinant human EGF in a mixed medium for 8 days.

[0086] (Evaluation of tight junction permeability) 5. A HEPES buffer solution containing a keratin penetration enhancer, 2 μM FITC-Dextran 4,000 (FD4K, manufactured by SIGMA), and the above-mentioned assembly extract (BG extraction) or ferulic acid was added to 50 μL of keratinocytes in the upper chamber, and CO 2 The culture was continued in an incubator. For the medium, Example 6 was prepared by adding the assembly extract (BG extraction) at a concentration of 0.12% (v / v) and MG-2070 (keratin penetration agent) at 1% (w / w). Also, Example 7 was prepared by adding the assembly extract (BG extraction) at a concentration of 0.03% (v / v) and MG-2070 (keratin penetration agent) at 0.25% (w / w) to the medium. Also, Example 8 was prepared by adding ferulic acid at a concentration of 125 μM and MG-753D (keratin penetration agent) at 0.25% (w / w) to the medium. Also, Example 9 was prepared by adding ferulic acid at a concentration of 500 μM and S-753D (keratin penetration agent) at 5% (w / w) to the medium. 6. At a certain time after the start of the culture in step 5 above (1 h to 61 h), 100 μL of the medium in the lower chamber was sampled each time. Also, 3 hours after the start of the culture in step 5 above, the remaining HEPES buffer solution in the upper chamber was removed. 7. The sampled 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 were 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 penetration enhancer and 2 μM FITC-Dextran 4,000 but not containing the assembly extract (BG extraction) and ferulic acid) was added in the above step 5. When the fluorescence intensity increased compared to the control sample, it indicated that FD4K with a molecular weight of 4,000 had penetrated the tight junction, that is, the tight junction had been relaxed.

[0087] The results of the permeability evaluation of the tight junction performed in the above flow are shown in Table 1 to Figure 4 below.

[0088] (Table 1) JPEG0007691564000002.jpg3731

[0089] (Table 2) JPEG0007691564000003.jpg2332

[0090] (Table 3) JPEG0007691564000004.jpg2330

[0091] (Table 4) JPEG0007691564000005.jpg3631

[0092] As shown in Table 1, in Example 6, after culturing after adding 0.12% (v / v) of assembly extract (BG extraction), a tendency for the fluorescence intensity to increase was confirmed. In particular, in the sampling results after 19 hours, 30 hours, and 43 hours of culturing, an increase in fluorescence intensity of 10% or more was confirmed compared to the control sample.

[0093] As shown in Table 2, in Example 7, after culturing after adding 0.03% (v / v) of assembly extract (BG extraction), a tendency for the fluorescence intensity to increase was confirmed. In particular, in the sampling results after 25 hours and 50 hours of culturing, an increase in fluorescence intensity of 10% or more was confirmed compared to the control sample.

[0094] As shown in Table 3, in Example 8, after culturing after adding 125 μM of ferulic acid, a tendency for the fluorescence intensity to increase was confirmed. In each sampling result after 1 hour, 18 hours, 48 hours, and 61 hours of culturing, an increase in fluorescence intensity of 10% or more was confirmed compared to the control sample.

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

Claims

1. Contains at least one of Swertia japonica extract and ferulic acid, and reduces the amount of claudin 1 (CLD1) Agents that reduce claudin levels.

2. Contains at least one of Swertia japonica extract and ferulic acid, and reduces the amount of claudin 4 (CLD4) Agents that reduce claudin levels.

3. A tight junction relaxing agent containing at least one of Swertia japonica extract and ferulic acid.

Images