Tight junction control agent, absorption promoting agent for useful substance, absorption promoting and leakage preventing agent for useful substance, and pharmaceutical composition, quasi-drug composition, cosmetic composition, and food composition

Abstract

To provide a novel compound capable of controlling tight junctions.SOLUTION: A tight junction control agent comprising at least one selected from the compounds represented by formulas (1) to (4), or at least one selected from pharmaceutically acceptable salts of the compounds represented by formulas (1) to (4).SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present disclosure relates to a tight junction regulator, an absorption enhancer for a useful substance that contains the regulator, an absorption enhancer and leakage preventer for a useful substance that contains the regulator, and a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, and a food composition that contain the regulator as an active ingredient. [Background technology]

[0002] To maintain homeostasis within the body, it is important to keep water, sugars, ions, and other substances contained within. Epithelial cells, which serve as a barrier between the outside world and the inside, have well-developed cell adhesion mechanisms that connect cells together. Of the several cell adhesion mechanisms present, the tight junction is the outermost. Tight junctions are formed by the strong interaction of membrane proteins called claudins between epithelial cells, preventing the free passage of water and ions. The function of such tight junctions is particularly important for moisturizing the skin and for the blood-brain barrier, which prevents foreign substances from entering the brain. Therefore, substances that regulate tight junction formation are attracting attention as targets for the development of cosmetics and pharmaceuticals.

[0003] Patent Document 1 lists the following compounds as compounds that relax tight junctions. [ka]

[0004] Furthermore, Patent Document 2 lists the following compounds as compounds that relax tight junctions. [ka] [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 190310 [Patent Document 2] Patent No. 7304049 Summary of the Invention [Problem to be solved by the invention]

[0006] The compounds described in Patent Documents 1 and 2 can relax tight junctions. However, even if a compound exhibiting a specific function is already known, it is preferable to have a wide range of compound options in consideration of industrial applications. [Means for solving the problem]

[0007] The disclosure of the present application has been made to solve the above-mentioned problems, and relates to the following: a tight junction regulator; an absorption enhancer for a useful substance that contains the regulator; an absorption enhancer and leakage-preventing agent for a useful substance that contains the regulator; and a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, and a food composition that contain the regulator as an active ingredient.

[0008] [1] At least one compound selected from the compounds represented by the following formulas (1) to (4), or At least one selected from pharmaceutically acceptable salts of compounds represented by the following formulas (1) to (4): A tight junction regulator comprising as an active ingredient. [ka] [2] The tight junction regulator according to [1] above, wherein the compound or pharmaceutically acceptable salt thereof represented by any one of formulas (1) to (3) has a tight junction relaxing function and a tight junction strengthening function. [3] The tight junction regulator comprises cherry bark extract, At least one of the compounds represented by the formulas (1) to (3) is contained as a component in the cherry bark extract. The tight junction regulator according to [2] above. [4] The tight junction regulator comprises yaba santa extract; The compound represented by formula (3) is contained as a component in the Yerba Santa extract. The tight junction regulator according to [2] above. [5] The tight junction regulator according to [2] above, wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function. [6] The tight junction regulator according to [3] above, wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function. [7] The tight junction regulator according to [4] above, wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function. [8] The tight junction regulator according to the above [1], wherein the compound represented by formula (4) or a pharmaceutically acceptable salt thereof has a tight junction relaxing function. [9] An absorption enhancer for a useful substance, comprising the tight junction regulator according to any one of [2] to [8] above as an active ingredient.

[10] An agent for promoting absorption and preventing leakage of a useful substance, comprising as an active ingredient the tight junction regulator according to any one of [2] to [7] above.

[11] The tight junction regulator according to any one of the above [2] to [8], further comprising a compound that is absorbed by relaxing the tight junction.

[12] The tight junction regulator according to any one of the above [2] to [8], which is used as a component of a pharmaceutical composition.

[13] The tight junction regulator according to any one of the above [2] to [8], which is used as a component of a quasi-drug composition.

[14] The tight junction regulator according to any one of [2] to [8] above, which is used as a component of a cosmetic composition.

[15] The tight junction regulator according to any one of [2] to [8] above, which is used as an ingredient of a food composition.

[16] A pharmaceutical composition comprising, as an active ingredient, the tight junction regulator according to any one of [2] to [8] above.

[17] A quasi-drug composition comprising, as an active ingredient, the tight junction regulator according to any one of [2] to [8] above.

[18] A cosmetic composition comprising, as an active ingredient, the tight junction regulator according to any one of [2] to [8] above.

[19] A food composition comprising, as an active ingredient, the tight junction regulator according to any one of [2] to [8] above. [Effects of the Invention]

[0009] The compounds represented by formulas (1) to (4) or pharmaceutically acceptable salts thereof can regulate tight junctions. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a photograph showing the results of immunostaining when 10 μM SAK, 10 μM ISO, and 100 μM NAR were added to cells in Example 1. [Figure 2] FIG. 2 shows photographs illustrating the results of Western blotting when 10 μM SAK, 10 μM ISO, and 100 μM NAR were added to cells in Example 1, and a graph illustrating the expression level of claudin-2 (CLD-2) protein. [Figure 3] FIG. 3 is a graph showing the expression level of CLD-2 mRNA when 10 μM SAK, 10 μM ISO, and 100 μM NAR were added to cells in Example 1. [Figure 4] Figure 4 shows photographs illustrating the results of Western blotting when varying concentrations of SAK, ISO, and NAR were added to cells in Example 1, and a graph illustrating the expression level of claudin-2 (CLD-2) protein. [Figure 5]FIG. 5 shows photographs illustrating the results of Western blotting when ERD was added to cells at varying concentrations in Example 1, and a graph illustrating the expression level of claudin-2 (CLD-2) protein. [Figure 6] FIG. 6 is a photograph showing the results of immunostaining when 100 μM SAK, 100 μM ISO, and 100 μM ERD were added to cells in Example 1. [Figure 7] FIG. 7 shows photographs illustrating the results of Western blotting when 100 μM SAK, 100 μM ISO, and 100 μM ERD were added to cells in Example 1, and a graph illustrating the expression level of claudin-2 (CLD-2) protein. [Figure 8] FIG. 8 is a graph showing the expression level of CLD-2 mRNA when 100 μM SAK, 100 μM ISO, and 100 μM ERD were added to cells in Example 1. [Figure 9] FIG. 9 is a graph showing the results of measuring the retention time of the extracted cherry bark extract by HPLC in Example 2. [Figure 10] FIG. 10 is a graph showing the results of measuring the retention time of the extracted Yerba Santa extract by HPLC in Example 2. [Figure 11] FIG. 11 is a photograph showing the results of immunostaining when the extracted cherry bark extract and Yerba Santa extract were added to cells in Example 2. [Figure 12] Figure 12 shows photographs illustrating the results of Western blotting when varying concentrations of extracted cherry bark extract and Yerba Santa extract were added to cells in Example 2, and a graph showing the expression level of claudin 2 (CLD-2) protein. [Figure 13] FIG. 13 is a graph showing the results of measuring the retention time of the cherry bark extract extracted by HPLC in Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0011]

[0023] Below, detailed descriptions are given of each embodiment of a tight junction regulator (hereinafter sometimes simply referred to as "regulator"), an absorption enhancer for a useful substance containing the regulator (hereinafter sometimes simply referred to as "absorption enhancer"), an absorption enhancer and leakage preventer for a useful substance containing the regulator (hereinafter sometimes simply referred to as "absorption enhancer and leakage preventer"), a pharmaceutical composition containing the regulator as an active ingredient (hereinafter sometimes simply referred to as "pharmaceutical composition"), a quasi-drug composition containing the regulator as an active ingredient (hereinafter sometimes simply referred to as "quasi-drug composition"), a cosmetic composition containing the regulator as an active ingredient (hereinafter sometimes simply referred to as "cosmetic composition"), and a food composition containing the regulator as an active ingredient (hereinafter sometimes simply referred to as "food composition").

[0012] The control agent according to the embodiment is At least one compound selected from the compounds represented by the following formulas (1) to (4), or At least one selected from pharmaceutically acceptable salts of compounds represented by the following formulas (1) to (4): It is characterized by containing as an active ingredient.

[0013] [ka]

[0014] The compound represented by the above formula (1) is called sakuranetin, a flavonoid found in cherry bark, rice leaves, propolis, etc. Sakuranetin is also known to promote adipocyte differentiation and glucose uptake into adipocytes.

[0015] The compound represented by the above formula (2) is called isosakuranetin, and is a flavonoid contained in citrus fruits such as oranges and grapefruits.

[0016] The compound represented by the above formula (3) is called naringenin, and is a flavonoid contained in citrus fruits such as oranges and grapefruits, tomatoes, and the like.

[0017] The compound represented by the above formula (4) is called eriodictyol, a type of flavonoid extracted from the plant Yerba Santa (Eriodictyon californicum, also known as Yerba Santa. Hereinafter, we will refer to it as Yerba Santa.) Eriodictyol is known to be a taste-modifying substance (a substance that has the effect of distorting the sense of taste).

[0018] In this specification, the term "pharmaceutically acceptable salt" refers to a salt that has the effectiveness of the above-mentioned compound and is not biologically harmful. For example, but not limited to, it refers to a salt formed by binding the above-mentioned compound with an acid or a base. The pharmaceutically acceptable salt may exist as various solvates, for example, with water, methanol, ethanol, dimethylformamide, etc.

[0019] Each of the compounds or pharmaceutically acceptable salts represented by the above formulas (1) to (3) (hereinafter, the compounds or pharmaceutically acceptable salts may be collectively referred to simply as "compounds") possesses both tight junction relaxation and tight junction strengthening functions, as shown in the examples described below. More specifically, when the compound concentration is low, it has the tight junction strengthening function, and when the compound concentration is high, it has the tight junction weakening function. In other words, each of the compounds represented by the above formulas (1) to (3) can be described as (a) a compound that has the tight junction strengthening function at a concentration lower than the tight junction weakening function, or (b) a compound that, when the tight junction function without the compound is set to 0, has the tight junction strengthening function (greater than 0) as the compound concentration is gradually increased, then becomes the same as the tight junction function without the compound despite containing the compound, and then has the tight junction weakening function (less than 0).

[0020] In addition, when a compound having a tight junction-relaxing function is used to relax tight junctions and allow a useful substance to be taken up by cells (in other words, taken up inside the body, which is a collection of cells; the following description of "taken up into cells" is similar and therefore omitted), if the tight junctions are left relaxed, the taken-up useful substance or intracellular substance may leak out of the cell (in other words, leak out to the outside of the body, which is a collection of cells; the following description of "leakage from cells" is similar and therefore omitted). Therefore, after the useful substance has been taken up into the cell, the relaxed tight junctions may optionally be strengthened again with a compound that strengthens (promotes the formation of) tight junctions. Examples of compounds that strengthen tight junctions are described in Patent Document 1.

[0021] However, when using the compound described in Patent Document 1, the compound that relaxes tight junctions and the compound that strengthens (promotes) tight junctions are different. Therefore, when relaxing and then strengthening tight junctions during the uptake of a useful substance, two types of compounds must be used.

[0022] On the other hand, the compounds (control agents) described in formulas (1) to (3) disclosed in the present application have both the function of loosening and strengthening tight junctions. Therefore, unlike Patent Document 1, a single compound can both loosen and strengthen tight junctions. Furthermore, the compounds (control agents) described in formulas (1) to (3) have a tight junction strengthening effect at a concentration lower than the tight junction strengthening effect. Therefore, for example, when a control agent is added to a shampoo together with a useful substance at a concentration sufficient to loosen tight junctions, (a) tight junctions are relaxed during hair washing, allowing the useful substance to be taken up by cells; (b) when the concentration of the compound contained in the control agent decreases during rinsing with shampoo after hair washing, the tight junctions are strengthened, thereby (c) preventing leakage of the useful substance and intracellular substances taken up by cells and preventing the entry of harmful substances from the outside.

[0023] Now, consider a compound (hereinafter referred to as "compound X") that strengthens tight junctions at a higher concentration than that that weakens them. Also consider the case where compound X is added to a pharmaceutical composition, quasi-drug composition, cosmetic composition, or food composition (hereinafter, these may be collectively referred to as "composition"). In this case, the useful substance must first be taken up by cells, and therefore the concentration of compound X is set to a concentration that weakens tight junctions. However, after tight junctions are weakened using a composition containing compound X at a concentration that weakens tight junctions, in order to strengthen tight junctions again, it is necessary to separately add compound X or another compound that strengthens tight junctions.

[0024] On the other hand, the compounds (control agents) disclosed in the present application represented by formulas (1) to (3) have a lower concentration of compound that strengthens tight junctions than the concentration that weakens tight junctions. Therefore, after tight junctions are weakened using a composition containing a compound (control agent) at a concentration that weakens tight junctions, tight junctions can be strengthened simply by diluting the composition. This improves the convenience of the composition for users.

[0025] The compound represented by formula (4) or a pharmaceutically acceptable salt thereof has a tight junction relaxing function, as will be shown in the Examples below.

[0026] As described above, all of the compounds represented by formulas (1) to (4) have a tight junction control function (relaxation function), and therefore the regulator according to the embodiment can be used as an absorption enhancer for useful substances.

[0027] Furthermore, the control agent containing the compound described in formulas (1) to (3) has the function of preventing useful substances from leaking out of cells after being taken up into the cells, and therefore can be used as an agent for promoting absorption of useful substances and preventing leakage.

[0028] Examples of dosage forms of the regulator, absorption enhancer, and absorption enhancer and leakage prevention agent include thin films, powders, pills, tablets, injections, suppositories, emulsions, capsules, granules, and liquids (including tinctures, liquid extracts, spirits, suspensions, and lemonades). Furthermore, various ingredients used in ordinary pharmaceuticals, quasi-drugs, cosmetics, and foods, such as oily ingredients, emulsifiers, moisturizers, thickeners, medicinal ingredients, antibiotics, antibacterial agents, hormones, supplements, preservatives, powders, pH adjusters, UV absorbers, and antioxidants, can be appropriately blended within the scope that does not impair the effects disclosed herein.

[0029] The regulator according to the embodiment can be used as a component of pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, food compositions, and the like.

[0030] In this specification, the terms "drugs," "quasi-drugs," and "cosmetics" are defined in accordance with the Pharmaceutical Affairs Act. More specifically, "drugs" refers to products intended for use in the diagnosis, treatment, or prevention of illness (disease). In this specification, "drugs" refers to pharmaceuticals for both humans and animals. Furthermore, "quasi-drugs" refers to products not used for active treatment, but used to prevent discomfort such as nausea, heat rash, and sores. They are also used for cosmetic purposes, such as preventing bad breath, body odor, and hair loss, as well as for hair growth and removal. They have a mild effect on the human body. Furthermore, "cosmetics" refers to products intended to cleanse, beautify, and enhance the attractiveness of the human body, alter appearance, or maintain healthy skin or hair. The above-mentioned regulations regarding "drugs," "quasi-drugs," and "cosmetics" are based on Japan's Act on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc. In countries other than Japan, the provisions of laws corresponding to or similar to the "Act on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc." Furthermore, "food" is not particularly limited as long as it is ingestible by humans or animals, and examples include fresh foods, animal foods, plant foods, fungal foods, processed foods, health foods, beverages, and seasonings. Furthermore, the term "useful substance" used herein refers to (1) the above-mentioned "drugs," "quasi-drugs," "cosmetics," and "foods" themselves, or compounds contained in the above-mentioned "drugs," "quasi-drugs," "cosmetics," and "foods" that exert their effects when taken up into cells, and (2) compounds not classified as the above-mentioned "drugs," "quasi-drugs," "cosmetics," or "foods" but that do not adversely affect the human body when taken up into cells.

[0031] When the regulator disclosed in the present specification is used as an absorption promoter or an absorption promoter and leakage preventive agent for a useful substance, it may be used in combination with the useful substance to be taken up into cells.

[0032] The "pharmaceutical composition" disclosed in the present specification may be, for example, a "drug" such as a transdermal drug, a transmucosal drug such as a nasal or enteral drug, an injectable drug, an ophthalmic drug, or an inhaled drug, to which the "control agent" disclosed in the present specification is added as an active ingredient.

[0033] The "pharmaceutical composition" disclosed in the present specification may be, for example, a "quasi-drug" such as a mouth freshener, medicated toothpaste, medicated soap, medicated cosmetic, antiperspirant spray, underarm odor suppressant, bath additive, medicated cream, baby powder, hair growth agent, hair dye, etc., to which the "control agent" disclosed in the present specification is added as an active ingredient.

[0034] The "cosmetic composition" disclosed in the present specification may be, for example, a "cosmetic" such as a basic cosmetic product such as emulsion, cream, lotion, essence, pack, or face wash; a makeup cosmetic product such as lipstick, foundation, liquid foundation, or pressed makeup powder; or a cleansing cosmetic product such as face wash, body shampoo, or soap; to which the "control agent" disclosed in the present specification is added as an active ingredient.

[0035] The "food composition" disclosed in the present specification may be, for example, a "food" such as the above-mentioned fresh foods, animal foods, plant foods, fungal foods, processed foods, health foods, beverages, seasonings, etc., to which the "control agent" disclosed in the present specification is added as an active ingredient.

[0036] The compounds represented by formulas (1) to (4) contained in the control agents according to the above-mentioned embodiments may be isolated or synthesized compounds contained as raw materials, may be contained as components in an extract extracted from a plant, or may be contained as a combination of compounds and extracts contained as raw materials.

[0037] As shown in the examples below, cherry bark extract contains compounds represented by formulas (1) to (3) as ingredients. In other words, it can be said that cherry bark extract contains at least one compound selected from the compounds represented by formulas (1) to (3). Therefore, for example, when a prescription lists one of the compounds represented by formulas (1) to (3) as an active ingredient, compounds represented by the other formulas may be optional additional ingredients. Of course, all of the compounds represented by formulas (1) to (3) may be active ingredients. Furthermore, as shown in the examples below, among the ingredients contained in cherry bark extract, the content of sakuranetin represented by formula (1) is higher than isosakuranetin represented by formula (2) and naringenin represented by formula (3). For example, in order to adjust the content of the active ingredient contained in the regulator, one or more compounds selected from the compounds represented by formulas (1) to (3) may be further added to the regulator in addition to the cherry bark extract.

[0038] Furthermore, Yerba Santa extract contains naringenin represented by formula (3). The regulator may contain only Yerba Santa extract, or one or more compounds selected from the compounds represented by formulas (1) to (3) may be further added to the regulator. Furthermore, the regulator according to the embodiment may contain cherry bark extract and Yerba Santa extract.

[0039] There are no particular limitations on the production method (extraction method) of cherry bark extract, as long as the extract contains the above-mentioned compounds. Although not limited, it is preferable to first perform a first extraction step on the raw cherry bark using water as the solvent, and then perform a second extraction step on the cherry bark residue after the first extraction step using methanol as the solvent. The above-mentioned compounds are extracted into the methanol solvent. As shown in the Examples and Comparative Examples described below, when the extraction step was performed directly from the raw cherry bark using methanol as the solvent without performing the first extraction step, almost no compounds represented by formulas (1) to (3) were extracted. On the other hand, when the first extraction step was first performed using water as the solvent and then the second extraction step was performed using methanol as the solvent, more compounds represented by formulas (1) to (3) could be extracted from cherry bark. In other words, the production method disclosed in the embodiments is a novel production method (extraction method).

[0040] In the production method (extraction method), the extract obtained in the second extraction step may optionally be subjected to a third extraction step using ethyl acetate and water as solvents. By performing the third extraction step, some of the unnecessary compounds other than the target compounds represented by formulas (1) to (3) are transferred to the water, thereby reducing the concentration of impurities contained in the cherry bark extract.

[0041] Although detailed description will be omitted, the Yerba Santa extract can be produced by the same procedure as the above-mentioned method for producing cherry bark extract, except that Yerba Santa is used as the raw material.

[0042] Cherry bark extract is known as an antitussive and expectorant, and Yamazakura bark extract, which is considered to be equivalent to cherry bark, is known as a cosmetic ingredient. Yerba Santa extract is also known as a cosmetic ingredient. Because products using cherry bark extract and Yerba Santa extract are already on the market, safety tests have already been conducted. Therefore, using cherry bark extract and / or Yerba Santa extract in the regulator according to the embodiment has the effect of shortening the time required for market launch.

[0043] The following examples are provided to specifically explain each embodiment, but these examples are provided merely as a reference for specific aspects and are not intended to limit or restrict the scope of the invention. [Example]

[0044] Example 1 [Reagents and Experimental Procedures] (1) Compound The compounds used were those represented by the formulas (1) to (4). The compounds represented by formulas (1), (2), and (4) were purchased from Funakoshi Co., Ltd. The compound represented by formula (3) was purchased from Tokyo Chemical Industry Co., Ltd. In the following, sakuranetin represented by formula (1) may be abbreviated as "SAK," isosakuranetin represented by formula (2) as "ISO," naringenin represented by formula (3) as "NAR," and eriodictyol represented by formula (4) as "ERD." Similar abbreviations are used in the drawings.

[0045] (2) Cell culture The cells used were the canine kidney tubular epithelial cell line MDCKII (Mardin-Darby Canine Kidney Cell Strain II, ECACC: Catalogue number 00062107), which was cultured in D-MEM medium (Waco) containing 10% FBS (Corning Japan) and 1% penicillin / streptomycin (Corning Japan).

[0046] (3) Antibodies and reagents The primary antibody used in immunostaining, rabbit anti-CLD2 antibody, was purchased from Sigma-Aldrich Co. The secondary antibody, Cy3-labeled anti-rabbit IgG antibody, was purchased from Sigma-Aldrich Co. The primary antibody used in Western blotting, rabbit anti-CLD2 antibody, was purchased from Sigma-Aldrich. The mouse anti-β-actin antibody was purchased from Wako Pure Chemical Industries, Ltd. The secondary antibody, HRP-labeled anti-rabbit IgG antibody, was purchased from Promega. The HRP-labeled anti-mouse IgG antibody was also purchased from Promega.

[0047] (4) Immunostaining of cells Place a cover glass in each well of a 6-well dish and add 30x10 4MDCKII cells were seeded and incubated at 37°C in a 5% CO2 environment for 24 hours. Then, SAK of formula (1) and ISO of formula (2) were mixed in D-MEM medium to a concentration of 10 μM or 100 μM with a DMSO concentration of 0.1%, and then incubated at 37°C in a 5% CO2 environment for 48 hours. NAR of formula (3) and ERD of formula (4) were added to the cells to a concentration of 100 μM with a DMSO concentration of 0.1%, and then incubated at 37°C in a 5% CO2 environment for 48 hours. After incubation, the coverslips were washed twice with D-MEM medium without FBS or antibiotics. Then, cells were fixed on the coverslips by adding 4% paraformaldehyde in phosphate buffer (Nacalai Tesque) and incubating for 10 minutes at room temperature. After washing once with PBS, permeabilization solution (0.5% Triton 20 in PBS) was added and incubated for 5 minutes at room temperature. After washing three times with PBS-T (0.1% Tween 20 in PBS), blocking solution (3% BSA in PBS-T) was added and incubated for 1 hour at room temperature. A diluted primary antibody solution in blocking solution was then placed on parafilm, and a coverslip was placed on top with the cell fixation side facing the coverslip. The coverslips were then incubated overnight at 4°C in a humid environment. The coverslips were then removed and washed twice with PBS-T. A diluted secondary antibody solution in blocking solution was then placed on parafilm, and a coverslip was placed on top with the cell fixation side facing the coverslip. The coverslips were then incubated for 1 hour at room temperature in a dark environment. The coverslips were then removed and washed twice with PBS-T. After that, DAPI (4',6-diamidino-2-phenylindole dihydrochloride) solution (DOJINDO, (1:1000)) diluted in PBS-T was applied and incubated for 5 minutes at room temperature in the dark. After washing three times with PBS-T, the coverslips were mounted with mounting medium. These samples were observed under a microscope using an Olympus IX71 microscope. Cells exposed to 0.1% DMSO were used as a control.

[0048] (5) Western blotting Place a cover glass in each well of a 6-well dish and add 30x10 4MDCKII cells were seeded and incubated at 37°C, 5% CO2 for 24 hours. SAK (1) and ISO (2) were then mixed in D-MEM medium to concentrations of 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM, with DMSO at a concentration of 0.1%. The mixture was added to the cells and incubated at 37°C, 5% CO2 for 48 hours. NAR (3) was mixed at concentrations of 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM, and ERD (4) was mixed at concentrations of 1 μM, 10 μM, 50 μM, 75 μM, and 100 μM. After incubation, the cells were washed twice with D-MEM medium without FBS or antibiotics, and then 100 μL of SDS-sample buffer was added. The cells were scraped with a cell scraper (IWAKI). The scraped cells were sonicated (on time 30 sec / off time 30 sec, total 3 min) using a Cosmo Bio Bioruptor to prepare cell extract samples. The collected cell extract samples were loaded onto a 15% polyacrylamide gel and electrophoresed for 30 minutes. After electrophoresis, the gel was recovered and immersed in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). A PVDF (Polyvinylidene difluoride) membrane (ATTO) and filter paper were then permeated with the transfer buffer. The sample proteins were transferred to the PVDF membrane at 30 V, 24 mA, for 1 hour, after which the PVDF membrane was permeated with blocking solution (5% skim milk in TBS-T) for 1 hour. The PVDF membrane was then washed three times with TBS-T and then immersed in a diluted primary antibody solution diluted in blocking solution at 4°C overnight. The PVDF membrane was then washed three times with TBS-T and then immersed in a diluted secondary antibody solution diluted in blocking solution at room temperature for 1 hour. The PVDF membrane was then washed three times with TBS-T and incubated for 1 minute with Chemi-Lumi One Super (Nacalai Tesque). Proteins transferred to the PVDF membrane were detected using ATTO's Lumino Graph 1. A cell extract sample exposed to 0.1% DMSO was used as a control.

[0049] (6) PCR primers The primers were prepared by Hokkaido System Science Co., Ltd. <cld-2> Forward Primer: 5'-CGCTCCGACTACTATGACTCCT-3' (SEQ ID NO: 1) Reverse Primer: 5'-GGCCTTGGAGAGCCTCTAGT-3' (SEQ ID NO: 2) <gapdh> Forward Primer: 5'-CAACTCCCTCAAGATTGTCAGCAA-3 (SEQ ID NO: 3) Reverse Primer: 5'-CATGGATGACTTTGGCTAGAGGA-3 (SEQ ID NO: 4)

[0050] (7) q-PCR Place a cover glass in each well of a 6-well dish and place 30x10 4 MDCKII cells were seeded and incubated at 37°C in a 5% CO2 environment for 24 hours. SAK of formula (1) and ISO of formula (2) were then mixed in D-MEM medium to concentrations of 10 μM and 100 μM, with a DMSO concentration of 0.1%, and added to the cells. The mixture was then incubated at 37°C in a 5% CO2 environment for 48 hours. NAR of formula (3) and ERD of formula (4) were added to the cells to concentrations of 100 μM and 0.1% DMSO, and the mixture was then incubated at 37°C in a 5% CO2 environment for 48 hours. RNA purification was performed using Qiagen's RNeasy Plus Mini Kit. After incubation, the cells were washed twice with PBS and then scraped with a cell scraper (IWAKI) after adding 600 μL of RLT buffer (containing 10 μL / 1 mL β-mercaptoethanol). The scraped cells were sonicated for 30 seconds using a Cosmo Bio Bioruptor and then mixed with 600 μL of 70% ethanol. This mixture was added to a 2 mL collection tube with a gDNA Eliminator spin column and centrifuged at 8000 x g for 15 seconds. The flow-through was discarded. Next, 350 μL of RW1 buffer was added and centrifuged at 8000 x g for 15 seconds. The flow-through was discarded. Next, 10 μL of DNase and 70 μL of RDD buffer were added to the spin column and allowed to stand at room temperature for 15 minutes. Next, 350 μL of RW1 buffer was added and centrifuged at 8000 x g for 15 seconds. The flow-through was discarded. Next, 500 μL of RPE buffer was added and the mixture was centrifuged at 8000 × g for 15 seconds. After discarding the flow-through, the mixture was centrifuged again at 8000 × g for 2 minutes. The 2 mL collection tube was then replaced with a new one and centrifuged at 8000 × g for 1 minute. The mixture was then replaced with a 1.5 mL collection tube, 30 μL of RNase-free water was added, and the mixture was centrifuged at 8000 × g for 1 minute. This was used as the RNA sample. The purified RNA sample was adjusted to 100 ng / μL. Then, 1 μL of the RNA sample was mixed with 2 μL of 5x RT Master Mix (Toyobo Co., Ltd.) and 7 μL of nuclease-free water to make a total volume of 10 μL. The adjusted solution was used for reverse transcription. The reaction time and temperature are shown below. 37℃, 15 minutes 50℃, 5 minutes 98℃, 5 minutes 15℃, hold

[0051] A reaction mixture was prepared using the cDNA obtained by reverse transcription. The reaction mixture was prepared by mixing 100 ng of cDNA with 10 μL of THUNDERBIRD SYBR qPCR MIX (Toyobo Co., Ltd.), 6 pmol of forward primer, and 1 reverse primer, and adjusting the total volume to 20 μL with RNase-free water. The reaction mixture was then used to perform a qPCR reaction. The temperature and time are shown below. (a) 95°C, 1 minute (b)95℃, 10 seconds (c) 60℃, 30 seconds The above (a) → (c) was repeated 45 cycles. As a control, a cell extract sample exposed to 0.1% DMSO was used.

[0052] [Experimental Results] Figure 1 shows the results of immunostaining for 10 μM SAK, 10 μM ISO, and 100 μM NAR. Figure 2 shows the results of Western blot analysis for 10 μM SAK, 10 μM ISO, and 100 μM NAR. Figure 3 shows the results of q-PCR analysis for 10 μM SAK, 10 μM ISO, and 100 μM NAR. Figure 4 shows the results of Western blot analysis for varying concentrations of SAK, ISO, and NAR. Figure 5 shows the results of Western blot analysis for varying concentrations of ERD. Figure 6 shows the results of immunostaining for 100 μM SAK, 100 μM ISO, and 100 μM ERD. Figure 7 shows the results of Western blot analysis for 100 μM SAK, 100 μM ISO, and 100 μM ERD. Figure 8 shows the results of q-PCR analysis for 100 μM SAK, 100 μM ISO, and 100 μM ERD.

[0053] As shown in Figure 1, the white areas between cells were significantly darker with 10 μM SAK, 10 μM ISO, and 100 μM NAR than with the control DMSO, indicating strengthening of tight junctions. Therefore, we investigated the expression levels of claudin-2 (CLD-2) protein, a major component of tight junctions, and CLD-2 mRNA. As shown in Figures 2 and 3, the expression levels of CLD-2 protein and mRNA were both higher with 10 μM SAK, 10 μM ISO, and 100 μM NAR than with DMSO.

[0054] Next, as is clear from Figure 4, SAK, ISO, and NAR have the ability to relax and strengthen tight junctions, and it was confirmed that the tight junction strengthening effect was obtained at a lower concentration than the tight junction relaxation effect. On the other hand, as is clear from Figure 5, as for ERD, the tight junction relaxation effect was observed at higher concentrations, but the tight junction strengthening effect was not observed.

[0055] Furthermore, as shown in Figure 6, the white areas between cells were clearly thinner in the 100 μM SAK, 100 μM ISO, and 100 μM ERD treatments than in the control DMSO, indicating that tight junctions were relaxed. Therefore, we investigated the expression levels of claudin-2 (CLD-2) protein, a major component of tight junctions, and CLD-2 mRNA. As shown in Figures 7 and 8, the expression levels of CLD-2 protein and mRNA were both lower at concentrations of 100 μM SAK, 100 μM ISO, and 100 μM ERD than at DMSO.

[0056] <Example 2> (1) Cherry bark extract Cherry bark, "Tochimoto no Ohi," was purchased from Tochimoto Tenkaido Co., Ltd. and ground to powder using a coffee mill. 15 mL of Milli-Q water was added to 1 g of cherry bark and stirred at room temperature for 8 hours. After removing the water extract, 15 mL of Milli-Q water was added to the cherry bark residue and stirred for an additional 16 hours. After removing the extract, 15 mL of methanol was added to the cherry bark residue and stirred at room temperature for 8 hours. After removing the methanol extract, 15 mL of methanol was added to the residue and stirred for an additional 16 hours. 30 mL of the methanol extract from 24 hours was mixed and distilled under reduced pressure using a rotary evaporator (EYELA). Half of the resulting solid was dried under reduced pressure to obtain the methanol extract. 20 mL of ethyl acetate and 20 mL of Milli-Q water were added to the remaining half of the solid and separated using a separatory funnel. The ethyl acetate layer and aqueous layer were concentrated using a rotary evaporator and dried under reduced pressure to obtain the ethyl acetate extract and aqueous extract, respectively.

[0057] (2) Extraction of Yerba Santa extract Yerba Santa extract was extracted in the same manner as in "(1) Extraction of cherry bark extract" above, except that "Yerba Santa" purchased from Natural Rhythm, a herbal tea store run by Uprising Co., Ltd., was used instead of cherry bark, to obtain a methanol extract and an ethyl acetate extract.

[0058] (3) Confirmation of ingredients contained in cherry bark extract and Yerba Santa extract The dried powders of the extracts (methanol extract and ethyl acetate extract) obtained in (1) and (2) above were dissolved in 10 μL of d6-DMSO, followed by the addition of 1 mL of 20% acetonitrile. Trifluoroacetic acid (TFA) was added to a final concentration of 0.1%, and the solution was passed through a syringe filter (Minisart, pore size: 0.45 μm (SARTORIUS)) to prepare a sample for HPLC. The sample was passed through an ODS column (Inertsil®) connected to a Primaide HPLC system (Hitachi) and separated and eluted using a solvent: 0.5% TFA in HO, 0.5% TFA in 80% CH3CN, gradient: 1.375% / min (17.5% to 72.5%). Additionally, authentic samples of the compounds represented by formulas (1) to (3) (obtained from the same source as in Example 1) believed to be contained in the extract were dissolved in the same manner as the extract, and their retention times were confirmed by HPLC. For comparison, the dried powder of the extract extracted with water before the methanol extraction was used as the water extract, and the retention time was confirmed by HPLC in the same manner as the above-mentioned methanol extract and ethyl acetate extract.

[0059] Figure 9 shows the results for the cherry bark extract. A indicates the aqueous extract (before methanol extraction), B indicates the methanol extract, and C indicates the ethyl acetate extract. Of the numbers assigned to the peaks in Figure 9C, comparison with the retention times of authentic samples confirmed that 3 corresponds to naringenin, 4 to sakuranetin, and 5 to isosakuranetin. As is clear from comparing the peaks in Figure 9A with those in B and C, the naringenin and isosakuranetin peaks were barely visible in the aqueous extract (A), whereas the naringenin and isosakuranetin peaks were larger in the methanol extract (B) and ethyl acetate extract (C). While sakuranetin was detected in the aqueous extract (A), the amount of sakuranetin in the methanol extract (B) and ethyl acetate extract (C) was significantly greater.

[0060] Furthermore, when comparing the methanol extract (B) with the ethyl acetate extract (C), the contents of naringenin, sakuranetin, and isosakuranetin were not significantly different, suggesting that most of these compounds were transferred from the methanol extract to the ethyl acetate extract. Meanwhile, the peak number 1 and the peak at approximately 6 minutes of elution were smaller in the ethyl acetate extract (C) than in the methanol extract (B). These results confirm that performing an ethyl acetate extraction process on the methanol extract can reduce the content of unwanted compounds other than those represented by formulas (1) to (3) disclosed in the present application; in other words, it can increase the proportion of compounds represented by formulas (1) to (3) among the total compounds in the cherry bark extract. The sakuranetin content calculated from the HPLC peak area was 1.98% in the aqueous extract (A) and 22.11% in the ethyl acetate extract (C).

[0061] Figure 10 shows the results for the Yerba Santa extract. A indicates the water extract (before methanol extraction), B indicates the methanol extract, and C indicates the ethyl acetate extract. Of the numbers assigned to the peaks in Figure 10C, comparison with the retention times of authentic samples confirmed that 3 is the naringenin peak. As is clear from Figures 10A-C, no naringenin peak was observed in the water extract (A), but peaks were observed in the methanol extract (B) and ethyl acetate extract (C). Furthermore, comparison of the methanol extract (B) with the ethyl acetate extract (C) confirmed that the peaks at elution times of approximately 8 to 27 minutes were smaller.

[0062] (4) Cell experiments Cell experiments were conducted using ethyl acetate extracts of cherry bark extract and Yerba Santa extract (hereinafter referred to simply as "extract"). The extract concentration was standardized based on the total amount of flavonoids contained in the extract, which has a molecular weight of 302 for quercetin (quercetin equivalent concentration). The standardization formula is shown below. JPEG2025158954000006.jpg29168

[0063] The experiment was carried out in the same manner as in "(2) Cell culture" to "(5) Western blotting" in Example 1 above, except that ethyl acetate extract was added at a d6-DMSO concentration of 0.1% to achieve the concentrations shown in Figures 11 and 12 described below.

[0064] The results of immunostaining are shown in Figure 11. In the photographs of cells exposed to 25 μM cherry bark extract and 10 μM Yerba Santa extract, the white areas between cells clearly became darker, confirming increased localization of CLD-2 at cell adhesion sites. Furthermore, exposure to 100 μM Yerba Santa extract revealed weak fluorescence of the CLD-2 antibody in the nucleus. This is thought to be the result of the antibody accumulating in the nucleus with nowhere to go. On the other hand, with 100 μM cherry bark extract, a pattern of CLD-2 localization was observed that was relatively similar to that of the control.

[0065] Figure 12 shows the Western blot results and graphs based on the results. Figure 12B shows the results for cherry bark extract, and Figure 12C shows the results for Yerba Santa extract. Both graphs have an n of 5. As can be seen from Figures 12B and 12C, after 48 hours of exposure to cherry bark extract and Yerba Santa extract, the CLD-2 protein levels increased compared to the control at concentrations of 10 to 75 μM and 10 to 50 μM. With increasing concentrations of cherry bark extract, the CLD-2 levels gradually decreased, reaching levels nearly equal to those of the control at 100 μM. The concentrations shown in Figure 12B are different from those shown in Figure 4 and represent the concentrations of flavonoids contained in cherry bark extract. As mentioned above, the sakuranetin content of the ethyl acetate extract (C) was 22.11%, which means that the sakuranetin content of the 100 μM sample in Figure 12B is 22.11 μM. The ethyl acetate extract (C) contains naringenin and isosakuranetin in addition to sakuranetin. Furthermore, based on the experimental results of varying the concentration of compounds shown in Figure 4, it is clear that increasing the concentration of cherry bark extract beyond 100 μM reduces the amount of CLD-2 compared to the control. Meanwhile, with Yerba Santa extract, the amount of CLD-2 decreased sharply from 75 μM onwards, and at 100 μM, the amount of CLD-2 was lower than the control. Furthermore, at 25 μM with cherry bark extract and 10 μM with Yerba Santa extract, the amount of LD-2 was approximately double that of the control.

[0066] These results confirm that cherry bark extract and Yerba Santa extract both relax and strengthen tight junctions, depending on the concentration. The cell experiments described above were conducted using the ethyl acetate extract (C). However, as is clear from the results shown in Figure 9, the methanol extract of cherry bark (B) contains approximately the same amounts of sakuranetin, naringenin, and isosakuranetin as the ethyl acetate extract (C). Furthermore, as is clear from the results shown in Figure 10, the methanol extract of Yerba Santa (B) contains approximately the same amount of naringenin as the ethyl acetate extract (C). Therefore, it is clear that similar results to those shown in Figures 11 and 12 would be obtained if cell experiments were conducted using the methanol extract (B) of cherry bark extract and Yerba Santa extract instead of the ethyl acetate extract (C).

[0067] <Comparative Example 1> When preparing cherry bark extract, the first extraction step using water as the solvent was omitted, and the second extraction step was directly performed using methanol as the solvent. Specifically, a methanol extract and an ethyl acetate extract were obtained using the same procedure as in "(1) Extraction of Cherry Bark Extract" in Example 2 above, except that the step of adding Milli-Q water and stirring was omitted. Next, the obtained methanol extract and ethyl acetate extract were used to check their HPLC retention times using the same procedure as in "(3) Confirmation of Components Contained in Cherry Bark Extract and Yerba Santa Extract" in Example 2. For comparison, the HPLC retention times of the aqueous layer obtained when separating the ethyl acetate extract were also checked using the same procedure.

[0068] The results are shown in Figure 13. A shows the methanol extract, B shows the ethyl acetate extract, and C shows the result of the aqueous layer during separation. The peaks indicated by ▼ in Figure 13A and B are the peaks of sakuranetin, based on a comparison with the retention time of the authentic sample. From these results, it was confirmed that the production method disclosed in this application can effectively extract an extract containing components that regulate tight junctions from cherry bark. [Industrial Applicability]

[0069] The compounds represented by formulas (1) to (4) disclosed in the present application have the function of relaxing tight junctions. Furthermore, the compounds represented by formulas (1) to (3), as well as cherry bark extract and Yerba Santa extract, have both the function of relaxing and strengthening tight junctions. Therefore, tight junction regulators containing the compounds represented by formulas (1) to (4) and cherry bark extract and Yerba Santa extract can be used as absorption enhancers for useful substances, agents for enhancing absorption and preventing leakage of useful substances, pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, and food compositions, and are therefore useful in the medical, cosmetic, and food industries.< / gapdh>

Claims

1. At least one compound selected from the compounds represented by the following formulas (1) to (4), or At least one selected from pharmaceutically acceptable salts of compounds represented by the following formulas (1) to (4): A tight junction regulator comprising as an active ingredient. 【Chemical 1】

2. 2. The tight junction regulator according to claim 1, wherein the compound or pharmaceutically acceptable salt thereof represented by any one of formulas (1) to (3) has a tight junction relaxing function and a tight junction strengthening function.

3. the tight junction regulator comprises cherry bark extract, At least one of the compounds represented by formulas (1) to (3) is contained as a component in the cherry bark extract. The tight junction regulator according to claim 2.

4. the tight junction regulator comprises Yerba Santa extract; The compound represented by formula (3) is contained as a component in the Yerba Santa extract. The tight junction regulator according to claim 2.

5. The tight junction regulator according to claim 2 , wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function.

6. The tight junction regulator according to claim 3 , wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function.

7. The tight junction regulator according to claim 4, wherein the tight junction strengthening function is exhibited at a lower concentration than the tight junction relaxing function.

8. The tight junction regulator according to claim 1, wherein the compound represented by formula (4) or a pharmaceutically acceptable salt thereof has a tight junction relaxing function.

9. An absorption enhancer for a useful substance, comprising the tight junction regulator according to any one of claims 2 to 8 as an active ingredient.

10. An agent for promoting absorption and preventing leakage of useful substances, comprising the tight junction regulator according to any one of claims 2 to 7 as an active ingredient.

11. The tight junction regulator according to any one of claims 2 to 8, further comprising a compound that is absorbed by relaxing tight junctions.

12. The tight junction regulator according to any one of claims 2 to 8, which is used as a component of a pharmaceutical composition.

13. The tight junction regulator according to any one of claims 2 to 8, which is used as a component of a quasi-drug composition.

14. The tight junction regulator according to any one of claims 2 to 8, which is used as a component of a cosmetic composition.

15. The tight junction regulator according to any one of claims 2 to 8, which is used as an ingredient of a food composition.

Images