Composition for promoting tight junction formation

Abstract

To provide a composition for promoting tight junction formation that promotes formation of tight junctions between epithelial tissue cells.SOLUTION: A composition for promoting tight junction formation for promoting formation of tight junctions between epithelial tissue cells, contains triterpenes and skim milk and / or wheat gluten, thereby solving the problem to be solved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting tight junction formation. [Background technology]

[0002] The human body maintains an optimal internal environment by covering the surface of each organ with a layer of cells with barrier function. The barrier of the cell layer is formed by cells binding to each other via adhesion devices called tight junctions, which surround the cells in a belt-like fashion (Non-Patent Document 2). The intestinal tract is an organ that absorbs orally ingested substances, and tight junctions expressed in intestinal epithelial cells prevent the entry of harmful foreign substances. Under normal conditions, intestinal tight junctions physically prevent the entry of harmful foreign substances, pathogenic bacteria, allergens, etc., but if they are disrupted, they can penetrate. A condition in which intestinal tight junctions are impaired is called leaky gut, and can cause various diseases. Therefore, substances that promote the formation of tight junctions are needed. Tight junctions are intercellular adhesion structures that prevent substances from passing freely through gaps between cells in epithelial tissues, which are the cell layers on the surface of the body, including the intestinal tract, skin, respiratory, digestive, and urinary organs, and the inner surface of body cavities such as vascular endothelium. For example, skin is a structure that covers the surface of the body of vertebrates and functions as a boundary between the body and the outside world. From the outside, skin consists of the epidermis, dermis, and subcutaneous tissue. The epidermis is made up of four layers: the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. Cells in the granular layer are called SG1, SG2, and SG3 cells, respectively, and tight junctions are formed in SG2 cells. The skin barrier function provided by tight junctions has two functions: one is to restrict the movement of water from the inside to the outside of the body and retain moisture, and the other is to protect against the invasion of pathogens and allergens from the outside world. Thus, tight junctions in epithelial cells are important as a barrier to prevent the invasion of foreign substances, and it is desirable to protect their formation. As tight junction formation promoters, Patent Document 3 discloses a composition containing glucooligosaccharide as an active ingredient, and Patent Document 4 discloses sodium dl-α-tocopheryl phosphate and its derivatives. Triterpenes are a group of compounds widely distributed in the plant kingdom and are classified into oleanane-type triterpenes, ursane-type triterpenes, lupane-type triterpenes, hopane-type triterpenes, etc. Plant-derived triterpenes have recently attracted attention as functional components with physiological activities. R1-barrigenol, a type of oleanane triterpene, and its derivatives such as glycosides and acylated derivatives are natural components found in tea flowers and Pittosporum tobira leaves, and are known to have antibacterial activity (Non-Patent Document 1), oxytocin receptor activation activity (Patent Document 1), and microglial activation inhibitory effect (Patent Document 2). However, there have been no reports that triterpenes are involved in promoting the formation of tight junctions. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-072837 [Patent Document 2] Japanese Patent Publication No. 2022-183626 [Patent Document 3] Japanese Patent Publication No. 2020-002096 [Patent Document 4] Japanese Patent Application Laid-Open No. 2007-210948 [Non-patent literature]

[0004] [Non-Patent Document 1] Oh JH, et al. Molecules. 19(3):3607-3616, 2014 Published 2014 Mar 24. doi:10.3390 / molecules19033607 [Non-patent document 2] Nakamura S et al. Nat Commun 10, 816 (2019). https: / / doi.org / 10.1038 / s41467-019-08760-7 [Non-patent document 3] Srinivasan B et al. J Lab Autom, 20(2), 107-126 (2015) Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a composition for promoting tight junction formation, which promotes the formation of tight junctions between epithelial tissue cells. [Means for solving the problem]

[0006] The present invention is as follows. [1] A composition for promoting tight junction formation, which contains triterpenes and skim milk and / or wheat gluten, and is used to promote the formation of tight junctions between epithelial tissue cells. [2] The triterpene is R1-valigenol of the following formula I: Formula I TIFF2025183783000001.tif6072, composition of [1]. [3] The composition according to [1] or [2], wherein the epithelial tissue cells are intestinal epithelial cells. [4] The composition according to any one of [1] to [3], wherein the mass of the skim milk is 4 or more when the mass of the triterpenes is taken as 1. [5] The composition according to any one of [1] to [4], wherein the mass of wheat gluten is 4 or more when the mass of triterpenes is taken as 1. [6] A food product for promoting the formation of tight junctions between epithelial tissue cells, comprising the composition according to any one of [1] to [5]. [7] A food product according to [6] for the prevention, alleviation, or treatment of symptoms or diseases through promoting the formation of tight junctions between epithelial tissue cells. [8] A pharmaceutical composition for promoting the formation of tight junctions between epithelial tissue cells, comprising the composition according to any one of [1] to [5]. [9] The pharmaceutical composition according to [8] for preventing, alleviating, or treating symptoms or diseases through promoting the formation of tight junctions between epithelial tissue cells.

[10] A method for promoting the formation of tight junctions using the composition according to any one of [1] to [5]. [Effects of the Invention]

[0007] The composition containing the triterpenes of the present invention and skim milk and / or wheat gluten has the effect of promoting tight junction formation, and can provide a composition for promoting tight junction formation that is highly effective in preventing the invasion of allergens, bacteria, and microorganisms into the gastrointestinal mucosa and epithelial tissue. DETAILED DESCRIPTION OF THE INVENTION

[0008] The composition for promoting tight junction formation of the present invention contains triterpenes and skim milk and / or wheat gluten. In the present invention, triterpenes are compounds with a basic skeleton of 30 carbon atoms, and a representative example thereof is a pentacyclic triterpene. Here, a pentacyclic triterpene is a type of triterpene, a five-ring compound consisting of six isoprene units, and basically has 30 carbon atoms, but also includes those with an increased or decreased carbon number due to rearrangement, oxidation, elimination, or alkylation during the biosynthesis process. These compounds can be obtained by extraction and purification from natural plants using known methods, or by artificially obtaining them through chemical synthesis or the like. Commercially available products can also be suitably used. Pentacyclic triterpenes are generally classified according to their skeletons. Examples include oleanane-type triterpenes, ursane-type triterpenes, lupane-type triterpenes, hopane-type triterpenes, selatan-type triterpenes, friedelane-type triterpenes, taraxerane-type triterpenes, taraxastan-type triterpenes, multiflorane-type triterpenes, and germanican-type triterpenes. Among the pentacyclic triterpenes, representative examples of oleanane-type triterpenes include R1-valigenol, maslinic acid, and oleanolic acid. The triterpenes may be used as a single component or a mixture of several components, and may also be used in the form of pharmacologically acceptable salts and / or derivatives.

[0009] In the present invention, the triterpene is preferably an oleanane-type triterpene, more preferably R1-valigenol of the following formula I: Formula I The file is TIFF2025183783000002.tif6072.

[0010] R1-valigenol is a type of oleanane triterpene and the aglycone of saponin found in tea. It is also known to have antibacterial activity (Non-Patent Document 1), oxytocin receptor activation activity (Patent Document 1), and microglial activation inhibitory effect (Patent Document 2). The R1-valigenol used in the present invention does not exhibit its effects in the form of tea saponin, which is a glycoside; it must be in the aglycone form, obtained by separating the sugar moiety from the glycoside by enzymatic reaction, acid hydrolysis, or the like.

[0011] In the present invention, R1-valigenol can be purified from an extract obtained by extraction from the flowers and leaves of tea, which are naturally occurring materials. Tea is the tea plant (scientific name: Camellia sinensis) of the Theaceae family, Camellia genus. For example, tea flowers or leaves can be used as the extract sample. In the present invention, there are no particular limitations on the storage state, crushing method, variety, or place of origin of these extract samples.

[0012] The extract can be obtained by an extraction method known to those skilled in the art, such as organic solvent extraction, supercritical fluid extraction, heated and pressurized extraction, etc. These extraction methods may be used alone or in combination. In the present invention, a particularly preferred extraction method is organic solvent extraction. Organic solvent extraction can be performed using a common technique known to those skilled in the art. Specifically, water and an organic solvent are first added to a pulverized or dried fragmented sample, and the mixture is extracted at room temperature or with heating. Examples of extraction solvents include, but are not limited to, ethanol, methanol, ethyl acetate, acetone, and water. These solvents may be used alone or in combination. Ethanol is preferred from the viewpoint of safety in use with food, etc. The mass of the solvent to be added is preferably 1 to 100 times, more preferably 5 to 50 times, the mass of the sample to be extracted; for example, 10 times the mass may be used. One example of an extraction method involves mixing tea flowers or leaves with the extraction solvent and stirring for 1 to 5 hours at room temperature or refluxing for 1 to 5 hours at the boiling temperature of the extraction solvent. The extract is then filtered or centrifuged to remove sample residue, and the extract is concentrated by vacuum or ultrafiltration. This is followed by acid or alkali hydrolysis. The extract can also be dried by standard methods such as drying, freeze-drying, or spray-drying. R1-valigenol can be purified from the extract obtained as described above by a combination of appropriate separation and purification methods, such as liquid-liquid partitioning, organic solvent precipitation, various column chromatography methods (e.g., HPLC, silica gel chromatography, molecular sieve chromatography, ion exchange chromatography, reversed-phase chromatography, etc.), and crystallization.

[0013] In the present invention, skim milk refers to skim milk powder obtained by removing milk fat from whole milk and then removing almost all of the water from the skim milk to form a powder, but it may also be in the form of concentrated skim milk obtained by concentrating skim milk or skim milk liquid obtained by dissolving skim milk powder in water. It may also be semi-skim milk obtained by removing at least a portion of the milk fat from whole milk.

[0014] Wheat gluten is a so-called wheat protein concentrate, which is made by kneading wheat flour and water to form a viscoelastic dough with developed gluten (a type of protein produced in wheat endosperm, glutenin and gliadin, which absorb water and form a network).The dough is then washed to remove soluble components such as starch, resulting in a higher wheat protein content.The wheat gluten commonly used in the food manufacturing industry is concentrated to about 70-95% wheat protein, and is often in the form of a powder. The method for producing wheat gluten in the present invention can be any method commonly used for extracting gluten from wheat flour. For example, 55 parts by weight of water is added to 100 parts by weight of wheat flour and kneaded. The resulting dough ball is then left in a 30°C water bath for 20 minutes, then washed in water to remove starch, yielding a wheat protein concentrate. The resulting raw wheat protein concentrate (raw wheat gluten) is then dried in a vacuum dryer and pulverized to a powder, yielding a dried wheat protein concentrate (wheat gluten) with a protein content of 70% or more. Gluten dried at low temperatures to preserve the properties of raw gluten is sometimes referred to as vital gluten. However, there is no particular limitation on the wheat gluten used in the present invention, and vital gluten may also be used. Commercially available products may also be used.

[0015] In the composition for promoting tight junction formation of the present invention, the mass of skim milk and / or wheat gluten is 4 or more relative to the mass of triterpenes, assuming that the mass is 1. As the mass of wheat gluten relative to the mass of triterpenes increases, tight junction formation tends to be more strongly promoted, and there is no particular upper limit, but from the economical standpoint and in consideration of the dosage limit of the composition for promoting tight junction formation, preferably the mass of skim milk and / or wheat gluten is 1,000 or less relative to the mass of triterpenes. More preferably, the mass of skim milk and / or wheat gluten is 4 to 950, even more preferably 40 to 850, and even more preferably 400 to 800 relative to the mass of triterpenes.

[0016] In the present invention, tight junctions are intercellular adhesion structures that prevent substances from passing freely through gaps between cells in epithelial tissues, which are cell layers on the surfaces of the body, such as the skin, respiratory tract, digestive tract, and urinary tract, and on the inner surfaces of body cavities, such as vascular endothelium. The composition for promoting tight junction formation of the present invention promotes the formation of tight junctions between epithelial tissue cells. Preferably, the epithelial tissue cells are intestinal epithelial cells. It is widely known that the state of intercellular tight junction formation can be quantitatively evaluated by measuring the transepithelial electrical resistance (TEER, hereinafter simply referred to as resistance) of a cultured epithelial cell model (see Non-Patent Document 3: Srinivasan B et al. J Lab Autom, 20(2), 107-126 (2015)). TEER can be measured, for example, using a Millicell ERS-2 resistance measurement system (Merck). The Millicell ERS-2 resistance measurement system measures the integrity of a monolayer-cultured cell sheet by electrical resistance. Resistance is used as an indicator of tight junction formation in the intercellular space, and a higher resistance indicates more promoted tight junction formation.

[0017] In the present invention, the composition for promoting tight junction formation may contain known carriers and various base materials in addition to triterpenes, skim milk, and / or wheat gluten, but preferably consists solely of triterpenes, skim milk, and / or wheat gluten. The composition for promoting tight junction formation may be used as is, or, as described below, may be used in the form of a food or pharmaceutical composition containing the composition for promoting tight junction formation.

[0018] The present invention can be used orally or parenterally in the form of cosmetics, foods, beverages, etc. containing the composition for promoting tight junction formation, and is preferably used as a food. The composition for promoting tight junction formation of the present invention can be prepared as a health food, a food with functional claims, a food for specified health uses, a nutritional supplement, a food with nutritional function claims, a food for special dietary uses (e.g., a food for patients), a food labeled for reducing disease risk, a health supplement, a supplement, etc.

[0019] The supplement may be in the form of, for example, a tablet, pill, capsule (including hard capsule, soft capsule, and microcapsule), powder, granule, fine granule, lozenge, liquid (including syrup, milk, and suspension) together with various additives commonly used in the manufacture of supplements. It may also be in the form of a liquid, semi-liquid, or solid, a paste, or added to general foods and beverages.

[0020] There are no particular limitations on the foods that can be blended with the compound, but examples include carbohydrate-containing foods and beverages such as rice, rice cakes, noodles, bread, and pasta; various sweets such as Western sweets such as cookies and cakes, Japanese sweets such as buns and yokan, candies, gum, and frozen desserts and ice creams such as yogurt and pudding; various beverages such as juice, soft drinks, milk drinks, tea drinks, functional drinks, nutritional supplements, and non-alcoholic beer; alcoholic beverages such as beer and low-malt beer; foods and beverages such as soup, miso soup, and clear soup; processed products using eggs, and processed products (including delicacies) made from seafood (squid, octopus, shellfish, eel, etc.) and meat (including organ meats such as liver); dashi, soy sauce, mirin, and other seasonings.

[0021] Furthermore, the food of the present invention can contain other ingredients such as nutritional supplements in addition to the composition for promoting tight junction formation, provided that the effects of the present invention are not impaired. Such ingredients include vitamins (e.g., vitamin A, B vitamins, vitamin E, vitamin C, vitamin D, vitamin K, niacin, pantothenic acid, folic acid, etc.), carotenoids (e.g., β-carotene, lycopene, fucoxanthin, etc.), minerals (e.g., seaweed components, CCM, heme iron, iron salts, whey calcium, fermented lactate calcium, bovine bone calcium, coral calcium, eggshell calcium, etc.), various plants, and their extracts, purified products, and fractions. (e.g., plantain, chlorella, spirulina, garlic, ginkgo leaf, gymnema, eucommia leaf, perilla leaf, Job's tears, soy globulin, rutin, green tea extract, theanine, polyphenols, licorice, yucca, soy saponin, caffeine, watermelon extract, mushroom extract, Garcinia cambogia extract, etc.), microorganisms and their growth factors and microbial products (e.g., lactic acid bacteria, yeast, lactic acid bacteria growth factors, etc.), dietary fiber and its enzymatic degradation substances (e.g., apple fiber, corn fiber, starch-derived dietary fiber, indigestible dextrin, guar gum enzyme hydrolysate, sweet potato fiber, soybean fiber, seaweed fiber, mushroom fiber, tea fiber, acidic polysaccharides, plant mucilage, wheat bran, etc.), animal bodies and their extracts, refined products, hydrolysates and products (e.g., royal jelly, propolis, oyster extract, chitin, chitosan, taurine, collagen, gelatin, etc.), various oligosaccharides (e.g., galactooligosaccharides, Examples include xylooligosaccharides, soybean oligosaccharides, fructooligosaccharides, isomaltooligosaccharides, lactoferrin oligosaccharides, etc.), lipids (e.g., unsaturated fatty acids (DHA, EPA, etc.), phospholipids, Salatrim, etc.), various proteins and protein hydrolysates (e.g., corn protein, soy protein, TMP (total milk protein), lactalbumin, casein, whey, glutathione, soy peptides, egg white peptides, glutamine peptides, etc.), and wheat germ such as defatted germ.

[0022] The composition for promoting tight junction formation of the present invention can be formulated with auxiliary agents into any form to form a pharmaceutical composition that can be administered orally. For example, oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharmaceutically acceptable carriers by techniques commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives. For example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting wax, and cocoa butter can be used as carriers.

[0023] Oral preparations can be prepared by adding excipients (e.g., lactose, sucrose, starch, mannitol), disintegrants (e.g., calcium carbonate, carboxymethylcellulose calcium), binders (e.g., pregelatinized starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose), or lubricants (e.g., talc, magnesium stearate, polyethylene glycol 6000) to the active ingredient, compressing the mixture, and then coating it by a known method, if necessary, for taste masking, enteric coating, or sustained release. Examples of coating agents that can be used include ethyl cellulose, hydroxymethyl cellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, and Eudragit (Rohm, Germany, methacrylic acid-acrylic acid copolymer).

[0024] The intake or administration amount of the composition for promoting tight junction formation of the present invention can be determined appropriately depending on the concentration of triterpenes, dosage form, age, weight, and sex of the recipient, and, if administered before exercise, the exercise load. For example, if the dosage form is a tablet, it is preferably taken with water. The administration interval can be determined appropriately, and may be, for example, before meals, after meals, between meals, before exercise, or after exercise. The oral dosage of the composition for promoting tight junction formation of the present invention is typically 1 mg to 5000 mg / day, preferably 5 mg to 1000 mg / day, and more preferably 10 mg to 100 mg / day, in terms of the daily intake of triterpenes by a human (body weight 50 kg).

[0025] The food or pharmaceutical composition of the present invention can prevent the invasion of allergens, bacteria, and microorganisms into the gastrointestinal mucosa or epithelial tissue by promoting the formation of tight junctions between epithelial tissue cells using the composition for promoting tight junction formation, and can be used to prevent, alleviate, or treat symptoms or diseases mediated by the promotion of tight junction formation between epithelial tissue cells. Examples of such symptoms or diseases include, but are not limited to, leaky gut syndrome. [Example]

[0026] EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0027] Test Example 1: Tight junction evaluation test (1) Sample solution preparation To evaluate the effects of skim milk, wheat gluten, and bovine serum albumin (BSA) on tight junction formation by the triterpene R1-valigenol (hereinafter sometimes abbreviated as R1), 15 formulations were established as shown in Table 1. R1-valigenol was dissolved in DMSO to a concentration of 20 mM to prepare a 20 mM R1 solution, and each additive was dissolved in Hank's balanced salt solution (HBSS) as shown in Table 1 to prepare a sample solution. The final concentration of R1-valigenol was 25 μg / mL.

[0028] Table 1 TIFF2025183783000003.tif157170 * The amount of additive is the mass of the solid ingredients and does not include water.

[0029] (2) Cellular testing Human colon cancer-derived cells (Caco-2) were cultured in DMEM medium (containing 10% FBS) in a 37°C, 5% CO2 incubator. 2.0 × 10 cells were placed in the insert well of a 12-well Transwell plate (Corning). 5 Cells were seeded at a cell density of 1000 cells / well. They were cultured for approximately 14 days in a 37°C, 5% CO2 incubator to form a monolayer. Medium was replaced every 3–4 days. After washing the outside of the insert wells three times and the inside three times with HBSS, 500 μL of sample solution was added to the insert wells, and 1500 μL of HBSS was added to the outer wells. After culturing for 2 hours in a 37°C, 5% CO2 incubator, electrical resistance was measured using a Millicell ERS-2 Resistivity Measurement System (Merck). Three to four samples were used for each formulation, and the average values ​​were calculated. The results are shown in Table 2.

[0030] Table 2 TIFF2025183783000004.tif117149

[0031] Formulas 12 to 15, in which skim milk was added alone, showed electrical resistance values ​​equivalent to that of Formula 2, but Formulas 3 to 6, in which a composition of skim milk and R1-valigenol was added, showed higher electrical resistance values ​​than Formula 2. When skim milk at a concentration of 0.01 to 2% (w / v) was co-added with R1-valigenol, an effect of promoting tight junction formation was observed. Furthermore, when a composition of R1-valigenol and wheat gluten was added, the resistance value also increased, and the effect of promoting tight junction formation was observed at 0.01 to 2% (w / v) of wheat gluten. From the above, when the mass ratio of R1-valigenol in the composition is 1, the effect of promoting tight junction formation was observed when the mass ratio of skim milk is 4 or more or when the mass ratio of wheat gluten is 4 or more.

Claims

1. A composition for promoting tight junction formation, which contains triterpenes and skim milk and / or wheat gluten, and is used to promote the formation of tight junctions between epithelial tissue cells.

2. The triterpenes are represented by the formula I below: 1 -Valigenol Formula I 2. The composition of claim 1, wherein

3. The composition according to claim 1 , wherein the epithelial tissue cells are intestinal epithelial cells.

4. The composition according to claim 1, wherein the mass of the skim milk is 4 or more when the mass of the triterpenes is 1.

5. The composition according to claim 1, wherein the mass of wheat gluten is 4 or more when the mass of triterpenes is 1.

6. A food for promoting the formation of tight junctions between epithelial tissue cells, comprising the composition according to any one of claims 1 to 5.

7. The food product according to claim 6 for preventing, alleviating, or treating symptoms or diseases through promoting the formation of tight junctions between epithelial tissue cells.

8. A pharmaceutical composition for promoting the formation of tight junctions between epithelial tissue cells, comprising the composition according to any one of claims 1 to 5.

9. The pharmaceutical composition according to claim 8, for preventing, alleviating, or treating symptoms or diseases via promoting the formation of tight junctions between epithelial tissue cells.

10. A method for promoting the formation of tight junctions using the composition according to any one of claims 1 to 5.

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