Intestinal barrier improver

Abstract

To provide a novel intestinal tract barrier improver.SOLUTION: An intestinal tract barrier improver that consists of one or more kinds of amino acid selected from the group consisting of D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, and D-serine, or one or more kinds of component selected from the group consisting of mogroside V, humulone, 5,7-dimethoxy flavone, 5- hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a novel agent for improving an intestinal barrier. [Background technology]

[0002] The digestive tract, such as the intestine, is important not only for absorbing necessary nutrients from food, but also from the viewpoint of a barrier that separates the outside world from the inside of the body to protect against the invasion of foreign substances such as allergens. When the intestinal barrier is damaged, harmful substances that should be eliminated are absorbed from the intestine, causing various adverse effects. Such damage to the intestinal barrier is also called leaky gut, and it has been suggested that it is related to various diseases such as food allergy, immune disease, inflammatory bowel disease, irritable bowel syndrome, infection, obesity, diabetes, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), metabolic syndrome, cognitive decline, and depression. Furthermore, it has been suggested that damage to the barrier function of the intestine is related to various skin diseases. For example, it has been reported that in pediatric atopic dermatitis patients, the severity of symptoms is correlated with intestinal permeability, and that in psoriasis patients, intestinal permeability is higher than that of healthy people (Patent Documents 1 to 6, Non-Patent Documents 1 to 9).

[0003] In recent years, by improving the intestinal barrier function and improving the intestinal condition, it is expected that the health of the whole body and skin, the treatment and prevention of diseases, and cosmetic effects can be maintained, and ingestion of prebiotics such as oligosaccharides and probiotics such as lactic acid bacteria has been proposed to improve the intestinal barrier function. The present inventors have also shown that Peony extract has an effect of improving the intestinal barrier function (Patent Document 1). It has also been reported that compounds having gallic acid residues, polyphenols such as flavan-3-ol polymers, nobiletin, kaempferol, quercetin, and myricetin (Patent Documents 2-4, Non-Patent Documents 1-3), and unsaturated fatty acid metabolic intermediates (Patent Document 5), etc. have an effect of improving the intestinal barrier function, and that tangeretin has an effect of reducing colitis (Non-Patent Document 4). Furthermore, it has been reported that proline, serine, and threonine have an effect of healing the intestinal mucosa (Patent Document 6), and that D-alanine, L-glutamine, glutamine, and whey protein have an effect of improving the intestinal barrier function (Non-Patent Documents 5-7). Under these circumstances, further substances that are highly effective in improving the intestinal barrier function are desired. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-140946 A [Patent Document 2] International Publication No. 2019 / 131772 [Patent Document 3] International Publication No. 2019 / 131767 [Patent Document 4] International Publication No. 2019 / 131759 [Patent Document 5] International Publication No. 2014 / 129384 [Patent Document 6] International Publication No. 2016 / 116585 [Patent Document 7] Patent No. 4291628 [Non-patent literature]

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel intestinal barrier improving agent. [Means for solving the problem]

[0007] As a result of extensive research into various substances, the inventors discovered a component that is highly effective as an intestinal barrier improving agent and completed the following invention. (1) An intestinal barrier improving agent comprising one or more amino acids selected from the group consisting of D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, and D-serine. (2) An intestinal barrier improver comprising one or more components selected from the group consisting of mogroside V, humulone, 5,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone. (3) A composition for improving intestinal barrier function, comprising the intestinal barrier improving agent according to (1) or (2) as an active ingredient. Effect of the Invention

[0008] By administering the intestinal barrier improving agent of the present invention, the intestinal barrier function can be improved. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the TER values ​​of Caco-2 cells at each time point when various D-amino acids (50 μM) or a control (PBS) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Diagram 2] FIG. 2 shows the TER values ​​of Caco-2 cells 24 hours after the addition of various L-amino acids (50 μM) or control (PBS), expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Diagram 3]FIG. 3 shows the TER values ​​of Caco-2 cells at each time point when various components (100 μM) or a control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Figure 4] FIG. 4 shows the TER values ​​of Caco-2 cells at each time point when various components including 5,7-dimethoxyflavone (100 μM) or control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Diagram 5] FIG. 5 shows the TER values ​​of Caco-2 cells at each time point when various components including 5,7-dimethoxyflavone (100 μM) or control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Figure 6] FIG. 6 shows the TER values ​​of Caco-2 cells at each time point when mogroside V, humulone (100 μM), or control (DMSO) was added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Figure 7] FIG. 7 shows the TER values ​​of Caco-2 cells at each time point when various methoxyflavones (100 μM) or control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention provides an intestinal barrier improving agent comprising one or more D-amino acids selected from the group consisting of D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, and D-serine, or one or more components selected from the group consisting of mogroside V, humulone, 5,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone.

[0011] The administration of the intestinal barrier improving agent of the present invention can improve the intestinal barrier function. The improvement of the intestinal barrier function is useful for treating and / or preventing various diseases and disorders caused by the impairment of the intestinal barrier function, such as intestinal diseases such as colitis and Crohn's disease, immune diseases such as celiac disease, obesity, diabetes, NAFLD, NASH, metabolic syndrome, allergic diseases, cognitive decline, and depression, as well as for beauty, such as improving the skin condition.

[0012] The present invention also encompasses D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, D-serine, mogroside V, humulone, 5,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone for use in improving intestinal barrier function; a method for improving intestinal barrier function comprising administering the above amino acid or component as an active ingredient to a subject in need of improvement of intestinal barrier function; and use of the above amino acid or component for producing an intestinal barrier improver.

[0013] The intestinal barrier function refers to the function of the intestinal epithelial cells that separate the outside world from the inside of the body to restrict the intrusion of foreign substances such as allergens and harmful substances. For example, there is a structure called tight junction (TJ) in the intercellular space of intestinal epithelial cells, which contributes to maintaining the intestinal barrier function by controlling the permeation of substances between cells. It has also been reported that sugars and intestinal bacterial flora are also involved in the intestinal barrier function. If the intestinal barrier function is impaired, undesirable substances may permeate the intestine, causing adverse effects in the body, such as inflammation (Patent Document 1, Non-Patent Documents 2, 3, 7-9).

[0014] Improving intestinal barrier function may include preventing and / or treating disorders of the intestinal barrier function as described above, maintaining the intestinal barrier in a normal state, forming / repairing / regulating the function of tight junctions, protecting the intestinal mucosa, promoting the formation of a mucus layer, etc.

[0015] Intestinal barrier function can be evaluated by various methods, for example, by measuring the urinary lactulose and mannitol concentrations after ingestion of mannitol (M), a marker of easy intestinal permeability, and lactulose (L), a marker of poor permeability, and comparing the lactulose / mannitol (L / M) ratio, and it is known that the L / M ratio 2.5-4 hours after ingestion reflects the barrier function of the small intestine (Patent Document 1). In addition, a method for measuring the barrier function of the intestine by measuring the transepithelial electric resistance (TER) value between digestive epithelial Caco-2 cells is also known (Patent Document 1, Non-Patent Documents 2, 3, 8, 9).

[0016] D-amino acids are metabolites of intestinal bacteria and have been reported to be important for neurotransmission, renal protection, etc., but the present inventors have demonstrated that D-amino acids such as D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, and D-serine exert particularly strong intestinal barrier function.

[0017] When using D-amino acids as active ingredients in the present invention, they may be obtained from natural products such as animals and plants by various methods such as fermentation, enzymatic methods, extraction methods, acid decomposition methods, etc., or may be chemically synthesized. The D-amino acids may be in a form contained in natural products or may exist in a form of a mixture with L-amino acids, but they may be separated by the method described in Patent Document 7, etc.

[0018] Mogroside V is a type of glycoside derived from cucurbitane contained in the fruits of Momordica swingle (Momordica grosvenorii, Siraitia grosvenorii, Momordica grosvenorii) and has the following structure (CAS number: 88901-36-4). Mogroside V is known as a sweetening component, and has been reported to have anticancer effects in recent years. Mogroside V may be in the form of a natural product such as a Momordica extract, or may be chemically synthesized. [ka]

[0019] Humulone (also known as α-lupulic acid) is a compound found in hops (Humulus lupulus L.) and has the following structure (CAS number: 26472-41-3). Humulone is known as a bitter component, and has been reported to have bactericidal and anti-inflammatory effects. Humulone may be in the form of a natural product such as a hop extract, or it may be chemically synthesized. [ka]

[0020] 5,7-Dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone are polymethoxyflavonoids having the following structures: [Table 1]

[0021] Polymethoxyflavonoids have been reported to have cancer-suppressing and anti-inflammatory effects, and are effective in preventing and treating Alzheimer's disease. The present inventors have demonstrated that 5,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4',7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3',4'-tetramethoxyflavone, and 3,5,7,3',4'-pentamethoxyflavone exhibit particularly high intestinal barrier function.

[0022] 5,7-Dimethoxyflavone is found in black ginger (Kaempferia parviflora, also known as black turmeric) and other plants. 5-Hydroxy-7-methoxyflavone is found in black ginger, Kaempferia chinensis and other plants. 5-Hydroxy-3,4',7-trimethoxyflavone is found in black ginger and the leaves of plants of the Meliaceae family. 5,7,4'-Trimethoxyflavone is found in black ginger, the peel of mandarin (Citrus miaray) and the leaves of plants of the Thymelaeaceae family (Aquilaria sinensis). 5,7,3',4'-Tetramethoxyflavone is found in the leaves and stems of black ginger, Moon Orange (Murraya paniculata) and the leaves of plants of the Thymelaeaceae family. 3,5,7,3',4'-Pentamethoxyflavone is found in black ginger, the leaves and stems of Moon Orange and mandarin peel. The polymethoxyflavonoid of the present invention described above may be in a form contained in natural products such as extracts of the above-mentioned plants, other animals and plants, or may be chemically synthesized.

[0023] When using a plant body as an active ingredient of the present invention, the plant body may be in any form, such as a method in which the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are finely divided by methods such as shredding, crushing, cutting, crushing, grinding, homogenizing, mixing, etc., and then dried, a method in which the plant is dried and then finely divided, or an extract extracted by any method. When using an extract, the extraction method and the form of the extract are optional as long as they do not impair the effects of the present invention, but an extract obtained by an extraction method using a lower alcohol such as ethanol, an organic solvent such as hexane, or a mixed solvent of these such as hexane / ethanol can be used. However, the extraction method is not limited to solvent extraction, and may be a commonly used method known in the art.

[0024] The active ingredient of the present invention may be used after conventional purification treatment such as sterilization, washing, filtration, decolorization, deodorization, etc., and may be used after concentration or dilution as necessary. The active ingredient of the present invention may be used alone or in combination of two or more kinds.

[0025] Furthermore, the intestinal barrier improving agent and composition of the present invention can be used in combination with additives selected arbitrarily as necessary within the scope of not impairing the effects of the present invention. The additives can include excipients and the like.

[0026] The excipient may be any one that is normally used when the desired form is obtained, and examples thereof include starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, and cyclodextrin, crystalline celluloses, sugars such as lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharides, and emulsified oligosaccharides, and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients may be used alone or in combination of two or more.

[0027] Furthermore, the intestinal barrier improving agent and composition of the present invention may contain other ingredients, as necessary, appropriately selected from known ingredients such as colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, preservatives, pH adjusters, oils, powders, water, alcohols, chelating agents, flavorings, various medicinal ingredients, antiseptics, neutralizing agents, etc. In order to further enhance the effects of the present invention, one or more other ingredients, such as various lactic acid bacteria, sugars, other intestinal barrier improving agents, etc. may be used in combination.

[0028] The amount of active ingredient to be taken is not particularly limited, but when a D-amino acid is used, a human weighing 60 kg should preferably take 50 to 5,000 mg, more preferably take 100 to 1,000 mg, and even more preferably take 100 to 500 mg per day; when mogroside V, humulone, or polymethoxyflavonoid is used, a human weighing 60 kg should preferably take 0.1 to 5,000 mg, more preferably take 0.5 to 1,000 mg, and even more preferably take 1 to 500 mg per day.

[0029] The content of the active ingredient in the intestinal barrier improving agent and composition of the present invention is not particularly limited, but when a D-amino acid is used, it is preferably incorporated in an amount to be ingested per day of 50 to 5,000 mg, more preferably 100 to 1,000 mg, and even more preferably 100 to 500 mg. When mogroside V, humulone, or polymethoxyflavonoid is used, it is preferably incorporated in an amount to be ingested per day of 0.1 to 5,000 mg, more preferably 0.5 to 1,000 mg, and even more preferably 1 to 500 mg.

[0030] The form of the intestinal barrier improving agent and composition of the present invention can be selected arbitrarily from liquid, solid, granular, particulate, paste, gel, etc., depending on the active ingredient, application, and other conditions. Furthermore, the composition containing the intestinal barrier improving agent of the present invention may be in the form of a dry powder, a beverage such as tea or soft drink, a tablet or capsule such as a supplement, a food composition such as a processed food, or a pharmaceutical composition such as a medicine. The composition can be produced by a conventional method in a formulation appropriately combined with an excipient, a carrier and / or a diluent, etc., and other ingredients depending on the dosage form.

[0031] In addition, the administration routes of the intestinal barrier improving agent and composition of the present invention include, but are not limited to, oral, nasal, enteral, and the like. EXAMPLES

[0032] The present invention will now be described in more detail with reference to examples, although the present invention is not limited thereto.

[0033] Sample preparation: Each sample was prepared as described below. (1) Preparation of D-amino acids Samples of the D-amino acids shown in FIG. 1 and the L-amino acids shown in FIG. 2 were prepared by dissolving the reagents (Nacalai Tesque, Inc.) in PBS (pH 7.4, Gibco) at a concentration of 50 mM. (2) Preparation of other ingredients A total of 33 types of ingredients shown below, including the components of the present invention and various polyphenols including myricetin, nobiletin, and tangeretin, which are known to have intestinal barrier function, were prepared by dissolving them in DMSO (manufactured by Fujifilm) at a concentration of 200 mM. [Table 2]

[0034] Cell culture: Human intestinal epithelial cells, Caco-2 (HTB-37), were purchased from American Type Cell Culture (Rockville, MD, USA) and cultured under standard conditions as previously reported (Non-Patent Documents 2, 3). Cells were cultured at 0.25 × 10 cells / well on the permeable polyester membrane of a transwell insert (diameter 12 mm, pore size 0.4 μM; Corning, Cambridge Corning, Cambridge, MA, USA). 6 cells / cm 2 The cells were seeded at a density of 100 μg / ml, and all tests were performed 14 days after seeding. The cells were between passage numbers 55 and 65, and the medium was changed every 3 days. After culturing for 14 days, each component was added as described below, and the intestinal barrier function was measured.

[0035] Measurement of intestinal barrier function: The intestinal barrier function was evaluated by measuring the TJ barrier function of the intestinal epithelium by measuring the transepithelial electrical resistance (TER) of Caco-2 monolayer cells in a Transwell insert, based on the description in Non-Patent Documents 8 and 9. The TER of the monolayer cells was 1000-1300 Ω cm 2On the day of the test, the test medium was changed to Hank's balanced salt solution (glucose (+)) only for the test using L / D amino acids, and the test was performed about 6 hours later. Each L / D amino acid compound (50 μMol / L medium), other components (100 μMol / L medium), or control (PBS or DMSO) was added to the apical well, and the cells were incubated for 48 hours. This experiment was based on previous reports (Non-Patent Documents 2, 3) in which 100 μM kaempferol, myricetin, and quercetin showed protective effects on TJ barrier function 48 hours after addition. TER was measured using a Millicell-ERS system (Millipore, Bedford, MA, USA) before and 12, 24, and 48 hours after addition of the compounds.

[0036] Statistical analysis: All values ​​are expressed as mean ± SEM (n = 3). TER at each time point was expressed as a percentage (%) of the initial value before addition. Statistical analysis was performed using the Dunnett test with 4Step Excel Statistics 4th Edition (OMS Publishing). P < 0.05 (vs. Control, Dunnett test) was considered significant and is indicated with an * in the figure.

[0037] The results for D-amino acids are shown in Figure 1, and the results for L-amino acids are shown in Figure 2. As can be seen in Figure 1, the TER was significantly higher in Caco-2 cells to which the D-amino acids of the present invention had been added than in the control, indicating that the intestinal barrier function was improved. Furthermore, it can be seen that the D-amino acids of the present invention are superior to D-alanine (D-Ala), which is known to improve intestinal barrier function. On the other hand, as is clear from Figure 2, the TER of Caco-2 cells did not increase significantly even when various L-amino acids were added, and no significant improvement in intestinal barrier function was observed.

[0038] Figures 3 to 7 show the results of using a total of 33 kinds of components. No significant increase in TER was observed for the seven kinds of components 1 to 7 shown in Figure 3, and the result was significantly lower for component 5. Figure 4 shows the results of a total of seven kinds of components including nobiletin, tangeretin, and 5,7-dimethoxyflavone. It can be seen that the 5,7-dimethoxyflavone of the present invention shows a very high increase in TER compared to nobiletin and tangeretin, which have been reported to have intestinal barrier functions. Figure 5 shows the results of a total of three kinds of components including myricetin and 5,7-dimethoxyflavone. It can be seen that 5,7-dimethoxyflavone has a particularly excellent intestinal barrier function. Figure 6 shows the results of humulone and mogroside V. Figure 7 shows the results of the polymethoxyflavone of the present invention, and all polymethoxyflavones showed a significantly high increase in TER.

[0039] This example confirmed that the present invention has a very excellent effect of improving the intestinal barrier function, even when compared with known components that improve the intestinal barrier function. It has been shown that impairment of the intestinal barrier function affects various diseases and conditions (Patent Documents 1-6, Non-Patent Documents 1-10). Therefore, the intestinal barrier improving agent of the present invention is effective for treating and / or preventing various diseases caused by impairment of the intestinal barrier function, and for cosmetics such as improving skin conditions.

Claims

[Claim 1] A composition for improving the intestinal barrier comprising 5,7-dimethoxyflavone.

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