Intestinal tract barrier improver

Abstract

To provide a novel intestinal tract barrier improver.SOLUTION: A composition for improving the intestinal tract barrier comprising mogroside V.SELECTED DRAWING: Figure 6

Description

[Technical Field]

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

[0002] The digestive tract, including the intestine, is important not only for absorbing essential nutrients from food but also for serving as a barrier between the outside world and the body, protecting against the invasion of foreign substances such as allergens. When the intestinal barrier is impaired, harmful substances that should normally be eliminated are absorbed from the intestine, resulting in various adverse effects. Such intestinal barrier impairment, also known as leaky gut, has been implicated in various diseases, including food allergies, immune disorders, inflammatory bowel disease, irritable bowel syndrome, infections, obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic syndrome, cognitive decline, and depression. Furthermore, impaired intestinal barrier function has also been implicated in various skin diseases. For example, it has been reported that intestinal permeability correlates with the severity of symptoms in pediatric patients with atopic dermatitis, and that intestinal permeability is higher in patients with psoriasis compared to healthy individuals (Patent Documents 1-6, Non-Patent Documents 1-9).

[0003] In recent years, improving intestinal barrier function and intestinal health has been expected to contribute to the maintenance of overall health and skin condition, the treatment and prevention of diseases, and cosmetic benefits. To improve intestinal barrier function, the intake of prebiotics such as oligosaccharides and probiotics such as lactic acid bacteria has been proposed. The present inventors have also demonstrated that Peucedanum japonicum extract has the effect of improving intestinal barrier function (Patent Document 1). It has also been reported that compounds containing gallic acid residues, flavan-3-ol polymers, polyphenols such as nobiletin, kaempferol, quercetin, and myricetin (Patent Documents 2-4, Non-Patent Documents 1-3), and unsaturated fatty acid metabolic intermediates (Patent Document 5) have the effect of improving intestinal barrier function, and that tangeretin has the effect of alleviating colitis (Non-Patent Document 4). Furthermore, it has been reported that proline, serine, and threonine have the effect of healing the intestinal mucosa (Patent Document 6), and that D-alanine, L-glutamine, glutamine, and whey protein have the effect of improving intestinal barrier function (Non-Patent Documents 5-7). Under these circumstances, there is a need for further substances that are highly effective in improving intestinal barrier function. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-140946 [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 agent for improving an intestinal barrier. [Means for solving the problem]

[0007] As a result of extensive research into various substances, the inventors have discovered a component that is highly effective as an intestinal barrier improver, and have completed the following invention. (1) An intestinal barrier improver characterized by 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 characterized by 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. [Effects of the Invention]

[0008] By administering the intestinal barrier improving agent of the present invention, the intestinal barrier function can be improved. [Brief explanation 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 control (PBS) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Figure 2] FIG. 2 shows the TER values of Caco-2 cells 24 hours after addition of various L-amino acids (50 μM) or control (PBS), expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). [Figure 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] Figure 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). [Figure 5] Figure 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 INVENTION

[0010] The present invention provides an intestinal barrier improver characterized by 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] Administration of the intestinal barrier improving agent of the present invention can improve intestinal barrier function, which is useful for treating and / or preventing various diseases and disorders caused by impaired 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 cosmetic purposes such as improving 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 any of the above amino acids or components as an active ingredient to a subject in need of such improvement; and use of the above amino acids or components for producing an intestinal barrier improver.

[0013] The intestinal barrier function refers to the ability of intestinal epithelial cells, which separate the outside world from the body, to restrict the penetration of foreign substances such as allergens and harmful substances. For example, structures called tight junctions (TJs) exist in the intercellular spaces of intestinal epithelial cells, which contribute 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. When the intestinal barrier function is impaired, unwanted substances can 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 assessed by various methods, including measuring the urinary lactulose and mannitol concentrations after ingestion of mannitol (M), a marker of high intestinal permeability, and lactulose (L), a marker of low intestinal permeability, and comparing the lactulose / mannitol (L / M) ratio. It is known that the L / M ratio 2.5-4 hours after ingestion reflects small intestinal barrier function (Patent Document 1). Another method for measuring intestinal barrier function is known, which involves measuring the transepithelial electrical resistance (TER) between Caco-2 gastrointestinal epithelial cells (Patent Document 1, Non-Patent Documents 2, 3, 8, and 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 a D-amino acid is used as an active ingredient of the present invention, it may be obtained from natural products such as animals and plants by various methods such as fermentation, enzymatic methods, extraction methods, acid hydrolysis methods, etc., or it may be chemically synthesized. The D-amino acid may be in a form contained in natural products or may exist in the form of a mixture with an L-amino acid, but it may also be separated by the method described in Patent Document 7, etc.

[0018] Mogroside V is a glycoside derivative of cucurbitane found in the fruit of Momordica swingle (Siraitia grosvenorii, Momordica grosvenorii) and has the following structure (CAS number: 88901-36-4). Mogroside V is known as a sweetening component and has recently been reported to have anticancer properties. Mogroside V can be found in the form of a natural product, such as Momordica fruit extract, or 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 antibacterial and anti-inflammatory effects. Humulone can be found in natural forms such as hop extracts or 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 each having the following structure: [Table 1]

[0021] Polymethoxyflavonoids have been reported to have anti-cancer and anti-inflammatory effects, and to be effective in preventing and treating Alzheimer's disease. However, 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), etc. 5-Hydroxy-7-methoxyflavone is found in black ginger, Kaempferia parviflora, etc. 5-Hydroxy-3,4',7-trimethoxyflavone is found in black ginger and the leaves of plants in the Meliaceae family. 5,7,4'-Trimethoxyflavone is found in black ginger, mandarin (Citrus miaray) peel, and the leaves of plants in the Thymelaeaceae family (Aquilaria sinensis). 5,7,3',4'-Tetramethoxyflavone is found in black ginger, the leaves and stems of Moon Orange (Murraya paniculata), and the leaves of plants in the Thymelaeaceae family. 3,5,7,3',4'-Pentamethoxyflavone is found in black ginger, Moon Orange leaves and stems, mandarin peel, etc. 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 a plant is used as an active ingredient of the present invention, the plant may be in any form, such as by shredding, crushing, cutting, crushing, grinding, homogenizing, mixing, or other methods of comminuted fractions of the whole plant or various parts (e.g., leaves, flowers, roots) and then drying them; by comminuted fractions of the dried plant; or by extracts extracted by any method. When an extract is used, the extraction method and form of the extract may be any as long as they do not impair the effects of the present invention. Extracts obtained by extraction using lower alcohols such as ethanol, organic solvents such as hexane, or mixtures of these solvents, such as hexane / ethanol, can be used. However, the extraction method is not limited to solvent extraction, and conventional methods known in the art may also be used.

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

[0025] Furthermore, the intestinal barrier improving agent and composition of the present invention can be used in combination with additives selected arbitrarily as needed, as long as the effects of the present invention are not impaired. Additives that can be used include excipients and the like.

[0026] The excipient may be any one that is commonly used when forming the desired form, 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 can be used alone or in combination of two or more.

[0027] Furthermore, the intestinal barrier improver and composition of the present invention can contain other ingredients, as needed, that are 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. Furthermore, to further enhance the effects of the present invention, one or more other ingredients, such as various lactic acid bacteria, sugars, other intestinal barrier improvers, etc., may be used in combination.

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

[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 taken per day of 50 to 5,000 mg, more preferably 100 to 1,000 mg, and even more preferably 100 to 500 mg, and when mogroside V, humulone, or polymethoxyflavonoid is used, it is preferably incorporated in an amount to be taken 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 forms of the intestinal barrier improver and composition of the present invention can be selected as desired depending on the active ingredient, intended use, and other conditions, such as liquid, solid, granular, particulate, paste, or gel. Furthermore, compositions containing the intestinal barrier improver of the present invention may be in the form of dry powders, beverages such as tea and soft drinks, tablets and capsules such as supplements, food compositions such as processed foods, or pharmaceutical compositions such as medicines. The compositions can be prepared using conventional methods in a formulation appropriately combined with excipients, carriers and / or diluents, and other ingredients depending on the dosage form.

[0031] Furthermore, 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. [Example]

[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 reagents manufactured by Nacalai Tesque in PBS (pH 7.4, manufactured by Gibco) at a concentration of 50 mM. (2) Preparation of other ingredients A total of 33 ingredients shown below, including the ingredients 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 described (Non-Patent Documents 2, 3). Cells were cultured at 0.25 × 10 cells permeable polyester membrane of a transwell insert (12 mm diameter, 0.4 μM pore size; 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. Cells were used at passage numbers between 55 and 65, and the medium was changed every 3 days. After 14 days of culture, each component was added as described below, and 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 Transwell inserts, based on the description of 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 (+)) for the L / D amino acid test only, and the test was performed approximately 6 hours later. Each L / D amino acid compound (50 μM / L medium), other components (100 μM / L medium), or control (PBS or DMSO) was added to the apical wells, and the cells were incubated for 48 hours. This experiment was based on previous reports showing that 100 μM kaempferol, myricetin, and quercetin exhibited protective effects on TJ barrier function 48 hours after addition (Non-Patent Documents 2, 3). TER was measured using a Millicell-ERS system (Millipore, Bedford, MA, USA) before and 12, 24, and 48 hours after compound addition.

[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 by an * in the figures.

[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 compared to the control, indicating improved intestinal barrier function. Furthermore, 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 addition of various L-amino acids did not significantly increase the TER of Caco-2 cells, and no significant improvement in intestinal barrier function was observed.

[0038] Figures 3 to 7 show the results using a total of 33 ingredients. No significant increase in TER was observed for the seven ingredients 1 to 7 listed in Figure 3, and ingredient 5 even showed a significantly lower result. Figure 4 shows the results for a total of seven ingredients, including nobiletin, tangeretin, and 5,7-dimethoxyflavone. It can be seen that 5,7-dimethoxyflavone of the present invention exhibits a significantly higher increase in TER than nobiletin and tangeretin, which have previously been reported to have intestinal barrier function. Figure 5 shows the results for a total of three ingredients, including myricetin and 5,7-dimethoxyflavone. It can be seen that 5,7-dimethoxyflavone exhibits particularly excellent intestinal barrier function. Figure 6 shows the results for humulone and mogroside V. Figure 7 shows the results for the polymethoxyflavones of the present invention, and all polymethoxyflavones exhibited significantly higher increases in TER.

[0039] This example demonstrates that the present invention has a significantly superior effect of improving intestinal barrier function, even compared to known components that improve intestinal barrier function. Impairment of intestinal barrier function has been shown to affect various diseases and conditions (Patent Documents 1 to 6, Non-Patent Documents 1 to 10). Therefore, the intestinal barrier improving agent of the present invention is effective in treating and / or preventing various diseases caused by impairment of intestinal barrier function, as well as for cosmetic purposes such as improving skin condition.

Claims

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

Images