JP7467138B2 - Intestinal barrier improver - Google Patents

Description

The present invention relates to a novel intestinal barrier improving agent.

The digestive tract, including the intestines, is important not only for absorbing necessary nutrients from food, but also from the perspective 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. If 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 allergies, immune diseases, inflammatory bowel disease, irritable bowel syndrome, infections, 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 intestinal barrier function is related to various skin diseases. For example, it has been reported that in pediatric patients with atopic dermatitis, the severity of symptoms is correlated with intestinal permeability, and that in psoriasis patients, intestinal permeability is higher than in healthy people (Patent Documents 1 to 6, Non-Patent Documents 1 to 9).

In recent years, it is expected that improving the intestinal barrier function and improving the condition of the intestine will maintain the health of the whole body and skin, treat and prevent diseases, and have cosmetic effects. In order to improve the intestinal barrier function, it has been proposed to take prebiotics such as oligosaccharides and probiotics such as lactic acid bacteria. The 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 with 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). In addition, 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, there is a need for further substances that are highly effective in improving intestinal barrier function.

JP 2018-140946 A International Publication No. 2019/131772 International Publication No. 2019/131767 International Publication No. 2019/131759 International Publication No. 2014/129384 International Publication No. 2016/116585 Patent No. 4291628

Xiong Y. et. al., Mol Nutr Food Res. 2015 May;59(5):829-42 Suzuki, T. et al., J. Nutr. 2011, 141, 87-94 Suzuki, T. et al., J. Nutr. 2009, 139, 965-74 Su-Hyeon Eun et al., Planta Med 2017;83:527-533 Miyauchi, E. et al., Biosci. Biotechnol. Biochem., 76 (2), 283-288, 2012 Jaya Benjamin et. al., Digestive Diseases and Sciences 57(4):1000-12 ・ 2012 R. K. Rao et. al., J Epithel Biol Pharmacol. 2012 January ; 5(Suppl 1-M7): 47-54 Al-Sadi, R. et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2011, 300, G1054-64 Donato, K. A. et al., Microbiology 2010, 156, 3288-97

The objective of the present invention is to provide a novel intestinal barrier improving agent.

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.

By administering the intestinal barrier improving agent of the present invention, the intestinal barrier function can be improved.

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). 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). 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). FIG. 4 shows the TER values of Caco-2 cells at each time point when various components including 5,7-dimethoxyflavone (100 μM) or a control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). FIG. 5 shows the TER values of Caco-2 cells at each time point when various components including 5,7-dimethoxyflavone (100 μM) or a control (DMSO) were added, expressed as a percentage (%) of the initial value before addition (%, vs 0 hr). 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, as a percentage (%) of the initial value before addition (%, vs 0 hr). FIG. 7 shows the TER values of Caco-2 cells at each time point when various methoxyflavones (100 μM) or a control (DMSO) were added, expressed as a percentage of the initial value before addition (%, vs 0 hr).

The present invention provides an intestinal barrier improving agent 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.

By administering the intestinal barrier improving agent of the present invention, the intestinal barrier function can be improved. Improvement of the intestinal barrier function is useful for the treatment and/or prevention of various diseases and disorders caused by disorders 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 purposes such as improving skin condition.

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.

The intestinal barrier function refers to the function of the intestinal epithelial cells, which 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).

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.

Intestinal barrier function can be evaluated by various methods. For example, it can be evaluated 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. 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).

D-amino acids are metabolites of intestinal bacteria and have been reported to be important in 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.

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, and acid decomposition methods, or may be chemically synthesized. They may be in a form contained in natural products or may exist in the form of a mixture with L-amino acids, but the D-amino acids may be separated by the method described in Patent Document 7, etc.

Mogroside V is a type of glycoside derived from cucurbitane contained in the fruits 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 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.

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 can be found in the form of a natural product such as a hop extract, or it can be chemically synthesized.

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:

Polymethoxyflavonoids have been reported to have cancer-suppressing and anti-inflammatory effects, and to be 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 exert particularly strong intestinal barrier function.

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 and Kaempferia japonica. 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 polymethoxyflavonoids of the present invention described above may be in a form contained in natural products such as extracts of the above-mentioned plants and other animals and plants, or may be chemically synthesized.

When using a plant body as an active ingredient of the present invention, the plant may be in any form, such as a method in which the whole plant or various parts (leaves, flowers, roots, etc.) 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 arbitrary 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 mixture of these solvents 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.

The active ingredient of the present invention may be used after conventional purification treatment such as sterilization, washing, filtration, decolorization, and deodorization, 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 kinds.

Additives may be selected and used in combination with the intestinal barrier improving agent and composition of the present invention as needed, as long as the effects of the present invention are not impaired. Additives may include excipients, etc.

Any excipient that is normally used when forming the desired form may be used, 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.

In addition, the intestinal barrier improving agent and composition of the present invention may contain other ingredients, such as colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, preservatives, pH adjusters, oils, powders, water, alcohols, chelating agents, fragrances, various medicinal ingredients, antiseptics, neutralizing agents, and other known ingredients, which may be appropriately selected and used as needed. 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, and the like, may be used in combination.

The amount of active ingredient to be taken is not particularly limited, but when using D-amino acids, a person weighing 60 kg should preferably take 50 to 5000 mg per day, more preferably 100 to 1000 mg, and even more preferably 100 to 500 mg, and when using mogroside V, humulone, or polymethoxyflavonoids, a person weighing 60 kg should preferably take 0.1 to 5000 mg per day, more preferably 0.5 to 1000 mg, and even more preferably 1 to 500 mg.

The content of the active ingredient in the intestinal barrier improving agent and composition of the present invention is not particularly limited, but when D-amino acids are used, they are preferably incorporated in an amount of 50 to 5000 mg, more preferably 100 to 1000 mg, and even more preferably 100 to 500 mg per day, and when mogroside V, humulone, or polymethoxyflavonoids are used, they are preferably incorporated in an amount of 0.1 to 5000 mg, more preferably 0.5 to 1000 mg, and even more preferably 1 to 500 mg per day.

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 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 manufactured by a conventional method in a formulation appropriately combined with excipients, carriers and/or diluents, etc., and other ingredients depending on the dosage form.

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, etc.

Next, the present invention will be described in more detail with reference to examples. Note that the present invention is not limited to these examples.

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 manufactured by Nacalai Tesque in PBS (pH 7.4, manufactured by Gibco) at a concentration of 50 mM.
(2) Preparation of other components The components of the present invention, as well as 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.

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 seeded at a density of 0.25 × ¹⁰⁶ cells/ ^cm2 on the permeable polyester membrane of a transwell insert (12 mm diameter, 0.4 μM pore size; Corning, Cambridge Corning, Cambridge, MA, USA), and all tests were performed 14 days after seeding. Cells were between passage numbers 55 and 65, and the medium was changed every 3 days. After 14 days of culture, each component was added and the intestinal barrier function was measured as described below.

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 ² (data not shown). On 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 and 3) in which 100 μM kaempferol, myricetin, and quercetin showed a protective effect on the TJ barrier function 48 hours after addition. TER was measured with a Millicell-ERS system (Millipore, Bedford, Mass., USA) before and 12, 24, and 48 hours after compound addition.

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 Dunnett's test with 4Step Excel Statistics 4th Edition (OMS Publishing). P < 0.05 (vs. Control, Dunnett's test) was considered significant and is indicated with an * in the figure.

The results for D-amino acids are shown in Figure 1, and the results for L-amino acids 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 had been 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.

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

This example confirms that the present invention has a highly superior effect of improving intestinal barrier function, even when compared with known components that improve intestinal barrier function. (Patent Documents 1-6 and Non-Patent Documents 1-10 show that intestinal barrier function disorders affect various diseases and conditions.) Therefore, the intestinal barrier improving agent of the present invention is effective in treating and/or preventing various diseases caused by intestinal barrier function disorders, as well as for beauty purposes such as improving skin conditions.

Claims (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,
The intestinal barrier improving agent, wherein the intestinal barrier improvement is an improvement in the tight junction barrier function of the intestinal epithelium .

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