JP2021120359A - Intestinal tract barrier improver - Google Patents

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

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

The digestive tract, such as the intestinal tract, is important not only for absorbing necessary nutrients from food, but also for the barrier that separates the outside world from the inside of the body so as to protect it from the invasion of foreign substances such as allergens. When the barrier of the intestine is damaged, harmful substances that should be eliminated are absorbed from the intestine, which has various adverse effects. Such disorders of the intestinal barrier, also called leaky gut, include food allergies, immune disorders, inflammatory bowel disease, irritable bowel syndrome, infections, obesity, diabetes, NAFLD (non-alcoholic steatohepatitis), and NASH ( It has been suggested to be associated with various diseases such as non-alcoholic steatohepatitis), metabolic syndrome, cognitive decline, and depression. Furthermore, it has been suggested that impaired barrier function of the intestinal tract is associated with various skin diseases. For example, it has been reported that in pediatric atopic dermatitis patients, the severity of symptoms correlates with intestinal permeability, and in psoriasis patients, intestinal permeability is higher than in healthy subjects (Patent Document 1). ~ 6, Non-Patent Documents 1-9).

In recent years, by improving the intestinal barrier function and improving the intestinal condition, it is expected to maintain the health of the whole body and skin condition, treat and prevent diseases, and have a beauty effect. Ingestion of prebiotics such as oligosaccharides and probiotics such as lactic acid bacteria has been proposed. In addition, the present inventors have shown that the button bow extract has an effect of improving the intestinal barrier function (Patent Document 1). Compounds with gallic acid residues, flavan-3-ol polymers, polyphenols such as nobiletin, kaempferol, quercetin, myricetin (Patent Documents 2-4, Non-Patent Documents 1-3) and unsaturated fatty acid metabolism intermediates (Patent Documents) It has also been reported that 5) and the like have an effect of improving the intestinal barrier function, and tangeretin has an effect of reducing colitis (Non-Patent Document 4). Furthermore, proline, serine, and threonine have an intestinal mucosal healing effect (Patent Document 6), and D-alanine, L-glutamine, glutamine, and whey protein have an intestinal barrier function improving effect (Non-Patent Documents 5 to 5). 7) has also been reported. Under such circumstances, a further substance having a high effect of improving the intestinal barrier function has been desired.

Japanese Unexamined Patent Publication No. 2018-140946 International Publication No. 2019/131772 International Publication No. 2019/131767 International Publication No. 2019/131 759 International Publication No. 2014/129384 International Publication No. 2016/116585 Japanese 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

An object of the present invention is to provide a novel intestinal barrier improving agent.

As a result of diligent research on various substances, the inventors have found a component having a high effect as an intestinal barrier improving agent, and have completed the following inventions.
(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) Mogroside V, Humlon, 5,7-dimethoxyflavon, 5-hydroxy-7-methoxyflavon, 5-hydroxy-3,4', 7-trimethoxyflavon, 5,7,4'-trimethoxyflavon, It is characterized by consisting of one or more components selected from the group consisting of 5,7,3', 4'-tetramethoxyflavon and 3,5,7,3', 4'-pentamethoxyflavon. Intestinal barrier improver.
(3) A composition for improving the intestinal barrier function, which comprises the intestinal barrier improving agent according to (1) or (2) as an active ingredient.

The intestinal barrier function can be improved by administration of the intestinal barrier improving agent of the present invention.

FIG. 1 shows the TER value of Caco-2 cells at each time when various D-amino acids (50 μM) or Control (PBS) were added as a ratio (%) of the initial value before addition (%, vs 0hr). .. FIG. 2 shows the TER value of Caco-2 cells 24 hours after the addition of various L-amino acids (50 μM) or Control (PBS) as a ratio (%) of the initial value before addition (%, vs 0hr). ). FIG. 3 shows the TER value of Caco-2 cells at each time when various components (100 μM) or Control (DMSO) were added as a ratio (%) of the initial value before addition (%, vs 0hr). FIG. 4 shows the TER value of Caco-2 cells at each time when various components (100 μM) containing 5,7-dimethoxyflavone or Control (DMSO) were added as a ratio (%) of the initial value before addition. (%, vs 0hr). FIG. 5 shows the TER value of Caco-2 cells at each time when various components (100 μM) containing 5,7-dimethoxyflavone or Control (DMSO) were added as a ratio (%) of the initial value before addition. (%, vs 0hr). FIG. 6 shows the TER value of Caco-2 cells at each time when mogroside V, humulone (100 μM), or Control (DMSO) was added as a percentage (%) of the initial value before addition (%, vs 0hr). ). FIG. 7 shows the TER value of Caco-2 cells at each time when various methoxyflavones (100 μM) or Control (DMSO) were added as a ratio (%) of the initial value before addition (%, vs 0hr).

The present invention relates to 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 mogloside V, humlon, 5 , 7-Dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4', 7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3', 4' Provided is an intestinal barrier improving agent comprising one or more components selected from the group consisting of -tetramethoxyflavon and 3,5,7,3', 4'-pentamethoxyflavon.

The intestinal barrier function can be improved by administration of the intestinal barrier improving agent of the present invention. Improvement of intestinal barrier function includes 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. It is useful for beauty such as treatment and / or prevention of various diseases and disorders caused by disorders, and improvement of skin condition.

The present invention also presents D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, D-serine, mogloside V, humron, 5,7-dimethoxyflavon, 5 for use in improving intestinal barrier function. -Hydroxy-7-methoxyflavone, 5-hydroxy-3,4', 7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3', 4'-tetramethoxyflavone, and 3 , 5,7,3', 4'-Pentamethoxyflavon; Method for improving intestinal barrier function including administration of the above amino acid or ingredient as an active ingredient to a subject who needs improvement of intestinal barrier function; It also includes the use of the above amino acids or components to produce barrier improvers.

The intestinal barrier function is a function in which epithelial cells of the intestinal tract that separate the outside world from the living body limit the invasion of foreign substances such as allergens and harmful substances. For example, the intercellular space of intestinal epithelial cells has a structure called a tight junction (TJ), which contributes to the maintenance of the intestinal barrier function by controlling the permeation of substances between cells. There are also reports that sugars and bacterial flora in the intestine are also involved in the intestinal barrier function. When the intestinal barrier function is impaired, unwanted substances may permeate the intestine and cause adverse effects on the body such as inflammation (Patent Document 1, Non-Patent Documents 2, 3, 7-9).

Improving intestinal barrier function means preventing and / or treating the above-mentioned disorders of intestinal barrier function, maintaining the intestinal barrier in a normal state, forming / repairing / regulating tight junctions, and intestinal function. It may include protecting the mucosa, promoting the formation of a mucous layer, and the like.

The intestinal barrier function can be evaluated by various methods, for example, the urinary lactulose concentration after ingestion of the intestinal permeable marker mannitol (M) and the imperviously permeable marker lactulose (L). It can be evaluated by measuring the mannitol concentration and comparing the lactulose / mannitol (L / M) ratio, and it is known that the L / M ratio 2.5-4 h after ingestion reflects the barrier function of the small intestine (Patent). Reference 1). In addition, a method of measuring the barrier function of the intestinal tract by measuring the transepithelial electric resistance (TER) value between gastrointestinal epithelial Caco-2 cells is also known (Patent Document 1, Non-Patent Document 1). 2, 3, 8, 9).

D-amino acids are intestinal bacterial metabolites and have been reported to be important for neurotransmission, kidney protection, etc., but the present inventors have reported that D-aspartic acid, D-cysteine, D-glutamine, D. -It was revealed that D-amino acids such as glutamic acid and D-serine exert a particularly high intestinal barrier function.

When D-amino acid is used as the active ingredient of the present invention, it may be obtained from natural products such as animals and plants by various methods such as fermentation method, enzyme method, extraction method and acid decomposition method, or it may be chemically synthesized. .. It may be in the form contained in a natural product or in the form of a mixture with L-amino acid, but D-amino acid may be separated by the method described in Patent Document 7.

Mogroside V is a kind of glycoside of cucurbitane derivative contained in Luo Han Guo (Momordica swingle, Siraitia grosvenorii, Momordica grosvenorii) fruit and the like, and is a compound having the following structure (CAS number: 88901-36-4). Mogroside V is known as a sweetening component, and its anticancer activity has been reported in recent years. Mogroside V may be in the form of a natural product such as Luo Han Guo extract or may be chemically synthesized.

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

5,7-dimethoxyflavon, 5-hydroxy-7-methoxyflavon, 5-hydroxy-3,4', 7-trimethoxyflavon, 5,7,4'-trimethoxyflavon, 5,7,3', 4 '-Tetramethoxyflavon and 3,5,7,3', 4'-pentamethoxyflavon are polymethoxyflavonoids having the following structures, respectively.

Polymethoxyflavonoids have been reported to be effective in suppressing cancer, anti-inflammatory effects, and preventing or treating Alzheimer's disease. , 5-Hydroxy-3,4', 7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,3', 4'-tetramethoxyflavone, and 3,5,7,3' It was revealed that 4'-pentamethoxyflavone has a particularly high intestinal barrier function.

5,7-Dimethoxyflavone is contained in black ginger (Kaempferia parviflora, also known as black kaempferia) and the like. 5-Hydroxy-7-methoxyflavone is contained in black ginger, Masuchi and the like. 5-Hydroxy-3,4', 7-trimethoxyflavone is contained in leaves of black ginger, mahogany plant and the like. 5,7,4'-Trimethoxyflavone is contained in black ginger, mandarin (Citrus miaray) pericarp, leaves of Thymelaeaceae (Aquilaria sinensis) and the like. 5,7,3', 4'-Tetramethoxyflavon is contained in the leaves and stems of black ginger, Murraya paniculata, and the leaves of Thymelaeaceae plants. 3,5,7,3', 4'-pentamethoxyflavone is contained in black ginger, leaves and stems of orange jessamine, mandarin peel and the like. The polymethoxyflavonoids of the present invention described above may be in the form contained in natural products such as the above-mentioned extracts of plants and other animals and plants, or may be chemically synthesized.

When a plant is used as the active ingredient of the present invention, the whole plant or various parts (leaves, flowers, roots, etc.) of the plant are shredded, crushed, cut, crushed, mashed, homogenized, mixed, etc., and then subdivided. It can be in any form, such as a method of drying the plant, a method of drying the plant and then subdividing it, or an extract extracted by an arbitrary method. When an extract is used, the extraction method and the form of the extract are arbitrary as long as the effects of the present invention are not impaired, but a lower alcohol such as ethanol, an organic solvent such as hexane, or a mixed solvent thereof such as hexane / ethanol may be used. The extract obtained by the extraction method used 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 being subjected to conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization, and may be used after being concentrated or diluted as necessary. In addition, the active ingredient of the present invention may be used alone or in combination of two or more.

Further, in the intestinal barrier improving agent and the composition of the present invention, additives can be arbitrarily selected and used in combination as long as the effects of the present invention are not impaired. Excipients and the like can be included as the additive.

The excipient may be any as long as it is usually used in the desired form, for example, starches such as wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin, crystalline celluloses, etc. Examples include sugars such as lactose, glucose, sugar, reduced maltose, water candy, fructo-oligosaccharides 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.

In addition, the intestinal barrier improving agent and composition of the present invention can be used as necessary for other components such as colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, and gelling agents. Known agents, antioxidants, preservatives, pH adjusters, oils, powders, water, alcohols, chelating agents, fragrances, various medicinal ingredients, preservatives, neutralizing agents and the like can be appropriately selected and used. Further, in order to further enhance the effect of the present invention, one or more other components such as various lactic acid bacteria, sugars, other intestinal barrier improving agents and the like may be used in combination.

The intake of the active ingredient is not particularly limited, but when D-amino acid is used, it is preferable to ingest 50 to 5000 mg, more preferably 100 to 1000 mg, and 100 to 1000 mg per day for a person weighing 60 kg. It is more preferable to take 500 mg, and when using mogloside V, humron, or polymethoxyflavonoid, in a person weighing 60 kg, it is preferable to take 0.1 to 5000 mg per day, and it is more preferable to take 0.5 to 1000 mg. It is preferable to take 1 to 500 mg, and it is more preferable to take 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 acid is used, it is preferably 50 to 5000 mg, more preferably 100 to 1000 mg, and even more preferably 100 to 100 mg per day. When mogroside V, humulone, or polymethoxyflavonoid is used in an amount of 500 mg to be ingested, it is preferably formulated in an amount of 0.1 to 5000 mg, more preferably 0.5 to 1000 mg, and further preferably 1 to 500 mg per day. ing.

The form of the intestinal barrier improving agent and the composition of the present invention can be arbitrarily selected depending on the conditions such as the active ingredient and the application such as liquid, solid, granular, granular, paste, and gel. Further, 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 product. It may be a pharmaceutical composition. The composition can be produced by a conventional method with a formulation appropriately combined with an excipient, a carrier and / or a diluent and the like and other components according to the dosage form.

The route of administration of the intestinal barrier improving agent and the composition of the present invention includes, but is not limited to, oral, nasal, enteral and the like.

Next, the present invention will be described in more detail by way of examples. The present invention is not limited thereto.

Sample preparation:
Each sample was prepared by the method described below.
(1) Preparation of D-amino acids For the samples of D-amino acids shown in Fig. 1 and L-amino acids shown in Fig. 2, a reagent manufactured by Nakaraitesk Co., Ltd. was added to PBS (pH 7.4, manufactured by Gibco Co., Ltd.) at a concentration of 50 mM. Prepared by dissolving in.
(2) Preparation of other components The components of the present invention and various polyphenols including myricetin, nobiletin, and tangeretin, whose intestinal barrier function is known, and the following 33 types of components in total are DMSO (manufactured by Fujifilm). Prepared by dissolving in 200 mM.

Cell culture:
Caco-2 (HTB-37), a human intestinal epithelial cell, was purchased from American Type Cell Culture (Rockville, MD, USA) and cultured under the standard conditions previously reported (Non-Patent Documents 2 and 3). Cells were seeded on a permeable polyester membrane of transwell inserts (12 mm diameter, 0.4 μM pore size; Corning, Cambridge Corning, Cambridge, MA, USA) at a density of ^{0.25 × 10 6} cells / cm ^{2 for all.} The test was conducted 14 days after sowing. Cells with passages between 55 and 65 were used 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.

Measurement of intestinal barrier function:
The intestinal barrier function was evaluated by measuring the TJ barrier function of the intestinal epithelium by transepithelial electrical resistance (TER) of Caco-2 monolayer cells of Transwell insert based on the description of Non-Patent Documents 8 and 9. The TER of monolayer cells was 1000-1300 Ω · cm ² (data not shown). On the day of the test, the test medium was changed to Hanks solution (glucose (+)) only for the test using L / D amino acids, and the test was carried out about 6 hours after that. 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 wells on the apical side, and the cells were incubated for 48 hours. This experiment was based on previously reported that 100 μM kaempferol, myricetin, and quercetin had a protective effect on TJ barrier function 48 hours after addition (Non-Patent Documents 2 and 3). TER was measured with the Millicell-ERS system (Millipore, Bedford, MA, USA) before and at 12, 24, and 48 hours after the addition of the compound.

Statistical analysis:
All values were expressed as mean ± SEM (n = 3). Moreover, the TER at each time point was expressed as a ratio (%) of the initial value before addition. Statistical analysis was performed by using the Dunnet test using 4Step Excel Statistics 4th Edition (OMS Publishing). P <0.05 (vs Control, Dunnet test) was regarded as significant and is indicated by * in the figure.

The results of D-amino acid are shown in FIG. 1, and the results of L-amino acid are shown in FIG. As can be seen in FIG. 1, it can be seen that in the Caco-2 cells to which the D-amino acid of the present invention was added, the TER was significantly higher than that of Control, and the intestinal barrier function was improved. Furthermore, it can be seen that the D-amino acid of the present invention is superior to D-alanine (D-Ala), which is known to have an effect of improving the intestinal barrier function. On the other hand, as is clear from FIG. 2, the addition of various L-amino acids did not significantly increase the TER of Caco-2 cells, and no significant improvement in the intestinal barrier function was observed.

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

From this example, it was confirmed that the present invention has a very excellent intestinal barrier function improving effect even when compared with a component having a known intestinal barrier function improving. Disorders of intestinal barrier function have been shown to affect various diseases and conditions, Patent Documents 1 to 6 and Non-Patent Documents 1 to 10). Therefore, the intestinal barrier improving agent of the present invention is effective for cosmetology such as treatment and / or prevention of various diseases caused by impaired intestinal barrier function and improvement of skin condition.

Claims (2)

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. Mogloside V, Humlon, 5,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,4', 7-trimethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7 An intestinal barrier comprising one or more components selected from the group consisting of, 3', 4'-tetramethoxyflavones, and 3,5,7,3', 4'-pentamethoxyflavones. Improving agent.

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