JP2020007255A - Intestinal barrier function improving agent - Google Patents

Abstract

To provide intestinal barrier function improving agents characterized by comprising Senna obtusifolia seeds.SOLUTION: Senna obtusifolia seed can provide excellent intestinal barrier function improving agent because of having a mucin production promoting action, antimicrobial peptide production promoting action, tight junction production promoting action. It can also provide an intestinal barrier function improving agent with a rough skin improving effect for internal use, or a food composition for improving intestinal barrier function, and can be used as quasi-drugs and medicines containing Senna obtusifolia seed having these effects.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to an intestinal barrier function-improving agent containing shrimp grass seeds. More specifically, the present invention relates to an intestinal barrier function enhancer whose intestinal barrier function improving action is promotion of mucin production, promotion of antimicrobial peptide production, and promotion of tight junction production. Further, the present invention relates to an intestinal barrier function-improving agent having a skin roughness improving effect for internal use, and a food composition for improving intestinal barrier function.

There is a monolayer of intestinal epithelial cells inside the intestinal tract, which is a part of the digestive tract, that is, on the lumen side through which food digests pass. Intestinal epithelial cells have not only an absorption function that takes in various nutrients in digested foods, but also a barrier function that prevents harmful microorganisms, toxins, and allergens from entering the living body, or a function that protects the living body called intestinal immunity. It is known. When the barrier function of the intestinal tract is disrupted, foreign substances such as bacteria, toxins, and allergens may penetrate and invade the living body, and may cause diseases such as inflammatory bowel disease, liver disease, and food allergy (Patent Document 1).

As the intestinal barrier function, mucus, antimicrobial peptides, and tight junctions are mainly known (Non-Patent Document 1).

The main component of mucus is a glycoprotein called mucin produced by intestinal epithelial cells. Mucus covering the mucous membrane of the digestive tract lumen is important as a protective factor against the invasion of foreign microorganisms and foreign substances by maintaining the lubricity of the mucosal surface. However, it is known that due to various stimuli in daily life such as tobacco, alcohol, various drugs, or stress, mucus generation becomes insufficient, mucin protecting the mucous membrane decreases, and the mucous membrane is damaged ( Patent Document 2). Therefore, increasing the amount of mucin produced by intestinal epithelial cells enhances the barrier function of the intestinal tract, and as a result, is important for exerting a biological defense function of protecting the living body from injury.

The antimicrobial peptide is a peptide produced by intestinal epithelial cells and having an antibacterial action, and is one of the biological defense functions for suppressing the growth of pathogenic microorganisms. Human-derived antimicrobial peptides belong to the defensin family (α-defensin, β-defensin, etc.), cathelicidin (LL-37), dermcidin, LEAP-1 (hepcidin), LEAP-2, and Regenerating Islet-derived family. Peptides (Reg peptides) are known. Increasing the amount of antimicrobial peptides produced by intestinal epithelial cells is important for suppressing the growth of pathogenic microorganisms, thereby enhancing the intestinal barrier function and exerting a biological defense function for protecting the living body from injury.

Tight junction is a protein produced by intestinal epithelial cells that not only adheres adjacent intestinal epithelial cells to each other, but also controls the permeation of substances by sealing the gap between cells, thus allowing the It is important to maintain the adhesion. For tight junctions produced by intestinal epithelial cells, a complex protein group composed of proteins such as transmembrane proteins occludin and claudin, TJP1, and TJP2 is known (Non-Patent Document 2). It is considered that when the tight junction function of the intestinal tract decreases, food allergens and pathogenic microorganisms invade the living body and contribute to inflammatory bowel disease and various infectious diseases. Increasing the production of tight junctions of intestinal epithelial cells is important for enhancing the intestinal barrier function, preventing inflammatory bowel diseases such as enteritis, or various infectious diseases, and exerting a biological defense function of protecting the living body from injury.

In addition, improving the intestinal barrier function by promoting mucin production, antimicrobial peptide production, and tight junction production promotes good bacteria such as bifidobacteria present in the intestine, resulting in a healthy intestinal flora. It is known that keeping diarrhea and constipation improves. Furthermore, by keeping the intestinal flora healthy, it is expected to have an effect of protecting the living body from various infectious diseases and an effect of improving rough skin, acne and pimples.

Ebisugusa has a scientific name of Cassia obtusifolia, and has recently been referred to as Senna obtusifolia. Ebisugusa seeds, called Kamekiko, have been used as a medicine to restore eyesight in China. It is also known as hub tea (Non-Patent Document 3). As pharmacological actions, adiponectin increase (Patent Document 3), periodontal disease preventing action (Patent Document 4), and the like are known, but the effect of improving the intestinal barrier function of Ebisugus seeds is not known.

JP 2014-210718 A JP-A-2006-169119 JP 2011-63542 A Japanese Patent No. 2986991

Kyoko Takahashi, Journal of Intestinal Bacteriology, 25, p. 213-219 (2011) Makoto Shimizu, Protein Nucleic Acid Enzyme, 44, p. 874-880 (1999) Nankodo, Tokyo Herbal Medicine Association, New Jyowa-Kankan Collection, p. 38-39 (1973)

An object of the present invention is to provide an intestinal barrier function-improving agent, characterized by containing shrimp grass seeds. In detail, an intestinal barrier function enhancer is an intestinal barrier function enhancer that promotes mucin production, antimicrobial peptide production, and tight junction production. It is to provide a food composition for improvement.

As the seeds of the ibis grass used in the present invention, sesame grass (Cassia obtusifolia or Senna obtusifolia), which is a leguminous plant, or seeds of Cassia tora or Senna tora, a closely related species, can be used. As the ibis grass seed, the Chinese medicine name is referred to as Kayoko, and the seed of a plant used for Kayoko can also be used.

The shrimp grass seed used in the present invention can be used as it is, and if necessary, a seed that has been subjected to a treatment such as drying, pulverization, and shredding can also be used. In addition, an extract obtained by extracting the shrimp grass seeds as they are or by subjecting them to the above treatment can also be used. As a solvent to be extracted, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene) Glycols, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, petroleum ether, etc.), ethers (ethyl ether, tetrahydrofuran, propyl) Ether, etc.). These solvents may be used alone or in combination of two or more. In the present invention, the extraction solvent is preferably a polar solvent such as water or a lower alcohol, particularly preferably water.

The above extract may be used as it is, or may be used, if necessary, after concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation and the like. Further, the extracted solution may be subjected to a treatment such as concentration and drying, spray drying and freeze drying, and used as a dried product.

The ingestion amount of the prawn seeds used in the present invention can be appropriately adjusted depending on the administration form, purpose of use, age, body weight, and the like. The daily intake of Ebisugrass seeds per adult is 0.1 to 1000 mg, preferably 1 to 100 mg, in terms of dry matter of Ebisugrass seeds, which can be taken orally once to several times a day. In some cases, an amount smaller than the above-mentioned intake range may be sufficient, and in other cases, it may be necessary to ingest beyond the range. The method of adding the medicinal component in the formulation may be added in advance or during the production, and may be appropriately selected in consideration of workability.

The intestinal barrier function improving agent of the present invention can be used as foods, quasi-drugs, and pharmaceuticals. As foods, it can be used as granules, tablets, hard capsules, soft capsules, drinks, jellies and the like. For quasi-drugs and pharmaceuticals, parenteral injections and suppositories such as oral powders, granules, tablets, sugar-coated tablets, capsules, syrups, pills, suspensions, solutions, and emulsions It can be used as such.

The intestinal barrier function-enhancing agent of the present invention may be used as it is, as long as it does not impair the effect, and, as long as the effect is not impaired, an ordinary food, a quasi-drug or an excipient used for pharmaceuticals, Components such as agents, preservatives, binders, disintegrants, hydrocarbons, fatty acids, alcohols, esters, pH adjusters, preservatives, and fragrances can also be included. Furthermore, it can contain components such as vitamins, saccharides, and proteins. In order to achieve the object of the present invention, it is desirable to contain components such as bifidobacteria, lactic acid bacteria, oligosaccharides, indigestible dextrin, and lactoferrin.

The intestinal barrier function-improving agent of the present invention can be used to suppress or restore a decrease in intestinal barrier function. Therefore, it can be used for preventing or ameliorating inflammatory bowel disease, Crohn's disease, ulcerative colitis and the like, which are diseases caused by a decrease in intestinal barrier function.

The intestinal barrier function-improving agent containing the shrimp grass seed of the present invention has a mucin production promoting action, an antibacterial peptide production promoting action, a tight junction production promoting action, and a rough skin improving action.

The present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. The percentages of the contents shown in the examples represent% by weight.

Hereinafter, an example of the production of a solvent extract using shrimp grass seeds will be described.

Production Example 1 Hot water extract of Ebisugusa seeds After adding 2000 mL of purified water to 100 g of crushed seeds of Ebisugusa and extracting the mixture at 95 to 100 ° C for 2 hours, the filtrate is concentrated and freeze-dried to give a hot water extract of Ebisugusa seeds. 7.0 g were obtained.

Production Example 2 Ebisugrass Seed 30% Ethanol Extract Purified water 1400 mL and ethanol 600 mL were added to 100 g of crushed Ebisugrass seed, extracted at room temperature for 5 days, and the filtrate was concentrated to dryness to give a 30% ethanol extract of Ebisugusa seed. 5.1 g were obtained.

Production Example 3 Ebisugusa seed 50% ethanol extract Purified water 1000 mL and ethanol 1000 mL were added to 100 g of a crushed Ebisugusa seed, extracted at room temperature for 5 days, and the filtrate was concentrated to dryness to give a 50% ethanol extract of Ebisugusa seed. 3.8 g were obtained.

Production Example 4 Ebisugrass Seed Ethanol Extract Ethanol 2000 ml was added to 100 g of crushed Ebisugrass seeds, extracted at room temperature for 7 days, and the filtrate was concentrated to dryness to obtain 1.2 g of Ebisugusa seed ethanol extract.

Formulation Example 1 Granule Formulation Content (%)
1. Shrimp seed hot water extract (Production Example 1) 0.05
2. Sucrose 50.00
3. Corn starch 49.95
[Production Method] Components 1 to 3 were granulated by fluidized bed granulation (spray liquid was water) to obtain granules.
[Usage] One packet (1.5 g) is ingested per day.

Formulation Example 2 Granule Formulation Content (%)
1. Shrimp grass seeds hot water extract (Production Example 1) 0.01
2. Bifidobacterium powder 10.00
3. Lactic acid bacteria powder 5.00
4. Milk oligosaccharide 50.00
5. Indigestible dextrin 33.99
6. Pullulan 1.00
[Production Method] Components 1 to 5 were granulated by fluidized-bed granulation (the spray liquid dissolves pullulan in water).
[Usage] One packet (1.5 g) is ingested daily.

Formulation Example 3 Tablet Formulation Content (%)
1. Ebisugusa seed 30% ethanol extract (Production Example 2) 0.1
2. Lactic acid bacteria powder 2.0
3. Milk oligosaccharide 30.0
4. Corn starch 55.9
5. Crystalline cellulose 10.0
6. Glycerin fatty acid ester 2.0
[Production method] Components 1 to 5 are mixed, 10% of water is added as a binder, extruded, granulated, and dried. Ingredient 6 is added to the molded granules, mixed and tableted. 0.5 g per tablet.
[Usage] Take 4 tablets daily.

Formulation Example 4 Powder Formulation Content (%)
1. Ebisugusa seed 50% ethanol extract (Production Example 3) 0.01
2. Bifidobacterium powder 10.00
3. Corn starch 25.00
4. Crystalline cellulose 64.99
[Production method] Components 1 to 4 are mixed to prepare a powder.
[Usage] One packet (3 g) is ingested daily.

Formulation Example 5 Jelly Formulation Content (%)
1. Ebisugrass seed ethanol extract (Production Example 4) 0.001
2. Milk oligosaccharide 1.000
3. Carrageenan 2.000
4. Gelatin 1.000
5. Sucrose 25,000
6. Purified water 70.999
[Production method] Components 1 to 6 are mixed, boiled under heating, poured into a jelly mold, and cooled.
[Usage] One (100 g) per day.

Formulation Example 6 Beverage Formulation Content (%)
1. Shrimp grass seeds hot water extract (Production Example 1) 0.01
2. Maltitol 5.00
3. Citric acid 1.00
4. Fragrance 0.50
5. Purified water 93.49
[Production method] Components 1 to 4 are dissolved in a part of component 5 with stirring. Add the rest of component 5 and mix, heat to 90 ° C. and fill into a 30 mL glass bottle.
[Usage] Ingest 1 bottle (30 mL) per day.

Test Example 1 Mucin, Antimicrobial Peptide and Tight Junction Production-Promoting Action of Intestinal Epithelial Cells on Intestinal Epithelial Cells After incubating intestinal epithelial cells Caco-2 for 14 days in DMEM containing 1% non-essential amino acid and 10% FBS, Ebisu seed hot water The extract (Preparation Example 1) was added. In the fluorescent immunostaining, cells were fixed with 4% paraformaldehyde after 24 hours from the addition of the sample, stained with tight junction (TJP2, manufactured by Bioss), and DAPI (manufactured by Roche), and the fluorescence intensity per cell number was compared. For gene expression analysis, a cDNA solution was prepared from RNA extracted 24 hours after the addition of the sample, and gene expression analysis was performed by a real-time PCR method using SYBR Green. Gene expression was analyzed for mucin (MUC2), antimicrobial peptide (REG1A), and tight junction (TJP2). At that time, the expression level was calculated from the ratio of the target gene to GAPDH using GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) as an internal standard. The primers used for gene expression analysis are as follows.

Primer set for MUC2 (SEQ ID NO: 1) CAATAACCTGCACCTGCAAGAG
(SEQ ID NO: 2) CCGTCAAAGTCGTAGTAGTTTCC
Primer set for REG1A (SEQ ID NO: 3) GTTTGATGCAGATCTCTTATTGCC
(SEQ ID NO: 4) GACATTTGAAGTCATCAGTGCC
Primer set for TJP2 (SEQ ID NO: 5) AAAGCAGGCGAACGAAGAG
(SEQ ID NO: 6) CGAAGATCTTGACTCCCAAGC
Primer set for GAPDH (SEQ ID NO: 7) TGCACCACCAACTGCTTTAGC
(SEQ ID NO: 8) TCTTCTGGGTGGCAGTGATG

Table 1 shows the results of the fluorescent immunostaining of Test Example 1. The addition of the hot water extract of Ebisu grass seeds increased the fluorescence intensity of TJP2, indicating that the hot water extract of Ebisu grass seeds had an effect of promoting the production of tight junctions.

Table 2 shows the results of the gene expression analysis of Test Example 1. The gene expression level of MUC2, REG1A, and TJP2 was increased by the hot water extract of Ebisugusa seeds.

In addition, the same effect was obtained in any case where the hot water extract of Ebisu grass seed of Test Example 1 was replaced with the E. ginseng seed extract of Production Examples 2, 3, and 4. From the above results, the effect of promoting mucin production, the activity of promoting the production of antimicrobial peptides, and the activity of promoting the production of tight junctions were observed in the seeds of Ebisugusa.

Test Example 2 Inhibitory Effect of Ebisugus Seeds on Intestinal Barrier Function on Intestinal Epithelial Cells Intestinal epithelial cells Caco-2 were seeded on a 12-well transwell and cultured for 14 days in DMEM containing 1% non-essential amino acids and 10% FBS. , TNFα and a hot water extract of Ebisugrass seed were added, and 72 hours later, transepithelial electric resistance (TER) was measured as an index of the barrier function. In gene expression analysis, cells were collected 24 hours after the addition of TNFα and the hot water extract of Ebisugusa seeds, and analyzed for tight junctions (TJP2) and inflammatory cytokines (IL-6) in the same manner as in Test Example 1. The primers used for gene expression analysis are as follows.

Primer set for TJP2 (SEQ ID NO: 5) AAAGCAGGCGAACGAAGAG
(SEQ ID NO: 6) CGAAGATCTTGACTCCCAAGC
Primer set for IL-6 (SEQ ID NO: 9) ATGGCTGAAAAAGATGGATGCT
(SEQ ID NO: 10) GCTCTGGCTTGTTCCTCACTACTC
Primer set for GAPDH (SEQ ID NO: 7) TGCACCACCAACTGCTTTAGC
(SEQ ID NO: 8) TCTTCTGGGTGGCAGTGATG

Table 3 shows the results of the transepithelial electric resistance in the case where TNFα or TNFα + Ebisugusa seed hot water extract was added in Test Example 2 where the unadded transepithelial electric resistance was 100. Shrimp grass seed hot water extract increased transepithelial electrical resistance reduced by TNFα. From the above results, it was clarified that Ebisugusa seeds have an excellent inhibitory action on intestinal barrier function decrease.

Table 4 shows the results of the gene expression analysis in Test Example 2. The suppression of TJP2 decrease by TNFα and suppression of increase of IL-6 were observed by the hot water extract of Ebisugusa seeds.

Test Example 3 Intestinal barrier function improving effect of Ebisu grass seeds using mice Five-week-old male Balb / c mice were divided into two groups, a control group and a Ebisu grass seed administration group. The seed group was allowed to freely ingest a powdered feed containing 1.2% of a hot water extract of Ebisu grass seeds for 14 days. RNA was extracted from large intestine tissue, and gene expression analysis was performed in the same manner as in Test Example 1. The gene expression was analyzed for mucin (MUC2), antimicrobial peptide (REG1A), and tight junction (TJP2, CLDN4), and the expression level was calculated from the ratio of the target gene and GAPDH as an internal standard. The primers used for gene expression analysis are as follows.

Primer set for MUC2 (SEQ ID NO: 11) CACTCCCTCTTCACCCTCCAG
(SEQ ID NO: 12) GCCCTGTTGTGGTCTTTTGAG
Primer set for REG1A (SEQ ID NO: 13) GCAGTGGGTCTCTCGTTTTCTC
(SEQ ID NO: 14) CTTGTATCCTGTGTTTGAAGTCAG
Primer set for TJP2 (SEQ ID NO: 15) AAGGAAAGATGGGAAGGGATGG
(SEQ ID NO: 16) TGTCTTCAAAGTCACTGATGAG
Primer set for CLDN4 (SEQ ID NO: 17) GTCATCAGCATCATCGTGGG
(SEQ ID NO: 18) CCACCATAGGGTTGTAGAAGTC
Primer set for GAPDH (SEQ ID NO: 19) GTCATCAGCATCATCGTGGG
(SEQ ID NO: 20) CCACCATAGGGTTGTAGAAGTC

Table 5 shows the results of the gene expression analysis of Test Example 3. The gene expression level of MUC2, REG1A, TJP2, and CLDN4 was increased by the hot water extract of Ebisugusa seeds.

Test Example 4 Suppression of Intestinal Barrier Function Reduction of Ebisugrass Seeds Using a DSS Colitis Model A DSS (dextran sulfate sodium) -induced mouse was used as a model for inflammatory bowel disease. Dextran sodium sulfate (manufactured by MP Biochemicals) was dissolved in water and ingested to 5-week-old male Balb / c mice. In the test, three groups were provided: a control group, a colitis group, and a colitis + shrimp seed group. The colitis + Ebisugrass seed group was allowed to freely ingest a powdered feed mixed with a 1.2% Ebisugrass seed hot water extract 5 days before DSS administration. From the day after the DSS administration, the body weight was measured once a day, and the stool properties were visually evaluated for score. The score of the stool properties was 0 point (normal), 1 point (loose stool), 2 points (diarrheal stool), 3 points (watery stool) for diarrhea, and 0 point (normal), 1 point (blood mixed with stool) 2 for bloody stool. Points (blood adhered to anus) 3 points (always bleeding from anus). The sum of the diarrhea and bloody stool scores was calculated as the colitis score per individual. On the 9th day of DSS administration, the length of the large intestine tissue was measured and defined as the large intestine length. In addition, gene expression analysis of large intestine tissue was performed in the same manner as in Test Example 1. Tight junctions (TJP2, CLDN4) and inflammatory cytokines (IL-6, TNFα) were analyzed, and the target gene and the internal standard GAPDH were analyzed. The expression level was calculated from the ratio.

Primer set for TJP2 (SEQ ID NO: 15) AAGGAAAGATGGGAAGGGATGG
(SEQ ID NO: 16) TGTCTTCAAAGTCACTGATGAG
Primer set for CLDN4 (SEQ ID NO: 17) GTCATCAGCATCATCGTGGG
(SEQ ID NO: 18) CCACCATAGGGTTGTAGAAGTC
Primer set for IL-6 (SEQ ID NO: 21) ACTCCCAACAGACCTGTCTATACCA
(SEQ ID NO: 22) TCCACGATTTCCCAGAAGACA
Primer set for TNFα (SEQ ID NO: 23) GCCAGCCGATGGGGTTGTA
(SEQ ID NO: 24) GGCAGCCCTTGTCCCTTTGA
Primer set for GAPDH (SEQ ID NO: 19) TGGAGAAACCTGCCAAGTATAG
(SEQ ID NO: 20) CCCTCAGATGCCTGCTTTCA

Table 6 shows the results of weight change, Table 7 shows the results of colitis score, and Table 8 shows the results of colon length in Test Example 4. Ingestion of the hot water extract of Ebisugusa seeds showed the effects of suppressing weight loss, suppressing colitis score, and reducing colon length in DSS-induced colitis mice.

Table 9 shows the results of the gene expression analysis of Test Example 4. Ingestion of the hot water extract of Ebisu grass seed suppressed the reduction of TJP2 and CLDN4 and the increase of IL-6 and TNFα in DSS-induced colitis mice. From the above results, it can be expected that the seed of Ebisugusa has the effect of suppressing the decrease in tight junction production and the effect of suppressing inflammation, and the effect of suppressing the intestinal barrier function of the seed of Ebisugusa and preventing colitis.

Test Example 5 Effect of Improving Roughness of Ebisugrass Seeds Twenty-two men and women suffering from rough skin were ingested a granule containing the hot water extract of Ebisugrass seed of Formulation Example 1 for 3 months. As a result, the keratin moisture content of the skin and the ability to retain epidermal moisture increased. Further, when a questionnaire survey was conducted on the skin roughness improving effect, as shown in Table 10, an extremely excellent skin roughness improving effect was recognized.

The present invention provides an intestinal barrier function-improving agent containing a seed of prawn and having an effect of promoting mucin production, promoting antimicrobial peptide production, and promoting tight junction production. In addition, it can provide an intestinal barrier function improving agent having an action of improving rough skin for internal use, or a food composition for improving intestinal barrier function, and can be used for foods, quasi-drugs, and pharmaceuticals containing shrimp seeds having these effects. Available.

Claims (6)

An intestinal barrier function-improving agent, characterized by containing shrimp seeds. 2. The intestinal barrier function improving agent according to claim 1, wherein the intestinal barrier function improving effect is a mucin production promoting effect. The intestinal barrier function improving agent according to claim 1, wherein the intestinal barrier function improving effect is an antibacterial peptide production promoting effect. The intestinal barrier function improving agent according to claim 1, wherein the intestinal barrier function improving effect is a tight junction production promoting effect. An intestinal barrier function enhancer for internal use, which has a skin roughening improving effect, which comprises a lobster seed. A food composition for improving intestinal barrier function, comprising the intestinal barrier function improving agent according to any one of claims 1 to 5.