JP2013063941A - Prophylactic or improving agent of irritable bowel syndrome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a prophylactic or improving agent of irritable bowel syndrome not accompanying inflammations such as stress-related large intestine hyperkinesia and stress-related large intestine hyperesthesia.SOLUTION: The prophylactic or improving agent of irritable bowel syndrome contains one or more selected from a grape seed, cordyceps, a peanut seed coat, and extracts thereof as active ingredients.

Description

  The present invention relates to a preventive or ameliorating agent for irritable bowel syndrome.

  Diarrhea includes non-inflammatory diarrhea without inflammation, such as irritable bowel syndrome (IBS) and functional diarrhea, in addition to inflammatory diarrhea such as infection with pathogenic microorganisms and inflammatory bowel disease. It is believed that stress is greatly involved in the development of these non-inflammatory diarrhea, and it has been reported that stress exacerbates the symptoms (Non-Patent Documents 1-3). In recent years, stress has been reported to cause increased colonic motility (diarrhea) and hypersensitivity of the large intestine through corticotropin-releasing factor (CRF) secretion in the hypothalamic paraventricular nucleus (Non-patent Document 4).

  Conventionally, as a method for treating such irritable bowel syndrome, symptomatic therapy for alleviating symptoms such as diarrhea has been carried out as drug therapy in addition to psychotherapy and diet therapy. For example, serotonin (5-HT) receptor antagonists such as Ondansetron, opioid receptor agonists such as Trimebutine and Loperamide have been administered. Modulation of receptors in the myenteric plexus improves colonic motility (diarrhea), and pharmacotherapy for controlling the cause has not yet been established.

  On the other hand, grape seeds contain proanthocyanidins and have anti-allergic and anti-inflammatory effects (Patent Document 1), Cordyceps has immune regulation and blood pressure lowering effects (Patent Document 3), peanuts (peanuts) It has been reported that the seed coat has a collagenase activity inhibitory action (Patent Document 1).

  However, it is not known at all that grape seeds, cordyceps and peanut seed coats are effective in preventing or ameliorating irritable bowel syndrome.

JP 2001-278792 A JP 2002-145757 A JP-A-8-157388

Minerva Med. 2004; 95: 443-450. Gut 2004; 53: 1102-1108 Curr Psychiatry Rep. 2004; 6: 210-215 Irritable bowel syndrome In search of brain-intestinal interaction 2006; 9-15

  The present invention relates to providing a preventive or ameliorating agent for irritable bowel syndrome that is not accompanied by inflammation such as stressed colonic hyperactivity and stressful colonic hypersensitivity.

  As a result of examining the influence of stress on colon function, the present inventor has found that degranulation of mast cells in the large intestine is involved in stress-induced decrease in colon function without inflammation, and mast cell stabilization and stress properties. It has been clarified that there is a positive correlation between improvement of colonic hyperactivity and improvement of colonic hypersensitivity (see Japanese Patent Application No. 2010-053066, Reference Examples below). As a result of further research, it was found that grape seed extract, cordyceps extract and peanut seed coat extract have a mast cell stabilizing action and a colonic hypersensitivity improving action, which is an irritable bowel syndrome. It was found useful for the prevention or improvement of

That is, the present invention relates to the following 1) -2).
1) A preventive or ameliorating agent for irritable bowel syndrome comprising one or more selected from grape seeds, cordyceps, peanut seed coats and extracts thereof as active ingredients.
2) The preventive or ameliorating agent according to 1) above, wherein the irritable bowel syndrome is stressful colonic hyperactivity or stressful colonic hypersensitivity.

  According to the present invention, food, medicine, quasi-drug, feed, or a combination thereof can be used which can prevent or ameliorate irritable bowel syndrome such as stressed colonic hyperactivity and stressful colonic hypersensitivity. Material can be provided.

The graph which shows the influence which stress load has on large intestine perception. The graph which shows the effect of the mast cell stabilizer with respect to stress-related large intestine hypersensitivity.

In the present invention, “grape seeds” include grapes belonging to the genus Grapeaceae (V. vinifera, V. labursca, V. amurensis, V. coignetiae, V. rupestris, V. riparia, V. berlandieri, V. champini). Etc.) seeds.
“Peanut seed coat” includes seed coat of leguminous peanut (Arachis hypogaea LINNE), and the seed includes astringent skin, thin skin, cuticle, and the like.
Cordyceps sinensis, Cordyceps militaris, Cordyceps nutans, Cordyceps sobolifera, Cordyceps sobolifera in the genus Cordyceps. And other species are included. Any species can be used in the present invention, but Cordyceps sinensis parasitic on larvae (Hepialus armoricanus) is preferred. It is preferable to use the fruit body or the complex thereof for the cordyceps.
Such grape seeds, cordyceps and peanut seed coats can be used as dried or pulverized products thereof, or extracts thereof, but are preferably used as extracts.

As the extract, various solvent extracts obtained by known extraction methods such as extracting grape seeds, cordyceps or peanut seed coats at room temperature or under heating or using an extraction device such as a Soxhlet extractor. , A diluted solution thereof, a concentrated solution thereof or a dried powder thereof.
Known extraction methods include, for example, immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, and microwave extraction.

  Examples of the extraction solvent include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate, ethyl acetate, and the like. Esters of: chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether These may be used singly or in combination of two or more, and may be repeated by changing the solvent. Among these, it is preferable to use water; alcohols such as methanol, ethanol, propanol and butanol, and it is particularly preferable to use water and a mixed solution of ethanol, for example, 20 to 80 v / v% ethanol water.

  Extraction uses, for example, 1 to 10 parts by mass of a solvent with respect to 1 part by mass of grape seed, cordyceps or peanut seed coat, and is 0 to 70 ° C., preferably 10 to 30 ° C. for several hours to several weeks, preferably 12 hours to It can be carried out by immersion or heating under reflux for 2 days. It can also be obtained by steam distillation.

  The above extract can be used as it is, but it can also be used by diluting, concentrating or lyophilizing the extract and preparing it in a powder or paste form.

  Moreover, you may use the plant of this invention, or its extract in mixture of 2 or more types. Moreover, you may use a commercial item other than the said extraction processed material.

  The extract is preferably used after removing inactive impurities by a known technique such as liquid-liquid distribution, solid-liquid distribution, filtration membrane, activated carbon, absorption resin, ion exchange resin and the like. As the solvent used at this time, those exemplified for the extraction solvent may be used. Moreover, you may use these, after giving processes, such as a deodorizing and a decoloring, by a well-known method as needed. Moreover, when the extract is further purified, the known techniques and methods may be used.

Irritable bowel syndrome (IBS) generally has symptoms of abdominal pain or abdominal discomfort mainly in the lower abdomen, bowel movements such as constipation or diarrhea, and improvement of abdominal symptoms due to defecation. It is defined as a disease in which there are no organic diseases or blood biochemical abnormalities that can be explained, and symptoms persist chronically (Japanese Clinical 2006; 8: 1415). Such irritable bowel syndrome is not accompanied by inflammation, and is distinguished from inflammation-associated diarrhea and hypersensitivity, that is, inflammatory diarrhea and hypersensitivity such as infection with pathogenic microorganisms and inflammatory bowel disease. From this point of view, the irritable bowel syndrome (IBS) in the present invention may be referred to as non-inflammatory irritable bowel syndrome.
When stress is applied, corticotropin-releasing factor (CRF) is released in the brain. It is considered that CRF stimulates the sacral parasympathetic nerve and stimulates acetylcholinergic neurons in the myenteric plexus to cause constriction of the cricoid muscles (Japanese clinical 2006; 64: 1406). 1408). At the same time, stress is thought to cause colonic hypersensitivity through the secretion of CRF (Non-Patent Document 4). As described above, the irritable bowel syndrome of the present invention includes increased colonic motility (diarrhea) and colonic hypersensitivity caused by physical or mental stress. In addition, for diseases conventionally referred to as convulsive colon, convulsive colitis, neurocolitis, neurological diarrhea, mucinous colitis, functional colitis, colonic neurosis, irritated colon syndrome, inflammation, etc. These are included unless accompanied by.
As described above, stress-induced decrease in colon function without inflammation is related to degranulation of mast cells in the large intestine, stabilizing mast cells, improving stress-induced hyperactivity of the colon, and improving colon hypersensitivity. Has a positive correlation (see Japanese Patent Application No. 2010-053066, Reference Examples below).

Grape seed extract, cordyceps extract and peanut seed coat extract stabilize mast cells and significantly reduce the AWR score in the colon expansion test using rats, as shown in the Examples below. Has the effect of improving hypersensitivity.
Therefore, the grape seed, cordyceps, peanut seed coat or an extract thereof of the present invention can be used for the prevention or amelioration of irritable bowel syndrome by ingestion or administration to animals including humans, and non-inflammatory hypersensitivity. It can be a preventive or ameliorating agent for bowel syndrome.

The preventive or ameliorating agent for irritable bowel syndrome may itself be a human or veterinary drug or quasi-drug for the prevention or amelioration of irritable bowel syndrome, or the drug, pharmacy It may be a material or a preparation used by blending with a foreign product, food or feed.
The food also has the concept of prevention or improvement of irritable bowel syndrome, prevention or improvement of stressed colonic hyperactivity, prevention or improvement of stressful colonic hypersensitivity, etc. Functional foods such as food, food for the sick or food for specified health use are included.

Examples of the dosage form of the above-mentioned pharmaceutical containing the grape seed, cordyceps, peanut seed coat or extracts thereof according to the present invention include tablets, capsules, granules, powders, syrups, enteric solvents, troches, drinks, etc. Oral administration or parenteral administration by injection, suppository, percutaneous absorption agent, external preparation and the like can be mentioned.
In order to prepare pharmaceutical preparations of such various dosage forms, the grape seeds, cordyceps, peanut seed coats or extracts thereof of the present invention alone or other pharmaceutically acceptable excipients ( Sorbitol, glucose, lactose, dextrin, starch and other sugars, calcium carbonate and other inorganic substances, crystalline cellulose, distilled water, sesame oil, corn oil, olive oil, rapeseed oil, etc.), binders, lubricants, extenders, disintegrants, interfaces Use an appropriate combination of activators, lubricants, dispersants, suspending agents, emulsifiers, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, antioxidants, bacterial inhibitors, etc. Can do.
Among these dosage forms, the preferred form is oral administration, and the content of the grape seed, cordyceps, peanut seed coat of the present invention or the extract thereof in the preparation for oral administration is generally 0.001 to 50 mass. %, Preferably 0.01 to 10% by mass.

Examples of the form of the food containing the grape seeds, cordyceps, peanut seed coats or extracts thereof of the present invention include processed foods such as bread, noodles, etc., processed rice foods such as rice cakes and cooked rice, biscuits , Cakes, jellies, chocolate, rice crackers, ice cream confectionery, tofu, processed foods such as processed foods, soft drinks, fruit juices, milk drinks, carbonated drinks, yogurt, cheese, butter, milk Dairy products such as soy sauce, sauce, miso, mayonnaise, dressing, meat storage such as ham, bacon, sausage, meat storage processed food, marine processed food such as hampen, chikuwa, canned fish, cooking oil and frying oil Etc. In addition, oral enteral nutritional foods such as capsules, concentrated liquid foods, natural liquid foods, semi-digested nutritional foods, ingredient nutritional foods, drink nutritional foods, etc. It is also possible to adopt a form such as
Examples of the feed include feed for small animals used for rabbits, rats, mice and the like, feed for pet foods used for dogs, cats, small birds, squirrels, and the like.
In order to prepare various forms of food and feed, the grape seeds, cordyceps, peanut seed coats or extracts thereof of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives Fragrances, stabilizers, colorants, antioxidants, humectants, thickeners and the like can be used in appropriate combinations. The content of the grape seeds, cordyceps, peanut seed coats or extracts thereof of the present invention in the food is generally preferably 0.001 to 50% by mass, preferably 0.01 to 10% by mass as the solid content concentration. % Is more preferable.

The intake / dosage of the grape seeds, cordyceps, peanut seed coats or extracts thereof of the present invention in the above-mentioned pharmaceuticals, quasi drugs, foods or feeds are not particularly limited as long as an effect is obtained. In addition, the intake / dose may vary according to the condition, weight, sex, age or other factors of the subject, but the daily administration / intake per adult (60 kg) may be the grape of the present invention. As seeds, cordyceps, peanut seed coats or extracts thereof (in terms of dry matter), for example, it is preferably 0.001 to 10,000 mg, more preferably 0.01 to 5000 mg, and particularly preferably 0.3 to 3000 mg. The preparation can be ingested / administered according to an arbitrary ingestion / administration plan, but is preferably divided into once to several times a day and continuously ingested / administered for several weeks to several months.
In addition, the subject of ingestion or administration of the above-mentioned pharmaceutical, quasi-drug or food is not particularly limited as long as it is required, such as irritable bowel syndrome, stressed colonic hyperactivity (diarrhea), and colonic hypersensitivity. Humans and non-human mammals for the purpose of prevention or improvement or treatment are preferred. In addition, the said subject person also includes the person who has the symptom of irritable bowel syndrome, the person who may be there, and the person who expects the prevention of the disease and symptom.

Reference example 1
<Analysis of the effects of stress on blood components>
1. Method Four groups of male Wistar rats (9-10 weeks old: CLEA Japan) were raised in an assembly cage under CE-2 diet (CLEA Japan) free feeding for 7 days, and there was no weight difference between the groups. Into groups (N = 10 / group). Group 2 was reared under free feeding and group 2 was fasted for 20 hours.
In each of the free feeding group and the fasting group, the shoulder, forelimb and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and a part of the forelimb movement was restricted (partial restraint stress, stress group). After standing in an individual cage for 2 hours under wakefulness, the animals were released from fixation. In the control group, one group each of a free feeding group and a fasting group was treated with anesthesia by inhalation of isoflurane, and then left in an individual cage for 2 hours under awakening.
Blood was collected from the abdominal aorta under anesthesia, and blood histamine, serotonin, (ELISA method: H
isamine EIA Kit; Oxford Biomedical Research, Serotonin ELISA; DRG), CRF amount (ELISA method: Mouse / Rat CRF-HS_ELISA Kit, Yauchihara Institute), blood cytokine amount (IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, TNF-α) (microbead array: Millipore) were each quantified.

Male Wistar rats (9 weeks old: Japan Claire) were reared in a collection cage for 7 days under free feeding of CE-2 diet (Japan Claire) and then divided into 2 groups so that there was no weight difference between each group (N = 3 / group).
In the stress group, the shoulders, forelimbs and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and some movements of the forelimbs were restricted. After standing in an individual cage for 2 hours under wakefulness, the animals were released from fixation. After euthanasia by whole blood collection from the abdominal aorta under anesthesia, 7 sections of the colon (1 cm downstream from the cecal colonic transition area and 1 cm length divided into 7 parts) were removed, and each section was 0.5 mL maintenance P / Ringer. Incubated in solution (60 μM) for 60 minutes. The supernatant was collected, and the amount of histamine released from the colon tissue was quantified by ELISA (Histamine EIA Kit; Oxford Biomedical Research).

2. Results When partial restraint stress was applied, the blood IL-4, IL-6, and IL-13 levels tended to be higher in both fasting and non-fasting compared to the control group (no stress loading) (Table 1). ). The amount of IL-1 and IL-2 in the blood at the time of fasting tended to be low in the stress load group. Other cytokines and CRF levels were not different between the two groups.
When partial restraint stress was applied, the blood histamine level (Table 2) was significantly higher than the control group (no stress load), and the blood serotonin level tended to increase (Table 2). Moreover, the amount of histamine released from the excised colon by stimulation with Substance P was significantly high (Table 3).
In addition, each point was shown by the average +/- standard error, and Student's t-test with respect to the control group was performed about the statistically significant difference between groups (* p <0.05).

Reference example 2
<Analysis of the effect of stress on large intestine exercise>
1. Method Male Wistar rats (9-10 weeks old: Japan Claire) were reared in an assembly cage for 7 days under free feeding of CE-2 diet (Japan Claire), and then 2 groups so that there was no difference in body weight between groups. Into groups (N = 10 / group). In group 1 (stress group), under isoflurane inhalation anesthesia, the shoulders, forelimbs, and chest were lightly fixed with tape to limit some of the forelimb movements. It was left to stand in an individual cage for 2 hours under awakening, and the number of excreted feces during that time was measured (Williams CL. Et al. Gastroenterology 1988; 94: 611-621). The number of feces excreted is an index of large intestine movement. The control group was subjected to isoflurane inhalation anesthesia and then left in an individual cage for 2 hours under awakening.
Male WsRC-+ / + (wild-type) rats, WsRC-Ws / Ws (mast cell deficient) rats (9-12 weeks old: Japan SLC) in a collective cage under CE-2 diet (Japan Claire) free feeding Then, the animals were divided into groups so that there was no difference in body weight between the groups (N = 9 animals / group, average body weight 260 g).
In the stress group, the shoulders, forelimbs and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and some movements of the forelimbs were restricted. It was left in an individual cage for 2 hours under awakening, and the number of excreted feces during that period was measured.

2. Results When the Wistar rats were subjected to partial restraint stress, the number of excreted feces was significantly increased as compared to the control group (no stress load) (Table 4).
In wild-type (+ / +) rats, the number of excreted feces was significantly increased by partial restraint stress loading compared to the control group (no stress loading) (Table 5). This increase in the number of defecations due to stress loading was not observed in mast cell-deficient (Ws / Ws) rats.
In addition, each point was shown by the average +/- standard error, and Student's t-test with respect to the control group was performed about the statistically significant difference between groups (*** p <0.001).

Reference Example 3 Effect analysis of stress on large intestine perception <Influence analysis of stress on large intestine perception>
1. Method Male Wistar rats (8-10 weeks old: Japan Claire) fasted for 20 hours were grouped in a cage so that there was no difference in body weight between each group of CE-2 diet (Japan Claire) (N = 8 / Swarm).
In the stress group, the shoulders, forelimbs and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and some movements of the forelimbs were restricted. After standing in an individual cage for 2 hours under wakefulness, the animals were unfixed and colon perception was evaluated by a colonic dilatation test using the previously reported method (Winston J et al. Gastroenterology 2007; 132: 615-627). . That is, a balloon catheter (silicone Foley catheter, 2.0 mm (6 Fr), Toray Medical) was inserted 4 cm transanally into the large intestine of anesthetized rats. After awakening, the large intestine was gradually expanded by water injection into the balloon catheter in the measurement cage. The “Abdominal Withdrawal Reflex (AWR) score” in each expansion capacity shown below was evaluated blindly, and colonic perception was evaluated semi-quantitatively.
Male WsRC-+ / + (wild type) rats, WsRC-Ws / Ws (mast cell deficient) rats (9-13 weeks old: Japan SLC), which were fasted for 20 hours, were collected in a CE-2 diet (Japan). Claire) After free-feeding, the animals were divided into groups (N = 8-12 / group, average body weight 250 g) so that there was no difference in body weight between groups.
Under isoflurane inhalation anesthesia, the shoulders, forelimbs and chest were lightly fixed with tape to limit some of the forelimb movements. After standing in an individual cage for 2 hours under wakefulness, the restraint of the animal was released, and colonic perception was evaluated by a colonic dilatation test.

≪Abdominal Withdrawal Reflex (AWR) Score≫
0: no behavioral response (no change)
1: brief head movements followed by
immobility (moves the head and then stops)
2: construction of abdominal muscle
without lifting of abdomin (abdominal muscle contraction)
3: Lifting of abdome (raise the abdomen)
4: body arching and lifting of
pelvic structure (archs the body and lifts the pelvis)

2. Results When Wistar rats were subjected to partial restraint stress, they showed a large AWR response with less expansion capacity compared to the control group (no stress loading) (FIG. 1).
Wild type (+ / +) rats had significantly higher AWR scores in each expansion volume due to partial restraint stress loading compared to the control group (no stress loading) (FIG. 1). This increase in AWR response due to stress loading was not observed in mast cell-deficient (Ws / Ws) rats (FIG. 1).
In addition, each point is shown by the average +/- standard error, and about the statistical significance difference between groups, Mann-Whitney U test with respect to the control group was performed (* p <0.05, ** p <0.01, *** p <0.001).

Reference example 4
<Effects of various drugs on stress-induced colonic hyperactivity>
1. Method Male Wistar rats (9 weeks old: Japan Claire) were reared in an assembly cage for 5 days under free feeding of CE-2 diet (Japan Claire), and then grouped so that there was no weight difference between the groups (N = 10 / group).
The stress group consists of various drugs (suspended in methylcellulose aqueous solution (Wako Pure Chemical Industries, 0.5 w / v% methylcellulose 400 solution, 0.5 w / v%)) or methylcellulose 0.5 under isoflurane inhalation anesthesia. A w / v% aqueous solution was administered intragastrically (4 mL / kg body weight), and left in an individual cage for 1 hour under awakening. In the control group, a 0.5 w / v% aqueous solution of methylcellulose was intragastrically administered (4 mL / kg body weight). In the stress group, the shoulder, forelimbs, and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and some movement of the forelimbs was restricted. It was left in an individual cage for 2 hours under awakening, and the number of excreted feces during that period was measured.

2. Results In the drug non-administration group (methylcellulose administration), the number of feces excreted significantly increased compared to the control group due to partial restraint stress load. Before 5-HT3 receptor antagonist, mast cell stabilizer, doxantrazole, 5-HT4 receptor agonist, opioid receptor agonist, opioid receptor agonist When administered, the increase in the number of defecations due to stress load was significantly suppressed (Table 7).
In addition, each point was shown by the average +/- standard error, and about the statistically significant difference between groups, Scheffe test was performed (* p <0.05, *** p <0.001).

Reference Example 5
<Effects of various drugs on steady-state colonic motility>
1. Method Male Wistar rats (9 weeks old: Japan Claire) were reared in an assembly cage for 5 days under free feeding of CE-2 diet (Japan Claire), and then grouped so that there was no weight difference between the groups (N = 5 / group).
Various drugs shown in Table 6 were administered into the stomach (4 mL / kg body weight), and were raised in an individual cage for 12 hours (dark period) under arousal, and the number of excreted feces during that period was measured. In the control group, a 0.5 v / w% aqueous solution of methylcellulose was intragastrically administered (4 mL / kg body weight).

2. Results When the opioid receptor agonist (loperamide) was pre-administered, the number of feces excreted was significantly smaller than that in the control group (methylcellulose administration) (Table 8). On the other hand, mast cell stabilizer, doxantrazole, 5-HT3 receptor antagonist (ondansetron), 5-HT4 receptor agonist (mosapride), and opioid receptor agonist (trimebutine) have an effect on the number of excreted feces I couldn't.
In addition, each point was shown by the average +/- standard error, and Scheffe test was performed about the statistically significant difference between groups (*** p <0.001).

Reference Example 6
<Effects of mast cell stabilizer on stress-induced colorectal hypersensitivity>
1. Method Male Wistar rats (8-10 weeks old: Japan Claire) fasted for 20 hours were grouped in a cage so that there was no difference in body weight between each group of CE-2 diet (Japan Claire) (N = 8 / Swarm).
In the mast cell stabilizer administration group, under isoflurane inhalation anesthesia, mast cell stabilizer (doxantrazole, suspended in methylcellulose solution) was intragastrically administered (5 mg / kg body weight), and at the same time, the shoulder, forelimbs and chest were lightly taped. Fixed and limited some forelimb movement. In the control group, methylcellulose 0.5 w / v% aqueous solution was intragastrically administered (4 mL / kg body weight), and at the same time, the shoulders, forelimbs and chest were lightly fixed with tape to restrict a part of the forelimb movement. After standing in an individual cage for 2 hours under wakefulness, the animals were released from the fixation, and colonic perception was evaluated by a colonic dilatation test as in Reference Example 3.

2. Results In the Doxantrazole-treated rats, the AWR score in each expansion volume was significantly lower than that in the control group (methylcellulose administration) (FIG. 2).
In addition, each point is shown by the average +/- standard error, and about the statistical significance difference between groups, Mann-Whitney U test with respect to the control group was performed (** p <0.01, *** p <0. 001).

  From the above results, it was considered that the stabilization of mast cells contributes to the improvement of stress-induced hyperactivity of the large intestine and the improvement of stress-induced hypersensitivity of the large intestine.

Reference Example 7
<Construction of screening system for mast cell stabilizer>
1. Methods After growing rat basophilic leukemia cells RBL-2H3 cells (ATCC) in MEM medium containing 10% fetal bovine serum (FBS), penicillin (100 units / mL), streptomycin (100 μg / mL). 24-well plate, RBL-2H3 cells were seeded at 8 × 10 ³ cells / well (N = 3), 10% fetal bovine serum (FBS), penicillin (100 units / mL), streptomycin (100 μg / ML) in RPMI 1640 medium for 3 days. After washing twice with PBS (−), incubation was performed for 15 minutes with compound 48/80 (0 to 150 μg / mL phenol red, serum, and antibiotic-free RPMI medium).
Similarly, RBL-2H3 cells cultured for 3 days in RPMI 1640 medium were incubated at room temperature for 30 minutes in RPMI medium containing doxantrazole (0-400 μM), and then doxantrazole (0-400 μM) +15 μg / mL compound 48/80 Incubated for 15 minutes at room temperature in RPMI medium (no phenol red, serum, antibiotics).
50 μL of culture supernatant from each well, then 500 μL of 0.1% T in each well
Riton X-100 was added to lyse the cells, and 50 μL each was collected. To this, 50 μL of 0.2 M citrate buffer (pH 4.5) containing 2 mM p-nitrophenyl N-acetyl-β-D-glucosamine, which is a substrate of β-hexosaminidase, was added and reacted at 37 ° C. for 2 hours. It was. After completion of the reaction, after stopping with 150 μL 1M Tris buffer (pH 9.0), the absorbance at 405 nm was measured with a microplate reader, the intracellular and extracellular β-hexosaminidase activity was measured, and β-hexosaminidase was measured. Secretion (degranulation) (OD supernatant / (OD supernatant + OD cell lysate) × 100 (%)) was calculated.

2. Results Compound 48/80 promoted degranulation in a concentration-dependent manner (Table 9). doxantrazole inhibited degranulation in a concentration-dependent manner (Table 10).
In addition, each point is shown by the average +/- standard error, and Fisher's PLSD was performed about the statistically significant difference between groups (** p <0.01, *** p <0.001).

Example 1 Preparation of various extracts (1) Grape seed extract A water-containing ethanol extract of Chinese grapevine (Vitis vinifera) seeds (product name grape seed extract (manufactured by Ningbo Green Health)) was mixed with 50% ethanol water. The test sample was dissolved at a concentration of 1% (w / v).
(2) Peanut seed coat extract Arachis hypogaea seed coat extract (product name "peanut seed coat extract", manufactured by Tokiwa Phytochemical Research Institute) in 1% concentration (w / v) in 50% ethanol water ) To obtain a test sample.
(3) Cordyceps extract Extract concentration of cordyceps (a fruiting body or insect body of a bat larva infected with Fuyumushinakusatake) in 70% ethanol aqueous solution (product name: Cordyceps extract, manufactured by Suntory Ltd.) The test sample was concentrated to 1%.

Example 2 Mast Cell Stabilizing Action Methods After growing rat basophilic leukemia cells RBL-2H3 cells (ATCC) in MEM medium containing 10% fetal bovine serum (FBS), penicillin (100 units / mL), streptomycin (100 μg / mL). 24-well plate, RBL-2H3 cells were seeded at 8 × 10 ³ cells / well (N = 3), 10% fetal bovine serum (FBS), penicillin (100 units / mL), streptomycin (100 μg / ML) in RPMI 1640 medium for 2 days. After washing twice with PBS (−), each sample (0.001%) prepared in Example 1 was incubated for 30 minutes in RPMI1640 medium, and then sample 1 or 2 (0.001%) And RPMI medium (without phenol red, serum, antibiotics) containing compound 48/80 (50 μg / mL) for 15 minutes.
50 μL of the culture supernatant was added from each well, and then 500 μL of 0.1% Triton X-100 was added to each well to lyse the cells and 50 μL was collected. To this, 50 μL of 0.2 M citrate buffer (pH 4.5) containing 2 mM p-nitrophenyl N-acetyl-β-D-glucosamine, which is a substrate of β-hexosaminidase, was added and reacted at 37 ° C. for 2 hours. It was. After completion of the reaction, after stopping with 150 μL 1M Tris buffer (pH 9.0), the absorbance at 405 nm was measured with a microplate reader, the intracellular and extracellular β-hexosaminidase activity was measured, and β-hexosaminidase was measured. Secretion (degranulation) (OD supernatant / (OD supernatant + OD cell lysate) × 100 (%)) was calculated. Doxantrazole (400 μM) was used as a positive control.

2. Results Grape seed extract, cordyceps extract and peanut seed coat extract are RPMI medium (phenol red, serum, antibiotics) with solvent control (compound 48/80 (50 μg / mL) for β-hexosaminidase secretion rate. The relative value when 1) was 1 was 0.8 or less, and had a mast cell stabilizing effect (Table 11).

Example 3 Effect of Mast Cell Stabilizer on Stress-induced Increased Colonic Movement Method Male Wistar rats (9 weeks old: Japan Claire) were reared in an assembly cage for 5 days under free feeding of CE-2 diet (Japan Claire), and then grouped so that there was no weight difference between the groups (N = 6 / group).
The stress group is each test sample prepared in Example 1 under isoflurane inhalation anesthesia (suspended in methylcellulose aqueous solution (Wako Pure Chemical Industries, 0.5 w / v% methylcellulose 400 solution, 0.5 w / v%)) or Methylcellulose 0.5 w / v% aqueous solution was intragastrically administered (4 mL / kg body weight) as shown in Table 12, and left in an individual cage for 1 hour under arousal. In the control group, a 0.5 w / v% aqueous solution of methylcellulose was intragastrically administered (4 mL / kg body weight). In the stress group, the shoulder, forelimbs, and chest were lightly fixed with tape under isoflurane inhalation anesthesia, and some movement of the forelimbs was restricted. It was left in an individual cage for 2 hours under awakening, and the number of excreted feces during that period was measured.

2. Results In the drug non-administration group (methylcellulose administration), the number of feces excreted significantly increased compared to the control group due to partial restraint stress load. When grape seed extract, cordyceps extract, and peanut seed coat extract were pre-administered, the increase in the number of defecations due to stress load was significantly suppressed (Table 13).
In addition, each point was shown by the average +/- standard error, and about the statistically significant difference between groups, Scheffe test was performed (* p <0.05, *** p <0.001).

Example 4 Effect of Mast Cell Stabilizer on Regular Colon Movement Method Male Wistar rats (9 weeks old: Japan Claire) were reared in an assembly cage for 5 days under free feeding of CE-2 diet (Japan Claire), and then grouped so that there was no weight difference between the groups (N = 4 / group).
Each test sample prepared in Example 1 was administered intragastrically (4 mL / kg body weight) as shown in Table 14, and was raised in an individual cage under awakening for 12 hours (dark period). The number of excreted feces during that period Was measured. In the control group, a 0.5 v / w% aqueous solution of methylcellulose was intragastrically administered (4 mL / kg body weight).

2. Results When pre-administered grape seed extract, cordyceps extract, or peanut seed coat extract, no effect was observed on the number of feces excreted relative to the control group (Table 15).
In addition, each point was shown by the average +/- standard error, and Scheffe test was performed about the statistically significant difference between groups (*** p <0.001).

  From the above results, it was considered that the grape seed extract, cordyceps extract, and peanut seed coat extract, which have a mast cell stabilizing effect, contribute to the improvement of stress-induced increase in colonic motility.

Claims (2)

  A preventive or ameliorating agent for irritable bowel syndrome comprising one or more selected from grape seeds, cordyceps, peanut seed coats and extracts thereof.   The preventive or ameliorating agent according to claim 1 or 2, wherein the irritable bowel syndrome is stressed colonic hyperactivity or stressful colonic hypersensitivity.

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