JP2007077122A - Inflammatory intestinal disease-preventing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inflammatory intestinal disease-preventing agent which contains a food-originated ingredient and is useful for safely and effectively preventing inflammatory intestinal diseases represented by ulcerative colitis. <P>SOLUTION: Various experiments related to a plant-originated proanthocyanidin have been carried out, and researches have zealously been tried from biochemical and medical viewpoints. Consequently, it has been found that the vegetable proanthocyanidin is useful as an agent for preventing the inflammatory intestinal diseases represented by the ulcerative colitis. The present invention relates to the inflammatory intestinal disease-preventing agent, characterized by containing the plant-originated proanthocyanidin as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a prophylactic agent for inflammatory bowel disease.

Food can be broadly divided into three functions. There are a primary function that is a nutrition function, a secondary function that is a preference function, and a tertiary function that is a biological regulation and disease prevention function. The tertiary function of food is neutralization and detoxification of coexisting harmful substances by continuously ingesting ingredients in food over a long period of time, action of various physical condition functions, life support, health promotion, etc. It is a regulation function for higher life activity.

Specifically, it is defined as a function related to biological rhythm regulation by food, regulation of absorption function, arousal and sedation of nerves, biological defense such as strengthening and regulation of immune function, aging suppression and the like. Examples of its physiological activity include antioxidant activity (inhibition of lipid peroxidation, radical scavenging, ulcer prevention, arteriosclerosis prevention), antibacterial activity, suppression of blood pressure increase, anti-inflammatory activity, antiallergic activity, hair growth promoting activity, antimutagenicity Carcinogenesis suppression, antitumor activity, etc. have been reported.

On the other hand, animals eat food and replenish water to obtain the energy and body materials necessary to maintain life. In this way, the digestive tract plays a role until food is taken into the body, digested and absorbed, and finally unnecessary substances are excreted. The digestive organ includes not only the stomach and intestine but also the mouth (oral cavity) for taking food and the liver for storing and processing nutrients. The part of the digestive tract that serves as a passage for food and water is called the digestive tract. The gastrointestinal tract begins in the oral cavity and extends to the pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum) large intestine, and anus, and the total length extends to about 6 m. The food passes through this digestive tract and is digested and absorbed. Eventually, the residue of the fluid (unnecessary waste) becomes feces in the large intestine and is excreted. In particular, in the large intestine, water and electrolytes are absorbed, and it works to transport those that have not been digested and absorbed or waste products to the anus.

Ulcerative colitis is a disease of the large intestine where erosions and ulcers occur in the mucosa (innermost layer) of the large intestine, and is classified as inflammatory bowel disease together with Crohn's disease and the like. It is said that there are about 1 million patients in the United States, and the number of patients with ulcerative colitis in Japan is reported to be 77,073 (from the number of medical certificates issued for specific diseases in FY2002). Approximately 5,000 people have increased. Up to now, the causes of intestinal bacteria, the immune system that originally protects the body from external enemies, or abnormal autoimmune reactions, or changes in dietary habits have been considered. Is not clear.

As an experimental model of this disease, it is known to induce ulcerative colitis by administering dextran sulfate sodium to mice. Mice administered with dextran sulfate sodium induce ulcerative colitis, not only atrophy of the large intestine but also inflammation and loss of intrinsic structure leading to weight loss and even debilitating death.

For ulcerative colitis, medical treatment with drugs is in principle performed. At present, there is no medical treatment that completely cures ulcerative colitis, but there are effective drug treatments that suppress intestinal inflammation. The purpose of treatment is to control abnormal symptoms by suppressing abnormal inflammation of the large intestine mucosa. For the medical treatment of ulcerative colitis, 5-aminosalicylic acid (5-ASA) preparations, corticosteroids, blood cell component removal therapy, and immunosuppressants are used.

Proanthocyanidins are single molecules of polyphenols that are biosynthesized as plant secondary metabolites (including flavonoids such as catechin, flavone, anthocyanin, isoflavone, and chalcone, and phenol carboxylic acids such as caffeic acid, chlorogenic acid, ellagic acid, and gallic acid. Is a kind of a compound having a plurality of phenolic hydroxyl groups in it, and has a structure in which two or more molecules of catechins are bonded. Apple, pear, peach, grape, barley, guava, hop, red bean, pine bark, etc. It has been reported to exist in plants.

As a technique relating to the treatment or prevention of ulcerative colitis, Patent Document 1 describes that proanthocyanidin-containing ginseng extract preparation exhibits an excellent therapeutic effect on ulcerative colitis. Proanthocyanidins and Panax ginseng extracts each exhibit a remarkable effect when used in combination rather than alone. Proanthocyanidins are thought to have been effective in peripheral vasodilatory effect, removal of active oxygen, and anti-inflammatory effect. Specific examples of proanthocyanidins include proanthocyanidins derived from French coastal pine bark. Although the therapeutic effect is examined in the administration test to patients with ulcerative colitis, the preventive effect is not mentioned.

Patent Document 2 discloses croton spp. Useful for the treatment and prevention of secretory diarrhea. Or calophilum spp. An enteric coating formulation of a proanthocyanidin polymer composition isolated from is described. It mainly describes the treatment and prevention of secretory diarrhea caused by Vibrio cholerae, enterotoxin Escherichia coli, Shigella, etc., but non-infectious etiology is related to nonspecific diarrhea, ulcerative colitis, inflammatory bowel disease syndrome However, there is no description in the examples.

Non-patent document 1 describes that catechin exhibits an inhibitory action in an experiment for inducing ulcerative colitis by DSS and is useful for prevention of ulcerative colitis. However, catechin and the proanthocyanidins of the present invention are There are differences between monomers and polymers, and they are materially different.

In addition, the conventional technologies related to plant-derived proanthocyanidins other than the treatment or prevention of ulcerative colitis include Patent Document 3 (Prophylactic / therapeutic agent for diseases caused by APC gene mutation), Patent Document 4 (Antioxidant), Patent Document 5 (prophylaxis and treatment agent for urinary tract infection), Patent Document 6 (bacterial toxin neutralizer), Patent Document 7 (apoptosis inducer), Patent Document 8 (immunomodulator) and the like.

Moreover, as prior art regarding the treatment or prevention of ulcerative colitis other than the above, Patent Document (N-adamantylmethyl derivative and intermediate), Patent Document 10 (Inosine Compound (Salt)), Patent Document 11 (Phosphate Derivative) ), Patent Document 12 (antibody against CD23), Patent Document 13 (polyamino acid skeleton, non-steroidal anti-inflammatory agent), Patent Document 14 (Sucralfate preparation), and the like.
JP 2004-75587 A Special table 2001-524938 JP 2001-64172 A Special table 2001-500546 Special table 2001-515860 JP 2004-155719 A JP-A-2005-75790 JP2005-82497 International application number PCT / SE02 / 002057 International application number PCT / US02 / 009335 International application number PCT / JP00 / 001005 International application number PCT / GB99 / 001434 International application number PCT / US96 / 007563 JP 08-268895 A Hidetoshi Yamazaki et al., “Effects of catechins in DSS colitis model”, Experimental Vol. 2, pp150-152 (2001)

An object of the present invention is to provide a safe and effective preventive agent for an inflammatory bowel disease represented by ulcerative colitis by a food-derived component.

Under these circumstances, the present inventors conducted various experiments on plant-derived proanthocyanidins, and as a result of diligent research from a biochemical / medical point of view, plant proanthocyanidins represented ulcerative colitis. It was found useful as a preventive agent for inflammatory bowel disease. That is, the present invention is intended to induce colitis by continuously administering to a mouse an apple procyanidin fraction obtained by purifying a polyphenol from a plant, particularly an apple, and further purifying a proanthocyanidin fraction therefrom. It has been found that the effects of sodium dextran sulfate administered to the patient are reduced, and various phenomena due to ulcerative colitis are reduced.

Therefore, the present invention provides a prophylactic agent for inflammatory bowel disease comprising plant proanthocyanidins as an active ingredient. In the present invention, the plant proanthocyanidins may be derived from apple, pear, peach, grape, barley, guava, hop, red bean, or pine bark. In addition, ingestion of plant-derived proanthocyanidins as an inflammatory bowel disease is a safe and effective way of taking food from the viewpoint of safety. Prophylactic and therapeutic agents can be provided. Also, its safety has been reported.

According to the present invention, a prophylactic agent for inflammatory bowel disease comprising plant proanthocyanidins as an active ingredient is provided. According to the present invention, it is particularly useful as a preventive agent for ulcerative colitis, and can be used as a pharmaceutical, food, feed and beverage.

It is an object of the present invention to provide a novel highly safe inflammatory bowel disease preventive agent characterized by comprising plant-derived proanthocyanidins as an active ingredient, and a pharmaceutical and functional food / beverage containing the same. .
The present invention is described in detail below. In the present invention, the proanthocyanidin component used as an active ingredient of the intestinal immune tolerance inducer may be a commercially available product or one extracted and separated directly from a plant. The proanthocyanidins referred to in the present invention are condensed tannins existing in a plant body, that is, a mixture of compounds obtained by condensing or polymerizing 4 → 8 or 4 → 6 using flavan-3-ols as a structural unit. These produce anthocyanidins such as cyanidin, delphinidin and pelargonidin by acid treatment. In the present invention, it is a proanthocyanidin such as a polymer procyanidin, prodelphinidin, propelargonidin or the like having 2 to 15 mer of the above structural unit.

For example, the methods described in JP-A-7-285876, JP-A-2000-16951 and JP-A-2002-87978 can be used for extraction and purification of proanthocyanidin components from apple fruit. As the raw material apple, unripe apple fruit may be used as described in JP-A-7-285876, or wild apple (Crab Apple) is used as described in Japanese Patent Application No. 2000-277228. You may do it.

First, an extract is obtained based on the method of JP-A-7-285876. Specifically, after washing the apple fruit, the juice can be obtained by crushing and pressing as it is or while adding sulfurous acid, and the clarified fruit juice can be prepared by centrifugation, filtration or the like. The clarified juice may be appropriately concentrated by a known method. As a method for extracting the crude apple polyphenol component, the obtained fruit juice may be used as a raw material, but the fruit is mixed with alcohols, crushed, soaked as it is, extracted while being compressed or heated to reflux, and then alcohol. Then, centrifugation and filtration, or partitioning and filtration with an organic solvent such as hexane and chloroform may be performed to obtain a clarified extract.

Subsequently, the said extract is refine | purified by the method of Unexamined-Japanese-Patent No. 2000-16951. Specifically, the polyphenol component is obtained by passing the clarified juice or clarified extract through a column packed with an adsorbent capable of selectively adsorbing polyphenol, for example, a styrene divinylbenzene synthetic adsorption resin, an anion exchange resin, or the like. To adsorb. Next, after washing the column with distilled water, the polyphenol component can be eluted and recovered by passing an alcohol solution of 20 to 100%, preferably 30 to 60%, through the column. When alcohol is distilled off from the obtained alcohol solution fraction, a crude apple polyphenol fraction is obtained. This crude apple polyphenol fraction contains components as shown in FIG.

Further, the crude apple polyphenol fraction is treated by the method disclosed in JP-A-2002-87978 to obtain a proanthocyanidin fraction. Specifically, the obtained crude polyphenol fraction can be separated and purified into a procyanidin 2 to 5mer fraction and a hexamer or more fraction by solid-liquid extraction using methyl acetate as a liquid phase. The fraction of procyanidin hexamer or more that is not extracted into methyl acetate is distilled off by a known method. The procyanidin 2-5 mer fraction extracted into methyl acetate is dissolved in distilled water after the extraction solution is concentrated by a known method. Furthermore, the procyanidin 2-5 mer fraction can be separated and purified according to the degree of polymerization (by molecular weight) by normal phase chromatography to obtain a procyanidin oligomer having a uniform degree of polymerization.

As proanthocyanidins, those obtained by a synthesis method can also be used.
The proanthocyanidin preparation thus prepared can be used in pharmaceuticals as a prophylactic agent for inflammatory bowel disease. As a pharmaceutical composition, you may mix with the conventional inflammatory bowel disease therapeutic agent. The pharmaceutical agent containing the prophylactic agent for inflammatory bowel disease can be made into oral preparations such as tablets, powders, granules, capsules and syrups by a known method. This orally administered agent includes excipients, disintegrants, binders, lubricants, surfactants, alcohols, water, water-soluble polymers, sweeteners, flavoring agents, It can be produced by a commonly used method by adding a sour agent, a pharmaceutical carrier and the like.

The daily dose for adults to obtain the effect of preventing inflammatory bowel disease with the preventive agent for inflammatory bowel disease of the present invention is 100 to 2500 mg as apple extract or apple proanthocyanidin, preferably 150 to 1500 mg, more preferably 150 to 1000 mg, particularly 150 to 750 mg is preferable.

When using the prophylactic agent for inflammatory bowel disease of the present invention, the blood concentration of proanthocyanidins is more likely to be taken in a smaller number of apple extracts or apple proanthocyanidins per day in terms of absorption of polyphenols. It becomes high and it is easy to express the action of proanthocyanidins.

The preventive agent for inflammatory bowel disease of the present invention can be used by adding as a food additive to foods in general, including beverages. For example, soups, beverages (juice, liquor, mineral water, coffee, tea, non-alcoholic beer Etc.), confectionery (gum, candy, chocolate, snacks, jelly, etc.), noodles (soba, udon, ramen, etc.), alcoholic beverages (beer, sparkling liquor, cocktail, Chuhai, shochu, sake, whiskey, brandy, wine, etc.) ).
EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited at all by these Examples.

Homogenized 3 kg of unripe apple (Fuji) collected in June in Hirosaki, Japan, in the presence of potassium pyrosulfite (0.1% w / w) The pulverized product was kept at 4 ° C. for 24 hours. Fruit juice obtained by pressing the pulverized material was collected, clarified by centrifugation (3500 g), and filtered through a glass filter. The filtrate (1.8 L) was applied to a column (diameter 25 mm × 285 mm) packed with styrene-divinylbenzene synthetic adsorption resin Sepa beads SP-850 (manufactured by Mitsubishi Kasei). After washing with distilled water (300 ml) to remove water-soluble components, the 80% ethanol fraction (200 ml) was removed with ethanol and freeze-dried to obtain a crude apple polyphenol fraction. When this crude apple polyphenol fraction was analyzed by reversed-phase high performance liquid chromatography, chlorogenic acids (about 20%), phloretin glycosides (about 5%), flavonols (about 15%), proanthocyanidins (About 50%) and other browning substances (about 10%). Furthermore, as a result of analysis by MALDI-TOF / MS, these proanthocyanidins were confirmed to be dimer to 15 mer consisting of catechin and epicatechin which are flavan-3-ols, and were high molecular polyphenols. [M. Ohnishi-Kameyama et al., Mass Spectrometry , 11, 31-36, 1997].

Furthermore, the crude apple polyphenol fraction was dissolved in distilled water, adjusted to pH 6.5 with 5N sodium hydroxide, and packed in a column packed with styrene-divinylbenzene synthetic adsorption resin Diaion HP-20ss (manufactured by Mitsubishi Kasei). Provided. After washing the column with distilled water, the fraction dissolved in 25% ethanol was concentrated and freeze-dried to obtain a proanthocyanidin fraction. [T. Shoji, et al., J. Argic. Food Chem., 51, 386-3813 (2003)]. The apple-derived proanthocyanidin fraction thus obtained was used as a sample for evaluating the influence on ulcerative colitis described later.

Test Example 1: Influence on the intestinal tract immune system in free oral intake The apple-derived proanthocyanidin fraction obtained in Example 1 was used in the test by the following method. In order to investigate the possibility of acting on ulcerative colitis, mice were allowed to freely ingest the apple-derived anthroprocyanidin fraction and the effects were examined.

That is, 2.5% dextran was obtained by drinking 6-week-old male C57BL6 mice with pure proanthocyanidin fractions dissolved in pure water at concentrations of 0.1%, 0.3% and 1.0% as drinking water. A sodium sulfate (Dextran Sulfate Sodium, abbreviated as DSS) aqueous solution was ingested freely for 14 days before the start of the change. Free intake of 2.5% aqueous dextran sulfate solution was for 4 days, drinking water was then changed to pure water, and the breeding was continued for 20 days. The control group was a group in which pure water not containing the apple-derived proanthocyanidin fraction was freely drunk and the same ulcerative colitis was induced with a 2.5% aqueous dextran sulfate solution. With this test, the dose of the apple-derived proanthocyanidins fraction can be examined.

Test example 2
Next, 6 days old male C57BL6 mice in which apple-derived proanthocyanidin fraction was dissolved in pure water at a concentration of 1.0% were used as drinking water for 3 days from the start of the change to 2.5% dextran sodium sulfate aqueous solution. It was ingested freely for 7 days and 14 days in advance. Free intake of 2.5% aqueous dextran sulfate solution was for 4 days, drinking water was then changed to pure water, and the breeding was continued for 20 days. The control group was a group in which pure water not containing the apple-derived proanthocyanidin fraction was freely drunk and the same ulcerative colitis was induced with a 2.5% aqueous dextran sulfate solution. By this test, the pre-administration period when 1% apple-derived proanthocyanidin fraction aqueous solution is used as drinking water can be examined.

By the above method in Test Example 1, the effect of free oral intake on inflammatory bowel disease in ulcerative colitis model mice of apple-derived proanthocyanidin fraction was examined. First, in the influence of each concentration of the apple-derived proanthocyanidin fraction during the pre-administration period of 14 days, the body weight, which is the effect of ulcerative colitis due to dextran sulfate sodium, was 0.3% in the apple-derived proanthocyanidin group compared to the control group. And 1.0% group significantly suppressed its weight loss. In addition, the survival rate of the apple-derived procyanidins of 0.3% and 1.0% greatly improved, and the survival rate of the 1.0% group was 100% (Fig . 1). 2). Furthermore, the photograph of the large intestine of a control group and a 1% apple origin proanthocyanidin pre-administration group is shown in FIG. It was found that colon atrophy caused by dextran sulfate sodium was suppressed by pre-administration of apple-derived proanthocyanidins. From these results, it was suggested that a certain concentration of proanthocyanidins is necessary for the prevention of ulcerative colitis.

Next, the effect of ulcerative colitis due to sodium dextran sulfate was observed when the pre-administration period at a concentration of 1.0% of the apple-derived proanthocyanidin fraction was 3 days, 7 days, and 14 days by the above-described method in Test Example 2. In the apple-derived proanthocyanidin group of 1.0% compared to the control group, the 7-day and 14-day groups for pre-administration suppressed the weight loss. Regarding the survival rate, the survival rate was 40% in the control group depending on the period of the pre-administration of 1.0% apple-derived proanthocyanidins, but 60% in the pre-administration group for 3 days and 7% in the pre-administration group. The survival rate was greatly improved, with 80% in the daily group and 100% in the 14-day group (Fig . 1 and Fig . 4). From these results, it was suggested that long-term pre-administration at a certain concentration of proanthocyanidins is effective in preventing ulcerative colitis.

Formulation Example (1) Tablets Example 1 apple-derived proanthocyanidin fraction 2.0 g
Lactose 15.0g
Magnesium stearate 3.0g
Total 20g
Each said weight part was mixed uniformly and it was set as the tablet according to the conventional method.
(2) Capsule Example 1 apple-derived proanthocyanidin fraction 2.0 g
Lactose 15.0g
Magnesium stearate 3.0g
Total 20g
Each said weight part was mixed uniformly, and it was set as the capsule according to the conventional method.
(3) Powder, granule Apple-derived proanthocyanidin fraction of Example 1 4.0 g
Lactose 10.0g
Starch 6.0g
Total 20g
Each said weight part was mixed uniformly, and it was set as the powder and the granule according to the conventional method.
(4) Sucrose 15.0 g
Citric acid 0.2g
Fragrance 0.1g
Sodium ascorbate 0.05g
Apple-derived proanthocyanidin fraction of Example 1 0.5 g
94.45 g of water
Using each component of each of the above parts by weight, it was made into a candy according to a conventional method.
(5) Juice concentrated mandarin orange juice 15.0 g
Fructose 4.5g
Citric acid 0.2g
Fragrance 0.1g
Dye 0.2g
Sodium ascorbate 0.05g
Apple-derived proanthocyanidin fraction of Example 1 0.5 g
49.45 g of water
Using each component of each of the above parts by weight, juice was obtained according to a conventional method.
(6) Barley tea Barley tea 1.0g
599 g of water
Total 600g
Using each component of each of the above parts by weight, after extracting barley tea components according to a conventional method,
Sodium ascorbate 0.36g
3.0 g of apple-derived proanthocyanidins fraction of Example 1
To make barley tea.

The structural formula of procyanidins contained in the apple-derived proanthocyanidin extract fraction obtained in Example 1 is shown. It is a graph which shows the inhibitory effect according to density | concentration of the apple origin proanthocyanidins in an ulcerative colitis model mouse. It is a figure which shows the effect | action of the apple origin proanthocyanidins with respect to the colitis by DSS administration. It is a graph which shows the inhibitory effect according to the pre-administration period of the apple origin proanthocyanidins in an ulcerative colitis model mouse.

Claims (5)

An agent for preventing inflammatory bowel disease, comprising plant-derived proanthocyanidins as an active ingredient. The inflammatory bowel disease preventive agent according to claim 1, wherein the plant is apple, pear, peach, grape, barley, guava, hop, red bean, or pine bark. The agent for preventing inflammatory bowel disease according to claim 1 or 2, wherein the proanthocyanidins are procyanidins. It is a pharmaceutical, The inflammatory bowel disease preventive agent of any one of Claims 1-3 characterized by the above-mentioned. It is a functional food or a drink, The inflammatory bowel disease preventive agent of any one of Claims 1-3 characterized by the above-mentioned.