JPH11116484A - Prophylactic and therapeutic agent and drink and food for inflammatory bowel disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject prophylactic and therapeutic agent having therapeutic effects on inflammatory bowel diseases and suppressing effects on exacerbation of rethema by including a galactooligosaccharide as an active ingredient therein. SOLUTION: This prophylactic and therapeutic agent is obtained by including a galactooligosaccharide Gal-(Gal)n -Glc [Gal is a galactose residue; (n) is 1-4]}, and preferably a bacterium of the genus Bifidobacterium (e.g. Bifidobacterium breve) as active ingredients. When the gallactooligosaccharide is continuously administered, the daily dose thereof is preferably about 5-10 g for an adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic / therapeutic agent for inflammatory bowel disease for preventing and treating inflammatory bowel disease by oral administration, and a food and drink. Specifically, the present invention relates to a prophylactic / therapeutic agent for inflammatory bowel disease containing galacto-oligosaccharide, and a food or drink.

[0002]

2. Description of the Related Art Inflammatory Bowel Dise
ase) has a large number of patients in Europe and the United States, and the incidence in Japan has been increasing in recent years. This inflammatory bowel disease is a disease that frequently occurs in young people, and is divided into ulcerative colitis and Crohn's disease.

[0003] Ulcerative colitis occurs in the lamina propria of the large intestine.
Crohn's disease mainly affects the entire colon and ileum. Many efforts have been made to elucidate the pathology of these diseases as one of the designations of the Ministry of Health and Welfare's Intractable Disease, but the cause is not yet clear. The pathology of the disease is such that hot water and ulcers are formed on the intestinal mucosa, and symptoms such as weight loss, diarrhea, and bloody stool appear.

[0004] In this disease, an active period with severe symptoms and a remission period with mild symptoms are usually repeated. The distinction between the active phase and the remission phase is mainly made by observing the lesion with an endoscope. Diagnosis is also made using the amount of inflammatory substances (myeloperoxidase: MPO) produced by neutrophils as an index. Is possible.

[0005] The appearance and deterioration of such pathological conditions have also been suggested to involve abnormalities of the host immune system and involvement of intestinal bacteria. For example, in patients with ulcerative colitis, increased mucin degrading activity in the large intestine has been reported.

[0006] At present, no drug or treatment has been found to completely cure the disease, but salazopyrin (generic name: salazosulfapyridine (Sal)
icylazosulfapyridine)) has been used. According to the pharmacotherapy guidelines for ulcerative colitis plaques specified by the Ministry of Health and Welfare, it is important to use prednisolone enema therapy earlier than salazopyrine monotherapy. (Ministry of Health and Welfare Research Group on Intestinal Digestion and Absorption Disorders, 1983, 198
4, p. 11).

[0007] Recently, urinastin (Tada Masahiro et al .: New treatment of intractable ulcerative colitis, Journal of colorectal anal, 4
2: 578.1989), Fish oil EPA (eicosapentaenoic acid).
(Salomon P. et al .: Treatment of ulcerative cohtis
acid-an open trial-.J. Clin. Gastroenterol. 12:15
7.1990), bifidobacteria (Japanese Patent Application Laid-Open No. 7-126177).
Publication), fructooligosaccharides (Digestion & Absorptio)
n, Vo 1.16 NO.1 1993) has been reported to be effective for ulcerative colitis, but it is not an established treatment for humans.

[0008] For this reason, the purpose of medical treatment is to shorten the active period as much as possible and to promptly introduce it into the remission period. As a treatment method actually used in hospitals and the like, dietary guidance such as feeding a high-protein, high-calorie, high-vitamin diet with less residue has become mainstream.

[0009]

On the other hand, inflammatory bowel disease is characterized by dextran sodium sulfone (dextran sodium sulfone).
ate: DSS) can be caused artificially. Further, by adjusting the dose of DSS, an acute or chronic mouse ulcerative colitis model or the like can be created. It is also possible to confirm the therapeutic effect. Furthermore, as described above, it is also possible to search for a substance that suppresses the deterioration of the pathological condition from the remission phase to the active phase by using an endoscopic diagnosis, MPO activity in intestinal fluid or feces as an index.

[0010] Accordingly, an object of the present invention is to find a prophylactic / therapeutic agent for inflammatory bowel disease or aggravation of the pathological condition, that is, an agent which prevents the transition from the remission phase to the active phase, based on the above findings. It is in. In addition, the purpose of this agent is to search for a substance which is excellent in safety and palatability as well as its pharmaceutical effect and which does not burden the patient even when used for dietary guidance. The present invention has been found that co-administration of a galactooligosaccharide and a bacterium belonging to the genus Bifidobacterium can further prevent and treat inflammatory bowel diseases, and can provide an inflammatory bowel containing these as an active ingredient. It is intended to provide an agent for preventing and treating diseases and food.

[0011]

DISCLOSURE OF THE INVENTION The present invention for solving the above-mentioned problems is based on the finding that galacto-oligosaccharide, which is one of the oligosaccharides, has a therapeutic effect on inflammatory bowel disease and an inhibitory effect on deterioration of pathological conditions. It is. That is, the present invention
It is intended to provide a prophylactic / therapeutic agent for inflammatory bowel disease containing galacto-oligosaccharide as an active ingredient.

[0012] Galactooligosaccharides are contained in foods and beverages even at present, and are excellent in both safety and palatability. Therefore, the present invention also provides a food for preventing or treating inflammatory bowel disease, comprising galacto-oligosaccharide as an active ingredient, which can be suitably used even during dietary guidance.

The prophylactic / therapeutic agent for inflammatory bowel disease according to the first aspect of the present invention contains galacto-oligosaccharide as an active ingredient.

The inflammatory bowel disease preventive / therapeutic agent according to the second aspect of the present invention further contains a Bifidobacterium bacterium as an active ingredient.

The food and drink for preventing and treating inflammatory bowel disease according to the third aspect of the present invention contains galacto-oligosaccharide.

The food for preventing or treating inflammatory bowel disease according to the fourth aspect of the present invention further contains a Bifidobacterium bacterium.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The galactooligosaccharide in the present invention is represented by the general formula Gal- (Gal) n-Glc (where Gal is a galactose residue, Glc is a glucose residue, and n is an integer of 1 to 4). Which is a major component of breast milk oligosaccharides and is known to be a growth promoting factor of a useful bacterium bifidobacterium that inhabits the human intestine.

The production of galactooligosaccharides is usually carried out by an enzymatic method. That is, galacto-oligosaccharide is obtained as a product of a decomposition reaction of lactose by β-galactosidase derived from bacteria and the like, and a transfer reaction of glucose and galactose generated by the decomposition to lactose. As β-galactosidase, any one can be used as long as it can catalyze the decomposition / transfer reaction as described above by combining one or two or more thereof. Among them, those derived from Bacillus circulans are preferable. Also, the production method is not particularly limited, and those obtained by an enzymatic method or any other method can be used.

The myeloperoxidase (MPO) used for evaluation in the examples of the present invention is an inflammatory substance produced mainly by neutrophils, and when its amount is measured, it is a disease state, especially in remission. And whether it is in the active period. Usually, when measuring MPO activity in a mouse model or the like, feces are used as a specimen. However, in an experiment conducted on humans, an endoscope washing solution (Lavage solution: LS) obtained by washing at the time of endoscopic diagnosis was used as a specimen. The LS expresses the pathological condition of the lesion site most clearly, and is suitable as an index for grasping the pathological condition in a state fresher than the feces.

In the present invention, mouse and human hegaractooligosaccharides were administered, and the effect on inflammatory bowel disease was examined. The pathology of mice is determined by weight loss, rectum hypertrophy, MPO activity in feces, and the amount of IgG in feces (increases when the symptoms of colitis are severe). The results and the MPO activity contained in the patient's LS were used as indices.

In an experiment using mice, it was confirmed that galactooligosaccharide has a preventive effect on acute and chronic ulcerative colitis induced by DSS. In addition, the effects of administration to humans on patients differed depending on the condition of the patient before administration, that is, whether the patient was in remission or active. Although the amount of MPO in the intestinal fluid of the patient who was in the active phase before administration was significantly reduced, the amount of MPO of the patient who was in the remission phase did not change during the administration period. However, the MPO level increased in the group that simply provided dietary guidance. At this time, the result of diagnosis by the endoscope and MP
O concentration was well correlated.

In an experiment using mice, it was significantly found that co-administration of galacto-oligosaccharide and Bifidobacterium bacterium had a stronger effect of weight reduction and improvement of MPO activity than administration of galacto-oligosaccharide alone. Not shown though.

On the other hand, although the mechanism by which galactooligosaccharides prevent and treat inflammatory bowel disease is not clear,
The findings already obtained by the present inventors suggest that Bacteroides bacteria are involved. In addition, as described above, the association with mucin-degrading bacteria and Bifidobacterium bacteria has also been suggested, and it is considered that these are combined to contribute to improvement of the disease state and the like.

Since galactooligosaccharides are originally used as foods, there is no problem in both safety and palatability, and they may be directly administered orally. In addition, if necessary, excipients, binders, disintegrants, lubricants, coating agents, emulsifiers, dispersants, solvent stabilizers and the like are appropriately added, and tablets, granules, powders, powders, and capsules are added. It may be used in the form of a formulation.

When galacto-oligosaccharide is continuously administered, it is preferably 5 g to 10 g per day for an adult, particularly preferably 5 g to 10 g per day.
g is good.

[0026] As the Bifidobacterium bacteria to be used in combination with galactooligosaccharide in the present invention, propagation of the human body is desirable Bifidobacterium breve (Bi
didobacterium breve ), Bididobacterium longum ( Bididobacterium longum ) and the like. In addition, you may use together with the Lactobacillus genus bacteria and the Enterococcus bacterium which are expected to reproduce by galactooligosaccharide.

[0027]

【Example】

Embodiment 1 FIG. Preparation of galactooligosaccharide (BOS) First, lactose as a raw material is dissolved in warm water, and then cooled and pH
Preparation is performed, β-galactosidase derived from Bacillus circulans is added, and an enzyme reaction is performed. When the decomposition reaction of lactose and the transfer reaction of monosaccharide by the enzyme are performed, the enzyme is inactivated by heating. The solution thus obtained is subjected to filtration, ion exchange chromatography and the like to purify the galactooligosaccharide.

Embodiment 2 FIG. Examination of the preventive effect of galactooligosaccharide on ulcerative colitis in mice The inflammation inhibitory effect of the administration of galactooligosaccharide was examined under the following conditions. (2-1) Animal: Balb / c mouse (Clea Japan, ♂ and SLC,
♂: 8 to 10 weeks old).

(2-2) Galactooligosaccharide: BOS (described in Example 1) was used.

(2-3) Experiment contents: A group in which acute colitis was induced by administering DSS (molecular weight: 50, OOO, 5%) for 7 days after BOS was administered for 10 days, and 6% by 1% DSS. The preventive effect of BOS on each group that induced chronic colitis by weekly administration was examined. In order to clarify the dose at which the effect of BOS appears, the dose of BOS was 6 mg, 60 mg, and 120 mg.
(/ mouse / day). Physiological saline was administered to the control, and the number of mice was 4 each.

(2-4) Evaluation method: In the evaluation of the acute colitis group, changes in body weight and MPO activity in feces were used as indices. For evaluation of the chronic colitis group, the weight of the large intestine and IgG activity in feces were used. In addition, in order to clarify the relationship between colitis and intestinal flora, and changes in intestinal flora caused by BOS administration, the fecal flora and fecal organic acids were measured.

Measurement of Change in Body Weight The body weight of the mice was measured every two days after the induction of colitis. The results are shown in FIG. Measurement of colon weight After killing the mice in both the BOS administration group and the control group,
The ratio of colon weight to individual body weight was measured. The results are shown in FIG.

Measurement of Fecal MPO Activity MPO activity was measured on the third and seventh days from the start of 5% DSS administration using the method of Krawisz et al. First, feces diluted 5 times were homogenized with a polytron homogenizer, frozen at -30 ° C for 1 hour, and sonicated. After melting,
After centrifugation at 15,000 rpm for 15 minutes, the supernatant was washed with 0.167 mg / ml o-dianisidine hydrochloride (Sigma) and 0.0005
% Of hydrogen belloxide, and the MPO activity was measured using the absorbance at 455 nm as an index. The results are shown in FIG.

Measurement of Fecal IgG Activity The amount of IgG in feces six weeks after the start of 1% DSS administration was measured using a sandwich ELISA method. First, an anti-mouse IgG antibody (Zymed
Co., Ltd.), blocking and washing were performed, and stool diluted serially was reacted at 37 ° C. for 1 hour. This was washed several times with Triton-X containing PBS, and then reacted with a peroxidase-labeled anti-mouse IgG antibody (Zymed). Further, after washing, a substrate solution was added, and the amount of IgG was measured using the color of the substrate as an index. FIG. 4 shows the results.

Search for Fecal Microflora Search was performed before, after and after administration of BOS. Total anaerobic bacteria, total aerobic bacteria, Bcteroidaceae , Lacto
To search for bacillus and Enterobacteriaceae , blood G
AM (Nissui), TSA (BBL), NBG
T (manufactured by Nissui), LBS (manufactured by BBL), DHL
(Manufactured by Nissui) was used. The search was performed by pooling mouse feces. For the measurement of the number of anaerobic mucin-degrading bacteria, a medium consisting of saline, hemin, and pig stomach mucin was used. FIG. 5 shows the results.

(2-5) Effect of BOS administration As shown in the weight change of each group in FIG. 1, DSS was observed in all groups.
Body weight loss was observed after administration (after 10 days).
In the group to which 120 mg was administered, weight loss was suppressed as compared with the control group. As shown in FIG. 2, the ratio of the large intestine weight to the individual body weight was significantly higher in the control group than in the administration group.

Further, as shown in FIG. 3, the fecal MPO activity was higher on the third day of DSS administration than on the control group.
There was a significant decrease in the group administered with 60 mg, and a significant decrease was observed in all groups pre-administered with BOS on day 7 of DSS administration. As shown in FIG. 4, the amount of IgG was significantly reduced in the administration group. Further, as shown in FIG. 5, the number of anaerobic mucin-degrading bacteria in the control group did not change, and total aerobic bacteria, Entero
bacteriaceae number of bacteria tended to increase after DSS administration. The number of other searched bacteria did not change during the experiment.

Embodiment 3 FIG. Examination of the combined effect of galactooligosaccharide and bifidobacterium bacteria on ulcerative colitis in mice Comparison of the inflammation-suppressing effect of galacto-oligosaccharide alone and in combination with bifidobacterium bacteria under the following conditions investigated.

(3-1) Animal: Balb / c mouse (Clear Japan,
♂ and Charles River, ♂: 8 to 10 weeks old) 5 animals were used.

(3-2) Galactooligosaccharide: BOS (described in Example 1) was used.

(3-3) Experiment contents: A group to which BOS or B. breve alone was administered, a group to which BOS and B. breve were co-administered, and a control group to which physiological saline was administered. The dose of BOS is 60 mg (/ mo
use / day). B. breve, a live bacterial preparation of BBG-01 , was suspended in 10 ml of sterile distilled water, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was further centrifuged at 3000 rpm for 10 minutes. The precipitate was suspended in 1 ml of sterile distilled water, and B. breve was administered. At this time, the bacterial count of B. breve was 10 ⁸ (/ mouse / day).

Each administration group was composed of BOS, B. breve or B
OS and B. breve were administered 7 days before DSS (molecular weight 50, OOO,
(4%) administration (7 days) induced inflammation. Saline was administered to the control, and n = 5 for each group. The control and the BOS alone administration group were started first, and then the B. breve alone administration group and the BOS and B. breve co-administration group were started. A control group was also provided for the group in which the experiment was started with a delay.

(3-4) Evaluation method: MPO activity in feces was used as an index. MPO activity was measured on days 2, 4, and 6 from the start of DSS administration. The method is the same as in the second embodiment. As a result, as shown in FIG. 6, MPO activity was increased by administration of BOS and B. breve on the second day after DSS administration.
It was significantly suppressed on the fourth day.

Embodiment 4 FIG. To study human漬瘍colitis patients combined effect of galactooligosaccharide and Bifidobacterium bacteria to human漬瘍colitis, BOS and BBG-O1 (B. Breve
1O ^9/1 capsule) was administered and the one year continuously, was examined the effect. The conditions are as follows.

(4-1) Cases Administration group: (administration of BOS, BBG-01) 29, (5 patients with poor oral administration) Control group: (dietary instruction group) 29 * Both groups are treated normally.

(4-2) Galactooligosaccharide: BOS was used.

[0047] (4-3) Experimental contents:. BOS in the active phase and 29 patients with ulcerative colitis in remission phase and ^{BBG-O1 (B breve 10 9} /1
) Was administered continuously for one year, and endoscopic examination and MP
The effect on the lesion by the O activity was examined. The dose is B
The OS was about 5.5 g / day, and the BBB-01 was 3 packets / day. The ELISA method was used to measure the MPO activity.

(4-4) Effect of combined use of BOS and BBG-O1 Results of endoscopy 1 year after the start of administration (excluding those with poor oral administration)
Are shown in Table 1 below. FIG. 7 shows the amount of MPO (including those with poor oral administration) before and after treatment of patients with ulcerative colitis in the active and remission phases. In addition, regarding the results in FIG. 7, FIG. 8 shows a case where the disease state of the patient before the treatment was in the active phase, and FIG.

[0049]

[Table 1]

As shown in FIGS. 8 and 9, the amount of MPO in the intestinal fluid of the patient who was in the active phase before treatment was significantly reduced, but the MPO level of the patient who was in the remission phase did not change during the administration period. . However, the MPO level increased in the group that simply provided dietary guidance. At this time, it is understood that the result of the diagnosis by the endoscope and the MPO concentration were well correlated.

[0051]

According to the galacto-oligosaccharide of the present invention,
The prophylactic / therapeutic agent for inflammatory bowel disease and its food can be supplied at low cost and stably. Since this is also effective in improving the condition from the active phase to the remission phase, it can be used regardless of the patient's symptoms. In addition, since galactooligosaccharides are also preferable in terms of flavor, they can be suitably used in the case of continuous administration, such as during dietary guidance.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the results of the effect of pre-BOS administration on the weight change of DSS-induced colitis mice, in which the vertical axis represents the weight change of the mouse (ratio to the weight at the first administration), and the horizontal axis represents the BOS administration. Later time (day). In the figure, a black triangle is a control, a square is BOS 6 mg, a black circle is BOS 60 mg, and a circle is BOS.
Shows the 120 mg group.

FIG. 2 is an explanatory diagram showing the results of measuring the ratio (%) of the weight of the large intestine to the body weight of the BOS-administered group mouse and the control group mouse after the experiment.

FIG. 3 is an explanatory diagram showing the results of effects on fecal MPO activity in DSS-induced colitis mice administered before BOS, FIG. A shows MPO activity of each group on day 3 of DSS administration, and FIG. The MPO activity of each group on day 7 of administration is shown.

FIG. 4 is an explanatory diagram showing the measurement results of IgG activity in feces.

FIG. 5 is an explanatory diagram showing search results of fecal flora before, after and after administration of BOS. Figure a shows the total number of anaerobic bacteria, Figure b shows the total number of aerobic bacteria, and Figure c shows Enterobacteriaceae.
ae , d is Bacteroidaceae , e is Lactobacillus , f
The figure shows the transition of each of the anaerobic mucin-degrading bacteria.

FIG. 6: MP in feces in DSS-induced colitis mice by pre-administration of BOS or Bifidobacterium.
It is explanatory drawing which shows the effect on O activity, FIG. A shows the comparison of a control and BOS single administration, FIG. B shows a control, B.breve .
A comparison of BOS alone and B. breve is shown.

FIG. 7 is an explanatory diagram showing measurement results of MPO levels before and after treatment of ulcerative colitis patients in active and remission phases.

FIG. 8 is an explanatory diagram showing the results of measuring the amount of MPO before and after treatment of a patient with ulcerative colitis in the active phase.

FIG. 9 is an explanatory diagram showing the results of measuring the amount of MPO before and after treatment of a patient with ulcerative colitis in remission.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ryuichiro Tanaka 1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Akemi Imaoka 1-1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo No. Yakult Honsha (72) Inventor Hiromi Setoyama 1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha

Claims (4)

[Claims] 1. An agent for preventing or treating inflammatory bowel disease, comprising galacto-oligosaccharide as an active ingredient. 2. The prophylactic / therapeutic agent for inflammatory bowel disease according to claim 1, further comprising a Bifidobacterium bacterium as an active ingredient. 3. A food or drink for preventing or treating inflammatory bowel disease, which comprises galacto-oligosaccharide. 4. The food or drink for the prevention and treatment of inflammatory bowel disease according to claim 3, further comprising a Bifidobacterium bacterium.