TW200836754A - Prophylactic/therapeutic agent for inflammatory bowel disease - Google Patents

Abstract

Disclosed are: an agent for inhibiting the production of active oxygen from an intestinal epithelium cell; an agent for inhibiting the production of IL-8 from an intestinal epithelium cell; and a prophylactic and therapeutic agent for inflammatory bowel disease and/or irritable bowel syndrome, each of which comprises a fermentation product, a culture supernatant and/or a culture of a bacterium belonging to the genus Bacillus as an active ingredient. The bacterium belonging to the genus Bacillus is preferably Bacillus subtilis, more preferably Bacillus subtilis C-3102 (FERM BP-1096). The agent for inhibiting the production of active oxygen from an intestinal epithelium cell, the agent for inhibiting the production of IL-8 from an intestinal epithelium cell, and the prophylactic and therapeutic agent for inflammatory bowel disease and/or irritable bowel syndrome can be administered through an oral route and are useful for the treatment and prevention of diseases including ulcerative colitis, Crohn's disease and irritable bowel syndrome.

Description

200836754 IX. Description of the Invention: [Technical Field] The present invention provides a novel prophylactic and therapeutic agent for inflammatory glandular gland diseases and/or allergic intestinal syndrome, especially by intestinal epithelial cells. ... stimulated active oxygen production inhibitors derived from intestinal epithelial cells and sputum production inhibitors derived from intestinal epithelial cells. The prophylactic and therapeutic agent for inflammatory bowel disease and/or allergic powder syndrome of the present invention can be administered by the oral route to prevent inflammatory bowel disease such as ulcerative colitis and Crohn's disease in the human digestive tract. 'Also prompts allergic bowel syndrome related to inflammation, etc., to improve the bloody stools, blood in the stool, abdominal pain, weight loss, and loss of appetite accompanying these diseases, and to prevent, treat or alleviate the constipation associated with such diseases. , diarrhea, flatulence, abdominal pain and many other symptoms. [Prior Art] Representative diseases of inflammatory bowel disease (IBD) include ulcerative colitis (uc) and gram-negative disease (CD), which have a large number of patients in Europe and the United States. There is also an increasing trend. These diseases are mainly caused by a group of young people aged 20 years old who develop a large amount of inflammation or cancer in the intestines to present abdominal pain or abdominal writing. As symptoms, it is reported that there are delays in growth of school-age children, rectal prolapse, iron deficiency and even anemia, fatigue and burnout, fever, weight loss, etc. It is unknown that the cause of frequent conversion to colon cancer is unknown. A refractory disease with recurrence and remission. Analysis of clinical knowledge or the use of gene knockout mouse models may suggest that in the tissue damage of these diseases, the antigen present in various intestinal lumens, including intestinal bacteria, will produce a mucosal immune defect. 127277.doc 200836754 Also, it can be seen that from the other side, the activation of clinically active cells, resulting in the excessive production of inflammatory cytokines in inflammatory phagocytes, has not yet established an effective treatment. For each case, the use of nutritional therapy and drug therapy or immunosuppressive agents using a tranexamine or a steroid agent, a derivative, or the like, or a combination therapy thereof is discussed. On the other hand, the prevalence of diseases of irritable bowel syndrome (IBS, Irritable bowel syndrome) can be estimated as the work of the general population ~i,!

Among the major civilized countries, allergic bowel syndrome has a greater impact on the increase in medical expenses. In addition, due to the decline in allergic intestinal syndrome (QOL, qUality Gf life), the resulting economic loss is also a large m treatment method, currently undergoing cardiac and rational control, while implementing anticholinergic agents, laxatives , enteral agents, mucous membrane paralysis agents, autonomic nervous regulators, anti-anxiety agents, antidepressants, sleep drugs, antipsychotics and other phased drug therapy corresponding to symptoms. However, the side effects of these drugs are a problem. Molecular biological methods can also be applied to the treatment of inflammatory bowel diseases, and anti-cytokine therapy for eliminating inflammation can be applied. Although these therapies are more effective, they must be aware of their side effects. Regarding the onset and recurrence of inflammatory bowel disease, it is generally believed that the cause is the multiplication effect of genetic factors and the influence of environmental factors such as diet or intestinal bacteria, and it is considered that the immune regulation caused by these is often caused by diseases. Great impact. In recent years, the causal relationship between intestinal g, especially non-pathogenic Enterobacter bacteria and inflammatory bowel disease has been reported extensively, which can effectively treat diseases without side effects of prebiotics and probiotics (pr〇bi〇). The tics) is believed to restore the balance of bacteria in the intestines or to control the mucosal defense system as a new strategy in the treatment of 127277.doc 200836754. In addition, it is reported that the lactic acid bacteria Lactobacillus kawaii has a defensive effect on the symptoms of ulcerative colitis (Japanese Patent Special Open 2 Team No. 95963). / (4) For the pathogenesis of these inflammatory bowel diseases, the immune system such as the abnormality of cytokines accompanying the destruction of the mucosal immune system is often unknown, and the mechanism is almost unclear. However, it has been clarified that in the active intestinal mucosa, tumor necrosis factor (tnf♦ Ge: Baisujiang-Bu^(4) interferon sputum and other inflammatory cytokines produced by giant sputum cells will be produced. The inhibitory effect of inflammatory cytokines such as TNF-c^IL_8 has the effect of regulating the immune system, and it can improve laparotomy or abdominal pain and symptoms for all the above-mentioned intestinal diseases including allergic intestinal tract. ^ According to the above The development of a inflammatory bowel disease is expected to be a safe and effective inflammatory bowel disease (IV) therapeutic agent. The references cited in the present specification are as follows. The contents disclosed in these documents are all as this specification. The inventors of the present invention are not limited to the prior art of the present specification. [Patent Document 1] Japanese Patent Laid-Open No. Hei. No. Hei. No. 2-95963. It was found that the subfamily of Bacillus subtilis, especially the cell of C_3102 and its sputum product, had anti-inflammation activity and inhibition of IL_8 production in the (4) epithelial cells. In order to complete the 127277.doc 200836754 invention, the present invention provides inhibition of active oxygen production from intestinal epithelial cells using the yeast product of the genus Bacillus, the culture liquid or the k-culture as an active ingredient. J and an inhibitory agent derived from intestinal epithelial cells. From another point of view, too chromium. This is a kind of bacterial yeast product, culture supernatant, or culture of the genus Bacillus. A prophylactic and therapeutic agent for inflammatory 2 diseases and/or allergic intestinal syndromes, belonging to the genus Bacillus subtilis, which is a better bacterium of the genus Pole, and more preferably B. subtilis C_3i 〇 2 (ERM BP 1 096). X, inflammatory bowel disease is preferably ulcerative colitis. In the present specification, the term "culture" means that the bacteria, the culture medium and the acid yeast are obtained by cultivating the bacteria belonging to the genus Bacillus in a suitable medium. a mixture of substances. The so-called "fermented product" means that the part other than the bacteria in the culture includes a preparation obtained by removing the g body from the culture. Further, the "acid yeast product" also includes the self-culture extract. In addition, the "fermented product" in the case of using a liquid medium is called "culture supernatant". In addition, "fermented product" and π solution in culture are included. It can be obtained by a method commonly used in the field of technology, such as filtration or centrifugation, to remove bacteria from the culture. Further, the fermentation product and the culture supernatant are obtained. When it is obtained by operation such as centrifugation, it does not mean that the cells or fragments thereof are completely absent. [Embodiment] Bacillus bacteria (including Bacillus subtilis) have been treated with 127277.doc 200836754 since ancient times. People's eating habits are closely related, and there is a lot of information about their functionality', but there is no report on the effect of preventing or treating inflammatory bowel disease. In the present invention, as shown in the following examples, it was confirmed that the culture belonging to the genus Bacillus showed a therapeutic and preventive effect of ulcerative colitis in the DSS (Dextran Sulfate sodium) model. and then,

It is well known that temporary colorectal inflammation changes the sensory function of the organ and precedes the symptoms of irritable bowel syndrome. Therefore, TNbs (Tnmtrobenzene Sulf0nic Acid, III) is used in a rat test system for evaluating the autonomic nervous response caused by colorectal dilation. Nitrobenzenesulfonic acid) acts as an inducer of inflammation (Adam B, et al., 2006, Pain 123(1-2): 179-86). This reagent is also used in the test system for ulcerative colitis in the same manner as DSS (Charles 〇 Elson, et al., 1995, Gastroenterology 1〇9: 1344-1367) 'It can be considered that the present invention belongs to the genus Bacillus The culture also shows therapeutic and preventive effects on allergic intestinal syndrome. In the present invention, it is clarified that Bacillus subtilis (II) 1 () 2 has an effect of preventing or even treating inflammatory bowel disease and allergic intestinal syndrome. Moreover, the culture supernatant of the hairline C - 3 10 2 showed an effect of inhibiting the active oxygen and the active oxygen produced by the human intestinal epithelial cells. As shown in the following example, the subculture of the culture supernatant of the C = auditory strain was pretreated. In the epithelial cells, TMΡ·« stimulated the expression of IL_8 was inhibited, thus showing anti-inflammatory effects... and TNF When -α is preliminarily subjected to the reaction caused by the π liquid at 37t, the TMFa stimulation is generated and the performance of the image is suppressed. Thus, 127277.doc 200836754 shows that the factor secreted extracellularly by C-3102 is involved in the action of TNF-α which neutralizes inflammatory cytokines. The agent for preventing and treating inflammatory bowel disease and/or allergic intestinal syndrome according to the present invention, which comprises a culture of a bacterium belonging to the genus Bacillus, preferably a culture of Bacillusius Sllbtilis. Active ingredients. The bacteriological properties of Bacillus subtilis are disclosed in Bergey, s Manual μ Bacteriology V〇i. U (1986) and the like, and specifically, for example, have the following features. (1) Gram-positive (2) Formation of oval-shaped spores (3) Bacilli (4) Exercise ·· (5) Aerobic (6) Catalase: positive (7) Development at 50QC: + (8) Development at pH 5.7: + (9) Use of citrate: + (1〇) Whether or not to produce acid from sugar: arabinose, glucose, xylose, mannitol: + (11) VP Reaction: + (12) Hydrolysis of starch: + (13) Reducibility of nitrate: + (14) Formation of hydrazine: - (15) Hydrolysis of gelatin: + 127277.doc • 11- 200836754 (16) Cool protein Hydrolysis: + (17) Formation of a film in liquid medium: + (18) Coagulation of milk: _ (19) Protein adenosis of milk: + as an inflammatory bowel disease and/or allergic bowel of the present invention For the prevention and treatment of syndromes, the use of dry, ® C 3102^ (A: dry bacteria, for example, ·····························

RP 1〇〇^ ^ w Nine, the registration number FERM BP-Na, the date of registration is called the next day). Bacillus subtilis can be used, for example, by the 专 专 专 沬馑 寻 寻 和 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 63 生物 生物 生物 生物 生物 生物 生物 生物Nutrients such as source and inorganic substances are cultured. As the carbon source, it may be a base or a solid (tetra) source, for example, glucose, fructose, sucrose = a slave that can be deuterated, and a helium gas, a tower η 庶 庶 sugar, a starch, a molasses, etc. Examples include peptone, casein, and ammonium sulfate. Further, it is also possible to add a pot of a phosphate, a potassium, a sulphur, a sulphate, a sodium, an iron, a bell, a clock, a pot of a vitamin, an activator, and the like. In addition, in addition to these, the beans, the yin, the 杳笙% ώ attack, and the nurturing base can also be cultured using a good material of the natural substance. As a culture condition, it is preferable to use a sour culture medium or a cultivating device, such as a sour fermenter, a cultivating device, or the like. It is good to raise and simmer 3 (four) 20~5〇 °C, especially good is PH value 5 / day for 12 hours ~ 7 days 'cultivation initial PH value is better 疋 PH value 5~9, especially good The positive value is 6~8. The culture thus obtained can also be used directly after the culture of Hans 127277.doc -12· 200836754. Alternatively, the cells may be isolated from the culture by filtration, centrifugation, extraction, or the like to prepare a fermentation product or a culture supernatant. Further, an excipient or the like may be added to the culture, the fermentation product or the culture supernatant to prepare a preparation such as a dry powder, granules or tablets. In a particularly good manner, 'Bacillus subtilis is cultivated using natural oils and other substances such as soybean oil residue, boiled soybeans, boiled bean rice, wheat rice, wheat noodles, boiled corn, and other cereals. The cells are isolated from the culture and can be directly added to the food.

The prophylactic and therapeutic agent for the heart disease and/or allergic intestinal syndrome can be administered in the form of a liquid, a powder, a granule, a lozenge, or the like, and can also be added as a food additive to a food or beverage. Ingestion. The beverage 艮' may, for example, be a beverage, a confectionery, a paste, a bread, a processed fish product, a dairy product or the like. The prophylactic and therapeutic agents for inflammatory bowel diseases and/or allergic intestinal syndromes of the present invention are added to these various food materials, and can be used as health drinks, health foods or functional foods.

The contents of all patents and references explicitly cited in this specification are incorporated herein by reference. In addition, the claimant’s priority claim is based on the claim of the Japanese version of the Japanese patent application No. 2〇〇6_329596, which is uncovered in the mouthpiece of the Japanese Patent Application No. 2〇〇6_329596. This is cited as part of this specification. [Embodiment] However, the present invention is not limited to the following, and the present invention will be described in more detail based on the embodiments. I27277.doc -13· 200836754 Example 1 - Verification of the effect of Bacillus subtilis C-3102 strain on human colonic epithelial cells In the following examples, Bacillus subtilis C-3102 strain was used as an example of a bacterium belonging to the genus Bacillus ( Institute of Industrial Engineering, Institute of Biotechnology, registered number FERM BP-1096, deposited on December 25, 1985. The soybean culture of Bacillus subtilis C-3 102 strain has the effect of improving intestinal bacterial growth, increasing 'weight, preventing infection, strengthening egg shell, improving meat quality, improving stool odor, etc., and is used as an additive (Japan) Patent Special Fair 4-24022). • As a health benefit of this strain, it is known that the intestinal action is reduced and the intestinal decay products are reduced. (Intestinal Bacteriology Society, Vol. 18, No. 2, 93-99 (2004)). Bacillus subtilis C-3102 has a feature of a fragment of about 700 bps which can be amplified by PCR using the PCR primers of the following sequence 1 and sequence 2. Other B. subtilis does not cause amplification by the PCR primer. A fragment of about 700 bps which proliferates in B. subtilis C-3 102 has a characteristic of no homology with the sequence of amylase and can be clearly distinguished from other Bacillus subtilis.歹丨 j 1 : 5'-GCCCCGCACATACGAAAAGACTGGCTGAAA-3, (SEQ ID NO: 1), SEQ ID NO: 2, 5, -GGATCCCACGTTGTGATTAAAAGCAGCGAT-3, (SEQ ID NO: 2) Further, Bacillus subtilis C-3 102 strain has the following properties: (1) Does not have plastid DNA. (2) Preparation of genomic DNA, digestion with restriction enzyme Not I or Sfi I, and digestion pattern when separated by agarose electrophoresis is shown in Fig. 1. 127277.doc -14- 200836754 (3) Production of B.cerous antibacterial substances. (4) For ampicillin, chloramphenicol, ciprofloxacin 'erythromycin, Jiantianmycin, ketomycin, linezolid, quinupristin/dalofopine, rifampicin, streptomycin Tetracycline, trimethoprim, and vancomycin were not tolerated (minimum inhibitory concentrations were 0.03 to 4 pg/ml). With respect to the components of live bacteria, dead bacteria and culture supernatant of Bacillus subtilis C-3102 strain, inhibition of inflammation of colonic epithelial cells induced by TNF-α was examined using induction of active oxygen and cytokines as indicators. Materials and Methods (Α) Cells The cell line used was a 细胞84 cell line derived from human colorectal cancer. As a medium, culture was carried out using a DMEM/HAM-F12 (1:1) mixed medium (DF) supplemented with 5% fetal calf serum (Fcs), 1 〇〇 gg/mi streptomycin, and 100 U/ml penicillin. As the T84 cells, cells which were seeded in a 24-well cell culture dish or a 35 mm culture dish and cultured for 2 days under a 5% c〇2_ atmosphere were used. (B) Preparation of C-3102 freeze-dried bacterial powder C-3102 strain was isolated and cultured in TS medium (Tripticase Soy Broth: BBL, +2% agar) at 37 ° C for one night, and the colonies that appeared were inoculated. In 5 ml of TS broth (removing the agar medium in the above medium), one night of culture at 37 ° C was used as a production of a fermentation broth. 3 ml of the production of the fermentation broth was added to the TS broth of 3 〇〇, and the mixture was shaken at 37 ε for one night, and then collected by centrifugation (7000 rpm, 2 〇 min). The granules were washed with sterile water and centrifuged again to obtain granules. 127277.doc -15· 200836754 The system was freeze-dried after one night of freezing. The bacterial powder thus obtained was prepared by the three methods shown below. (i) c_3i〇2 live bacteria (L) added serum-free training to the bacterial powder, and dissolved and dissolved for 15 minutes using a vortex mixer. (2) C-31〇2 dead bacteria (HK) ··丨(10) After 5 minutes of treatment, centrifuge at MOOxg for 15 minutes, add DF to the granules again, and stir and dissolve using a vortex mixer. (3) c_3i〇2 culture supernatant (CM) ···0·2 μιη The filter was filtered in a serum-free medium for 24 hours to culture the bacterial culture supernatant. (C) C-3102 treatment method and conditions were cultured in a PBS, 37C, 5% C〇2 gas atmosphere. After 2 days, the 84 cells were washed 5 times and exchanged for serum-free DF. After 丨 hours, add C-31〇2 live bacteria (L) at a concentration of 〇~2 cfu per t84 cells. Dead bacteria (HK). The culture supernatant (CM) was added with a supernatant containing C-3 102 viable bacteria for 24 hours in an amount equivalent to the concentration of 〇~2 cfu/cell. (D) TNF -α treatment conditions Human recombinant TNF_a (R&D systems) was added to T84 cells cultured in 24-well cell culture plates or 3 5 mm culture dishes at 20 ng/ml. (E) Ultra oxygen Ion (〇2·) assay cell line was cultured in a 24-well cell culture dish and treated with C-3 102 live bacteria (L), dead bacteria (HK) or culture supernatant (CM) for 2 hours. Further, TNF-a stimulation was carried out. After 24 hours, the cells were washed 5 times with HBSS, and cytochrome C (1 mg/ml) + PMA solution was added thereto, and culture was carried out for 1 hour at 37 ° C under a 5% CCV atmosphere. The absorbance of the reaction solution (550 nm) was measured, and the remaining cell line of 127277.doc -16-200836754 was washed three times with HBSS, and the protein was determined by the Lowry method. The amount of 〇2_ produced per well was calculated and nmol was obtained. /mg-protein/h is indicated.

(F) RT-PCR Cells were cultured in PBS, cultured in 35 mm culture, and TNF-α-treated cells were washed three times, and RNA was extracted using ISOGEN (Nippongene, Toyama). The obtained RNA (1 pg) was subjected to reverse transcription using a TAKARA RT-PCR kit (TAKARA, Tokyo). The resulting reverse transcription product was synonymous with primer 5, -TTGGCAGCCTTCCTGATTTCT-3, (SEQ ID NO: 3); antisense tweezers, 5'-TTTCCTTGGGGTCCAGACAGA-3' (SEQ ID NO: 4); glyceraldehyde triphosphate dehydrogenase ( The synonymous primer of GAPDH), 5'-TCATGACCACAGTCCATGCCATCACT-3丨 (SEQ ID NO: 5), antisense bow | sub 5-GCCTGCTTCACCACCTTCTTGATGT-3, (Sequence 歹 ij No. 6) was used as a primer set for IL-8 for PCR. Results (A) For the treatment of live bacteria with enhanced 02_production after TNF-0C stimulation, T84 cells cultured in 24-well cell culture plates were used in the live bacteria (L) of 0~2 cfu/cell. After 2 hours of treatment, 20 ng/ml of TNF-a was added. The sputum/production from T84 cells, although enhanced by TNF-a stimulation, did not reveal the effects of prior treatment with live bacteria (L). The cells were damaged at 2 cfu/cell, which was thought to be due to a decrease in 02-production due to a decrease in the number of cells (Fig. 1). From the above results, it can be suggested that the previous treatment of live bacteria (L) does not affect the 02 caused by TNF-a stimulation, resulting in enhancement. (B) For the live bacteria, the dead bacteria 127277.doc -17- 200836754 and the culture supernatant before the stimulation with TNF-a, the effect of the treatment is second, for the dead bacteria (HK) and the culture supernatant ( CM) conducted the same study. T84 cells cultured in 24-well culture dishes were pretreated for 2 hours in live bacteria (L), dead bacteria (HK) and culture supernatant (CM) of c·5 cfu/cell, and added 20 ng/ Ml TNF-α. 〇2- is enhanced by TNF-a stimulation (Μ) compared to untreated (N). The effect of the treatment before 2 hours was investigated, and as a result, it was not affected in the living bacteria and produced. The cell damage of the dead bacteria (ΗΚ) is strong, and cell detachment occurs, and sputum cannot be measured. However, in the culture supernatant (CM), the sputum caused by TNF-a stimulation was significantly inhibited (Fig. 2). (C) For the culture supernatant which was stimulated by TNF_a, the amount of production was increased. The effect of the treatment was as follows. The concentration of the inhibitory effect of the culture supernatant (CM) was examined. The butyl 84 cells cultured in a 24-well culture dish were pretreated for 2 hours in the culture supernatant (CM) of (1)...~丨cfu/ceU, and 2 ng/ml of TNF_a was added. 〇2· is enhanced by TNF-a stimulation (M) compared to untreated (N). From the culture supernatant 〇 〇 5 cfu / ceU concentration can be seen to produce a potent inhibitory effect (Figure 3). From the above results, it was suggested that the culture supernatant (CM) of C-3 102 inhibited the production of TNF-ct by TNF-ct stimulation in T84 cells. (D) The effect of living bacteria, dead bacteria, and culture supernatants expressing IL_8 mRNA by TNF_ stimulation, and then studying the live bacteria, dead bacteria, and culture of inflammatory cytokine il_8 mRNA by TNF-stimulation. The effect is treated before the clear liquid. In 127277.doc -18- 200836754 T84 cells cultured in 35 mm culture dishes were supplemented with 20 ng/ml of TNF-α, and the expression of IL-8 mRNA was continued using RT-PCR. When TNF-α stimulation was applied to T84 cells, the expression of IL-8 mRNA was observed after 1 hour, and a peak was formed after 3 hours. Add TNF-a at a concentration of 20 ng/ml to cells pre-treated for 2 hours with live bacteria (L), dead bacteria (HK), and culture supernatant (CM) at a concentration of c·5 cfu/cell. IL-8 mRNA was analyzed 3 hours later. In the cells treated with live bacteria (L) and culture supernatant (CM) for 2 hours alone, the expression of IL-8 mRNA was not observed, and it was observed in cells treated with dead bacteria (HK) for 2 hours alone. The performance of IL-8 mRNA. The reason is considered to be that no anti-inflammatory component is produced in the dead bacteria (HK). Further, the same experiment was repeated, and the following results were obtained: strongly suggest that the expression of IL-8 mRNA was inhibited in the pretreatment with the culture supernatant (CM), and the product had an anti-inflammatory action. From the above results, it was confirmed that the culture supernatant (CM) has an inhibitory effect on IL-8 mRNA expression by stimulation with TNF-a. (E) Inhibition effect of IL-8 mRNA pre-treated with culture supernatant (CM) Further, the effect of inhibiting the expression of IL-8 mRNA by pretreatment of the culture supernatant (CM) was examined in detail. After pretreating T84 cells in the culture supernatant (CM) for 2 hours, the cells were washed, and TNF-a (CM_re) was added, and the expression of IL-8 mRNA in the cells treated as above was not inhibited. From this, it can be suggested that the culture supernatant (CM) does not act on T84 cells. Further, in a case where the culture supernatant (CM) was treated at 100 ° C for 5 minutes (b-CM), T84 cells were pretreated for 2 hours, and TNF-a was added to analyze the expression of IL-8 mRNA, but No inhibition effect was observed. From this situation, it is suggested that the factors in the culture supernatant (CM) are secreted from 127277.doc -19- 200836754. 1 5 The possibility of α activity (Fig. 4) The knowledge of the knowledge of the sea can be compared with + · , RMA ^ . The beauty of the liquid (CM) inhibition ratio -8

The mechanism of m expression is different from the existing anti-inflammatory factor of s〇D u忒 peptide temple.

Whether the pre-game 2 ^ itself acts directly on the 7-pulse "to conduct research. The supernatant is cultured in advance with ·α, and cultured for 2 hours to obtain a reaction (CM+TNF♦ is added to m cells, to W

m table line analysis (8). In the pre-mixed culture supernatant and TNF a culture, the expression of IL-8 mRNA was inhibited. In the case where DF and TNF__# were cultured for 2 hours in 3 passages (M+ pulse a), no inhibitory effect was observed. Further, even if the culture supernatant and τΝρ· a ' were simultaneously added, the expression of IL_8 mRNA was not completely inhibited. From the above results, no factors in the culture supernatant secreted to C-31〇2 acted on tnf_a, but inhibited the expansion of inflammation (Fig. 5). Example 2: Effect on ulcerative colitis model As an ulcerative colitis model animal, (4) rats in which glucan sulfate was taken up. In 5 kg of soybean oil granules, add 5 kg of tap water to 121 (: 35 bacteria for 120 minutes, and take the pre-cultured culture solution of Bacillus subtilis C-310 (FERM BP-1096). When it is cultured at 37. under the armpit, it is dried and pulverized to obtain c_3102 soybean culture. It is mixed into CE-2 (Clea) Clea powder feed (about 1%) (2·5χ1) 〇8 efu/g) Male Sprague Dawley (IGS) rats of 5 weeks old, group (7), were given free access to the feed for about 1 week. The control group was inoculated with a pulverizer, a sputum granule, and a slag granule, which was mixed with the test group to the CE 2 powder 127277.doc -20-200836754 feed. During the feeding of the control feed and the test feed, the drinking water was administered (3./.w/v) with sodium sulphate (molecular weight 5, sulfur content 15.0 to 20.0%) and Wako Pure Chemical Industries). Heterogeneous traits During the test, the body weight was measured. • One or eight long-term sees if 8 (=NC-l IL_8: Panapham)

The animals were sacrificed on the next day of the day, and the spleen weight, colon length and cecal weight were dissected. The change in body weight is shown in Fig. 6, the fecal trait is shown in Fig. 7, and the ?" in plasma is shown in Fig. 8. Further, the anatomical data are shown in Table 1. Further, the figure of Fig. 7 indicates the number of days of administration relative to the control group, and the test feed group has a significant difference under P<(U)1 (Bon Franny correction X2 test). Further, ☆ of Fig. 7 indicates the number of days of administration with respect to the control group, and the test feed group has a difference of (4) under ρ < 0. (5) (Bon Franny correction χ 2 test). [Table 1]

** Ρ&lt;0.01 &gt; * ρ&lt;0.05 There was no difference between the two groups regarding the amount of water consumed and the amount of food intake. For the body weight, the test feed group changed more than the control group, and a significant difference was observed on the final day (ρ&lt;0·05 )(Figure 6). Further, regarding the fecal traits, blood was observed on the control group from the second day, and significant differences in fecal traits were observed on the test feed group from the fourth day, the fifth day, the sixth day, the seventh day, and the eighth day (Fig. 7). The length of the colon of the test feed group was significantly longer (ρ &lt; 0·01). The cecal weight S after washing every 1 〇〇g of rat body weight was also significantly reduced (? &lt; 0 05) (Table U. Again, regarding the plasma in the plasma _8, 127277.doc • 21 · 200836754 on 4 tests _ The tendency of the material group to decrease (Fig. 8) suggests that the inflammatory reaction may be inhibited. It is confirmed by the results of sputum and the like that C-3102 has an excellent defense effect on the condition of colitis induced by DSS. The confirmation test for the active ingredient of the ulcerative colitis model is in Example 2, and since it is confirmed that the c_31〇2 soybean culture has an anti-inflammatory effect, it is observed whether the active ingredient is present in the cells or the fermentation product, and the culture supernatant component. In addition, the c_31〇2 soybean culture was mixed with water, centrifuged, and the obtained water extract was heat-treated to inactivate the protease-active component to observe whether the effect affected the anti-inflammatory effect. The animals and test methods used were the same as in Example 2. The rats were set to 1 group and 4 rats were used as test feeds to prepare CE 2 powder feed, and (1) 1% C-3102 soybean culture was added. The original <Figure 9 C-31〇 2, Fig. 10 C-3102>, (2) The same amount of c_31〇2 soybean cultures as described above (1) are mixed with water, and centrifuged to obtain an aqueous extract. The aqueous extract is freeze-dried. Further, the addition is &lt;Fig. 9 protease +, Fig. 10 extract&gt;, (3) The same amount of c_3102 soybean culture as the above (1) is mixed with water, and centrifuged to obtain an aqueous extract. The water extract was heated for 5 minutes, then lyophilized, and then added. [Fig. 9 Protease 'Fig. 10 Heated extract>, (4) The same amount of C_3102 as the above (1) The soybean culture is mixed with water, and centrifuged to obtain an aqueous extract. The remaining residue is freeze-dried (Fig. 9 residue, j|i〇 residue), 127277.doc -22· 200836754 (5) Adding 1% of soybean granules to the granules &lt;Show&gt; The contents of Fig. 9 and the feed of Fig. 10 are forked. For the control group, the control feed used in Example 2 was administered. The same, the protease activity was measured using a PW Assay Kit (PIERCE). The change in body weight is shown in Fig. 9, and The change in the traits is shown in Figure 1. There was no significant change in body weight between the groups. The addition of 1% to the mixed C-3102 soybean culture was added by the addition; the 7 components were separated from the water by centrifugation. The obtained water extract is cooled; the group of the feed of the east dryness (the above (2) has protease activity) is lighter than other groups, and the heat treatment (the above (3) has no protease activity) Since it is the same as the control group (controlled by (5) above), it is considered that the active ingredient exhibiting an anti-inflammatory effect is heat-inactivated. As shown in the above Examples 2 and 3, it can be seen that in the model test of the colitis, 'by adding 1% of the 〇31〇2 soybean culture to the feed, the body weight change, the incidence of bloody stool and blood in the stool, and the large intestine tissue can be reduced. The anti-inflammatory action, plasma IL-8 concentration, etc., thereby inhibiting colitis. Further, in the third embodiment, the water extract of the soybean culture of the C. crispii c_3丨〇2 strain was confirmed to have an anti-father effect, and this action was inactivated by heating the water extract. From these conditions, the culture, the fermentation product, and the culture supernatant of the C-3 102 strain have an inhibitory effect against the inflammation of the intestinal epithelial cells induced by TNF-α stimulation. According to the above results, by using the bacteria of Bacillus subtilis C-3 102 or the culture thereof, the acid yeast product, and the culture supernatant, an inflammatory bowel disease and/or an allergic bowel which can be used and effective can be provided. Prevention of the syndrome. Treatment agent. 127277.doc -23- 200836754 [Simplified Schematic Description] Fig. 1 shows the effect of pretreatment of C-3102 viable bacteria in a cell line derived from human colorectal cancer for enhancing the production of 〇2 by stimulation with TNF_a. Fig. 2 shows the effect of treatment of c-3102 live bacteria, dead bacteria, and culture supernatant α in the cell line derived from human colorectal cancer, which is used to stimulate the production of sputum. Fig. 3 shows C-3102 cultured on a cell line derived from human colorectal cancer, which is enhanced by the stimulation of TNF_a, and is cultured. π on the lips, the shadow of the night w treatment W ring. Fig. 4 shows the effect of pretreatment on 活3102 live bacteria, dead bacteria, and culture supernatant of IL-8 mRNA stimulated by TNF_a in a cell line derived from human colorectal cancer. Fig. 5 shows a study of the effect of C-3102 culture supernatant on the effect of TNF_ai. Fig. 6 shows changes in body weight of Dss_induced ulcerative colitis model rats fed with c_3 102 soybean culture feed. Fig. 7 shows the occurrence of symptoms such as bloody stools and blood in the stool of a model of DSS-induced ulcerative colitis in which a feed supplemented with a C-3 102 soybean culture was administered.

Fig. 8 shows the sputum expression rate in plasma of a DSS-induced ulcerative colitis model to which a feed of C-3 102 soybean culture was added. Fig. 9 is a graph showing changes in body weight of a dSS-induced ulcerative colitis model rat administered with a feed supplemented with c_31〇2 soybean culture. Fig. 10-1 shows the incidence of symptoms such as bloody stools and blood in the stool of a rat model of ulcerative colitis induced by 127277.doc -24-200836754 DSS administered with a supplement of c_3102 strain soybean culture. Fig. 10-2 shows the incidence of symptoms such as bloody stools and blood in the stool of a model of ulcerative colitis induced by DSS administered with a C-3102 strain of soybean culture.

127277.doc -25- 200836754 Sequence Listing &lt;110> Riker Co., Ltd. &lt;12〇&gt; Inflammatory Bowel Disease Prevention and Treatment Agent &lt;130&gt; PCP-90Q7WO &lt;140&gt; 096146528 &lt;141&gt; 2007-12-06 &lt;150>"P 2006-329596 &lt;150 2006-12-06 &lt;160&gt; 6 &lt;170>Patenln version 3.1 &lt;210>&lt;210 &lt;212&gt;&lt;213 〉

30 DNA Bacillus subtilis

&lt;400〉 1 gccccgcaca tacgaaaaga ctggctgaaa

&lt;210&gt; 2 &lt;211&gt; 30 &lt;212&gt; DNA &lt;m&gt; Bacillus subtilis &lt;400&gt; 2 ggatcccacg ttgtgatlaa aagcagcgat &lt;210> 3 &lt;2I1&gt; 21 &lt;212> DNA &lt;213> Homo sapiens &lt;400&gt; 3 ttggcagcct tcctgatttc t

&lt;210> 4 &lt;211> 21 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 4 tttccttggg gtccagacag a &lt;210> 5 &lt;211> 26 &lt;212&gt; DNA &lt;213> Homo sapiens &lt;400&gt; 5 tcatgaccac agtccatgcc atcact &lt;210&gt; 6 &lt;211&gt; 25 &lt;2!2&gt; DMA &lt;213> Homo sapiens &lt;400&gt; 6 gcctgcttca ccaccttctt gatgt 127277 - Sequence Listing.doc

Claims (1)

200836754 X. Patent application scope: 1 · An active oxygen production inhibitor derived from intestinal epithelial cells, which is an active ingredient of a bacterium derived from a bacterium belonging to the genus Cupula. 2' is an active oxygen production inhibitor according to claim 1, wherein the microorganism belonging to the genus Bacillus is Bacillus licheniformis. 3. The active oxygen production inhibitor according to claim 1 or 2, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP_1096). 4. An IL-8 production inhibitor derived from intestinal epithelial cells, which is an active ingredient of a bacterium derived from a bacterium belonging to the genus Bacterium. 5. The inhibitor of IL-8 production according to claim 4, wherein the bacterium belonging to the genus Bacillus is Bacillus licheniformis. 6. An inhibitor of il-8 production according to the item 4 or 5, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP-1096). A prophylactic and therapeutic agent for inflammatory bowel disease and/or irritable bowel syndrome, which is an active ingredient of a bacterium belonging to the genus Bacillus. A preventive and/or oral therapeutic agent for an inflammatory bowel disease and/or an allergic intestinal syndrome according to Item 7, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. The prophylactic and therapeutic agent for the catastrophic fat disease and/or allergic intestinal syndrome of the seventh or eighth month, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP-1096) 〇l〇. Item 7 to 9 for the prevention and treatment of inflammatory bowel disease and/or irritable bowel syndrome, wherein the inflammatory bowel disease is ulceration 127277.doc 200836754 11 · an intestinal epithelial cell The active oxygen generating inhibitor is a culture supernatant of a bacterium belonging to the genus Bacillus as an active ingredient. 12·If requested! The active oxygen generating inhibitor, wherein the genus belonging to the genus Bacillus is Bacillus subtilis. 13. The active oxygen production inhibitor according to claim u or 12, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP-1096). 14. A ratio-inducing inhibitor derived from intestinal epithelial cells, which comprises a culture supernatant of a bacterium belonging to the genus Bacillus as an active ingredient. 15. If the claim 142IL_8 produces an inhibitor, the bacterium belonging to the genus Bacillus is Bacillus licheniformis. 16. The inhibitor of claim 14 or 15 wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FFRM BP-1096). A preventive and therapeutic agent for a sputum intestinal disease and/or an allergic intestinal syndrome, which comprises a culture supernatant of a bacterium belonging to the genus Bacillus as an effective component. 1 8 · As requested! A prophylactic and therapeutic agent for inflammatory bowel disease and/or allergic intestinal syndrome, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. 19. The prophylactic and therapeutic agent for inflammatory bowel disease and/or irritable bowel syndrome according to claim 17 or 18, wherein the bacterium belonging to the genus Bacillus is Bacillus licheniformis c_ 3102 (FERM BP-1096) 〇 20. The prophylactic and therapeutic agent for inflammatory bowel disease and/or allergic intestinal syndrome according to any one of claims 17 to 19, wherein the inflammatory bowel disease is ulcerative colitis. An active oxygen production inhibitor derived from intestinal epithelial cells, which is a culture of a bacterium belonging to the genus 127277.doc 200836754. 22. The active oxygen production inhibitor of claim 21, wherein the genus belonging to the genus Bacillus is a bacterium. 23_ The active oxygen production inhibitor of claim 21 or 22, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP-1096). 24. An inhibitor of _8 production from intestinal epithelial cells, which is a culture of bacteria belonging to the genus Bacillus as an active ingredient. 25. If the claim 24iIL-8 produces an inhibitor, the bacterium belonging to the genus Bacillus is Bacillus subtilis. 26. The IL-8 producing inhibitor according to claim 24 or 25, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis C-3102 (FERM BP_1096). 27. A prophylactic and therapeutic agent for inflammatory bowel diseases and/or allergic intestinal syndromes, wherein a culture of bacteria belonging to the genus Bacillus is used as an active ingredient. 28. The prophylactic and therapeutic agent for inflammatory bowel disease and/or allergic intestinal syndrome according to claim 27, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. • 29. The prophylactic and therapeutic agent for inflammatory bowel disease and/or irritable bowel syndrome according to claim 27 or 28, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis c_ 3102 (FERM BP-1096) 〇. 30· The prophylactic and therapeutic agent for inflammatory bowel disease and/or allergic intestinal syndrome according to any one of claims 27 to 29, wherein the inflammatory bowel disease is ulcerative colitis. 127277.doc