JP2006001922A - Allergy inhibitory composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a substance having a safer and more effective allergic disease inhibitory activity for preventing and treating the allergic diseases safely and more effectively. <P>SOLUTION: The subject allergy inhibitory composition is provided by using levan which is experienced as a foodstuff, especially produced by Bacillus Natto, as an active ingredient for inhibiting the production of IgE and IL-4 and promoting the production of IL-12 inhibiting the differentiation to Th2 cells. The allergy inhibitory composition includes a medicine, beverage and foodstuff capable of preventing, moderating or treating the allergy. The amount of daily effective oral dose of the levan is approximately 50-1,000 mg, and preferably approximately 120-600 mg. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides an allergy-suppressing composition containing levan, particularly levan produced by Bacillus natto, as an active ingredient as a highly safe and highly effective substance having an allergy-inhibiting action, and enables prevention and treatment of allergies Related to making.

  Allergic diseases are immunodeficiencies such as bronchial asthma, atopic dermatitis, and allergic rhinitis, and hay fever, which has been sought to increase in recent years, is one of allergic rhinitis.

  Immune cells include T cells and B cells, and B cells make antibodies. On the other hand, it is helper T cells (Th cells) that control antibody production. Cytokines such as interferon (IFN) and interleukin (IL) produced in Th cells play a role in transmitting signals between the two.

  Furthermore, Th cells are broadly classified into Th1 cells and Th2 cells depending on the type of cytokine produced, and it has been found that Th2 cells producing IL-4 are involved in so-called allergies such as hay fever and green fish allergy. Yes.

  That is, when Th2 cells are activated by allergens, Th2 cells release IL-4 and IL-13 to promote production of immunoglobulin E (IgE) from B cells, and at the same time, IL-3 and IL- It is said that the action of 5 or the like acts on the differentiation proliferation and survival extension of mediators such as eosinophils and basophils (for example, see Non-Patent Document 1).

  Furthermore, it is known that differentiation into Th2 cells is promoted by IL-2 and conversely suppressed by IL-12.

  Therefore, it is possible to confirm the allergic inhibitory effect by examining the effect of suppressing the production of IgE and IL-4 and the effect of promoting the production of IL-12 that suppresses differentiation into Th2 cells.

  Conventional allergy therapeutic agents are broadly classified into mediator release inhibitors, histamine H1 antagonists, thromboxane inhibitors, leukotriene antagonists, Th2 cytokine inhibitors, etc., most of which differentiate into Th2 cells. There are few basic therapeutic drugs that are symptomatic treatments that deal with subsequent symptoms, such as suppressing the differentiation into Th2 cells themselves. In addition, many of these allergic drugs have severe side effects, and most of them require a certain amount of time before the effects are manifested (for example, see Non-Patent Document 1).

  In view of these, research on allergy suppression using microorganisms has also been conducted, for example, anti-allergic active substances mainly composed of fermented products obtained by treating soybeans, wheat, citrus fruits, fruits, etc. with filamentous fungi or natto enzymes. (For example, refer to Patent Document 1), and microbial cell degradation products having immunomodulating activity obtained by dissolving cell walls of Bacillus natto, Bacillus genus bacteria, lactic acid bacteria, etc. (for example, refer to Patent Document 2), etc. However, in these cases, the active main substance showing the allergy suppressing effect has not been identified, and the main substance has not been clarified.

On the other hand, the present invention has been found for the first time that levan has an excellent allergy-inhibiting action, but levan is one of the natto bacteria products and is also known to be contained in natto. However, the anti-tumor activity is only known as the physiological function of levan (for example, see non-patent document 3). It was not known. Moreover, its administration method and dose were not known at all.
“ASTHMA”, Vol. 13, No. 1, p. 15-19, 2000 “Bulletin of Faculty of Home Economics, Fukuoka Women's University”, Volume 16, p. 1-5, 1984 “International Journal of Biological Macromolecules”, 27, p. 245-247, 2000 JP 2003-327540 A Japanese Patent Laid-Open No. 2000-4830

  The present inventors have aimed to provide a safer and more effective substance having an allergic disease inhibitory action to prevent and treat allergic diseases safely and more effectively.

  The present invention has been made to achieve the above object, and the present inventors have considered that conventional allergy treatment drugs are often symptomatic treatments, and there are few basic treatment drugs. We set a new technical challenge to develop an allergy prevention / treatment composition that is more effective and safer than the basic treatment of allergic reactions by suppressing differentiation into Th2 cells.

  In order to solve the above technical problem, the present inventors have focused on microorganisms from the viewpoint of safety as a result of examination from various directions, and among them, one of natto bacteria and natto bacteria-utilized foods that have been used for a long time as food. I focused on natto.

  In this way, the present inventors have extensively searched for food materials and food-related microorganisms that are considered to be safer, and as a result, have strong IgE production in levan produced by Bacillus natto who has experience eating foods. And the effect of suppressing IL-4 production and the effect of promoting the production of IL-12 that suppresses differentiation into Th2 cells were found, and the present invention could be completed.

That is, this invention relates to the allergy suppression composition which uses levan as an active ingredient, and is shown next.
(1) An allergy-suppressing composition comprising levan as an active ingredient.
(2) An allergy-suppressing composition, characterized in that levan is produced by Bacillus natto.
(3) Add saline to natto and stir, add ethanol or ethanol water to the supernatant of the resulting suspension, and treat each of the resulting precipitate with water and ethanol or ethanol water A method for producing an allergy-suppressing composition, characterized in that levan prepared by a method comprising a step of dialysis of the solution of the final precipitate against water and drying is used as an active ingredient.
(4) At least selected from (A) natto as a raw material in (3), (B) levan as a final product, and (C) at least one product generated in the process from natto to production of levan. An allergy-suppressing composition comprising levan as an active ingredient, characterized by comprising one as an active ingredient.
(5) An allergy suppressant containing levan as an active ingredient.
(6) A food or drink product having an allergy-suppressing activity comprising levan as an active ingredient.
(7) The food / beverage product according to (6) above, wherein the food / beverage product is natto.
(8) An allergy-suppressing method characterized by orally ingesting 50 mg to 1000 mg of levan per day.
(9) A method for suppressing allergy, which comprises orally ingesting 120 to 600 mg of levan per day.
(10) The method for suppressing allergy according to the above (8) or (9), wherein leban is orally ingested by ingesting natto.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the allergy suppression composition which uses highly safe levan as an active ingredient.

  The allergy-suppressing composition according to the present invention includes pharmaceuticals and foods and drinks that reduce and / or suppress allergies, including prevention and / or treatment of allergies (including nutritional foods, foods for specified health use, and functional foods). Therefore, the present invention can provide an allergy suppressing agent containing levan as an active ingredient and a food or drink having allergy suppressing activity. Moreover, this invention can also provide at least one of the IL-12 production induction composition, IL-4 production suppression composition, and IgE production suppression composition which contain levan as an active ingredient. In addition, a pharmaceutical and food-drinks are also included in these compositions.

  As a levan used as an active ingredient in the present invention, a levan-containing product can be used in addition to a purified levan. Any levan-containing material can be used as long as it contains levan, but for example, a product produced in each step of separating and collecting levan from natto as a raw material can be used as a levan-containing material. Natto is also included in the levan content. Therefore, natto can also be used as the allergy-suppressing composition according to the present invention, and is particularly useful as an allergy-suppressing food for the purpose of prevention.

  In addition, according to the present invention, a method for suppressing allergy by orally ingesting levan was provided, and as a result of further research, the ingestion amount of levan was successfully identified. In addition, natto, which is a levan-containing material, can be used as levan, and the present invention also provides a method for suppressing allergy by ingesting natto, for example, as a functional food.

Hereinafter, the present invention will be described in detail.
In the present invention, levan produced by plants and microorganisms is used.
Levan is a polysaccharide composed of D-fructose in which fructose (fructose) is linked by β-2,6, and is a kind of fructan.

  In plants, levan is found in monocotyledonous plants, in particular leaves and stems of the grass family, has a molecular weight of about 25 to 50 fructose residues which are constituent sugars, and glucose is bound to the reducing end.

  Among microorganisms, various bacteria such as Bacillus subtilis including Bacillus subtilis and Bacillus megaterium (Bacillus megaterium IFO 3003), acetic acid bacteria (Gluconacetobacter xylum, etc.), lactic acid bacteria Many bacteria are known to produce levan, such as those used in food production such as Leuconostoc, which provides sucrose directly to fructose by the action of sucrose-6-fructosyltransferase. This is because the body has the ability to synthesize fructose polymers with 2 → 6 bonds, and therefore, natto contains levan produced by Bacillus natto. Yes.

  Examples of Levan-producing bacteria include Bacillus natto as described above. Bacillus natto is classified into Bacillus subtilis, but it can produce the characteristics of natto such as sticky material (stringent material), and is a bacterium that forms the main body in natto fermentation, Moreover, since it has characteristics such as requiring biotin for growth, it is classified as Bacillus nutto, or Bacillus subtilis var. Natto or Bacillus subtilis (natto) may be distinguished from Bacillus subtilis. Examples of Bacillus natto include Bacillus Natto IFO 3009, Bacillus subtilis IFO 3335, IFO 3336, and IFO 13169, and various Bacillus natto can be widely used.

Specific examples include the O-2 strain isolated from commercial natto and the r22 strain (see, for example, Japanese Patent Application Laid-Open No. 2000-224982). by Takahashi bacteria (T ₃ strains, TUA culture collection chamber) various Bacillus natto such or Miyagino bacteria (Miyagino natto Seisakusho) can be appropriately used.

  In the present invention, any leban can be used as long as it is a levan, regardless of the origin of production. Among them, those produced during natto have been eaten as food and have been confirmed to be safe. Therefore, it is more preferable.

  When these levans are used, the intake varies depending on the age, symptom level, etc., but is not particularly limited. However, it is usually preferable to take about 50 mg to 1000 mg / day, more preferably about 120 to 600 mg / day. And in this invention, it is also possible to use natto instead of levan, and in that case, the intake of natto may be defined in consideration of the amount of levan contained in natto. Normally, the content of leban in natto is about 3 mg / g natto, so if you eat 1 pack (40 g / pack) of natto per day, you will consume 120 mg of leban. Moreover, it is also possible to use Levan and natto together if desired.

  In addition, when taking for the purpose of health or prevention or health maintenance over a long period of time, it may be taken in a smaller amount than the above range. Moreover, since this active ingredient does not have a special problem about safety | security, it can also be ingested in a larger quantity than the said range. In fact, as a result of a 10-day acute toxicity test using mice, no death was observed even after oral administration of 1000 mg / Kg.

  When using this composition as food / beverage products, this active ingredient can be used as it is, or it can be used together with other food / beverage products or food / beverage product components according to conventional methods.

  The composition according to the present invention using the active ingredient may be solid (powder, granule, granule, etc.), paste, liquid or suspension, but sweetener, acidulant, vitamin and other drinks. Various ingredients commonly used in production may be used to formulate a health drink, or may be formulated into a supplement dosage form according to conventional methods.

  In addition, when using natto as a composition according to the present invention, natto contains about 3 mg of levan per gram natto, so when using natto with 40 g per pack, 1 natto per day is used. If you eat, you can eat a sufficient amount of levan.

  In addition, when labeling the efficacy of this active ingredient in food, for example, hay fever, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic gastroenteritis, atopic dermatitis, contact dermatitis, gin machine, drug allergy It is possible to attach a display indicating that it is effective.

  Therefore, the present invention contains levan as an active ingredient and has at least one action of preventing, alleviating, and treating allergies such as hay fever, bronchial asthma, and allergic rhinitis as described above. The natto is newly provided with an indication that it is used for allergy suppression, and the natto according to the present invention can be sufficiently distinguished from known natto. In addition, the present invention has succeeded in specifying the oral intake of levan and / or its contents (for example, natto), and provides a method for suppressing allergies based on this. This is one mode of use or utilization of functional foods.

  When the present invention is used as a pharmaceutical, it may be ingested as a powder, or dissolved in water and ingested as an aqueous solution. The ingestion method can also be administered through general administration routes such as oral administration, rectal administration, intravenous injection, and infusion.

  In the case of oral administration, in addition to administering the composition itself, it may be mixed with a pharmaceutically acceptable carrier, excipient, diluent, and used as a powder, granule, tablet, capsule, troche, etc. . However, since solid powders and tablets may take time to absorb, oral administration of the composition itself is desirable. In this case, it may be administered as a solution described above together with a suitable additive, for example, a salt such as sodium chloride, a pH adjusting agent, and a chelating agent. When used as an injection, an appropriate buffer or isotonic agent or the like added and dissolved in sterile distilled water may be used.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

Example 1 (Method for purifying levan from natto)
2 L of 5% saline was added to 1 kg of commercially available natto and suspended with a stirring rod. The supernatant obtained by centrifugation (7000 rpm, 20 minutes) was collected, 4 L of 99.5% ethanol was added, and the resulting precipitate was wound up and collected with a glass rod. After washing 3 times with 30% ethanol, it was dissolved in 1 L of deionized water, and 5% cetablone (cetyltrimethylammonium bromide) was added until no new precipitation occurred. The supernatant of centrifugation (7000 rpm, 20 minutes) was put into a dialysis tube, dialyzed against running water for 2 days, 3 volumes of 99.5% ethanol was added, and the resulting precipitate was collected with a glass rod. This solution was dialyzed against deionized water and then freeze-dried to obtain 0.5 g of levan powder.

Example 2 (Measurement of IL-12 Production Inducing Activity)
Mouse macrophage-derived J744.1 cells were prepared in RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.) containing 5% fetal calf serum to a concentration of 5 × 10 ⁵ cells / ml, and 300 μl each was dispensed onto a microplate. . 30 μl of 10 to 100 μg / ml solution of levan purified from natto and reagent levan (from Serratia levanicum: manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto and cultured at 37 ° C. for 8 hours under 5% CO ₂ condition. After completion of the culture, the amount of IL-12 in the centrifugal supernatant of each cell was measured with an enzyme immunoassay kit (genzyme, manufactured by TENHNE).

  50 mM phosphate buffer: pH 7.4 (hereinafter, PBS) and 0.02 M NaCl solution were used as controls, and LPS (E. coli O55-derived lipopolysaccharide Sigma) 1 μg / ml solution was used as a positive control. 30 μl of these were added. The above results are shown in FIG.

  From the results of FIG. 1 (in the figure, the Levan bar graph shows levan at 10 μg / ml on the left side and 100 μg / ml on the right side), all levans increase IL-12 production in a concentration-dependent manner. From these facts, it was confirmed that levan had an activity to induce IL-12 production from macrophages.

Example 3 (IL-4 and IgE production inhibitory effect)
3-week-old BALB / c mice were sensitized by intraperitoneal administration of 0.5 mg / ml ovalbumin (OVA). The first intraperitoneal administration day was defined as day 0, and 1 mg of the natto levan prepared in the Examples was orally administered on the first day (that is, one day before administration), the fourth day, the ninth day, and the 14th day. PBS was administered as a control.

  Sensitization with albumin was similarly performed on the 12th day, and was performed twice in total. On day 29, mice were sacrificed, and spleen cells were measured for IL-4 production ability and OVA-specific serum IgE levels.

IL-4 production ability was measured by preparing a spleen cell suspension from the spleen immediately after killing the mice of each test group, treating with 0.16 M ammonium chloride to remove erythrocytes, and 5% fetal calf serum, 2 mM. The suspension was suspended in RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.) containing glutamine, 0.05 mM 2-mercaptoethanol, 100 u / ml penicillin, and 100 μg / ml streptomycin to a concentration of 5 × 10 ⁵ cells / ml.

  This was dispensed into a microplate, 100 μg of OVA was added to each well, and cultured at 37 ° C. for 40 hours under 5% CO 2 condition. The amount of IL-4 in the supernatant of the culture broth was measured with an enzyme immunoassay kit (manufactured by genzyme Diagnostic). The above results are shown in FIG.

  The amount of OVA-specific serum IgE was measured by enzyme immunization (ELISA). That is, a 0.1 mg / ml OVA solution (50 mM carbonate buffer: dissolved in pH 9.6) was dispensed in 300 μl portions on a microplate and allowed to stand overnight at 4 ° C. for immobilization. Subsequently, the well was washed with 10 mM PBS containing 0.1% Tween 20 (hereinafter, PBS-0.1% Tween), and then 1.5% gelatin solution (50 mM carbonate buffer: dissolved in pH 9.6) was added for 30 minutes. Left at room temperature.

  Then, after washing with PBS-0.1% Tween, a serum sample appropriately diluted with PBS-0.1% Tween containing 3% polyethylene glycol 6000 was added and left at room temperature for 20 minutes. Each well was washed with PBS, peroxidase-conjugated anti-IgE antibody (manufactured by Nordic) was added, and the mixture was further allowed to stand at room temperature for 2 hours.

  Thereafter, each well was washed with PBS, a color developing solution (200 mM citrate-phosphate buffer: pH 5.0 containing 40 mg% o-phenylenediamine and 12% hydrogen peroxide) was added, and the mixture was allowed to stand at room temperature for 1 hour. 2N sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured. The above results are shown in FIG.

  From the results in FIG. 2, it was confirmed that IL-4 production was suppressed by levan, and from the results in FIG. 3, the effect of lowering serum IgE was confirmed. It was confirmed that there is an effect of suppressing the onset of

Example 4 (effect of natto)
Levan prepared in Example 1 was added to commercial natto (50 g natto / 1 pack, manufactured by Mitsukan), and 3 mg (3 mg leban natto), 6 mg (6 mg leban natto), 15 mg (15 mg leban natto) per 1 g natto ) Natto containing was prepared. On the other hand, soybeans were cooked according to the method for preparing boiled beans for natto to prepare steamed soybeans.

  After freeze-drying these natto and steamed soybeans, each 3% by weight was added to the basic feed (CRF-1, manufactured by Oriental Yeast Co., Ltd.), steamed soybean feed, 3 mg levan natto feed, 6 mg levan natto feed, A 15 mg levan natto feed was prepared.

  Subsequently, 6-week-old BALB / c mice were divided into 4 groups of 6 mice, and each group of mice was sensitized with OVA as in Example 3.

  In addition, from the two weeks before sensitization, each group of the above feeds was given to 4 groups of mice and allowed to be freely ingested into a steamed soybean feed group, a 3 mg levan natto feed group, a 6 mg levan natto feed group, and a 15 mg levan natto feed group.

  During such breeding, one mouse consumed an average of 0.7 g of each feed per day, and therefore, the 3 mg levan natto feed group fed 60 ng / day of levan. In addition, in the 6 mg levan natto feed group, 120 ng / day of levan was fed, and in the 15 mg levan natto feed group, 300 ng / day of levan was fed.

  The mice were sacrificed on the 29th day of breeding, and the IL-4 production amount and the OVA-specific serum IgE amount of the spleen cells were measured in the same manner as in Example 3.

  The amount of IL-4 produced in each group is shown in FIG. 4. In the 3 mg levan natto feed group, it can be confirmed that levan suppresses the production ability of IL-4, and further the IL-4 production ability in a dose-dependent manner. I was able to confirm that it was suppressed. This confirms that levan suppresses allergic reactions in mice in a dose-dependent manner.

  In addition, Table 1 shows the measurement results of the OVA-specific IgE production amount of each group. It should be noted that the IgE production amount of each Leban natto feed group was compared with the IgE production amount of the steamed soybean feed group by a t-test method (for example, “Stat. Sci.”, 6 Volume, p. 100-116, 1995), and the p value was calculated. It can be determined that there is a significant difference when the p value is less than 0.05.

  As shown in Table 1, it was confirmed that levan also suppressed IgE production ability in a dose-dependent manner, and levan was confirmed to suppress allergic reactions in mice in a dose-dependent manner.

  Based on the results of the above mice, when the average human body weight is 60 kg with respect to the average mouse body weight of 30 g, and the human effect area is converted to body weight, the 3 mg leban natto feed group shows 120 mg / day levan, In the 6 mg levan natto feed group, 240 mg / day levan was fed, and in the 15 mg levan natto feed group, levan was fed 600 mg / day.

  Therefore, it was confirmed that the effective area of oral intake of levan in humans is 120 mg / day to 600 mg / day.

  Furthermore, since a sufficient effect is obtained even in the 3 mg levan natto feed group (corresponding to 120 mg / day ingestion in humans) which is the minimum levan content, the effect area in humans is 50 mg / day to 1000 mg / day. It was recognized as about one day.

It is the figure which showed the production induction activity of IL-12 (interleukin-12) of levan of various origins. It is a figure which shows IL-4 production inhibitory activity of a levan. It is a figure which shows IgE production inhibitory activity of a levan. It is a figure which shows IL-4 production inhibitory activity of levan containing natto.

Claims (10)

  An allergy-suppressing composition comprising levan as an active ingredient.   An allergy-suppressing composition, characterized in that levan is produced by Bacillus natto.   Saline was added to natto and stirred, ethanol or ethanol water was added to the supernatant of the obtained suspension, and the obtained precipitate was repeatedly dissolved in water and precipitated with ethanol or ethanol water, A method for producing an allergy-suppressing composition, comprising levan prepared by a method comprising a step of dialysis of a solution of a final precipitate against water and drying.   The active ingredient is at least one selected from (A) natto as a raw material, (B) leban as a final product, and (C) at least one product produced in the process from natto to production of levan in claim 3. An allergy-suppressing composition comprising levan as an active ingredient.   An allergy suppressant containing levan as an active ingredient.   A food and drink having allergy-suppressing activity comprising levan as an active ingredient.   The food / beverage product according to claim 6, wherein the food / beverage product is natto.   A method for suppressing allergy, which comprises orally ingesting 50 mg to 1000 mg of levan per day.   A method for suppressing allergy, which comprises orally ingesting 120 mg to 600 mg of levan per day.   The method for suppressing allergy according to claim 8 or 9, wherein levan is orally ingested by eating natto.

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