JP2008231094A - Antiallergic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an antiallergic agent having an antiallergic action by IgE antibody production inhibition and using a safe naturally occurring lactic acid bacterium. <P>SOLUTION: The antiallergic agent comprises a lactic acid bacterium having a high IgE antibody production inhibition action and an antiallergic action and belonging to Leuconostoc mesenteroides, preferably lactic acid bacterium Leuconostoc RIE strain deposited as accession number FERM P-21110 as an active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to an antiallergic agent containing the lactic acid bacterium Leuconostoc mesenteroides , which has an antiallergic effect by suppressing IgE antibody production.

  The immune system is a system that protects the living body from bacteria and viruses that enter the human body, or tumors that occur in the body. On the other hand, nutritional components necessary for a living body such as food can cause tolerance and can be actively taken into the living body. However, the immune system produces an allergic reaction as a result of an oversensitive response. Allergic diseases are roughly classified into four types, from type I to type IV, depending on the mechanism of action. Pollen allergic rhinitis, perennial rhinitis, bronchial asthma and the like are said to be type I allergy mediated by immunoglobulin E (IgE). Atopic dermatitis is also called a combined type of type I and type IV. In addition, type I, type II, and type IV are considered to be involved in the development of food allergy, and allergic diseases related to type I are increasing in Japan.

  Type I allergic symptoms are triggered by antigen-presenting cells biased in T cell differentiation in response to the invasion of antigens such as pollen, and the induction of IgE antibody production by B cells. Thereafter, when specific cells such as mast cells to which antibodies are bound are subjected to antigen invasion again, it is believed that allergic symptoms occur due to release of granules from these cells and release of inflammatory substances such as histamine. Therefore, for the purpose of preventing and treating allergies, there are a wide range of target action points from the initial antigen presentation stage to the final degranulation stage, and antiallergic agents focusing on each action point are proposed. Has been. A target for suppressing antigen presentation is a hawthorn hot water extract (see Patent Document 1). In addition, many of the foods and drinks represented by colostrum (see Patent Document 2), lactic acid bacteria KW3110 strain (see Patent Document 3) and the like are those whose action point is to improve differentiation balance of helper T cells (Th1 / Th2 balance improvement). Things have been proposed. Further, strictinin, a tea leaf component, has been proposed as a material for suppressing the IgE class switch (see Patent Document 4). These are intended to suppress allergic reaction by suppressing IgE antibody production as a result. However, none of them has been directly evaluated for the ability to produce IgE antibody in a living body, or even if it has been evaluated, no statistical evaluation has been performed, so it has not been confirmed whether the final purpose has been achieved. The lactic acid bacteria KW3110 strain has been statistically evaluated for its suppression of IgE production in the living body, and shows a significant effect. However, it is assumed that oral administration for a considerably long period of 100 days or more is usually more effective in mice. New material is anxious.

JP 09-143090 A JP 2006-96752 A JP 2005-137357 A JP 2005-198664 A

  An object of the present invention is to provide an antiallergic agent containing a safe naturally-derived lactic acid bacterium (plant lactic acid bacterium) as an active ingredient, which has an antiallergic effect by suppressing IgE antibody production.

As a result of various studies to achieve the above-mentioned object, the present inventors have found that an antiallergic agent containing a lactic acid bacterium belonging to Leuconostoc mesenteroides isolated from galling has a high IgE antibody production-inhibiting action. The inventors have found that the present invention is achieved and have completed the present invention.
That is, the present invention provides an antiallergic agent containing, as an active ingredient, a lactic acid bacterium belonging to Leuconostoc mesenteroides, which has a high IgE antibody production inhibitory action and an antiallergic action.
The present invention also provides an antiallergic agent wherein the lactic acid bacterium is Leuconostoc mesenteroides RIE strain (Accession No. FERM P-21110).

  The lactic acid bacteria belonging to Leuconostoc mesenteroides contained in the antiallergic agent of the present invention have a high IgE antibody production inhibitory action, and this action brings about an antiallergic action. Therefore, prevention and treatment of allergic diseases can be achieved by taking a preparation containing the lactic acid bacteria. The lactic acid bacteria are excellent in safety because they are separated from natural food materials with food experience.

  The lactic acid bacterium Leuconostoc mesenteroides RIE strain has been deposited at the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depositary Center, and its deposit number is FERM P-21110.

The mycological properties of the lactic acid bacterium Leuconostoc mesenteroides RIE are shown below.
Bacterial morphology when cultured for 18 hours at 30 ° C. using MRS liquid medium (DIFCO) (1) Bacterial morphology Cocci (2) Gram staining Positive (3) Motility None (4) Spore None (5) No catalase (6) facultative anaerobic (7) metabolism of glucose Convert to 50% or more lactic acid (8) Growth temperature range Growth is observed at 15-45 ° C (9) Lactic acid fermentation Heterotype (10) Optical rotation of lactic acid D
(11) Fermentability of carbohydrates Glycerol is negative, D-arabinose is negative, L-arabinose is positive, ribose is positive, D-xylose is positive, galactose is positive, glucose is positive, fructose is positive, mannose is positive, rhamnose Is negative, mannitol is positive, sorbitol is negative, α-methyl D glucoside is positive, amygdalin is positive, esculin is positive, salicin is positive, cellobiose is positive, maltose is positive, lactose is positive, melibiose is positive, sucrose is positive Positive for trehalose, negative for inulin, negative for melezitose, positive for raffinose, negative for starch, positive for gluconic acid.

  Since the lactic acid bacterium Leuconostoc mesenteroides RIE is isolated from a material with abundant food experience (steel pickled), it can be safely used as an antiallergic agent.

  The lactic acid bacterium Leuconostoc mesenteroides RIE strain can be used as an antiallergic agent by formulating various pharmaceutically acceptable carriers, excipients, other additives and other ingredients as they are or as necessary. It can be used as an agent. The allergic agent may be based on various animal and plant substances fermented using the lactic acid bacterium Leuconostoc mesenteroides RIE.

  The antiallergic agent of the present invention contains, as an active ingredient, a lactic acid bacterium belonging to Leuconostoc mesenteroides having a high IgE antibody production inhibitory action, particularly preferably the above leuconostock mesenteroides RIE strain.

  The suppression of IgE antibody production, for example, reduces the allergen recognition ability of antigen-presenting cells, improves the balance of differentiation of helper T cells (Th1 / Th2 balance), which are immunocompetent cells biased toward Th2 production, Which can be achieved by acting on a receptor on the B cell and inhibiting the transmission of information from the T cell to the B cell, etc., but having the above high IgE antibody production inhibitory activity used in the antiallergic agent of the present invention. The action mechanism of lactic acid bacteria belonging to mesenteroides is not particularly limited as long as it suppresses IgE antibody production and has an antiallergic action by suppressing the IgE antibody production. Here, “having a high IgE antibody production inhibitory effect” preferably refers to a state in which IgE antibody production is suppressed at a statistically significant level of 5% as compared to the group not taking an antiallergic agent.

It is preferable to evaluate the IgE antibody production inhibitory action of lactic acid bacteria by the following method.
Lactic acid bacteria are cultured in MRS medium or the like, and a feed containing 0.1 to 20% by mass of the cultured lactic acid bacteria is sensitized with OVA (ovalbumin) to be ingested for a certain period of time, and the feed does not contain lactic acid bacteria The inhibitory effect of IgE antibody production is evaluated in comparison with an allergy model animal (group without addition of lactic acid bacteria). It is known that the blood IgE content is higher than usual when allergies develop.

  The content of lactic acid bacteria in the antiallergic agent of the present invention may be any amount as long as it can suppress an increase in the production amount of IgE antibody. It can be changed appropriately depending on the symptoms and age and gender. When the antiallergic agent of the present invention is orally administered or ingested, it is preferably contained so that the daily dose or intake per person is 1 mg to 20 g.

  EXAMPLES Next, examples are given to describe the present invention more specifically, but the present invention is not limited to the following examples.

Example 1 and Comparative Examples 1 to 4
(Lactic acid bacteria used)
Among plant lactic acid bacteria isolated from botanical materials with abundant Japanese dietary experience, such as pickled pickles, pickled bitter gourd, pickled kimchi, yeast, etc. 5 typical strains shown in Table 1 were selected on the basis of the fact that a plurality of strains were found and that the growth was high in a normal medium. The RIE shown in Table 1 is the Leuconostoc mesenteroides RIE strain of the present invention.

(Preparation of lactic acid bacteria sample)
Each lactic acid strain shown in Table 1 cultured for 48 hours at 37 ° C. in MRS medium containing 10 μg / ml cycloheximide was collected by centrifugation, washed 3 times with sterile water, and then suspended in sterile water. And autoclaving at 121 ° C. for 30 minutes. These were freeze-dried to obtain lactic acid bacteria samples.

Test example 1
(Evaluation of IgE antibody production inhibitory effect in allergic model mice of lactic acid bacteria group)
1. Preparation of Feed Basic Feed [AIN-93 (Standard Purified Feed for Nutrition Research Using Mice and Rats by the National Institute of Nutrition) shown below as lactic acid bacteria samples prepared in Example 1 and Comparative Examples 1-4 The feed containing the lactic acid bacteria was prepared respectively. As a control group, a basic feed without lactic acid bacteria was also prepared. The raw materials for the basic feed were all manufactured by Oriental Yeast Co., Ltd. After mixing the lactic acid bacteria sample, the feed was obtained by adding water and forming into a cylindrical shape.
2. Animal Experiment Schedule The weight of 5-week-old BALB / c mice was measured, and grouped into 6 animals in each group using the stratified continuous randomization method with the body weight as an index. After grouping, ingestion of the basic feed and feed containing lactic acid bacteria prepared in 1 above was started. In addition, acclimatization processing (one week) was performed prior to the body weight measurement, and during this period, all mice were allowed to freely ingest basic food.
All mice were intraperitoneally administered OVA-alum (OVA 50 μg, aluminum hydroxide 1 mg / animal) on the 14th and 28th days after the start of feed intake to induce allergic conditions. The 14th day after the start of feed intake was defined as the 0th day of sensitization.
After 0 days, 22 days, 28 days, and 35 days after sensitization, 100 μl of blood was collected from the orbital venous plexus. After blood collection, centrifugation was performed at 12,000 rpm for 5 to 10 minutes, and serum was collected.
3. Antibody quantification The amount of OVA-specific IgE antibody and the amount of total IgE antibody in the collected serum were quantified by ELISA.
4). Experimental Results The amount of OVA-specific IgE antibody produced 22 days after sensitization is shown in FIG. The amount of IgE antibody production on the vertical axis in FIG. 1 indicates the amount of antibody production relative to a self-made standard antibody. FIG. 2 shows the total IgE antibody production on day 22 after sensitization. The unit on the vertical axis in FIG. 2 is a unit in which the antibody amount is converted with 1.0 ng / ml = 1 unit. As a result of statistical analysis by Dunnett's method, the production of OVA-specific IgE antibody was significantly suppressed in the group fed the lactic acid bacterium-containing feed prepared in Example 1 and the lactic acid bacterium AYB-containing feed prepared in Comparative Example 1. (Significance level 5%). It was also revealed that the production of total IgE antibody was significantly suppressed (significant level 5%) in the group fed with the feed containing lactic acid bacteria RIE prepared in Example 1. From this, it was proved that the antiallergic agent containing Leuconostoc mesenteroides RIE strain has a high IgE antibody production inhibitory action.

(Basic feed composition)
Casein 20.0 mass%
Corn starch 50.5% by mass
Sucrose 10.0% by mass
Lard 10.0% by mass
Cellulose powder 5.0% by mass
AIN-93 mineral mixture 3.5% by mass
AIN-93 vitamin mix 1.0% by mass

Test example 2
(Evaluation of nasal discharge suppression effect of allergic rhinitis model guinea pig by feeding lactic acid bacteria group)
1. Animal Experiment Schedule The body weights of 20 4-week-old male guinea pigs were measured and divided into groups so that the average body weight and variance of 10 animals in each group were almost equal by random sampling. After grouping, solid feed LRC4 (manufactured by Oriental Yeast Co., Ltd.) within 5 months after production was placed in a feeder and allowed to ingest freely. In the lactic acid bacteria administration group, the lactic acid bacteria sample prepared in Example 1 was orally administered twice a day from the next day for a total of 28 days (1000 mg / kg / day).
In addition, a thin-shaft swab soaked in a 10% TDI solution was applied to both nasal vestibules of all guinea pigs for 10 seconds, and this operation was repeated once a day for 5 days from the next day of grouping for sensitization. I did it. Thereafter, breeding was continued without sensitization, and nasal discharge was induced 4 weeks after the final sensitization. Nasal discharge was induced by applying a thin-shaft swab soaked in 5% TDI solution in contact with both nasal vestibules for 10 seconds.
2. Measurement of nasal discharge The nasal discharge for 15 minutes from the start of nasal discharge induction was absorbed into absorbent cotton, placed in a sealed micro tube, and its weight was measured.
3. 3. Experimental Results As shown in FIG. 3, the leuconostok / mesenteroides RIE strain administration group showed that the amount of nasal discharge associated with the development of allergic rhinitis was significantly reduced (significant level 1%) compared to the control group. It was confirmed that the antiallergic agent containing Leuconostoc mesenteroides RIE strain has an effect of alleviating allergic symptoms by oral administration, that is, exhibits an antiallergic action.

Test example 3
(Evaluation of improvement effect of hay fever symptoms in humans by intake of lactic acid bacteria)
1. Preparation of Test Agent Using the lactic acid bacteria sample prepared in Example 1, Test Agent 1 (low dose), Test Agent 2 (high dose) and placebo having the composition and shape shown in Table 2 were prepared.

2. Test method The subjects were 44 patients with cedar IgE class 2 (blood IgE concentration of 0.70 to 3.49 UA / ml) or 3 (blood IgE concentration of 3.50 to 17.49 UA / ml). About these subjects, in addition to the medicine prescribed by the doctor, the above-mentioned test agent 1 (low dose), test agent 2 (high dose) or placebo was randomly taken. The number of subjects who took Test Agent 1 (low dose), Test Agent 2 (high dose) or placebo and the cedar IgE class were as follows. However, the test subjects were ingested in a state where it was unknown which test agent was ingested.
Patients taking Test Agent 1 (low dose): 12 patients (Class 2: 6 people, Class 3: 6 people)
Patients taking Test Agent 2 (high dose): 14 patients (Class 2: 7 people, Class 3: 7 people)
Patients who took placebo: 18 people in total (Class 2: 8 people, Class 3: 10 people) The intake period of the test agent was 12 weeks (84 days) from the start date of cedar pollen scattering.
3. Evaluation method Evaluation was made based on a symptom severity score (hereinafter referred to as “SS”) and a used drug score (hereinafter referred to as “MS”).
SS evaluated each subject about the sneezing seizure, nasal discharge, nasal congestion, and the difficulty level of daily life according to the criteria shown in Table 3. For each group, the average value of evaluation points was calculated every week for SS.
In addition, MS gave a score to each drug as shown in Table 4 according to “2005 Nasal Allergy Examination Guidelines” for the drugs (excluding the test drug) taken by each subject during the test drug intake period. For each group, the average value of the scores of drugs taken every week was calculated as MS.
The sum of the above SS and MS is the SMS (symptom mediation).
score), the SMS fluctuation amount during the test agent intake period was calculated based on the SMS value of the first week, and the improvement effect of hay fever symptoms by the test agent intake was evaluated.

4). Test result The transition of the SMS fluctuation amount during each test agent intake period is shown in FIG. Graph A in FIG. 4 is a graph showing the transition of the amount of cedar pollen scattered in the test site, and the scale on the right vertical axis shows the amount of pollen scattered. As is clear from FIG. 4, the test agent 2 (high dose) intake group had a significantly lower SMS value compared to the placebo intake group within the entire intake period of the test agent. In addition, in the test agent 1 (low dose) intake group, is the SMS value significantly lower than that in the placebo intake group except for 2 weeks before and after the cedar pollen scattering peak (5 weeks from the start date of cedar pollen scattering)? At least showed a downward trend. From this, it was confirmed that the antiallergic agent containing Leuconostoc mesenteroides RIE strain has an effect of improving hay fever symptoms. Furthermore, the antiallergic agent of the present invention is expected to improve symptoms in immune diseases such as atopic dermatitis, perennial allergic rhinitis, bronchial asthma as well as hay fever.

Test example 4
(Stability test of lactic acid bacteria sample)
Test agent 1 (low dose), test agent 2 (high dose) and placebo used in Test Example 3 were stored at 80 ° C. for 8 weeks and stored at 45 ° C. and 75% humidity for 8 weeks. Was evaluated by measuring the production amount of IFN-γ as a marker for Th1 cells and the production amount of IL-4 as a marker for Th2 cells by the following test method. The measurement results are shown in FIGS.

Test method Spleens were collected from ovalbumin (OVA) -specific T cell receptor-expressing mice (DO11.10 mice), ground, passed through a cell strainer to obtain a spleen cell suspension, and red blood cells were lysed with an ammonium chloride solution. I let you. The spleen cells were washed twice with 10 ml of RPMI medium manufactured by Nihon Suisan with 5% of fetal calf serum: FCS (Fetal Calf Serum), and finally suspended in 1 ml of RPMI medium containing 5% FCS. The number of cells in the suspension was counted, diluted with RPMI medium containing 5% FCS to 2.5 × 10 ⁶ cells / ml, and 100 μl of each well was placed in a 96-well plate.
Prepare a sample in RPMI medium containing 5% FCS to 40 μg / ml, add 50 μl of the sample to the 96-well plate, add 50 μl of RPMI medium containing 2 mg / ml of OVA, and in a CO2 incubator for 7 days. Cultured.
As a control, 50 μl of RPMI medium containing 5% FCS and 50 μl of RPMI medium containing 2 mg / ml of OVA were added to the control wells and treated in the same manner.
The culture supernatant was collected, the amount of IFN-γ antibody was quantified by ELISA, the amount of IFN-γ produced was examined, and compared with the value in the control group. In addition, the amount of IL-4 antibody was quantified, the amount of IL-4 produced was examined, and compared with the value in the control group.

  As is clear from FIGS. 5 and 6, after storage, both Test Agent 1 (low dose) and Test Agent 2 (high dose) produced a high amount of IFN-γ, whereas IL-4 was produced. The amount was low. From this, even after storage, it is confirmed that Test Agent 1 (low dose) and Test Agent 2 (high dose) both have excellent Th1 / Th2 balance improving activity, that is, have storage stability. It was.

  Next, examples of the antiallergic agent of the present invention will be given, but the present invention is not limited to the following examples.

Example 2 (tablets)
Sample of lactic acid bacteria prepared in Example 1 5 g
Corn starch 10 g
Lactose 40 g
Carboxymethylcellulose calcium 8 g
Microcrystalline cellulose 27 g
Polyvinylpyrrolidone 7 g
Magnesium stearate 3 g
Total 100 g
Microcrystalline cellulose, corn starch, lactose and carboxymethylcellulose calcium are mixed with the lactic acid bacteria sample, and then an aqueous solution of polyvinylpyrrolidone is added as a binder and granulated by a conventional method. To this, magnesium stearate as a lubricant is added and mixed, and then tableted into 100 mg tablets.

Example 3 (hard capsule)
10 g of lactic acid bacteria sample prepared in Example 1
Microcrystalline cellulose 55 g
Corn starch 25 g
Lactose 30 g
Polyvinylpyrrolidone 4 g
Magnesium stearate 1 g
Total 125 g
The above ingredients are granulated by a conventional method and then filled into gelatin hard capsules.

Example 4 (powder)
50 g of lactic acid bacteria sample prepared in Example 1
Microcrystalline cellulose 600 g
Corn starch 300 g
Polyvinylpyrrolidone 50 g
Total 1000 g
The above ingredients are mixed and powdered by a conventional method.

Example 5 (granule)
10 g of lactic acid bacteria sample prepared in Example 1
Lactose 130 g
Corn starch 87 g
Polyvinylpyrrolidone 8 g
L-menthol 15 g
Light anhydrous silicic acid 5 g
Total 255g
In the above formulation, a lactic acid bacterium sample, lactose, corn starch and a polyvinylpyrrolidone aqueous solution are mixed and heated and granulated with stirring in a granulator. After cooling, it is sieved to a particle size of 500 μm or less, L-menthol is added, silicic anhydride is added, mixed and packaged (1.0 g) to give granules.

It is a figure which shows the production amount of OVA specific IgE antibody in the serum on the 22nd day after sensitization in Test Example 1. It is a figure which shows the total IgE antibody production amount in the serum on the 22nd day after the sensitization in Test Example 1. It is a figure which shows the amount of nasal discharge of the guinea pig which induced the allergic rhinitis which orally administered the RIE strain | stump | stock for 28 days in Test Example 2. It is a figure which shows transition of the SMS fluctuation amount in each test agent intake period in Test Example 3. It is the figure which showed the IFN-gamma (Th1 cell marker cytokine) production amount of each test agent after the preservation | save in Test Example 4. It is the figure which showed the IL-4 (Th2 cell marker cytokine) production amount of each test agent after the preservation | save in Test Example 4.

Claims (2)

An antiallergic agent containing, as an active ingredient, a lactic acid bacterium belonging to Leuconostoc mesenteroides , which has a high IgE antibody production inhibitory action and has an antiallergic action.   The antiallergic agent according to claim 1, wherein the lactic acid bacterium is Leuconostoc mesenteroides RIE strain (Accession No. FERM P-21110).

Images