JP2001288093A - IgE ANTIBODY PRODUCTION REDUCING AGENT AND ALLERGIC CONSTITUTION AMELIORANT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain both an IgE antibody production reducing agent and an allergic constitution ameliorant capable of not only carrying out the prophylaxis/ therapy of allergies by reducing the IgE antibody production but also correcting the Th1/Th2 cell balance and ameliorating the allergic constitution by enhancing the production of interleukine-12 (IL-12)/or interferon-γ(IFN-γ). SOLUTION: The IgE antibody production reducing agent and allergic constitution ameliorant comprise raffinose as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an IgE antibody production-reducing agent and an allergic constitution ameliorating agent which are effective for the prevention and treatment of allergy, and more particularly to an IgE antibody production-reducing agent comprising raffinose as an active ingredient. , IgE
The present invention relates to an antibody production reducing food composition, an allergic constitution improving agent, and an allergic constitution improving food composition.

[0002]

2. Description of the Related Art Allergy is a disease that is originally caused by attacking a foreign enemy (foreign body), causing the immune system working as a self-defense function to become abnormally hypersensitive and attacking its own tissue. Examples of allergies include conditions such as anaphylaxis, bronchial asthma, allergic rhinitis, urticaria, atopic dermatitis, drug allergies, food allergies, and hay fever. Antibodies called immunoglobulin E (IgE) play a central role in the development of such allergic conditions.

[0003] The mechanism of the onset of allergy involving IgE antibodies is considered as follows. That is, various foreign antigens such as food antigens such as eggs, milk and soybeans and inhalation pits such as mites and pollen enter the body, are taken up by antigen presenting cells, and present antigens to CD4 ⁺ T (helper T) cells. I do. The helper T cells that recognize the antigen presented by the antigen presenting cells interact with the B cells and differentiate and proliferate the B cells into antibody-producing cells.

[0004] The helper T cells are mainly classified into type I helper T cells (Th1 cells) that induce cellular immunity and type II helper T cells (Th2 cells) that induce humoral immunity, depending on the type of cytokines produced. It is classified into two types (Mosmann, TR et al .: J. Immunol., 136, 2348-235).
7, 1986). The cytokines produced by Th1 cells include interleukin 2 (IL-2), interferon γ
(IFN-γ), TGF-β, and Th2 cells
L-4, IL-5, IL-6, IL-10, IL-13
To produce It is known that naive T cells that have not been sensitized to antigens differentiate into Th1 cells when they act on antigen-presenting cells and IL-12, and differentiate into Th2 cells when they act on antigen-presenting cells and IL-4.

B cells that have undergone the interaction of Th2 cells involved in allergic reactions mature and proliferate into antibody-producing cells,
Producing IgM antibodies and then IgG1 antibodies,
It produces E antibodies. On the other hand, B cells that have undergone the interaction of Th1 cells are IgM antibodies, IgG2a antibodies,
It matures and proliferates into antibody-producing cells that produce IgG2b and IgG3 antibodies. The most important function of Th1 cells is
The purpose of the present invention is to activate macrophages by the action of IFN-γ produced thereby to induce cellular immunity. The produced IgE antibody binds to the surface of mast cells (mast cells). When a specific antigen binds to the IgE antibody, mast cells are activated and inflammatory substances such as histamine are released.
Allergic symptoms are induced. In addition, chemotactic factors released from mast cells attract eosinophils with inflammatory effects,
In addition to inflammation, interleukin 4 (IL-4) and IL-13, also produced by mast cells, further promote IgE antibody production by B cells.

[0006] Th1 cells and Th2 cells have an aspect of inhibiting each other's actions, for example, the IF1 produced by Th1 cells.
N-γ is known to suppress the production of IgE antibodies. In a healthy state, the balance of Th1 / Th2 cells is maintained, and no IgE antibody is produced against the invading antigen. However, in allergic patients, the balance of Th1 / Th2 cells is disturbed, and the invading antigen is better than the Th1 cell. It is also considered that Th2 cells are in a predominantly responsive state, and that they are likely to be susceptible to induction of allergy involving IgE antibodies.

[0007] For the treatment of allergy, antihistamines, steroids, antiallergic agents and the like are used as drug therapy. In addition, in order to suppress the invasion of the antigen itself, there is a countermeasure such as wearing a mask with respect to a diet therapy for restricting the intake of the causal food or an aspirated antigen. However,
It is the current state of the art that no safe and satisfactory measures have been developed in both prevention and treatment.

[0008] On the other hand, oligosaccharides are sugars in which 2 to 10 monosaccharides are bound, and reach the lower gastrointestinal tract without being digested and absorbed, and proliferate the bifidobacteria inhabiting there and assist the intestinal function. There is. Recently, it has been used in lactic acid bacteria drinks, soft drinks, canned coffee and the like. Raffinose is a type of oligosaccharide that is assimilated by bifidobacteria and lactobacilli. It is widely distributed in the plant world, including beets, and has a white, needle-like crystal form. Raffinose is known to induce the growth of bifidobacteria, suppress the growth of harmful bacteria such as Escherichia coli and C. perfringens, and improve the intestinal flora.

[0009] However, it has not been known that raffinose reduces the production of IgE antibodies, corrects the disorder of Th1 / Th2 cell balance, and improves or prevents allergy based on them, in the immune response to the antigen. .

[0010]

In recent years, the number of allergic patients has been increasing, and it is said that about 30% of Japanese suffer from allergy. However, the above-mentioned drug treatments are all coping treatments, do not improve the fundamental allergic constitution, and have some side effects due to the drugs. Countermeasures such as diets also place a considerable burden on patients and their families.

[0011]

Means for Solving the Problems The present inventors have studied from various aspects to achieve the above object, and as a result, the causes of allergic diseases are caused by changes in the environment such as eating habits, living environment and natural environment, and stress. In view of the fact that the cause has not yet been determined, it is not only possible to reduce allergic factors in vivo but also to reduce the allergic constitution. I came to the realization that there was a fundamental need for improvement.

Therefore, the present inventors focused on IgE antibodies which play a central role in the onset of allergy, and conducted intensive screening for the effective components of the IgE antibody reducing agent for the purpose of reducing IgE antibodies. As a result, among the many natural products, oligosaccharides, particularly raffinose, have obtained useful new knowledge that oral administration reduces IgE antibody production.

Further, from the viewpoint of improving allergic constitution, the reduction of IgE antibodies by raffinose was described.
As a result of extensive research, they have also discovered for the first time that raffinose enhances the production of IL-12 and IFN-γ. That is, oral administration of raffinose increases IL-12 production of antigen presenting cells such as macrophages and dendritic cells, and this IL-12 acts to induce differentiation of naive helper T cells into Th1 cells, Th
It was confirmed that IFN-γ production of one cell was enhanced. It was also confirmed that the macrophages were activated by the action of IFN-γ, and the production of IL-12 was further increased. IFN-γ is a function of Th2 cell
It works to suppress gE antibody production.

That is, raffinose not only reduces the production of IgE antibody, which is an important factor in the onset of allergy, but also corrects the allergic constitution in which Th2 cell response to antigen is dominant in Th1 / Th2 cell balance. However, the present inventors have obtained a novel and extremely useful finding that the response of Th1 cells can be predominantly induced.

The present invention is based on the above-mentioned useful new findings,
As a result of further research, it has been completed, and is an IgE production-reducing agent containing raffinose as an active ingredient and Th1 /
The basic technical idea is to improve the allergic constitution by correcting the Th2 cell balance, and according to the present invention, it is highly safe, can reduce IgE antibody production, and can further improve the allergic constitution. An object of the present invention is to provide a prophylactic and / or therapeutic agent and a food composition that can be administered.

The agent for reducing the production of IgE antibodies and the agent for improving an allergic constitution according to the present invention are prepared by adding raffinose as an active ingredient to an inorganic or organic carrier or a pharmaceutical excipient commonly used in the art, and adding solids to the mixture according to a conventional method. In semi-solid or liquid form, it is formulated into parenteral preparations such as enteral preparations and external preparations in addition to oral preparations. In the case of oral administration, as the administration form, for example, tablets, capsules, granules, powders,
Syrup, gargle and the like. These various preparations are known in the art for pharmaceuticals such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending agents, coating agents and the like, which are commonly used in pharmaceuticals. It can be formulated with auxiliaries. In the case of food, it can be mixed with juice or the like to form a liquid composition,
It can also be mixed into biscuits or the like to form a solid composition.

The amount used varies depending on symptoms, age, body weight, administration method, dosage form and the like, but usually 0.5 to 20 g, preferably 3 to 10 g per day for an adult can be orally administered. Since the active ingredient according to the present invention is of natural origin and is used as a food, there is no particular problem with regard to toxicity, and the amount of 6,000 mg / (kg body weight) per day for rats is high. Did not show any acute toxicity. Therefore, if necessary, it may be used in an amount larger than the above range. However, since raffinose is an indigestible saccharide, it may be used in 10 days a day.
When administered more than g, osmotic diarrhea may accompany some people.

As for the active ingredient, it is optimal to use purified raffinose, but if desired, for example, in the case of some oral administration preparations and foods, secondary raffinose is used in the production process of sugar beet. It is also possible to use processed syrups with a slightly reduced degree of purification, such as processed syrups, and raffinose-containing oligosaccharide mixtures produced from soybean whey. Hereinafter, examples of the present invention will be described.

[0019]

Example 1 In an animal test, ovalbumin (OV
A) The effect of oral administration of raffinose on blood antibody titers induced by administration was investigated. Test animals include:
A T cell receptor gene that specifically recognizes OVA (O
VA _323-339 specific I-A ^d restricted T cell clone 7-3
-7 TCR α chain and β chain genes), and a transgenic mouse (hereinafter referred to as a Tg mouse) having a genetic background almost identical to that of a BALB / c mouse was used.

(1) Method Twenty-two 6-week-old Tg mice were divided into two groups (11 males per group, 5 females and 6 females), and the control group contained a basic diet (20% casein, 4 corn starches).
8.17%, α-starch 9%, sucrose 5%, cellulose 5%, soybean oil 6%, mineral mixture 5%,
1.3% vitamin mixture, 0.23 choline chloride
% And methionine 0.3%), and the raffinose group was fed with raffinose-added feed (basic feed with corn starch changed to 43.17% and raffinose 5% added). Two weeks later, drinking water is 2% OV from tap water
The solution was switched to the aqueous solution containing A and continued until the eighth week. From the 8th week after the start of the test, drinking water was used as tap water, and from the 12th week, feeds in which 8% casein of each test feed was replaced with OVA were fed. During the test period, blood was collected from the tail vein with time, and the total IgE antibody concentration in the serum, the OVA-specific IgE antibody titer, and the OVA-specific IgG1 antibody side were measured by ELISA.

(2) Results As shown in FIGS.
From the second week onward, total IgE antibody concentration in serum, OV
The A-specific IgE antibody titer and the OVA-specific IgG1 antibody titer increased remarkably, but all the analytical items in the raffinose group were lower than those in the control group. Therefore, it was revealed that raffinose suppresses the production of an antigen-specific IgE antibody that plays a central role in the onset of allergy.
The IgG1 antibody is an antibody produced together with an IgE antibody by antibody-producing cells that have differentiated and proliferated under the action of Th2 cells. In the raffinose group, the number of OVA-specific IgE antibody-producing antibody-producing cells or the antibody-producing ability was reduced in the Tg mice to which raffinose was administered because the OVA-specific IgG1 antibody and the IgE antibody were reduced. it seems to do.

[0022]

Example 2 In a mouse test, the effect of oral administration of raffinose on the immune response induced by ovalbumin was investigated to examine the IL-12 and IFN-γ produced by mouse immune cells. IL-12 is secreted by antigen-presenting cells such as macrophages and dendritic cells and has the function of differentiating naive helper T cells not sensitized to antigens into Th1 cells. Further, it is known that IFN-γ produced by Th1 cells activates macrophages and suppresses the action of Th2 cells, thereby suppressing the production of IgE.

(1) Method Sixteen-week-old female BALB / c mice were divided into two groups, and the control group was a basic feed (see Example 1), and the raffinose group was a raffinose-added feed (see Example 1). Were fed respectively.

After breeding for 2 weeks, the mice were sacrificed, and the Xiaoyang Bayer plate tissues were excised from each mouse. Bayer plate is 10% FC
Collagenase (S) in RPMI medium supplemented with S (Nissui Pharmaceutical)
igma) at a concentration of 1 mg / ml was stirred and reacted for about 1 hour to prepare a single-cell suspension of Bayer plate cells, and washed several times with RPMI medium supplemented with 10% FCS to obtain antigen-presenting cells.

In addition, two 10-week-old female Tg mice (see Example 1) bred on the basic diet were sacrificed in the same manner, and their spleens were removed. The spleen was ground in a 10% FCS-supplemented RPMI medium, two animals were combined, and washed several times to prepare a single cell suspension. From this spleen single cell suspension, helper T cells expressing the CD4 molecule were transferred to a magnetic cell separation system (M
The cells were fractionated using iltenyi biotec) and used as helper T cells. The obtained helper T cells are naive T cells that are not antigen-sensitized.

Antigen-presenting cells, helper T cells, and antigen (OVA) were added to each well of a 48-well cell culture plate as shown in Table 1, and cultured in a 5% CO ₂ atmosphere at 37 ° C. for 48 hours. After completion of the culture, the culture supernatant was collected, and the cytokine concentration was measured using an ELISA method.

Table 1 Immune cells and antigen addition in each test group in cell culture ─ Test area Bayer plate cell helper T cell antigen (OVA) 2 × 10 ⁶ cells / well 5 × 10 ⁵ cells / well 1 mg / well ──────────────────── Ａ A ○ ○ ○ B ○ ○ − C ○ − ○ ────────────────────────各 Add each cell and antigen to each well, and add 10% FCS
Make up to 1 ml with PMI. “○” indicates addition, and “−” indicates no addition.

(2) Results As shown in FIG. 4, the raffinose group had better I in the test plots A, B and C than the control group.
The concentration of L-12 was significantly higher. In test zone C,
Since the IL-12 concentration in the raffinose group was significantly higher, the administration of raffinose caused the IL-
It was found that the production of -12 increased. FIG.
As shown in the figure, the IFN-γ concentration was also significantly higher in the test group A in the raffinose group. This IFN-γ reflects the action of IL-12 produced by the antigen-presenting cells to differentiate and proliferate the naive T cells into Th1 cells against the presented OVA antigen, and reflect the IFN-γ produced by the Th1 cells. ing. That is, in the raffinose group, the production of IL-12 of the antigen-presenting cells was increased, and the action of this IL-12 caused the Th1 cells to predominantly differentiate and proliferate, increasing the production of IFN-γ.

In addition, the fact that the IL-12 concentration in the test plot A in FIG. 4 is higher than that in the test plots B and C means that IFN-γ produced by Th1 cells activates macrophages, which are antigen-presenting cells. This shows that IL-12 production is increasing.

That is, orally administered raffinose is
It apparently enhances 1L-I2 production of antigen presenting cells, and thus supports the differentiation and proliferation of Th1 cells more than Th2 cells against ovalbumin antigen, and significantly increases the production of IFN-γ, which suppresses IgE production. It became.

In allergic patients, the Th1 / Th2 cell balance is predominant in Th2 cells, and it is considered that IgE-related allergy is likely to be induced. However, administration of raffinose causes differentiation of Th1 cells. It has become clear that it is effective in supporting proliferation, correcting the Th1 / Th2 cell balance, and improving allergic constitution.

[0032]

Example 3 400 parts by weight of crystalline glucose, 10 parts by weight of raffinose, 7 parts by weight of citric acid, Na-Casein
Using 7 parts by weight of ate, 5 parts by weight of ascorbic acid, and 3 parts by weight of hardened oil, a tablet for reducing IgE antibody production was produced according to a conventional method.

[0033]

EXAMPLE 4 Using 50 parts by weight of raffinose, 20 parts by weight of purified calcium carbonate, 178 parts by weight of lactose, and 2 parts by weight of magnesium stearate, a mixture thereof was used in an amount of 25 parts.
Fill 0 capsules at 0 mg each, and 50 capsules per capsule
A capsule for allergic constitution improvement containing mg of raffinose was produced.

[0034]

According to the present invention, by using raffinose, an excellent effect of reducing IgE antibody production and an effect of improving allergic constitution are exhibited, and the prevention or treatment of allergic diseases is effectively performed. In addition, the composition according to the present invention is particularly safe, so that it can be safely administered to infants and infants, and can be administered for a long period of time. It can be used for a long time as a food composition.

[Brief description of the drawings]

FIG. 1 shows changes in total IgE in serum in Tg mice administered with OVA.

FIG. 2 shows changes in OVA-specific IgE in serum in Tg mice administered with OVA.

FIG. 3 shows changes in OVA-specific IgG1 in serum in Tg mice to which OVA was administered.

FIG. 4 shows a comparison of IL-12 production. However, * indicates that there is a significant difference between the groups at the 5% level.

FIG. 5 shows a comparison of IFN-γ production. However, * indicates that there is a significant difference between the groups at the 5% level.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) // C07H 3/06 C07H 3/06 (72) Inventor Tsutomu Arisuka Tsutomu Inadacho Minami 9 Line Nishi13, Obihiro City, Hokkaido Address Japan Research Institute of Sugar Beet Sugar Co., Ltd. MA04 NA14 ZB13 ZC02

Claims (5)

[Claims] 1. An allergic constitution ameliorating agent comprising raffinose as an active ingredient. 2. An agent for reducing IgE antibody production, comprising raffinose as an active ingredient. 3. An allergic constitution-improving food composition comprising raffinose as an active ingredient. 4. An IgE antibody production reducing food composition comprising raffinose as an active ingredient. 5. The improvement in constitution according to claim 1 or 3, wherein
The allergic constitution improving agent or the allergic constitution improving food composition according to claim 1 or 3, which is obtained by enhancing the production of N-γ and / or IL-12.