JP2001288094A - Cellular immunopotentiator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cellular immunopotentiator capable of enhancing the production of interleukin-12 (IL-12) or interferon-γ (IFN-γ) capable of activating cells such as macrophage, killer T cells or NK cells and enhancing the cellular immune potency by administering raffinose. SOLUTION: This cellular immunopotentiator comprises the raffinose as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cell-mediated immunity enhancer effective for prevention / treatment of various infectious diseases and various tumor diseases, and more particularly, to a cell-mediated immunity enhancer containing raffinose as an active ingredient. And a food composition for enhancing cellular immunity.

[0002]

[Unknown technology] The immune system in humans and animals recognizes self and non-self (foreign body), eliminates non-self microorganisms, virus-infected cells, and tumor cells, and is important for the maintenance of life. Has a role. The mechanism of immunity can be broadly classified into cell-mediated immunity, in which immune cells themselves attack and eliminate foreign substances,
There is humoral immunity that eliminates foreign substances with antibodies.

[0003] Cellular immunity protects against intracellular parasitic microorganisms such as killing against intracellular parasites such as Mycobacterium tuberculosis and Salmonella, fungi such as Candida, and protozoa such as Carini and malaria. Involved in the destruction and removal of tumors and the destruction of tumor cells.

[0004] Macrophages, killer T cells, and NK cells are immune cells responsible for the above-mentioned cellular immunity. Interferon γ produced by helper T cells (IFN-
γ) and interleukin 2 (IL-2) differentiate pre-killer T cells into killer T cells, and IFN-γ has the effect of activating macrophages and killer T cells and enhancing damage activity. In addition to IL-12 produced by macrophages, NK cells are activated, and naive helper T cells not sensitized to antigen are converted to IFN-γ-producing helper T cells.
It works to differentiate cells.

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 which inhabit 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 Escherichia coli and C. perfringens, and improve the intestinal flora.

[0006] However, IFN-γ, a cytokine that activates immune cells (macrophages, killer T cells, NK cells) of the cellular immune system, which are responsible for protection against infection with pathogenic microorganisms and viruses and destruction of tumor cells, etc. And IL
It was not previously known that the production of -12 was enhanced by the administration of raffinose.

[0007]

In recent years, the average life expectancy has been prolonged due to the improvement of nutritional status and the improvement of the medical system. However, immunity decreases with aging. Infections are often fatal and countermeasures are strongly required. To address this, new antitumor agents, vaccines and antibiotics have been energetically developed, but none have been developed yet. In addition, cytokine therapy, such as direct subcutaneous administration of cytokines such as IFN-γ that activate macrophages and killer T cells that have anti-tumor, anti-viral and anti-microbial infection effects, has been developed. In order to do so, dosing must be continued,
There is also a problem in that side effects such as fever and headache occur.

[0008]

Means for Solving the Problems The present inventors have studied from various aspects to achieve the above object. As a result, the present inventors have found that, instead of the above-described coping therapy, the cellular immunity inherently possessed by the living body is not used. I came to realize the need to increase the safety.

Therefore, the present inventors conducted intensive screening for an active ingredient that enhances cellular immunity of a living body. As a result, a useful new finding that oral administration of oligosaccharides, particularly raffinose, among many natural products, enhances cellular immunity was obtained.

The present invention is based on the above-mentioned useful new findings,
As a result of further research, it has been completed.According to the present invention, it is highly safe, effective in protecting against infection with pathogenic microorganisms and viruses, suppressing and destroying the growth of tumor cells, and can be administered orally. A novel cellular immunity enhancer and a food composition.

The cellular immunity enhancer according to the present invention is prepared by adding raffinose as an active ingredient to an inorganic or organic carrier or a pharmaceutical excipient commonly used in a solid, semisolid or liquid form according to a conventional method. It is formulated into parenteral preparations such as enteral preparations and external preparations in addition to oral preparations. In the case of oral administration, examples of the administration form include tablets, capsules, granules, powders, syrups, gargles 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, or mixed into biscuits or the like to form a solid composition.

The amount used depends on the symptoms, age, body weight, administration method, dosage form, etc., but usually 0.5 to 20 g, preferably 3 to 10 g per day for an adult can be administered orally. Since the active ingredient according to the present invention is of natural origin and used as a food, there is no particular problem in toxicity, and the amount of 6,000 mg / (kg body weight) per day for rats 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.

[0013] As for the active ingredient, it is optimal to use purified raffinose, but if desired, for example, in the case of some oral preparations and foods, it may be necessary to use a supplement 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.

[0014]

Example 1 In a mouse test, the effect of oral administration of raffinose on the cellular immune system was examined to examine the IL-12 produced by mouse immune cells. IL-
Numeral 12 secretes macrophages and dendritic cells, activates NK cells, and differentiates naive helper T cells not sensitized to antigens into IFN-γ-producing helper T cells.

(1) Method Ten 3-week-old female BALB / c mice were divided into two groups, and a control group was treated with a basic diet (casein 20%, corn starch 48.17%, α-
Starch 9%, sucrose 5%, cellulose 5%, soybean oil 6%, mineral mixture 5%, vitamin mixture 1.3%, choline chloride 0.23%, methionine 0.
3%), and the raffinose group was fed with raffinose-added feed (corn starch was changed to 43.17% of the basic feed and raffinose was added at 5%).

After breeding for 2 weeks, the mice were sacrificed, and the Bayer plate tissue of Koyo was 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 reacted with stirring for about 1 hour, and a single cell suspension washed several times with RPMI medium supplemented with 10% FCS was used as a Bayer plate cell.

Each Bayer plate cell was added to a 48-well cell culture plate at 2 × 10 ⁶ cells / well, and 10% FCS added.
The total volume was 1 ml in PMI medium. This is 5% C
The cells were cultured in an O ₂ environment at 37 ° C. for 48 hours. After cultivation,
The culture supernatant was collected, and the concentration of IL-12 was measured using an ELISA method.

(2) Results As shown in FIG. 1, the concentration of IL-12 was significantly higher in the raffinose group than in the control group. From these results, it was revealed that administration of raffinose increases the action of inducing IFN-γ production of helper T cells and the production of IL-12 having an NK cell activating action.

[0019]

Example 2 IL-12 production was induced by administration of raffinose, and IFN-
Whether gamma production was enhanced was examined by a mouse test.

(1) Method Six BALB / c mice, 3 weeks old, 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 Bayer plate tissue of Koyo was excised from each mouse.

Moreover, helper T cells recognize ovalbumin (OVA) specific T cell receptor gene (OVA _323-339 specific I-A ^d restricted T cell clone 7
-3-7 TCR α-chain and β-chain genes) were prepared from spleen cells of a transgenic mouse (hereinafter referred to as a Tg mouse) having a genetic background almost identical to that of a BALB / c mouse.

That is, two 7-week-old female Tg mice bred on the basic feed were sacrificed and their spleens were removed. 1 spleen
Trituration was performed in RPMI medium supplemented with 0% FCS, and two mice were combined and washed several times to prepare a single cell suspension. Helper T expressing CD4 molecule from this spleen single cell suspension
The cells were fractionated using a magnetic cell separation system (Miltenyi biotec) and used as helper T cells. The obtained helper T cells are naive T cells that are not antigen-sensitized.

Bayer plate cells, helper T cells, and antigens (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 concentrations of IL-2, IL-12, and IFN-γ were measured by ELISA.

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.

As shown in FIG. 2, the raffinose group produced higher IL-12 production in test groups A, B and C than in the control group. The IFN-γ production was also higher in the raffinose group, as shown in test plot A in FIG. These results indicate that administration of raffinose increased the production of IL-12 from macrophages and dendritic cells.
This indicates that IFN-γ production of helper T cells upon antigen stimulation was induced and enhanced by the action of -12.

In FIG. 2, in the test plot A, the test plot B,
The higher IL-12 production than C indicates that macrophages and the like were activated by IFN-γ produced by helper T cells, and that the production of IL-12 was further increased.

As shown in FIG. 4, there was no difference in IL-2 production between the groups.

From the above results, it can be seen that administration of raffinose enhances 1L-I, a cytokine that enhances cellular immunity.
2 and IFN-γ production were found to be induced and enhanced.

[0030]

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 enhancing cellular immunity was produced according to a conventional method.

[0031]

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 enhancing cellular immunity containing mg of raffinose was produced.

[0032]

According to the present invention, the use of raffinose enhances cellular immunity and effectively prevents / treats various infectious diseases and various tumor diseases. In addition, the composition according to the present invention is particularly safe, so that it can be administered from infants to the elderly and can be administered for a long period of time, so that it can be used to prevent various infectious diseases and various tumor diseases. It can be used for a long period of time as a health drug or food composition.

[Brief description of the drawings]

FIG. 1 shows a comparison of IL-12 production.

FIG. 2 shows a comparison of IL-12 production.

FIG. 3 shows a comparison of IFN-γ production.

FIG. 4 shows a comparison of IL-2 production.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/00 A61P 37/00 // C07H 3/06 C07H 3/06 (72) Inventor Tsutomu Arisuka Hokkaido 13 Ina-machi, Nishi 9 Line, Odahiro-shi, Nippon Beet Sugar Manufacturing Co., Ltd. (72) Inventor Koji Sayama 13-West, Ina-machi, South 9 Line, Obihiro City, Hokkaido F T-term (reference) 4B018 LB01 LB08 MD31 ME07 ME08 ME11 4C057 BB04 4C086 AA01 AA02 EA01 MA52 NA14 ZB07 ZB26 ZB33 ZB35

Claims (3)

[Claims] 1. A cellular immunity enhancer comprising raffinose as an active ingredient. 2. A food composition for enhancing cellular immunity, comprising raffinose as an active ingredient. 3. The cellular immunity according to claim 1 or 2, wherein the enhancement of the cellular immunity according to claim 1 or 2 is due to enhancement of the production of IFN-γ and / or IL-12. An enhancer or a cellular immunity enhancing food composition.