JPH1149767A - Improving agent of immunological function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an improving agent of immunological function capable of normalizing an immunological function and heightening an infection resistance by including tocotrienol as an active ingredient. SOLUTION: This medicine comprises, as an active ingredient, one or more kinds of tocotrienols obtained by e.g. extracting from a vegetable oil such as a palm oil or chemically synthesizing, and is useful for prevention, improvement, remission and treatment of various immunodeficiency syndromes or infectious diseases such as cancer, infectious diseases, asthma, articular rheumatism, autoimmune disease, sepsis, pneumonia, cold syndrome, diarrhea, meningitis or other viral diseases. The medicine is also usable as a component of food additives, auxiliary nutrient foods, and functionally effective foods. It is administered by dividing 0.1 to 5,000 mg into 1 to 4 doses per day in case of an adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel immunological function-improving agent which contains one or more tocotrienols as an active ingredient and is effective against various immunodeficiencies.

[0002]

BACKGROUND OF THE INVENTION In recent years, advances in immunology have been rapid, and various diseases are considered to be caused by immune dysfunction. For example, cancer, bacterial infection, asthma, rheumatoid arthritis,
Autoimmune diseases and the like can be mentioned as diseases caused by immune dysfunction. As for infectious diseases, in addition to simple infectious diseases caused only by invasion of pathogenic bacteria, an increase in complicated infectious diseases accompanied by various serious underlying diseases has become a serious problem. For example, infections associated with cancer are the most troublesome clinical problems. Carrying cancer causes systemic and local reduction of resistance, and secondary infections are secondary to secondary infections under immunocompromised conditions. Infections associated with cancer initially include respiratory infections, urinary tract infections,
There are many fetal tract infections and skin infections, and late stage pneumonia and sepsis. Regarding the mechanism of complications of infection associated with this tumor, the following process is common.

[0003] That is, with the development of leukemia, malignant lymphoma, and cancer, normal tissues and cells are impaired, especially lymphocyte cells and granulocyte cell function are reduced. It is believed that. In such cases, the curative effect of the administration of the antibiotic is poor, and often leads to repeated infections, bacterial replacement, and intractable infections. Therefore, cure is hardly expected with conventional antibiotics and chemotherapeutic agents alone,
It is impossible to treat without improving the biological defense function, and the development of a drug that enhances the biological defense function has been desired. on the other hand,
Antibiotics play a central role in bacterial infection of animals such as livestock and poultry, and in fact, the emergence of various antibiotics has reduced serious infections caused by pathogenic bacteria. However, the abuse of antibiotics in the livestock industry has led to an increase in residual and resistant bacteria in livestock and marine products, as well as bacterial alternation,
It has become a social problem. That is, since the host has a remarkably reduced ability to defend against infection and has a damaged repair function against infectious diseases, the infectious disease is hardly cured and easily re-infected. In addition, spontaneous infections (opportunistic infections) reduce livestock productivity and the losses are large.

[0004]

[Problems to be solved by the present invention] Under the circumstances described above, it is necessary to enhance the activity of the host's immune function and enhance the defense function of the living body.

[0005]

BACKGROUND OF THE INVENTION Anim. Sci., 68, 4303-4309, 1990 states that supplementation of vitamin E (tocopherol) and selenium after vaccination against brucellosis in cattle had effects on several immune parameters. Is described. J.
Nutr., 124, 2024-2032, 1994, describe that the nutritional status and immune response of mice that became murine AIDS were normalized by vitamin E supplementation. However, these prior arts relate to tocopherol, not tocotrienol.

[0006]

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors have long and intensively studied a drug that normalizes an immune function and enhances a protective function of a living body.
More than one species of tocotrienol has been found to be effective as a preventive / therapeutic agent for diseases caused by immune dysfunction in humans and animals, particularly as a protective agent against human and animal infectious diseases,
The present invention has been completed. That is, since the compound of the present invention has a function of normalizing the immune function of humans and animals and increasing the resistance to infection, it is useful as a preventive / therapeutic agent for diseases due to immune dysfunction in humans and animals, and a protective agent against various infectious diseases. is there. The tocotrienol according to the present invention is at least one kind represented by the following chemical formula.

[0007]

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[0008] Each tocotrienol can be obtained, for example, by extraction from a vegetable oil such as palm oil or by chemical synthesis. The tocotrienol in the present invention is not limited to a single isomer, but also includes a mixture, and the mixing ratio is not limited. The present invention relates to various immunodeficiencies or infectious diseases, such as cancer, infectious diseases, asthma, rheumatoid arthritis, autoimmune diseases, sepsis, pneumonia, cold syndrome, diarrhea, meningitis or other viral infections. It is useful for prevention, improvement, improvement, reduction or treatment. When the composition according to the present invention is used as a food additive, a supplemental nutritional food or a component of a functional food, it can be added to an arbitrary concentration according to a conventional method.

When the composition of the present invention is used as a pharmaceutical for the above-mentioned diseases, it can be administered orally or parenterally. Parenterally, it is generally administered as an injection, such as intravenous administration. The dose does not vary greatly depending on age, gender, body weight, patient sensitivity, administration method, administration timing, interval and characteristics, drug delivery and dosage form, type of active ingredient, and the like. Usually, one or more tocotrienols are administered in an amount of 0.1 mg to 5000 mg, preferably 0.5 mg / day for an adult.
mg to 3000 mg, more preferably 1 mg to 2000 mg, is usually administered in one to four minutes.

[0010] For example, preparation of injections, suppositories, sublingual tablets, tablets, capsules and the like can be carried out according to a conventional method. That is, in the production of injections, the active ingredients are mixed and, if necessary, a pH adjuster, a buffer, a suspending agent,
A solubilizing agent, a stabilizing agent, an isotonic agent, a preservative and the like can be added, and the preparation can be made by a conventional method. In this case, if necessary, a freeze-dried preparation can be prepared by a conventional method.
Specific examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate. Specific examples of the solubilizer include hydrogenated polyoxyethylene castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester. Specific examples of the stabilizer include sodium sulfite, sodium metasulfite, and ether.
Specific examples of the preservative include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.

[0011]

【The invention's effect】

Effect of d-α-tocopherol or tocotrienol mixture on rat immune function

(1) The compound used, d-α-tocopherol, was obtained from Tama Seikagaku Co., Ltd. (Yamanashi Prefecture)
Purchased from. A tocotrienol mixture comprising the following components was provided by Lion Corporation (Tokyo). ────────────────── d-α-tocotrienol 28.3% d-β-tocotrienol 4.8% d-γ-tocotrienol 50.0% d-δ-tocotrienol 15.9% ──── ──────────────

Twin 80 used for the enzyme antibody immunoassay (ELISA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka). 0.05
% TWIN 80-containing PBS (TPBS) was used as an ELISA plate washing solution. Blocking solution (brand name; Block Ace)
Was used for blocking ELISA plates using a solution purchased from Snow Brand Milk Products Co., Ltd. (Sapporo) and diluted 4 times with ultrapure water. For dilution of the sample and the antibody, a solution diluted 10-fold with ultrapure water was used. As the solid-phase antibody dilution solution, 50 mM-NaHCO ₃ -Na ₂ CO ₃ (pH 9.6) was used.

As a solid phase antibody, an anti-rat mouse monoclonal antibody (α-chain specific) was obtained from Zymed (San Francisco, CA, USA) and a goat anti-rat IgE (ε-chain specific).
Affinity purified goat anti-rat IgG F (ab ') 2 fragment and affinity purified goat anti-rat IgM F (ab') 2 fragment (μ chain specific) from Bethyl (MO, Texas, USA)
(West Chester, Pennsylvania, USA). These solid phase antibodies are 50 mM NaHCO ₃ -Na ₂ CO ₃ (pH
It was diluted 1000 times using 9.6) and used.

[0015] The enzyme-labeled antibody is peroxidase (POD).
Labeled mouse anti-rat IgA and biotin-labeled mouse anti-rat IgE were purchased from Zymed, and POD-labeled avidin was purchased from Daco (Glos
trup, Denmark), a POD-labeled affinity-purified goat anti-rat IgG F (ab ') 2 fragment and a POD-labeled affinity-purified goat anti-rat IgM F (ab') 2 fragment were purchased from Cappel. IgA and IgM enzyme-labeled antibodies are diluted 1000 times, IgG
And biotin antibody diluted 2000 times, avidin antibody 5000
A two-fold dilution was used. For dilution of these antibodies,
A 4-fold dilution of ace was used.

The coloring substrate solution is 0.006% -H ₂ O ₂ /0.2M-citrate buffer solution (pH 4.0): ultrapure water: 6 mg / ml 2,2′-aniso-bis (3-ethylbenzonin) -6-sulfonic acid) diammonium salt (ABTS) solution = 10: 9: 1. A 1.5% -oxalic acid solution was used as a reaction stopping solution. ELISA plates were purchased from Nunc. For the absorbance measurement, Tosoh
An MRP-A4i microplate spectrophotometer was used.

(2) Experimental Animals Four-week-old male Sprague-Dawley rats were used and purchased from SEAC Yoshitomi. For pre-feeding, NMF was purchased from Oriental Yeast Co., Ltd. and used. Dietary fat used was safflower oil manufactured by Linol Oils and Fats Co., Ltd., and quercetin was purchased and used from Nacalai Tesque, Inc.

(3) Feeding Sprague-Dawley rats were fed with NMF and preliminarily reared for one week. Thereafter, the rats were divided into 4 groups of 10 rats each, and fed with AIN-93 prepared diets having different compositions. Diet composition is% by weight
With casein 20, safflower oil 10, vitamin mix 1.
0, mineral mix 3.5, Choline bitartarate 0.25,
L-lysine 0.3, cellulose 5, corn starch 36.8, gelatinized corn starch 13.2 and sucrose 10 were used. This was used as a control diet, and the other three groups received d-α-tocopherol, a tocotrienol mixture, and quercetin, respectively.
1% was added, and the added portion was given a diet from which corn starch was subtracted.

(4) Efficacy test After preliminary breeding, a feeding experiment for 3 weeks was performed, and on the day of sacrifice, blood was collected from the tail vein of the rat and killed under ether anesthesia. The obtained venous blood was centrifuged at 10,000 × G for 20 minutes to collect the serum, which was frozen and stored at −30 ° C., and the antibody titer in the serum was measured by an enzyme antibody (ELISA) method. After sacrifice, spleen and mesenteric lymph nodes (MLN) were collected. The spleen or MLN was transferred to a dish containing RPMI1640 medium to remove adhering fat, and the tissue was crushed with a slide glass.
This cell suspension was centrifuged at 350 × g for 5 minutes, and the obtained cell pellet was resuspended in ROMI1640 medium. This washing operation
Repeat three times and gently overlay the cell suspension on 4 ml LSM.
Lymphocyte bands were collected by centrifugation at 1,000 × g for 30 minutes.
This lymphocyte fraction was suspended in RPMI1640 medium, and after the above washing operation was performed three times, it was suspended in RPMI1640 medium containing 10% FBS. The cell number was measured with a hemocytometer, 2 x 10 ⁶ cells / m
and adjusted for culture. 1 ml of this lymphocyte suspension was added to a 24-well cell culture plate, and cultured at 37 ° C. for 24 or 48 hours under aeration of 5% CO ₂ . After completion of the culture, the culture solution was collected in a microtube, and centrifuged at 400 × g for 5 minutes to collect the culture supernatant, which was frozen and stored at −30 ° C. for use in measuring the amount of antibody.

(5) Results The results are shown in Table 1-3 (mean ± standard error). In each table, between different symbols, ie, “a and b” or “b and a”, indicates that there is a statistically significant difference (p <0.05), and between partially different symbols, ie, “a And ab "or" ab and a "indicate that there is a statistical difference (p <0.1).

Table 1 Effect of Diet on Serum Immunoglobulin Concentration (See Fig. 1) / ml) (ng / ml) (mg / ml) (μg / ml) ───────────────────────── control 26.2 ± 4.8 1.3 ± 0.3 1.4 ± 0.7 283 ± 45 a ab ─────────────────────────Tocopherol 50.2 ± 10.8 0.7 ± 0.3 1.8 ± 0.2 200 ± 29 ba ─── ──────────────────────Tocotrienol 31.6 ± 4.2 1.7 ± 0.2 1.3 ± 0.3 245 ± 27 ab b ───────────── ────────────

Table 2 Effect of diet on immunoglobulin production of MLN lymphocytes (see Fig. 2) (ng / ml) (ng / ml) (ng / ml) ─────────────────────── (24 hour) Control 3.5 ± 0.9 5.6 ± 0.2 2.6 ± 0.1 aaa ───────────────────── Tocopherol 15.2 ± 1.1 6.0 ± 0.1 2.6 ± 0.1 baa ──────────────── ───── Tocotrienol 15.3 ± 3.9 8.2 ± 0.8 1 ± 0.4 bbb ─────────────────────── (48 hour) Control 4.0 ± 1.1 5.5 ± 0.3 2.7 ± 0.1 aaa ───────────────────── Tocopherol 20.9 ± 3.2 6.4 ± 0.4 3.8 ± 0.2 baa ────────────── ─────── Tocotrienol 19.9 ± 3.6 9.3 ± 0.5 8.6 ± 0.3 bbb ──── ──────────────────

Table 3 Effect of diet on immunoglobulin production of spleen lymphocytes (see FIG. 3) (ng / ml) (ng / ml) (ng / ml) ─────────────────────── (24 hour) Control ND 12.3 ± 2.6 28.5 ± 5.0 a ────────────────────── Tocopherol ND 7.7 ± 1.7 24.6 ± 3.0 a ─────────────────── ─── Tocotrienol ND 12.6 ± 0.5 70.3 ± 4.8 b ─────────────────────── (48 hour) Control ND 17.3 ± 5.0 52.8 ± 10.9 a ── ──────────────────── Tocopherol ND 11.1 ± 2.2 49.5 ± 5.2 a ───────────────────── ─ Tocotrienol ND 20.9 ± 3.2 155.2 ± 16.9 b ─────────────────────── N.D .; not detected

From Table 1-3, it can be seen that tocotrienol has an excellent immune function-improving or enhancing effect, preventing, improving, improving various immunodeficiencies or infectious diseases.
It is clear that it is useful for reduction or treatment.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of diet on serum immunoglobulin concentration. (Mean ± standard error)

FIG. 2 shows the effect of diet on MLN lymphocyte production of immunoglobulin. (Mean ± standard error)

FIG. 3 is a graph showing the effect of diet on the production of immunoglobulins of spleen lymphocytes. (Mean ± standard error)

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/35 ADU A61K 31/35 ADU ADZ ADZ

Claims (4)

[Claims] 1. An immune function improving agent comprising one or more tocotrienols as an active ingredient. 2. The immunodeficiency or infectious disease is one selected from cancer, bacterial infection, asthma, rheumatoid arthritis, autoimmune disease, sepsis, pneumonia, cold syndrome, diarrhea or meningitis. The immune function improving agent according to the above. 3. A composition for improving immune function, comprising at least one tocotrienol. 4. A method for treating immunodeficiency or infectious disease, comprising administering one or more tocotrienols to a human.