JP2001323001A - beta-GLUCAN HAVING ACTIVITY FOR ENHANCING IMMUNITY AND FORMED INTO THE LOW MOLECULAR ONE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a low-molecular-weight water-soluble β-glucan capable of promoting the in-vivo production of a cytokine exemplified by TNF, capable of enhancing the activities and useful for proliferation of various kinds of infectious disease and development of tumor by enhancing the productivity of antibody or the whole of immunological activities. SOLUTION: This water-soluble β-glucan formed into the low-molecular one has activity for enhancing immunity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for enhancing immunity,
The present invention relates to a water-soluble β-glucan having an action of preventing the occurrence of various bacteria and viral infections and cancer, and a food material, a cosmetic material, a pharmaceutical material and a processed product of the material containing the water-soluble β-glucan as an active ingredient.

[0002]

2. Description of the Related Art The living body is protected from the attack of microorganisms such as bacteria and viruses or tumors and the like generated in the living body mainly by the action of the immune system. In recent years, attention has been focused on research on influenza prevention or cancer treatment using a biological response modifier (hereinafter abbreviated as BRM) having an effect on an immune function and enhancing it.

[0003] The immunopotentiating effect of BRM is based on the fact that BRM acts on various cells in a living body to induce tumor necrosis factor (TN).
This is caused by activation and induction of the production of substances generally referred to as cytokines such as F), interleukins and interferons. These derived substances are:
Acts on immunocompetent cells to activate the immune system. Among the cytokines, TNF is released from monocytes and macrophages, and is known to have a cell growth effect and an antiviral effect. As interleukins,
The presence of IL1 to IL12 is known. Of which IL
No. 1 has a molecular weight of 17,500 produced mainly from monocytes and macrophages due to infection, inflammation, various immune reactions, etc.
Is a peptide hormone. Three types of interferons are known: interferon-α, interferon-β, and interferon-γ. Interferon-γ is a glycoprotein having a molecular weight of about 20,000, and has been attracting attention as an immunoregulatory factor by acting on antiviral activity, activation of immune-competent cells such as macrophages and natural killer cells, and induction of differentiation.

Research has been conducted to isolate and purify these cytokines or to prepare them by genetic recombination and administer them to apply them to the treatment of viral infections or cancers. Work in concert and exert their effects for the first time, and the balance of each cytokine is important, and there is no way to find a way to administer many cytokines in a well-balanced manner. At present, there is great expectation for BRM used to overcome these problems.

[0005] Known BRMs include polysaccharides derived from microorganisms, yeast cells, lactic acid bacteria cells, and the like as cell wall components. It is also known that lentinan, a polysaccharide extracted from shiitake mushroom, and β-glucans of other bile fungi are effective. However, these microorganisms and bile fungi require time and labor for culture and require special equipment. In addition, the extraction of β-glucan or other polysaccharides derived from these microorganisms and bile fungi requires complicated operations, requires a complicated purification step, requires a great deal of cost and operation time, and results in a BR.
There is a problem that M becomes expensive.

[0006] With regard to the BRM action of water-soluble β-glucan, that is, the immunopotentiating action, the same action has been observed in cultures of bile fungi or hot water extracts of fruiting bodies. However, in these mushrooms, a high molecular weight β-glucan having a weight average molecular weight of 2,000,000 to 200,000 has an immunopotentiating effect. It is known that relatively high molecular weight β-glucan having a weight average molecular weight of more than 100,000 derived from gramineous plants has an immunopotentiating effect, but the immunopotentiating effect is not satisfactory, and the weight average The immunopotentiating effect of β-glucan whose molecular weight has been reduced to 100,000 or less has not been known.

[0007] β-glucan, which has been recognized as having an immunopotentiating effect, has a weight-average molecular weight exceeding 100,000. Generally, high molecular weight β-glucan has high viscosity and is difficult to dissolve in water. Therefore, when used as a material for foods, cosmetics, pharmaceuticals, etc., a heating operation or a long-time stirring operation is required, and there are problems such as an increase in cost for heat-induced denaturation and molecular modification.
There is a need for lower molecular weight β-glucan that retains its functionality.

Accordingly, it is an object of the present invention to provide a T
Provide low-molecular weight water-soluble β-glucan that promotes the production of NF and other cytokines, enhances their action, and enhances antibody production ability or overall immunity to help prevent various infectious diseases and tumor development. Is to do.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, obtained a low-molecular-weight water-soluble β-glucan obtained by converting a high-molecular-weight β-glucan into a low-molecular-weight compound. Have a stronger immunopotentiating effect than β-glucan of high molecular weight, and have reached the present invention.

That is, the present invention provides a low molecular weight water-soluble β-glucan having an immunopotentiating effect.

The present invention also provides the water-soluble β-glucan having a weight average molecular weight of 100,000 or less.

Further, the present invention provides the above water-soluble β-glucan, wherein the ratio of the content of β-glucan having a molecular weight of less than 100,000 is 80% by weight or more of the total β-glucan.

[0013] The present invention also provides the above water-soluble β-glucan, wherein the β-glucan is derived from a plant of the grass family.

The present invention also provides the water-soluble β-glucan, wherein the grass is a barley or oats.

The present invention also provides a food material, a cosmetic material, a pharmaceutical material and a processed product of the material, which contain the water-soluble β-glucan.

Further, the present invention provides the above-mentioned raw material and the processed product containing 0.1 to 90% by weight of the above-mentioned water-soluble β-glucan.

Further, the present invention provides the above-mentioned material and the processed product further containing a lactic acid bacterium or a lactic acid bacterium cell component.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The low molecular weight water-soluble β of the present invention
Glucan has an antibody production enhancing effect or an immunopotentiating effect of promoting production of cytokines, particularly TNF, and is particularly suitable as a material for foods, cosmetics, pharmaceuticals and the like.

The β-glucan of the present invention is preferably derived from a plant, particularly a grass plant, and is most suitable as a food material. Examples of grasses include rice, wheat, corn, barnyard millet, millet, millet, barley, oats (crow oats), rye, and the like, and preferably barleys. , Oats, and more preferably barley. These gramineous plants include those of a non-glutinous line expressing waxy protein and synthesizing amylose and those of a glutinous line lacking the action of this protein. Can be.

The water solubility of the low molecular weight water-soluble β-glucan of the present invention means a property of being completely dissolved in hot water at 65 ° C. The property of completely dissolving is that a sample is added so as to be 1% by weight with respect to distilled water, and after stirring and dissolving for 10 minutes, no precipitate is visually observed at 660 nm.
In the turbidity measurement by the absorbance of O., the water dissolved in O. D. The property whose value is 0.4 or less. Since the water-soluble β-glucan of the present invention has such water solubility, it is easy to handle as a material for food, cosmetics, pharmaceuticals, and the like,
Processing time is not required, and costs can be reduced.

The water-soluble β-glucan of the present invention has a low molecular weight. The term "low molecular weight" as used herein means that it is water-soluble and has an immunopotentiating effect. Among them, water-soluble β-glucan having a weight-average molecular weight of 100,000 or less is excellent in immunity-enhancing action and is also excellent as a material for foods, cosmetics, pharmaceuticals and the like. Among them, those having a weight average molecular weight of 5,000 to 100,000 are preferable. Further, those having a weight average molecular weight of 10,000 to 60,000 are more preferable.

The water-soluble β-glucan of the present invention has a molecular weight of 10
The ratio of the β-glucan content of less than 10,000 is preferably 80% by weight or more, more preferably 90% by weight or more, more preferably 95% by weight or more, and most preferably 100% by weight of the total β-glucan.

The low-molecular-weight water-soluble β-glucan of the present invention can be obtained directly from a raw material such as a gramineous plant, and can also be obtained by reducing the molecular weight of a high-molecular-weight β-glucan extracted and purified from the raw material. it can.

As a method for obtaining a high molecular weight β-glucan from cereals, for example, a method of producing a waxy barley as a raw material and extracting it with water (Japanese Patent Publication No. 4-11197), or a method of producing a barley or oat as a raw material As a weight average molecular weight of 100,000 to 1 by alkali extraction, neutralization, and alcohol precipitation.
How to get a million β-glucan (Japanese Patent Publication No. 6-83652)
Publication). Since β-glucan obtained by these production methods has a high molecular weight, it has high viscosity and it is difficult to re-dissolve in water as it is. The molecular weight of these high molecular weight β-glucan can be reduced to obtain the low molecular weight water-soluble β-glucan of the present invention.

As a method for lowering the molecular weight of high-molecular weight β-glucan, any known polysaccharide hydrolysis reaction can be used. For example, it is known that a water-soluble polysaccharide is hydrolyzed by heating under pressure in the presence of an acid, and this can be used to lower the molecular weight of high-molecular-weight β-glucan. It is also effective to reduce the molecular weight using a hydrolysis reaction by an enzyme. As the enzyme, 1 / 3β glucanase or the like can be used. As described above, the low molecular weight water-soluble β-glucan of the present invention can be obtained from the high-molecular-weight β-glucan extracted and purified from the raw material. Further, the low-molecular-weight water-soluble β-glucan of the present invention can also be obtained by directly extracting from raw cereal grains by the method described in WO98 / 13056 or the like.

Among the low molecular weight water-soluble β-glucans of the present invention, the most preferable one is barley-derived low molecular weight water-soluble β-glucan, which is excellent in immunity-enhancing action and is particularly useful in the body. The effect of promoting the production of cytokines such as TNFs, interleukins, interferons, and the like, and enhancing the immunity such as antibody production by the action is excellent. It is remarkably superior to β-glucan having a molecular weight of 10,000 or more.

When the low molecular weight water-soluble β-glucan of the present invention is used in a material such as foods, cosmetics and pharmaceuticals or a processed product of such a material, it is contained in an amount of 0.1 to 90% by weight. Is preferred. Although the immunopotentiating effect of the low-molecular-weight water-soluble β-glucan of the present invention can be sufficiently expressed alone, it is preferable to use it in combination with lactic acid bacteria or lactic acid bacteria cell components, because the immunopotentiating effect is further increased.

[0028]

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified,% in the examples is by weight, and the molecular weight is a weight average molecular weight.

Example 1 (Production of Low-Molecular Water-Soluble β-Glucan) Commercial barley was pulverized and passed through a 24-mesh sieve to obtain barley flour. 2.5 L (liter) of water was added to 500 g of the barley flour, heated to 55 ° C., and extracted with stirring for 2 hours. After the extraction, the mixture was centrifuged at 1,500 G for 10 minutes to obtain 2.2 L of the supernatant. Cool and freeze the supernatant,
It was left at -10 ° C for 24 hours. After thawing, the precipitated β-glucan was separated by filtration and dried by heating at 80 ° C. to obtain 15 g of β-glucan.
A glucan crude extract was obtained. After adding 300 ml of water to the β-glucan crude extract and completely dissolving it at 90 ° C.,
It was cooled to 20 ° C. and stored frozen for 24 hours. After thawing, the precipitated β-glucan was separated by filtration, freeze-dried, and
A glucan purified product (sample A) was obtained.

This sample A was added to distilled water so as to have a concentration of 1%, heated to 65 ° C., and allowed to stand for 10 minutes to be dissolved to form a transparent aqueous solution. Absorbance at 660 nm was measured using an O.D. D. A value of 0.127 was shown. Next, the molecular weight of Sample A was measured by gel filtration chromatography as follows. Sample A was added to distilled water to a concentration of 1% and dissolved in boiling water. Gel separation column Supero at a temperature of 50 ° C for separation
Using se6HR (manufactured by Pharmacia), Shodex pullulan standard solution P-82 (manufactured by Showa Denko KK) was used as a molecular weight marker. Using distilled water as eluent, flow rate 0.6 ml
/ Min. The elution fraction was monitored with a refractometer (RI). As a result of measuring the molecular weight of sample A from the molecular weight of the pullulan standard, the molecular weight was distributed in the range of 100,000 to 5,000, and the peak having a molecular weight of 100,000 or more was slight (3%
Below), the content increased as the molecular weight decreased, and the main component was 40,000 in molecular weight. Β-glucan having a molecular weight of 5,000 or less was slight (5% or less).

Comparative Example 2 (Production of High-Molecular-Weight β-Glucan) Commercial barley was pulverized and passed through a 24-mesh sieve to obtain barley flour. 2.5 L of water was added to 500 g of the barley flour, and 5
After heating to 5 ° C., the mixture was stirred and extracted for 30 minutes. After the extraction, the mixture was centrifuged at 1,500 G for 10 minutes to obtain 2.2 L of the supernatant. The supernatant was cooled, frozen and kept at -10 ° C for 24 hours.
Left for hours. After thawing, the precipitated β-glucan was separated by filtration and dried by heating at 80 ° C. to obtain 5 g of a β-glucan crude extract. 500 ml of water was added to the β-glucan crude extract, and the mixture was boiled to dissolve, cooled to −20 ° C., and stored frozen for 24 hours. After thawing, the precipitated β-glucan was separated by filtration and freeze-dried to obtain 3.6 g of purified β-glucan (sample B).

This sample B was added to distilled water so as to have a concentration of 1%, heated to 65 ° C., and attempted to dissolve for 10 minutes. However, sample B was found to precipitate but not completely dissolved. 66
The absorbance at 0 nm was measured. D. Value 1.74
showed that. Next, the molecular weight of Sample B was measured by gel filtration chromatography as follows. Sample B
Was added to distilled water to a concentration of 1% and dissolved in boiling water. Gel separation column Supero at 50 ° C for separation
Using se6HR (manufactured by Pharmacia), Shodex pullulan standard solution P-82 (manufactured by Showa Denko KK) was used as a molecular weight marker. Using distilled water as eluent, flow rate 0.6 ml
/ Min. The elution fraction was monitored with a refractometer (RI). As a result, the molecular weight of Sample B was measured from the molecular weight of the standard pullulan. As a result, the molecular weight distribution was distributed in the range of 500,000 to 150,000, and the main component was 300,000. Low molecular weight β-glucan having a molecular weight of 100,000 or less was not observed.

Test Example 1 (Variation in the number of cells in the peritoneal cavity) First, the sample A (low-molecular-weight water-soluble β-glucan of the present invention) obtained in Example 1 was treated with a 10 mM phosphate buffer (pH
6.9) The cells were dissolved in 100 μl and administered in an amount of 0.32 to 20 mg to the peritoneum of a 6-week-old female ICR mouse. Six hours later, peritoneal cells were collected, and the number of cells was counted under a microscope. As a result, the number of cells increased in proportion to the dose, and was 2 to 3 times higher than that of the non-administered mice. When the ratio of neutrophil count to total cell count was counted,
The dose increases with the dose.
The increase was 20-fold with the administration of mg and 30-fold with the administration of 5 mg. The proportion of neutrophils in the increased cells exceeded 70% of the increased cells at the 1 mg dose. Purified lentinan derived from shiitake mushroom extract, a clinically used immunopotentiator, is 1 m
g, the number of cells was 3 times, the number of neutrophils was 30 times, and the ratio was 8
It was 0%. In the case of administration of OK-432 derived from a bacterium, the amount of 0.
At 2 mg administration, the cell count was 5 times, neutrophils were 60 times, and the ratio was 9
It was 0%.

Next, the sample A was administered, and the qualitative change of the cells over time was examined. 10 mg of the sample A
Was administered to the abdominal cavity of the mouse, and the cells were collected 6, 12, 24, 48, and 72 hours later, and the numbers of neutrophils, macrophages, and lymphocytes, which are immunocompetent cells, were counted. As a result, the total cell number reached a maximum in 24 hours, and was 10 times that of the non-administration. Neutrophils reached a maximum after 24 hours, decreased sharply after 48 hours, and were almost normal after 72 hours. Macrophages increased after 12 hours and peaked at 48 hours. Lymphocyte counts also peaked after 48 hours. Lentinan had the highest total cell number after 12 hours and then decreased. Neutrophil counts were greatest after 6 hours and decreased thereafter. The number of neutrophils did not decrease in sample A until 24 hours and was more persistent than lentinan. Changes in macrophages and lymphocytes in mice administered with lentinan were almost the same as those in Sample A.
In addition, the count of the above-mentioned cell number was performed by applying abdominal cells to a slide glass, staining the nucleus, and categorizing neutrophils, macrophages, and lymphocytes based on morphological changes. In addition, microscopic observation of the cells revealed morphological characteristics considered to be the result of active phagocytosis of macrophages and dramatic decrease in neutrophils in the cell observation 48 hours after the administration of Sample A. Such changes are caused by Lentinan, O
K-432 and others were not recognized, and were specifically recognized by the administration of the sample A.

Test Example 2 (TN produced from cells in the abdominal cavity)
Fluctuation in F Amount) TNF-α production ability was evaluated using peritoneal cells obtained from mice to which sample A obtained in Example 1 was administered.
First, the sample A was added to a 10 mM phosphate buffer (pH
6.9) The solution was dissolved in 100 μl, and administered to a 6-week-old female ICR mouse abdominally in a concentration of 0 to 10 mg. Three days later, the peritoneal cells were collected, washed with a serum-free culture solution, and then added to each well with the same culture solution so as to be 5 × 105 cells. Further, lactic acid bacteria cells were added to each well to a concentration of 20 μg / ml, and the total volume was adjusted to 200 μl for 2 hours 37
Left at ℃. The concentration of TNF-α in the culture supernatant was determined by ELISA.
A measurement kit (TNF-2 ELI manufactured by ENDOGEN)
SAKIT EN-2601-90). As a result, when 0.3 mg of the sample A was administered, 51 mg was administered.
pg / ml, TNF produced in a concentration-dependent manner as 128 pg / ml at 1 mg administration and 141 pg / ml at 3 mg administration
The concentration of -α increased. 25 pg / ml when not added
And the production amount was specific to the sample A.

The time course of TNF-α production was evaluated using peritoneal cells obtained from the mouse to which the sample A was administered. 3 mg of sample A was dissolved in 100 μl of 10 mM phosphate buffer (pH 6.9), and ICR mouse 6
Weekly females were administered intraperitoneally. After 0 to 7 days, the peritoneal cells were collected, washed with a serum-free culture medium, and added to each well with the same culture medium.
It adjusted so that it might become x105 cells, and was added. Further, lactic acid bacteria cells were added to each well at a concentration of 20 μg / ml to make a total volume of 200 μl, and left at 37 ° C. for 2 hours. The TNF-α concentration of the culture supernatant was measured using an ELISA measurement kit (TNF-2 ELISA Kit manufactured by ENDOGEN).
EN-2601-90). As a result, immediately after the administration of the sample A, 18 pg / ml, 84 pg / ml after 1 day, 130 pg / ml after 3 days, 121 pg / ml after 5 days, 98 pg / ml after 7 days, and TN
The production of F-α increased daily, and its production ability was maintained for 7 days.

Test Example 3 A sample A obtained in Example 1 and a sample B obtained in Comparative Example 1 were administered to mice, and the effect of enhancing antibody production was analyzed. First, 1 mg of bovine / plasma gamma globulin as an antigen was dissolved in 1 ml of PBS (phosphate buffer) to prepare a 1 mg / ml antigen solution. Sample A
Alternatively, 500 μg of B was added to 1 ml of PBS and dissolved by heating. The sample A was instantaneously dissolved by heating to 50 ° C. The sample B was not dissolved by heating to 50 ° C., but was left at 90 ° C. for 30 minutes to be dissolved.
An equal amount of the antigen solution and the sample solution are mixed, and 1
Each dose of 00 μl was administered. The mouse is Balbc mouse 4
Using a female of the age and a female, each group consisted of 5 animals. Two weeks later, a booster immunization was performed in the same manner as the first time. In addition, 0.5 mg / ml of bovine / plasma gamma globulin was dissolved in 1 ml of PBS to 0.5 mg / ml, and a group administered with 100 μl of mice was used as a control. Blood was collected two weeks after the booster immunization, and the difference in the total antibody production (IgG, IgM, IgA antibody amounts) was measured by ELISA. In the ELISA, 10 g of bovine / plasma gamma globulin used for immunization were first used.
The plate was coated on a 96-microplate (Maxi Soap, manufactured by Nunc Corporation) at a concentration of μg / ml, and a diluted solution of serum was reacted at 37 ° C. for 1 hour, and then an anti-mouse immunoglobulin antibody labeled with peroxidase was further added. 2
Reaction was carried out at 37 ° C. for 1 hour as a secondary antibody. Next, an orthophenylenediamine solution (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a coloring agent, left at 37 ° C. for 10 minutes, stopped the reaction with 2M sulfuric acid, and measured the absorbance at 490 nm.

Next, blood collected from the mouse was centrifuged to obtain serum, which was then multiplied by a factor of 10 to 5120 to 0.2 times.
PBS-Twee containing% BSA (bovine serum albumin)
n solution (Tween 20 which is a sorbitan monoester)
Was dissolved in PBS to a concentration of 0.05%) and the antibody titer was measured. As a result, the anti-bovine / plasma gamma-globulin antibody titer of the serum diluted 2560-fold was determined to be O.D. D. When compared as an average value, the sample A administration group (n = 5) was 0.703 ± 0.1, the sample B administration group (n = 5) was 0.491 ± 0.075, and the control group (n = 5).
5) was 0.315 ± 0.090. From this test, the antibody titer of the sample A administration group was higher than that of the sample B administration group, and the antibody production enhancing effect of the sample A was confirmed.

[0039]

Industrial Applicability The low molecular weight water-soluble β-glucan of the present invention promotes the production of TNF and other cytokines in the body, enhances its action, and enhances the antibody-producing ability or the overall immunity. It helps to prevent various infections and tumors.

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Claims (8)

[Claims] 1. A low molecular weight water-soluble β-glucan having an immunopotentiating effect. 2. The water-soluble β-glucan according to claim 1, having a weight average molecular weight of 100,000 or less. 3. The water-soluble β-glucan according to claim 1, wherein the ratio of the content of β-glucan having a molecular weight of less than 100,000 is 80% by weight or more of the total β-glucan. 4. The water-soluble β-glucan according to claim 1, wherein the β-glucan is derived from a gramineous plant. 5. The water-soluble β-glucan according to claim 4, wherein the gramineous plant is barley or oats. 6. The water-soluble β according to claim 1,
Food materials, cosmetic materials or pharmaceutical materials containing glucans or processed products of these materials. 7. 0.1 to 90% by weight of water-soluble β-glucan
The raw material or processed product according to claim 6, which contains. 8. The material or processed product according to claim 6, further comprising a lactic acid bacterium or a lactic acid bacterium cell component.