JP4812157B2 - Pharmaceutical material containing low molecular weight β-glucan with immunopotentiating action - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a water-soluble β-glucan that has an action of enhancing immunity and preventing the occurrence of various bacteria, viral infections and cancer, and a food material, a cosmetic material, and a pharmaceutical containing the water-soluble β-glucan as an active ingredient The present invention relates to materials and processed products of those materials.
[0002]
[Prior art and problems to be solved by the invention]
The living body is protected from attacks of microorganisms such as bacteria and viruses, or tumors generated in the living body mainly by the action of the immune system. In recent years, research on influenza prevention or cancer treatment using a biological response modifier (hereinafter abbreviated as BRM) having an effect of acting on and enhancing immune functions has attracted attention.
[0003]
First, BRM acts on various cells in the body to activate the production of substances generally called cytokines such as tumor necrosis factor (hereinafter abbreviated as TNF), interleukins, and interferons. It is caused by being induced. These induced substances act on immunocompetent cells and activate the immune system. Among cytokines, TNF is released from monocytes and macrophages and is known to exhibit cell proliferation and antiviral effects. As interleukins, the presence of IL1 to IL12 is known. Among them, IL1 is a peptide hormone having a molecular weight of 17500, which is mainly produced from monocytes and macrophages due to infection, inflammation, various immune reactions and the like. 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 attracted attention as an immunoregulatory factor by acting on antiviral action, activation of immune cells such as macrophages and natural killer cells, and induction of differentiation.
[0004]
Studies have been conducted to isolate and purify these cytokines, or to prepare them by gene recombination, and administer each of them to apply virus infection treatment or cancer treatment. The balance of each cytokine is important, the method of administering many cytokines in a balanced manner has not yet been found, and cancer treatment has not been achieved. At present, expectations are high for BRM used for the purpose of overcoming these problems.
[0005]
As BRM, yeast cells, lactic acid bacteria and the like are known as microorganism-derived polysaccharides or cell wall components. It is also known that Lentinan, a shiitake-extracted polysaccharide, or β-glucans of other bile fungi are also effective.
However, these microorganisms and gallbladder are time-consuming to culture and require special equipment. In addition, the extraction of β-glucan or other polysaccharides derived from these microorganisms or bile fungi is complicated in operation, complicated in the purification process, takes a lot of cost and operation time, and the resulting BRM is expensive. There is a problem.
[0006]
Regarding the action of BRM of water-soluble β-glucan, that is, the immunopotentiating action, the same action has been observed in the bile fungus culture or the hot water extract of fruiting bodies.
However, in these mushrooms, a high molecular weight β-glucan having a weight average molecular weight of 2 million to 200,000 has an immunopotentiating action.
Moreover, it is known that β glucan having a relatively high molecular weight derived from a Gramineae plant having a weight average molecular weight exceeding 100,000 has an immunopotentiating action, but the immunopotentiating action is not satisfactory, and the weight average It was unknown about the immunopotentiating action of β-glucan having a molecular weight reduced to 100,000 or less.
[0007]
The β-glucan that has been confirmed to have an immunopotentiating action has a high molecular weight with a weight average molecular weight exceeding 100,000. In general, high molecular weight β-glucan is highly viscous and difficult to dissolve in water. Therefore, when used as a raw material for food, cosmetics, pharmaceuticals, etc., there are problems such as heating operation and long-time stirring operation, and there are problems such as high cost for denaturation by heat and molecular modification. There is a demand for β-glucan having a lower molecular weight and retaining the above.
[0008]
Therefore, the object of the present invention is to promote the production of cytokines including TNF in the body, enhance its action, and enhance the antibody production ability or the overall immune action, thereby preventing various infectious diseases and tumor development. It is to provide a low molecular weight water-soluble β-glucan.
[0009]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have compared low molecular weight water-soluble β-glucan obtained by lowering high molecular weight β-glucan to high-molecular-weight β-glucan. As a result, the present inventors have found that it has a stronger immunity enhancing action, and reached the present invention.
[0010]
That is, the present invention provides a pharmaceutical material for immune enhancement, which contains a barley flour- derived water-soluble β-glucan whose weight average molecular weight is reduced to 5000 to 100,000 as an active ingredient. To do.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
The low molecular weight water-soluble β-glucan of the present invention has an antibody enhancing effect or an immune enhancing effect that promotes the production of cytokines, particularly TNF, and is particularly suitable as a material for foods, cosmetics, and pharmaceuticals.
[0019]
The β-glucan of the present invention is preferably derived from a plant, particularly a grass family plant, and is most suitable as a food material. Examples of gramineous plants include rice, wheat, corn, barnyard millet, millet, millet, barley, oats (crown), rye, etc., preferably barley Oats, more preferably barley. Among these gramineous plants, there are those of the silkworm strain that expresses the waxy protein and synthesizes amylose, or those of the hatched strain that lacks the action of this protein. Can do.
[0020]
The water solubility of the low molecular weight water-soluble β-glucan of the present invention refers to the property of completely dissolving in 65 ° C. warm water. The property of complete dissolution is that the sample was added to 1% by weight with respect to distilled water, stirred and dissolved for 10 minutes, and then no precipitate was visually confirmed. In the turbidity measurement by absorbance at 660 nm, O. D. The 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 raw material for foods, cosmetics, pharmaceuticals, etc., and it does not take time and effort for processing, and the cost can be reduced.
[0021]
The water-soluble β-glucan of the present invention has a low molecular weight. The term “low molecular weight” as used herein may be water-soluble and have an immunopotentiating action. Among these, water-soluble β-glucan having a weight average molecular weight of 100,000 or less is particularly excellent as an immunopotentiating 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.
[0022]
In the water-soluble β-glucan of the present invention, the content ratio of β-glucan having a molecular weight of less than 100,000 is preferably 80% by weight or more of the total β-glucan, more preferably 90% by weight or more, and further preferably 95% by weight or more. 100% by weight is most preferred.
[0023]
The low molecular weight water-soluble β-glucan of the present invention can be obtained directly from raw materials such as gramineous plants, and can also be obtained by reducing the molecular weight of high molecular weight β-glucan extracted and purified from raw materials.
[0024]
Examples of a method for obtaining high molecular weight β-glucan from cereals include, for example, a method of producing a waxy barley as a raw material by water extraction (Japanese Patent Publication No. 4-11197), or barley and oat as a raw material. There is a method of obtaining β-glucan having a weight average molecular weight of 100,000 to 1,000,000 by extraction, neutralization and alcohol precipitation (Japanese Patent Publication No. 6-83651). Since β-glucan obtained by these production methods has a high molecular weight, it is highly viscous and difficult to re-solubilize in water. The low molecular weight water-soluble β-glucan of the present invention can be obtained by reducing the molecular weight of these high molecular weight β-glucans.
[0025]
As a method for reducing the molecular weight of high molecular weight β-glucan, any known hydrolysis reaction of polysaccharides can be used. For example, water-soluble polysaccharides are known to hydrolyze by heating under pressure in the presence of an acid, and this can be used to reduce the molecular weight of high molecular weight β-glucan. Further, it is effective to lower the molecular weight by utilizing an enzymatic hydrolysis reaction, and 1/3 β-glucanase or the like can be used as the enzyme. 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.
Moreover, the low molecular weight water-soluble β-glucan of the present invention can also be obtained by directly extracting from raw material grains by the method described in WO98 / 13056 International Publication.
[0026]
Among the low molecular weight water-soluble β-glucan of the present invention, the most preferable one is barley-derived low molecular weight water-soluble β-glucan, which has an excellent immune enhancing action, particularly TNFs in the body, It promotes the production of cytokines such as interleukins and interferons, and is excellent in the effect of enhancing immunity such as antibody production by its action. Among these effects, the antibody production enhancing action has a weight average molecular weight of 100,000 or more. It is remarkably superior to high molecular weight β-glucan.
[0027]
In addition, when the low molecular weight water-soluble β-glucan of the present invention is used in materials such as foods, cosmetics, and pharmaceuticals or processed products of these materials, it is preferably contained in an amount of 0.1 to 90% by weight. .
Moreover, the immunopotentiating action of the low molecular weight water-soluble β-glucan of the present invention can be sufficiently expressed alone, but it is preferable to use it together with lactic acid bacteria or lactic acid bacteria cell components since the immunopotentiating action is further increased.
[0028]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited by these Examples. Unless otherwise specified,% in the examples is by weight, and the molecular weight is a weight average molecular weight.
[0029]
Example 1 (Production of low molecular weight water-soluble β-glucan)
Commercial barley was pulverized and passed through a 24 mesh sieve to make barley flour. 2.5 L (liter) of water was added to 500 g of the barley flour, heated to 55 ° C., and extracted by stirring for 2 hours. After extraction, the mixture was centrifuged at 1500 G for 10 minutes to obtain 2.2 L of supernatant. The supernatant was cooled, frozen and 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 a β-glucan crude extract. 300 ml of water was added to this β-glucan crude extract and completely dissolved at 90 ° C., then cooled to −20 ° C. and stored frozen for 24 hours. After thawing, the precipitated β-glucan was separated by filtration and lyophilized to obtain 10 g of a purified product of β-glucan (sample A).
[0030]
This sample A was added to distilled water so as to be 1%, heated to 65 ° C., and allowed to stand for 10 minutes to dissolve and become a transparent aqueous solution. Absorbance at 660 nm is O.D. with distilled water as a control. D. A value of 0.127 was indicated.
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. For the separation, a gel filtration column Superose 6HR (manufactured by Pharmacia) was used at a temperature of 50 ° C., and Shodex pullulan standard solution P-82 (manufactured by Showa Denko KK) was used as a molecular weight marker. Using distilled water as the eluent, a flow rate of 0.6 ml / min. The elution fraction was monitored with a refractometer (RI). As a result of measuring the molecular weight of the sample A from the molecular weight of the pullulan standard, the molecular weight is distributed in the range of 100,000 to 5,000, the peak having a molecular weight of 100,000 or more is slight (3% or less), and the content decreases as the molecular weight decreases. The main component was 40,000 molecular weight. In addition, β-glucan having a molecular weight of 5000 or less was slight (5% or less).
[0031]
Comparative Example 2 (Production of high molecular weight β-glucan)
Commercial barley was pulverized and passed through a 24 mesh sieve to make barley flour. Water (2.5 L) was added to 500 g of the barley flour, heated to 55 ° C., and extracted by stirring for 30 minutes. After extraction, the mixture was centrifuged at 1500 G for 10 minutes to obtain 2.2 L of supernatant. The supernatant was cooled, frozen and 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 5 g of a crude extract of β-glucan. The β-glucan crude extract was added with 500 ml of water, boiled and dissolved, 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).
[0032]
Sample B was added to distilled water so as to be 1%, heated to 65 ° C., and dissolution was attempted for 10 minutes. Sample B was found to be precipitated but not completely dissolved. When the absorbance at 660 nm was measured, O.D. D. A value of 1.74 was indicated.
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. For the separation, gel filtration column Superose 6HR (manufactured by Pharmacia) was used at 50 ° C., and Shodex pullulan standard solution P-82 (manufactured by Showa Denko KK) was used as a molecular weight marker. Using distilled water as the eluent, a flow rate of 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 pullulan standard, and as a result, the molecular weight distribution was distributed to a molecular weight of 500,000 to 150,000, and the main component was a molecular weight of 300,000. No low molecular weight β-glucan having a molecular weight of 100,000 or less was observed.
[0033]
Test Example 1 (Change in the number of cells in the ascites)
First, sample A (low molecular weight water-soluble β-glucan of the present invention) obtained in Example 1 was dissolved in 100 μl of 10 mM phosphate buffer (pH 6.9), and ICR mouse 6 in an amount of 0.32 to 20 mg. It was administered to the abdominal cavity of a week-old female, and after 6 hours, ascites 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, which was 2-3 times that of untreated mice. When the ratio of the number of neutrophils to the total number of cells increased was counted, it increased according to the dose, and increased approximately 10 times by 0.32 mg administration, 20 times by 1 mg administration, and 30 times by 5 mg administration. The percentage of neutrophil count in the increased cells exceeded 70% of the increased cells at 1 mg administration. Purified lentinan derived from Shiitake extract, an immunopotentiator used clinically, was administered at 1 mg, and the number of cells was 3 times, the number of neutrophils was 30 times, and the rate was 80%. In the case of administration of bacteria-derived OK-432, the cell number was 5-fold, neutrophil was 60-fold, and the ratio was 90% when 0.2 mg was administered.
[0034]
Next, the sample A was administered, and qualitative changes in cells over time were examined. 10 mg of the sample A was administered to the mouse ascites, and cells were collected after 6, 12, 24, 48 and 72 hours, and the number of neutrophils, macrophages and lymphocytes as immunocompetent cells was counted. As a result, the total number of cells reached a maximum at 24 hours, which was 10 times that of untreated cells. Neutrophils reached a maximum after 24 hours, then decreased rapidly after 48 hours, and were almost normal after 72 hours. Macrophages increased after 12 hours and showed a maximum at 48 hours. The lymphocyte count also showed a maximum after 48 hours.
With lentinan, the total cell number reached a maximum after 12 hours and then decreased. The neutrophil count reached its maximum after 6 hours and then decreased. The number of neutrophils did not decrease in the sample A until 24 hours, and was more persistent than lentinan. Changes in macrophages and lymphocytes in lentinan-administered mice were almost the same as in sample A. The number of cells was counted by applying peritoneal cells on a slide glass, nuclear staining, and classifying neutrophils, macrophages and lymphocytes according to morphological changes.
Further, by microscopic observation of the cells, morphological characteristics considered to be a result of the active phagocytosis of macrophages and a dramatic decrease in neutrophils were observed in the cell observation 48 hours after administration of the sample A. Such a change was not observed in lentinan, OK-432 and others, but was observed specifically by administration of the sample A.
[0035]
Test Example 2 (Fluctuation in the amount of TNF produced from intraperitoneal cells)
Using the ascites cells obtained from the mice administered with sample A obtained in Example 1, the ability to produce TNF-α was evaluated. First, the sample A was dissolved in 100 μl of 10 mM phosphate buffer (pH 6.9), and administered to the ICR mouse 6-week-old female as 0 to 10 mg ascites. Three days later, ascites cells were collected, washed with a serum-free culture solution, and added to each well with the same culture solution to 5 × 10 5 cells. Furthermore, lactic acid bacteria were added to each well so as to be 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 EN-2601-90 manufactured by Endogen). As a result, the concentration of TNF-α produced in a concentration-dependent manner was 51 pg / ml after administration of 0.3 mg of Sample A, 128 pg / ml after 1 mg administration, and 141 pg / ml after 3 mg administration. In the case of no addition, it was 25 pg / ml, and the production amount was specific to the sample A.
[0036]
Moreover, the time-dependent change of TNF- (alpha) production ability was evaluated using the ascites cells obtained from the mouse | mouth which administered the said sample A. FIG. 3 mg of Sample A was dissolved in 100 μl of 10 mM phosphate buffer (pH 6.9) and administered ascites to 6-week-old female ICR mice. After 0 to 7 days, ascites cells were collected, washed with a serum-free culture solution, and added to each well with the same culture solution so as to have 5 × 10 5 cells. Furthermore, lactic acid bacteria were added to each well so as to be 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 EN-2601-90 manufactured by Endogen). As a result, 18 pg / ml immediately after administration of Sample A, 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 the amount of TNF-α produced is The production capacity was maintained for 7 days.
[0037]
Test example 3
Sample A obtained in Example 1 and 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. 500 μg of the sample A or B was added to 1 ml of PBS and dissolved by heating. Sample A was instantly dissolved by heating to 50 ° C. Sample B was not dissolved by heating to 50 ° C., but was allowed to stand at 90 ° C. for 30 minutes for dissolution. The antigen solution and the sample solution were mixed in an equivalent amount, and 100 μl each was administered to the mouse ascites. The mice were Balbc mice, 4 weeks old, female, and each sample consisted of 5 mice. Two weeks later, booster immunization was performed in the same manner as the first time. A group in which 0.5 mg of bovine plasma gamma globulin was dissolved in 1 ml of PBS to give 0.5 mg / ml and 100 μl was administered to mice was used as a control. Blood was collected 2 weeks after the booster immunization, and the difference in total antibody production (IgG, IgM, IgA antibody amounts) was measured by ELISA. In the ELISA, first, bovine / plasma gamma globulin used for immunization was plate-coated at a concentration of 10 μg / ml on a 96 microplate (manufactured by NUNK, maxi soap), and a serum dilution was applied at 37 ° C. for 1 hour. After the reaction, an anti-mouse immunoglobulin antibody labeled with peroxidase was further reacted as a secondary antibody at 37 ° C. for 1 hour. Next, using an orthophenylenediamine solution (manufactured by Wako Pure Chemical Industries, Ltd.) as a color former, the mixture was allowed to stand at 37 ° C. for 10 minutes, the reaction was stopped with 2M sulfuric acid, and the absorbance at 490 nm was measured.
[0038]
Next, the blood collected from the mouse was centrifuged to obtain serum, and this was obtained in a PBS-Tween solution (Tween 20 which is a sorbitan monoester) containing 0.2% BSA (bovine serum albumin) x10 to x5120 times. Was dissolved in PBS so as to be 0.05%), and the antibody titer was measured. As a result, the anti-bovine / plasma gamma globulin antibody titer of the serum diluted 2560 times was determined as O.D. D. When compared as an average value, the sample A administration group (n = 5) is 0.703 ± 0.1, the sample B administration group (n = 5) is 0.491 ± 0.075, and the control group (n = 5). ) Was 0.315 ± 0.090.
From this test, the antibody titer in the sample A administration group was higher than that in the sample B administration group, and the antibody production enhancement effect of the sample A was observed.
[0039]
【The invention's effect】
The low-molecular-weight water-soluble β-glucan of the present invention promotes the production of cytokines including TNF in the body, enhances its action, and enhances the antibody-producing ability or the overall immune action, thereby causing various infectious diseases and the like. It helps to prevent tumor development.

Claims (4)

A pharmaceutical material for immunity enhancing action, which contains, as an active ingredient, water-soluble β-glucan derived from barley flour having a weight average molecular weight reduced to 5000 to 100,000 .   The pharmaceutical material according to claim 1, wherein the content ratio of β-glucan having a molecular weight of less than 100,000 is 80% by weight or more of the total β-glucan. The pharmaceutical material according to claim 1 or 2 , comprising 0.1 to 90% by weight of water-soluble β-glucan. Furthermore, the pharmaceutical raw material of any one of Claims 1-3 containing a lactic acid bacterium or a lactic-acid-bacteria cell component.