WO2009110486A1

WO2009110486A1 - Inducer for inducing a th1 cell-dominant condition from a th2 cell-dominant condition

Info

Publication number: WO2009110486A1
Application number: PCT/JP2009/054011
Authority: WO
Inventors: 宏彰難波; 有紀増田
Original assignee: 株式会社雪国まいたけ
Priority date: 2008-03-04
Filing date: 2009-03-04
Publication date: 2009-09-11
Also published as: JP2009209092A

Abstract

Disclosed is an inducer that would enable the provision of products such as medical preparations, cosmetic products, foods, and beverages that can improve, treat, or prevent allergic disorders or other inflammatory diseases caused by a Th2 cell-dominant condition, by improving the balance between Th1 cells and Th2 cells and inducing a Th1 cell-dominant condition from a Th2 cell-dominant condition. The invention was completed based on knowledge obtained in studies regarding helper T-cells, which are immunocompetent cells, pertaining to the effects of Grifola frondosa extract and components derived from Grifola frondosa in improving the balance between Th1 cells and Th2 cells, and inducing a Th1 cell-dominant condition.

Description

Inducer from Th2 cell dominant state to Th1 cell dominant state

The present invention improves the disorder of the balance between type 1 helper T cells (hereinafter abbreviated as Th1 cells) and type 2 helper T cells (hereinafter abbreviated as Th2), and changes from a state predominantly Th2 cells to a state predominantly Th1 cells. The present invention relates to an induced maitake extract, a polysaccharide-protein complex derived from maitake, and a polysaccharide derived from maitake.

¡Allergic diseases are currently increasing year by year due to environmental changes. It was said that hay fever was rare in Japan before or even before the war, but recently cedar pollinosis alone has more than 10% of the population. The prevalence of allergic rhinitis including hay fever is also younger.

On the other hand, in the past, atopic dermatitis was a childhood illness, and it often improved and improved with growth. However, it is estimated that the incidence of adults is increasing recently. In this way, allergic diseases are definitely increasing in Japan, and it has been pointed out that in Europe and the United States, it has been increasing at a rate of 20-50% in 10 years, and this increase is a phenomenon not only in Japan but also in developed countries. It can be seen (Hideoki Ogawa et al .: Journal of the Japan Medical Association Vol. 133, No. 4, pp. 488-489 (2006) Non-Patent Document 1).

Th1 / Th2 cell balance is disrupted in patients with atopic dermatitis, whose incidence of onset and recurrence in adults is increasing due to factors such as changes in the living environment, dietary habits, and stress. IgE is produced by IL-4, which is a cytokine produced by this Th2 cell, and inflammation is enhanced. On the other hand, IFN-γ, which is a cytokine of Th1 cells, is said to suppress the activation of Th2 cells, decrease the production of IgE, and work anti-inflammatory.

The present inventor has a strong interest as a research material and has been conducting research for many years since Maitake has a rich food experience in Japan and is produced in a factory managed in Japan. Further, it has been found that the sugar-protein complex extracted from the fruit body and mycelium of maitake has an immunopotentiating action and an antitumor action (JP 9-238697 A, Patent Document 1) (JP 2007- No. 31665, Patent Document 2).

On the other hand, the inventor of the present invention has an immunosuppressive effect on extracts from maitake fruit bodies and mycelia (Japanese Patent Laid-Open No. 6-31934, Patent Document 3) and β-1,6 bonds derived from maitake. It has also been found that proteoglucan having α-1,4 branched chain as a chain is useful for treatment and prevention of non-insulin-dependent diabetes (Japanese Patent Laid-Open No. 10-182702, Patent Document 4).
JP-A-9-238697 JP 2007-31665 A Japanese Patent Laid-Open No. 6-312934 Japanese Patent Laid-Open No. 10-182702 Hideki Ogawa et al .: Journal of the Japan Medical Association Vol. 133, No. 4, 488-489 (2006)

The present invention improves the Th1 cell / Th2 cell balance disorder and induces a Th2 cell dominant state to a Th1 cell dominant state, thereby causing allergic diseases and other inflammations caused by the Th2 cell dominant state. The objective is to provide pharmaceuticals, cosmetics, food and drink, and other products that can improve, treat, or prevent sexually transmitted diseases.

While the present inventor is conducting research on helper T cells, which are immunocompetent cells, the balance of Th1 cells / Th2 cells is improved in the maitake extract and maitake-derived components, leading to a Th1 cell-dominated state. The present invention was completed upon finding out that it has a function.

That is, the present invention is (1) hot water extraction of the fruit body or mycelium of maitake, adding alcohol to the extracted water-soluble fraction, and collecting the substance floating or adhering to the liquid surface or in the liquid after standing An agent for improving the balance of Th1 cells / Th2 cells, which is characterized by using the maitake extract obtained as an active ingredient, from a Th2 cell dominant state to a Th1 cell dominant state,
(2) Improves the disruption of the Th1 / Th2 cell balance, characterized by using a polysaccharide-protein complex obtained by purifying the maitake extract of (1) by chromatography as an active ingredient. An inducer from a dominant state to a Th1 cell dominant state,
(3) In the polysaccharide-protein complex according to (2), the disturbance of Th1 cell / Th2 cell balance, characterized in that the ratio of sugar to protein is 65:35 to 90:10, and Th2 cells An inducer from a dominant state to a Th1 cell dominant state,
(4) The polysaccharide-protein complex according to (2) or (3), wherein the polysaccharide is a main component, the sugar portion is a β-1,6 bond as a main chain, and α and β-1,4 bonds are branched. Or an agent for improving the balance of Th1 cells / Th2 cells, which is characterized by being a glucan having any one of them as a branched chain, from a state predominantly Th2 cells to a state predominantly Th1 cells,
(5) Improves the disruption of Th1 cell / Th2 cell balance, characterized by comprising a polysaccharide obtained by treating the polysaccharide-protein complex described in (2) with a proteolytic enzyme, and Th2 cells An inducer from a dominant state to a Th1 cell dominant state,
(6) The polysaccharide according to (5), wherein the polysaccharide is a glucan having a β-1,6 bond as a main chain and α and β-1,4 bonds as a branched chain or one of them as a branched chain. Improve the balance of Th1 / Th2 cell balance, and induce from Th2 cell dominant state to Th1 cell dominant state,
(7) A pharmaceutical comprising the inducer according to any one of (1) to (6),
(8) An agent for preventing / ameliorating or treating an allergic disease, comprising the inducer according to any one of (1) to (6),
(9) The atopic dermatitis preventive / ameliorating or treating agent, wherein the allergic disease according to (8) is atopic dermatitis,
(10) A cosmetic comprising the inducer according to any one of (1) to (6),
(11) A food or drink comprising the inducer according to any one of (1) to (6),
(12) A pet food, livestock feed or livestock feed additive characterized by containing the inducer according to any one of (1) to (6).

In the present invention, the maitake may be any of maitake (Grifola frondosa), white maitake (Grifola albicans Imaz.), Choreimaitake (Dendropolyporus umbellatus), etc. Thus, the dried product can be used as it is, in the state of being appropriately cut or in powder form. In order to increase the extraction efficiency, it is preferable to use a dry powder. As a drying method, any of sun, warm air, hot air, infrared rays, freeze-drying and the like can be used.

As a hot water extraction method, it is performed at 50 to 135 ° C. for 15 minutes to 3 hours. In order to perform in a short time, the extraction is performed at a pressure of 100 ° C. or higher, for example, at about 120 ° C. under a pressure of 1 to 2 atm.

As the water, distilled water, purified water, ion exchange water, tap water, etc. can be used. For example, about 4 to 50 times the volume of dry maitake is used as a guide. When using raw maitake, use about 2 to 20 times the weight.

As the alcohol to be added to the obtained hot water extraction solution, methanol, ethanol, propanol or the like can be used, but it is preferable to use ethanol. The alcohol is added so that the final volume concentration of the alcohol is 10 to 80%, preferably 30 to 60%. After the addition, if left at a temperature of 1 to 45 ° C, preferably 1 to 25 ° C for 1 to 20 hours, substances appear floating on the liquid surface or in the liquid or adhering to the wall surface of the container. Gather, centrifuge, crush with a mesh, etc.

The collected substance has a dark black appearance, is positive for anthrone reaction and Raleigh reaction, and contains sugar and protein. Although it can be used as it is, it can be further purified by chromatography. Chromatography includes commonly used ones such as ion exchange chromatography and gel filtration, and also includes high performance liquid chromatography (HPLC) using an open column, high performance pump, UV monitor, etc. in terms of equipment. Is done. As the exchanger, those commonly used can be used, but agarose and cellulose are particularly preferable. The final product is light yellow.

The polysaccharide-protein complex in the present invention means a substance consisting of a polysaccharide and a protein or peptide, and includes any binding mode or structure of both components. .

The physical properties and properties of the polysaccharide-protein complex obtained as described above are as follows.

Appearance: Light yellow liquid color reaction: Anthrone and Raleigh reaction positive sugar to protein ratio: 65:35 to 90:10
Average molecular weight: 200,000 to 550000
When the polysaccharide-protein complex was treated with a proteolytic enzyme and the polysaccharide obtained by removing the protein or peptide was acid-degraded with hydrochloric acid, glucose was obtained, and it was confirmed that the polysaccharide was a glucan. Furthermore, as a result of tests such as methylation by the box protection method and enzymatic degradation by α-glucosidase and β-glucosidase, the glucan which is a polysaccharide has β-1,6 bonds as the main chain, and α and β-1, It was confirmed to be a glucan having 4 bonds as a branched chain or any one of them as a branched chain.

The disease-ameliorating effect of the polysaccharide-protein complex obtained as described above was examined using an atopic dermatitis model mouse.

Sensitization and induction were performed by applying Picryl chloride to the back of female NC / Nga mice, and the onset of atopic dermatitis was confirmed by dermatitis findings and increased blood IgE levels. Next, three days before the sensitization, the polysaccharide-protein complex of the present invention was applied every day, and the dermatitis findings were examined 45 days after the induction. Decreased by significant difference. On the other hand, in the spleen, there was a decrease in the expression level of IL-4 mRNA, a Th2 cytokine, and an increase in the expression level of IFN-γ, a Th1 cytokine, in the inguinal lymph nodes. However, no change was observed in the ratio of CD4 ⁺ CD25 ⁺ Regulatory T cells having immunosuppressive function in the spleen.

As a result of the above tests, the maitake-derived polysaccharide-protein complex of the present invention significantly reduces the amount of IgE production in the blood by significantly inducing the response of Th1 cells, which is caused by allergic diseases and other Th2 cell-dominated conditions. It was found to be effective in the prevention, amelioration or treatment of diseases.

Specific examples of allergic diseases include atopic dermatitis, allergic rhinitis, hay fever, urticaria, contact dermatitis, allergic bronchial asthma, food allergy, drug allergy, allergic conjunctivitis, hypersensitivity pneumonia Anaphylaxis and the like, but are not limited thereto.

Improves the disruption of Th1 / Th2 cell balance using the maitake extract of the present invention, the maitake-derived polysaccharide-protein complex, and the maitake-derived polysaccharide as active ingredients. Can be administered to humans or animals in various dosage forms containing carriers, excipients, and other additives used in pharmaceutical preparations.

The object of the present invention can be administered orally or parenterally. For oral administration, it can be applied to tablets, granules, powders, pills, capsules, liquids, syrups, and inhaled aerosols. For parenteral administration, external preparations, suppositories, injections and the like can be applied. Inhaled aerosol is used for atopic asthma, and as an external preparation for atopic dermatitis.

The object of the present invention is soluble in water, the liquidity of the aqueous solution is also neutral to weakly acidic, mild according to the pH of the skin, and as an external preparation or cosmetic, for example, an ointment, cream, emulsion, liquid It can be applied as an aerosol agent, and also as a soap, shampoo, body shampoo, bathing agent. In addition to pharmaceuticals, quasi-drugs and cosmetics include skin care and hair care products. Allergic diseases caused by Th2 cell predominance, such as atopic dermatitis and allergic rhinitis, often follow a chronic course, and external preparations and cosmetics are useful for the prevention, improvement, and treatment of these diseases. It is a form.

The object of the present invention can be used as a food or drink or mixed with a food or drink. The foods and drinks in the present invention are not limited to beverages such as milk and drinks, or foods that are eaten daily, but also insurance functional foods including so-called health foods, nutritional supplements, nutritional functional foods and specific functional foods Furthermore, all foods included in the category of food and drink such as foods for special needs including foods for the sick and foods for the elderly are included.

The object of the present invention can be appropriately used as pet food, feed or in addition to allergic diseases in pets and livestock, and other diseases caused by Th2 cell predominance.

The maitake extract, the maitake-derived polysaccharide-protein complex and the maitake-derived polysaccharide of the present invention can improve the balance of Th1 cells / Th2 cells and induce a Th1 cell dominant state to a Th1 cell dominant state. Can prevent, ameliorate, or treat allergic diseases and other diseases caused by Th2 cell-dominated conditions, and is highly safe.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-053831 which is the basis of the priority of the present application.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the examples.

(1) Extraction method 1 L of purified water was added to 150 g of dried powder of maitake fruit bodies, and hot water extraction was performed at 121 ° C. for 30 minutes under pressure in an autoclave to obtain 850 ml of an extract. Ethanol is added to the extract so that the final volume concentration is 30 to 60%, and the mixture is allowed to stand at around 4 ° C for 12 hours, and the substance floating on the liquid surface or in the liquid or adhering to the container is centrifuged. A dark black liquid substance was collected.

(2) Purification method After the collected extract was dissolved in purified water, it was applied to a DEAE-SepharoseCL-6B (3 × 60 cm) column and eluted with 0.0125 M Tris-HCl buffer. Two peaks appeared on the chromatogram. The peak having a short retention time from the time of sample injection was designated as peak 1, and the peak having a long retention time was designated as peak 2. Each peak portion was collected.

(3) Physical property test 1) After washing the peak 1 and peak 2 as purified products obtained in (2) above with 8M urea solution, a single peak was shown by schlieren analysis. Was identified.

2) The purified product obtained in (2) above was treated with a trypsin (pig pancreas-derived) solution (pH 6.8) at 37 ° C. for 2 hours, and then further purified with a protease ( Aspergillus oryzae- derived) solution (pH 8.2). Time processed. The polysaccharide fraction excluding the protein obtained by the treatment as described above was treated with 3M HCl at 100 ° C. for 60 minutes. As a result, it was confirmed that only glucose as a sugar was detected as a free substance and the polysaccharide was glucan.

3) As a result of analyzing the glucan and protein obtained as a result of the above 2) by anthrone and Raleigh methods, respectively, it was confirmed that the ratio of sugar to protein was as follows.

Peak 1 Sugar: Protein = 65: 35
Peak 2 sugar: protein = 90: 10
In order to analyze the chemical structure of the glucan obtained in 2) above, methylation was performed by the box guard method and then GLC analysis was performed, and the results shown in Table 1 were obtained from the peak areas.

2,3-di-methyl glucose with 1,4- and 6-position carbon bonds has 2 moles, and 2,3,4-tri-methyl glucose with bonds at 1,6-position has 2 moles and bonds with 1-position 1 mol of 2,3,4,6-tetra-methyl glucose was obtained.

Next, as a result of enzymatic degradation of the glucan obtained in 2) above with α-, β-glucosidase, the release of glucose was recognized and no significant decrease in molecular weight was observed with any enzyme treatment. Therefore, it was recognized that it was a branched chain that was cleaved by these enzymes.

Based on the above results, the polysaccharide part of the polysaccharide-protein complex is a glucan having β-1,6 bonds as the main chain and α and β-1,4 bonds as branched chains or one of them as branched chains. It turned out to be.

(4) Molecular weight As a result of examining the polysaccharide-protein complex and polysaccharide obtained as described above by gel filtration chromatography,
Average molecular weight of polysaccharide-protein complex: 200,000 to 550000
Average molecular weight of polysaccharide (glucan): 150,000 to 500,000
It was confirmed that.

Animal test The sensitization and induction were performed by applying picryl chloride to the back of female NC / Nga mice, and the onset of atopic dermatitis was confirmed by dermatitis findings and increased blood IgE levels.

Next, from 3 days before sensitization, physiological saline as a control, 4 mg / kg / day of each of the peak 1 and peak 2 substances obtained in Example 1 were applied to the back of the mouse every day, and 21 days and 33 days after induction, The dermatitis findings were examined on the 36th and 45th days. The results are shown in Table 2. As compared with the control, the substance of the present invention had a low score value and significantly suppressed the exacerbation of the symptoms, indicating that the onset of atopic dermatitis was small.

Table 3 shows the measurement results of the IgE level in blood on day 36 after induction of sensitization. Compared with the control, the substance of the present invention significantly reduces IgE production and suppresses the release of histamine from mast cells.

On the other hand, Table 4 shows the results of examining IL-4 mRNA expression level, which is a cytokine of Th2 cells, and IFN-γ mRNA expression level, which is a cytokine of Th1 cells, in the spleen on the 36th day after sensitization induction. In the application group of the substance of the present invention, the expression level of IL-4 is clearly lower than that of the control, and the expression level of IFN-γ is increased. It was shown that it led to.

All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims

Maitake extract obtained by hot water extraction of the fruit body or mycelium of maitake, adding alcohol to the extracted water-soluble fraction, and collecting the substance floating or adhering to the liquid surface or in the liquid after standing An agent for improving Th1 cell-dominated state to Th1 cell-dominated state by improving disturbance of Th1 cell / Th2 cell balance, characterized by using as an active ingredient.
3. Thy cell / Th2 cell balance disorder characterized by using a polysaccharide-protein complex obtained by purifying the maitake extract of claim 1 by chromatography as an active ingredient, and a predominant state of Th2 cells To induce Th1 cell predominance.
3. The polysaccharide-protein complex according to claim 2, wherein the ratio of sugar to protein is 65:35 to 90:10, and the disorder of Th1 cell / Th2 cell balance is improved, and Th2 cell predominates. To induce Th1 cell predominance.
4. The polysaccharide-protein complex according to claim 2 or 3, wherein the polysaccharide is a main component, the sugar portion is a β-1,6 bond as a main chain, and α and β-1,4 bonds are branched or any one thereof. An inducer from a Th2 cell-dominated state to a Th1 cell-dominated state by improving disturbance of the Th1 / Th2 cell balance, characterized by being a glucan having a branched chain.
3. Improves the balance of Th1 cell / Th2 cell balance, which is characterized by using a polysaccharide obtained by treating the polysaccharide-protein complex according to claim 2 with a proteolytic enzyme as an active ingredient. To induce Th1 cell predominance.
6. The polysaccharide according to claim 5, wherein the polysaccharide is a glucan having a β-1,6 bond as a main chain and α and β-1,4 bonds as a branched chain or one of them as a branched chain. An agent that improves the balance of cells / Th2 cells and leads from Th2 cells to Th1 cells.
A pharmaceutical comprising the inducer according to any one of claims 1 to 6.
An agent for preventing / ameliorating or treating allergic diseases, comprising the inducer according to any one of claims 1 to 6.
A preventive / ameliorating or treating agent for atopic dermatitis, wherein the allergic disease according to claim 8 is atopic dermatitis.
A cosmetic comprising the inducer according to any one of claims 1 to 6.
A food or drink comprising the inducer according to any one of claims 1 to 6.
A pet food, livestock feed or livestock feed additive comprising the inducer according to any one of claims 1 to 6.