JP5882635B2 - Maitake-derived polymeric α-glucan - Google Patents

Description

  The present invention relates to a novel Maitake-derived macromolecular α-glucan and the prevention and treatment of colds such as influenza caused thereby.

  As for the extract of Maitake, various actions are known by the development efforts of the present applicants. For example, Japanese Patent Application Laid-Open No. 7-69913 (Patent Document 1) for the effect of improving AIDS disease, Japanese Patent Application Laid-Open No. 9-238697 (Patent Document 2) for the anti-tumor action, and Japanese Patent Application Laid-Open No. 2000- No. 119650 (Patent Document 3), NO production inducing action is disclosed in JP-A No. 2001-172194 (Patent Document 4), and substances having anti-influenza virus activity are disclosed in JP-A No. 2005-145934 (Patent Document 5). ) And Japanese Patent Application Laid-Open No. 2008-106018 (Patent Document 6).

  There are effective preventive measures such as vaccines for preventing influenza. However, since the antigenicity of the influenza virus is likely to change, it is said that the initial effect may not always be expected due to fluctuations in the effect. On the other hand, in addition to rapid diagnostic methods, therapeutic drugs have also made significant progress, and oseltamivir phosphate, zanamivir hydrate, etc. exert excellent effects on influenza. However, the emergence of resistant viruses and side effects due to their use are reported, and these treatments are not without problems.

  Once an outbreak occurs, influenza, which has a rapidly expanding tendency on a global scale, can be used to prevent or treat influenza from available natural products or its extracts without being satisfied with existing prevention and treatment methods. The search for effective substances is considered to be very significant, and research and search for effective substances against influenza contained in maitake cultivated in large quantities has been continued.

  Prior literature relating to Maitake influenza includes Patent Document 5, Patent Document 6, and Japanese Patent Application Laid-Open No. 2008-273638 (Patent Document 7) relating to the applicant's application. Patent Document 5 relates to an extract fraction mainly composed of maitake β-glucan, and Patent Document 6 relates to a glycoprotein or glycoprotein complex-containing fraction of maitake having a molecular weight of 30,000 to 100,000. Patent Document 7 relates to an adjuvant for an inactivated antigen derived from a virus or pathogenic fungus mainly composed of a hot water extract of a mycelium of a mushroom. In addition, maitake mushrooms and influenza viruses are exemplified as viruses, but it is unclear what the hot water extract of maitake is, and this extract is administered together with a vaccine that is an inactivated antigen of the virus. It induces the production of secretory IgA antibodies specific for viruses, and cannot be used alone for treatment or prevention.

  On the other hand, for α-glucan contained in maitake, hot-water extract of maitake fruit bodies, α-D-glucan and β-D-glucan are separated by gel filtration and affinity chromatography using Con A-Sepharose, The obtained α-D-glucan has a molecular weight of 1,000,000, and as a result of methylation analysis, it was α- (1 → 4) -D-glucan having α- (1 → 6) branched chain. No antitumor activity was observed for Shizuoka University Faculty of Agriculture No. 35 49-61p (1985) (Non-Patent Document 1).

  Furthermore, the fruit body of maitake was extracted with water and hot water, and fractionated purification was performed with DEAE-cellulose column and Sepharose CL-4B column chromatography to obtain a polysaccharide presumed to be α-glucan. Agricultural Research Bulletin (54): 199-203p (1989) (Non-Patent Document 2). The weight average molecular weight of the polysaccharide is about 1.3 million, and the (1 → 4) bond is the main chain (1 → 6). It is reported in Starch Science Vol. 38, No. 3, 263-265p, (1991) (Non-patent Document 3) that it is an α-glucan branched by a bond.

  Furthermore, in Journal of Pharmacy and Pharmacology 59, 575-582p (2007) (Non-patent Document 4), an α-glucan having a molecular weight of about 400,000 to 450,000 obtained from a hot water extract of a maitake fruiting body has an antidiabetic effect. Although it is described, there is no description about the structure of α-glucan.

  None of the α-glucans described in Non-Patent Documents 1 to 4 have a low molecular weight, and no effect on influenza is described.

Japanese Patent Laid-Open No. 7-69913 JP-A-9-238697 JP 2000-119650 A JP 2001-172194 A JP 2005-145934 A JP 2008-106018 A JP 2008-273446 A

Shizuoka University Faculty of Agriculture research report 35 49-61p (1985) Gifu University Agricultural Research Bulletin (54): 199-203p (1989) Starch Science Vol.38 No.3 263-265p (1991) Journal of Pharmacy and Pharmacology 59, 575-582p (2007)

  It is an object of the present invention to find a new component from maitake, develop an extraction / purification method thereof, search for an effective action, and further use it for foods and drinks, pharmaceuticals, feed additives, and the like.

  As a result of repeated research on components in maitake other than β-glucan, which is widely known as an active ingredient in maitake, the present inventors have found that α-glucan of a new polymer, specifically (1 → 4) bond. Successful extraction of a polymer α-glucan having a polysaccharide structure branched as a main chain with a (1 → 6) bond, and the α-glucan of the polymer and a fraction containing the same are used to prevent colds such as influenza. The present invention was completed by finding it effective for treatment.

  Incidentally, when an excellent antitumor activity was discovered in β-glucan or sugar-protein complex (sugar portion is β-D-glucan) contained in maitake, its molecular weight was about 1 million or at most about 2 million. A focus has been on polymers.

However, the present invention relates to α-glucan having a structure different from that of β-glucan, and the molecular weight is distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, and the weight average molecular weight is also This is 8.5 × 10 ⁶ , which is completely different from the conventional research on maitake-containing substances. After performing a treatment to suppress or prevent the action of the enzyme such as freeze-drying maitake, a fraction extracted with hot water was obtained and purified. As a result, (1 → 4) bond of the polymer was used as the main chain (1 → 6) As a result of obtaining α-glucan having a polysaccharide structure branched by a bond and searching for the active action of the high-molecular α-glucan and its extracted fraction, it is effective for the prevention and treatment of colds such as influenza. Knowing and solving the problems.

That is, the present invention provides (1) α-glucan derived from maitake and having a molecular weight distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, or an extract containing the α-glucan,
(2) α-glucan derived from maitake and having a molecular weight distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method and having a weight average molecular weight of about 8.5 × 10 ⁶ Or its extracted fraction,
(3) A polysaccharide derived from maitake and having a molecular weight distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, and having a (1 → 4) bond as a main chain and branched by a (1 → 6) bond Α-glucan or an extract containing the same, characterized by having a structure;
(4) The molecular weight derived from maitake is distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, the weight average molecular weight is approximately 8.5 × 10 ⁶ , and (1 → 4) bond Α-glucan having a polysaccharide structure branched as a main chain by (1 → 6) bond, or an extract containing the α-glucan,
(5) The α-glucan according to any one of (1) to (4) above, which is effective for the prevention and treatment of colds such as influenza, or an extract containing the α-glucan,
(6) The fruit body or mycelium of maitake is extracted with hot water after treatment or means for inhibiting or preventing the action of an enzyme that affects α-glucan contained therein. The method for producing α-glucan according to any one of the above (1) to (4) or an extract containing the same,
(7) The fruit body or mycelium of maitake is obtained by extracting with hot water after performing treatment / means to inhibit or block the action of the enzyme that affects α-glucan contained therein. The obtained fraction is then purified using any one of a fractional precipitation method, a chromatographic method, a gel filtration method, an ultrafiltration method, etc., or an appropriate combination of these methods (1) ) To (4) according to any one of the methods for producing α-glucan or an extract containing the same,
(8) Treatment / means for inhibiting or preventing the action of an enzyme that affects α-glucan are frozen storage, freeze-drying, vacuum (decompression) storage, vacuum (decompression) drying, freeze-vacuum (decompression) drying, and The method for producing α-glucan or an extract containing the α-glucan according to the above (6) or (7), which is one of heat drying or a combination thereof,
(9) The above-mentioned (1) to (1), wherein fruit bodies or mycelia of maitake are immediately extracted with hot water so that an enzyme that affects α-glucan contained therein does not work. 4) A method for producing α-glucan or an extract containing the α-glucan according to any one of
(10) A fraction obtained by immediately extracting a fruit body or mycelium of maitake with hot water so that an enzyme that affects α-glucan contained therein does not work, and then fractional precipitation Any one of the above methods (1) to (4), wherein any one of the method, chromatographic method, gel filtration method, ultrafiltration method, etc., or a combination of these methods is used as appropriate Of α-glucan or an extract fraction containing the same,
(11) In the fractional precipitation method, alcohol is added to an aqueous solution of an extract fraction obtained by extracting maitake fruit bodies or mycelium with hot water or an aqueous solution of the fraction, to 20 to 30% by volume. (7) or (7), wherein the precipitate formed by removing the resulting precipitate and then adding alcohol to the supernatant to 40 to 60% by volume is collected. (10) The production method of α-glucan or an extract containing the α-glucan according to any one of
(12) The fruit body or mycelium of maitake is freeze-dried and then extracted with hot water, and alcohol is added to the extracted fraction or an aqueous solution of the fraction so as to be 20 to 30% by volume of the whole. (1) to (1) above, wherein the precipitate formed is removed by adding the alcohol to the supernatant to 40 to 60% by volume of the total and then collecting the deposited precipitate. 4) A method for producing α-glucan or an extract containing the α-glucan according to any one of
(13) A food or drink comprising the α-glucan according to any one of (1) to (4) above or an extract containing the α-glucan,
(14) A pharmaceutical product for humans or animals comprising the α-glucan according to any one of (1) to (4) above or an extract containing the α-glucan,
(15) The present invention relates to a feed or a feed additive comprising the α-glucan according to any one of (1) to (4) or an extract containing the α-glucan.

In the present invention, a maitake is a narrowly defined genus of mushrooms ( Grifola frondosa ), white maitake ( Grifola albicans ), choreimaitake ( Grifola umbellata ), and mushrooms ( Grifola gigantea ). It means that both can be used. In addition, fruit bodies and mycelia of these maitake mushrooms can be used. Recently, fruit bodies of maitake ( Grifola frondosa ) and white maitake ( Grifola albicans ) can be artificially cultivated. From the viewpoint of securing stable raw materials, it is preferable to use the two fruit bodies.

`` Maitake fruit bodies and mycelium '' in the present invention can be used to mean any raw, dry product, or dry powder, but they contain enzymes that affect α-glucan, In order to collect the polymer α-glucan in the present invention as efficiently as possible
(1) After performing treatment / means to inhibit or block the action of an enzyme that affects α-glucan contained in maitake, it is extracted with hot water,
(2) In order to prevent the degradation of α-glucan before the action of the enzyme that affects α-glucan contained in maitake, it is cut or crushed immediately after collection, and then extracted with hot water so that the enzyme does not act It is important to use raw maitake that has been harvested as much as possible and has not passed.

  Enzymes that affect α-glucan include enzymes that break down α-glucan into glucose, enzymes that form branches, enzymes that cause transfer, enzymes that generate disaccharides (trehalose) or oligosaccharides, etc. As can be seen, an enzyme having a large effect is an enzyme that degrades α-glucan into glucose. The present inventors have confirmed that the peak area was reduced to 56% by HPLC using a gel filtration column when the harvested raw maitake was allowed to stand at room temperature for 24 hours. Moreover, when water was added to the freeze-dried powder and allowed to stand at 36 ° C. for 30 hours, it was confirmed that the peak area was similarly reduced to 30%.

  The treatment for inhibiting or blocking an enzyme that affects α-glucan is one of frozen storage, freeze-drying, vacuum (reduced pressure) storage, vacuum (reduced pressure) drying, freeze-vacuum (reduced pressure) drying, and heat drying, or Although the combination can be carried out, freeze drying, vacuum (reduced pressure) drying, and heat drying are preferable in order to facilitate the extraction process. Furthermore, freeze-drying (reduced pressure) drying is particularly preferable from the viewpoint of reliably preventing the action of the enzyme and reducing the processing time.

In any case of cryopreservation, freeze-drying, vacuum (reduced pressure) storage, vacuum (reduced pressure) drying, freeze-vacuum (reduced pressure) drying, generally commercially available or used equipment can be used.
Although it does not specifically limit about freezing temperature, Preferably it is -20 degrees C or less, and -30 degreeC--40 degreeC is especially preferable. Further, the degree of decompression may be appropriately selected in consideration of the performance of the device.

For heat drying, the heating temperature is preferably 60 ° C. or more, particularly around 80 ° C., but taking into consideration the state of raw materials, heating time, etc., for example, under the temperature conditions of roasting (approximately 150 ° C. to 240 ° C.) Heating is also possible. In addition, as a method for heating and drying, for example, a method using a rapid hot air dryer, a rotary dryer, a microwave (microwave oven), a roaster or the like can be used.
Prior to drying and extraction, it is preferable to pulverize or powder. Specifically, particles of about 25 to 60 mesh pass are desirable to improve extraction efficiency, but an extract can be obtained with other particle sizes. Is possible.

  Prior to extraction, removal of components and substances that interfere with extraction can be appropriately performed depending on the condition of the extraction object. For example, for the purpose of degreasing, treatment with ethanol, benzene, petroleum ether, acetone, ether or the like can be performed, but it is not always essential.

  The extraction method is performed with hot water. Enzymes that affect α-glucan in maitake work from 55 ° C. to 60 ° C., but the function declines at temperatures exceeding 60 ° C. Therefore, the hot water in the present invention specifically exceeds 60 ° C. Think of it as temperature water. In carrying out, preferably, hot water of 80 ° C. or higher is mixed, and the mixture can be appropriately heated under normal pressure or under pressure according to a conventional method. For example, extraction is performed at 80 to 125 ° C. for 5 minutes to several hours. In order to perform efficiently in a short time, it is preferable to perform extraction at about 100 ° C. or higher, for example, at 120 to 125 ° C. under pressure using a pressure cooker for about 30 minutes to 1 hour.

“Water” means tap water, deionized water (ion exchange water), distilled water, reverse osmosis water, and the like.
The obtained extract may be transferred to the purification step as it is, cooled to room temperature, concentrated and used as an extract, or dried and used as an extract powder. It is easy to use a dry extract powder, and any means such as spray drying and freeze drying can be used. However, by drying, it becomes a raw material for α-glucan purification and can be stored for a long time by preventing moisture absorption.

  The obtained extracted fraction can be purified by using a fractional precipitation method, a chromatographic method, a gel filtration method, an ultrafiltration method, or the like, or an appropriate combination of these methods.

  As methods for fractional precipitation of polysaccharides, methods using an organic solvent such as alcohol and acetone are known, and these methods are possible. In particular, it is preferable to use alcohol. Examples of alcohol include methanol, ethanol, and propanol. A lower alcohol such as butanol can be used.

  In chromatographic methods, for example, ion exchange chromatography or affinity chromatography using DEAE-cellulose or DEAE-Sephadex columns, and in gel filtration methods, gel filtration agents such as Sephadex and acrylamide are limited. As an external filtration method, for example, a commercially available apparatus using an ultrafiltration membrane such as cellulose or cellulose acetate can be used.

  Although the separation / purification method for obtaining the α-glucan of the present invention or the extract containing the same has been exemplified, a simple and economical method has been developed by utilizing the alcohol fractionation method. To do.

First, 1) Addition of the same amount (50%) or more of alcohol to the extracted fraction obtained by extraction with hot water or the dried product aqueous solution, the crude α-glucan is precipitated and centrifuged. Can be collected by separation. Low molecular weight substances can be transferred to the supernatant and removed.
2) Next, dissolve the resulting precipitate completely in water, and add 20-30 volumes of alcohol to this aqueous solution so that the target precipitate begins to precipitate and other fractions do not settle. %, Preferably 24 to 26%, and the precipitates / precipitates produced by standing are removed. The precipitate / precipitate can be removed by a method such as centrifugation.
3) Since the desired α-glucan is contained in the obtained supernatant, alcohol is added to the whole so that the target precipitate begins to precipitate and other fractions do not precipitate. The precipitate is collected by allowing it to stand to be 40 to 60% by volume, preferably 40 to 45%, and depositing.

This precipitate was α-glucan, the molecular weight was distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, and the weight average molecular weight was 8.5 × 10 ⁶ , which was known as a maitake component. A polysaccharide structure that is a huge polymer body that has nothing to do, and that has a (1 → 4) bond as the main chain and a (1 → 6) bond branched by specific rotation, box protection method and proton NMR measurement. It was confirmed that it was an α-glucan having a high purity.
A partial diagram of the structural formula of α-glucan is shown below.

  Of the steps 1) to 3), steps 2) and 3) are important and essential. In some cases, step 1) can be omitted, but preferably from 1) to 2) and 3). By proceeding, a desired high-purity α-glucan can be obtained.

Furthermore, for the α-glucan or high-purity fraction from which impurities have been removed, the precipitate is dispersed in about 50% ethanol, and the α-glucan is recovered again by centrifugation, and this operation is repeated until the target purity is reached. It can be repeated or obtained by conventional means such as chromatographic method, gel filtration method, ultrafiltration method and the like.
The α-glucan thus obtained can be dried and powdered as desired.

  As a control, the maitake-derived α-glucan of the present invention and an extract containing the same were infected with BALB / c mice, pupa, and 6 weeks old with influenza A virus (A / NWS / 33, H1N1 subtype). When the therapeutic effect was evaluated, it was found that the α-glucan and the extract fraction containing the α-glucan were effective in preventing and treating influenza.

  Such effectiveness can be applied to colds similar to influenza caused by, for example, rhinovirus, coronavirus, RS virus, herpangina virus and the like.

  From the above results, the maitake-derived α-glucan and the extract containing the same are effective in the prevention and treatment of the common cold such as influenza, and the food and drink for preventing infection such as viruses or reducing symptoms after illness, It can be used in combination with human or veterinary drugs and feed or feed additives.

  The food and drink in the present invention refers to beverages such as milk and drink and various processed foods that are eaten daily, nutritional supplements, health supplements, so-called health foods called supplements, nutritional functional foods and foods for specified health All special-purpose foods including functional foods and foods for elderly people are included.

  When used as health foods, supplements, functional foods, human and veterinary drugs, excipients, bulking agents are added as appropriate, and tablets, capsules, granules, powders, pills, glazes, troches, liquids for internal use It can be processed into various preparations such as suspensions for internal use, nasal drops, poultices and injections.

  What is necessary is just to mix | blend suitably with various feeds as it is, when using as a feed or a feed additive. It can also be dissolved in drinking water.

  High molecular weight α-glucan and extract fractions containing it are components in maitake that are widely used as food ingredients. They are polysaccharides with a structure similar to starch and glycogen. It can be used as a food for preventing cold or reducing symptoms after illness, a pharmaceutical for humans or animals, and a feed or feed additive.

The figure which shows the measurement molecular weight Mw distribution (plot) and retention time by the SEC-MALS method of the refinement | purification containing alpha-glucan obtained in Example 3. FIG. The figure which shows that the α-glucan contained at the beginning decreased when raw maitake was allowed to stand at room temperature for 24 hours after harvesting. The figure which shows that the alpha-glucan contained initially decreased, when freeze-dried maitake powder was suspended in water and left still at 36 ° C. for 30 hours. The figure which shows the change of the body weight of each group 3 days after the influenza infection of the alpha-glucan containing extraction fraction obtained in Example 2, and the alpha-glucan containing refinement | purification group administration group obtained in Example 3, and a control group mouse | mouth. . Lung and bronchoalveolar lavage fluid (BALF) 3 days after influenza infection of the α-glucan-containing extract fraction obtained in Example 2 and the α-glucan-containing purified fraction administration group obtained in Example 3 and the control group mice Of the amount of virus.

Hereinafter, examples will be shown in order to specifically explain the present invention, but it goes without saying that the present invention is not limited thereto.

[Example 1]
The stems and umbrellas of fresh maitake ( Grifola frondosa ) produced by artificial cultivation were frozen at −30 ° C. to −40 ° C. and dried using a vacuum freeze dryer. The inside of the dryer was decompressed to 20 Pa and dried in about one day. Next, the dried powder was pulverized with a mill, and a fine powder passing 25 mesh was obtained.

[Example 2]
10 kg of the freeze-dried maitake powder obtained in Example 1 was added to 210 l of ion-exchanged water that had been heated to 80 ° C. in advance, and hot water extraction was performed at 121 ° C. for 30 minutes. The extract was filtered using a filter press, the obtained extract solution was concentrated, and a dry powder (α-glucan-containing extract fraction) was obtained by spray drying.

[Example 3]
1 l of water was added to 100 g of dry powder of the maitake hot water extract obtained in Example 2 and dissolved completely, and ethanol was added with stirring to a final concentration of 50% by volume. After stirring for about 1 hour, the resulting suspended matter (50% ethanol precipitate) was obtained by centrifugation (5,000 rpm / 10 min). The obtained 50% ethanol precipitate was completely dissolved by adding 500 ml of water, ethanol was added with stirring to a final concentration of 25% by volume, and the resulting suspension (25% ethanol precipitate) was centrifuged ( 7,000 rpm / 30 min), and the supernatant was obtained.

  Ethanol was added to the 25% ethanol supernatant with stirring so that the final concentration was 42% by volume, and the resulting suspension (42% ethanol precipitate) was obtained by centrifugation (5,000 rpm / 5 min). 42% ethanol precipitate was dissolved in 100 ml of water, and the solution was frozen in a freezer at about -30 ° C., dried using a vacuum freeze-drying apparatus, pulverized and powdered (α-glucan-containing purified fraction) 30 g Got.

The physical properties, molecular weight and structural analysis of α-glucan obtained as described above are described below.
Appearance: Hydrolysis with colorless crystalline powder acid: only D-glucose was given.
Specific rotation: [α] _D ²³ = 165.8 ± 12.6 (c = 1.0, H ₂ O); n = 10
Molecular weight: Measured by SEC-MALS method (Size Exclusion Chromatography-Multi Angle Light Scattering :).

<Analysis conditions>
Sample preparation: α-glucan: dissolved at a concentration of 0.492 mg / ml, filtered through a 0.45 μm filter, then injected detector: multi-angle optical confusion detector (Wyatt Technology DAWN HERIOS II)
Column: Size exclusion chromatography; SEC (Shodex SB-806M HQ)
Eluent: 0.1M sodium nitrate aqueous solution Flow rate: 1.0ml / min
Injection volume: 100 μl
Column temperature: 40 ° C

<Measurement result>
The α-glucan sample was measured by multi-angle light scattering detection method, and the weight molecular weight was roughly distributed from 4 × 10 ⁶ to 7 × 10 ⁷ and the weight average molecular weight (Mw) was measured as 8.51 × 10 ⁶ (see FIG. 1).

Structural analysis:
The structural analysis of α-glucan obtained in Example 3 will be specifically described below. 60% sodium hydride (3.75 mmol, 150 mg) was washed with distilled hexane and reacted with distilled dimethyl sulfoxide (42.2 mmol, 3 ml) at 50 ° C. under an argon atmosphere for 3 hours to prepare a sodium methylsulfinyl carbanion solution. The above α-glucan-containing powder (10.8 mg) is dissolved in distilled dimethyl sulfoxide (1 ml), and after adding sodium methylsulfinyl carbanion solution (0.5 ml), methylation reaction with methyl iodide (8.03 mmol, 0.5 ml) Went. After confirming disappearance of the starting material spot by TLC analysis, the reaction was terminated by quenching with distilled water. The reaction solution was diluted with dichloromethane and washed 3 times with distilled water. The extract was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and dried with a vacuum pump. To the obtained residue (14.9 mg), 88% formic acid (1 ml) was added and hydrolysis was carried out at 100 ° C. After 1 hour, formic acid was removed by concentrating the reaction solution under reduced pressure. Next, 0.5N sulfuric acid solution (1 ml) was added to the residue and hydrolysis was carried out at 100 ° C. After 1 hour, the reaction solution was neutralized with barium carbonate, and insolubles were filtered off with celite. Thereafter, the obtained filtrate was concentrated under reduced pressure. The obtained residue (21.7 mg) was dissolved in a 50% methanol solution (1.5 ml), the pH was adjusted to 10-11 with concentrated aqueous ammonia, and sodium borohydride (0.528 mmol, 20 mg) was added. After 3 hours, disappearance of the raw material was confirmed by TLC analysis, and quenched with acetic acid. The reaction solution was azeotroped with methanol to remove the borate ester. The obtained residue was dissolved in pyridine (1 ml), and acetic anhydride (5.28 mmol, 0.5 ml) was added and reacted at 100 ° C. under an argon atmosphere. One hour later, disappearance of the raw materials was confirmed by TLC analysis, and quenched with methanol. Pyridine was removed by concentration under reduced pressure, and the resulting residue was diluted with ethyl acetate and washed successively with 1N hydrochloric acid solution, saturated sodium hydrogen carbonate solution, and saturated brine. The ethyl acetate phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and dried with a vacuum pump.

The obtained partial methylalditol acetate mixture (12.8 mg) was used for analysis by ¹ H-NMR (D ₂ O, HOD = 4.81 ppm). As a result of analyzing partial alditol acetate, 2,3-di-O-methyl isomer [Chemical Formula 2]: 2,3,6-tri-O-methyl isomer [Chemical Formula 3]: 2,3,4,6-tetra It was -O-methyl form [Chemical Formula 4]. Based on this finding, it was analyzed as a polysaccharide structure with a (1 → 4) bond as the main chain and branched by a (1 → 6) bond.

[Reference Example 1]
Immediately after harvesting the raw maitake from the medium, the fruit body was finely crushed with a multi blender, 50 g was weighed, 55 ml of distilled water was added, and hot water extraction was performed at 121 ° C. for 30 minutes. In addition, after harvesting raw maitake, the fruit bodies were allowed to stand at room temperature for 24 hours, and then hot water extraction was performed in the same manner. These extracts were subjected to HPLC analysis using a gel filtration column, and the areas of peaks identified as α-glucan by nuclear magnetic resonance and methylation analysis were compared. Compared to those extracted immediately (retention time: 8.369). Those that were allowed to stand at room temperature for 24 hours (retention time: 8.379) were reduced to 56% (FIG. 2).

[Reference Example 2]
100 ml of sterilized water was added to 5 g of freeze-dried maitake to prepare a suspension. The lid was covered with a silicon stopper, sufficiently suspended, and immediately extracted with hot water. Moreover, after suspending, after leaving still at 36 degreeC with an incubator for 30 hours, hot water extraction was performed at 121 degreeC for 30 minutes. These extracts were subjected to HPLC analysis using a gel filtration column, and the areas of peaks identified as α-glucan by nuclear magnetic resonance and methylation analysis were compared. As a result, they were extracted immediately after suspension (retention time: 8.462). ) And after standing for 30 hours at 36 ° C. (retention time: 7.962) decreased to 30% (FIG. 3).

  As can be seen from the above Reference Examples 1 and 2, the content of α-glucan is extremely reduced in the product extracted with hot water after standing for a long time as compared with the product extracted with hot water immediately.

[Test example] Influenza treatment effect evaluation method
(1) Virus (Influenza A virus: A / NWS / 33, H1N1 subtype, 2 ×
10 ⁴ PFU / 50 μl / mouse) are intranasally inoculated into mice (3 mice in each group).
(2) Samples were taken twice a day (9am, 6pm) from 7 days before infection to 3 days after infection.
Oral administration.
(3) The body weight of the mouse is measured twice on the day of infection (day 0) and 3 days after infection.
(4) Three days after infection, the lung and bronchoalveolar lavage fluid (BALF, using a plastic cannula, wash the airways with 0.8 ml of phosphate buffered saline (PBS) 5 times and collect the fluid) Collect.
(5) The bronchoalveolar lavage fluid is centrifuged (1,000 rpm, 5 minutes), and the supernatant is appropriately diluted and subjected to plaque assay to measure the amount of virus. In the case of lung, 1 μl of PBS per 1 mg is added, sonicated, centrifuged (5,000 rpm, 10 minutes), and the supernatant is appropriately diluted and used for plaque assay.
(Note) PFU: plaque forming unit, 1 PFU corresponds to one live virus.
Plaque method: Influenza virus quantification method. MDCK cells cultured in a monolayer on a cell culture dish are infected with an appropriately diluted sample, and then added to an agar medium and cultured for 2 days. Correspondingly, a spot-like cell dropout portion can be detected. This is called a plaque, and the number represents the amount of virus.

Experimental zone (n = 3)
# 1: Distilled water (control)
# 2: Oseltamivir phosphate (positive control) 0.2 mg / day / mouse
# 3: Extracted fraction containing α-glucan obtained in Example 2 0.5 mg / day / mouse
# 4: 〃 2 mg / day / mouse
# 5: 〃 5 mg / day / mouse
# 6: α-glucan-containing purified fraction obtained in Example 3 0.5 mg / day / mouse
# 7: 〃 2 mg / day / mouse
# 8: 〃 5 mg / day / mouse
(All solvents used sterile distilled water)

Result 1. Change in body weight (Figure 4)
Weight change is a very important indicator of the extent of influenza. In the present virus inoculation amount, in the experimental examples thus far, a change starts to appear 3 days after the infection, and the maximum weight loss is observed 7 to 8 days later.

  This time, the mice were disposed 3 days after the infection, and the body weight at that time was determined with the measured value on the day of infection as 100%. The numerical values shown in FIG. 4 are average values of three animals in each group. The control group (# 1) was slightly reduced to 96.5%, and the positive control (oseltamivir phosphate administration) group (# 2) was 101.8%, indicating no weight loss. When the α-glucan-containing extract fraction obtained in Example 2 was administered, the 0.5 mg and 2 mg administration groups (# 3, # 4) showed a similar weight loss as # 1, but a high dose of 5.0 mg was administered. In the group (# 5), it was 98.1%, and the decrease was slightly prevented. The α-glucan-containing purified fraction and administration group (# 6-8) also showed a dose-dependent body weight loss inhibitory effect, and in particular, no decrease was observed in # 8 (100.8%).

2. Virus growth (Table 1, Fig. 5)
The viral load 3 days after infection was measured by the plaque method. The unit of viral load in FIG. 5 is x 10 ⁴ PFU / 100 mg in the lung and x 10 ³ PFU / 100 μl in the bronchoalveolar lavage fluid. Oseltamivir phosphate significantly reduced viral load in bronchoalveolar lavage fluid (P <0.01) and lung (P <0.001) compared to control group (# 1). The α-glucan-containing extract fraction obtained in Example 2 significantly suppressed airway virus growth (P <0.05) at a high dose (# 5). The α-glucan-containing purified fraction obtained in Example 3 significantly reduced the viral load in the lung (P <0.05) and bronchoalveolar lavage fluid (P <0.01) at the high dose (# 8).

3. Evaluation Both the α-glucan-containing extract fraction obtained in Example 2 and the α-glucan-containing purified fraction obtained in Example 3 significantly suppressed airway virus growth at high doses. Weight loss was comparable to that of oseltamivir phosphate, a positive control. Both specimens, like oseltamivir phosphate, do not act directly on the influenza virus, but may act on some defense mechanism in vivo to show the above effect. Both of these specimens showed an effect at a dose of 5 mg / mouse / day (200 to 250 mg / kg, mouse weight 20 to 25 g / animal) in this test system, and human (adult) Therefore, it is extrapolated that the daily dose of 200 to 250 mg would be effective, and it is judged to be effective for the prevention and treatment of colds such as influenza.

  A novel and useful high molecular weight α-glucan derived from maitake and an extract fraction containing the same can be produced by the method of the present invention from maitake, which is mass-produced in large quantities as a foodstuff, to prevent colds such as influenza -It can be used safely and effectively for treatment, can be used as food and drink, human or veterinary medicine, and feed or feed additive, and can be supplied in a wide range and on a large scale.

Claims (15)

An α-glucan derived from maitake and having a molecular weight distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, or an extract containing the α-glucan. Α-glucan derived from maitake and distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method and having a weight average molecular weight of about 8.5 × 10 ⁶ or its content Extracted fraction. It has a polysaccharide structure derived from maitake and having a molecular weight distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, and having a (1 → 4) bond as the main chain and branched by a (1 → 6) bond. An α-glucan or an extract containing the same is characterized. The molecular weight derived from Maitake is distributed in the range of 4 × 10 ^{6 to} 7 × 10 ⁷ by the SEC-MALS method, the weight average molecular weight is about 8.5 × 10 ⁶ , and the (1 → 4) bond is the main chain. (1 → 6) An α-glucan having a polysaccharide structure branched by a bond or an extract containing the α-glucan.   The α-glucan or the extract containing the α-glucan according to any one of claims 1 to 4, which is effective for the prevention and treatment of colds such as influenza.   The fruit body or mycelium of maitake is extracted with hot water after treatment or means for inhibiting or preventing the action of an enzyme that affects α-glucan contained therein. The manufacturing method of the alpha-glucan in any one of Claims 1-4, or its containing fraction. An image obtained by extracting the fruit bodies or mycelium of maitake mushrooms with hot water after treating or inhibiting the action of enzymes that affect the α-glucan contained in them. The fraction is then purified using any one of a fractional precipitation method, a chromatographic method, a gel filtration method, an ultrafiltration method , or an appropriate combination of these methods. The manufacturing method of the alpha-glucan in any one of these, or its containing extraction fraction.   Treatments / means that inhibit or prevent the action of enzymes that affect α-glucan include cryopreservation, lyophilization, vacuum (decompression) storage, vacuum (decompression) drying, freeze-vacuum (decompression) drying, and heat drying. The method for producing an α-glucan or an extract containing the α-glucan according to claim 6 or 7, which is any one or a combination thereof.   The fruit body or mycelium of maitake is immediately extracted with hot water so that an enzyme that affects α-glucan contained therein does not work. A method for producing α-glucan or an extract fraction containing the same. The fraction obtained by immediately extracting the fruit bodies or mycelium of Maitake mushrooms with hot water so that the enzyme that affects the α-glucan contained therein does not work, The α-glucan according to any one of claims 1 to 4, wherein the α-glucan is purified by any one of a graph method, a gel filtration method, an ultrafiltration method , or a combination of these methods as appropriate. A method for producing the extracted fraction.   In the fractional precipitation method, alcohol is added to the extracted fraction obtained by extracting maitake fruit bodies or mycelia with hot water or the aqueous solution of the dried fraction so that the total amount of alcohol is 20 to 30% by volume. The method according to claim 7 or 10, wherein the precipitate formed is removed, and then the alcohol is added to the supernatant so as to be 40 to 60% by volume of the total, and the precipitated precipitate is collected. The manufacturing method of the alpha-glucan in any one or its containing extraction fraction.   The fruit body or mycelium of maitake is freeze-dried and extracted with hot water, and alcohol is added to the obtained extracted fraction or an aqueous solution of the dried fraction so that the total amount is 20 to 30% by volume. Any one of claims 1 to 4, wherein the precipitate is collected by adding alcohol to the supernatant so that the total amount is 40 to 60% by volume. A method for producing α-glucan or an extract fraction containing the same.   A food or drink comprising the α-glucan according to any one of claims 1 to 4 or an extract containing the α-glucan.   A pharmaceutical product for humans or animals comprising the α-glucan according to any one of claims 1 to 4 or an extract containing the α-glucan.   A feed or a feed additive comprising the α-glucan according to any one of claims 1 to 4 or an extract containing the α-glucan.

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