JP2014221899A - METHOD FOR EXTRACTING POLYSACCHARIDE COMPRISING β-GLUCAN AS MAIN COMPONENT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for extracting polysaccharides comprising β-glucan as a main component from a mushroom with high yield and excellent efficiency.SOLUTION: In a method for extracting polysaccharides comprising β-glucan as a main component, a mushroom is treated with water having a temperature within the range of about 140-180°C under a pressure within a range of about 2-3 MPa. The mushroom is preferably maitake mushroom. The mushroom to be used is preferably used after being pretreated with a solvent. The polysaccharides are soluble in water, so that they are easy to be ingested into a living body. Therefore, the polysaccharides can be utilized for pharmaceuticals, especially pharmaceuticals for prevention and/or treatment of tumors, quasi-drugs, cosmetics, health foods, health supplements, foods for specified health use, foods such as dietary supplements, or animal feeds.

Description

  The present invention relates to a method for extracting a polysaccharide mainly composed of β-glucan from mushrooms such as maitake.

Maitake contains various physiologically active substances, and in particular, polysaccharides such as β-glucan are known to have antitumor activity (Non-patent Document 1).
Until now, attempts have been made to extract physiologically active substances from maitake by combining various conditions such as solvent, liquidity, and temperature. However, polysaccharides that have antitumor activity have many poorly soluble components. It was difficult to obtain in a high yield. To extract polysaccharides mainly composed of β-glucan from maitake, extract the polysaccharides using water or alkaline aqueous solution, and then fractionate fractions mainly composed of β-glucan by alcohol precipitation or chromatography. -The method of refinement | purification has been performed (patent document 1, nonpatent literature 2).
Recently, basidiomycetes, a method for efficiently and selectively extracting and separating pigments and physiologically active substances produced in woody parts by basidiomycetes, especially wood decaying fungi, or pigments and physiologically active substances contained in cells The cell powder or woody powder containing the product is first treated with water vapor or hot water at 100 to 140 ° C. under an open pressure to extract and separate the readily soluble components of the product, and the residue is further removed at 100 ° C. A method of extracting and separating the hardly soluble component of the product by treating with pressurized hot water at a higher temperature is reported (Patent Document 2).

JP-A-9-238697 JP 2001-321191 A

Taku Mizuno, edited by Masaaki Kawai, “Mushroom Chemistry and Biochemistry”, 237-249, published by the Society Publishing Center, 2000 Ohno N, Suzuki I, Oikawa S, Sato K, Miyazaki T, Yadomae T, Antitumor Activity and Structural Characterization of Glucans Extracted from Cultured Fruit Bodies of Grifola frondosa, Chem. Pharm. Bull., 32 (3), 1142-1151 ( (1984)

  However, in the methods such as Patent Document 1 and Non-Patent Document 2 described above, the yield of polysaccharides such as β-glucan in the extract is low. Therefore, even if the extract is further purified, useful components are only present in a low yield. There is a problem that it cannot be obtained.

Therefore, as a result of intensive studies to solve the above problems, the present inventors have extracted polysaccharides mainly composed of β-glucan from mushrooms such as maitake mushrooms by extracting with water at a specific pressure and temperature. The present invention was completed by finding that it can be efficiently obtained in a high yield.
That is, the present invention treats mushrooms with water having a temperature in the range of about 140 to about 180 ° C. under a pressure in the range of about 2 to about 3 MPa. The present invention relates to a method for extracting saccharides.

  According to the present invention, polysaccharides mainly composed of β-glucan can be efficiently extracted at high yield from mushrooms such as maitake.

It is a figure showing the extraction flow of Example 1. FIG. It is a figure showing the extraction flow of the comparative example 1.

  In the present invention, the polysaccharide mainly composed of β-glucan is not only β-glucan itself but also a polysaccharide containing β-glucan, for example, β-glucan, saccharide other than D-glucose, and other than D-glucose. It refers to a heteropolysaccharide formed by binding a polysaccharide containing a saccharide, and a complex of β-glucan and protein.

Mushrooms that can be used in the present invention are not particularly limited, and examples include basidiomycetes and ascomycetes.
As the above basidiomycetes,
・ Oyster mushrooms, for example, Octopusaceae (Maitake, Shiromaitake, Tombaimaitake, Choreimaitake, Mustache, etc.), Hanabiratake (Hanaburitake, etc.);
Agaric, for example, oyster mushrooms (shiitake, eringi, oyster mushrooms, tomato oyster mushrooms, ushiratake mushrooms, tamogi mushrooms, etc.) , Moegitake family (nameko, Kuritatake, Numerisugitake, etc.), Agaricaceae (Agaricus, Tsukuritake (commonly known as mushrooms), Agaricus, etc.) ;
・ Agaricidae, for example, Coralhariaceae (Yamabushitake, etc.);
・ Jellyfish, for example, jellyfish (eg, jellyfish);
* Pleurotus jellyfish, for example, the family Aceraceae (such as the jellyfish);
Suppontake, for example, Suppontake department (such as Kinugasatake)
Etc. are exemplified.
As the above ascomycetes,
-Chawantake, for example, Amphiidae (such as Amigasatake);
・ Buckakkin eyes, for example, Bakakukinkin [Cycium caterpillar (generic name for Cordyceps genus)];
・ Lepidoptera, for example, Aceraceae [Truffles (generic name for Astragalus), etc.]
Etc. are exemplified.
Preferred are basidiomycetes, more preferred are mushrooms of the order mushrooms, still more preferred are mushrooms of the family Octeridae, most preferred maitake.

  In the present invention, the fruit body or the mycelium or spore can be used as the mushroom. The fruit body or its mycelium or spore may be processed as it is, but from the point of increasing the extraction efficiency of polysaccharides mainly composed of β-glucan, the fruit body or its mycelium or spore is crushed, It is preferable to use in the form of crushed pieces, fine pieces, powders, flakes, or pastes.

For the purpose of increasing the extraction efficiency of polysaccharides mainly composed of β-glucan, mushrooms are pretreated with an appropriate solvent before being subjected to the extraction process of the present invention, and fats and solubles contained in the mushrooms It is preferred to remove soluble components such as sugars. The solvent used at this time is not particularly limited as long as it can elute soluble components such as fats and soluble saccharides in mushrooms. For example, water or an organic solvent such as ethanol, methanol, or acetone is used. Water or ethanol can be preferably used. These solvents may be used alone or as a mixed solvent of two or more.
The pretreatment conditions, such as temperature and pressure, are not particularly limited. For example, the pretreatment can be performed at room temperature and normal pressure, but in order to promote the removal of soluble components, the pretreatment is performed under hot water, particularly under reflux. Is preferably performed. In this case, about 50-60% of the mushroom solids can be removed. The mixing ratio of the mushroom and the solvent is not particularly limited, and it is preferable to add about 500 to about 2000 parts by weight of the solvent with respect to 100 parts by weight of the mushroom from the viewpoint of increasing the removal of soluble components. The pretreatment time is not particularly limited, but is preferably about 1 to about 3 hours from the viewpoint of enhancing the removal of soluble components.

  The above pretreatment may be repeated to enhance the removal of soluble components. That is, the pretreatment may be repeated twice or more, preferably 3 to 5 times, by subjecting the pretreated mushroom to the pretreatment again before being subjected to the extraction step of the present invention. .

In the extraction process of the present invention, the aforementioned mushrooms are treated with subcritical water, ie, water at a temperature in the range of about 140 to about 180 ° C. under a pressure in the range of about 2 to about 3 MPa to produce β-glucan. Extract the main polysaccharide. Specifically, for example, water is added to mushrooms, the pressure is increased to a range of 2 to 3 MPa, the temperature is increased to a range of 140 to 180 ° C., and extraction is performed while stirring as necessary. .
In the extraction step, the temperature is preferably about 140 to about 160 ° C., and the pressure is preferably about 2 MPa in order to efficiently extract a polysaccharide mainly composed of β-glucan with high yield.
Further, the mixing ratio of the mushroom and water is not particularly limited, but in order to efficiently extract a polysaccharide mainly composed of β-glucan in a high yield, the solvent is added at about 1000 to about 100 parts by weight with respect to 100 parts by weight of the mushroom. It is preferred to add 3000 parts by weight, especially about 1500 parts by weight.
The extraction time is not particularly limited, but is preferably about 0.5 to about 3 hours, particularly about 1 hour in order to efficiently extract a polysaccharide mainly composed of β-glucan with high yield.

  After the above extraction step, the residue may be removed by subjecting a mixture of mushrooms and water to filtration, centrifugation, or the like to obtain an extract containing a polysaccharide mainly composed of β-glucan. Furthermore, you may obtain a concentrated extract by concentrating the obtained extract, for example, concentrate | evaporating under reduced pressure. The obtained extract or concentrated extract may be dried as it is, for example, freeze-dried or spray-dried to obtain a solid extract.

Specifically, the above-described extraction step may be performed as follows, for example. First, mushrooms and water are sealed in a pressure vessel. In order to increase the pressure inside the pressure vessel, a gas such as nitrogen gas is injected to obtain a high pressure state of about 2 to about 3 MPa, for example, about 2 MPa. While maintaining the high pressure state, the mixture of mushrooms and water inside the pressure vessel is stirred, heated to about 140 to about 180 ° C., and subjected to pressurized hot water extraction for about 1 hour.
When the above pressurized hot water extraction step is completed, the gas inside the pressure vessel is removed and returned to normal pressure, and the mixture of mushrooms and water is recovered. Next, the residue is removed from the mixture by a centrifuge or a glass filter to obtain an extract. Furthermore, the obtained extract can be dried to obtain a light brown solid extract. This solid extract is easily soluble in water and can contain a polysaccharide mainly composed of β-glucan at a high concentration (for example, about 30 to 50%).

  The extraction process of the present invention may be performed repeatedly in order to increase the yield of polysaccharides mainly composed of β-glucan. That is, the extraction process may be repeated twice or more, preferably 3 to 5 times, for example, by subjecting the residue to the extraction process again.

  The extract obtained in the present invention may be subjected to further purification steps. For example, a polysaccharide mainly composed of β-glucan obtained by further purifying an extract such as an extract, a concentrated extract, or a solid extract obtained in the extraction step using an alcohol precipitation method or a chromatography method. Can be obtained as a purified product containing a higher concentration of or a highly purified β-glucan. Or you may refine | purify using a ultrafiltration method.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Moreover, unless otherwise indicated,% means weight%.

Example 1
To 80 g of maitake powder, 800 mL of water was added and heated, followed by extraction under reflux for 1 hour. After extraction, the filtrate and residue were separated using a centrifuge. The same amount of hot water as the filtrate was added to the residue, and the same operation was repeated again. This operation was repeated once more, and the obtained filtrate and residue were dried to obtain a hot water extract and a hot water extraction residue.
Next, 30 g of hot water extraction residue and 450 mL of water were sealed in a pressure vessel, and nitrogen gas was injected (about 2 MPa). While stirring, extraction with pressurized hot water was performed at 160 ° C. for 1 hour.
After extraction, the internal gas was removed to return to normal pressure, and the sample was collected. Samples were separated from solids and liquids by a centrifuge. The solid content and the liquid part were dried to obtain a pressurized hot water extraction residue and a pressurized hot water extract, respectively. The extraction flow is shown in FIG.

Comparative Example 1
To 200 g of maitake powder, 1400 mL of an 80% ethanol aqueous solution was added and heated, followed by extraction at reflux for 1 hour. After extraction, the mixture was filtered through a glass filter to obtain a filtrate and a residue. The same amount of 80% ethanol aqueous solution as the filtrate was added to the residue, and the same operation was repeated again. This operation was repeated once more, and the obtained filtrate and residue were dried to obtain an 80% ethanol extract and an 80% ethanol extraction residue.
The 80% ethanol extraction residue was further subjected to hot water extraction. 1200 mL of water was added to 200 g of 80% ethanol extraction residue, heated, and extracted for 1 hour under reflux. After extraction, the mixture was filtered through a glass filter to obtain a filtrate and a residue. The same amount of hot water as the filtrate was added to the residue, and the same operation was repeated again. This operation was repeated once more, and the obtained filtrate and residue were dried to obtain a hot water extract and a hot water extraction residue.
The hot water extraction residue was further subjected to autoclave extraction. To 100 g of the hot water extraction residue, 700 mL of water was added and extracted in an autoclave at 120 ° C. for 1 hour. After extraction, the mixture was filtered through a glass filter to obtain a filtrate and a residue. The same amount of hot water as the filtrate was added to the residue, and the same operation was repeated again. This operation was repeated once more, and the obtained filtrate and residue were dried to obtain an autoclave extract and an autoclave extraction residue.
The autoclave extraction residue was further subjected to ammonia extraction. To 10 g of the autoclave extraction residue, 100 mL of a 10% aqueous ammonia solution was added and heated, followed by extraction at reflux for 1 hour. After extraction, the mixture was filtered through a glass filter to obtain a filtrate and a residue. The same amount of hot water as the filtrate was added to the residue, and the same operation was repeated again. The same operation was repeated until the filtrate became neutral, and extraction was performed 4 times in total. The obtained filtrate and residue were dried to obtain 10% ammonia extract and 10% ammonia extraction residue. The extraction flow is shown in FIG.

得られた結果は、実施例１については表１に、比較例１については表２にそれぞれ示した。 For Example 1 and Comparative Example 1 obtained above, the yield of each fraction and the β-glucan content were examined. “Yield” represents the ratio of the yield of each fraction to the amount of maitake powder used for extraction. The “β-glucan content” was determined based on the enzyme method adopted by the Japan Food Analysis Center. The enzymatic method is a method in which α-glucan is degraded by an enzyme and the remaining glucan is quantified as β-glucan. In addition, the ratio of β-glucan content of each fraction to β-glucan content of maitake powder, β-glucan extraction rate for the extract, β-glucan residual rate for the extraction residue, I asked for it.

The obtained results are shown in Table 1 for Example 1 and Table 2 for Comparative Example 1, respectively.

  From Table 1 and FIG. 1, in the two-stage extraction process, about 50% of β-glucan contained in maitake is contained in the pressurized hot water extract, and it can be extracted efficiently in Example 1. all right. On the other hand, it was found from Table 2 and FIG. 2 that even in the four-stage extraction process, most of the β-glucan contained in the maitake remained in the residue, and Comparative Example 1 showed low extraction efficiency.

The following items were evaluated for the pressurized hot water extract obtained in Example 1.
(1) Average molecular weight measurement
The average molecular weight of the pressurized hot water extract obtained in Example 1 was measured using the GPC method. The average molecular weight is shown as a relative value based on pullulan molecular weight. The results obtained are shown in Table 3.

(2) Molecular weight distribution measurement (ultrafiltration method)
About 0.5 g of the pressurized hot water extract obtained in Example 1 was weighed and dissolved in 50 mL of distilled water to prepare a sample solution. First, a PBQK membrane (fractionated molecular weight: 50,000) was used as an ultrafiltration membrane, ultrafiltration was carried out by the following method, and the collected filtrate (40 mL × 3) and the sample solution residue remaining in the cell ( 10 mL) were each lyophilized and weighed. Next, all the dried filtrates were combined and redissolved in 50 mL distilled water, and ultrafiltration was performed using a PBCC membrane (fractionated molecular weight: 5,000) as an ultrafiltration membrane. Similarly, the filtrate (40 mL × 3) and the sample solution residue (10 mL) remaining in the cell were collected, and after lyophilization, the weight was measured.
Through the above steps, the pressurized hot water extract obtained in Example 1 was divided into three fractions having a molecular weight of 50,000 or more, 5,000 to 50,000, and 5,000 or less. The results obtained are shown in Table 4. The percentages in the table indicate the ratio of the weight of each fraction after lyophilization to the weight of the pressurized hot water extract dissolved in distilled water.
(Ultrafiltration method)
An ultrafiltration membrane was sandwiched in an Amicon stirring cell (Model 8050 φ44.5 mm), and a sample solution was added to the cell. While stirring, nitrogen gas was blown in to apply pressure, and the filtrate that passed through the filtration membrane was recovered. When the filtrate reached 80% of the amount of sample solution added to the cell, nitrogen blowing and agitation were stopped. The filtrate was recovered, the same amount of distilled water as the recovered filtrate was added to the cell, and ultrafiltration was resumed. This was repeated twice.

(3) Determination of the sugar chain binding position For the fraction with a molecular weight of 50,000 or more obtained by the ultrafiltration method in (2) above, the amount ratio of 1-3, 1-4, and 1-6 bonds of the glucose binding moiety was determined. Measured by methylation analysis. The results obtained are shown in Table 5.

(4) Anti-tumor evaluation test 450 mg of the pressurized hot water extract obtained in Example 1 was suspended in 4.5 ml of distilled water for injection to prepare a sample solution having a concentration of 100 mg / ml. Moreover, TS-1 (trade name TS-1 (registered trademark) capsule 25, Taiho Pharmaceutical Co., Ltd.), a kind of commercially available antitumor agent, is dissolved in 12.5 ml of distilled water for injection to give 2 mg. A sample solution having a concentration of / ml was prepared.
Cryopreserved S-180 cells (derived from mouse Sarcoma 180) PBS (-) was centrifugally washed with, grafted Approximately ¹⁰⁶ mice (ICR type, 6 weeks old, female) intraperitoneally about 7 Ascites fluid containing tumor cells grown after 10 days was collected. Next, the collected ascites was injected into the ventral skin of another mouse (ICR line, 6 weeks old, female) in an amount of 10 ⁵ tumor cells / mouse to transplant tumor cells. Next, the mice transplanted with these tumor cells were divided into 3 groups of 7 mice, and distilled water, the pressurized hot water extract sample solution of Example 1 and the TS-1 sample solution were respectively added to each group. The doses shown in Table 6 were orally administered daily from the day of transplantation until the 19th day. On the 22nd day after transplantation, the mice were bled to death, the tumors were removed and their weights were measured. During the administration period, body weight was measured twice / week. Further, a test using an amount of 10 ⁶ tumor cells / mouse was similarly performed. The obtained results are shown in Table 6.

From Table 6, it was recognized that the tumor weight decreased in the group administered with the pressurized hot water extract obtained in Example 1 compared with the group administered with distilled water. Moreover, in the group administered with distilled water or the pressurized hot water extract of Example 1, the body weight of the mice increased steadily, and there was no case of drowning, but in the TS-1 administration group, the mice were up to 8 days after administration. Although the body weight increased, it turned to a decrease thereafter, and finally, 4 out of 7 animals were drowned.
From the above, it was confirmed that the pressurized hot water extract obtained in Example 1 has an antitumor effect against S-180 tumor and is excellent in safety.

According to the present invention, an extract containing a polysaccharide mainly composed of β-glucan at a high concentration can be obtained. Since the extract of the present invention contains a polysaccharide mainly composed of β-glucan at a high concentration, it can be advantageously used for high-purity purification of a polysaccharide mainly composed of β-glucan.
In addition, the extract of the present invention is water-soluble, so it can be easily taken into the living body, and it is a pharmaceutical, particularly a pharmaceutical for tumor prevention and / or treatment, a quasi-drug, a cosmetic, or a health food, health supplement, or food for specified health use. Or foods such as dietary supplements or animal feeds.

Claims (3)

  A method for extracting polysaccharides based on β-glucan, characterized in that mushrooms are treated with water at a temperature in the range of about 140 to about 180 ° C. under a pressure in the range of about 2 to about 3 MPa.   The extraction method according to claim 1, wherein the mushroom is maitake.   The extraction method according to claim 1 or 2, wherein mushrooms previously treated with a solvent are used.

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