JP2000159808A - Method for separating and purifying basidiomycota hypha extract - Google Patents
Method for separating and purifying basidiomycota hypha extractInfo
- Publication number
- JP2000159808A JP2000159808A JP10353919A JP35391998A JP2000159808A JP 2000159808 A JP2000159808 A JP 2000159808A JP 10353919 A JP10353919 A JP 10353919A JP 35391998 A JP35391998 A JP 35391998A JP 2000159808 A JP2000159808 A JP 2000159808A
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- JP
- Japan
- Prior art keywords
- fraction
- ethanol
- ion
- exchanger
- mushroom
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は有効成分の新規分
離、精製方法に関し、さらに詳しくは担子菌類菌糸体抽
出物から有効成分を分離、精製する新規な方法に関す
る。The present invention relates to a novel method for separating and purifying an active ingredient, and more particularly to a novel method for separating and purifying an active ingredient from a basidiomycete mycelium extract.
【０００２】[0002]
【従来の技術】古来より、マツタケ、シイタケ、エノキ
タケなどの担子菌類のキノコは食用されており、中には
サルノコシカケなどのように漢方薬として重用されてい
るものもある。2. Description of the Related Art Mushrooms of basidiomycetes such as Matsutake, Shiitake and Enokitake have been edible since ancient times, and some mushrooms, such as Sarno-koshitake, have been frequently used as herbal medicines.
【０００３】特に、シイタケ（Lentinus edodes）は日
本、中国の代表的な食用キノコであり、日本では約３０
０年前から人工栽培が行われてきた。日常食用にしてい
るキノコは子実体と呼ばれ、菌類が子孫を残すために胞
子を生じる生殖体であり、栄養体である菌糸細胞は地中
や原木中で長い時間をかけて成長し、菌糸体を形成す
る。[0003] In particular, shiitake mushroom (Lentinus edodes) is a typical edible mushroom in Japan and China, and about 30 in Japan.
Artificial cultivation has been performed for 0 years. Mushrooms that are consumed daily are called fruiting bodies, which are germ bodies that produce spores in order for fungi to leave their offspring, and vegetative mycelia grow over time in the ground and in raw wood, Form the body.
【０００４】シイタケは古くからさまざまな病気や症状
に効果があると言われてきたが、その薬理作用が解明さ
れてきたのは比較的最近である。シイタケ菌糸体抽出物
については、ラット、マウスでの発癌実験において、動
物の大腸、肝臓などの腫瘍形成及び移植腫瘍細胞の増殖
を抑制し、動物の生存率を上昇させた（N. Sugano eta
l., Cancer Letter, 17:109, 1982;鈴木康将ら、日本大
腸肛門病会誌、43:178, 1990など）、マイトジェン活性
を示した（T. Tabata et al., Immunopharmacology, 2
4:57, 1992; Y. Hibino et al., Immunopharmacology,
28:77, 1994など）、抗体産生を増強した（溝口靖紘
ら、肝胆膵、15:127, 1987）などの種々な報告がなされ
ている。この他にもシイタケ菌糸体抽出物には発根促
進、農作物の成長促進などの植物ホルモン作用（M. Mit
suhashi-Kato et al., Plant Cell Physiol. 26:221, 1
985）や抗植物ウイルス作用（小室康雄：農林水産省植
物ウイルス研報告, 1977）があることが報告されてい
る。[0004] Shiitake mushrooms have long been said to be effective against various diseases and symptoms, but their pharmacological actions have been elucidated relatively recently. Shiitake mushroom mycelium extract inhibited tumor formation and growth of transplanted tumor cells in the large intestine and liver of animals and increased the survival rate of animals in carcinogenicity experiments in rats and mice (N. Sugano eta).
l., Cancer Letter, 17: 109, 1982; Yasumasa Suzuki et al., Journal of the Japanese College of Colitis, 43: 178, 1990, etc., and showed mitogenic activity (T. Tabata et al., Immunopharmacology, 2
4:57, 1992; Y. Hibino et al., Immunopharmacology,
28:77, 1994), and various reports such as enhanced antibody production (Yasuhiro Mizoguchi et al., Hepatobiliary pancreas, 15: 127, 1987). In addition, the extract of Shiitake mushroom mycelium has phytohormonal effects such as promoting rooting and growing crops (M. Mit
suhashi-Kato et al., Plant Cell Physiol. 26: 221, 1
985) and anti-plant virus activity (Yasuo Komuro: Report of Plant Virus Research Institute, Ministry of Agriculture, Forestry and Fisheries, 1977).
【０００５】シイタケ菌糸体抽出物中の成分の分離・精
製については、様々な手法が試みられている。リグニン
抽出を目的とした方法では、エタノール不溶画分を疎水
クロマトグラフィーにより分離を行い、得られた画分を
更にゲルろ過クロマトグラフィ−によって分画を行って
いるが、その得られた分画物の分子量による分離精度は
不十分であった（S.Toda et.al.,Biochemical and Biop
hysical Research Communications 367-373 Vol.160,N
o.1,1998）。また、多糖を目的とした分離・精製につい
ては、エタノール不溶画分をゲルろ過クロマトグラフィ
ーによって分画しており、その後、この得られた主要画
分をイオン交換クロマトグラフィーで２回処理すること
よって中性多糖と酸性多糖を得ている。しかし、イオン
交換クロマトグラフィーを２度施すなどの手技的な制約
がある（N.Sugano et.al.,Immunopharmacology,57-63,2
4,1992）のが現状である。[0005] Various techniques have been tried for separation and purification of the components in the shiitake mushroom mycelium extract. In the method for lignin extraction, the ethanol-insoluble fraction is separated by hydrophobic chromatography, and the obtained fraction is further fractionated by gel filtration chromatography. Separation accuracy by molecular weight was insufficient (S. Toda et.al., Biochemical and Biop.
hysical Research Communications 367-373 Vol.160, N
o.1,1998). For separation and purification for the purpose of polysaccharide, the ethanol-insoluble fraction is fractionated by gel filtration chromatography, and then the obtained main fraction is treated twice by ion exchange chromatography. Neutral and acidic polysaccharides are obtained. However, there are technical restrictions such as performing ion exchange chromatography twice (N. Sugano et.al., Immunoopharmacology, 57-63, 2).
4,1992) is the current situation.
【０００６】[0006]
【発明が解決すべき課題】シイタケに代表される担子菌
類抽出物の薬理作用をさらに詳しく解明するには、担子
菌類菌糸体抽出物を効率よく分離、精製して各画分に分
けて薬理試験を行うことが必須である。本発明は担子菌
類菌糸体抽出物中の成分の新規な分離、精製方法を探索
する事を目的とする。In order to elucidate the pharmacological action of a basidiomycete extract represented by shiitake mushroom in more detail, a basidiomycete mycelium extract is efficiently separated and purified, and each fraction is separated into pharmacological tests. It is essential to do An object of the present invention is to search for a novel separation and purification method for components in a basidiomycete mycelium extract.
【０００７】[0007]
【課題を解決するための手段】本発明者らは上記課題を
解決するため、鋭意研究した結果、新規で効率よい担子
菌類菌糸体抽出物の分離、精製方法を発明した。Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies and as a result, have invented a novel and efficient method for separating and purifying a Basidiomycete mycelium extract.
【０００８】すなわち、本発明は、以下の工程を含んで
なる担子菌類菌糸体抽出物の分離、精製方法を提供す
る： １）担子菌類菌糸体抽出物にエタノールを加えてエタノ
ール不溶性画分と可溶性画分に分ける； ２）工程１で得られたエタノール不溶画分をイオン交換
体で処理し、イオン交換体非吸着画分とイオン交換体吸
着画分に分ける； ３）工程２で得られたイオン交換体非吸着画分をゲルろ
過クロマトグラフィーにかけて、ホモ多糖画分、ヘテロ
多糖画分及び低分子画分に分ける。That is, the present invention provides a method for separating and purifying a basidiomycete mycelium extract comprising the following steps: 1) adding ethanol to the basidiomycete mycelium extract to obtain an ethanol-insoluble fraction; 2) treating the ethanol-insoluble fraction obtained in step 1 with an ion exchanger, and separating it into an ion-exchanger non-adsorbed fraction and an ion-exchanger adsorbed fraction; 3) obtained in step 2 The fraction not adsorbed by the ion exchanger is subjected to gel filtration chromatography to separate it into a homopolysaccharide fraction, a heteropolysaccharide fraction and a low molecular fraction.
【０００９】本発明では、エタノール含有溶液による処
理で得られるエタノール不溶画分をまずイオン交換体で
処理し、ついでイオン交換体非吸着画分をゲルろ過クロ
マトグラフィーにかけて分子量に応じてさらに分離する
ことを特徴とする。In the present invention, the ethanol-insoluble fraction obtained by the treatment with the ethanol-containing solution is first treated with an ion exchanger, and then the ion-exchanger non-adsorbed fraction is subjected to gel filtration chromatography to further separate the fraction according to the molecular weight. It is characterized by.
【００１０】担子菌類としては、通常食用されているマ
ツタケ、シイタケ、エノキタケ、ヒラタケ、ナメコ、イ
グチ、シメジ、チチタケ、ヒメマツタケなどのマツタケ
目の茸類；漢方薬として利用され、あるいは食用として
用いられるコフキサルノコシカケ、ツガサルノコシカ
ケ、カワラタケ、マンネンタケ（霊芝）、マイタケなど
のサルノコシカケ目の茸類；食用されるキクラゲ、シロ
キクラゲなどのキクラゲ目又はシロキクラゲ目の茸類を
含む。Examples of basidiomycetes include matsutake mushrooms such as matsutake, shiitake, enokitake, oyster mushroom, nameko, iguchi, shimeji, ichitake mushroom, and himetake mushroom which are commonly used for food; And mushrooms of the order Salmonaceae, such as Tsugasaronokoshake, Kawaratake, Mannentake (Lingzhi), and Maitake;
【００１１】本発明の分離、精製方法で使用する担子菌
菌糸体抽出物とは、担子菌類を固体培地上で培養して得
られる菌糸体、好ましくは菌糸体を含む固体培地を水及
び酵素の存在下に粉砕、分解して得られる抽出物を言
う。The basidiomycete mycelium extract used in the separation and purification method of the present invention refers to a mycelium obtained by culturing basidiomycetes on a solid medium, preferably a solid medium containing mycelium, containing water and enzymes. An extract obtained by crushing and decomposing in the presence.
【００１２】担子菌類菌糸体抽出物は好ましくは以下の
方法により得られたものを使用するが、これに限定され
ない。すなわち、バガス（サトウキビのしぼりかす）と
脱脂米糠を基材とする固体培地上に担子菌類を接種し、
次いで菌糸体を増殖して得られる菌糸体を含む固体培地
を１２メッシュ通過分が３０重量％以下となるよう解束
し、この解束された固体培地に水およびセルラーゼ、プ
ロテアーゼまたはグルコシダーゼから選ばれる酵素の１
種またはそれ以上を、前記固体培地を３０〜５５℃の温
度に保ちながら添加するとともに、前記固体培地を前記
酵素の存在下に粉砕・すりつぶしてバガス繊維の少なく
とも７０重量％以上が１２メッシュ通過分であるように
し、次いで９５℃までの温度に加熱することにより酵素
を失活させるとともに滅菌し、得られた懸濁状液をろ過
することによって担子菌類菌糸体抽出物を得る。担子菌
類菌糸体抽出物はそのまま本発明の分離、精製方法に用
いてもよいが、これを濃縮、凍結乾燥して粉末として保
存し、使用時に種々の形態で使用するのが便宜的であ
る。例えば、シイタケ菌糸体抽出物を凍結乾燥して得ら
れる粉末は褐色粉末で、吸湿性があり、特異な味と臭い
をもつ。The basidiomycete mycelium extract is preferably one obtained by the following method, but is not limited thereto. In other words, basidiomycetes are inoculated on a solid medium based on bagasse (sugar cane pulp) and defatted rice bran,
Next, the solid medium containing the mycelium obtained by growing the mycelium is unbundled so that the amount passed through 12 mesh is 30% by weight or less, and the unbundled solid medium is selected from water and cellulase, protease or glucosidase. Enzyme 1
Seeds or more are added while maintaining the solid medium at a temperature of 30 to 55 ° C., and the solid medium is crushed and ground in the presence of the enzyme so that at least 70% by weight or more of bagasse fiber passes through 12 mesh. Then, the enzyme is inactivated and sterilized by heating to a temperature of up to 95 ° C., and the resulting suspension is filtered to obtain a basidiomycete mycelium extract. The basidiomycete mycelium extract may be used as it is in the separation and purification method of the present invention, but it is convenient to concentrate, freeze-dry and store it as a powder and use it in various forms at the time of use. For example, the powder obtained by freeze-drying a shiitake mushroom mycelium extract is a brown powder, has hygroscopicity, and has a unique taste and odor.
【００１３】このようにして得られる担子菌類菌糸体抽
出物を本発明の分離、精製方法に付す。本発明の方法の
概略を示したものが図１である。The basidiomycete mycelium extract thus obtained is subjected to the separation and purification method of the present invention. FIG. 1 schematically shows the method of the present invention.
【００１４】本発明の分離、精製方法を代表的担子菌類
であるシイタケ菌糸体抽出物の分離、精製によって例示
するが、本発明の方法はその他の担子菌類の抽出にも応
用できる。The separation and purification method of the present invention is exemplified by the separation and purification of a mushroom mycelium extract, which is a representative basidiomycete, but the method of the present invention can be applied to the extraction of other basidiomycetes.
【００１５】まず、本発明の分離、精製工程を行う前
に、好ましくはシイタケ菌糸体抽出物を適当な濃度の水
溶液として夾雑物を除去するために、固形粒子ろ過剤を
用いろ過する。ろ過剤としてはセライト５０３、セライ
ト５３５、セライト５４５などで、好ましくはセライト
５４５で処理して夾雑物を除去する。First, before performing the separation and purification steps of the present invention, preferably, a shiitake mushroom mycelium extract is filtered as an aqueous solution of a suitable concentration using a solid particle filter agent to remove impurities. As a filtering agent, Celite 503, Celite 535, Celite 545, or the like is preferably used, and treatment is preferably performed with Celite 545 to remove impurities.
【００１６】本発明の第１工程では、シイタケ菌糸体抽
出物にエタノール（好ましくは４容）を加えて、エタノ
ール不溶性画分（以下においてエタノール不溶画分とい
うこともある）と可溶性画分に分ける。エタノール不溶
画分には粗多糖類が含まれている。In the first step of the present invention, ethanol (preferably 4 volumes) is added to the shiitake mushroom mycelium extract to separate into an ethanol-insoluble fraction (hereinafter sometimes referred to as an ethanol-insoluble fraction) and a soluble fraction. . The ethanol-insoluble fraction contains a crude polysaccharide.
【００１７】第２工程では、第１工程で得られたエタノ
ール不溶画分を蒸留水に再溶解し、エタノール不溶画分
をイオン交換体で処理し分離する。分離する方法はカラ
ム法とバッチ法があり、好ましくはカラム法によって分
離する。水で溶出したときに吸着されないで流出する非
吸着画分（以下においてイオン交換体非吸着画分という
こともある）と、イオン交換体に吸着され、0.05-3MのN
aCl、好ましくは2M NaClで溶離する吸着画分（以下にお
いてイオン交換体吸着画分ということもある）に分け
る。イオン交換体としては、例えばジエチルアミノエチ
ル・合成樹脂イオン交換体（Ｃｌ―型）、ジエチルアミ
ノエチル・セルロース、ジエチルアミノエチル・アガロ
ース、ジエチル−（２−ヒドロキシプロピル）アミノエ
チル・セルロース、ジエチル−（２−ヒドロキシプロピ
ル）アミノエチル・アガロースなどの陰イオン交換体を
使用でき、好ましくはジエチルアミノエチル−トヨパー
ルイオン交換体（東ソー株式会社製）である。第２工程
で得られるクロマトグラムの一例を図２に示す。In the second step, the ethanol-insoluble fraction obtained in the first step is redissolved in distilled water, and the ethanol-insoluble fraction is treated with an ion exchanger and separated. Separation methods include a column method and a batch method, and preferably separation is performed by a column method. A non-adsorbed fraction which is not adsorbed and eluted when eluted with water (hereinafter also referred to as an ion-exchanger non-adsorbed fraction) is adsorbed by the ion exchanger and has a concentration of 0.05-3M N
The adsorbed fraction is eluted with aCl, preferably 2M NaCl (hereinafter sometimes referred to as an ion exchanger adsorbed fraction). Examples of the ion exchanger include diethylaminoethyl / synthetic resin ion exchanger (Cl- type), diethylaminoethyl cellulose, diethylaminoethyl agarose, diethyl- (2-hydroxypropyl) aminoethyl cellulose, and diethyl- (2-hydroxy Anion exchangers such as propyl) aminoethyl agarose can be used, and preferably a diethylaminoethyl-toyopearl ion exchanger (manufactured by Tosoh Corporation). FIG. 2 shows an example of the chromatogram obtained in the second step.
【００１８】本発明の第３工程では、第２工程で得られ
たイオン交換体非吸着画分を蒸留水に再溶解し、ゲルろ
過カラムクロマトグラフィーにかけて分子量に応じて分
画する。ゲルろ過剤としては例えば、デキストランゲ
ル、ポリアクリルアミドゲル、アガロースゲル、ポリビ
ニルゲル、セルロースゲルなどを使用することができ、
好ましくはセファロースＣＬ−６Ｂ、セファクリルＳ−
３００などがある。また、溶出液としては、0−0.5MのN
aClを使用することが出来、好ましくは0.2M NaClを用い
ることが出来る。この操作によって、分子量の大きい順
にホモ多糖画分、ヘテロ多糖画分及び低分子画分を得
た。第３工程で得られるクロマトグラムの一例を図３に
示す。In the third step of the present invention, the non-adsorbed fraction of the ion exchanger obtained in the second step is redissolved in distilled water and subjected to gel filtration column chromatography to fractionate according to the molecular weight. As the gel filtration agent, for example, dextran gel, polyacrylamide gel, agarose gel, polyvinyl gel, cellulose gel and the like can be used,
Preferably Sepharose CL-6B, Sephacryl S-
300 and the like. The eluate used was 0-0.5M N
aCl can be used, and preferably 0.2M NaCl can be used. By this operation, a homopolysaccharide fraction, a heteropolysaccharide fraction, and a low-molecular-weight fraction were obtained in the descending order of molecular weight. FIG. 3 shows an example of the chromatogram obtained in the third step.
【００１９】シイタケ菌糸体抽出物から本発明の方法で
分離された各画分について、成分組成及び構成糖の組成
を分析した。Each of the fractions separated from the shiitake mushroom mycelium extract by the method of the present invention was analyzed for the component composition and the constituent sugar composition.
【００２０】本発明の方法で分離、精製されるシイタケ
菌糸体抽出物の各画分は本願と同日に同一出願人によっ
て出願された特許出願に示すように、種々の顕著な薬理
作用を示した。Each fraction of the shiitake mushroom mycelium extract separated and purified by the method of the present invention exhibited various remarkable pharmacological actions as shown in a patent application filed by the same applicant on the same day as the present application. .
【００２１】本発明を以下の実施例によりさらに詳しく
説明するが、本発明の範囲はこれに限定されない。本発
明の方法を種々変更、修飾して使用することが当業者に
は可能であり、これらも本発明の範囲に含まれる。The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. It is possible for those skilled in the art to use the method of the present invention with various changes and modifications, and these are also included in the scope of the present invention.
【００２２】[0022]
【実施例】実施例１：シイタケ菌糸体抽出物の調製法 バガス９０重量部、米糖１０重量部からなる固体培地に
純水を適度に含ませた後に、シイタケ種菌を接種し、温
度および湿度を調節した培養室内に放置し、菌糸体を増
殖させた。菌糸体が固体培地に蔓延した後、バガス基材
の繊維素を解束し、１２メッシュ通過分が２４重量％以
下となるようにした。この解束された培地1.0kgに、純
水3.5Ｌを加え、４０℃に保ちながら精製セルラーゼ2.0
ｇを加えて培地含有混合物とした。EXAMPLES Example 1 Method for Preparing Shiitake Mushroom Mycelium Extract A solid medium consisting of 90 parts by weight of bagasse and 10 parts by weight of rice sugar was made to contain moderately pure water. Was allowed to stand in a conditioned culture room to grow mycelium. After the mycelium spread on the solid medium, the fibrous material of the bagasse base material was unbundled so that the amount passed through the 12 mesh was 24% by weight or less. To 1.0 kg of the unbundled medium, 3.5 L of pure water was added.
g to give a medium-containing mixture.
【００２３】次いで培地含有混合物を変速付ギヤーポン
プにより循環させながら、固体培地にギヤー部分におい
て粉砕およびすりつぶしを２００分間程度加えバカス繊
維の約８０重量％が１２メッシュ通過分となるようにし
た。培地含有混合物の粉砕およびすりつぶしは、該混合
物の温度を徐々に上昇させながら行った。その後培地含
有混合物をさらに加熱して、９０℃として３０分間放置
した。９０℃への加熱により、酵素を失活せしめ、かつ
殺菌を施した。得られた培地含有混合物を６０メッシュ
ろ布を用いてろ過してシイタケ菌糸体抽出物とし、濃縮
した後、凍結乾燥粉末を得た。Next, while the medium-containing mixture was circulated by a gear pump having a variable speed, the solid medium was crushed and ground in the gear portion for about 200 minutes so that about 80% by weight of the Bacas fiber passed 12 mesh. Grinding and grinding of the medium-containing mixture was performed while gradually increasing the temperature of the mixture. Thereafter, the medium-containing mixture was further heated to 90 ° C. and left for 30 minutes. By heating to 90 ° C., the enzyme was inactivated and sterilized. The obtained culture medium-containing mixture was filtered using a 60-mesh filter cloth to obtain a shiitake mushroom mycelium extract, which was concentrated to obtain a lyophilized powder.
【００２４】実施例２：第１工程 実施例１で得られたシイタケ菌糸体抽出物乾燥粉末１２
ｇを水２Ｌに溶解し、セライト５４５で処理して夾雑物
を除去した。これにエタノール８Ｌを加えて攪拌、静置
して生じた沈殿を遠心分離し、エタノール不溶画分を得
た。エタノール不溶画分は粗多糖類画分であり、シイタ
ケ菌糸体抽出物乾燥粉末に対し、２０％（凍結乾燥重
量）であった。Example 2 First step Dry powder 12 of Shiitake mushroom mycelium extract obtained in Example 1
g was dissolved in 2 L of water and treated with Celite 545 to remove impurities. 8 L of ethanol was added thereto, and the mixture was stirred and allowed to stand. The resulting precipitate was centrifuged to obtain an ethanol-insoluble fraction. The ethanol-insoluble fraction was a crude polysaccharide fraction, and was 20% (lyophilized weight) based on the dry powder of Shiitake mushroom mycelium extract.
【００２５】実施例３：第２工程 実施例２で得られたエタノール不溶画分１ｇを蒸留水２
００ｍＬに溶解し、ジエチルアミノエチル−トヨパール
イオン交換カラム（2.5 x 50 cm：東ソー株式会社製）
のクロマトグラフィーにかけて、蒸留水で溶出した。カ
ラムに吸着されないイオン交換体非吸着画分が流出した
後、2M NaClでカラムに吸着されたイオン交換体吸着画
分を溶離した。溶離して得た各フラクションフェノール
-硫酸法およびLowry法により波長４９０ｎｍ及び６６０
ｎｍで分光学的に測定したクロマトグラムを図２に示
す。得られたイオン交換体非吸着画分は47.5ｍｇ、イオ
ン交換体吸着画分は51.5ｍｇであった。Example 3 Second Step 1 g of the ethanol-insoluble fraction obtained in Example 2 was distilled water 2
After dissolving in 00 mL, diethylaminoethyl-Toyopearl ion exchange column (2.5 x 50 cm: manufactured by Tosoh Corporation)
And eluted with distilled water. After the non-ion exchanger non-adsorbed fraction that was not adsorbed to the column flowed out, the ion exchanger-adsorbed fraction adsorbed to the column was eluted with 2M NaCl. Each fraction phenol obtained by elution
-Wavelength 490nm and 660 by sulfuric acid method and Lowry method
The chromatogram measured spectroscopically in nm is shown in FIG. The obtained ion exchanger non-adsorbed fraction was 47.5 mg, and the ion exchanger adsorbed fraction was 51.5 mg.
【００２６】実施例４：第３工程 実施例３で得られたイオン交換体非吸着画分（４１７．
５ｍｇ）を蒸留水（１４ｍＬ）に溶解し、2.5 x 120 cm
のカラムに０．2Ｍ ＮａＣｌで平衡化したセファロー
スＣＬ−６Ｂ（ファルマシア社製）を充填したクロマト
グラフィーにかけて、0.2M NaClで溶出した。この操作
によって、分子量の大きい順にホモ多糖画分、ヘテロ多
糖画分及び低分子画分を得た。この操作で得られたクロ
マトグラムを図３に示す。得られたホモ多糖画分は10.0
2ｍｇ、ヘテロ多糖画分は63.0ｍｇ、低分子画分は11.3
ｍｇであった。Example 4: Third step The non-adsorbed fraction of the ion exchanger obtained in Example 3 (417.
5 mg) in distilled water (14 mL) and 2.5 x 120 cm
Column was packed with Sepharose CL-6B (Pharmacia) equilibrated with 0.2 M NaCl, and eluted with 0.2 M NaCl. By this operation, a homopolysaccharide fraction, a heteropolysaccharide fraction, and a low-molecular-weight fraction were obtained in the descending order of molecular weight. FIG. 3 shows the chromatogram obtained by this operation. The obtained homopolysaccharide fraction was 10.0
2mg, heteropolysaccharide fraction 63.0mg, low molecular fraction 11.3
mg.
【００２７】実施例５：各画分の組成 上記実施例で得られたシイタケ菌糸体抽出物の各画分に
ついて、シイタケ菌糸体抽出物に対する収率（％）、デ
キストランを標準とするゲルろ過法による分子量測定、
フェノール-硫酸法による糖質分析、Lowry法によるタン
パク質分析、および没食子酸を標準とするFolin-Denis
法によるポリフェノール分析を実施した。得られた結果
を表１に示す。Example 5: Composition of Each Fraction The yield (%) based on the shiitake mushroom mycelium extract of each fraction of the shiitake mushroom mycelium extract obtained in the above example, and a gel filtration method using dextran as a standard Molecular weight measurement by
Carbohydrate analysis by phenol-sulfuric acid method, protein analysis by Lowry method, and Folin-Denis using gallic acid as standard
Polyphenol analysis by the method was performed. Table 1 shows the obtained results.
【００２８】[0028]
【表１】 [Table 1]
【００２９】実施例６：分画成分の構成糖組成 加水分解管（３ｍＬ用、Pierce社製）に上記実施例で得
られた各画分１ｍｇをとり、４Ｍ トリフルオロ酢酸を
１ｍＬ加えて真空シールし、１００℃で３時間、酸加水
分解を行った。加水分解液をナスフラスコに移して粘稠
なオイル状になるまで減圧濃縮（ロータリーエバポレー
ター）し、純水２ｍＬに溶解した後、０．４５μｍフィ
ルターでろ過し、高速液体クロマトグラフィーを用いた
ポストカラムラベル法により構成単糖の定量を行った。
アミノ糖は亜硝酸-indole法、ウロン酸はカルバゾール
法を用いて測定した。得られた結果を表２に示す。Example 6: Constituent sugar composition of fraction components 1 mg of each fraction obtained in the above example was taken in a hydrolysis tube (for 3 mL, manufactured by Pierce), and 1 mL of 4M trifluoroacetic acid was added thereto, followed by vacuum sealing. Then, acid hydrolysis was performed at 100 ° C. for 3 hours. The hydrolyzed solution was transferred to an eggplant flask and concentrated under reduced pressure (rotary evaporator) until it became a viscous oil. After dissolving in 2 mL of pure water, the solution was filtered through a 0.45 μm filter, and post-column using high performance liquid chromatography. The constituent monosaccharides were quantified by the labeling method.
Amino sugar was measured by the nitrite-indole method, and uronic acid was measured by the carbazole method. Table 2 shows the obtained results.
【００３０】[0030]
【表２】 [Table 2]
【００３１】[0031]
【発明の効果】本発明の方法を用いると、担子菌類菌糸
体抽出物から各種薬理作用を持つ画分を極めて効率よ
く、また良好な分離能で得ることができる。According to the method of the present invention, fractions having various pharmacological actions can be obtained very efficiently from basidiomycete mycelium extract with good separation ability.
【図１】本発明の分離、精製方法の概略を示す図であ
る。FIG. 1 is a diagram showing an outline of a separation and purification method of the present invention.
【図２】本発明の第２工程であるイオン交換カラムクロ
マトグラフィーの結果を示す図である。FIG. 2 is a view showing the results of ion exchange column chromatography which is the second step of the present invention.
【図３】本発明の第３工程であるゲルろ過カラムクロマ
トグラフィーの結果を示す図である。FIG. 3 is a view showing the results of gel filtration column chromatography which is the third step of the present invention.
フロントページの続き (72)発明者 杉野 玲子 大阪府大阪市淀川区三津屋南３−13−35 小林製薬株式会社内 (72)発明者 白銀 英樹 大阪府大阪市淀川区三津屋南３−13−35 小林製薬株式会社内 (72)発明者 竹内 豊実 大阪府大阪市淀川区三津屋南３−13−35 小林製薬株式会社内 Ｆターム(参考） 4B065 AA71X BD16 BD18 BD27 CA44 4C088 AA02 AA03 AA04 AA05 AA06 AA07 AA08 AC17 BA04 4C090 AA01 AA04 BA01 BA61 BA91 BC18 CA13 CA15 CA18 DA09 DA23 Continued on the front page (72) Inventor Reiko Sugino 3-13-35 Mitsujinaminami, Yodogawa-ku, Osaka, Osaka Inside Kobayashi Pharmaceutical Co., Ltd. (72) Inventor Hideki Shirogane 3-13-35 Mitsumayaminami, Yodogawa-ku, Osaka, Osaka Kobayashi Pharmaceutical Co., Ltd. (72) Inventor Toyomi Takeuchi 3-13-35, Mitsujinaminami, Yodogawa-ku, Osaka-shi, Osaka F-term (reference) 4B065 AA71X BD16 BD18 BD27 CA44 4C088 AA02 AA03 AA04 AA05 AA06 AA07 AA08 AC17 BA04 4C090 AA01 AA04 BA01 BA61 BA91 BC18 CA13 CA15 CA18 DA09 DA23
Claims (4)
出物の分離、精製方法： １）担子菌類菌糸体抽出物をエタノールを含有する溶液
で処理することによりエタノール不溶性画分と可溶性画
分に分ける； ２）工程１で得られたエタノール不溶性画分をイオン交
換体で処理し、イオン交換体非吸着画分とイオン交換体
吸着画分に分ける； ３）工程２で得られたイオン交換体非吸着画分をゲルろ
過クロマトグラフィーにかけて、ホモ多糖画分、ヘテロ
多糖画分及び低分子画分に分ける。1. A method for separating and purifying a Basidiomycete mycelium extract comprising the following steps: 1) An ethanol-insoluble fraction and a soluble fraction are obtained by treating a Basidiomycete mycelium extract with a solution containing ethanol. 2) treating the ethanol-insoluble fraction obtained in step 1 with an ion exchanger, and separating it into an ion-exchanger non-adsorption fraction and an ion-exchanger adsorption fraction; 3) the ion obtained in step 2 The fraction not adsorbed by the exchanger is subjected to gel filtration chromatography to separate it into a homopolysaccharide fraction, a heteropolysaccharide fraction and a low molecular fraction.
タケ、ヒラタケ、ナメコ、イグチ、シメジ、チチタケ、
コフキサルノコシカケ、ツガサルノコシカケ、カワラタ
ケ、マンネンタケ（霊芝）、マイタケ、キクラゲ、シロ
キクラゲ、ヒメマツタケから選択される請求項１記載の
方法。2. The basidiomycetes are matsutake mushroom, shiitake mushroom, enokitake mushroom, oyster mushroom, nameko, iguchi, shimeji mushroom, chitake mushroom,
The method according to claim 1, wherein the method is selected from Kofukisaronokoshika, Tsugasarunokoshake, Kawaratake, Mannentake (Lingzhi), Maitake, Jellyfish, Shiroki jellyfish, and Himematsutake.
出物の分離、精製方法： １）シイタケ菌糸体抽出物をエタノールを含有する溶液
で処理することによりエタノール不溶性画分と可溶性画
分に分ける； ２）工程１で得られたエタノール不溶性画分をイオン交
換体で処理し、イオン交換体非吸着画分とイオン交換体
吸着画分に分ける； ３）工程２で得られたイオン交換体非吸着画分をゲルろ
過クロマトグラフィーにかけて、ホモ多糖画分、ヘテロ
多糖画分及び低分子画分に分ける。3. A method for separating and purifying a shiitake mushroom mycelium extract comprising the following steps: 1) treating a shiitake mushroom mycelium extract with a solution containing ethanol to obtain an ethanol-insoluble fraction and a soluble fraction; 2) treating the ethanol-insoluble fraction obtained in step 1 with an ion exchanger to separate it into an ion-exchanger non-adsorbed fraction and an ion-exchanger adsorbed fraction; 3) the ion exchanger obtained in step 2 The non-adsorbed fraction is subjected to gel filtration chromatography to separate into a homopolysaccharide fraction, a heteropolysaccharide fraction and a low molecular fraction.
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|---|---|---|---|
| JP10353919A JP2000159808A (en) | 1998-11-27 | 1998-11-27 | Method for separating and purifying basidiomycota hypha extract |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10353919A JP2000159808A (en) | 1998-11-27 | 1998-11-27 | Method for separating and purifying basidiomycota hypha extract |
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| WO2005027952A1 (en) * | 2003-09-17 | 2005-03-31 | Ssi Co., Ltd | Composition exerting physiological activity via biological immune mechanism |
| WO2006126488A1 (en) * | 2005-05-25 | 2006-11-30 | Unitika Ltd. | Cauliflower mushroom extract |
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