JP2000157205A - Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereof - Google Patents
Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereofInfo
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- JP2000157205A JP2000157205A JP10353920A JP35392098A JP2000157205A JP 2000157205 A JP2000157205 A JP 2000157205A JP 10353920 A JP10353920 A JP 10353920A JP 35392098 A JP35392098 A JP 35392098A JP 2000157205 A JP2000157205 A JP 2000157205A
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- fraction
- ethanol
- extract
- shiitake mushroom
- mycelium extract
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明はシイタケ菌糸体抽出
物から得られる新規画分及びこれを含む肝障害防御剤に
関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel fraction obtained from a Shiitake mushroom mycelium extract and a liver damage-protecting agent containing the same.
【0002】[0002]
【従来の技術】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300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菌糸体を形成する。2. Description of the Related Art Shiitake (Lentinus edodes) is
It is a typical edible mushroom in China, and has been cultivated artificially in Japan for about 300 years. Mushrooms that are consumed daily are called fruiting bodies, and they are germ bodies that produce spores because fungi leave their offspring, and vegetative mycelium cells form mycelia over a long period of time in the ground and in raw wood. I do.
【0003】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、抗体を介する
ADCC(antibody-dependent cell-mediated cytotox
icity)による免疫学的肝細胞障害に抑制効果を示した
(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.Mitsuhashi-Kato et al., Plant Cell Physiol. 2
6:221, 1985)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)のあることが報告さ
れている。[0003] Shiitake has long been said to be effective for various diseases and symptoms, but it has been relatively recently that its pharmacological action has been elucidated. 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, etc.), which enhance antibody production and allow antibody-mediated ADCC (antibody-dependent cell-mediated cytotox
Various reports have been reported, such as those showing an inhibitory effect on immunological hepatocellular injury due to icity) (Yasuhiro Mizoguchi et al., Hepatobiliary pancreas, 15: 127, 1987). In addition, the extract of shiitake mushroom mycelium also has phytohormonal effects such as promotion of rooting and growth of crops (M. Mitsuhashi-Kato et al., Plant Cell Physiol.
6: 221, 1985) and anti-plant virus activity (Yasuo Komuro: Report of the Institute of Plant Virus Research, Ministry of Agriculture, Forestry and Fisheries, 1977).
【0004】[0004]
【発明が解決すべき課題】本発明の目的は、シイタケ菌
糸体抽出物から新規な画分を得て、その薬理作用をさら
に詳しく解明し、シイタケ菌糸体抽出物分画物の新しい
医薬用途および/又は保健用途を探索することである。SUMMARY OF THE INVENTION An object of the present invention is to obtain a novel fraction from Shiitake mushroom mycelium extract, elucidate its pharmacological action in more detail, and obtain a new pharmaceutical use of the shiitake mushroom mycelium extract fraction. And / or exploring health applications.
【0005】[0005]
【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、シイタケ菌糸体抽出
物をエタノール含有溶液で処理することにより得られる
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)をイオン交換体で処理し水で溶出したときに
吸着されずに流出する、シイタケ菌糸体抽出物の分画物
が肝障害に対して顕著な防御効果を示すことを発見して
本発明を完成した。Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the insoluble fraction obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution (hereinafter referred to as ethanol-insoluble fraction) Fraction, which is treated with an ion exchanger and eluted with water. The fraction of Shiitake mushroom mycelium extract, which flows out without adsorption, has a remarkable protective effect against liver damage. Discovered and completed the present invention.
【0006】すなわち、本発明は、シイタケ菌糸体抽出
物のエタノール不溶画分をイオン交換体で処理し水で溶
出したときに吸着されずに流出する、シイタケ菌糸体抽
出物の分画物を提供する。[0006] That is, the present invention provides a fraction of Shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the mushroom mycelium extract is treated with an ion exchanger and eluted with water. I do.
【0007】本発明はさらに、シイタケ菌糸体抽出物の
エタノール不溶画分をイオン交換体で処理し水で溶出し
たときに吸着されずに流出する、シイタケ菌糸体抽出物
の分画物を含む肝障害防御剤を提供する。[0007] The present invention further provides a liver containing a fraction of Shiitake mushroom mycelium extract, which is not adsorbed when the ethanol-insoluble fraction of the shiitake mushroom mycelium extract is treated with an ion exchanger and eluted with water. Provide a protective agent.
【0008】本発明の肝障害防御剤は、シイタケ菌糸体
抽出物のエタノール不溶画分をイオン交換体で処理し水
で溶出したときに吸着されずに流出する、シイタケ菌糸
体抽出物の分画物及び任意成分として薬剤的に許容でき
る担体を含む、肝障害の治療用及び/又は予防用組成物
の形であってよい。[0008] The agent for protecting liver damage according to the present invention is a fraction of a shiitake mushroom mycelium extract which flows out without being adsorbed when an ethanol-insoluble fraction of a shiitake mushroom mycelium extract is treated with an ion exchanger and eluted with water. It may be in the form of a composition for the treatment and / or prophylaxis of liver disorders, comprising a product and optionally a pharmaceutically acceptable carrier.
【0009】また、本発明の肝障害防御剤は、食品の形
であってもよく、飲料の形であってもよい。[0009] The liver injury protective agent of the present invention may be in the form of a food or a beverage.
【0010】さらに、本発明の肝障害防御剤は、これら
の形態に何ら限定されるものではない。Furthermore, the liver injury protective agent of the present invention is not limited to these forms.
【0011】[0011]
【発明の実施の形態】本発明において、シイタケ菌糸体
抽出物とは、シイタケ菌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。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a shiitake mushroom mycelium extract refers to a mycelium obtained by culturing shiitake mushroom on a solid medium, preferably a solid medium containing the mycelium in the presence of water and an enzyme. An extract obtained by pulverization and decomposition.
【0012】シイタケ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シイタケ菌を接種
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・擂潰してバカス繊維の少な
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シイタケ菌糸体抽出物を得る。シイ
タケ菌糸体抽出物はそのまま本発明の防御剤に用いても
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。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 shiitake mushroom mycelium extract preferably used is one obtained by the following method, but is not limited thereto. That is, Shiitake mushrooms are inoculated on a solid medium based on bagasse (sugar cane squeezer) and defatted rice bran, and then a solid medium containing mycelium obtained by growing mycelium is 30 weight per 30 mesh passing. % Of water and one or more enzymes selected from cellulase, protease or glucosidase, and the solid medium is mixed with 30 to 55% of water.
The solid medium is crushed and crushed in the presence of the enzyme so that at least 70% by weight of the Bacass fiber passes through 12 mesh, and then the solid medium is heated to 95 ° C. The enzyme is inactivated by heating and sterilized, and the resulting suspension is filtered to obtain a shiitake mushroom mycelium extract. Shiitake mushroom mycelium extract may be used as it is in the protective agent of the present invention, but it is concentrated, freeze-dried and stored as a powder,
It is convenient to use it in various forms at the time of use. The powder obtained by freeze-drying is a brown powder, is hygroscopic and has a unique taste and odor.
【0013】このようにして得られるシイタケ菌糸体抽
出物はフェノール-硫酸法による糖質分析により糖質を
15−50%、好ましくは20−40%(w/w)、Lo
wry法によるタンパク質分析によりタンパク質を10−
40%、好ましくは13−30%(w/w)、没食子酸
を標準とするFolin-Denis法によりポリフェノールを1
−5%、好ましくは2.5−3.5%(w/w)含む。
シイタケ菌糸体抽出物にはそのほかに脂質約0.1%、
繊維約0.4%、灰分約20%を含む。The thus-obtained shiitake mushroom mycelium extract contains 15-50%, preferably 20-40% (w / w) of saccharide by Lo sugar analysis by a phenol-sulfuric acid method.
10- protein by wry protein analysis
40%, preferably 13-30% (w / w) of polyphenol by the Folin-Denis method using gallic acid as a standard.
-5%, preferably 2.5-3.5% (w / w).
Shiitake mushroom mycelium extract also contains about 0.1% lipid,
Contains about 0.4% fiber and about 20% ash.
【0014】また、シイタケ菌糸体抽出物の構成糖組成
(%)は以下の通りであった:キシロース:15.2;
アラビノール:8.2;マンノース:8.4;グルコー
ス:39.4;ガラクトース:5.4;アミノ糖:1
2.0;ウロン酸:11.3。The constituent sugar composition (%) of the shiitake mushroom mycelium extract was as follows: xylose: 15.2;
Arabinol: 8.2; Mannose: 8.4; Glucose: 39.4; Galactose: 5.4; Amino sugar: 1
2.0; uronic acid: 11.3.
【0015】本発明によるシイタケ菌糸体抽出物のエタ
ノール不溶画分をイオン交換体で処理し水で溶出したと
きに吸着されずに流出する、シイタケ菌糸体抽出物の分
画物(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)は、上述したシイタケ菌糸体抽出物、好ましく
はシイタケ菌糸体抽出物粉末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(好ましく
はセライト545)を用いてろ過した後、 1)シイタケ菌糸体抽出物にエタノールを加えて不溶性
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
体で処理し水で溶出したときに吸着されずに流出するイ
オン交換体非吸着画分と、カラムに吸着され0.05-3MのN
aCl、好ましくは2M NaClで溶離する画分(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、こ
とによって得られる。[0015] The fraction of Shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the Shiitake mushroom mycelium extract according to the present invention is treated with an ion exchanger and eluted with water (hereinafter referred to as ion exchange) The non-body-adsorbed fraction is sometimes used as a solid particle filter agent (preferably, a shiitake mushroom mycelium extract, preferably a shiitake mushroom mycelium extract powder as an aqueous solution of an appropriate concentration to remove impurities. After filtration using Celite 545), 1) ethanol is added to the Lentinus edodes mycelium extract to separate it into an insoluble fraction and a soluble fraction; 2) the ethanol-insoluble fraction obtained in step 1 is treated with an ion exchanger A non-adsorbed non-adsorbed fraction which is not adsorbed when eluted with water and adsorbed to the column with 0.05-3M N
aCl, preferably a fraction eluted with 2M NaCl (hereinafter sometimes referred to as an ion-exchanger-adsorbed fraction).
【0016】上記1)の工程では、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
ル不溶画分と可溶性画分に分ける。エタノール不溶画分
は粗多糖類を含む画分であり、主成分として糖質を5
4.3%、タンパク質を14.6%含む。In the above step 1), ethanol (preferably 4 volumes) is added to the Lentinus edodes mycelium extract to separate into an ethanol-insoluble fraction and a soluble fraction. The ethanol-insoluble fraction is a fraction containing a crude polysaccharide, and contains saccharide as a main component.
Contains 4.3%, 14.6% protein.
【0017】エタノール不溶画分はシイタケ菌糸体抽出
物から20%の収率で得られた。エタノール不溶画分の
構成糖組成(%)は以下の通りであった:キシロース:
15.0;アラビノース:9.3;マンノース:12.
9;グルコース:28.7;ガラクトース:10.7;
アミノ糖:12.5;ウロン酸:10.9。The ethanol-insoluble fraction was obtained from the shiitake mushroom mycelium extract in a yield of 20%. The constituent sugar composition (%) of the ethanol-insoluble fraction was as follows: xylose:
15.0; Arabinose: 9.3; Mannose: 12.
9; glucose: 28.7; galactose: 10.7;
Amino sugar: 12.5; uronic acid: 10.9.
【0018】次いで、2)の工程では、1)で得られた
エタノール不溶画分を蒸留水に再溶解し、イオン交換体
で処理し、吸着されないで流出するイオン交換体非吸着
画分と、カラムに吸着され、0.05-3MのNaCl、好ましく
は2MのNaClで溶離するイオン交換体吸着画分に分ける。
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)のカラム式であ
る。第2工程で得られるクロマトグラムの一例を図1に
示す。Next, in the step 2), the ethanol-insoluble fraction obtained in the step 1) is redissolved in distilled water, treated with an ion exchanger, and an ion-exchanger non-adsorbed fraction flowing out without being adsorbed; The adsorbent fraction is adsorbed on the column and eluted with 0.05-3M NaCl, preferably 2M NaCl.
As an ion exchanger, for example, diethylaminoethyl
Anions such as synthetic resin ion exchanger (Cl- type), diethylaminoethyl cellulose, diethylaminoethyl agarose, diethyl- (2-hydroxypropyl) aminoethyl cellulose, and diethyl- (2-hydroxypropyl) aminoethyl agarose An exchanger can be used, and a column type of a diethylaminoethyl-toyopearl ion exchanger (manufactured by Tosoh Corporation) is preferable. FIG. 1 shows an example of the chromatogram obtained in the second step.
【0019】本発明の分画物(イオン交換体非吸着画
分)はシイタケ菌糸体抽出物から換算して9.5%の収
率で得られた。イオン交換体非吸着画分は糖質21.6
%、タンパク質2.6%、ポリフェノール0.1%(い
ずれもw/w)を含んでいた。The fraction of the present invention (the fraction not adsorbed by the ion exchanger) was obtained in a yield of 9.5% in terms of Shiitake mushroom mycelium extract. The fraction not adsorbed by the ion exchanger had a sugar content of 21.6.
%, 2.6% protein, and 0.1% polyphenol (all w / w).
【0020】イオン交換体非吸着分画の構成糖組成
(%)は以下の通りであった:マンノース:14.3;
グルコース:63.9;ガラクトース:21.8;アミ
ノ糖:3.6;ウロン酸:1.1。The constituent sugar composition (%) of the non-adsorbed fraction of the ion exchanger was as follows: mannose: 14.3;
Glucose: 63.9; galactose: 21.8; amino sugar: 3.6; uronic acid: 1.1.
【0021】一方、イオン交換体吸着画分はシイタケ菌
糸体抽出物から換算して10.3%の収率で得られた。
イオン交換体吸着画分は糖質47.4%、タンパク質1
8.2%、ポリフェノール2.7%(いずれもw/w)
を含んでいた。On the other hand, the ion-exchanger-adsorbed fraction was obtained in a yield of 10.3% in terms of Shiitake mushroom mycelium extract.
The ion-exchanger-adsorbed fraction contains 47.4% of carbohydrate and 1 protein
8.2%, Polyphenol 2.7% (all w / w)
Was included.
【0022】イオン交換体吸着画分の構成糖組成(%)
は以下の通りであった:キシロース:23.2;アラビ
ノース:13.5;マンノース:18.7;グルコー
ス:31.9;ガラクトース:12.7;アミノ糖:
9.1;ウロン酸:3.0。Saccharide composition (%) of the ion-exchanger adsorbed fraction
Was as follows: xylose: 23.2; arabinose: 13.5; mannose: 18.7; glucose: 31.9; galactose: 12.7;
9.1; uronic acid: 3.0.
【0023】本発明によるシイタケ菌糸体抽出物のエタ
ノール不溶画分をイオン交換体で処理し水で溶出したと
きに吸着されずに流出する、シイタケ菌糸体抽出物の分
画物(イオン交換体非吸着画分)の肝障害に対する防御
効果を以下の方法により試験したところ、ラット肝細胞
を用いるin vitro試験において、顕著な防御効果が観察
された。The ethanol-insoluble fraction of the shiitake mushroom mycelium extract according to the present invention is treated with an ion exchanger and eluted with water. When the protective effect of the adsorbed fraction) on liver damage was tested by the following method, a remarkable protective effect was observed in an in vitro test using rat hepatocytes.
【0024】本発明の防御剤は、治療剤及び/又は予防
剤及び/又は保健用剤である。本発明の防御剤は、セフ
ロキサジン、セフロキシムナトリウム、ラタモキセフナ
トリウムなどのセフェム系、硫酸アミカシンなどのアミ
ノ配糖体系、アクラルビシンなどの抗腫瘍系、リファン
ピシンなどの抗酸菌抗生物質系、テトラサイクリン系、
マクロライド系、ペニシリン系などの抗生物質、中枢神
経用薬である、アスピリンなどの解熱鎮痛消炎系、クロ
キサゾラムなどの精神神経系、バルプロ酸ナトリウムな
どの抗てんかん剤系、ハロタンなどの全身麻酔剤系、フ
ェノバルビタールなどの催眠鎮静系、総合感冒薬系など
の薬剤の投与によって引き起こされる肝障害、またこれ
らの他に、ピンドロールなどの不整脈用系、トラビジル
などの血管拡張剤系、塩酸ニカルジピンなどの循環器官
用系、塩酸ラベタロールなどの降圧剤系、クロフィブラ
ートなどの動脈硬化用系で知られる循環器用薬、さらに
テガフールなどの代謝拮抗剤系やエストラサイトなどの
ホルモン剤やスルファメトキサゾールなどのサルファ
剤、イソニアジドなどの抗結核剤、ノルフロキサシンな
どの合成抗菌剤などの薬剤の投与により引き起こされる
肝障害、さらには、生活環境中に存在するビスフェノー
ル、フタル酸エステル、塩素化合物、アルキルフェノー
ル、アルコール類などの環境有害物質によって引き起こ
される肝障害に有用であるのみならず、免疫学的肝障
害、ウィルス性肝障害、脂肪肝、肝癌、アルコール性肝
障害、肝硬変などに有効である。従って、シイタケ菌糸
体抽出物のエタノール不溶画分をイオン交換体で処理し
水で溶出したときに吸着されずに流出する、シイタケ菌
糸体抽出物の分画物及び任意成分として薬剤的に許容で
きる担体を含む、肝障害の治療用及び/又は予防用組成
物として使用することができる。The protective agent of the present invention is a therapeutic agent and / or a prophylactic agent and / or a health agent. The protective agent of the present invention includes cefemazines such as cefloxazine, cefuroxime sodium and latamoxef sodium, aminoglycosides such as amikacin sulfate, antitumor systems such as aclarubicin, mycobacterial antibiotics such as rifampicin, and tetracyclines. ,
Antibiotics such as macrolides and penicillins, drugs for central nervous system, antipyretic analgesic and anti-inflammatory systems such as aspirin, mental and nervous systems such as cloxazolam, antiepileptic agents such as sodium valproate, and general anesthetics such as halothane , Hepatic disorders caused by administration of drugs such as sedative hypnotics such as phenobarbital, general cold remedy, and arrhythmia such as pindolol, vasodilators such as travidil, and circulation of nicardipine hydrochloride Organ system, antihypertensive system such as labetalol hydrochloride, cardiovascular drugs known in the arteriosclerotic system such as clofibrate, antimetabolites such as tegafur, hormonal agents such as estrasite, and sulfamethoxazole Anti-tuberculosis drugs such as sulfa drugs, isoniazid, and synthetic antibacterial drugs such as norfloxacin It is useful not only for liver damage caused by the administration of drugs, but also for liver damage caused by environmental harmful substances such as bisphenol, phthalate ester, chlorine compound, alkylphenol, and alcohol present in the living environment. Liver disease, viral liver injury, fatty liver, liver cancer, alcoholic liver injury, cirrhosis, and the like. Therefore, the ethanol-insoluble fraction of the Lentinula edodes mycelium extract is treated with an ion exchanger and eluted with water. It can be used as a composition for treating and / or preventing liver damage, comprising a carrier.
【0025】投与経路は経口投与が最も好ましいが、静
脈内投与、皮下投与などであってもよい。経口投与に適
した製剤には、錠剤、カプセル剤、散剤、顆粒剤、溶液
剤、シロップ剤などが含まれるが、これに限定されな
い。The administration route is most preferably oral administration, but may be intravenous administration, subcutaneous administration and the like. Formulations suitable for oral administration include, but are not limited to, tablets, capsules, powders, granules, solutions, syrups and the like.
【0026】薬剤的に許容できる担体には、当業界で公
知の適当な賦形剤、結合剤、崩壊剤、滑沢剤、着香料、
着色剤、溶解補助剤、懸濁剤、コーティング剤などを含
むが、これに限定されない。Pharmaceutically acceptable carriers include suitable excipients, binders, disintegrants, lubricants, flavoring agents, and the like known in the art.
Examples include, but are not limited to, colorants, solubilizing agents, suspending agents, coating agents, and the like.
【0027】本発明の防御剤の投与量は患者の年齢、体
重、症状、投与経路などを考慮して医師、薬剤師、栄養
士などにより決定される。本発明の防御剤に含まれるシ
イタケ菌糸体抽出物は食品として使用されてきたもので
あり、極めて安全であるところから、投与量を厳しく限
定する必要はないが、通常イオン交換体非吸着画分に換
算して1日あたり3mg−600mg、好ましくは30
mg−200mgである。さらに、肝障害の原因となる
薬剤と併用して投与しても支障はない。The dose of the protective agent of the present invention is determined by a physician, pharmacist, dietitian or the like in consideration of the patient's age, body weight, symptoms, administration route and the like. The shiitake mushroom mycelium extract contained in the protective agent of the present invention has been used as a food, and since it is extremely safe, it is not necessary to strictly limit the dose, but usually the ion-exchange non-adsorbed fraction 3 mg-600 mg per day, preferably 30
mg-200 mg. Furthermore, there is no problem even when administered in combination with a drug that causes liver damage.
【0028】本発明の防御剤は、食品の形で提供するこ
ともできる。好ましい食品としては顆粒、麺類、キャン
ディー、ゼリー、クッキーなどが挙げられる。さらに、
本発明の防御剤は、飲料の形で提供することもできる。
このような食品、飲料にはシイタケ菌糸体抽出物の他
に、ビタミン剤、カルシウムなどの無機成分、キトサン
などの食物繊維、大豆抽出物などの蛋白質、レシチンな
どの脂質、乳糖などの糖類などを追加してもよい。The protective agent of the present invention can be provided in the form of a food. Preferred foods include granules, noodles, candies, jellies, cookies and the like. further,
The protective agent of the present invention can also be provided in the form of a beverage.
Such foods and beverages contain, in addition to shiitake mushroom mycelium extract, vitamins, inorganic components such as calcium, dietary fiber such as chitosan, proteins such as soybean extract, lipids such as lecithin, and sugars such as lactose. May be added.
【0029】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.
【0030】[0030]
【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 was added to obtain a mixture containing the medium.
【0031】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
て粉砕および擂潰作用を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擂潰は、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 circulating the medium-containing mixture with a gear pump having a variable speed, the solid medium was subjected to a pulverizing and crushing action in the gear portion for about 200 minutes so that about 80% by weight of the Bacass fiber passed 12 mesh. The pulverization and crushing of the medium-containing mixture were 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.
【0032】実施例2:エタノール不溶画分の調製 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、沈殿物を得た。沈殿画分
(エタノール不溶分画)は粗多糖類画分であり、シイタ
ケ菌糸体抽出物に対し、20%(凍結乾燥重量)であっ
た。Example 2 Preparation of Ethanol Insoluble Fraction Shiitake Mushroom Mycelium Extract Dry Powder 12 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 a precipitate. The precipitate fraction (ethanol-insoluble fraction) was a crude polysaccharide fraction, which was 20% (lyophilized weight) with respect to the shiitake mushroom mycelium extract.
【0033】実施例3:イオン交換クロマトグラフィー 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をフェノー
ル−硫酸法およびLowry法により波長490nm及び6
60nmで分光学的に測定したクロマトグラムを図1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 Ion Exchange Chromatography 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 obtained by elution was subjected to phenol-sulfuric acid method and Lowry method at wavelengths of 490 nm and 6 nm.
The chromatogram measured spectrophotometrically at 60 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.
【0034】実施例4:組成分析 実施例1で得たシイタケ菌糸体抽出物、実施例2で得た
エタノール不溶画分、実施例3で得られたイオン交換体
非吸着画分及びイオン交換体吸着画分について、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 4: Composition analysis Shiitake mushroom mycelium extract obtained in Example 1, ethanol-insoluble fraction obtained in Example 2, ion-exchanger non-adsorbed fraction obtained in Example 3, and ion exchanger The adsorbed fraction was subjected to carbohydrate analysis by the phenol-sulfuric acid method, protein analysis by the Lowry method, and polyphenol analysis by the Folin-Denis method using gallic acid as a standard. Table 1 shows the obtained results.
Shown in
【0035】[0035]
【表1】 [Table 1]
【0036】実施例5:構成糖組成 加水分解管(3mL用、Pierce社製)に実施例1で得た
シイタケ菌糸体抽出物、実施例2で得たシイタケ菌糸体
抽出物エタノール不溶画分、実施例3で得られたイオン
交換体非吸着画分及びイオン交換体吸着画分を、それぞ
れ1mg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 5: Constituent sugar composition Hydrolyzate tube (for 3 mL, manufactured by Pierce) was put into the shiitake mushroom mycelium extract obtained in Example 1, the shiitake mushroom mycelium extract obtained in Example 2 ethanol-insoluble fraction, 1 mg of each of the ion-exchanger-non-adsorbed fraction and the ion-exchanger-adsorbed fraction obtained in Example 3 was taken, 1 mL of 4M trifluoroacetic acid was added, and the mixture was vacuum-sealed and subjected to acid hydrolysis at 100 ° C. for 3 hours. Was. Transfer the hydrolyzed liquid to an eggplant flask and concentrate under reduced pressure (rotary evaporator) until it becomes a viscous oil.
After dissolving in 2 mL of pure water, the solution was filtered with a 0.45 μm filter, and the constituent monosaccharide was quantified by a post-column labeling method using high performance liquid chromatography. Amino sugar was measured by the nitrite-indole method, and uronic acid was measured by the carbazole method. Table 2 shows the obtained results.
【0037】[0037]
【表2】 [Table 2]
【0038】実施例6:初代培養肝細胞を用いるin vit
ro試験 1)ラット肝細胞の分離及び初代培養 ラット(Wistar、雄、6−8週齢)の肝細胞を分離し、
37℃、5%CO2条件下で一晩培養した。 2)肝細胞のCCl4及び被験物質による処理 1)で調製した肝細胞に、CCl4(DMSO溶液:肝
障害誘導物質)及び被験物質(イオン交換体非吸着画
分)を種々の濃度で調製した無血清培地を500μl加
え、37℃、5%CO2条件下で培養した。 3)GOT活性測定 培養液をエッペンドルフチューブに移し、10000r
pm、5分間、室温にて遠心した。この培養液上清中の
GOT活性をGOT測定試薬(リキテックGOT IF
CC:ベーリンガーマンハイム社製)を用い、測定し
た。被験物質を加えていない培養液上清のGOT活性の
平均値を100としたときの各培養液上清のGOT活性
の割合を求め、肝障害強度とした。その結果を図2に示
す。Example 6 In Vitro Using Primary Cultured Hepatocytes
ro test 1) Isolation and primary culture of rat hepatocytes Hepatocytes of rat (Wistar, male, 6-8 weeks old) were isolated,
The cells were cultured overnight at 37 ° C. and 5% CO 2 . 2) Treatment of hepatocytes with CCl 4 and test substance To the hepatocytes prepared in 1), CCl 4 (DMSO solution: liver injury inducer) and test substance (ion exchanger non-adsorbed fraction) were prepared at various concentrations. 500 μl of the serum-free medium thus obtained was added, and the cells were cultured at 37 ° C. under 5% CO 2 conditions. 3) GOT activity measurement Transfer the culture to an Eppendorf tube and
pm for 5 minutes at room temperature. The GOT activity in the culture supernatant was measured using a GOT measurement reagent (Liquitec GOT IF
CC: Boehringer Mannheim). The ratio of the GOT activity of each culture supernatant was determined when the average value of the GOT activity of the culture supernatant to which the test substance was not added was taken as 100, and the ratio was defined as liver injury intensity. The result is shown in FIG.
【0039】図から明らかなように、シイタケ菌糸体抽
出物のイオン交換体非吸着画分は肝障害防御効果が観察
された。As is clear from the figure, the ion-exchanger-non-adsorbed fraction of the shiitake mushroom mycelium extract was observed to have a protective effect against liver damage.
【0040】[0040]
【発明の効果】本発明のシイタケ菌糸体抽出物のエタノ
ール不溶画分をイオン交換体で処理し水で溶出したとき
に吸着されずに流出する、シイタケ菌糸体抽出物の分画
物は顕著な肝障害の防御効果を有しており、肝障害の予
防、治療に使用できる。本発明の防御剤は、肝障害の原
因となる薬剤の投与と併用することができ、また副作用
がないことから、安全に使用でき、大きな産業上の利用
可能性が期待できる。EFFECTS OF THE INVENTION The fraction of the shiitake mushroom mycelium extract which is not adsorbed when the ethanol-insoluble fraction of the shiitake mushroom mycelium extract of the present invention is treated with an ion exchanger and eluted with water is remarkable. It has a protective effect on liver damage and can be used for prevention and treatment of liver damage. The protective agent of the present invention can be used in combination with the administration of a drug that causes liver damage, and has no side effects. Therefore, it can be used safely, and great industrial applicability can be expected.
【図1】分離の第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. 1 is a diagram showing the results of ion exchange column chromatography, which is the second step of separation.
【図2】ラット初代培養肝細胞を用いるin vitro試験に
おける、本発明の分画物の肝障害防御試験の結果を示す
グラフである。FIG. 2 is a graph showing the results of a liver damage protection test of the fraction of the present invention in an in vitro test using primary cultured rat hepatocytes.
フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) A61K 35/84 A23L 2/00 F (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) 4B017 LC03 LG19 LP01 LP03 LP08 4B018 LB08 LE03 MD83 ME14 MF01 MF06 MF07 4C088 AA08 AC17 BA05 BA12 BA16 CA14 CA19 MA16 MA52 NA14 ZA75 Continuation of the front page (51) Int.Cl. 7 Identification symbol FI Theme coat II (Reference) A61K 35/84 A23L 2/00 F (72) Inventor Reiko Sugino 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka-shi, Osaka Kobayashi Inside Pharmaceutical Co., Ltd. (72) Inventor Hideki Shirogane 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka, Osaka Prefecture Inside Kobayashi Pharmaceutical Co., Ltd. (72) Toyomi Takeuchi 3-13-35 Minitsuya Minami, Yodogawa-ku, Osaka, Osaka Kobayashi Pharmaceutical Co., Ltd. F-term (reference) 4B017 LC03 LG19 LP01 LP03 LP08 4B018 LB08 LE03 MD83 ME14 MF01 MF06 MF07 4C088 AA08 AC17 BA05 BA12 BA16 CA14 CA19 MA16 MA52 NA14 ZA75
Claims (6)
液で処理して得られるエタノール不溶性画分をイオン交
換体で処理し水で溶出したときに吸着されずに流出す
る、シイタケ菌糸体抽出物の分画物。An ethanol-insoluble fraction obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution is treated with an ion exchanger and eluted with water. Fractions.
液で処理して得られるエタノール不溶性画分をイオン交
換体で処理し水で溶出したときに吸着されずに流出す
る、シイタケ菌糸体抽出物の分画物を含む肝障害防御
剤。2. A method according to claim 1, wherein the ethanol-insoluble fraction obtained by treating the Lentinus edodes mycelium extract with an ethanol-containing solution is treated with an ion exchanger and eluted with water. Liver injury protective agent containing fractions.
液で処理して得られるエタノール不溶性画分をイオン交
換体で処理し水で溶出したときに吸着されずに流出す
る、シイタケ菌糸体抽出物の分画物及び任意成分として
薬剤的に許容できる担体を含む、肝障害の治療用及び/
又は予防用組成物である請求項2記載の防御剤。3. An extract of a shiitake mushroom mycelium extract, which is obtained by treating a shiitake mushroom mycelium extract with an ethanol-containing solution, treating the ethanol-insoluble fraction with an ion exchanger, and eluted with water when eluted with water. For the treatment of liver damage and / or comprising a fraction and optionally a pharmaceutically acceptable carrier.
3. The protective agent according to claim 2, which is a prophylactic composition.
剤。4. The protective agent according to claim 2, which is administered orally.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10353920A JP2000157205A (en) | 1998-11-27 | 1998-11-27 | Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10353920A JP2000157205A (en) | 1998-11-27 | 1998-11-27 | Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
JP2000157205A true JP2000157205A (en) | 2000-06-13 |
Family
ID=18434119
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP10353920A Withdrawn JP2000157205A (en) | 1998-11-27 | 1998-11-27 | Fractionated substance of extract from mycelium of lentinus edodes sing. and use thereof |
Country Status (1)
Country | Link |
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JP (1) | JP2000157205A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005072763A1 (en) * | 2004-01-30 | 2005-08-11 | Kobayashi Pharmaceutical Co., Ltd. | Liver fibrosis inhibitor |
-
1998
- 1998-11-27 JP JP10353920A patent/JP2000157205A/en not_active Withdrawn
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005072763A1 (en) * | 2004-01-30 | 2005-08-11 | Kobayashi Pharmaceutical Co., Ltd. | Liver fibrosis inhibitor |
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