JPWO2006126488A1 - Hanabira bamboo extract - Google Patents
Hanabira bamboo extract Download PDFInfo
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- JPWO2006126488A1 JPWO2006126488A1 JP2007517815A JP2007517815A JPWO2006126488A1 JP WO2006126488 A1 JPWO2006126488 A1 JP WO2006126488A1 JP 2007517815 A JP2007517815 A JP 2007517815A JP 2007517815 A JP2007517815 A JP 2007517815A JP WO2006126488 A1 JPWO2006126488 A1 JP WO2006126488A1
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- hanabiratake
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- antitumor
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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Abstract
優れた薬理活性を有し、消化管から吸収され易い比較的低分子量の抗腫瘍成分、ならびに、その成分を有効成分とする抗腫瘍剤、化粧品及び健康食品を提供する。ハナビラタケの子実体又は菌糸体を凍結乾燥の後、微粉末化し、その粉末を溶媒抽出処理し、抽出物を分画処理することにより得られる分子量が500〜10000の範囲内の成分を主成分として含有するハナビラタケ抽出物組成物及びそれを有効成分とする抗腫瘍性組成物、化粧品及び健康食品。Provided are an antitumor component having a relatively low molecular weight that has excellent pharmacological activity and is easily absorbed from the digestive tract, and an antitumor agent, cosmetics, and health food containing the component as an active ingredient. The fruit body or mycelium of garlic mushroom is freeze-dried and then pulverized, the powder is subjected to solvent extraction treatment, and the extract is fractionated to obtain a component having a molecular weight in the range of 500 to 10,000 as a main component. Containing Hanabiratake extract composition, antitumor composition, cosmetic and health food containing the composition as an active ingredient.
Description
本発明は、ハナビラタケの子実体又は菌糸体から得られる比較的低分子量の成分を主成分とするハナビラタケ抽出物並びにそれを有効成分として含有する抗腫瘍剤、化粧品及び健康食品に関するものである。 The present invention relates to an extract of Hanabira bamboo mainly composed of a relatively low molecular weight component obtained from the fruit body or mycelium of Hanabira bamboo, and an antitumor agent, a cosmetic and a health food containing the same as an active ingredient.
キノコ類は古くから食用として利用されているが、近年、その成分の生理活性が明らかにされてきている。なかでも、クレスチン、レンチナン、シゾフィラン(いずれも商品名)等は、医薬品としてその有用性が認められおり、また、アガリクス、メシマコブ、霊芝等は免疫賦活作用や抗腫瘍作用を期待して、子実体や菌糸体の乾燥物あるいは抽出物を健康食品素材として利用することが試みられている。例えば、マンネンタケは、子実体又はその抽出物を健康食品の原料として利用することが試みられている(例えば、特許文献1、2及び3参照)。 Mushrooms have been used for food since ancient times, but in recent years, the physiological activity of the components has been clarified. Among them, krestin, lentinan, and schizophyllan (all trade names) are recognized as useful drugs, and agaricus, mesimacob, ganoderma, etc. Attempts have been made to use dried or extracted substances or mycelia as health food materials. For example, Mannentake has been tried to use fruit bodies or extracts thereof as raw materials for health foods (see, for example, Patent Documents 1, 2, and 3).
一方、これらキノコ類に属するハナビラタケ(Sparassis crispa)は、カラマツ等に生えるキノコであって、歯ごたえが良く、その純白な色合いと葉牡丹のような形態が特徴である、非常に僅少な食用キノコである。これまで、このハナビラタケは成長が遅く人工栽培は非常に困難であるとされてきたが、近年、比較的短期間で栽培可能な新しい栽培方法が確立され、商業規模での供給が可能となっている(例えば、特許文献4及び5参照)。 On the other hand, Hanabiratake (Sparasis crispa) belonging to these mushrooms is a very small edible mushroom that grows in larch etc., has a good texture and is characterized by its pure white color and leafy peony. . Up until now, it has been said that this bamboo shoot has slow growth and is extremely difficult to cultivate artificially. (For example, see Patent Documents 4 and 5).
そのハナビラタケの子実体は、抗腫瘍活性を有することがこれまでに明らかになっている(例えば、非特許文献1、2参照)。そして、その有効成分は、ハナビラタケの乾燥重量の40〜50%程度を占めるβ−1,3−グルカンであるといわれている(例えば、特許文献6参照)。
キノコ類の抗腫瘍活性の主体とされていたβグルカンについては、分子量が数百万程度と大きく、一般的には必ずしも消化管からの吸収が良いとはいえない。そこで、より低分子で消化管からの吸収が良好な抗腫瘍成分が強く望まれていた。 Β-glucan, which has been regarded as the main antitumor activity of mushrooms, has a large molecular weight of about several millions and is generally not necessarily well absorbed from the digestive tract. Therefore, an antitumor component having a lower molecular weight and good absorption from the gastrointestinal tract has been strongly desired.
本発明は、ハナビラタケ子実体又は菌糸体由来の低分子抗腫瘍成分並びにそれを有効成分とする抗腫瘍剤、化粧品及び健康食品を提供することを目的とするものである。 An object of the present invention is to provide a low molecular weight antitumor component derived from a fruit body or a mycelium of an alga, and an antitumor agent, a cosmetic and a health food containing the same as an active ingredient.
本発明者らは、上記課題を達成するために鋭意検討を重ねた結果、ハナビラタケの子実体又は菌糸体の溶媒抽出物より得られる、βグルカンをほとんど含有しない低分子成分が良好な抗腫瘍活性を有することを見出し、本発明に至った。 As a result of intensive studies to achieve the above-mentioned problems, the present inventors have obtained a low molecular weight component containing almost no β-glucan, which is obtained from a fruit extract or a mycelium extract of Hanabiratake, and has a good antitumor activity. As a result, the present invention was reached.
すなわち、本発明は、ハナビラタケの溶媒抽出物であって、主成分が分子量500〜10000の範囲内の成分であるハナビラタケ抽出物を要旨とするものであり、別の発明は、ハナビラタケの溶媒抽出物から、透析膜を用いて主成分が分子量500〜10000の範囲内の成分を分画して得られるハナビラタケ抽出物を要旨とするものであり、また別の発明は、ハナビラタケの溶媒抽出物から、ゲル濾過法により主成分が分子量500〜10000の範囲内の成分を分画して得られるハナビラタケ抽出物を要旨とするものであり、また別の発明は、ハナビラタケの溶媒抽出物から、エタノール沈澱を行なうことにより主成分が分子量500〜10000の範囲内の成分を分画して得られるハナビラタケ抽出物を要旨とするものである。また他の発明は、前記したハナビラタケ抽出物を有効成分として含有する抗腫瘍剤を要旨とするものであり、また別の発明は前記したハナビラタケ抽出物を有効成分として含有する化粧品を要旨とするものであり、さらに別の発明は前記したハナビラタケ抽出物を有効成分として含有する健康食品を要旨とするものである。
本発明は、上記ハナビラタケの溶媒抽出物を対象に投与することによる腫瘍の治療方法または腫瘍細胞の増殖抑制方法も提供し、さらに、抗腫瘍剤の製造のための上記ハナビラタケの溶媒抽出物の使用も提供する。That is, the present invention is a solvent extract of Hanabiratake, the main component of which is a component having a molecular weight in the range of 500 to 10,000, and another invention is a solvent extract of Hanabiratake. In addition, the ginseng extract is obtained by fractionating components having a molecular weight of 500 to 10,000 using a dialysis membrane, and another invention is based on the solvent extract of Hanabiratake, The gist of the present invention is that of an extract of Hanabiratake obtained by fractionating components having a molecular weight of 500 to 10,000 by gel filtration, and another invention relates to ethanol precipitation from a solvent extract of Hanabiratake. A ginseng extract obtained by fractionating a component having a molecular weight of 500 to 10,000 as a main component by carrying out the present invention. Another aspect of the invention is a gist of an antitumor agent containing the above-mentioned Hanabiratake extract as an active ingredient, and another invention is a gist of a cosmetic containing the above-mentioned Hanabiratake extract as an active ingredient. Yet another invention is summarized as a health food containing the above-mentioned Hanabiratake extract as an active ingredient.
The present invention also provides a method for treating a tumor or a method for inhibiting the growth of tumor cells by administering the above-mentioned solvent extract of Hanabiratake to a subject, and further uses the solvent extract of Hanabiratake for the production of an antitumor agent. Also provide.
本発明によれば、分子量が500〜10000の範囲の低分子成分であるため、経口投与した場合に消化管からの吸収に優れ、その結果抗腫瘍活性が向上するという効果を奏する。 According to the present invention, since it is a low molecular weight component having a molecular weight in the range of 500 to 10,000, when administered orally, it is excellent in absorption from the gastrointestinal tract, resulting in an effect of improving antitumor activity.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
ハナビラタケは、標高1000メートル以上のカラマツ等の針葉樹林に特異的に発生するキノコであり、発見することが困難なために「幻のキノコ」と言われてきた。これまで、その栽培は難しく、一般にはあまり知られていなかったが、近年、人工栽培方法が確立され、量産が可能となった。 Hanabiratake is a mushroom that occurs specifically in coniferous forests such as larch at an altitude of 1000 meters or more, and has been said to be a “phantom mushroom” because it is difficult to find. Until now, its cultivation has been difficult and generally not well known, but in recent years, an artificial cultivation method has been established and mass production has become possible.
本発明で用いられるハナビラタケ子実体は、その製造方法は特に限定されるものではなく、天然ものでも、人工栽培により得られたものであっても良い。人工栽培の方法としては、従来から知られている人工栽培用の菌床を作製することにより行なうことができる(詳細は、特開平11−56098号公報、特開2002−369621号公報及び特開2002−125460号公報、米国特許6,212,822などを参照)。 The manufacturing method of the fruit body used in the present invention is not particularly limited, and may be natural or artificially cultivated. Artificial cultivation can be performed by preparing a conventionally known fungal bed for artificial cultivation (for details, see Japanese Patent Application Laid-Open Nos. 11-56098, 2002-369621, and Japanese Patent Application Laid-Open No. 2002-369621). 2002-125460, US Pat. No. 6,212,822, etc.).
また、本発明で用いられるハナビラタケ菌糸体は、従来の液体培養法によって得ることができる。培地に使用する炭素源としては、グルコース等の単糖の他、デキストリン、グリセロール等、通常用いられる炭素源が使用できる。窒素源としては無機または有機窒素源が使用できるが、生育速度の観点からは有機窒素源を用いるほうが好ましい。また、必要に応じて微量元素やビタミン等の生育因子を添加することは通常の培養と何ら変わりはない。培養温度は15℃〜30℃、pHは2.5〜8.0が好ましい。培地成分には不溶成分を添加することが均一に生育させることができることから好ましい。培養期間は菌株により、数日から数週間程度に設定されうる。 The mycelium mycelium used in the present invention can be obtained by a conventional liquid culture method. As the carbon source used in the medium, a commonly used carbon source such as dextrin and glycerol can be used in addition to a monosaccharide such as glucose. An inorganic or organic nitrogen source can be used as the nitrogen source, but it is preferable to use an organic nitrogen source from the viewpoint of the growth rate. Moreover, adding growth factors such as trace elements and vitamins as needed is no different from normal culture. The culture temperature is preferably 15 ° C to 30 ° C, and the pH is preferably 2.5 to 8.0. It is preferable to add an insoluble component to the medium component because it can be grown uniformly. The culture period can be set to several days to several weeks depending on the strain.
このようにして得られたハナビラタケ子実体又は菌糸体は次の抽出工程に移されるが、その際のハナビラタケの形態は特に限定されるものではなく、そのままの状態で、あるいは凍結乾燥物や乾燥物を微粉末化したものであっても良い。なかでも、乾燥物を微粉末化したものが抽出効率の点で好ましい。 The fruit body or mycelium obtained in this way is transferred to the next extraction step, but the form of the flower is not particularly limited, and is used as it is or in a freeze-dried product or a dried product. May be finely powdered. Among these, a powdered dry product is preferable from the viewpoint of extraction efficiency.
抽出工程で用いられる抽出溶媒としては、水、メタノール、エタノール、イソプロピルアルコール、ブタノール、グリセリン、エチレングリコール、1,3−ブチレングリコール、アセトン、クロロホルム、酢酸エチル、ヘキサン、エーテルなどが使用できる。これらの中で、水が好ましく、中でも熱水がさらに好ましい。熱水による抽出方法は特に限定されるものではなく、例えば、ハナビラタケ子実体又は菌糸体の乾燥粉末に5〜100倍量の水を添加し、80〜100℃(高圧条件下では100〜121℃)で20分〜3時間、煮沸(加熱)する、あるいは、乾燥粉末に5〜100倍量の水を添加し、1〜3時間加熱還流する。それら熱水抽出処理を数回繰り返す等、いかなる方法でも構わない。 As the extraction solvent used in the extraction step, water, methanol, ethanol, isopropyl alcohol, butanol, glycerin, ethylene glycol, 1,3-butylene glycol, acetone, chloroform, ethyl acetate, hexane, ether, or the like can be used. Among these, water is preferable, and hot water is more preferable. The extraction method using hot water is not particularly limited. For example, 5 to 100 times the amount of water is added to the dried powder of the fruit body or mycelia, and 80 to 100 ° C. (100 to 121 ° C. under high pressure conditions). ) Boil (heat) for 20 minutes to 3 hours, or add 5 to 100 times the amount of water to the dry powder and heat to reflux for 1 to 3 hours. Any method may be used such as repeating the hot water extraction treatment several times.
このようにして得られた溶媒抽出物には、分子量が500〜10000の範囲内の成分が含有されているので、この低分子量の成分を溶媒抽出物から分画すればよい。分画する方法は特に限定されないが、例えば、透析膜を用いる方法、ゲル濾過法あるいはエタノール沈澱による方法などが挙げられる。具体的には以下のようにして行うことができる。 Since the solvent extract thus obtained contains components having a molecular weight in the range of 500 to 10,000, this low molecular weight component may be fractionated from the solvent extract. The method of fractionation is not particularly limited, and examples thereof include a method using a dialysis membrane, a gel filtration method, and a method using ethanol precipitation. Specifically, it can be performed as follows.
上記のようにして得られた溶媒抽出物を一旦凍結乾燥処理により乾燥物とした後、適当量の水に再溶解し、分画分子量が5,000〜10,000の範囲にある透析膜を用いて透析を行う。透析膜の具体例としては、フナコシ製、スペクトラバイオテックメンブレンシリーズ、スペクトラ/ポアCEシリーズ、同RCシリーズ等が挙げられる。透析外液には蒸留水を用いれば良いが、溶媒抽出物の乾燥物より調製する透析内液は、乾燥物の溶解性に応じて酢酸ナトリウム、水酸化ナトリウムや尿素等を適宜添加する。透析外液(蒸留水)を入れた容器に透析外液を満たして、両端を厳重に密封した透析膜(チューブ)を浸漬し、1〜3日静置、あるいは攪拌下で放置する。これにより透析外液中に分子量が500〜10000の範囲内の成分が得られる。なお、透析工程を数回繰り返すことにより、透析外液画分の回収量を増やすことも可能である。 The solvent extract obtained as described above is once lyophilized to obtain a dried product, which is then redissolved in an appropriate amount of water, and a dialysis membrane having a fractional molecular weight in the range of 5,000 to 10,000 is obtained. Dialyze using. Specific examples of the dialysis membrane include Funakoshi, Spectra Biotech Membrane Series, Spectra / Pore CE Series, and RC Series. Distilled water may be used as the dialysis outer solution, but sodium acetate, sodium hydroxide, urea, or the like is appropriately added to the dialysis internal solution prepared from the dried product of the solvent extract depending on the solubility of the dried product. A dialysis membrane (tube) tightly sealed at both ends is immersed in a container filled with dialysis solution (distilled water), and left at 1 to 3 days or left under stirring. Thereby, a component having a molecular weight in the range of 500 to 10,000 is obtained in the dialysis external solution. In addition, it is also possible to increase the collection amount of the dialysis external liquid fraction by repeating the dialysis step several times.
ゲル濾過法による方法は、上記と同様にして得られた溶媒抽出物の乾燥物を適当な濃度に溶解し、ゲル濾過用の担体を充填したガラスカラムに通すことにより、分子量が500〜10000の範囲内の成分を得ることができる。ここで用いるカラム担体としては、アマシャムファルマシアバイオテク社のセファクリルシリーズ、セファデックスシリーズ等が挙げられる。 In the gel filtration method, the dried product of the solvent extract obtained in the same manner as described above is dissolved in an appropriate concentration and passed through a glass column packed with a carrier for gel filtration, so that the molecular weight is 500 to 10,000. Components in the range can be obtained. Examples of the column carrier used here include Sephacryl series and Sephadex series of Amersham Pharmacia Biotech.
エタノール沈殿の方法は、上記と同様にして得られた溶媒抽出物を、75%エタノールに溶解し、不溶性の沈殿物を取除くことにより上澄液中に分子量が500〜10000の範囲内の成分を得ることができる。 In the ethanol precipitation method, a solvent extract obtained in the same manner as described above was dissolved in 75% ethanol, and the insoluble precipitate was removed to remove components in the supernatant having a molecular weight in the range of 500 to 10,000. Can be obtained.
このようにして得られた、分子量が500〜10000の範囲内、好ましくは分子量が2000〜8000の範囲内、さらに好ましくは分子量が4000〜6000の範囲内、最も好ましくは分子量が約5000の成分を主成分とするハナビラタケ溶媒抽出物は、以下の実施例に示すように高い抗腫瘍活性を持つことが明らかになった。これは、本成分が低分子量であるために消化管からの吸収が促進された、あるいは腸管における免疫活性が向上したことによるものと考えられる。
なお、本発明において分子量は、GPC法(カラム;UltrahydrogelGuard+120+500;Waters製、移動相;0.5Mりん酸緩衝液(pH11)、流速;0.5mL/分、検出;示差屈折率、推定分子量は各分子量のデキストランの保持時間より算出)により求めたものである。The component thus obtained has a molecular weight in the range of 500 to 10,000, preferably in the range of 2000 to 8000, more preferably in the range of 4000 to 6000, most preferably about 5000. As a result, it has been clarified that the Hanabiratake solvent extract as a main component has high antitumor activity as shown in the following examples. This is thought to be due to the fact that this component has a low molecular weight, so that absorption from the gastrointestinal tract is promoted or immune activity in the intestinal tract is improved.
In the present invention, the molecular weight is the GPC method (column; UltrahydroguardGuard + 120 + 500; manufactured by Waters, mobile phase; 0.5 M phosphate buffer (pH 11), flow rate: 0.5 mL / min, detection; differential refractive index, estimated molecular weight is Calculated from the retention time of molecular weight dextran).
本発明のハナビラタケ抽出物組成物の乾燥重量中、分子量500〜10000の範囲内の成分は、好ましくは80重量%〜100重量%、さらに好ましくは90重量%〜100重量%、最も好ましくは95重量%〜100重量%である。
また、本発明のハナビラタケ抽出物組成物は分子量が数百万程度のβグルカンをほとんど含有せず、本発明のハナビラタケ抽出物組成物の乾燥重量中、分子量が数百万程度のβグルカンの含有量は、5重量%以下、好ましくは、1重量%以下である。In the dry weight of the extract of the present invention, the components having a molecular weight in the range of 500 to 10,000 are preferably 80% to 100% by weight, more preferably 90% to 100% by weight, and most preferably 95% by weight. % To 100% by weight.
In addition, the garlic bamboo extract composition of the present invention contains almost no β-glucan having a molecular weight of about several million, and the dry weight of the garlic bamboo extract composition of the present invention contains β-glucan having a molecular weight of about several million. The amount is 5% by weight or less, preferably 1% by weight or less.
本発明の抗腫瘍性組成物、抗腫瘍剤、化粧品又は健康食品は、上記のようにして得られたハナビラタケ抽出物を有効成分として含有し、さらに製薬的に許容される担体、化粧用として許容される担体、又は、健康食品として許容される食品及び/または飲料を含有するものである。ハナビラタケ抽出物の含有量としては、ハナビラタケが食経験のある極めて安全なキノコであることから、特に限定されるものではないが、概ね、下限は予防または治療といった目的に応じた効果を発揮する量を、上限は服用のし易さ、経済性等の観点から実際的な量を基準とし、原料であるハナビラタケ子実体又は菌糸体の乾燥重量に換算して、成人1日あたり5mg〜500g、好ましくは50mg〜50gを摂取できるように含有量及び摂取量を設定すればよい。投与量は、本発明の組成物としては、乾燥重量に換算して、成人1日あたり0.25mg〜2.5g、好ましくは2.5mg〜2.5gを摂取できるように含有量及び摂取量を設定すればよい。本発明の抗腫瘍剤、化粧品、又は健康食品は、その形態は特に限定されるものではなく、錠剤状、カプセル状、粉末状、顆粒状、液状、懸濁液状、クリーム状等、あらゆる形態で利用できる。
本発明の抗腫瘍剤には製薬的に許容できる種々の担体を加えることができる。例えば、賦形剤、結合剤、崩壊剤、滑沢剤、着香剤、着色剤、甘味剤、矯味剤、溶解補助剤、懸濁化剤、乳化剤、コーティング剤を含むことができるが、これらに限定されない。本発明のハナビラタケ溶媒抽出物組成物を持続性、徐放性のものとしてもよい。
本発明の化粧料は、化粧品、医薬部外品、医薬品に用いられる水性成分、油性成分、植物抽出液、動物抽出液、粉末、界面活性剤、油剤、アルコール、pH調整剤、防腐剤、酸化防止剤、増粘剤、色素、香料等を必要に応じて適宜配合することにより調整される。さらに本発明の化粧料は、上記したもののほかに、美白剤、保湿剤、抗炎症剤、紫外線吸収剤等、他の有効成分を含ませるものであってもよい。
本発明の健康食品は、本発明のハナビラタケ溶媒抽出物組成物を飲食品の原材料に前記の配合量となるよう配合することにより、麺類、パン、キャンディー、ゼリー、クッキー、スープの形態とすることができる。さらに、鉄、カルシウム等の無機成分、種々のビタミン類、オリゴ糖、キトサン等の食物繊維、大豆抽出物等のタンパク質、レシチンなどの脂質、ショ糖、乳糖等の糖類を加えることもできる。
本発明の、ハナビラタケ抽出物の投与対象はその効果が発揮される限り特に限定されないが、好ましくは、温血動物であり、さらに好ましくは哺乳類であり、最も好ましくはヒトである。The antitumor composition, antitumor agent, cosmetic or health food of the present invention contains the extract of Hanabira bamboo obtained as described above as an active ingredient, and is further pharmaceutically acceptable carrier and cosmetically acceptable. Or a food and / or beverage acceptable as a health food. The content of the extract is not particularly limited because it is an extremely safe mushroom with food experience, but the lower limit is generally an amount that exerts an effect according to the purpose of prevention or treatment. The upper limit is 5 mg to 500 g per day for adults, preferably in terms of the dry weight of the fruit body or mycelium that is the raw material, based on the practical amount from the viewpoint of ease of taking, economy, etc. Should just set content and intake so that 50 mg-50 g can be ingested. As for the composition of the present invention, the content and the intake amount so that the adult can ingest 0.25 mg to 2.5 g, preferably 2.5 mg to 2.5 g per day, in terms of dry weight. Should be set. The form of the antitumor agent, cosmetic or health food of the present invention is not particularly limited, and it can be in any form such as tablets, capsules, powders, granules, liquids, suspensions, creams, etc. Available.
Various carriers that are pharmaceutically acceptable can be added to the antitumor agent of the present invention. For example, excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents, sweeteners, corrigents, solubilizers, suspending agents, emulsifiers, coating agents can be included. It is not limited to. It is good also as a long-lasting and sustained-release thing for the Hanabira bamboo solvent extract composition of this invention.
Cosmetics of the present invention are cosmetics, quasi drugs, aqueous components used in pharmaceuticals, oily components, plant extracts, animal extracts, powders, surfactants, oils, alcohols, pH adjusters, preservatives, oxidation It adjusts by mix | blending an inhibitor, a thickener, a pigment | dye, a fragrance | flavor, etc. suitably as needed. Furthermore, the cosmetic of the present invention may contain other active ingredients such as a whitening agent, a moisturizing agent, an anti-inflammatory agent, and an ultraviolet absorber in addition to the above.
The health food of the present invention is made into the form of noodles, bread, candy, jelly, cookies, soup by blending the extract of the solvent extract of the present invention into the raw materials of food and drink so as to have the above blending amount. Can do. Furthermore, inorganic components such as iron and calcium, various vitamins, dietary fibers such as oligosaccharides and chitosan, proteins such as soybean extract, lipids such as lecithin, saccharides such as sucrose and lactose can also be added.
The subject of the present invention is not particularly limited as long as its effect is exerted, but is preferably a warm-blooded animal, more preferably a mammal, and most preferably a human.
以下、実施例により本発明を詳細に説明するが、これらは本発明を何ら限定するものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit this invention at all.
製造例1〔ハナビラタケ子実体の製造〕
ハナビラタケ子実体を以下の方法により製造した。
カラマツの大鋸屑、小麦粉、栄養分(バナナ、蜂蜜、エビオス、ペプトン、塩化カルシウム、ハイポネックス)および水を、大鋸屑:小麦粉:栄養分:水=100:11.5:1.9:51の重量比で含む菌床基材を準備した。この菌床基材(520g)を850ccのポリプロピレン製の培養瓶に入れ、常法にしたがって培養瓶を滅菌した後、ハナビラタケの種菌(16g)を接種した。その後、この培養瓶を23℃の温度下、56日間培養することによりハナビラタケ子実体を収穫した。子実体の重量は培養瓶1本あたり140gであった。Production Example 1 [Manufacture of Hanabiratake fruit body]
Hanabira bamboo fruit bodies were produced by the following method.
Bacteria containing larch sawdust, flour, nutrients (bananas, honey, prawns, peptone, calcium chloride, hyponex) and water in a weight ratio of sawdust: flour: nutrients: water = 100: 11.5: 1.9: 51 A floor substrate was prepared. This fungus bed base material (520 g) was placed in an 850 cc polypropylene culture bottle, and the culture bottle was sterilized according to a conventional method, followed by inoculation with seeds of Hanabiratake (16 g). Thereafter, the fruit bodies of the bamboo leaf were harvested by culturing the culture bottle at a temperature of 23 ° C. for 56 days. The weight of the fruiting body was 140 g per culture bottle.
製造例2〔ハナビラタケ菌糸体の製造〕
ハナビラタケ菌糸体を以下の方法により製造した。
イーストエキス0.4%、グルコース2%、りん酸2水素カリウム0.1%、りん酸水素2ナトリウム0.1%となるように水に溶解し、1Nの塩化水素でpH5.0に調整した培地を500mL容三角フラスコに200mL入れ、常法にしたがって滅菌した。ハナビラタケの種菌を生育させた平板培地から径6mmの寒天片を打ち抜き、この液体培地に接種した。24℃の暗黒下、21日間振とう培養することによりハナビラタケ菌糸体を収穫した。菌糸体の乾燥重量は三角フラスコ1本あたり2gであった。Production Example 2 [Manufacture of agaric mycelium]
Hanabiratake mycelium was produced by the following method.
Yeast extract 0.4%, glucose 2%, potassium dihydrogen phosphate 0.1%, disodium hydrogen phosphate 0.1% dissolved in water and adjusted to pH 5.0 with
実施例1〔ハナビラタケ子実体低分子抗腫瘍成分の調製〕
上記の製造例1で得られたハナビラタケ子実体の乾燥物をブレンダーで粉末とし、そのうち50gに水2Lを加え、100℃、2時間の抽出処理を行なった。遠心分離により上澄みを回収し、沈澱物について同様の抽出操作をさらに2回繰り返した。回収した上澄みを凍結乾燥し、抽出物23g(ハナビラタケ子実体熱水抽出物;SCFHExとする)を得た。抽出物を再び500mLの水に溶解し、分子量8000カットの透析膜(フナコシ製、スペクトラバイオテックメンブレン ポア1.1)により透析を行なった。透析外液を回収し、凍結乾燥によりハナビラタケ子実体低分子抗腫瘍成分2.3g(SCFHLとする)を得た。Example 1 [Preparation of Hanabiratake fruiting body small molecule antitumor component]
The dried product of the fruit of the bamboo shoot obtained in Production Example 1 was powdered with a blender, 2 L of water was added to 50 g, and an extraction treatment was performed at 100 ° C. for 2 hours. The supernatant was collected by centrifugation, and the same extraction operation was further repeated twice for the precipitate. The collected supernatant was freeze-dried to obtain 23 g of extract (Hanaburatake fruiting body hot water extract; referred to as SCFHEx). The extract was dissolved again in 500 mL of water and dialyzed with a dialysis membrane having a molecular weight of 8000 cut (Funakoshi, Spectra Biotech Membrane Pore 1.1). The dialyzed external solution was collected, and 2.3 g (referred to as SCFHL) of a low molecular weight anti-tumor component of Hanabiratake fruit body was obtained by lyophilization.
実施例2〔ハナビラタケ菌糸体低分子抗腫瘍成分の調製〕
上記の製造例2で得られたハナビラタケ菌糸体の乾燥物をブレンダーで粉末とし、そのうち5gに水200mLを加え、100℃、1時間の抽出処理を行なった。遠心分離により回収した上澄みを凍結乾燥し、抽出物1.6g(ハナビラタケ菌糸体熱水抽出物;SCMHExとする)を得た。抽出物を再び200mLの水に溶解し、分子量8000カットの透析膜(フナコシ製、スペクトラバイオテックメンブレン ポア1.1)により透析を行なった。透析外液を回収し、凍結乾燥によりハナビラタケ菌糸体低分子抗腫瘍成分0.62g(SCMHLとする)を得た。Example 2 [Preparation of low-molecular weight anti-tumor component of Mytilus mycelium]
The dried material of the agaric mycelium obtained in Production Example 2 was powdered with a blender, 200 mL of water was added to 5 g of the dried product, and an extraction treatment was performed at 100 ° C. for 1 hour. The supernatant collected by centrifugation was freeze-dried to obtain 1.6 g of extract (Hanabiratake mycelium hot water extract; referred to as SCMHEx). The extract was dissolved again in 200 mL of water, and dialyzed with a dialysis membrane having a molecular weight of 8000 cut (Funakoshi, Spectra Biotech Membrane Pore 1.1). The dialyzed external solution was collected, and 0.62 g (referred to as SCMHL) of the low molecular weight anti-tumor component of the fly mycelia mycelia was obtained by lyophilization.
実施例3〔ハナビラタケ子実体低分子抗腫瘍成分の調製2〕
上記の製造例1で得られたハナビラタケ子実体の乾燥物をブレンダーで粉末とし、そのうち50gに水2Lを加え、100℃、2時間の抽出処理を行なった。遠心分離により上澄みを回収し、沈澱物について同様の抽出操作をさらに2回繰り返した。回収した上澄みを凍結乾燥し、得られた抽出物のうち1gを50mLの水に溶解し、セファデックスG−75のゲル濾過担体を充填したカラム(φ30mm×900mm、充填量500mL)に流速200mm/時でアプライし、引き続き蒸留水により展開を行なった。50mLずつ分画分取しGPC法(カラム;UltrahydrogelGuard+120+500;Waters製、移動相;0.5Mりん酸緩衝液(pH11)、流速;0.5mL/分、検出;示差屈折率、推定分子量は各分子量のデキストランの保持時間より算出)により分子量を確認し、分子量500以上、10000以下の各分を回収し、凍結乾燥によりハナビラタケ子実体低分子抗腫瘍成分0.076gを得た。Example 3 [Preparation 2 of small-sized anti-tumor component of Hanabiratake fruit body]
The dried product of the fruit of the bamboo shoot obtained in Production Example 1 was powdered with a blender, 2 L of water was added to 50 g, and an extraction treatment was performed at 100 ° C. for 2 hours. The supernatant was collected by centrifugation, and the same extraction operation was further repeated twice for the precipitate. The collected supernatant was freeze-dried, 1 g of the obtained extract was dissolved in 50 mL of water, and a flow rate of 200 mm / mm was applied to a column (φ30 mm × 900 mm, packing amount 500 mL) packed with Sephadex G-75 gel filtration carrier. It was applied at the same time and subsequently developed with distilled water. 50 mL fractions were collected and the GPC method (column; UltrahydroGuard + 120 + 500; manufactured by Waters, mobile phase; 0.5 M phosphate buffer (pH 11), flow rate: 0.5 mL / min, detection; differential refractive index, estimated molecular weight for each molecular weight The molecular weight was determined by calculating from the retention time of dextran, and each part having a molecular weight of 500 or more and 10,000 or less was collected, and 0.076 g of a small-sized antitumor component of Hanabiratake fruiting body was obtained by lyophilization.
比較例1〔アガリクスタケ子実体低分子抗腫瘍成分の調製〕
アガリクスタケ子実体の乾燥物をブレンダーで粉末とし、そのうち36gに水2Lを加え、100℃、2時間の抽出処理を行なった。遠心分離により上澄みを回収し、沈澱物について同様の抽出操作をさらに2回繰り返した。回収した上澄みを凍結乾燥し、抽出物25gを得た。抽出物を再び500mLの水に溶解し、分子量8000カットの透析膜(フナコシ製、スペクトラバイオテックメンブレン ポア1.1)により透析を行なった。透析外液を回収し、凍結乾燥によりアガリクスタケ子実体低分子抗腫瘍成分10.9g(ABFHL)を得た。Comparative Example 1 [Preparation of small body anti-tumor component of Agaricusutake fruit body]
The dried product of Agaricus stadium fruit body was powdered with a blender, and 2 L of water was added to 36 g, and extraction treatment was performed at 100 ° C. for 2 hours. The supernatant was collected by centrifugation, and the same extraction operation was further repeated twice for the precipitate. The collected supernatant was freeze-dried to obtain 25 g of an extract. The extract was dissolved again in 500 mL of water and dialyzed with a dialysis membrane having a molecular weight of 8000 cut (Funakoshi, Spectra Biotech Membrane Pore 1.1). The dialyzed external solution was collected and freeze-dried to obtain 10.9 g (ABFHL) of Agaricustake fruit body small molecule antitumor component.
試験例1〔βグルカンの定量〕
実施例1、2にしたがって調製したハナビラタケ熱水抽出物(SCFHEx、SCMHEx)、ハナビラタケ低分子抗腫瘍成分(SCFHL、SCMHL)について、コンゴーレッド法によるβグルカン量の定量を行なった。それぞれの2%水溶液を調製し、コンゴーレッド試薬を用いてβグルカンの定量を行なったところ、SCFExは5mg/mL(25%対固形分)、SCMExは1mg/mL(5%対固形分)のβグルカンが検出されたのに対し、分画処理して得られた低分子成分であるSCFHLには0.08mg/mL(0.4%対固形分)以下、SCMHLには0.03mg/mL(0.2%対固形分)以下のβグルカンしか検出されなかった。Test Example 1 [Quantification of β-glucan]
The amount of β-glucan was quantified by Congo red method for the hot water extract (SCFHEx, SCMHEx) and the low molecular weight antitumor component (SCFHL, SCMHL) prepared according to Examples 1 and 2. Each 2% aqueous solution was prepared, and β-glucan was quantified using Congo red reagent. SCFEx was 5 mg / mL (25% vs. solid content), SCMEx was 1 mg / mL (5% vs. solid content). β-glucan was detected, whereas SCFHL, which is a low molecular component obtained by fractionation, was 0.08 mg / mL (0.4% solids) or less, and SCMHL was 0.03 mg / mL. Only β-glucan below (0.2% solids) was detected.
なお、コンゴーレッド法によって定量されるβグルカンは、分子量が10万以上のものといわれる。 Note that β-glucan quantified by the Congo Red method is said to have a molecular weight of 100,000 or more.
このことから、SCFEx、SCMExに含まれていたβグルカン(分子量数万〜数十万)は透析膜による分画処理によって96%以上が除去され、SCFHL、SCMHLにはほとんど含まれないことが明らかになった。また、コンゴーレッドのメタクロマジー(色調の変化、吸収極大のシフト)(特開2000−217543号公報参照)も、SCFHL、SCMHLでは認められなかった。 From this, it is clear that 96% or more of β-glucan (molecular weight tens of thousands to several hundreds of thousands) contained in SCFEx and SCMEx is removed by fractionation treatment with a dialysis membrane, and is hardly contained in SCFHL and SCMHL. Became. Also, Congo red metachromy (change in color tone, shift in absorption maximum) (see JP 2000-217543 A) was not observed in SCFHL and SCMHL.
試験例2〔分子量分布の確認1〕
実施例1に従って調製したハナビラタケ子実体熱水抽出物(SCFHEx)、ハナビラタケ子実体低分子抗腫瘍成分(SCFHL)の分子量分布をGPC法(前記)により確認したところ、熱水抽出物のメインピークは5万前後の分子量を示したのに対し、低分子抗腫瘍成分は5000前後であった。チャートを図1に示した。Test Example 2 [Confirmation 1 of molecular weight distribution]
As a result of confirming the molecular weight distribution of Hanabiratake fruit body hot water extract (SCFHEx) and Hanabiratake fruit body low molecular weight antitumor component (SCFHL) prepared according to Example 1, the main peak of the hot water extract is While the molecular weight was around 50,000, the low molecular weight antitumor component was around 5000. The chart is shown in FIG.
試験例3〔分子量分布の確認2〕
実施例2に従って調製したハナビラタケ菌糸体熱水抽出物(SCMHEx)、ハナビラタケ菌糸体低分子抗腫瘍成分(SCMHL)の分子量分布をGPC法(前記)により確認したところ、熱水抽出物のメインピークは5万前後の分子量を示したのに対し、低分子抗腫瘍成分は5000前後であった。Test Example 3 [Confirmation of molecular weight distribution 2]
As a result of confirming the molecular weight distribution of Hanabiratake mycelium hot water extract (SCMHEX) and Hanabiratake mycelium low molecular weight antitumor component (SCMHL) prepared according to Example 2, the main peak of the hot water extract is While the molecular weight was around 50,000, the low molecular weight antitumor component was around 5000.
試験例4〔がん細胞の増殖抑制試験〕
実施例1、2で調製したハナビラタケ子実体熱水抽出物(SCFHEx)、ハナビラタケ菌糸体熱水抽出物(SCMHEx)、ハナビラタケ子実体低分子抗腫瘍成分(SCFHL)、ハナビラタケ菌糸体低分子抗腫瘍成分(SCMHL)、比較例1で調製したアガリクスタケ子実体低分子抗腫瘍成分(ABFHL)をりん酸バッファー(PBS、pH7.2)に溶解し、それぞれ10mg/mLの濃度に調整した。ヒト大腸がん細胞(Caco−2)、あるいは、ヒト白血病細胞(HL−60)をRPMI1640−10%ウシ血清培地(pH7.2)に60000個/mLの濃度となるように懸濁し、90μLずつ96穴プレートに分注した。上記試料をそれぞれ10μLずつ(ブランクにはPBSのみを10μL、ポジティブコントロールにはアクチノマイシンDを1μg/mLの濃度に調整したものを10μL)分注し、よく混和した後、5%炭酸ガス雰囲気下、37℃で48〜72時間培養し、生細胞数をCellCountingKit8(同仁化学研究所製)で計測し、ブランクの細胞増殖量と比較して、細胞増殖抑制率を算出した。Test Example 4 [Cancer Cell Growth Inhibition Test]
Hanabiratake fruit body hot water extract (SCFHEx), Hanabiratake mycelium hot water extract (SCMHEEx), Hanabiratake fruit body small molecule antitumor component (SCFHL), Hanabiratake mycelium small molecule antitumor component prepared in Examples 1 and 2 (SCMHL), Agaricustake fruit body small molecule antitumor component (ABFHL) prepared in Comparative Example 1 was dissolved in phosphate buffer (PBS, pH 7.2) and adjusted to a concentration of 10 mg / mL. Human colon cancer cells (Caco-2) or human leukemia cells (HL-60) are suspended in RPMI 1640-10% bovine serum medium (pH 7.2) to a concentration of 60,000 cells / mL, and 90 μL each. Dispense into 96-well plates. Dispense 10 μL each of the above samples (10 μL of PBS alone for the blank and 10 μL of the actinomycin D adjusted to a concentration of 1 μg / mL for the positive control), mix well, and then in a 5% carbon dioxide atmosphere The cells were cultured at 37 ° C. for 48 to 72 hours, the number of viable cells was measured with CellCounting Kit 8 (manufactured by Dojindo Laboratories), and the cell growth inhibition rate was calculated by comparison with the blank cell growth amount.
結果を表1に示した。 The results are shown in Table 1.
表1の結果から明らかなように、本発明のハナビラタケ低分子抗腫瘍成分は、がん細胞の増殖を抑制する効果を示し、その強度は単なる熱水抽出物と比べると有意に高くなっており、透析膜による分画が、がん細胞の増殖抑制効果を発揮するのに有効であることが明らかになった。 As is clear from the results of Table 1, the low molecular weight anti-tumor component of the present invention exhibits an effect of suppressing the growth of cancer cells, and its strength is significantly higher than that of a mere hot water extract. It was revealed that fractionation using a dialysis membrane is effective for exerting an inhibitory effect on the growth of cancer cells.
試験例5〔経口摂取による腫瘍増殖抑制試験〕
マウス(ICR:Jcl、6週齢、メス、10匹/群)の右上肢腋下部に、E・MEM無血清培地(pH7.2)に40000000個/mLの濃度となるように懸濁したマウス肉腫細胞(S180)を50μLずつ移植した。移植日より7日目から10日間、実施例1,2及び比較例1にしたがって調製したハナビラタケ子実体低分子抗腫瘍成分(SCFHL)、ハナビラタケ菌糸体低分子抗腫瘍成分(SCMHL)、アガリクスタケ子実体低分子抗腫瘍成分(ABFHL)、ならびに、ハナビラタケ熱水抽出物(SCFHEx、SCMHEx)の水溶液(濃度;18mg/mL)を50μLずつ(ブランクには水道水を50μL)経口投与した(体重1Kgあたり30mg投与)。移植日より35日間、3、4日おきに腫瘍サイズの計測を行ない経時変化を観察すると共に、最終日に腫瘍塊を摘出し、その重量より腫瘍増殖抑制率を算出した。腫瘍サイズはその長径×短径×短径÷2の計算式より算出した。Test Example 5 [Tumor Growth Inhibition Test by Ingestion]
A mouse (ICR: Jcl, 6 weeks old, female, 10 mice / group) suspended in an E.MEM serum-free medium (pH 7.2) at a concentration of 40000000 / mL at the lower right limbs Sarcoma cells (S180) were transplanted at 50 μL each. Hanabiratake fruit body small molecule antitumor component (SCFHL), Hanabiratake mycelium small molecule antitumor component (SCMHL), Agaricustake fruit body prepared from Examples 1 and 2 and Comparative Example 1 for 7 days from the day of transplantation An aqueous solution (concentration: 18 mg / mL) of a low molecular weight antitumor component (ABFHL) and an extract of hot water bamboo (SCFHEx, SCMHEEx) was orally administered at 50 μL each (50 μL of tap water in the blank) (30 mg per 1 kg body weight) Administration). The tumor size was measured every 35 days, 3 days, and 4 days from the date of transplantation, the change with time was observed, and the tumor mass was excised on the final day, and the tumor growth inhibition rate was calculated from its weight. The tumor size was calculated from the formula of its major axis × minor axis × minor axis ÷ 2.
結果を図2、表2に示した。 The results are shown in FIG.
図2、表2の結果から明らかなように、本発明のハナビラタケ低分子抗腫瘍成分は、がん細胞の増殖を抑制する効果を示し、その強度は単なる熱水抽出物と比べると、それぞれ10%程度高くなっており、透析膜による分画が、がん細胞の増殖抑制効果を発揮するのに有効であることが明らかになった。
本発明を詳細にまた特定の実施態様を参照して説明したが、本発明の精神と範囲を逸脱することなく様々な変更や修正を加えることができることは当業者にとって明らかである。
本出願は、2005年5月25日出願の日本特許出願(特願2005−152454)に基づくものであり、その内容はここに参照として取り込まれる。As is apparent from the results in FIG. 2 and Table 2, the low-molecular weight anti-tumor component of the present invention shows an effect of suppressing the growth of cancer cells, and its strength is 10 respectively compared with a simple hot water extract. It was revealed that fractionation with a dialysis membrane is effective for exerting a cancer cell growth inhibitory effect.
Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
This application is based on a Japanese patent application filed on May 25, 2005 (Japanese Patent Application No. 2005-152454), the contents of which are incorporated herein by reference.
本発明は、ハナビラタケの子実体又は菌糸体から得られる比較的低分子量の成分を主成分とするハナビラタケ抽出物並びにそれを有効成分として含有する抗腫瘍剤、化粧品及び健康食品を提供する。 The present invention provides an extract of Hanabira bamboo mainly composed of a relatively low molecular weight component obtained from the fruit body or mycelium of Hanabira bamboo, and an antitumor agent, a cosmetic and a health food containing the same as an active ingredient.
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JPH10298099A (en) * | 1997-04-23 | 1998-11-10 | Kozo Niwa | Cell-activating composition |
JP2000159808A (en) * | 1998-11-27 | 2000-06-13 | Kobayashi Pharmaceut Co Ltd | Method for separating and purifying basidiomycota hypha extract |
JP2000217543A (en) * | 1999-01-29 | 2000-08-08 | Nakajima Mitsuhiro | SPARASSIS CRISPA Fr. EXTRACT |
WO2001051070A1 (en) * | 2000-01-12 | 2001-07-19 | Life Science Laboratories Co., Ltd. | Physiologically active substance eem-s originating in mushrooms, process for producing the same and drugs |
WO2003051382A1 (en) * | 2001-12-14 | 2003-06-26 | Sundory Co., Ltd. | Method of inducing apoptosis and compositions therefor |
JP2004350620A (en) * | 2003-05-30 | 2004-12-16 | Unitika Ltd | Zinc replenishing food material |
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JPH10298099A (en) * | 1997-04-23 | 1998-11-10 | Kozo Niwa | Cell-activating composition |
JP2000159808A (en) * | 1998-11-27 | 2000-06-13 | Kobayashi Pharmaceut Co Ltd | Method for separating and purifying basidiomycota hypha extract |
JP2000217543A (en) * | 1999-01-29 | 2000-08-08 | Nakajima Mitsuhiro | SPARASSIS CRISPA Fr. EXTRACT |
WO2001051070A1 (en) * | 2000-01-12 | 2001-07-19 | Life Science Laboratories Co., Ltd. | Physiologically active substance eem-s originating in mushrooms, process for producing the same and drugs |
WO2003051382A1 (en) * | 2001-12-14 | 2003-06-26 | Sundory Co., Ltd. | Method of inducing apoptosis and compositions therefor |
JP2004350620A (en) * | 2003-05-30 | 2004-12-16 | Unitika Ltd | Zinc replenishing food material |
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