WO2005011407A1

WO2005011407A1 - Antitumor composition

Info

Publication number: WO2005011407A1
Application number: PCT/JP2004/011290
Authority: WO
Inventors: Takashi Kawai; Mayu Hashimoto; Kazuko Awazu; Shigetoshi Mizutani; Takeshi Sakai; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 2003-07-31
Filing date: 2004-07-30
Publication date: 2005-02-10
Also published as: JP2007166903A

Abstract

An antitumor composition comprising a fraction of a hot water-extract of Hypsizigus marmoreus fruit body which is soluble and cannot pass through an ultrafiltration membrane with a molecular weight cutoff of 100,000; an antitumor composition obtained by further treating a composition comprising a fraction of a hot water-extract of Hypsizigus marmoreus fruit body which is soluble and cannot passe through an ultrafiltration membrane with a molecular weight cutoff of 100,000 with an anion exchange resin and eluting; a food or a drink containing the above-described antitumor composition; a process for producing an antitumor composition characterized by comprising (1) the step of treating Hypsizigus marmoreus fruit body with hot water for 1 to 5 hours and removing insoluble matters from the hot-water extract thus obtained, and (2) the step of subjecting the soluble fraction obtained in the above step (1) to ultrafiltration of a molecular weight cutoff of 100,000; and a process for producing an antitumor composition characterized by comprising (1) the step of treating Hypsizigus marmoreus fruit body with hot water for 1 to 5 hours and removing insoluble matters from the hot-water extract thus obtained, (2) the step of subjecting the soluble fraction obtained in the above step (1) to ultrafiltration of a molecular weight cutoff of 100,000, and (3) the step of treating the composition obtained in the above step (2) with an anion exchange resin and then eluting.

Description

抗腫瘍性組成物技術分野 Technical field of antitumor composition

本発明はブナシメジ子実体を熱水処理し、不溶物を除去し、さらに排除分子量 In the present invention, the fruit body of Bunashimeji is treated with hot water to remove insolubles,

1 0万の限外ろ過を行うことにより得られ得る組成物、又は当該組成物を陰ィォン交換樹脂に供した後溶出させて得明られ得る組成物からなる抗腫瘍性組成物、及び該抗腫瘍性組成物を含む食品又は飲料に関するものである。 An antitumor composition comprising a composition obtainable by ultrafiltration of 100,000, or a composition obtainable by eluting the composition after applying it to an anion exchange resin, and And a food or beverage containing the antitumor composition.

書 book

背景技術 Background art

シィタケ、マイタケ、エノキタケ等の種々の食用担子菌が抗腫瘍性を有することは広く知られており、これらの子実体又は菌糸体よりこれまでに様々な抗腫瘍性組成物が調製されてきた。また、食用担子菌の一つであるブナシメジが抗腫瘍性を有することも明らかにされてきており、例えば、タンパク質含量 3〜2 0重量％、糖含量 2 0〜 5 0重量％を有する子実体熱水抽出物、及びその精製多糖体が（例えば、特開平 5— 3 0 6 2 3 3号公報）、さらには水又は親水性溶媒に懸濁後、加熱処理により得られる分子量 6， 0 0 0〜6 0， 0 0 0の生理活性物質 E E M— Sが抗腫瘍性を有することが明らかにされている（例えば、国際公開第 0 1 / 5 1 0 7 0号パンフレツト）。発明の開示 It is widely known that various edible basidiomycetes, such as shiitake, maitake, and enokitake, have antitumor properties, and various antitumor compositions have been prepared from these fruiting bodies or mycelia. . It has also been shown that Bunashimeji, one of the edible basidiomycetes, has antitumor properties. For example, a bacterium having a protein content of 3 to 20% by weight and a sugar content of 20 to 50% by weight. The actual hot water extract and the purified polysaccharide thereof (for example, Japanese Patent Application Laid-Open No. 5-306323) can be further suspended in water or a hydrophilic solvent and then subjected to a heat treatment to obtain a molecular weight of 6,0. It has been clarified that the bioactive substance EEM-S of 00 to 600,000 has antitumor properties (for example, WO 01/51070 pamphlet). Disclosure of the invention

各種食用担子菌の子実体が抗腫瘍性を有することは明らかとなつているが、効果を得るためには一定量を日常的に食すことが必要である。しかしながら、食用担子菌といえども味に癖があり、料理に工夫をしても日常的に一定量食すことは難しい。一方、ある種の担子菌から得られる抗腫瘍性、免疫賦活活性等を有する有効成分を配合した健康食品が広く市場に流通しているが、原料の担子菌が希少のため、生産される健康食品も非常に高価なものとならざるを得ない。 Although it is clear that the fruiting bodies of various edible basidiomycetes have antitumor properties, it is necessary to eat a certain amount on a daily basis to obtain the effect. However, even edible basidiomycetes have taste habits, and it is difficult to eat a fixed amount on a daily basis even if the dishes are devised. On the other hand, health foods containing active ingredients having antitumor properties, immunostimulatory activities, etc. obtained from certain basidiomycetes are widely distributed on the market, but are produced because the raw material basidiomycetes are scarce. Health food must also be very expensive.

本発明の課題は安価な食用担子菌が有する抗腫瘍性を損なわずに、しかも効率の良い方法で抽出 ·濃縮して得られた、経口摂取によりその効果を発揮し得る新規な抗腫瘍性組成物、及び該組成物を含有する食品又は飲料を提供するものである。 It is an object of the present invention to provide a novel antitumor agent that can exert its effects by oral ingestion, obtained by extracting and concentrating it by an efficient method without impairing the antitumor effect of inexpensive basidiomycetes. It is intended to provide a composition, and a food or beverage containing the composition.

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、ブナシメジ子実体の熱水抽出物に対し所定の処理を施すことにより、ブナシメジの子実体から、従来知られている抗腫瘍性成分とは異なる、経口摂取によりその効果を発揮し得る新規な抗腫瘍性組成物が得られることを見出し、本発明を完成した。 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result of subjecting the hot water extract of the fruit body of Bunashimeji to a predetermined treatment, the conventionally known antitumor activity from the fruit body of Bunashimeji The present inventors have found that a novel antitumor composition which can exert its effects by oral ingestion, which is different from the components, can be obtained, and completed the present invention.

すなわち本発明を概説すれば、本発明の第 1の発明は、ブナシメジ子実体の熱水抽出物中の、可溶性であって排除分子量 1 0万の限外ろ過膜を通過しない画分からなる抗腫瘍性組成物に関する。本発明の第 1の発明において、当該組成物としては、糖含量が 4 5〜6 0重量％、脂質含量が 2 0〜4 5重量％、タンパク質含量が 1〜 1 5重量％である抗腫瘍性組成物が例示される。 That is, to summarize the present invention, the first invention of the present invention is directed to an antibody comprising a soluble fraction in a hot water extract of Bunashimeji fruit body which does not pass through an ultrafiltration membrane having an excluded molecular weight of 100,000. It relates to a neoplastic composition. In the first invention of the present invention, the composition has a sugar content of 45 to 60% by weight, a lipid content of 20 to 45% by weight, and a protein content of 1 to 15% by weight. Antitumor compositions are exemplified.

本発明の第 2の発明は、ブナシメジ子実体の熱水抽出物中の、可溶性であって排除分子量 1 0万の限外ろ過膜を通過しない画分からなる組成物を、さらに陰ィオン交換樹脂に供した後、溶出させて得られ得る抗腫瘍性組成物に関する。本発明の第 2の発明において、当該組成物としては、糖含量が 5 0〜6 0重量％、脂質含量が 2 0〜4 0重量％、タンパク質含量が 5〜 2 0重量％である抗腫瘍性組成物が例示される。 The second invention of the present invention relates to a hot water extract of Bunashimeji fruiting body comprising a soluble fraction which does not pass through an ultrafiltration membrane having an excluded molecular weight of 100,000, further comprising an anion exchange resin. The present invention relates to an antitumor composition obtainable by eluting after subjecting the composition to an antitumor composition. In the second invention of the present invention, the composition has a sugar content of 50 to 60% by weight, a fat content of 20 to 40% by weight, and a protein content of 5 to 20% by weight. Antitumor compositions are exemplified.

本発明の第 3の発明は、本発明の第 1又は第 2の発明の組成物を含有する食品又は飲料に関する。 The third invention of the present invention relates to a food or beverage containing the composition of the first or second invention of the present invention.

本発明の第 4の発明は、（1 ) ブナシメジ子実体を 1〜 5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、及び（2 ) 工程（1 ) で得られた可溶性画分を排除分子量 10万の限外ろ過に供する工程を包含することを特徴とする抗腫瘍性組成物の製造方法に関する。本発明の第 4の発明において、抗腫瘍性組成物としては糖含量が 45〜60重量％、脂質含量が 20〜45重量％、タンパク質含量が 1〜15重量％でぁる組成物が例示される。 The fourth invention of the present invention comprises: (1) a step of removing insolubles from a hot water extract obtained by treating the fruit body of Bunashimeji with hot water for 1 to 5 hours; and (2) a step obtained by the step (1). Be And subjecting the soluble fraction to ultrafiltration having an excluded molecular weight of 100,000. In the fourth invention of the present invention, examples of the antitumor composition include a composition having a sugar content of 45 to 60% by weight, a lipid content of 20 to 45% by weight, and a protein content of 1 to 15% by weight. Is done.

本発明の第 5の発明は、（1) ブナシメジ子実体を 1〜 5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、（2) 工程（1) で得られた可溶性画分を排除分子量 10万の限外ろ過に供する工程、及び（3) 工程（2) で得られた組成物を陰イオン交換樹脂に供した後、溶出させる工程を包含することを特徴とする抗腫瘍性組成物の製造方法に関する。本発明の第 5の発明において、抗腫瘍性組成物としては、糖含量が 50〜60重量％、脂質含量が 20〜40 重量％、タンパク質含量が 5〜 20重量％の組成物が例示される。 The fifth invention of the present invention comprises: (1) a step of removing insolubles from a hot water extract obtained by treating a fruit body of Bunashimeji with hot water for 1 to 5 hours; (2) a step obtained by the step (1). Subjecting the soluble fraction to ultrafiltration with an excluded molecular weight of 100,000, and (3) subjecting the composition obtained in step (2) to an anion exchange resin, followed by elution. The present invention relates to a method for producing a characteristic antitumor composition. In the fifth invention of the present invention, examples of the antitumor composition include a composition having a sugar content of 50 to 60% by weight, a lipid content of 20 to 40% by weight, and a protein content of 5 to 20% by weight. .

本発明において、ブナシメジとしては、いずれも独立行政法人産業技術総合研究所特許生物寄託センター（〒305- 8566 日本国茨城県つくば市東 1丁目 1 番地 1中央第 6) に寄託されている、 Lyo phy l l um u 1 ma r i urn In the present invention, Bunashimeji has been deposited at the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture 305-8566, Japan). ll um u 1 ma ri urn

M- 8171 (FERM BP— 1415) (寄託日： 1986年 8月 23日）、又は Lyo phy l l um u lma r i um K- 0259 (FERM P - 1 2981) (寄託日： 1992年 6月 2日）が例示される。また、本発明の第 1、第 2 の発明において、処理温度が 80〜100°C、処理時間が 1〜 5時間の熱水処理が例示される。 M-8171 (FERM BP-1415) (Deposit date: August 23, 1986) or Lyo phyll ul umulma ri um K-0259 (FERM P-1 2981) (Deposit date: June 2, 1992) Day) is exemplified. In the first and second aspects of the present invention, a hot water treatment in which the treatment temperature is 80 to 100 ° C. and the treatment time is 1 to 5 hours is exemplified.

本発明により、以上の通りの、安価な食用担子菌が有する抗腫瘍性を有しており、経口摂取によりその効果を発揮し得る抗腫瘍性組成物、及び該組成物を含有する食品又は飲料を得ることができる。発明を実施するための最良の形態 According to the present invention, as described above, an antitumor composition which has the antitumor properties of inexpensive edible basidiomycetes and can exert its effects by oral ingestion, and a food or a food containing the composition, You can get a drink. BEST MODE FOR CARRYING OUT THE INVENTION

本発明の抗腫瘍性組成物はブナシメジ子実体に由来するものであるが、かかる組成物は、これまでにブナシメジより得られた抗腫瘍性の組成物と比較して、組成物中の構成成分が全く異なるものであり、特に脂質を通常 2 0重量％以上含有するという点で新規な組成物である。従来、ブナシメジの抗腫瘍性は、熱水抽出物中の分子量 6 0 0 0〜 6 0 0 0 0の画分により発揮されるものと思われていたところ、本発明者らは、意外にもブナシメジ子実体の熱水抽出物中の可溶性であつて排除分子量 1 0万の限外ろ過膜を通過しない画分こそ抗腫瘍性の発揮にとつて重要であることを初めて明らかにした。かかる知見は従来の知見からは全く予想外のことである。従って、本発明の組成物によれば、従来のブナシメジ由来の抗腫瘍性の組成物に比べ、より効果的な抗腫瘍効果の発現が期待できる。また、その原料であるブナシメジは安価であり、その処理工程も簡易であることから、本発明の組成物は従来のものと比べてより安価に製造することができ、従って、より汎用性に富む健康素材であるといえる。 Although the antitumor composition of the present invention is derived from the fruit body of Bunashimeji, the composition is compared with the antitumor composition obtained from Bunashimeji so far. The composition of the composition is completely different, especially a novel composition in that the lipid usually contains 20% by weight or more. Conventionally, it was thought that the antitumor property of Bunashimeji was exerted by the fraction having a molecular weight of 600 to 600 in the hot water extract, but the present inventors surprisingly found that For the first time, it has been clarified that the fraction in the hot water extract of Bunashimeji fruiting body that is soluble and does not pass through the ultrafiltration membrane having a molecular weight cut off of 100,000 is important for exhibiting antitumor properties. Such findings are completely unexpected from previous findings. Therefore, according to the composition of the present invention, a more effective antitumor effect can be expected as compared with the conventional antitumor composition derived from Bunashimeji. Moreover, since the raw material, Bunashimeji, is inexpensive and the processing steps are simple, the composition of the present invention can be manufactured at a lower cost than conventional ones, and therefore, is more versatile. It can be said that it is a healthy material.

本発明の第 1の発明である抗腫瘍性組成物は、ブナシメジ子実体の熱水抽出物中の可溶性であって排除分子量 1 0万の限外ろ過膜を通過しない画分からなる組成物であって、具体的には、ブナシメジ子実体を熱水処理して得られる熱水抽出物より、不溶物及び分子量 1 0万以下画分を除去して得られ得る組成物である。かかる組成物の製造方法については後述するが、該組成物による良好な抗腫瘍性の発現を促す観点から、該組成物の糖含量としては、好ましくは 4 5 ~ 6 0重量 %、脂質含量としては、好ましくは 2 0〜4 5重量％、タンパク質含量としては、好ましくは 1〜 1 5重量％である。また、該組成物のゥロン酸含量としては、特に限定はないが、好ましくは 0 . 0 1〜1 0重量％、より好ましくは 0 . 1〜 5重量％である。なお、本明細書における糖含量、脂質含量、タンパク質含量、及びゥ口ン酸含量は後述の実施例 8に記載の方法により測定することができる。また、本発明の第 2の発明である抗腫瘍性組成物は、本発明の第 1の発明の組成物を、さらに陰イオン交換樹脂に供した後、溶出させて得られ得る組成物である。かかる組成物は、本発明の第 1の発明である組成物中の抗腫瘍性成分が濃縮されたものである。前記と同様の観点から、該組成物の糖含量としては、好ましくは 50~60重量％、脂質含量としては、好ましくは 20〜40重量％、タンパク質含量としては、好ましくは 5〜20重量％である。また、ゥロン酸含量としては、特に限定はないが、好ましくは 0. 01〜10重量％、より好ましくは 0. ：!〜 5重量％である。 The antitumor composition according to the first invention of the present invention is a composition comprising a soluble fraction in a hot water extract of a fruit body of Bunashimejiji and not passing through an ultrafiltration membrane having an excluded molecular weight of 100,000. Specifically, it is a composition obtainable by removing insolubles and a fraction having a molecular weight of 100,000 or less from a hot water extract obtained by treating a fruit body of Bunashimeji with hot water. The method for producing such a composition will be described later, but from the viewpoint of promoting the expression of good antitumor properties by the composition, the sugar content of the composition is preferably 45 to 60% by weight, and the lipid content is preferably Is preferably 20 to 45% by weight, and the protein content is preferably 1 to 15% by weight. The content of peronic acid in the composition is not particularly limited, but is preferably from 0.1 to 10% by weight, more preferably from 0.1 to 5% by weight. The sugar content, lipid content, protein content, and humic acid content in this specification can be measured by the method described in Example 8 described later. Further, the antitumor composition according to the second invention of the present invention is a composition that can be obtained by subjecting the composition of the first invention of the present invention to an anion exchange resin and then eluting the composition. is there. Such a composition is a composition in which the antitumor component in the composition of the first invention of the present invention is concentrated. From the same viewpoint as above, the sugar content of the composition is preferably The amount is preferably 50 to 60% by weight, the lipid content is preferably 20 to 40% by weight, and the protein content is preferably 5 to 20% by weight. The content of peronic acid is not particularly limited, but is preferably 0.01 to 10% by weight, more preferably 0.1 to! 5% by weight.

本発明の第 1及び第 2の発明である組成物（以下、本発明の組成物という場合がある）が有する抗腫瘍性とは、具体的には、少なくとも、該組成物が個体の免疫を賦活すること、または直接腫瘍に作用すること等により、結果的に腫瘍の増殖を抑制することをいう。かかる抗腫瘍性は、後述の参考例 2に記載の方法に従つて評価することができる。本発明の組成物による抗腫瘍性は、個体が該組成物を経口摂取することにより個体内で発揮され得る。個体とは生体であれば特に限定されるものではないが、主としてヒトを含む哺乳動物をいう。 The antitumor properties of the compositions according to the first and second inventions of the present invention (hereinafter, sometimes referred to as the composition of the present invention) are specifically defined as at least when the compositions are immune to individuals. Activating, or acting directly on tumors, etc., resulting in suppression of tumor growth. Such antitumor properties can be evaluated according to the method described in Reference Example 2 below. The antitumor properties of the composition of the present invention can be exerted in an individual by orally ingesting the composition. The individual is not particularly limited as long as it is a living body, but mainly refers to mammals including humans.

本発明において原料として使用されるブナシメジは自然界においては秋期に種々の広葉樹の枯れ木に叢生あるいは離生しており、他のきのこと比較して形や歯切れのよい肉質のため、極めて美味なきのことして採食されてきた。また、近年ではォガクズに米糠やその他の栄養源を配合した培養基を用いて、ビン又は箱で栽培を行う菌床人工栽培法が確立され、季節に関係なく一年を通じて安定してきのこを収穫できるようになつている。 The beech shimeji used as a raw material in the present invention is crowded or incarcerated in the dead trees of various hardwoods in the autumn in nature, and is extremely delicious because of its shape and crispness compared to other mushrooms. It has been eaten by mushrooms. In recent years, an artificial cultivation method of cultivating in a bottle or a box using a culture medium in which rice bran and other nutrients are mixed with sawdust is established, and mushrooms can be stably harvested throughout the year regardless of the season. It is like that.

本発明に使用されるブナシメジは天然のものでも人工栽培品でもよいが、一年を通じて簡易かつ安価に入手可能であることから人工栽培品が好ましく、より好適には L y o p h y 1 1 um u 1 ma r i urn M- 8171 (FERM B P— 141 5) 、又は L y o p h y 1 1 um u 1 ma r i um K- 0259 (FERM P— 12981) が例示される。これらの菌株は、商品名「やまびこほんしめじ」もしくは「スーパーやまびこしめじ」として大量に市場に流通している。 The beech shimeji used in the present invention may be a natural one or an artificially cultivated product, but is preferably an artificially cultivated product because it is easily and inexpensively available throughout the year, and more preferably L yophy 1 1 um u 1 Examples are ma uri urn M-8171 (FERM BP-1415) and L yophy 11 umu 1 mari u K-0259 (FERM P-12981). These strains are marketed in large quantities under the trade name “Yamabiko Shimeji” or “Super Yamabiko Shimeji”.

子実体は生のもの（生鮮子実体）でも加熱乾燥、天日乾燥、凍結乾燥等で乾燥された子実体乾燥物でもよい。また子実体は株のままでも粉砕した後に熱水処理を行ってもよい。熱水処理とは、子実体をそのまま、もしくは上記記載のように処理したものに水を加え、一定時間加熱を行うことをいう。熱水とは、いわゆる加熱された水をいい、その温度は特に限定されるものではないが、水温は好適には後述の処理温度と同様の温度範囲である。熱水処理に用いる水としては、蒸留水、精製水、イオン交換水、水道水等を使用することができる。子実体として生鮮子実体を使用する場合は生鮮子実体 1重量部に対して好適には 1〜 1 0重量部、より好適には 1〜 5重量部の水を使用でき、また子実体乾燥物を使用する場合は乾燥物 1重量部に対して好適には 5〜 5 0重量部、より好適には 1 0〜2 5重量部の水を使用することができる。処理の条件には特に限定はないが、処理温度は好ましくは 8 0〜 1 0 0 °C、より好ましくは 9 0 ~ 1 0 0 °Cで、処理時間は好ましくは 1〜 5時間、得られる組成物の抗腫瘍性の発現を向上させる観点から、より好ましくは 2〜4時間の範囲で処理を行うのがよい。抽出処理は、静置して行っても、攪拌下に行ってもよい。以上の熱水処理によりブナシメジ子実体由来の熱水抽出物を得る。 The fruiting body may be a raw (fresh fruiting body) or a dried fruiting body dried by heat drying, solar drying, freeze drying, or the like. In addition, even if the fruiting body is a strain, it is pulverized and then treated with hot water May be performed. The hot water treatment means to add water to the fruit body as it is or to treat it as described above, and to heat it for a certain period of time. Hot water refers to so-called heated water, the temperature of which is not particularly limited, but the water temperature is preferably in the same temperature range as the processing temperature described below. As the water used for the hot water treatment, distilled water, purified water, ion-exchanged water, tap water, and the like can be used. When a fresh fruit body is used as the fruit body, 1 to 10 parts by weight, preferably 1 to 5 parts by weight of water can be used per 1 part by weight of the fresh fruit body, and the fruit body can be dried. When a product is used, preferably 5 to 50 parts by weight, more preferably 10 to 25 parts by weight of water can be used per 1 part by weight of the dried product. The processing conditions are not particularly limited, but the processing temperature is preferably 80 to 100 ° C, more preferably 90 to 100 ° C, and the processing time is preferably 1 to 5 hours. From the viewpoint of improving the expression of the antitumor property of the obtained composition, the treatment is more preferably performed for 2 to 4 hours. The extraction process may be performed while standing or under stirring. A hot water extract derived from the fruit body of Bunashimeji is obtained by the above hot water treatment.

上記で得られた熱水抽出物からの不溶物の除去は通常の方法で行えばよく、例えばろ過又は遠心分離により不溶物を除去することができる。ここで、不溶物とは、ろ過の場合、市販のろ紙（例えば、アドバンテック社製 N o . 2フィルター ) によりろ別される抽出物中の不溶性の成分を、遠心分離の場合、少なくとも 5 0 0 0 X gの遠心力により沈降し得る抽出物中の不溶性の成分をいう。そのようにして得られた可溶物、すなわち、熱水抽出物中の可溶性画分を次いで排除分子量 1 0万の限外ろ過に供することにより本発明の第 1の発明である抗腫瘍性組成物を得ることができる。すなわち、本発明の第 1の発明の組成物は、抗腫瘍性を有する分子量 1 0万を超える成分を主成分とする新規な組成物である。なお、本発明の組成物における分子量 1 0万とは限外ろ過に使用する限外ろ過膜の排除分子量により決まる分子量をいうが、前記可溶性画分を排除分子量 1 0万の限外ろ過に供した場合と同様な分子量範囲の画分を分取可能な、分子量 1 0万以下の画分の排除手段であれば、排除分子量 1 0万の限外ろ過の代わりに当該手段を任意に使用することができる。また、本発明の組成物において分子量 1 0万は前記の通り定義されるため、当該分子量の値は、本発明の組成物の構成成分の実際の分子量を示すものではない。例えば分子量が 1 0 0 0以下の物質であっても分子量 1 0万を超える高分子成分に吸着した状態で存在している場合、上記の限外ろ過により分子量 1 0万を超える画分に入ってくることがある。そのような分子量 1 0万以下の物質は不純物であるが、本発明の組成物は、そのような不純物を含む場合を排除するものではない。 The removal of insoluble matter from the hot water extract obtained above may be performed by a usual method, and for example, insoluble matter can be removed by filtration or centrifugation. Here, the insoluble matter means, in the case of filtration, an insoluble component in the extract which is filtered by a commercially available filter paper (for example, No. 2 filter manufactured by Advantech Co., Ltd.). Refers to insoluble components in the extract that can settle by centrifugal force of 0 X g. The solubilized material thus obtained, that is, the soluble fraction in the hot water extract, is then subjected to ultrafiltration with an exclusion molecular weight of 100,000 to obtain the antitumor activity of the first invention of the present invention. A composition can be obtained. That is, the composition of the first invention of the present invention is a novel composition containing, as a main component, an antitumor component having a molecular weight of more than 100,000. The molecular weight of 100,000 in the composition of the present invention refers to the molecular weight determined by the excluded molecular weight of the ultrafiltration membrane used for ultrafiltration. Fractions with a molecular weight of 100,000 or less can be fractionated in the same molecular weight range as when subjected to filtration. If it is a means for eliminating the minute, it can be used arbitrarily instead of the ultrafiltration having an excluded molecular weight of 100,000. In addition, since the molecular weight of 100,000 in the composition of the present invention is defined as described above, the value of the molecular weight does not indicate the actual molecular weight of the component of the composition of the present invention. For example, even if the substance has a molecular weight of 100,000 or less, if it exists in a state of being adsorbed on a polymer component having a molecular weight of more than 100,000, it enters the fraction having a molecular weight of more than 100,000 by the above ultrafiltration. May come. Such a substance having a molecular weight of 100,000 or less is an impurity, but the composition of the present invention does not exclude the case containing such an impurity.

一方、本発明の第 2の発明である抗腫瘍性組成物は、本発明の第 1の発明である上記組成物を、さらに陰イオン交換樹脂に供した後、溶出させて得られる。かかる処理は、第 1の発明である組成物を精製することを意図したものである。本発明の第 2の発明である組成物では本発明の第 1の発明である組成物に比べて抗腫瘍性成分が濃縮されており、より高い抗腫癟性の発現が期待できる。 On the other hand, the antitumor composition according to the second invention of the present invention is obtained by subjecting the above composition of the first invention of the present invention to an anion exchange resin and then eluting the composition. Such treatment is intended to purify the composition of the first invention. In the composition according to the second aspect of the present invention, the antitumor component is more concentrated than in the composition according to the first aspect of the present invention, and higher expression of antitumor properties can be expected.

陰イオン交換樹脂としては D E A E—セルロース、 T E A E—セルロース、 E C T E O L A—セルロース、 P A B—セルロース等を用いることができ、好ましくは D E A E—セル口ファイン（チッツ株式会社製）を用いることができる。上記第 1の発明である抗腫瘍性組成物を適切な開始緩衝液、例えば水、リン酸緩衝液等で平衡化した陰イオン交換樹脂を充填したカラムにかけ、前記緩衝液で非吸着画分を洗い出した後、緩衝液の塩濃度を上げて、好ましくは 4 0 O mM以上の N a C 1濃度で溶出させることにより、本発明の第 2の発明である抗腫瘍性組成物を得ることができる。 DAE-cellulose, TEAE-cellulose, ECTEOLA-cellulose, PAB-cellulose and the like can be used as the anion exchange resin, and preferably, DEAE-cell mouth fine (manufactured by Chitz Corporation) can be used. The antitumor composition according to the first aspect of the present invention is applied to a column filled with an anion exchange resin equilibrated with a suitable starting buffer, for example, water, a phosphate buffer, or the like, and non-adsorbed image is formed with the buffer. After washing out the components, the salt concentration of the buffer solution is increased, and elution is performed preferably at a NaC1 concentration of 40 O mM or more, thereby obtaining the antitumor composition according to the second invention of the present invention. be able to.

上記操作により得られた本発明の組成物はいずれも液性の組成物として得られるが、その後に、適宜、乾燥を行って乾燥物としてもよい。また、さらに当該乾燥物を粉末、錠剤等の任意の形態に加工してもよい。例えば、錠剤形態を有する本発明の組成物は、そのまま食品として使用することも可能である。さらに、本発明の組成物はそのまま食品素材として用いてもよく、他の食品素材、例えば増粘剤、甘味料、有機酸、栄養剤、香料、着色料等を組み合わせて食品素材、飲料素材として用いてもよい。 Any of the compositions of the present invention obtained by the above operation can be obtained as a liquid composition, and thereafter, may be appropriately dried to obtain a dried product. Further, the dried product may be processed into an arbitrary form such as a powder or a tablet. For example, the composition of the present invention having a tablet form can be used as it is as a food. Furthermore, the composition of the present invention may be used as it is as a food material, and other food materials, for example, Viscosity agents, sweeteners, organic acids, nutrients, flavors, coloring agents, etc. may be used in combination as food materials and beverage materials.

以上により、本発明の組成物が得られるが、本発明の組成物としては、良好な抗腫瘍性を発揮し得る観点から、処理温度が 8 0〜1 0 0で、処理時間が 1〜5 時間で熱水処理して得られたものである組成物が好適である。 As described above, the composition of the present invention is obtained. From the viewpoint of exhibiting good antitumor properties, the composition of the present invention has a treatment temperature of 80 to 100 and a treatment time of 1 to 5 Compositions obtained by hydrothermal treatment over time are preferred.

一方、同様の観点より、本発明の第 1の発明である組成物の製造方法としては、 ( 1 ) ブナシメジ子実体を 1〜5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、及び（2 ) 工程（1 ) で得られた可溶性画分を排除分子量 1 0万の限外ろ過に供する工程を包含する方法が、本発明の第 2の発明である組成物の製造方法としては、（1 ) ブナシメジ子実体を 1〜 5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、（2 ) 工程（1 ) で得られた可溶性画分を排除分子量 1 0万の限外ろ過に供する工程、及び（3 ) 工程（2 ) で得られた組成物を陰イオン交換樹脂に供した後、溶出させる工程を包含する方法が好適である。 On the other hand, from a similar point of view, the method for producing the composition according to the first invention of the present invention includes: (1) a hot water extract obtained by subjecting a fruit body of Bunashimeji to a hot water treatment for 1 to 5 hours; A second aspect of the present invention is a method comprising the steps of: (a) removing the soluble fraction and (b) subjecting the soluble fraction obtained in the step (1) to ultrafiltration with a molecular weight cut off of 100,000. (1) a step of removing insolubles from a hot water extract obtained by treating the fruit body of Bunashimejiji with hot water for 1 to 5 hours; (2) a step of removing the soluble substance obtained in step (1); Preferably, the method comprises a step of subjecting the fraction to ultrafiltration with an excluded molecular weight of 100,000, and a step of (3) subjecting the composition obtained in step (2) to an anion exchange resin and then eluting the fraction. It is.

本発明の食品又は飲料は、本発明の組成物を含有してなる、担子菌子実体の抗腫瘍作用を有する食品又は飲料であり、本発明の食品又は飲料を摂取、喫食することにより抗腫瘍性が発揮され生体の恒常性が維持されることにおいて特に有用である。 The food or beverage of the present invention is a food or beverage having the antitumor effect of the basidiomycete fruit body, which comprises the composition of the present invention, and has an antitumor effect by ingesting and eating the food or beverage of the present invention. Is particularly useful in maintaining the homeostasis of living organisms.

本発明の食品又は飲料は、特に限定するものではないが、例えば、穀物加工品 (例、小麦粉加工品、デンプン類加工品、プレミックス加工品、麵類、マカロニ類、パン類、あん類、そば類、麩、ビーフン、はるさめ、包装餅等）、油脂加工品（例、可塑性油脂、てんぷら油、サラダ油、マヨネーズ類、ドレッシング等）、大豆加工品（例、豆腐類、味噌、納豆等）、食肉加工品（例、ハム、ベーコン、プレスハム、ソーセージ等）、水産製品（例、冷凍すりみ、かまぼこ、ちくわ、はんぺん、さつま揚げ、つみれ、すじ、魚肉ハム、ソーセージ、かつお節、魚卵加工品、水産缶詰、つくだ煮等）、乳製品（例、原料乳、クリーム、ョーダルト、バタ一、チーズ、練乳、粉乳、アイスクリーム等）、野菜 ·果実加工品（例、ペースト類、ジャム類、漬物類、果実飲料、野菜飲料、ミックス飲料等）、菓子類（例、チョコレート、ビスケット類、菓子パン類、ケーキ、餅菓子、米菓類等）、アルコール類（例、日本酒、中国酒、ワイン、ウイスキー、焼酎、ゥォッ力、ブランデー、ジン、ラム酒、ビール、清涼アルコール飲料、果実酒、リキュ —ル等）、嗜好飲料（例、緑茶、紅茶、ウーロン茶、コーヒー、清涼飲料、乳酸飲料等）、調味料（例、しょうゆ、ソース、酢、みりん等）、缶詰 ·瓶詰 ·袋詰食品（例、牛飯、釜飯、赤飯、カレー、その他の各種調理済食品等）、半乾燥または濃縮食品（例、レバ一ペースト、その他のスプレツド、そば · うどんの汁、濃縮スープ類等）、乾燥食品（例、即席麵類、即席カレー、インスタントコ一ヒ一、粉末ジュース、粉末スープ、即席味噌汁、調理済食品、調理済飲料、調理済スープ等）、冷凍食品（例、すき焼き、茶碗蒸し、うなぎかば焼き、ハンバーグステーキ、シユウマイ、餃子、各種スティック、フルーツカクテル等）、固形食品、液体食品（例、スープ等）、香辛料類等の農産 ·林産加工品、畜産加工品、水産加工品等が挙げられる。 Although the food or beverage of the present invention is not particularly limited, for example, processed cereals (e.g., processed flour, processed starch, processed premix, varieties, macaroni, breads, bean jam, Buckwheat, fu, rice noodles, harusame, wrapped rice cake, etc.), processed fats and oils (eg, plastic fat, tempura oil, salad oil, mayonnaise, dressing, etc.), processed soybeans (eg, tofu, miso, natto, etc.), Processed meat products (eg, ham, bacon, pressed ham, sausage, etc.), fishery products (eg, frozen surimi, kamaboko, chikuwa, hampan, fish cake, fish cake, fish ham, sausage, bonito, fish egg processed products, Canned seafood, tsukudani, etc.), dairy products (eg, raw milk, cream, jordal) , Butter, cheese, condensed milk, powdered milk, ice cream, etc.), processed vegetables and fruits (eg, pastes, jams, pickles, fruit drinks, vegetable drinks, mixed drinks, etc.), confectionery (eg, Chocolate, biscuits, confectionery bread, cakes, rice cakes, rice crackers, etc.), alcohols (eg, sake, Chinese sake, wine, whiskey, shochu, shochu, brandy, gin, rum, beer, refreshing alcoholic beverages, Fruit drinks, liqueurs, etc.), favorite beverages (eg, green tea, black tea, oolong tea, coffee, soft drinks, lactic acid drinks, etc.), seasonings (eg, soy sauce, sauces, vinegar, mirin, etc.), canned food, bottled food, bags Filled foods (eg, beef rice, pot rice, red rice, curry, various other prepared foods, etc.), semi-dried or concentrated foods (eg, lever paste, other Spreads, buckwheat · udon juice, concentrated soups, etc., dried foods (eg, instant food, instant curry, instant rice, powdered juice, powdered soup, instant miso soup, cooked food, cooked beverages, etc.) Cooked soups), frozen foods (eg, sukiyaki, chawanmushi, eel kabayaki, hamburger steak, shiyumai, dumplings, various sticks, fruit cocktails, etc.), solid foods, liquid foods (eg, soups), spices, etc. Agricultural products ・ Processed forest products, processed livestock products, processed marine products, etc.

本発明の食品又は飲料は、その原料に本発明の組成物を用いて、公知の食品又は飲料の製造方法に従って製造することができる。該食品又は飲料中の本発明の組成物の含有量としては特に限定はないが、乾燥重量換算で、好ましくは 1〜1 0 0重量％、より好ましくは 1 0〜9 0重量％である。 The food or beverage of the present invention can be produced by using the composition of the present invention as a raw material according to a known method for producing a food or beverage. The content of the composition of the present invention in the food or beverage is not particularly limited, but is preferably 1 to 100% by weight, more preferably 10 to 90% by weight in terms of dry weight.

本発明の組成物を、マウスに対し体重 1 k g当り 1 g Z日で経口投与しても毒性は認められない。実施例 No toxicity was observed when the composition of the present invention was orally administered to mice at 1 g Z / kg body weight per day. Example

以下、本発明を実施例をもって詳細に説明するが本発明はこれらの実施例に何ら限定されるものではない。参考例 1 熱水可溶画分と不溶画分の調製 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Reference Example 1 Preparation of hot water soluble fraction and insoluble fraction

器内温を 60°Cに設定した熱風乾燥器を用いて、ブナシメジ Lyo p hy l 1 urn u 1 ma r i urn M— 8171 (FERM B P - 1415) の生鮮子実体を乾燥後、粉碎することにより子実体粉末を得た。該子実体粉末 500 gに 10 Lの純水を加えて 95°C、 3時間熱水処理を行った。熱水抽出物を室温まで冷却させた後、遠心分離（6500 G、 30分、室温）を行い、熱水可溶画分と熱水不溶画分を得た。得られた熱水可溶画分はエバポレー夕一にて濃縮後、熱水不溶画分はそのまま凍結乾燥を行うことにより、熱水可溶画分凍結乾燥物 210 g、熱水不溶画分凍結乾燥物 247 gを得た。これらの凍結乾燥物は下記実施例に用いる前に粉砕機を用いて粉末状にした。参考例 2 熱水可溶画分と不溶画分の腫瘍増殖抑制活性 Using a hot-air dryer with the inside temperature set at 60 ° C, the fresh fruit body of Bunashimeji Lyo phyl 1 urn u 1 ma ri urn M-8171 (FERM BP-1415) is dried and ground. A fruiting body powder was obtained. 10 L of pure water was added to 500 g of the fruit body powder, and subjected to a hot water treatment at 95 ° C for 3 hours. After the hot water extract was cooled to room temperature, it was centrifuged (6500 G, 30 minutes, room temperature) to obtain a hot water soluble fraction and a hot water insoluble fraction. The obtained hot water-soluble fraction was concentrated in an evaporator and the hot water-insoluble fraction was lyophilized to obtain 210 g of the freeze-dried hot-water-soluble fraction and 210 g of the hot-water-insoluble fraction. 247 g of a dried product were obtained. These freeze-dried products were pulverized using a pulverizer before use in the following Examples. Reference Example 2 Tumor growth inhibitory activity of hot water soluble fraction and insoluble fraction

I CRマウス（日本エスエルシー社）は、 5週齢の雌を購入し 6週齢で使用した。 S a r c oma— 180 (以下 S— 180 ) 腫瘍細胞は、 I CRマウスの腹腔に移植して腹水をつくり、 7日毎に別のマウスに移植して継代した。継代して 7日目の腹水を採取し、リン酸緩衝液を用いて、懸濁と遠心分離による洗浄を繰り返した後、同緩衝液に懸濁して細胞数を計測後、 1 X 10⁸個/ mLとなるように調整した。この 0. lmLを I CRマウスの右側腹部皮下に移植し、 7曰後に形成された固形腫瘍の大きさを測定した。腫瘍の大きさの平均が各群で均等になるように、また 1群 10匹となるようにマウスを 4群に群分けした。そのうちの 2群に対してそれぞれ、参考例 1で調製した 2種の凍結乾燥粉末を、別の 1群には参考例 1で調製した子実体粉末をサンプルとして通常の粉末飼料 C E— 2に混ぜてマウスに与えた。投与量は子実体粉末に換算して、粉末試料 CE— 2に対して重量比で 10 %混ぜて与えた場合と同等になるようにした。コント口一ル群には CE— 2のみをサンプルとして与えた。腫瘍の大きさは、 S— 180移植後 5週目に測定した。その結果を表 1に示す。なお、腫瘍の大きさは長径と短径を ICR mice (Japan SLC) purchased 5-week-old females and used them at the age of 6 weeks. S arc oma-180 (hereinafter S-180) tumor cells were transplanted into the abdominal cavity of ICR mice to create ascites, and transplanted to another mouse every 7 days for passage. The ascites was collected on day 7 after the passage, and the cells were repeatedly suspended and washed by centrifugation using a phosphate buffer, and then suspended in the same buffer, and the number of cells was counted. It was adjusted to 10 ⁸ cells / mL. This 0.1 mL was implanted subcutaneously into the right flank of ICR mice, and the size of the solid tumor formed after 7 was measured. Mice were divided into four groups so that the average tumor size was uniform in each group and that there were 10 mice per group. For each of the two groups, the two freeze-dried powders prepared in Reference Example 1 were mixed, and in another group, the fruiting body powder prepared in Reference Example 1 was mixed as a sample with ordinary powder feed CE-2. To the mice. The dose was converted to fruiting body powder, and was adjusted to be equivalent to that given when the powder sample CE-2 was mixed at a weight ratio of 10%. The control group received only CE-2 as a sample. Tumor size was measured 5 weeks after S-180 implantation. The results are shown in Table 1. The size of the tumor depends on the major axis and minor axis.

0 測定し、以下の計算式：腫瘍体積（mm³ ) = (長径） X (短径） ² /2 にしたがって体積を算出して比較した。また、腫瘍増殖抑制活性は、以下の計算式：腫瘍増殖抑制活性（％)= { (コントロール群の腫瘍体積一各サンプル投与群の腫瘍体積） /コントロール群の腫瘍体積 } X 100 にしたがって算出した。 0 Measured, the following calculation formula: Tumor volume (mm ³⁾ = (long diameter) was X (minor) to calculate the volume in accordance with ^2/2 compared. The tumor growth inhibitory activity was calculated according to the following formula: Tumor growth inhibitory activity (%) = {(tumor volume of control group-tumor volume of each sample administration group) / tumor volume of control group} × 100 did.

表 1 table 1

投与したサンプル腫瘍体積腫瘍増殖抑制活性 Administered sample Tumor volume Tumor growth inhibitory activity

(平均土標準誤差） (%) (Average soil standard error) (%)

CE- 2 (コントロール） 3212±370 CE-2 (control) 3212 ± 370

子実体粉未 2416±604 24. 8 Fruit body powder not yet 2416 ± 604 24.8

熱水不溶画分 2891±703 10. 0 Hot water insoluble fraction 2891 ± 703 10.0

熱水可溶面分 1447±511 55. 0 以上のようにブナシメジの子実体粉末を熱水処理した場合、可溶画分に S— 1 80腫瘍の増殖を抑制する作用が認められた。また、その抑制作用はもとの子実体粉末よりも強いものであった。実施例 1 熱水処理時間の検討 Hot water soluble surface fraction 1447 ± 511 55.0 When the fruit body powder of Bunashimeji was treated with hot water as described above, the soluble fraction was found to have the effect of suppressing the growth of S-180 tumor. The inhibitory effect was stronger than that of the original fruit body powder. Example 1 Examination of hot water treatment time

熱水処理時間を 1、 2、 3又は 5時間に変更した以外は参考例 1と同様の方法で調製することにより、 4種の熱水可溶画分の凍結乾燥粉末を得た。 250 gの子実体粉末よりそれぞれの熱水可溶画分の凍結乾燥粉末を約 1 1 O g得た。参考例 2と同じ条件で、 S— 180腫瘍を I CRマウスに移植し、 7日目に腫瘍の大きさの平均が各群間で均等になるように群分けして用いた。上記各画分の凍結乾燥粉末をサンプルとして通常の粉末飼料 CE— 2に混ぜてマウスに与えた。投与量は子実体粉末に換算して、粉末試料 CE— 2に対して重量比で 10%混ぜて与えた場合と同等になるように用いた。コント口一ル群には CE— 2のみをサンプルとして与えた。腫瘍の大きさは、 S— 180移植後 5週目に測定した。また、腫瘍の大きさと腫瘍増殖抑制活性は、参考例 2と同様に算出した。その結果を表 2に示す。表 2 Lyophilized powders of four types of hot water-soluble fractions were obtained by preparing in the same manner as in Reference Example 1 except that the hot water treatment time was changed to 1, 2, 3 or 5 hours. Approximately 11 Og of a lyophilized powder of each hot water-soluble fraction was obtained from 250 g of the fruiting body powder. Reference: Under the same conditions as in Example 2, S-180 tumors were transplanted into ICR mice, and The groups were used in such a manner that the average of the sizes was equal among the groups. The freeze-dried powder of each of the above fractions was mixed with a normal powder feed CE-2 as a sample and given to mice. The dose was converted to fruiting body powder and used so that it would be equivalent to the case where the powder sample CE-2 was mixed at a weight ratio of 10% and given. The control group received only CE-2 as a sample. Tumor size was measured 5 weeks after S-180 implantation. The tumor size and tumor growth inhibitory activity were calculated as in Reference Example 2. The results are shown in Table 2. Table 2

投与したサンプル腫瘍体積腫瘍增前抑制活性 Administered sample Tumor volume Tumor pre-inhibitory activity

(平均 ±標準誤差〉 (%) (Mean ± standard error) (%)

CE— 2 (コント口ール） 4230+917 CE-2 (control port) 4230 + 917

1時間熱水処理後の可溶面分 3132±673 26. 0 Soluble surface after 1 hour hot water treatment 3132 ± 673 26.0

2時間熱水処理後の可溶画分 2744±820 35. 1 Soluble fraction after 2 hours of hot water treatment 2744 ± 820 35.1

3時間熱水処理後の可溶画分 2240土 348 47. 0 Soluble fraction after 3 hours of hot water treatment 2240 Sat 348 47.0

5時間熱水処理後の可溶画分 3216+744 24, 0 すべてのブナシメジの熱水可溶画分に S— 180固形腫瘍に対する強い増殖抑制作用がみられ、中でも処理時間が 2時間と 3時間のものにおいて S— 180固形腫瘍に対するより強い増殖抑制作用がみられた。実施例 2 子実体粉末由来の分子量 10万超画分の調製 Soluble fraction after 5 hours of hot water treatment 3216 + 744 24, 0 All the hot water soluble fractions of Bunashimeji showed strong growth inhibition against S-180 solid tumors, and the treatment time was 2 hours. At 3 hours, a stronger inhibitory effect on the growth of S-180 solid tumors was observed. Example 2 Preparation of Fraction of Molecular Weight Over 100,000 from Fruiting Body Powder

器内温を 60°Cに設定した熱風乾燥器を用いて、ブナシメジ Lyo phy l 1 um u 1 ma r i urn M—8171 (FERM BP— 1415) の生鮮子実体を乾燥後、粉砕することにより子実体粉末を得た。該子実体粉末 500 gに 10 Lの純水を加えて 95°C、 3時間熱水処理を行った。熱水抽出物を室温まで冷却させた後、遠心分離（6500 G、 30分、室温）を行い、熱水可溶画分を得た。熱水可溶画分は排除分子量 10万のホロファイバー（アミコン社製）を装着した限外ろ過器により濃縮後、凍結乾燥し、粉碎器により粉碎することにより、分子量 10万超画分の凍結乾燥粉末約 20 gを得た。実施例 3 子実体粉末由来の分子量 10万超画分の腫瘍増殖抑制活性 Using a hot air dryer with the inside temperature set at 60 ° C, the fresh fruit body of Bunashimeji Lyo phyl 1 um u 1 ma uri urn M-8171 (FERM BP-1415) is dried and crushed. A substance powder was obtained. 10 L of pure water was added to 500 g of the fruit body powder, and subjected to a hot water treatment at 95 ° C for 3 hours. After cooling the hot water extract to room temperature, it was centrifuged (6500 G, 30 minutes, room temperature) to obtain a hot water soluble fraction. The hot water-soluble fraction is concentrated by an ultrafilter equipped with a hollow fiber of 100,000 exclusion molecular weight (manufactured by Amicon), freeze-dried, and pulverized by a pulverizer to obtain a fraction having a molecular weight of more than 100,000. About 20 g of freeze-dried powder was obtained. Example 3 Tumor growth inhibitory activity of a fraction with a molecular weight of more than 100,000 derived from fruiting body powder

参考例 2と同じ条件で、 S— 180腫瘍を I CRマウスに移植し、 7日目に腫瘍の大きさの平均が各群間で均等になるように 2群に群分けして用いた。そのうちの 1群に、実施例 2で調製した凍結乾燥粉末をサンプルとして通常の粉末飼料 CE— 2に混ぜてマウスに与えた。投与量は子実体粉末に換算して、粉末試料 C E— 2に対して重量比で 10 %混ぜて与えた場合と同等になるようにした。コントロ一ル群には CE— 2のみをサンプルとして与えた。腫瘍の大きさは、 S— 1 80移植後 5週目に測定した。また、腫瘍の大きさと腫瘍増殖抑制活性は、参考例 2と同様に算出した。その結果を表 3に示す。 Under the same conditions as in Reference Example 2, S-180 tumors were transplanted into ICR mice, and used on day 7 by dividing them into two groups so that the average size of the tumors became equal between the groups. . To one of the groups, the freeze-dried powder prepared in Example 2 was mixed with a normal powder feed CE-2 as a sample and fed to mice. The dose was converted to fruiting body powder so that it would be equivalent to the case where the powder sample CE-2 was mixed at a weight ratio of 10%. The control group received only CE-2 as a sample. Tumor size was measured 5 weeks after S-180 transplantation. The tumor size and tumor growth inhibitory activity were calculated as in Reference Example 2. The results are shown in Table 3.

表 3 Table 3

投与したサンプル腫疡体積腫瘍増殖抑制活性 Administered sample Tumor volume Tumor growth inhibitory activity

(平均土標準誤差〉 (%) (Average soil standard error) (%)

C E - 2 (コント口一ル） 5 1 3 1 ± 1 83 3 C E-2 (Control port) 5 1 3 1 ± 1 83 3

分子量 1 0万超面分 1 84 6土 5 90 6 4. 0 表 3に示すように、ブナシメジ熱水抽出物から得られた分子量 10万超画分において S— 180固形腫瘍の増殖を抑制する作用がみられた。実施例 4 生鮮子実体由来の分子量 10万超画分の調製 As shown in Table 3, the growth of S-180 solid tumors was observed in the fraction with a molecular weight of more than 100,000 obtained from Bunashimeji hot water extract. An inhibitory effect was observed. Example 4 Preparation of Fraction Over 100,000 from Fresh Fruit Entity

ブナシメジ Lyo phy l l um u lma r i um M— 817 1 (FER M BP— 1415) の生鮮子実体 2. 3 k gをホモジナイズしてペースト状にし、純水を加えて全量を 4. 6 Lとしたのち、 95 、 3時間熱水処理を行った。熱水抽出物を室温まで冷却させた後、遠心分離（6500 G、 30分、室温）を行い、熱水可溶画分 3 Lを得た。熱水不溶画分には再度純水を加えて全量を 3 Lとしたのち、 95° (：、 30分熱水処理し、熱水抽出物を室温まで冷却させた後、遠心分離（6500 G、 30分、室温）を行うことにより熱水可溶画分 1. 6 Lを得た。両熱水可溶画分を混合したのち、排除分子量 10万のホロファイバー (アミコン社製）を装着した限外ろ過器により濃縮後、凍結乾燥し、粉砕器により粉碎することにより、分子量 10万超画分の凍結乾燥粉末約 2. 4 gを得た。またブナシメジ L y 0 p h y 1 1 urn u 1 ma r i urn K一 0259 (FE RM P— 12981) の生鮮子実体 2. 5 k gから上記と同様の操作を行い、分子量 10万超画分の凍結乾燥粉末約 1. 8 gを得た。実施例 5 生鮮子実体由来の分子量 10万超画分の腫瘍増殖抑制活性 2.3 kg of fresh fruit body of Bunyoshimeji Lyo phy ll um ulma ri um M—817 1 (FER M BP—1415) was homogenized to form a paste, and pure water was added to make a total volume of 4.6 L. After that, hot water treatment was performed for 95, 3 hours. After cooling the hot water extract to room temperature, centrifugation (6500 G, 30 minutes, room temperature) was performed to obtain 3 L of a hot water soluble fraction. Pure water was again added to the hot water-insoluble fraction to make a total volume of 3 L, and then the mixture was treated with hot water at 95 ° (30 minutes, cooled to room temperature, and then centrifuged (6500 G , 30 minutes at room temperature) to obtain a hot water soluble fraction 1.6 L obtained. After mixing both hot water soluble fractions, the mixture is concentrated by an ultrafilter equipped with a hollow fiber with an excluded molecular weight of 100,000 (manufactured by Amicon), freeze-dried, and pulverized by a pulverizer to obtain a molecular weight. About 2.4 g of freeze-dried powder of more than 100,000 fractions was obtained. In addition, the same operation as above was performed from 2.5 kg of a fresh fruit body of Bunashimeji L y 0 phy 1 1 urn u 1 ma uri urn K-1 0259 (FE RM P-12981), and lyophilization of a fraction with a molecular weight of over 100,000 About 1.8 g of powder was obtained. Example 5 Tumor growth inhibitory activity of a fraction with a molecular weight of over 100,000 derived from a fresh fruit body

参考例 2と同じ条件で、 S— 180腫瘍を I CRマウスに移植し、 7日目に腫瘍の大きさの平均が各群間で均等になるように 3群に群分けして用いた。そのうちの 2群にそれぞれ、実施例 4で調製した 2種の凍結乾燥粉末をサンプルとして通常の粉末飼料 CE— 2に混ぜてマウスに与えた。投与量は子実体粉末に換算して、粉末試料 CE— 2に対して重量比で 10 %混ぜて与えた場合と同等になるように用いた。コントロール群には CE— 2のみをサンプルとして与えた。腫瘍の大きさは、 S— 180移植後 5週目に測定した。また、腫瘍の大きさと腫瘍抑制活性は、参考例 2と同様に算出した。その結果を表 4に示す。表 4 Under the same conditions as in Reference Example 2, S-180 tumors were transplanted into ICR mice, and used on day 7 by dividing them into three groups so that the average size of the tumors became equal among the groups. . To each of the two groups, mice were given the two freeze-dried powders prepared in Example 4 as samples and mixed with a normal powder feed CE-2. The dose was converted to fruiting body powder and used so that it would be equivalent to the case where the powder sample CE-2 was mixed at a weight ratio of 10% and given. The control group received only CE-2 as a sample. Tumor size was measured 5 weeks after S-180 implantation. In addition, tumor size and tumor-suppressing activity were calculated in the same manner as in Reference Example 2. The results are shown in Table 4. Table 4

サンプル投与群腫疡体積腫疡抑制活性 Sample administration group Tumor 疡 volume Tumor 疡 inhibitory activity

(平均土標準誤差） (%) (Average soil standard error) (%)

CE- 2 (コント口一ル） 2139±343 CE-2 (Control port) 2139 ± 343

L y o h y l l u m u 1 m a r 1 u m 1289±242 39. 7 L y o h y l l u m u 1 m a r 1 u m 1289 ± 242 39.7

M-8171生鮮子実体由来分子量 1 0万超画分 M-8171 Fresh fruit body origin Molecular weight fraction over 100,000

L y o p h y l l u m u 1 m a r i u m 1207±397 43. 6 L y o p h y l l u m u 1 m a r i u m 1207 ± 397 43.6

K-0259生鮮子実体由来分子: ¾ 1 0万超画分表 4に示すように、ブナシメジの生鮮子実体を熱水処理して得られた分子 0万超画分投与群において S— 180固形腫瘍増殖の抑制がみられた。実施例 6 ィォン交換樹脂分画物の調製器内温を 60°Cに設定した熱風乾燥器を用いて、ブナシメジ Lyophy l 1 urn u 1 ma r i um M— 8171 (FERM BP— 1415) の生鮮子実体を乾燥後、粉碎することにより子実体粉末を得た。該子実体粉末 500 に 10 Lの純水を加えて 95°C、 3時間熱水処理を行った。熱水抽出物を室温まで冷却させた後、遠心分離（6500 G、 30分、室温）を行い、熱水可溶画分 8 . 7 L (pH6. 2) を得た。該熱水可溶画分を排除分子量 10万のホロフアイバー（アミコン社製）を装着した限外ろ過器により濃縮することにより、分子量 10万超画分21^ (pH6. 4) を得た。該分子量 10万超画分に 60 OmLのエタノールを加え、かく拌し、不溶物を遠心分離（15000 G、 30分、室温 ) により取り除いた後、 8 X40 cmのカラムに 1 OmMの NaC 1を含む 10 mM リン酸ナトリウムバッファー（ρΗ7. 0) で平衡化した DEAE—セル口ファイン Α— 800 (チッソ株式会社製）を充填したクロマトグラフィーにかけ、同平衡化バッファーで洗浄した。次に、同平衡化バッファーの NaC 1濃度を 40 OmM又は 100 OmMとしたリン酸ナトリウムバッファー（ρΗ7. 0 ) で溶出し、それぞれを分取した。得られた 40 OmM N a C 1溶出画分及び 100 OmM NaC 1溶出画分は、排除分子量 1万のホロファイバー（アミコン社製）を装着した限外ろ過器により濃縮後、凍結乾燥し、粉砕器により粉砕することにより、 40 OmM NaC 1溶出画分の凍結乾燥粉末を 3. 9 g、 10 0 OmM Na C 1溶出画分の凍結乾燥粉末を 0. 9 g得た。実施例 7 イオン交換樹脂分画物の腫瘍増殖抑制活性 K-0259 Molecule derived from fresh fruit body: 超 100,000 fraction As shown in Table 4, molecules obtained by treating fresh fruit body of Bunashimeji with hot water S-180 solids Suppression of tumor growth was observed. Example 6 Preparation of ion exchange resin fraction After drying the fresh fruit body of Bunashimeji Lyophy l 1 urn u 1 mari um M-8171 (FERM BP-1415) using a hot air dryer with the inside temperature set to 60 ° C, the fruit body is obtained by grinding. A powder was obtained. 10 L of pure water was added to the fruiting body powder 500 and subjected to hot water treatment at 95 ° C. for 3 hours. After cooling the hot water extract to room temperature, it was centrifuged (6500 G, 30 minutes, room temperature) to obtain 8.7 L of hot water soluble fraction (pH 6.2). The hot water-soluble fraction was concentrated by an ultrafilter equipped with a holofiber having an excluded molecular weight of 100,000 (manufactured by Amicon) to obtain a fraction 21 ^ (pH 6.4) having a molecular weight of more than 100,000. After adding 60 OmL of ethanol to the fraction having a molecular weight of more than 100,000 and stirring the mixture, centrifugation (15000 G, 30 minutes, room temperature) removes insolubles, and then 1 OmM NaCl 1 was applied to an 8 × 40 cm column. The column was subjected to chromatography packed with DEAE-Cell Mouth Fine II-800 (manufactured by Chisso Corporation) equilibrated with the same 10 mM sodium phosphate buffer (ρ 7.0), and washed with the same equilibration buffer. Next, elution was carried out with a sodium phosphate buffer (ρ 7.0) in which the NaC 1 concentration of the equilibration buffer was 40 OmM or 100 OmM, and each was separated. The obtained 40 OmM NaC 1 eluted fraction and 100 OmM NaC 1 eluted fraction were concentrated by an ultrafilter equipped with a hollow fiber of 10,000 excluding molecular weight (manufactured by Amicon), lyophilized, By pulverizing with a pulverizer, 3.9 g of a lyophilized powder of a fraction eluted with 40 OmM NaC1 and 0.9 g of a lyophilized powder of a fraction eluted with 100 OmM NaC1 were obtained. Example 7 Tumor growth inhibitory activity of ion exchange resin fraction

参考例 2と同じ条件で、 S— 180腫瘍を I CRマウスに移植し、 7日目に腫瘍の大きさの平均が各群間で均等になるように 3群に群分けした。そのうちの 2 群にそれぞれ、実施例 6で調製した 2種の凍結乾燥粉末をサンプルとして通常の粉末飼料 CE— 2に混ぜてマウスに与えた。投与量は子実体粉末に換算して、粉末試料 C E— 2に対して重量比で 10 %混ぜて与えた場合と同等になるようにし表 6 Under the same conditions as in Reference Example 2, S-180 tumors were transplanted into ICR mice, and divided into three groups on the seventh day so that the average size of the tumors became equal among the groups. Two groups of the two freeze-dried powders prepared in Example 6 were mixed with a normal powdered feed CE-2 and fed to mice. Dosage should be converted to fruiting body powder so that it would be equivalent to that given by mixing 10% by weight with powder sample CE-2. Table 6

分画物名糖含量蛋白質含量脂質含量ゥロン酸含量 Fraction name Sugar content Protein content Lipid content Peronic acid content

(%) (%) (%) (%) (%) (%) (%) (%)

L y o p h y l l um u 1 m a r i u m 51 10 33 0. 6L y o p h y l l um u 1 m a r i u m 51 10 33 0.6

M- 8 1 71子実体粉末由来分子量 10万 M-8 1 71 Molecular weight derived from fruiting body powder 100,000

超面分 Super face

y o p h y l l um u 1 m a r i u m 54 3. 5 30 0. 8 y o p h y l l um u 1 m a r i u m 54 3.5 5.30 0.8

M— 8 171生鮮子実体由来分子量 10万 M—8 171 Molecular weight from fresh fruit body 100,000

超画分 Super fraction

L y o p h y l l um u l ma r i um 48 3 35 3. 2 L y o p h y l l um u l ma r i um 48 3 35 3.2

K- 0259生鮮子実体由来分子量 10万 K-0259 Fresh fruit body derived molecular weight 100,000

超画分 Super fraction

40 OmM N a C 1 溶出画分 55 16 30 2. 2 40 OmM Na C 1 Eluted fraction 55 16 30 2.2

産業上の利用可能性 Industrial applicability

本発明により、安価な食用担子菌が有する抗腫瘍性を有し、経口摂取によりその効果を発揮し得る抗腫瘍性組成物、及び該組成物を含有する食品又は飲料を得ることができる。よって、本発明は特に医薬や健康食品分野において特に有用である。 According to the present invention, it is possible to obtain an antitumor composition having the antitumor properties of inexpensive edible basidiomycetes and exerting its effects by oral ingestion, and a food or beverage containing the composition. . Therefore, the present invention is particularly useful in the field of medicine and health food.

7 た。コントロール群には CE— 2のみをサンプルとして与えた。腫瘍の大きさは S— 180移植後 5週目に測定した。また、腫瘍の大きさと腫瘍増殖抑制活性は、参考例 2と同様に算出した。その結果を表 5に Ιτ ^ 5 7 It was. The control group received only CE-2 as a sample. Tumor size was measured 5 weeks after S-180 implantation. The tumor size and tumor growth inhibitory activity were calculated in the same manner as in Reference Example 2. Table 5 shows the results as Ιτ ^ 5

投与したサンプル腫疡体積腫癍增殖抑制活性 Administered sample Tumor volume Tumor growth inhibitory activity

(平均士標 Φ誤差） (%) (Average Φ error) (%)

C E - 2 (コントロール） 3358± 101 5 C E-2 (control) 3358 ± 101 5

400 mM N a C 1溶出画分 1066± 312 68. 3 400 mM Na C 1 eluted fraction 1066 ± 312 68.3

1 000 mM N a C 1？容出画分 3051 ± 1034 9. 1 その結果、表 5に示すように 400mM NaC 1溶出画分に腫瘍増殖抑制活性が認められた。実施例 8 各画分の組成 1 000 mM Na C 1? Eluted fraction 3051 ± 1034 9.1 As a result, as shown in Table 5, a 400 mM NaC1 eluted fraction showed tumor growth inhibitory activity. Example 8 Composition of each fraction

上記実施例 2 4及び 6で得られた各分子量 10万超画分及び 40 OmM N aC l溶出画分について、糖含量、ゥロン酸含量、脂質含量及び蛋白質含量をそれぞれフエノール硫酸法〔アナリティカルケミストリー（An a 1 y t i c a 1 Ch em i s t r y) 、第 28巻、第 350頁（1956) 〕、カルバゾール硫酸法〔アナリティカルバイオケミストリー（An a 1 y t i c a 1 B i o c h em i s t r y) 、第 4巻、第 330頁（1962) 〕、クロ口ホルム一メタノール混液抽出法〔ジャーナルォブバイオロジカルケミストリー ( J ou r n a l o f B i o l o g i c a l Ch emi s t r y) 、第 22 6巻、第 497頁（1957) 〕、及び蛋白質測定キット（C o oma s s i e For each of the fractions having a molecular weight of more than 100,000 and the fraction eluted with 40 OmM NaCl obtained in the above Examples 24 and 6, the sugar content, the peronic acid content, the lipid content and the protein content were each determined by the phenol-sulfuric acid method [ Analytical Chemistry (An a 1 ytica 1 Chemistry), Vol. 28, p. 350 (1956)], Carbazole Sulfuric Acid Method [Analytical Biochemistry (An a 1 ytica 1 Biochemistry), Vol. , P. 330 (1962)], a method for extracting mixed forms of methanol with methanol [Journal of Biological Chemistry, Vol. 226, p. 497 (1957)], and proteins Measurement kit (C oma ssie

P r o t e i n As s ay Re a g e n t K i t P i e r c e社) を用いて測定した。その結果を表 6に示す。 The measurement was performed by using the method of “ProteinAssayReagentKitPiterc”. Table 6 shows the results.

16 16

Claims

請求の範囲 The scope of the claims

1. ブナシメジ子実体の熱水抽出物中の、可溶性であって排除分子量 10万の限外ろ過膜を通過しない画分からなる抗腫瘍性組成物。 1. An antitumor composition comprising a soluble, non-excluded molecular weight of 100,000 excluded ultrafiltration membrane in a hot water extract of Bunashimeji fruit body.

2. 糖含量が 45〜60重量％、脂質含量が 20〜45重量％、タンパク質含量が 1〜 15重量％である請求項 1記載の抗腫瘍性組成物。 2. The antitumor composition according to claim 1, wherein the sugar content is 45 to 60% by weight, the lipid content is 20 to 45% by weight, and the protein content is 1 to 15% by weight.

3. ブナシメジ子実体の熱水抽出物中の、可溶性であって排除分子量 10万の限外ろ過膜を通過しない画分からなる組成物を、さらに陰イオン交換樹脂に供した後、溶出させて得られ得る抗腫瘍性組成物。 3. The composition consisting of the soluble fraction of the hot-water extract of the fruit body of Bunashimeji, which does not pass through the ultrafiltration membrane with an excluded molecular weight of 100,000, is further applied to an anion exchange resin and eluted. Antitumor compositions that can be used.

4. 糖含量が 50〜60重量％、脂質含量が 20〜40重量％、タンパク質含量が 5〜 20重量％である請求項 3記載の抗腫瘍性組成物。 4. The antitumor composition according to claim 3, wherein the sugar content is 50 to 60% by weight, the lipid content is 20 to 40% by weight, and the protein content is 5 to 20% by weight.

5. ブナシメジが L y o p h y 1 1 um u 1 ma r i um M— 8171 ( FERM B P— 1415) 、又は Ly o p hy 1 1 um u 1 m a r i um K一 0259 (FERM P— 12981 ) である請求項 1〜 4いずれか 1項に記載の抗腫瘍性組成物。 5. Claims 1 to 5, wherein the benashimeji is Lyophy 1 1 umu 1 mari um M-8171 (FERM BP-1415) or Lyophy 11 umu 1 mari um K-1 0259 (FERM P-12981). 4. The antitumor composition according to any one of the above items 1.

6. 熱水抽出物が処理温度 80〜 100 ° (：、処理時間 1〜 5時間で熱水処理して得られたものである請求項 1〜 5のいずれ力、 1項に記載の抗腫瘍性組成物。 6. The antitumor according to any one of claims 1 to 5, wherein the hot water extract is obtained by performing a hot water treatment at a processing temperature of 80 to 100 ° (a processing time of 1 to 5 hours). Composition.

7. 請求項 1〜 6のいずれか 1項に記載の抗腫瘍性組成物を含有する食品又は飲料。 7. A food or beverage containing the antitumor composition according to any one of claims 1 to 6.

8. (1) ブナシメジ子実体を 1〜5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、及び（2) 工程（1) で得られた可溶性画分を排除分子量 10万の限外ろ過に供する工程を包含することを特徴とする抗腫瘍性組成物の製造方法。 8. (1) a step of removing insolubles from a hot water extract obtained by treating the fruit body of Bunashimeji with hot water for 1 to 5 hours; and (2) removing the soluble fraction obtained in step (1). A method for producing an antitumor composition, which comprises a step of subjecting to ultrafiltration having a molecular weight of 100,000 excluded.

9. 抗腫瘍性組成物が、糖含量が 45〜 60重量％、脂質含量が 20〜 45重量％、タンパク質含量が 1〜15重量％である組成物である請求項 8記載の抗腫瘍性組成物の製造方法。 9. The antitumor composition according to claim 8, wherein the antitumor composition has a sugar content of 45 to 60% by weight, a lipid content of 20 to 45% by weight, and a protein content of 1 to 15% by weight. A method for producing a hydrophilic composition.

10. (1) ブナシメジ子実体を 1〜5時間熱水処理して得られる熱水抽出物より、不溶物を除去する工程、（2) 工程（1) で得られた可溶性画分を排除分子量 10万の限外ろ過に供する工程、及び（3) 工程（2) で得られた組成物を陰イオン交換樹脂に供した後、溶出させる工程を包含することを特徴とする抗腫瘍性組成物の製造方法。 10. (1) The step of removing insolubles from the hot water extract obtained by treating the fruit body of Bunashimeji with hot water for 1 to 5 hours, (2) Excluding the soluble fraction obtained in step (1) And (3) subjecting the composition obtained in the step (2) to an anion exchange resin and then eluting it. A method for producing a hydrophilic composition.

1 1. 抗腫瘍性組成物が、糖含量が 50〜 60重量％、脂質含量が 20〜 40 重量％、タンパク質含量が 5〜20重量％である組成物である請求項 10記載の抗腫瘍性組成物の製造方法。 11. The antitumor composition according to claim 10, wherein the antitumor composition is a composition having a sugar content of 50 to 60% by weight, a lipid content of 20 to 40% by weight, and a protein content of 5 to 20% by weight. A method for producing the composition.

12. ブナシメジが L y o p h y 1 1 um u 1 m a r i u m M— 8171 (F E RM B P— 1415) 、又は L y o p h y 1 1 um u 1 m a r i um12. Bunashimeji is Lyophy 1 1 um u 1 ma a r i u m M— 8171 (F ERM B P— 1415) or Lyop h y 1 1 um u 1 m a r i um

K一 0259 (FERM P— 12981) である請求項 8〜11いずれか 1 項に記載の抗腫瘍性組成物の製造方法。 The method for producing an antitumor composition according to any one of claims 8 to 11, which is K-1259 (FERM P-12981).