JP6099360B2 - Immune function enhancer by polymer fraction of Anninkou extract - Google Patents
Immune function enhancer by polymer fraction of Anninkou extract Download PDFInfo
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- JP6099360B2 JP6099360B2 JP2012240606A JP2012240606A JP6099360B2 JP 6099360 B2 JP6099360 B2 JP 6099360B2 JP 2012240606 A JP2012240606 A JP 2012240606A JP 2012240606 A JP2012240606 A JP 2012240606A JP 6099360 B2 JP6099360 B2 JP 6099360B2
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Description
本発明は、アンニンコウ抽出液の高分子画分によるアレルギー疾患の治療や予防に関する。また本発明は、アンニンコウ抽出液の高分子画分、及び当該高分子画分の免疫機能の調整のための利用に関する。 The present invention relates to the treatment and prevention of allergic diseases by using a polymer fraction of an anninkou extract. The present invention also relates to a polymer fraction of an anninkou extract and use for adjusting the immune function of the polymer fraction.
アレルギー疾患の患者数は、先進国を中心に急激に増加している。日本の全人口の３人に1人が何らかのアレルギー疾患に罹患しており、近年はさらに増加が加速しているのが現状である。その増加の主体は、アレルギー性鼻炎（花粉症を含む）と喘息患者の増加によるものと考えられている。また、その重症化も進み、日常生活に著しい支障を慢性的にきたすことから 、国民の健康上重大な問題となっている。これらアレルギー疾患の治療には、抗アレルギー薬、抗ヒスタミン薬、副腎皮質ステロイド薬、気管支拡張薬、免疫抑制薬を用いた対症療法が中心である。これらの対処療法には、奏功性の向上や副作用の克服等の課題が指摘されている。さらに、難病性の喘息、アトピー性皮膚炎等は、これらのアレルギー治療薬では十分な治療効果が得られていないことも少なくない。このため、副作用を軽減した奏功性の高いアレルギー疾患治療法の確立が切望されている。 The number of patients with allergic diseases is increasing rapidly, especially in developed countries. One out of every three people in Japan suffers from some kind of allergic disease, and in recent years, the increase has accelerated further. The main reason for the increase is thought to be the increase in allergic rhinitis (including hay fever) and asthma patients. In addition, it has become a serious problem for people's health because it has become more serious and chronically has serious problems in daily life. Treatment of these allergic diseases is mainly symptomatic therapy using antiallergic drugs, antihistamines, corticosteroids, bronchodilators, and immunosuppressive drugs. These coping therapies have been pointed out issues such as improved response and overcoming side effects. Furthermore, intractable asthma, atopic dermatitis, etc., it is often the case that these allergy remedies do not provide sufficient therapeutic effects. Therefore, establishment of a highly effective allergic disease treatment method with reduced side effects is eagerly desired.
アンニンコウ（Grifola gargal）は、チリ・パタゴニア原産で杏仁様の芳香が特徴的な食用担子菌で、ヒダナシタケ目、サルノコシカケ科、マイタケ属に属する木材腐朽菌である。これまで抗酸化作用・破骨細胞抑制作用や糖尿病・肥満症改善効果など、アンニンコウは多彩な機能性を持つことが明らかになっている。
しかしながら、アンニンコウ抽出液の炎症、アレルギー、および喘息疾患に対する抑制効果は、これまで報告されておらず、いずれの文献にも記載されていない。
Anninkou (Grifola gargal) is an edible basidiomycete that is native to Chile and Patagonia and has a characteristic apricot-like fragrance. Up to now, it has been clarified that Anninkou has various functions such as antioxidative action, osteoclast inhibitory action, and diabetes / obesity improvement effect.
However, the inhibitory effect on the inflammation, allergy, and asthma disease of the Anninkou extract has not been reported so far and is not described in any literature.
本発明はこのような課題に鑑みてなされたものであり、アンニンコウの熱水抽出液の高分子画分によるアレルギー疾患の治療剤や予防剤を提供することを課題とする。また本発明は、アンニンコウ抽出液の高分子画分、及び当該高分子画分による免疫機能を調整するための薬剤を提供することを課題とする。 This invention is made | formed in view of such a subject, and makes it a subject to provide the therapeutic agent and preventive agent of an allergic disease by the high molecular fraction of the hot water extract of Anninkou. Moreover, this invention makes it a subject to provide the chemical | medical agent for adjusting the immune function by the high molecular fraction of the ginseng extract, and the said high molecular fraction.
本発明者らは、上記課題を解決するために鋭意研究を行った。本発明者らは、アンニンコウの熱水抽出液、その低分子画分および高分子画分を使用し、免疫応答細胞からのサイトカイン産生能等を検討した。また、卵白アルブミン（OVA）で誘導した気管支喘息モデルマウスにアンニンコウの熱水抽出液、その低分子画分および高分子画分を投与し、気道過敏性の測定および、脾臓、気管支肺胞洗浄液（BALF）、血液中の免疫機能に及ぼす影響を検討した。
その結果本発明者らは、アンニンコウの熱水抽出液の高分子画分に、IFN-γ、IL-12 及びIL-10 の産生促進作用、や制御性T細胞の増殖促進作用及びＮＫＴ細胞増殖抑制作用があることを明らかにした。また喘息マウスモデルにおいてIgE、IgG、MCP-1を強力に抑制し、肺組織における粘液過多分泌や炎症を抑制する機能を持ち、アレルギーおよび喘息疾患に対する抑制効果が認められた。
さらに本発明者らは、アンニンコウの熱水抽出液の高分子画分が、分子量400000以上の成分と分子量22800の2つの成分からなることを明らかにした。
本発明はこのような知見に基づくものであり、以下の〔１〕〜〔２０〕を提供する。
〔１〕食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方又はいずれか一方を含む、アレルギー疾患の治療及び予防の両方又はいずれか一方のための薬剤。
〔２〕食用キノコがＧｒｉｆｏｌａ属、Ｐｌｅｕｒｏｔｕｓ属、Ｌｅｎｔｉｎｕｌａ属、Ｐｈｏｌｉｏｔａ属、Ａｇｒｏｃｙｂｅ属、Ｌｅｕｃｏｐａｘｉｌｌｕｓ属、Ａｇａｒｉｃｕｓ属、Ｔｒｉｃｈｏｌｏｍａ属からなる群より選択される少なくとも一つのキノコである、〔１〕に記載の薬剤。
〔３〕食用キノコが、アンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）、マイタケ（Ｇｒｉｆｏｌａ ｆｒｏｎｄｏｓａ）、ヒラタケ（Ｐｌｅｕｒｏｔｕｓ ｏｓｔｒｅａｔｕｓ）、エリンギ（Ｐｌｅｕｒｏｔｕｓ ｅｒｉｎｇｉｉ）、シイタケ（Ｌｅｎｔｉｎｕｌａ ｅｄｏｄｅｓ）、ナメコ（Ｐｈｏｌｉｏｔａ ｎａｍｅｋｏ）、ヤナギマツタケ（Ａｇｒｏｃｙｂｅ ｃｙｌｉｎｄｒａｃｅａ）、オオイチョウタケ（Ｌｅｕｃｏｐａｘｉｌｌｕｓ ｇｉｇａｎｔｅｕｓ）、ヒメマツタケ（Ａｇａｒｉｃｕｓ ｂｌａｚｅｉ）、サウーバ（Ｔｒｉｃｈｏｌｏｍａ ｓｐ．）及びキッタリア（Ｃｙｔｔａｒｉａ ｅｓｐｉｎｏｓａｅ）からなる群より選択される少なくとも一つのキノコである、〔１〕又は〔２〕に記載の薬剤。
〔４〕食用キノコがアンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）である、〔１〕から〔３〕のいずれかに記載の薬剤。
〔５〕食用キノコがアンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）の子実体である、〔１〕から〔４〕のいずれかに記載の薬剤。
〔６〕アレルギー疾患が気管支喘息、アトピー性皮膚炎、アレルギー性鼻炎、気管支炎、花粉症、口内炎、腸炎、潰瘍、狭心症、リウマチ様関節炎からなる群より選択される、〔１〕から〔５〕のいずれかに記載の薬剤。
〔７〕食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方又はいずれか一方を含む、免疫機能調整剤。
〔８〕食用キノコがＧｒｉｆｏｌａ属、Ｐｌｅｕｒｏｔｕｓ属、Ｌｅｎｔｉｎｕｌａ属、Ｐｈｏｌｉｏｔａ属、Ａｇｒｏｃｙｂｅ属、Ｌｅｕｃｏｐａｘｉｌｌｕｓ属、Ａｇａｒｉｃｕｓ属、Ｔｒｉｃｈｏｌｏｍａ属からなる群より選択される少なくとも一つのキノコである、〔７〕に記載の免疫機能調整剤。
〔９〕食用キノコが、アンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）、マイタケ（Ｇｒｉｆｏｌａ ｆｒｏｎｄｏｓａ）、ヒラタケ（Ｐｌｅｕｒｏｔｕｓ ｏｓｔｒｅａｔｕｓ）、エリンギ（Ｐｌｅｕｒｏｔｕｓ ｅｒｉｎｇｉｉ）、シイタケ（Ｌｅｎｔｉｎｕｌａ ｅｄｏｄｅｓ）、ナメコ（Ｐｈｏｌｉｏｔａ ｎａｍｅｋｏ）、ヤナギマツタケ（Ａｇｒｏｃｙｂｅ ｃｙｌｉｎｄｒａｃｅａ）、オオイチョウタケ（Ｌｅｕｃｏｐａｘｉｌｌｕｓ ｇｉｇａｎｔｅｕｓ）、ヒメマツタケ（Ａｇａｒｉｃｕｓ ｂｌａｚｅｉ）、サウーバ（Ｔｒｉｃｈｏｌｏｍａ ｓｐ．）及びキッタリア（Ｃｙｔｔａｒｉａ ｅｓｐｉｎｏｓａｅ）からなる群より選択される少なくとも一つのキノコである、〔７〕又は〔８〕に記載の免疫機能調整剤。
〔１０〕食用キノコがアンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）である、〔７〕から〔９〕のいずれかに記載の免疫機能調整剤。
〔１１〕食用キノコがアンニンコウ（Ｇｒｉｆｏｌａ ｇａｒｇａｌ）の子実体である、〔７〕から〔１０〕のいずれかに記載の免疫機能調整剤。
〔１２〕免疫機能の調整がサイトカイン産生促進、ＩｇＥの産生抑制、ＭＣＰ−１の産生抑制、制御性T細胞の増殖促進及びＮＫＴ細胞増殖抑制からなる群より選択される、〔７〕から〔１１〕のいずれかに記載の免疫機能調整剤。
〔１３〕サイトカインがIFN-γ、IL-10及びIL-12からなる群より選択される、〔１２〕に記載の免疫機能調整剤。
〔１４〕高分子画分が以下の工程を含む方法によって得られる、〔１〕から〔６〕のいずれかに記載の薬剤又は〔７〕から〔１３〕のいずれかに記載の免疫機能調整剤；
（ａ）食用キノコの粉末を提供する工程、
（ｂ）工程（ａ）の粉末の熱水抽出液を提供する工程、及び
（ｃ）工程（ｂ）の熱水抽出液から分子量が6000以上の物質からなる画分を取得する工程。
〔１５〕高分子画分が分子量400000以上の物質及び分子量22800の物質の両方又はいずれか一方を含む、〔１４〕に記載の薬剤又は免疫機能調整剤。
〔１６〕以下（ａ）から（ｃ）の工程を含む方法によって調製される食用キノコの熱水抽出液の高分子画分であって、IFN-γ、IL-10又はIL-12の産生促進、ＩｇＥの産生抑制、ＭＣＰ−１の産生抑制、制御性T細胞の増殖促進及びＮＫＴ細胞増殖抑制からなる群より選択される少なくとも１つの活性を有する高分子画分；
（ａ）食用キノコの粉末を提供する工程、
（ｂ）工程（ａ）の粉末の熱水抽出液を提供する工程、及び
（ｃ）工程（ｂ）の熱水抽出液から分子量が6000以上の物質からなる画分を取得する工程。
〔１７〕工程（ｃ）の画分から分子量400000以上の物質及び分子量22800の物質の両方又はいずれか一方を抽出する工程をさらに含む、〔１６〕に記載の高分子画分。
〔１８〕〔１〕から〔６〕、〔１４〕及び〔１５〕のいずれかに記載の薬剤、〔７〕から〔１５〕のいずれかに記載の免疫機能調整剤、並びに〔１６〕又は〔１７〕に記載の高分子画分からなる群より選択される少なくとも１つを含む食品。
〔１９〕以下（ａ）から（ｃ）の工程を含む食用キノコの熱水抽出液の高分子画分であって、IFN-γ、IL-10又はIL-12の産生促進、ＩｇＥの産生抑制、ＭＣＰ−１の産生抑制、制御性T細胞の増殖促進及びＮＫＴ細胞増殖抑制からなる群より選択される少なくとも１つの活性を有する高分子画分の製造方法；
（ａ）食用キノコの粉末を提供する工程、
（ｂ）工程（ａ）の粉末の熱水抽出液を提供する工程、及び
（ｃ）工程（ｂ）の熱水抽出液から分子量が6000以上の物質からなる画分を取得する工程。
〔２０〕工程（ｃ）の画分から分子量400000以上の物質及び分子量22800の物質の両方又はいずれか一方を抽出する工程をさらに含む、〔１９〕に記載の方法。
The inventors of the present invention have intensively studied to solve the above problems. The present inventors examined the ability to produce cytokines from immune-responsive cells using the hot water extract of Anninkou, its low molecular fraction and high molecular fraction. In addition, bronchial asthma model mice induced with ovalbumin (OVA) were administered with hot water extract of Annin, its low and high molecular fractions, airway hypersensitivity measurement, spleen and bronchoalveolar lavage fluid ( BALF), the effect on blood immune function was examined.
As a result, the present inventors added IFN-γ, IL-12 and IL-10 production promoting action, regulatory T cell proliferation promoting action and NKT cell proliferation to the polymer fraction of the hot water extract of Anninkou. It was revealed that there is an inhibitory action. In the asthma mouse model, IgE, IgG, and MCP-1 were strongly suppressed, and they had functions to suppress mucus hypersecretion and inflammation in the lung tissue, and an inhibitory effect on allergy and asthma disease was observed.
Furthermore, the present inventors have clarified that the high molecular fraction of the hot water extract of Anninkou is composed of a component having a molecular weight of 400,000 or more and two components having a molecular weight of 22,800.
The present invention is based on such knowledge and provides the following [1] to [20].
[1] A drug for treatment and / or prevention of allergic diseases, comprising a hot water extract of edible mushrooms and / or a polymer fraction of the extract.
[2] The edible mushroom is at least one mushroom selected from the group consisting of Grifola, Pleurotus, Lentinula, Phoriota, Agrocybe, Leucopaxillus, Agaricus, and Tricholoma .
[3] Edible mushrooms include anemone (Grifola gargal), maitake (Grifola rondea), oyster mushroom (Pleurotus eringii) At least one mushroom selected from the group consisting of Leucopaxillus giganteus, Agaricus blazei, Saucho (Trichoroma sp.), And Cytaria espinosae [2] Drugs.
[4] The drug according to any one of [1] to [3], wherein the edible mushroom is a ginseng (Grifola gargal).
[5] The drug according to any one of [1] to [4], wherein the edible mushroom is a fruit body of an garlic (Grifola gargal).
[6] The allergic disease is selected from the group consisting of bronchial asthma, atopic dermatitis, allergic rhinitis, bronchitis, hay fever, stomatitis, enteritis, ulcer, angina, rheumatoid arthritis, [1] to [1] [5] The drug according to any one of [5].
[7] An immune function modifier comprising a hot water extract of edible mushrooms and / or a polymer fraction of the extract.
[8] The edible mushroom is at least one mushroom selected from the group consisting of the genus Grifola, the genus Pleurotus, the genus Lentinula, the genus Pholiota, the genus Agrocybe, the genus Leucopaxillus, the genus Agaricus, and the genus Tricholoma, [7] Function regulator.
[9] Edible mushrooms are garlic (Grifola gargal), maitake (Grifola rondea), oyster mushroom (Pleurotus eringii), shiitake (Lentinula edinae) At least one mushroom selected from the group consisting of Leucopaxillus giganteus, Agaricus blazei, Tricholoma sp. And Cytaria espinosae [8] Immune function regulator.
[10] The immune function regulator according to any one of [7] to [9], wherein the edible mushroom is an garlic (Grifola gargal).
[11] The immune function regulator according to any one of [7] to [10], wherein the edible mushroom is a fruit body of an garlic (Grifola gargal).
[12] The regulation of immune function is selected from the group consisting of cytokine production promotion, IgE production inhibition, MCP-1 production inhibition, regulatory T cell proliferation promotion and NKT cell proliferation inhibition, [7] to [11 ] The immune function regulator in any one of.
[13] The immune function regulator according to [12], wherein the cytokine is selected from the group consisting of IFN-γ, IL-10, and IL-12.
[14] The drug according to any one of [1] to [6] or the immune function regulator according to any one of [7] to [13], wherein the polymer fraction is obtained by a method comprising the following steps: ;
(A) providing an edible mushroom powder;
(B) a step of providing a hot water extract of the powder in step (a), and (c) a step of obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract of step (b).
[15] The drug or immune function regulator according to [14], wherein the high molecular fraction contains at least one of a substance having a molecular weight of 400,000 or more and a substance having a molecular weight of 22800.
[16] A high molecular fraction of an edible mushroom hot water extract prepared by a method comprising the following steps (a) to (c), and promoting production of IFN-γ, IL-10 or IL-12 A polymer fraction having at least one activity selected from the group consisting of suppression of IgE production, inhibition of MCP-1 production, promotion of proliferation of regulatory T cells, and inhibition of NKT cell proliferation;
(A) providing an edible mushroom powder;
(B) a step of providing a hot water extract of the powder in step (a), and (c) a step of obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract of step (b).
[17] The polymer fraction according to [16], further comprising a step of extracting one or both of a substance having a molecular weight of 400,000 or more and a substance having a molecular weight of 22800 from the fraction of step (c).
[18] The drug according to any one of [1] to [6], [14] and [15], the immune function regulator according to any one of [7] to [15], and [16] or [ [17] A food comprising at least one selected from the group consisting of polymer fractions according to [17].
[19] A high molecular fraction of an edible mushroom hot-water extract comprising the following steps (a) to (c), promoting the production of IFN-γ, IL-10 or IL-12, and suppressing the production of IgE A method for producing a polymer fraction having at least one activity selected from the group consisting of suppression of MCP-1 production, promotion of proliferation of regulatory T cells, and suppression of NKT cell proliferation;
(A) providing an edible mushroom powder;
(B) a step of providing a hot water extract of the powder in step (a), and (c) a step of obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract of step (b).
[20] The method according to [19], further comprising the step of extracting one or both of a substance having a molecular weight of 400,000 or more and a substance having a molecular weight of 22800 from the fraction of step (c).
本発明により、アンニンコウの熱水抽出液の高分子画分による免疫機能調整剤、並びにアレルギー疾患の治療及び予防の両方またはいずれか一方のための薬剤が提供された。アンニンコウは、従来より食品として使用されている。従って安全性が非常に高く、副作用のリスクが小さい治療あるいは予防効果が期待される。 INDUSTRIAL APPLICABILITY According to the present invention, an immune function regulator using a polymer fraction of a hot water extract of anninkou and a drug for treatment and / or prevention of allergic diseases are provided. Anninkou is conventionally used as a food. Accordingly, a therapeutic or preventive effect is expected that is very safe and has a low risk of side effects.
本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方を含む、アレルギー疾患の治療及び予防の両方又はいずれか一方のための薬剤に関する。また本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方を含む、免疫機能調整剤に関する。 The present invention relates to a medicament for treatment and / or prevention of allergic diseases, comprising a hot water extract of edible mushrooms and / or a polymer fraction of the extract. Moreover, this invention relates to the immune function regulator containing the hot water extract of an edible mushroom, and the high molecular fraction of the said extract, or any one.
食用キノコは、当該キノコの熱水抽出液または当該抽出液の高分子画分が抗アレルギー活性あるいは免疫機能調整活性を有する限り、特に限定されない。そのような食用キノコの例として、Ｇｒｉｆｏｌａ属、Ｐｌｅｕｒｏｔｕｓ属、Ｌｅｎｔｉｎｕｌａ属、Ｐｈｏｌｉｏｔａ属、Ａｇｒｏｃｙｂｅ属、Ｌｅｕｃｏｐａｘｉｌｌｕｓ属、Ａｇａｒｉｃｕｓ属、Ｔｒｉｃｈｏｌｏｍａ属からなる群より選択されるキノコが挙げられる。
このような食用キノコとして、より具体的には、
・アンニンコウ（学名：Ｇｒｉｆｏｌａ ｇａｒｇａｌ）
・マイタケ（学名：Ｇｒｉｆｏｌａ ｆｒｏｎｄｏｓａ）
・ヒラタケ（学名：Ｐｌｅｕｒｏｔｕｓ ｏｓｔｒｅａｔｕｓ）
・エリンギ（学名：Ｐｌｅｕｒｏｔｕｓ ｅｒｉｎｇｉｉ）
・シイタケ（学名：Ｌｅｎｔｉｎｕｌａ ｅｄｏｄｅｓ）
・ナメコ（学名：Ｐｈｏｌｉｏｔａ ｎａｍｅｋｏ）
・ヤナギマツタケ（学名：Ａｇｒｏｃｙｂｅ ｃｙｌｉｎｄｒａｃｅａ）
・オオイチョウタケ（学名：Ｌｅｕｃｏｐａｘｉｌｌｕｓ ｇｉｇａｎｔｅｕｓ）
・ヒメマツタケ（学名：Ａｇａｒｉｃｕｓ ｂｌａｚｅｉ）
・サウーバ（学名：Ｔｒｉｃｈｏｌｏｍａ ｓｐ．）
・キッタリア（学名：Ｃｙｔｔａｒｉａ ｅｓｐｉｎｏｓａｅ）
などを挙げることができるが、これらに限定されない。
上述の食用キノコの熱水抽出液または当該抽出液の高分子画分が抗アレルギー活性または免疫機能調整活性を有するか否かは、例えば、上述の食用キノコの熱水抽出液、または当該抽出液の高分子画分のＩｇＥの産生抑制作用、ＭＣＰ−１の産生抑制作用、サイトカイン産生促進活性、制御性T細胞の増殖促進活性及びＮＫＴ細胞増殖抑制作用などを、in vivoあるいはin vitroで測定することにより判定することができる。
The edible mushroom is not particularly limited as long as the hot water extract of the mushroom or the polymer fraction of the extract has antiallergic activity or immune function regulating activity. Examples of such edible mushrooms include mushrooms selected from the group consisting of the genus Grifola, Pleurotus, Lentinula, Phoriota, Agrocybe, Leucopaxillus, Agaricus, and Tricholoma.
As such edible mushrooms, more specifically,
・ Anninkou (scientific name: Grifola gargal)
・ Maitake (scientific name: Grifola frontosa)
・ Oyster mushroom (scientific name: Pleurotus osteoreas)
・ Eringhi (scientific name: Pleurotus eringii)
・ Shiitake (scientific name: Lentinula edodes)
・ Nameko (scientific name: Pholiota nameko)
・ Yanagi Matsutake (Scientific name: Agrocybe cylindracea)
・ Oitaketake (scientific name: Leucopaxillus giganteus)
・ Himematsutake (Scientific name: Agaricus blazei)
・ Sauber (scientific name: Tricholoma sp.)
・ Kittalia (scientific name: Cytaria espinosae)
However, it is not limited to these.
Whether the above-mentioned edible mushroom hot water extract or the polymer fraction of the extract has antiallergic activity or immune function regulating activity is determined by, for example, the above-mentioned edible mushroom hot water extract or the extract IgE production inhibitory activity, MCP-1 production inhibitory activity, cytokine production promoting activity, regulatory T cell proliferation promoting activity and NKT cell proliferation inhibiting activity, etc. are measured in vivo or in vitro. Can be determined.
サイトカインとしては、IFN-γ、IL-10及びIL-12などを挙げることができるがこれらに限定されない。対照と比較して、上述の食用キノコの熱水抽出液または当該抽出液の高分子画分がこれらサイトカインの産生を促進した場合、ＩＬ−１０や制御性T細胞の増殖を促進した場合、あるいはＮＫＴ細胞の増殖を抑制した場合、上述の食用キノコの熱水抽出液または当該抽出液の高分子画分は抗アレルギー活性あるいは免疫機能調整活性を有すると判定することができる。本発明において免疫機能調整活性とは、上述のサイトカインの産生を促進させる活性、あるいは制御性T細胞の増殖を促進させる活性、または、ＮＫＴ細胞増殖を抑制させる活性をいう。なおＮＫＴ細胞については、気道過敏症をひき起こす細胞であるという報告がある（Asuka Terashima , Hiroshi Watarai, Sayo Inoue, Etsuko Sekine, Ryusuke Nakagawa, Koji Hase , Chiaki Iwamura , Hiroshi Nakajima, Toshinori Nakayama and Masaru Taniguchi.A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity. J. Exp. Med.,205(12) 2727-2733 (2008)）。
あるいは、上述の食用キノコの熱水抽出液または当該抽出液の高分子画分をアレルギー性疾患の症状を有する対象に投与し、対象におけるこれらの症状を観察し、またはこれらの症状の指標となるマーカーを測定、検出することにより、抗アレルギー活性あるいは免疫機能調整活性を判定することもできる。アレルギー性疾患としては、後述の気管支喘息、アトピー性皮膚炎、アレルギー性鼻炎、気管支炎、花粉症、口内炎、腸炎、潰瘍、狭心症、リウマチ様関節炎などが挙げられるがこれらに限定されない。食用キノコの熱水抽出液または当該抽出液の高分子画分を投与しない対照と比較してこれらアレルギー性疾患の症状が抑制または緩和した場合、あるいは対照と比較してこれらの疾患の公知の診断マーカーの値が減少した場合、上述の食用キノコの熱水抽出液または当該抽出液の高分子画分は抗アレルギー活性あるいは免疫機能調整活性を有すると判定することができる。例えば喘息の場合、ＩｇＥの上昇や気道過敏性の亢進の有無を指標にこれらの活性を判定することができる。
Cytokines include, but are not limited to, IFN-γ, IL-10 and IL-12. Compared with the control, when the above-mentioned edible mushroom hot water extract or the polymer fraction of the extract promotes the production of these cytokines, promotes the proliferation of IL-10 or regulatory T cells, or When the growth of NKT cells is suppressed, it can be determined that the hot water extract of the above edible mushroom or the polymer fraction of the extract has antiallergic activity or immune function regulating activity. In the present invention, the immune function-modulating activity refers to an activity that promotes the production of the above-mentioned cytokine, an activity that promotes the proliferation of regulatory T cells, or an activity that suppresses NKT cell proliferation. NKT cells have been reported to cause airway hypersensitivity (Asuka Terashima, Hiroshi Watarai, Sayo Inoue, Etsuko Sekine, Ryusuke Nakagawa, Koji Hase, Chiaki Iwamura, Hiroshi Nakajima, Toshinori Nakayama and Masaru Taniguchi. A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity. J. Exp. Med., 205 (12) 2727-2733 (2008)).
Alternatively, the above-mentioned edible mushroom hot water extract or a high-molecular fraction of the extract is administered to a subject having symptoms of allergic disease, and these symptoms in the subject are observed or used as an indicator of these symptoms By measuring and detecting the marker, antiallergic activity or immune function regulating activity can also be determined. Examples of allergic diseases include, but are not limited to, bronchial asthma, atopic dermatitis, allergic rhinitis, bronchitis, hay fever, stomatitis, enteritis, ulcer, angina pectoris, rheumatoid arthritis and the like. Known symptoms of these diseases when symptoms or symptoms of these allergic diseases are suppressed or alleviated compared to controls that do not receive edible mushroom hot water extract or a high molecular fraction of the extract, or compared to controls When the marker value decreases, it can be determined that the hot water extract of the above edible mushroom or the polymer fraction of the extract has antiallergic activity or immune function regulating activity. For example, in the case of asthma, these activities can be determined using the presence or absence of increased IgE or increased airway hyperresponsiveness as an index.
上に挙げた食用キノコのうち、マイタケ、ヒラタケ、エリンギ、シイタケ、ナメコ、オオイチョウタケ及びヤナギマツタケの７種は、日本において食用として栽培されているキノコである。またアンニンコウ、ヒメマツタケ、サウーバ及びキッタリアの４種は、南アメリカに生息している食用キノコである。 Among the edible mushrooms listed above, the seven species of maitake, oyster mushrooms, eringi, shiitake mushrooms, nameko, oyster mushrooms and willow matsutake are mushrooms cultivated for food in Japan. The four species of Anninkou, Himematsutake, Sauba and Kittaria are edible mushrooms that inhabit South America.
本発明においては、食用キノコとして、特にアンニンコウが好ましい。アンニンコウは、菌糸体及び子実体のいずれも使用することができるが、本発明においては子実体が好ましい。
アンニンコウは、南アメリカのパタゴニア地方のＮｏｔｏｆａｇｕｓ ｓｐ．の枯れ木や倒木に発生する担子菌類ヒダナシタケ目サルノコシカケ科マイタケ属に属する木材腐朽菌である。近年、菌床栽培も可能となった。アンニンコウの菌床栽培は、当業者であれば、例えば特開２００７−２０５６０に開示の方法にしたがって行うことができる。あるいは、例えば以下の組成からなる培地にアンニンコウ種菌（例えば、株式会社岩出菌学研究所保存のＩｗａｄｅ−ＧＧ０１０株）を接種後、例えば培養温度20℃、湿度70％の恒温室内で90日間培養する。そして、培養物を以下に示す低温処理および発生処理を行うことにより、アンニンコウの子実体を得ることができる。なお、アンニンコウ種菌の培養により、菌糸が培地全体に蔓延し、ベンズアルデヒドを主成分とする強い芳香が培養室全体に広がる。この強烈な芳香はアンニンコウに特徴的なものである。
（培地組成）
基材：ナラ類、クヌギ等の広葉樹おが屑
栄養剤：米ぬか、フスマ等
基材：栄養剤＝5：1（体積比）にて、混合し、含水率65％とするように、水を加える。
このように調整した培地は、培養ビンや培養袋に入れて、高圧下で滅菌し、冷却後に使用することができる。
（低温処理）
充分に蔓延した菌糸体に刺激を与える。刺激は光照射、低温であり、光刺激は、照度2000Ｌx 程度である。低温刺激として、室温を10℃に維持する。菌糸培養中にベンズアルデヒドを主成分とする強い芳香を放散するが、この芳香が急に激減する時期に刺激を与える（例えば培養90日後）。
このような刺激を与えると、原基が発生し、原基の表面が白から、灰色、黒色へ、変化する。変化した後に、発生処理を行う。
（発生処理）
例えば相対湿度９０〜１００％、温度15℃の条件で、子実体を発生させることができる。上記のように原基の表面が黒色化した後、培養袋を開封し、培地の空気の循環を促して、培地中の二酸化炭素の濃度を下げ、子実体を発生させることができる。種菌の接種から約１２０日で、培地２ｋｇからアンニンコウの子実体約400ｇ（生きのこ）を収穫できる。
In the present invention, an ninnin is particularly preferable as the edible mushroom. The mycelium and the fruit body can be used for the anninko, but the fruit body is preferred in the present invention.
Anninkou is notofagus sp. In Patagonia, South America. It is a wood-rotting fungus belonging to the genus Maitake of the family Basidiomycete, Hydrangea, which occurs in dead and fallen trees. In recent years, fungus bed cultivation has become possible. A person skilled in the art can cultivate the mycelium of anninkou, for example, according to the method disclosed in Japanese Patent Application Laid-Open No. 2007-20560. Alternatively, for example, after inoculating a culture medium having the following composition with an Annin species (for example, Iwade-GG010 strain preserved by Iwade Mycology Laboratory Co., Ltd.), for example, culturing in a constant temperature room at 20 ° C. and 70% humidity for 90 days . And the fruit body of Anninkou can be obtained by performing the low temperature process and generation | occurrence | production process which are shown below for a culture. In addition, by cultivation of the Annin species, hyphae spread throughout the entire medium, and a strong aroma mainly composed of benzaldehyde spreads throughout the culture chamber. This intense fragrance is characteristic of Anninkou.
(Medium composition)
Substrate: broadleaf sawdust such as oaks, cucumber, etc. Nutrient: rice bran, bran, etc. Substrate: Nutrient = 5: 1 (volume ratio), mixed and water added to a water content of 65%.
The medium thus prepared can be placed in a culture bottle or a culture bag, sterilized under high pressure, and used after cooling.
(Low temperature treatment)
Stimulates sufficiently spread mycelium. The stimulus is light irradiation and low temperature, and the light stimulus has an illuminance of about 2000 Lx. Maintain room temperature at 10 ° C as a cold stimulus. A strong fragrance mainly composed of benzaldehyde is diffused during mycelial culture, but a stimulus is given when the fragrance suddenly decreases sharply (for example, after 90 days of culture).
When such a stimulus is applied, a primordium is generated, and the surface of the primordium changes from white to gray or black. After the change, the generation process is performed.
(Occurrence process)
For example, fruit bodies can be generated under conditions of a relative humidity of 90 to 100% and a temperature of 15 ° C. After the surface of the primordial surface is blackened as described above, the culture bag is opened, the circulation of air in the medium is promoted, the concentration of carbon dioxide in the medium is lowered, and fruiting bodies can be generated. About 120 days after inoculation with the inoculum, about 400 g (living mushrooms) of carrots can be harvested from 2 kg of the medium.
本発明の「食用キノコの熱水抽出液」は、食用キノコに由来する１つの成分または複数の成分を含むものであって、食用キノコを熱水中で浸漬することによって得られるものから残渣を取り除くことによって得られるものであれば特に限定されない。例えば、食用キノコの熱水抽出物から残渣を取り除くことによって得られる上澄み液、当該上済み液の濃縮液、凍結乾燥品なども、抗アレルギー活性あるいは免疫機能調整機能が損なわれない限り、本発明の「食用キノコの熱水抽出液」に含まれる。 The “hot water extract of edible mushrooms” of the present invention contains one component or a plurality of components derived from edible mushrooms, and the residue is obtained by immersing the edible mushrooms in hot water. There is no particular limitation as long as it can be obtained by removing. For example, as long as the supernatant obtained by removing the residue from the hot water extract of edible mushrooms, the concentrated solution of the supernatant, lyophilized product, etc., as long as the antiallergic activity or immune function regulating function is not impaired, the present invention Of “edible mushroom hot water extract”.
食用キノコの熱水抽出物は、例えば、食用キノコの乾燥品を粉砕機で粉砕し、それを大量の熱水で煮込むことによって得ることができる。食用キノコの乾燥は、例えば、キノコに４０乃至５０℃（好ましくは４５℃前後）の温風を一昼夜あて、その後６０乃至８０℃（好ましくは７０℃前後）の温風を、例えば約１時間あてることにより行うことができるがこれに限定されない。食用キノコの乾燥品の粉末化は、例えば、乾燥後のキノコをミキサーで粉砕することによって行うことができる。 The hot water extract of edible mushrooms can be obtained, for example, by pulverizing a dried product of edible mushrooms with a pulverizer and boiling it with a large amount of hot water. The edible mushroom is dried by, for example, applying warm air of 40 to 50 ° C. (preferably around 45 ° C.) to the mushroom for a whole day and then applying warm air of 60 to 80 ° C. (preferably around 70 ° C.) for about 1 hour. However, the present invention is not limited to this. For example, the dried edible mushroom can be powdered by pulverizing the dried mushroom with a mixer.
このようにして得られる食用キノコの粉末は、大量の熱水で処理される。具体的には、例えば、食用キノコの乾燥粉末を熱水に入れ、温度を一定に保ちながら、食用キノコと熱水との混合物を攪拌することにより、食用キノコの熱水抽出物を取得することができる。熱水の温度は、例えば70℃から120℃、好ましくは80℃から110℃、より好ましくは85℃から100℃、特に好ましくは90℃から95℃を例示することができるがこれらに限定されない。また抽出時間は、例えば4時間から8時間、好ましくは5時間から7時間、特に好ましくは6時間程度を例示することができるがこれらに限定されない。また熱水抽出は、通常、中性、常圧の条件下で行うが、減圧、加圧条件下で行うこともできる。 The edible mushroom powder thus obtained is treated with a large amount of hot water. Specifically, for example, to obtain a hot water extract of edible mushrooms by putting a dry powder of edible mushrooms in hot water and stirring the mixture of edible mushrooms and hot water while keeping the temperature constant. Can do. Examples of the temperature of hot water include, but are not limited to, 70 ° C. to 120 ° C., preferably 80 ° C. to 110 ° C., more preferably 85 ° C. to 100 ° C., and particularly preferably 90 ° C. to 95 ° C. Examples of the extraction time include 4 hours to 8 hours, preferably 5 hours to 7 hours, and particularly preferably about 6 hours, but are not limited thereto. Hot water extraction is usually carried out under neutral and atmospheric conditions, but can also be carried out under reduced pressure and pressurized conditions.
本発明の「食用キノコの熱水抽出物の熱水抽出液」は、例えば、上述の方法にしたがって得られる食用キノコの熱水抽出物から残渣を取り除き、上澄み液を分離することにより取得することができる。上澄み液の分離は、例えばセライト濾過、薮田式ろ過、フィルタープレス、遠心分離、限外濾過などによって行うことができるがこれに限定されない。熱水抽出物から上澄み液の分離は、熱水抽出物を遠心分離し、食用キノコの乾燥粉末を沈澱させた後に行うことが好ましい。またこの工程は、上述の方法にしたがって得られる熱水抽出物を篩にかけることによって、食用キノコの乾燥粉末を分離・除去した後に行うこともできる。 The “hot water extract of the edible mushroom hot water extract” of the present invention is obtained, for example, by removing the residue from the edible mushroom hot water extract obtained according to the above-described method and separating the supernatant. Can do. The supernatant can be separated by, for example, celite filtration, Iwata filtration, filter press, centrifugation, ultrafiltration, etc., but is not limited thereto. The supernatant liquid is preferably separated from the hot water extract after centrifuging the hot water extract and precipitating a dry powder of edible mushrooms. Moreover, this process can also be performed after isolate | separating and removing the dry powder of an edible mushroom by sieving the hot water extract obtained according to the above-mentioned method.
上述の食用キノコと熱水の混合物の攪拌の工程と、それにより得られる熱水抽出物から上澄み液を分離する工程は、それぞれ、１回又は複数回行うことができる。あるいは、熱水抽出物から上澄み液を分離することにより得られる食用キノコの残渣に新たな熱水を加え、それにより得られる食用キノコの残渣と熱水の混合物を攪拌し、当該混合物（新たな熱水抽出物）から上澄み液を分離し、熱水抽出液を取得することもできる。本発明においては、食用キノコと熱水の混合物の攪拌時間を例えば３０分程度とした場合、食用キノコと熱水の混合物の攪拌と、それにより得られる熱水抽出物からの上澄み液の分離の工程を、好ましくは２乃至５回程度、特に好ましくは３又は４回行うことができる。 The step of stirring the mixture of edible mushrooms and hot water and the step of separating the supernatant from the hot water extract obtained thereby can be performed once or a plurality of times, respectively. Alternatively, fresh hot water is added to the edible mushroom residue obtained by separating the supernatant from the hot water extract, the resulting edible mushroom residue and hot water mixture is stirred, and the mixture (fresh It is also possible to separate the supernatant from the hot water extract) and obtain the hot water extract. In the present invention, when the stirring time of the mixture of edible mushrooms and hot water is about 30 minutes, for example, stirring of the mixture of edible mushrooms and hot water and separation of the supernatant from the hot water extract obtained thereby The step can be performed preferably about 2 to 5 times, particularly preferably 3 or 4 times.
本発明においては、食用キノコの熱水抽出液を濃縮して使用することもできる。濃縮は、例えば逆相高速液体クロマトグラフィ（逆相ＨＰＬＣ）を使用して行うことができる。また本発明の熱水抽出液は、凍結乾燥又は噴霧乾燥した形態ものとして使用することもできる。凍結乾燥、噴霧乾燥は、この分野で通常行われている条件及び方法で行うことができる。本発明においては、熱水抽出液の濃縮や凍結乾燥、噴霧乾燥を複数回行うことができる。 In the present invention, the hot water extract of edible mushrooms can be concentrated and used. Concentration can be performed, for example, using reverse phase high performance liquid chromatography (reverse phase HPLC). The hot water extract of the present invention can also be used in a freeze-dried or spray-dried form. Freeze-drying and spray-drying can be carried out under conditions and methods usually used in this field. In the present invention, the hot water extract can be concentrated, freeze-dried or spray-dried a plurality of times.
本発明においては、「食用キノコの熱水抽出液の高分子画分」及びその濃縮物あるいは乾燥産物を、アレルギー疾患の治療や予防、又は対象における免疫機能の調整のために使用することが好ましい。
本発明において高分子画分とは、食用キノコの熱水抽出液を限外濾過膜などの濾過膜を使用して高分子側と低分子側に分画した際に、高分子側に得られる画分をいう。あるいは本発明の「食用キノコの熱水抽出液の高分子画分」は、濾過膜を使用してある分子量を境として食用キノコの熱水抽出液を分画した場合に、膜を透過しない画分と言い換えることもできる。本発明の高分子画分は、抗アレルギー活性あるいは免疫機能調整機能を有する限り特に限定されるものではないが、例えば、分子量１０００、２０００、３０００、４０００、５０００、６０００、７０００、８０００、９０００、１００００、２００００、３００００、４００００、５００００から選択される２点の分子量を下限（「〜以上、又は、〜より高い）及び上限（〜以下、又は、〜より低い）とする範囲により示される分子量の範囲にある物質を分離するための分離膜によって、高分子側に分離される画分であることが好ましい。本発明においては、分子量６０００を好ましい例として挙げることができるがこれに限定されない。
このような高分子画分は、食用キノコの熱水抽出液を、上述の分子量の範囲内にある分子の分離に使用可能な濾過膜を使用する限外濾過などによって取得することができる。上澄み液に含まれる成分を分離膜で分画するに際し、上澄み液は、そのまま使用してもよいし、ある程度濃縮した後に使用してもよい。あるいは、上澄み液を一旦凍結乾燥や噴霧乾燥等の方法で粉末化し、その粉末を水に溶解させて得られる水溶液の形態で使用してもよい。
In the present invention, it is preferable to use the “polymer fraction of edible mushroom hot water extract” and its concentrate or dried product for the treatment or prevention of allergic diseases or the adjustment of immune function in a subject. .
In the present invention, the polymer fraction is obtained on the polymer side when the hot water extract of edible mushrooms is fractionated into a polymer side and a low molecule side using a filtration membrane such as an ultrafiltration membrane. Say a fraction. Alternatively, the “polymer fraction of edible mushroom hot water extract” of the present invention is a fraction that does not permeate the membrane when the edible mushroom hot water extract is fractionated at a certain molecular weight using a filtration membrane. It can be paraphrased as minutes. The polymer fraction of the present invention is not particularly limited as long as it has an antiallergic activity or an immune function regulating function. For example, the molecular weight is 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, Two molecular weights selected from 10,000, 20000, 30000, 40000, and 50000 are molecular weights indicated by a range having a lower limit ("~ or higher or higher" and an upper limit (~ lower or lower). A fraction separated on the polymer side by a separation membrane for separating substances in the range is preferred, and in the present invention, a molecular weight of 6000 can be mentioned as a preferred example, but the present invention is not limited thereto.
Such a polymer fraction can be obtained by, for example, ultrafiltration using a filtration membrane that can be used to separate molecules within the molecular weight range of the edible mushroom hot water extract. When fractionating the components contained in the supernatant with a separation membrane, the supernatant may be used as it is or after being concentrated to some extent. Alternatively, the supernatant may be once powdered by a method such as freeze drying or spray drying, and the powder may be used in the form of an aqueous solution obtained by dissolving the powder in water.
本発明のアレルギー疾患には、特定の抗原に対す過剰反応（アレルギー反応）が関与するあらゆる疾患が含まれる。アレルギー反応の分類としてI型、II型、III型、IV型が知られている。本発明のアレルギー疾患は、これらのアレルギー反応が関与する疾患である限り特に限定されない。アレルギー性疾患の例として、気管支喘息、アトピー性皮膚炎、アレルギー性鼻炎、気管支炎、花粉症、口内炎、腸炎、潰瘍、狭心症、リウマチ様関節炎などを示すことができるがこれらに限定されない。
また本発明のアレルギー性疾患には、自己免疫疾患も含まれる。自己免疫疾患とは、自己の体を構成する物質を抗原として、免疫反応が起こる疾患をいう。自己免疫疾患としては関節リウマチや全身性エリテマトーデス、シェーグレン症候群などの全身性自己免疫疾患、ギラン・バレー症候群やバセドウ病などの臓器特異性自己免疫疾患が挙げられるがこれらに限定されない。
Allergic diseases of the present invention include all diseases involving an overreaction (allergic reaction) against a specific antigen. As types of allergic reactions, type I, type II, type III, and type IV are known. The allergic disease of the present invention is not particularly limited as long as these allergic reactions are involved. Examples of allergic diseases include, but are not limited to, bronchial asthma, atopic dermatitis, allergic rhinitis, bronchitis, hay fever, stomatitis, enteritis, ulcer, angina, rheumatoid arthritis and the like.
The allergic diseases of the present invention also include autoimmune diseases. An autoimmune disease refers to a disease in which an immune reaction occurs using substances constituting the body of the antigen as an antigen. Autoimmune diseases include, but are not limited to, systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjogren's syndrome, and organ-specific autoimmune diseases such as Guillain-Barre syndrome and Graves' disease.
本発明の食用キノコの熱水抽出液、当該抽出液の高分子画分は、サイトカイン産生促進、制御性T細胞の増殖促進などの免疫調整機能を有する。サイトカインとしては、IFN-γ、IL-10及びIL-12などが挙げられるがこれらに限定されない。サイトカイン産生促進の有無、制御性T細胞の増殖促進の有無は、実施例に記載の方法の他、例えば次の文献に記載の方法にしたがって行うことができる。
・TakagiＴ., Taguchi O., Toda M., Boveda Ruiz D., Gil Bernabe P., D'Alessandro-Gabazza C. N., Miyake Y., Kobayashi T., Aoki S., Chiba F., Yano Y., Conway E. M., Munesue S., Yamamoto Y., Yamamoto H., Suzuki K., Takei Y., Morser J. and Gabazza E. C. Inhibition of Allergic Bronchial Asthma by Thrombomodulin Is Mediated by Dendritic Cells. Am J Respir Crit Care Med, 183, 31-42.(2011)
・Boveda-Ruiza D., D'Alessandro-Gabazzaa C. N., Masaaki Toda M., Takagi T., Naito M., Matsushima Y., Matsumoto T, Kobayashi T., Gil-Bernabe P., Chelakkot-Govindalayathila A-L., Miyake Y., Yasukawa A., Morser J., Taguchi O., Gabazza E.C. Differential role of regulatory T cells in early and late stages of pulmonary fibrosis. Immunobiology, 22 May (2012).
・Ioannis Kotsianidis, Evangelia Nakou, Irene Bouchliou, Argyrios Tzouvelekis, Emmanouil Spanoudakis, Paschalis Steiropoulos, Ioannis Sotiriou, Vassilis Aidinis, Dimitrios Margaritis, Costas Tsatalas, and Demosthenes Bouros Global．Impairment of CD41CD251FOXP31 Regulatory T Cells in Idiopathic Pulmonary Fibrosis, Am J Respir Crit Care Med.2009 Jun 15;179(12):1121-30．
The hot water extract of the edible mushroom of the present invention and the polymer fraction of the extract have immunomodulating functions such as promotion of cytokine production and promotion of proliferation of regulatory T cells. Cytokines include, but are not limited to, IFN-γ, IL-10 and IL-12. Whether or not cytokine production is promoted and whether or not regulatory T cell proliferation is promoted can be performed according to, for example, the method described in the following document, in addition to the methods described in the Examples.
・ Takagi T., Taguchi O., Toda M., Boveda Ruiz D., Gil Bernabe P., D'Alessandro-Gabazza CN, Miyake Y., Kobayashi T., Aoki S., Chiba F., Yano Y., Conway EM, Munesue S., Yamamoto Y., Yamamoto H., Suzuki K., Takei Y., Morser J. and Gabazza EC Inhibition of Allergic Bronchial Asthma by Thrombomodulin Is Mediated by Dendritic Cells. Am J Respir Crit Care Med, 183, 31-42. (2011)
・ Boveda-Ruiza D., D'Alessandro-Gabazzaa CN, Masaaki Toda M., Takagi T., Naito M., Matsushima Y., Matsumoto T, Kobayashi T., Gil-Bernabe P., Chelakkot-Govindalayathila AL., Miyake Y., Yasukawa A., Morser J., Taguchi O., Gabazza EC Differential role of regulatory T cells in early and late stages of pulmonary fibrosis. Immunobiology, 22 May (2012).
・ Ioannis Kotsianidis, Evangelia Nakou, Irene Bouchliou, Argyrios Tzouvelekis, Emmanouil Spanoudakis, Paschalis Steiropoulos, Ioannis Sotiriou, Vassilis Aidinis, Dimitrios Margaritis, Costas Tsatalas, and Demosthenes Bouros Global. Impairment of CD41CD251FOXP31 Regulatory T Cells in Idiopathic Pulmonary Fibrosis, Am J Respir Crit Care Med. 2009
また本発明は、以下の工程を含む方法によって得られる食用キノコの熱水抽出液の高分子画分を含む、アレルギー疾患の治療及び予防の両方又はいずれか一方のための薬剤に関する。また本発明は、以下の工程を含む方法によって得られる食用キノコの熱水抽出液の高分子画分を含む、免疫機能調整剤に関する。
（ａ）食用キノコの粉末を提供する工程、
（ｂ）工程（ａ）の粉末の熱水抽出液を提供する工程、及び
（ｃ）工程（ｂ）の熱水抽出液から分子量が6000以上の物質からなる画分を取得する工程。
食用キノコの粉末、当該粉末の熱水抽出液、熱水抽出液から分子量が6000以上の物質からなる画分取得方法は上に述べた。食用キノコとしてはアンニンコウの子実体が好ましいがこれに限定されない。本発明の薬剤又は免疫機能調整剤は、分子量400000以上の物質及び分子量22800の物質の両方又はいずれか一方を含む。あるいは、分子量400000以上の物質及び分子量22800の物質の両方又はいずれか一方からなる。これらの物質は、例えば食用キノコの熱水抽出液の高分子画分をゲル濾過クロマトグラフィーにより分子量に応じて分画することにより取得することができる。各画分を構成する物質の分子量は、分子量マーカーはカラムを通して溶出させることによって判定することができる。
Moreover, this invention relates to the chemical | medical agent for the treatment and / or prevention of an allergic disease containing the high molecular fraction of the hot-water extract of the edible mushroom obtained by the method including the following processes. Moreover, this invention relates to the immune function regulator containing the high molecular fraction of the hot-water extract of the edible mushroom obtained by the method including the following processes.
(A) providing an edible mushroom powder;
(B) a step of providing a hot water extract of the powder in step (a), and (c) a step of obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract of step (b).
The edible mushroom powder, the hot water extract of the powder, and the method for obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract are described above. An edible mushroom is preferably a fruit body of Anninkou, but is not limited thereto. The drug or immune function regulator of the present invention contains a substance having a molecular weight of 400,000 or more and / or a substance having a molecular weight of 22,800. Or it consists of a substance with a molecular weight of 400000 or more and / or a substance with a molecular weight of 22800. These substances can be obtained, for example, by fractionating a polymer fraction of a hot water extract of edible mushrooms according to molecular weight by gel filtration chromatography. The molecular weight of the substance constituting each fraction can be determined by eluting the molecular weight marker through a column.
また本発明は、以下の工程を含む方法によって得られる食用キノコの熱水抽出液の高分子画分に関する。さらに本発明は、以下の工程を含む食用キノコの熱水抽出液の高分子画分の製造方法に関する。
（ａ）食用キノコの粉末を提供する工程、
（ｂ）工程（ａ）の粉末の熱水抽出液を提供する工程、及び
（ｃ）工程（ｂ）の熱水抽出液から分子量が6000以上の物質からなる画分を取得する工程。
本発明の高分子画分は、サイトカイン産生促進、ＩｇＥの産生抑制、ＭＣＰ−１の産生抑制、制御性T細胞の増殖促進及びＮＫＴ細胞増殖抑制などの免疫調整機能を有する。また本発明の高分子画分は、対象に投与した場合に、アレルギー性疾患の治療効果および/または予防効果を示す。
The present invention also relates to a polymer fraction of an edible mushroom hot water extract obtained by a method comprising the following steps. Furthermore, this invention relates to the manufacturing method of the high molecular fraction of the hot-water extract of an edible mushroom including the following processes.
(A) providing an edible mushroom powder;
(B) a step of providing a hot water extract of the powder in step (a), and (c) a step of obtaining a fraction comprising a substance having a molecular weight of 6000 or more from the hot water extract of step (b).
The polymer fraction of the present invention has immunomodulating functions such as cytokine production promotion, IgE production inhibition, MCP-1 production inhibition, regulatory T cell proliferation promotion and NKT cell proliferation inhibition. In addition, the polymer fraction of the present invention exhibits a therapeutic effect and / or a preventive effect for allergic diseases when administered to a subject.
本発明の薬剤又は免疫機能調整剤は、単独でヒトおよび動物におけるアレルギー性疾患の治療および/または予防に用いることができる。あるいは医薬品や食品に通常使用されうる他の成分と混合して経口投与することもできる。また、アレルギー性疾患の治療および/または予防効果が知られている他の化合物や微生物等と併用することもできる。
アレルギー性疾患の治療や予防の効果は、例えば気管支喘息であれば、実施例に示すように、気道過敏性試験やBALF中の免疫応答反応の測定などによって確認することができる。あるいは、血中のIgE濃度を指標に確認することができる。その他のアレルギー性疾患についても、当業者であれば、それぞれの疾患において確立された方法により治療効果や予防効果を判定することができる。
The agent or immune function regulator of the present invention can be used alone for the treatment and / or prevention of allergic diseases in humans and animals. Or it can also be orally administered by mixing with other ingredients that can be usually used in pharmaceuticals and foods. It can also be used in combination with other compounds, microorganisms, and the like that are known to be effective in treating and / or preventing allergic diseases.
For example, in the case of bronchial asthma, the effect of treatment or prevention of allergic diseases can be confirmed by an airway hypersensitivity test, measurement of immune response in BALF, and the like. Alternatively, the IgE concentration in blood can be confirmed using an index. Regarding other allergic diseases, those skilled in the art can determine the therapeutic effect and the preventive effect by a method established for each disease.
本発明の薬剤又は免疫機能調整剤は、食用キノコの熱水抽出液および当該抽出液の高分子画分の両方またはいずれか一方を主成分とするものであることが好ましい。本発明において「主成分とする」とは、当該薬剤又は免疫機能調整剤が目的とする活性（たとえば抗アレルギー活性あるいは免疫機能調整活性）の３０％以上、好ましくは５０％以上、さらに好ましくは７０％以上が、食用キノコの熱水抽出液および当該抽出液の高分子画分の両方またはいずれか一方によって達成されることをいう。 It is preferable that the chemical | medical agent or immune function regulator of this invention is what has as a main component the hot water extract of an edible mushroom and the polymer fraction of the said extract, or any one. In the present invention, “mainly comprising” means 30% or more, preferably 50% or more, more preferably 70% of the target activity (for example, antiallergic activity or immune function regulating activity) of the drug or immune function regulator. % Or more is achieved by the hot water extract of edible mushrooms and / or the polymer fraction of the extract.
本発明の薬剤又は免疫機能調整剤の投与量は、投与経路、ヒトを含む投与対象動物の年齢、体重、症状など、種々の要因を考慮して、適宜設定することができる。本発明はこれに限定されないが、成人１日当たり、熱水抽出液の凍結乾燥粉末の量に換算して、１０乃至２００ｍｇ／ｋｇ体重程度であることが好ましく、２０乃至１００ｍｇ／ｋｇ体重程度であることがさらに好ましい。また熱水抽出液の高分子画分の摂取量は、特に限定されないが、成人１日当たり、高分子画分の凍結乾燥粉末の量に換算して、２乃至３０ｍｇ／ｋｇ体重程度であることが好ましく、４乃至１５ｍｇ／ｋｇ体重であることがさらに好ましい。しかしながら、長期間に亘って治療および/または予防の目的で摂取する場合には、上記範囲よりも少量であってもよい。あるいは、本発明の薬剤又は免疫機能調整剤の有効成分は、食品としての安全性について問題がないので、上記範囲よりも多量に使用することもできる。
また本発明の薬剤又は免疫機能調整剤は、経口投与又は非経口投与（筋肉内、皮下、静脈内、坐薬、経皮、経胃管、経皮吸収等）のいずれでも投与することができる。また本発明の薬剤又は免疫機能調整剤は、湿布や塗布などの方法により局所投与することもできる。
The dose of the drug or immune function regulator of the present invention can be appropriately set in consideration of various factors such as the route of administration and the age, weight, and symptoms of animals to be administered including humans. Although the present invention is not limited to this, it is preferably about 10 to 200 mg / kg body weight, preferably about 20 to 100 mg / kg body weight in terms of the amount of lyophilized powder of the hot water extract per day for an adult. More preferably. Further, the intake of the polymer fraction of the hot water extract is not particularly limited, but it may be about 2 to 30 mg / kg body weight in terms of the amount of lyophilized powder of the polymer fraction per day for an adult. It is preferably 4 to 15 mg / kg body weight. However, when ingested for the purpose of treatment and / or prevention over a long period of time, the amount may be smaller than the above range. Alternatively, the active ingredient of the drug or immune function regulator of the present invention has no problem with food safety, and can be used in a larger amount than the above range.
The drug or immune function regulator of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, transgastric tube, transdermal absorption, etc.). Moreover, the chemical | medical agent or immune function regulator of this invention can also be locally administered by methods, such as a poultice and application | coating.
本発明の薬剤又は免疫機能調整剤の投与形態としては、その目的や投与経路等に応じて剤型を選択することができ、例えば錠剤、被覆錠剤、丸剤、カプセル剤、顆粒剤、細粒剤、散剤、液剤、懸濁剤、乳剤、シロップ剤、注射剤、坐剤、浸剤、煎剤、チンキ剤等が挙げられる。これらの各種製剤は、常法に従って主薬に対して必要に応じて充填剤、増量剤、賦形剤、結合剤、保湿剤、崩壊剤、界面活性剤、滑沢剤、着色剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの医薬の製剤技術分野において通常使用しうる既知の補助剤を用いて製剤化することができる。また、この医薬製剤中に着色剤、保存剤、香料、風味剤、甘味剤等や他の医薬品を含有させてもよい。 As a dosage form of the drug or immune function regulator of the present invention, a dosage form can be selected according to its purpose, administration route, and the like. For example, tablets, coated tablets, pills, capsules, granules, fine granules Agents, powders, solutions, suspensions, emulsions, syrups, injections, suppositories, dip agents, decoction, tinctures and the like. These various preparations are prepared according to conventional methods as necessary with respect to the active ingredient, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. The pharmaceutical composition can be formulated using known adjuvants that can be usually used in the pharmaceutical preparation technical field, such as solubilizing agents, suspension agents, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.
また本発明は、本発明の薬剤又は免疫機能調整剤を構成する食用キノコの熱水抽出液および当該抽出液の高分子画分の両方またはいずれか一方を含む、アレルギー性疾患の治療及び／又は予防用飲食品組成物を提供する。また本発明は、食用キノコの熱水抽出液および当該抽出液の高分子画分の両方またはいずれか一方を含む免疫機能調整用の飲食品組成物を提供する。 The present invention also provides a therapeutic and / or allergic disease comprising a hot water extract of edible mushrooms and / or a high molecular fraction of the extract constituting the drug or immune function regulator of the present invention. Provided is a preventive food and beverage composition. Moreover, this invention provides the food-drinks composition for immune function adjustment containing the hot-water extract of an edible mushroom, and the high molecular fraction of the said extract, or any one.
本発明の飲食品組成物は、保健機能食品や病者用食品にも適用することができる。保健機能食品制度は、内外の動向、従来からの特定保健用食品制度との整合性を踏まえて、通常の食品のみならず錠剤、カプセル等の形状をした食品を対象として設けられたもので、特定保健用食品（個別許可型）と栄養機能食品（規格基準型）の２種類の類型からなる。本発明の薬剤又は免疫機能調整剤を構成する食用キノコの熱水抽出液および当該抽出液の高分子画分の両方又はいずれか一方を含有する特定保健用食品等の特別用途食品や栄養機能食品を直接摂取することによりアレルギー性疾患に対する治療及び／又は予防が可能となる。 The food and beverage composition of the present invention can also be applied to health functional foods and foods for the sick. The health functional food system was established not only for regular foods but also for foods in the form of tablets, capsules, etc., based on domestic and foreign trends and consistency with the conventional food system for specified health use. It consists of two types of food for specified health use (individual permission type) and functional food for nutrition (standard type). Food for special use such as food for specified health use and / or nutritional functional food containing hot water extract of edible mushroom and / or polymer fraction of the extract constituting the drug or immune function regulator of the present invention It is possible to treat and / or prevent allergic diseases by directly ingesting.
本発明の飲食品組成物は、具体的には、各種飲食品に本発明の薬剤又は免疫機能調整剤を添加し、これを摂取してもよい。本発明の飲食品組成物は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方をそのまま使用し、あるいは他の食品ないし食品成分と混合するなど、飲食品組成物における常法にしたがって使用できる。また、その性状についても、通常用いられる飲食品の状態、例えば、固体状（粉末、顆粒状その他）、ペースト状、液状または懸濁状のいずれでもよい。 Specifically, the food / beverage product composition of the present invention may be ingested by adding the drug or immune function regulator of the present invention to various food / beverage products. The food / beverage product composition of the present invention uses a hot water extract of edible mushrooms and / or a polymer fraction of the extract as it is, or is mixed with other foods or food ingredients. It can be used according to conventional methods in the composition. Further, the properties thereof may be in the state of normally used foods and beverages, for example, solid (powder, granule, etc.), paste, liquid or suspension.
その他の成分についても特に限定されないが、本発明の飲食品組成物には、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等を主成分として使用することができる。タンパク質としては、例えば大豆タンパク質、鶏卵タンパク質、肉タンパク質等の動植物性タンパク質が挙げられる。糖質としては糖類、加工澱粉（テキストリンのほか、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維などが挙げられる。脂質としては、例えば、ラード、魚油等、これらの分別油、水素添加油、エステル交換油等の動物性油脂、パーム油、サフラワー油、コーン油、ナタネ油、ヤシ油、これらの分別油、水素添加油、エステル交換油等の植物性油脂などが挙げられる。ビタミン類としては、例えば、ビタミンＡ、カロチン類、ビタミンＢ群、ビタミンＣ、ビタミンＤ群、ビタミンＥ、ビタミンＫ群、ビタミンＰ、ビタミンＱ、ナイアシン、ニコチン酸、パントテン酸、ビオチン、イノシトール、コリン、葉酸などが挙げられ、ミネラル類としては、例えば、カルシウム、カリウム、マグネシウム、ナトリウム、銅、鉄、マンガン、亜鉛、セレン、乳清ミネラルなどが挙げられる。有機酸としては、例えば、リンゴ酸、クエン酸、乳酸、酒石酸などが挙げられる。これらの成分は、２種以上を組み合わせて使用することができる。これらは合成品、これらを多く含む食品由来のもののいずれであってもよい。 Other ingredients are not particularly limited, but the food and beverage composition of the present invention contains water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like as main components. Can be used. Examples of the protein include animal and plant proteins such as soybean protein, chicken egg protein, and meat protein. Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of lipids include lard, fish oil, etc., fractionated oils thereof, hydrogenated oils, animal oils such as transesterified oils, palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used in combination of two or more. These may be either synthetic products or those derived from foods containing a large amount of these.
本発明の薬剤又は免疫機能調整剤または飲食品組成物を構成する食用キノコの熱水抽出液又は当該抽出液の高分子画分の量は、その目的、用途（薬剤、飲食品組成物）に応じて任意に定めることができる。本発明はこれに限定されないがその含量としては、全体量に対して通常、０．００１〜１００％（ｗ／ｗ）、特に０．０１〜１００％（ｗ／ｗ）が好ましい。 The amount of the hot water extract of edible mushrooms or the polymer fraction of the extract constituting the drug, immune function regulator or food / beverage composition of the present invention depends on its purpose and use (drug, food / beverage composition). It can be arbitrarily determined depending on the situation. Although this invention is not limited to this, As the content, 0.001 to 100% (w / w) is preferable normally with respect to the whole quantity, Especially 0.01 to 100% (w / w) is preferable.
また本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方を動物に経口投与する工程を含む、アレルギー性疾患の治療及び予防の両方又はいずれか一方のための方法に関する。また本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方を動物に経口投与する工程を含む、免疫機能の調整方法に関する。食用キノコの熱水抽出液及び/又は当該抽出液の高分子画分が投与される対象としては、哺乳動物が挙げられる。哺乳動物としてはヒト及びヒト以外の哺乳動物が挙げられ、好ましくはヒトやサルが挙げられ、より好ましくはヒトが挙げられる。
また本発明は、アレルギー性疾患の治療及び予防の両方又はいずれか一方のために使用するための、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方に関する。また本発明は、免疫機能の調整に用いるための、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方に関する。
あるいは本発明はアレルギー性疾患の治療及び予防の両方又はいずれか一方のための薬剤の製造における、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方の使用に関する。また本発明は、免疫機能調整剤の製造における、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方の使用に関する。
また本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方と薬学的に許容される担体を配合する工程を含む、アレルギー性疾患の治療及び予防の両方又はいずれか一方のための薬剤の製造方法に関する。さらに本発明は、食用キノコの熱水抽出液及び当該抽出液の高分子画分の両方またはいずれか一方と薬学的に許容される担体を配合する工程を含む、免疫機能調整剤の製造方法に関する。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
The present invention also provides a treatment and / or prevention of allergic diseases, comprising the step of orally administering an edible mushroom hot water extract and / or a polymer fraction of the extract to an animal. Relating to the method. The present invention also relates to a method for adjusting immune function, which comprises the step of orally administering to the animal either or both of a hot water extract of edible mushrooms and a polymer fraction of the extract. Mammals are examples of subjects to which the hot water extract of edible mushrooms and / or the polymer fraction of the extract are administered. Mammals include humans and mammals other than humans, preferably humans and monkeys, more preferably humans.
The present invention also relates to a hot water extract of edible mushrooms and / or a polymer fraction of the extract for use in the treatment and / or prevention of allergic diseases. The present invention also relates to a hot water extract of edible mushrooms and a polymer fraction of the extract for use in adjusting immune function.
Alternatively, the present invention relates to the use of a hot water extract of edible mushrooms and / or a polymeric fraction of the extract in the manufacture of a medicament for the treatment and / or prevention of allergic diseases. . The present invention also relates to the use of a hot water extract of edible mushrooms and / or a polymer fraction of the extract in the production of an immune function regulator.
The present invention also provides a method for treating and preventing allergic diseases, comprising a step of combining a hot water extract of edible mushrooms and / or a polymer fraction of the extract with a pharmaceutically acceptable carrier. The present invention relates to a method for producing a drug for both or either one. Furthermore, the present invention relates to a method for producing an immune function regulator, comprising a step of blending a pharmaceutically acceptable carrier with a hot water extract of edible mushrooms and a polymer fraction of the extract and either one of them. .
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
以下、実施例により本発明をより詳細に説明するが、本発明は、これら実施例に制限されるものではない。
〔実施例１〕免疫応用細胞からのサイトカイン産生能
１．アンニンコウからの有効成分の熱水抽出液の調製
（１）キノコの乾燥粉末の調整
アンニンコウ（株式会社岩出菌学研究所にて人工栽培したもの）の子実体（川出光生, 原田栄津子，西岡宏樹，目黒貞利：チリ産食用担子菌Grifola gargal,の菌床栽培―栽培に適した菌株のスクリーニング―日本きのこ学会誌, 17, 75-79.(2009)）に45℃の温風を一昼夜あて、その後、70℃の温風を1時間あてた。得られたアンニンコウ子実体乾燥品を粉砕機にて粉砕し、粉末とした。
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to these Examples.
[Example 1] Ability to produce cytokines from immune-applied cells
1. Preparation of hot water extract of active ingredients from Anninkou
(1) Preparation of dry powder of mushrooms Fruit bodies of Anninkou (manufactured by Iwade Mycology Research Institute, Inc.) (Mitsuo Kawade, Eitsuko Harada, Hiroki Nishioka, Sadatoshi Meguro: Chilean edible basidiomycete, Grifola gargal, Bacteria bed cultivation-Screening for suitable strains for cultivation-Journal of the Japan Mushroom Society, 17, 75-79. (2009)), 45 ° C warm air was applied all day and night, and then 70 ° C warm air was applied for 1 hour. . The obtained dried anninko fruit body was pulverized by a pulverizer to obtain a powder.
（２）熱水抽出および限外濾過膜による分画
アンニンコウ子実体乾燥粉末200kgを200メッシュで篩、水21キロリットルを混合し、90〜95℃で6時間熱水抽出した。薮田式ろ過圧搾機にて、セライト濾過することで、抽出残渣を除去し、熱水抽出液（上澄み液）を得た。得られた熱水抽出液は、旭化成ケミカルズ株式会社製の限外濾過膜（旭化成UFモジュールマイクロ―ザRACP；分子量：6000）を用い、23-27℃、循環流量5-7l/minにて,限外ろ過を行い、分子量6,000以上の高分子画分（GHF）、および分子量6,000以下の低分子画分（GLF）を得た。これを減圧条件下で濃縮、121℃、20分で高圧滅菌し、ついで、株式会社宝製作所製の真空凍結乾燥器を用いて、凍結乾燥を行った（図１）。
(2) Extraction with hot water and fractionated anninko fruit body dry powder by ultrafiltration membrane Sieve with 200 mesh and 21 kiloliters of water were mixed, and hot water extraction was performed at 90-95 ° C. for 6 hours. The extraction residue was removed by celite filtration with a Kamata filter press, and a hot water extract (supernatant) was obtained. The obtained hot water extract was prepared using an ultrafiltration membrane (Asahi Kasei UF Module Micro-the RACP; molecular weight: 6000) manufactured by Asahi Kasei Chemicals Corporation at 23-27 ° C and a circulation flow rate of 5-7 l / min. Ultrafiltration was performed to obtain a high molecular fraction (GHF) having a molecular weight of 6,000 or more and a low molecular fraction (GLF) having a molecular weight of 6,000 or less. This was concentrated under reduced pressure, autoclaved at 121 ° C. for 20 minutes, and then lyophilized using a vacuum lyophilizer manufactured by Takara Manufacturing Co., Ltd. (FIG. 1).
２．免疫応答細胞からのサイトカイン産生能の測定
（１）マウス脾臓細胞刺激試験１（ＩＦＮ−γ、及びＩＬ−10の測定）
ＢＡＬＢ／ｃマウスから脾臓を無菌的に取り出し、10％（Ｖ／Ｖ）熱不活性化ウシ胎児血清（ＦＣＳ）を加えたＲＰＭI−1640培地中で脾臓細胞懸濁液を調整した。ナイロンフィルターでろ過したろ液を1,500rpm(回転／分)、４℃、5分間の遠心分離に供し、脾臓細胞のペレットを回収した。このペレットを0.144Ｍ塩化ナトリウム−0.017Ｍトリス塩酸緩衝液（ｐＨ７．６５）に再懸濁し、37℃で２．５分間、５％ＣＯ２下でインキュベートして赤血球を溶血させ、最終的に実験に用いる脾臓細胞（４×１０６/ml）を調製した。上記で得た脾臓細胞に熱水抽出液（GF：2％）、低分子画分（GLF：0.2％）、高分子画分（GHF：0.2％）を最終容量250μｌになるように加え、３７℃、５％CO2下で48時間共培養した。
培養後、上清中のIFN-γ、IL-10をELISA法によって検出し、その濃度を決定した。アンニンコウ抽出液の代替にリン酸緩衝生理食塩水（PBS；ｐH7.4）と共培養した脾臓細胞をコントロールとした（図２）。
図２に示すように、GHFは、GF、GLFと比較しIFN-γの産生を強力に誘導した。しかし、IL-10については、GF、GHF 、GLFによる産生量は同程度あり、GHFと他の抽出液であるGF、GLFとの間に大きな差は認められなかった。IFN-γは、Th1型のサイトカインであり、免疫アジュバントとして感染防御、抗腫瘍、アレルギー改善効果などが期待される。IL-10は、免疫抑制効果として皮膚の炎症、自己免疫疾患の改善効果などが期待できる。
2. Measurement of cytokine-producing ability from immune-responsive cells
(1) Mouse spleen cell stimulation test 1 (measurement of IFN-γ and IL-10 )
Spleens were removed aseptically from BALB / c mice and spleen cell suspensions were prepared in RPMI-1640 medium supplemented with 10% (V / V) heat-inactivated fetal calf serum (FCS). The filtrate filtered through a nylon filter was subjected to centrifugation at 1,500 rpm (rotation / min) and 4 ° C. for 5 minutes to collect a spleen cell pellet. This pellet was resuspended in 0.144 M sodium chloride-0.017 M Tris-HCl buffer (pH 7.65) and incubated at 37 ° C. for 2.5 minutes under 5% CO 2 to hemolyze erythrocytes and finally experimented. Spleen cells (4 × 10 6 / ml) to be used for the preparation were prepared. To the spleen cells obtained above, a hot water extract (GF: 2%), a low molecular fraction (GLF: 0.2%), and a high molecular fraction (GHF: 0.2%) were added to a final volume of 250 μl. ° C., for 48 hours co-cultured under 5% CO 2.
After the culture, IFN-γ and IL-10 in the supernatant were detected by ELISA, and their concentrations were determined. As a control, spleen cells co-cultured with phosphate buffered saline (PBS; pH 7.4) were used as a control instead of the ginseng extract (FIG. 2).
As shown in FIG. 2, GHF strongly induced IFN-γ production compared to GF and GLF. However, IL-10 produced almost the same amount of GF, GHF, and GLF, and no significant difference was observed between GHF and other extracts, GF and GLF. IFN-γ is a Th1-type cytokine, and is expected to provide infection protection, antitumor, allergy improvement effects and the like as an immune adjuvant. IL-10 is expected to have an effect of improving skin inflammation and autoimmune diseases as an immunosuppressive effect.
（２）マウス脾臓細胞刺激試験２（ＩＦＮ−γ、及びＩＬ−10の測定）
ＢＡＬＢ／ｃマウスから脾臓を無菌的に取り出し、10％（Ｖ／Ｖ）熱不活性化ウシ胎児血清（ＦＣＳ）を加えたＲＰＭI−1640培地中で脾臓細胞懸濁液を調整した。ナイロンフィルターでろ過したろ液を1,500rpm(回転／分)、４℃、5分間の遠心分離に供し、脾臓細胞のペレットを回収した。このペレットを0.144Ｍ塩化ナトリウム−0.017Ｍトリス塩酸緩衝液（ｐＨ７．６５）に再懸濁し、37℃で２．５分間、５％ＣＯ２下でインキュベートして赤血球を溶血させ、最終的に実験に用いる脾臓細胞（１×１０７/ml）を調整した。上記で得た脾臓細胞を、まず熱水抽出液（GF：2％）、低分子画分（GLF：0.2％）、高分子画分（GHF：0.2％）を最終容量１mｌで、３７℃、５％CO2下で48時間共培養した。次に、脾臓細胞を洗浄してアンニンコウの熱水抽出液を除き、１×１０６個/mlに調整して、ラットIgG（Rat IgG）、抗ヒトIL-12 モノクローナル抗体（aIL-12）、抗ヒトIL-10モノクローナル抗体（aIL-10）（最終濃度０．５μg/mｌ）を含有するRPMI-1640培地にて、最終容量250μLで37℃、５％CO2下で再び、36時間培養した。
培養後、上清中のIFN−γ及びIL-10をELISA法によって検出し、その濃度を決定した。アンニンコウ抽出液の代替にPBS（ｐH７．４）と共培養した脾細胞をコントロール（Ab）とした（図３）。
図３に示すように、GHFは、抗IL-10抗体 との培養においてはINF-γの産生を促進させた。一方、抗IL-12抗体との培養においてはINF-γ産生が抑制された。このことから、GHFは、IL-12を介してIFN-γを産生させることが判明した。
(2) Mouse spleen cell stimulation test 2 (measurement of IFN-γ and IL-10)
Spleens were removed aseptically from BALB / c mice and spleen cell suspensions were prepared in RPMI-1640 medium supplemented with 10% (V / V) heat-inactivated fetal calf serum (FCS). The filtrate filtered through a nylon filter was subjected to centrifugation at 1,500 rpm (rotation / min) and 4 ° C. for 5 minutes to collect a spleen cell pellet. This pellet was resuspended in 0.144 M sodium chloride-0.017 M Tris-HCl buffer (pH 7.65) and incubated at 37 ° C. for 2.5 minutes under 5% CO 2 to hemolyze erythrocytes and finally experimented. Spleen cells (1 × 10 7 / ml) used for the preparation were prepared. The spleen cells obtained above were first prepared with a hot water extract (GF: 2%), a low molecular fraction (GLF: 0.2%), and a high molecular fraction (GHF: 0.2%) in a final volume of 1 ml at 37 ° C. Co-cultured for 48 hours under 5% CO 2 . Next, the spleen cells were washed, the hot water extract of Anninkou was removed, adjusted to 1 × 10 6 cells / ml, rat IgG (Rat IgG), anti-human IL-12 monoclonal antibody (aIL-12), In RPMI-1640 medium containing anti-human IL-10 monoclonal antibody (aIL-10) (final concentration 0.5 μg / ml), the cells were cultured again for 36 hours at 37 ° C. and 5% CO 2 in a final volume of 250 μL. .
After culture, IFN-γ and IL-10 in the supernatant were detected by ELISA, and their concentrations were determined. As a control (Ab), splenocytes co-cultured with PBS (pH 7.4) were used as an alternative to the ginseng extract (FIG. 3).
As shown in FIG. 3, GHF promoted INF-γ production in culture with anti-IL-10 antibody. On the other hand, INF-γ production was suppressed in the culture with anti-IL-12 antibody. From this, it became clear that GHF produces IFN-γ via IL-12.
（３）マウス腹腔マクロファージ（PEC）刺激試験（ＩＬ−12の測定）
C56BL/6マウスに２mlの10％チオグリコレート培地を腹腔内投与し、4日後にマウス腹腔内をPBSで洗浄することによりPECを回収した。細胞をRPMI-1640培地で清浄した後、４×１０6個/mlに調整した。このPECと、10％FCSを含有するRPMI-1640培地中で熱水抽出液（GF：2％）、低分子画分（GLF：0.2％）、高分子画分（GHF：0.2％）を加え、最終容量250μｌとし、5％CO2下、37℃で、4時間、8時間、12時間、24時間にて、共培養した。
培養後、上清中のIL-12をELISA法によって、検出し、その濃度を決定した。アンニンコウ抽出液の代替にPBS（ｐH７．４）と共培養したPECをコントロールとした（図４）。
図４に示すように、GHFは、共培養後8時間から樹状細胞（BMDC）からのIL-12産生促進作用が確認された。一方GF、GLFでは、活性は認められなかった。マクロファージはIL-12の最も効率的な産生細胞のひとつであり、IL-12は、NK細胞やT細胞に作用して、IFN-γの産生を誘導することが知られている。これによって、Th1が活性化され、Th1/Th2バランスの不均等が是正されると考えられる。
(3) Mouse peritoneal macrophage (PEC) stimulation test (measurement of IL-12)
2 ml of 10% thioglycolate medium was intraperitoneally administered to C56BL / 6 mice, and 4 days later, the mice were intraperitoneally washed with PBS to recover PEC. Cells were cleaned with RPMI-1640 medium and adjusted to 4 × 10 6 cells / ml. Add hot water extract (GF: 2%), low molecular fraction (GLF: 0.2%), high molecular fraction (GHF: 0.2%) in RPMI-1640 medium containing 10% FCS with this PEC The final volume was 250 μl, and the cells were co-cultured at 37 ° C. under 5
After culture, IL-12 in the supernatant was detected by ELISA, and its concentration was determined. As a control, PEC co-cultured with PBS (pH 7.4) was used as an alternative to the Anninkou extract (FIG. 4).
As shown in FIG. 4, GHF was confirmed to promote IL-12 production from dendritic cells (BMDC) from 8 hours after co-culture. On the other hand, no activity was observed in GF and GLF. Macrophages are one of the most efficient producers of IL-12, and IL-12 is known to act on NK cells and T cells to induce IFN-γ production. This is thought to activate Th1 and correct the uneven Th1 / Th2 balance.
〔実施例２〕気管支喘息モデルマウスを用いた動物実験
実施例１より、GHFは、マウス脾臓細胞に対してIFN-γ産生を促進し、マウス腹腔マクロファージに対してIL-12 産生を誘導する免疫調整作用等を有することが確認された。このことから実施例２では、GHFを使用して、動物実験を行った。
（１）動物
BALB/cマウス 雌（6週齢）を卵白アルブミン（OVA）にて感作した。予備飼育3日後から、これらモデルマウスを3群に分け、比較検討した（図５）。
[モデルマウス]
ポジティブコントロール：OVA非感作＋生理食塩水吸入群（SAL/SAL）
ネガティブコントロール：OVA感作＋通常飼料群（NOR/OVA）
GHF処理：OVA感作＋２％GHF含有飼料群 (GHF/OVA)
[Example 2] Animal experiment using bronchial asthma model mice From Example 1, GHF promotes IFN-γ production to mouse spleen cells and immunity induces IL-12 production to mouse peritoneal macrophages. It was confirmed that it has a regulating action and the like. Therefore, in Example 2, animal experiments were performed using GHF.
(1) Animals
BALB / c mice Females (6 weeks old) were sensitized with ovalbumin (OVA). Three days after the preliminary breeding, these model mice were divided into three groups for comparison (FIG. 5).
[Model mouse]
Positive control: OVA non-sensitized + saline inhalation group (SAL / SAL)
Negative control: OVA sensitization + normal feed group (NOR / OVA)
GHF treatment: OVA sensitization + 2% GHF containing feed group (GHF / OVA)
（２）工程
マウスにアレルギー抗原として卵白アルブミン（OVA）を体重20グラムあたり10マイクログラム腹腔内投与して初回免疫を行った。その７日後（一週間後）に、同様の措置をして、2回目の免疫を行い、同様にその7日後に3回目の免疫を行った。
一方、初回免疫後21日目から6日間毎日OVA吸入を実施してマウスを感作し、アレルギー性喘息を誘発した。OVA吸入最終日にアレルギー性喘息の評価として、気道洗浄液中の細胞数の測定、気道過敏性を測定した。その後、解剖し、気管支肺胞洗浄液（BALF）や血液（PLASMA）を収得し、脾臓を無菌的に取りだした。
(2) Process The mouse was first immunized by intraperitoneally administering 10 μg ovalbumin (OVA) per 20 g body weight as an allergic antigen. Seven days later (one week later), the same measures were taken, and the second immunization was performed. Similarly, the third immunization was performed seven days later.
On the other hand, mice were sensitized by inhaling OVA daily for 6 days from the 21st day after the first immunization to induce allergic asthma. As the evaluation of allergic asthma on the last day of OVA inhalation, the number of cells in the airway lavage fluid and the airway hypersensitivity were measured. After dissection, bronchoalveolar lavage fluid (BALF) and blood (PLASMA) were collected, and the spleen was aseptically removed.
（３）気道過敏性試験
最終卵白アルブミン（OVA）感作２４時間後、Penh値を測定した（図６）。
図６に示すように、免疫していないSAL/SAL群では、OVA吸入によって気道過敏性（Penh値）ほとんど増加しなかった。しかし、OVA感作したNOR/OVA群のメタコリンを処理した際Penhの値は、SAL/SAL群に比べて著しく増加した。NOR/OVA群と比較して、GHFを処理したGHF/OVA群は、5mg/mlのメタコリンを処理した際、Penh値は有意に減少した。また、10mg/mlのメタコリンを処理した際でも、同様に減少傾向にあった。
(3) Airway hypersensitivity test The Penh value was measured 24 hours after the final ovalbumin (OVA) sensitization (FIG. 6).
As shown in FIG. 6, in the non-immunized SAL / SAL group, airway hypersensitivity (Penh value) was hardly increased by inhalation of OVA. However, when treated with methacholine in the NOR / OVA group sensitized with OVA, the Penh value increased significantly compared to the SAL / SAL group. Compared with the NOR / OVA group, the GHF / OVA group treated with GHF significantly decreased the Penh value when treated with 5 mg / ml methacholine. In addition, even when 10 mg / ml methacholine was treated, there was a similar downward trend.
（４）BALFおよびPLASMA中の免疫応答反応
最終誘発48時間後、マウスに対してペントバルビタール（pentobarbital）を過量使用して致死させ、気管切開術を行った。PBSを肺に流入した後、導管カニューレ押入させることにより、気管支肺胞洗浄液（BALF）を収得した。BALFを遠心分離して、上清液を収集し、使用前に−８０℃で貯蔵した。BALF内のIgE（図７）、IgG（図８）、IL-10（図１１）量は、製造社の指示に従い、特異的マウスELISAキットで測定した。
また、気道切開術の後、心臓穿刺（cardiac puncture）によって血漿（PLASMA）を得た。PLASMAを遠心分離して、PLASMA中のIgE（図７）、IgG（図８）、IFN-γ（図１０）、MCP-1（図９）、IL-10（図１１）、IL-12（図１０）量は、製造社の指示に従い、特異的マウスELISAキットで測定した。図７、図８および図９に示したように、SAL/SAL群では、免疫グロブリンIgE（図７）、IgG（図８）や炎症性マーカーであるMCP-1（図９）レベルは低く、アレルギー症状や炎症は認められなかった。しかしNOR/OVA群では、それらサイトカインの上昇が顕著に認められアレルギー状態にあることが示されたが、GHF処理のGHF/OVA群では、PLASMAやBALFいずれのIgE、IgG値も抑制されており、MCP-1レベルは、著しく減少し、アレルギー抑制効果、抗炎症効果を示した。
(4) 48 hours after the final induction of immune response in BALF and PLASMA , the mice were killed using an overdose of pentobarbital and tracheotomy was performed. After inflowing PBS into the lung, bronchoalveolar lavage fluid (BALF) was obtained by pushing the conduit cannula. BALF was centrifuged and the supernatant was collected and stored at −80 ° C. before use. The amounts of IgE (FIG. 7), IgG (FIG. 8) and IL-10 (FIG. 11) in BALF were measured with a specific mouse ELISA kit according to the manufacturer's instructions.
In addition, plasma (PLASMA) was obtained by cardiac puncture after airway incision. PLASMA was centrifuged and IgE in PLASMA (FIG. 7), IgG (FIG. 8), IFN-γ (FIG. 10), MCP-1 (FIG. 9), IL-10 (FIG. 11), IL-12 ( FIG. 10) The amount was measured with a specific mouse ELISA kit according to the manufacturer's instructions. As shown in FIGS. 7, 8 and 9, in the SAL / SAL group, immunoglobulin IgE (FIG. 7), IgG (FIG. 8) and MCP-1 (FIG. 9) levels of inflammatory markers are low, There were no allergic symptoms or inflammation. However, in the NOR / OVA group, these cytokines were significantly elevated and allergic conditions were shown, but in the GHF / GHA-treated GHF / OVA group, both IgE and IgG levels of PLASMA and BALF were suppressed. In addition, MCP-1 levels were significantly reduced, showing allergy-suppressing and anti-inflammatory effects.
実施例１の細胞実験では、GHF中は、IL-12を介してIFN-γを産生する能力を持つことが認められた。図10に示すように、モデルマウスを使用した動物実験においても、IFN-γやIL-12の産生を促進することを確認できた。IL-12はアレルギー因子であるIgEなどの抑制に重要な働きを示していることから、GHFのアレルギー改善効果は、生体内においても、IL-12産生を介していることが示された。 In the cell experiment of Example 1, it was confirmed that GHF has the ability to produce IFN-γ via IL-12. As shown in FIG. 10, it was confirmed that the production of IFN-γ and IL-12 was promoted also in animal experiments using model mice. Since IL-12 has an important function in suppressing IgE, which is an allergic factor, it has been shown that the allergy-improving effect of GHF is also mediated by IL-12 production in vivo.
さらに図11に示すように、IL-10の値は、PLASMA、BALF のいずれにおいてもGHF/OVA群にて上昇しており、GHF 中には、 IL-10 の産生を促進する作用もあることが認められた。IL-10は、主にTh2細胞や制御性T細胞、活性化B細胞、単球、肥満細胞からも産生される。IL-10は、おもに単球系細胞に作用して炎症性サイトカインの産生を始めとする免疫機能を抑制的に制御するため、炎症の抑制に重要な役割を果たしている。
これらの結果より、GHFは、生体内において、IL-12産生促進作用だけでなく、強いIL-10産生促進作用も有することが明らかとなった。
Furthermore, as shown in Fig. 11, IL-10 levels increased in the GHF / OVA group for both PLASMA and BALF, and GHF also has an effect of promoting IL-10 production. Was recognized. IL-10 is also produced mainly from Th2 cells, regulatory T cells, activated B cells, monocytes, and mast cells. IL-10 plays an important role in the suppression of inflammation because it mainly acts on monocyte cells and suppresses immune functions including the production of inflammatory cytokines.
From these results, it became clear that GHF has not only an IL-12 production promoting action but also a strong IL-10 production promoting action in vivo.
（５）脾臓での免疫機能
解剖後に取りだした脾臓からT細胞、制御性T細胞（Treg細胞）及びＮＫＴ細胞増殖抑制など免疫細胞の割合を測定した。
T細胞に関してはほとんど差が見られなかったが、Treg細胞が、有意に増加していることが確認できた。Treg 細胞は、免疫システムの行き過ぎた応答の抑制において極めて重要な役割を果たすT細胞の一種であり、制御性T細胞は、喘息をはじめとしたアレルギー性炎症を制御している。このことから、Treg細胞の値を上昇させる作用をもつGHFは、異常な免疫反応を抑制することで、炎症を緩和し、アレルギー性喘息などの緩和効果を持つことが示された。また、気道過敏症をひき起こす細胞であるといわれているＮＫＴ細胞の増殖を、GHFは抑制することも明らかにした（図12）。
(5) The proportion of immune cells such as T cells, regulatory T cells (Treg cells) and NKT cell proliferation suppression was measured from the spleen taken out after dissection of immune functions in the spleen.
Although there was almost no difference regarding T cells, it was confirmed that Treg cells were significantly increased. Treg cells are a type of T cell that plays an extremely important role in suppressing the excessive response of the immune system, and regulatory T cells control allergic inflammation including asthma. From this, it was shown that GHF, which has an action of increasing the value of Treg cells, alleviates inflammation and suppresses allergic asthma by suppressing abnormal immune responses. It was also revealed that GHF suppresses the growth of NKT cells, which are said to cause airway hypersensitivity (FIG. 12).
以上のように、GHFは、Th1を活性化させるIFN-γ、およびIL-12の産生促進作用のほかに、アレルギー性炎症を制御するIL-10 の産生促進や制御性T細胞の増殖の促進、あるいは、ＮＫＴ細胞の増殖を抑制することで、卵白アルブミン（OVA）で誘発された喘息マウスモデルにおいてPLASMAやBALFで増加したIgE、IgGを強力に抑制し、アレルギー症状や気道過敏性を改善し、肺組織における粘液過多分泌を抑制する機能を持つことが認められた。
アレルギーの発症機構は病態に関わらず、アレルギー抗原に特異的なTh2細胞の活性化が引き金となっている。このため、アンニンコウ（GHF）の免疫抑制効果を考慮すると、喘息以外のアレルギー疾患、アトピー性皮膚炎などの疾患に対しても有効と考えられ、難治性アレルギー疾患の治療法につながる可能性がある。したがって、アンニンコウは、炎症、アレルギーおよび喘息疾患を予防、および治療するための治療薬、または、健康機能食品として使用できると思われる。
As described above, GHF promotes the production of IL-10 that regulates allergic inflammation and the proliferation of regulatory T cells in addition to the production of IFN-γ and IL-12 that activate Th1. Or, by suppressing the growth of NKT cells, IgE and IgG increased by PLASMA and BALF are strongly suppressed in an asthma mouse model induced by ovalbumin (OVA), and allergic symptoms and airway hypersensitivity are improved. It was observed that it has the function of suppressing mucus hypersecretion in lung tissue.
Regardless of the pathogenesis of allergy, the activation of Th2 cells specific for allergic antigens is the trigger. Therefore, taking into account the immunosuppressive effect of ginseng (GHF), it is considered effective against allergic diseases other than asthma and diseases such as atopic dermatitis, which may lead to treatment of intractable allergic diseases. . Therefore, it seems that Anninkou can be used as a therapeutic agent for preventing and treating inflammation, allergies and asthma diseases, or as a health functional food.
〔実施例３〕有効成分の構造
アンニンコウ高分子画分（GHF）に、どのような分子量をもつ物質が含まれるのか検討した。
（１）分析
GHF30mg/mlをゲルろ過クロマトグラフィー（充填剤：Sephacry S-300HR, 展開溶液：1/15Mリン酸緩衝駅）を用いて150滴ずつ流速1.0mL/min で120本分取した。そして各フラクションをフェノール硫酸法にて分光光度計490nmの吸光度を測定した。その後各フラクションを4つのフラクションに分け、濃縮し、凍結乾燥した。また、グルコース（濃度200μｇ/ml，100μｇ/ml，50μｇ/ml，10μｇ/ml，1μｇ/ml）で検量線を作成し、回収量を求めた。さらに、分子量マーカー（Mw788000, Mw212000, Mw112000, Mw22800, Mw18800, Mw5900）をカラムに流し、分子量を求めた。
（２）分子量
溶出量208mlと314mlに２つのピークが得られた。溶出量208ｍｌは分子量400000以上の物質であることがわかり、溶出量314ｍｌは分子量22800であることがわかった。すなわち、GHFは、分子量400000以上の成分と分子量22800の2つの成分からなることが判明した。
[Example 3] Structure of active ingredient It was investigated what kind of molecular weight the anninkou polymer fraction (GHF) contains.
(1) Analysis
120 gels of
(2) Two peaks were obtained at molecular weight elution amounts of 208 ml and 314 ml. The elution amount 208 ml was found to be a substance having a molecular weight of 400,000 or more, and the elution amount 314 ml was found to have a molecular weight of 22800. That is, it has been found that GHF is composed of two components having a molecular weight of 400,000 or more and a molecular weight of 22,800.
本発明により、アンニンコウの熱水抽出液の高分子画分を有効成分とする免疫機能強化剤が提供された。本発明の免疫機能強化剤は、IFN-γ、IL-10 およびIL-12の産生促進、ＩｇＥの産生抑制、ＭＣＰ−１の産生抑制、さらには、アレルギー性炎症を制御している制御性T細胞の増殖、及び気道過敏性の改善に関わるＮＫＴ細胞増殖抑制を通してアレルギー疾患の症状を緩和する。またアンニンコウは、従来より食品として使用されており、安全性が保障されている。従って本発明の免疫機能強化剤は、アレルギー性疾患の治療や予防の分野において有用である。 INDUSTRIAL APPLICABILITY According to the present invention, there is provided an immune function enhancing agent comprising a polymer fraction of a hot water extract of anninko as an active ingredient. The immune function enhancer of the present invention is a regulatory T that regulates IFN-γ, IL-10 and IL-12 production promotion, IgE production inhibition, MCP-1 production inhibition, and allergic inflammation. Alleviate symptoms of allergic diseases through suppression of NKT cell proliferation, which is associated with improvement of cell proliferation and airway hypersensitivity. Anninkou has been conventionally used as a food, and its safety is guaranteed. Therefore, the immune function enhancer of the present invention is useful in the field of treatment and prevention of allergic diseases.
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