JP4109572B2 - Cordyceps mycelium culture method and composition - Google Patents
Cordyceps mycelium culture method and composition Download PDFInfo
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- JP4109572B2 JP4109572B2 JP2003142665A JP2003142665A JP4109572B2 JP 4109572 B2 JP4109572 B2 JP 4109572B2 JP 2003142665 A JP2003142665 A JP 2003142665A JP 2003142665 A JP2003142665 A JP 2003142665A JP 4109572 B2 JP4109572 B2 JP 4109572B2
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- mycelium
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Description
【０００１】
【発明の属する技術分野】
本発明は、冬虫夏草菌糸体の培養方法、当該方法により得られた菌糸体 当該菌糸体より抽出されるエキス、及びそれらを含有する医薬組成物若しくは食品組成物に関する。
【０００２】
【従来の技術】
冬虫夏草は、不老長寿、滋養強壮の妙薬として、また、結核、黄疸等の治療薬として古来より珍重されてきた。近年においても、薬効や有効成分の研究によりその有用性が再認識され、医薬品や食品としての需要がさらに増大している。例えば、最近では、抗癌作用、国際公開第９６／００５８０号パンフレット（ＰＣＴ／ＪＰ９５／０１２９８）（特許文献１）に示されるような、強心、降圧、鎮咳、及び抗疲労作用、特開平１０−２４５３４０号公報（特許文献２）に示されるような強壮強精作用、また、特開平１０−２４５３４１号公報（特許文献３）に示されるような血糖降下作用他種々の薬効も報告されている。また、冬虫夏草の成分としては、抗菌作用を有するコルジセピン、心筋収縮抑制作用を有するＮ6−（２−ヒドロキシエチル）−アデノシン（ＨＥＡ）（フルヤ（Furuya T.）外，フィトケミストリー（Phytochemistry），第２２巻，第１１号，１９８３年，ｐ．２５０９）（非特許文献１）及び免疫抑制作用を有するミリオシン等が単離同定されている。
【０００３】
しかしながら、冬虫夏草の採取物は薬理活性のばらつきが非常に大きく、安定した活性を期待することができない。また、冬虫夏草自体を天然から大量に入手することは非常に困難である。
【０００４】
また、冬虫夏草の培養方法について、種々検討が行われ、例えば、特開平９−３２７２３２号公報（特許文献４）に示すように、培地中にさなぎ粉を含有する方法、特開平１０−４２６９１号公報（特許文献５）に示すように、蚕の蛹組成成分を主成分とする培地を用いる方法、また、特開平１０−１１７７７０号公報（特許文献６）に示すように、冬虫夏草の寄生した同一種の昆虫の抽出エキスを培地に添加する方法等が報告されている。しかしながら、これらの培養方法はいずれも培地組成を天然の冬虫夏草の栄養源に類似させただけであり、菌の状態は同じ培地を用いても環境因子に大きく影響されるため、培地組成を工夫するだけでは薬理活性及び含有成分を安定に有する冬虫夏草を得ることはできない。
【０００５】
さらに、特開平９−６２号公報（特許文献７）には、おが屑、コーンコブ粉砕物等の培地基材に、米糠、豆皮等の栄養材を混合した培地を使用し、屋内での環境（温度、湿度等）制御により冬虫夏草（子実体）を栽培する方法が記載されている。さらに、特開２００１−２６９０５４号公報（特許文献８）には、菌糸培養工程においては黄色若しくは赤色の光を照射し、子実体発生工程においては青色若しくは緑色の光を照射して、子実体の収穫量を向上させる冬虫夏草の栽培方法が記載されている。
しかし、冬虫夏草菌糸体培養の際に、特定の波長域にピークを有する光が、得られる菌糸体培養物の薬理活性や含有成分に与える影響について今まで研究されたものはなかった。
【０００６】
【特許文献１】
国際公開第９６／００５８０号パンフレット
【特許文献２】
特開平１０−２４５３４０号公報
【特許文献３】
特開平１０−２４５３４１号公報
【特許文献４】
特開平９−３２７２３２号公報
【特許文献５】
特開平１０−４２６９１号公報
【特許文献６】
特開平１０−１１７７７０号公報
【特許文献７】
特開平９−６２号公報
【特許文献８】
特開２００１−２６９０５４号公報
【非特許文献１】
フルヤ（Furuya T.）外，フィトケミストリー（Phytochemistry），第２２巻，第１１号，１９８３年，ｐ．２５０９
【０００７】
【発明が解決しようとする課題】
本発明は、薬理活性及び含有成分を安定に有する冬虫夏草菌糸体を効率的に得ることができる、冬虫夏草菌糸体の培養方法、当該方法により得られた菌糸体、当該菌糸体より抽出されるエキス、及びそれらを含有する医薬組成物若しくは食品組成物を提供することを目的とする。また、本発明は、冬虫夏草菌糸体の薬理作用の増強方法の提供をも目的とする。
【０００８】
【課題を解決するための手段】
本発明者らは、上記目的を達成するために鋭意研究を重ねた結果、冬虫夏草は、例えばコルジセプス属等のように子実体よりも菌糸体の方に薬理活性及び有効成分の含有量を豊富に持つ場合も多いことから、活性に遜色がなければ、子実体よりも菌糸体の方が培養日数も短く培養条件も調節しやすいことに着目した。さらに本発明者らは研究の末、接種した菌に３５０〜５５０ｎｍの波長域にピークをもつ光を連続又は間欠的に照射することで、薬理活性及び含有成分を安定に有する冬虫夏草菌糸体を効率よく得ることができることを見出し、なおかつこの培養方法により得られる冬虫夏草菌糸体は薬理活性及び／又は有効成分の含有量が増大していることを見出して、本発明を完成させるに至った。
【０００９】
すなわち、本発明は以下の通りである。
（１）培地に菌を接種し、３５０〜５５０ｎｍの波長域にピークをもつ光を連続又は間欠的に照射することを特徴とする冬虫夏草菌糸体の培養方法、
（２）培地が、穀類、又は穀類及び酵母若しくはその抽出物を含む上記（１）記載の培養方法、
（３）冬虫夏草がコルジセプス（cordyceps）属に属することを特徴とする上記（１）記載の培養方法、
（４）上記（１）記載の培養方法により得られた冬虫夏草菌糸体、
（５）上記（４）記載の冬虫夏草菌糸体より抽出されるエキス、
（６）上記（１）記載の培養方法により得られた冬虫夏草菌糸体、及び／又は当該菌糸体より抽出されるエキスと、医薬として許容できる担体を含む医薬組成物、
（７）上記（１）記載の培養方法により得られた冬虫夏草菌糸体、及び／又は当該菌糸体より抽出されるエキスを含有してなる食品組成物、
（８）培地に菌を接種し、３５０〜５５０ｎｍの波長域にピークをもつ光を連続又は間欠的に照射することを特徴とする、冬虫夏草菌糸体の薬理作用の増強方法、及び、
（９）培地が、穀類、又は穀類及び酵母若しくはその抽出物を含む上記（８）記載の増強方法。
【００１０】
【発明の実施の形態】
以下、本発明を詳述する。
本発明の対象となる冬虫夏草は、子嚢菌類（Ascomycota）、核菌網（Pyrenomycetes）、麦角菌目（Clavicipitales）、麦角菌科（Clavicipitaceae又はHypocreaceae）に属し、完全世代と不完全世代とを有する微生物である。例えば、コルジセプス（Cordyceps）属（例えばサナギタケ（C.militaris）、ウスキサナギタケ（C.takaomontana）等）等が挙げられる。子実体よりも菌糸体の方に薬理活性及び／又は有効成分の含有量を豊富に持つことから、コルジセプス属が好ましい。これらの菌は、日本、台湾、中国、ネパール等に分布し、発生時期は３〜１１月である。ガの蛹、幼虫等に寄生して養分を摂取して増殖し、虫の死骸より子実体を発生する。
【００１１】
本発明に係る冬虫夏草菌糸体の培養方法は、穀類、又は穀類及び酵母若しくはその抽出物を含む培地で栽培することが好ましい。穀類には、一般に人間の主食となる作物が含まれ、例えば米、米糠、粟、麦（小麦、大麦、ハト麦等）、蕎麦、ヒエ、キビ、トウモロコシ、アマランサス、豆類等が挙げられる。酵母としては、例えば乾燥ビール酵母、パン用酵母、清酒用酵母、ワイン用酵母等が挙げられる。酵母は、そのまま用いることもできるが、水、熱水、あるいは、有機溶媒等で抽出した酵母の抽出物（酵母エキス）を用いることもできる。穀類、又は穀類及び酵母若しくはその抽出物に、さらに、豆皮、おから等の豆類、サナギ粉、魚粉、煮干粉砕物等の動物紛のいずれか１つ又は複数を添加してもよい。また、おが屑、コーンコブ粉砕物等の培地基材を用いて、これに穀類、又は穀類及び酵母若しくはその抽出物を加えたものでもよく、さらに上記の豆類、動物紛等を添加してもよい。穀類を用いることで、菌糸増殖の支持体と栄養源を兼ねる。また、酵母あるいはその抽出物は、アミノ酸、ミネラルを豊富に含むので、少量で効率的な増殖が得られる。また、穀類や酵母は、入手しやすく、成分的にも比較的安定した培地組成である。
【００１２】
穀類の配合量は、培地成分（後述の水等を含まない成分）の全重量の通常５．０〜５５．０重量％、好ましくは１０．０〜３５．０重量％である。酵母を用いる場合は、その配合量は、培地成分の全重量の通常１．０〜１０．０重量％、好ましくは４．０〜７．０重量％である。酵母の抽出物を用いる場合は、その配合量は、培地成分の全重量の通常０．０１〜３．０重量％、好ましくは０．０２〜１．０重量％である。
さらに豆類、動物粉等を添加する場合は、その配合量は、培地成分の全重量の通常１．０〜１０．０重量％、好ましくは４．０〜７．０重量％である。また、おが屑、コーンコブ粉砕物等の培地基材を添加する場合は、その配合量は、培地成分の全重量の通常５．０〜５０．０重量％、好ましくは６．０〜２０．０重量％である。
上記の培地のうち、麦（割麦）と乾燥ビール酵母とからなる培地が好ましく、麦（割麦）１４０〜１６０ｇ、乾燥ビール酵母２０〜４０ｇの割合で配合した培地がさらに好ましい。具体的には、例えば、麦（割麦）１５０ｇ：乾燥ビール酵母３０ｇ：水３００ｍｌ、麦（割麦）１４０ｇ：乾燥ビール酵母４０ｇ：水３００ｍｌ、または麦（割麦）１６０ｇ：乾燥ビール酵母２０ｇ：水３００ｍｌの割合で配合した培地等が好ましい。
上記の成分を含有する培地は、従来公知の方法により作製することができ、例えば、これらの培地成分全重量に対して、水等を、通常５０．０〜８５．０重量％、好ましくは６０．０〜７５．０重量％に調製し、高圧蒸気滅菌器にて殺菌することにより作成することができる。
【００１３】
本発明では、培養条件として上記に例示されるような培地に菌を接種し、３５０〜５５０ｎｍの波長域にピークをもつ光（主に青色若しくは緑色の光）を連続又は間欠的に照射することで、薬理活性及び含有成分を効率的に安定的に有することが可能となった。
子実体発生に有効な光は青色域の光であることが知られており、前記特開２００１−２６９０５４号公報（特許文献８）に示すように、菌糸培養工程においては、黄色若しくは赤色、また、子実体発生工程においては、青色若しくは緑色を用いることで、子実体の収穫量が向上している。
しかしながら、菌糸体で薬理活性や含有成分を効率的かつ安定的に有する培養方法としては、菌糸の接種直後から、３５０〜５５０ｎｍ、好ましくは３５０〜５３０ｎｍ、さらに好ましくは４００〜５００ｎｍの波長域にピークを持つ光を連続又は間欠的に照射することが望ましいことを見出した。波長域は、３５０ｎｍ未満では、菌糸の増殖を阻害する傾向が強まり、また、５５０ｎｍを超えると、薬理活性や含有成分の低下を招く。
【００１４】
３５０〜５５０ｎｍの波長域等にピークを持つ光とは、光源から照射される光のうち、可視光域の波長における光の中で、相対発光強度が最も強い強度を示す光の波長が３５０〜５５０ｎｍの波長域にある光のことをいう。３５０〜５５０ｎｍの波長域等にピークを持つ光を照射し得る光源としては、例えば蛍光管では、カラー蛍光ランプの青色、緑色、ブラックライト等があり、また、発光ダイオードでは、青又は緑の発光ダイオードが挙げられるが、これらに限定されず、同等域にピークのある光源であれば何れも用いることができる。
照度は、通常１０ｌｕｘ以上、好ましくは１２００〜５０００ｌｕｘである。間欠的に光を照射する場合には、照射する時間と暗所に置く時間が１：１であることが好ましい。例えば、照射時間が３０分である場合は暗所時間を３０分とし、照射時間を１２時間とした場合は暗所時間を１２時間とする。
【００１５】
菌の接種は、特に限定されず従来公知の方法で行うことができるが、例えば野生の菌から純粋分離した菌株を、寒天培地または液体培地に接種した菌株を接種することにより行うことができる。
培養温度は、通常１５〜３０℃、好ましくは１９〜２３℃、培養湿度は、通常６０〜９５％、好ましくは８５〜９５％である。培養期間は、通常１〜２ヶ月、好ましくは４〜６週間である。
【００１６】
ハナサナギタケ（I.Japonica）等のイサリア属は、無性胞子を有する子実体を形成するが、子実体の方に薬理活性及び／又は有効成分を有する場合も多く、大島らは、ハナサナギタケ子実体に薬理活性を有するセスキテルペン系の新規成分を見出している（特願２００２−５２１４０号）。一方、サナギタケ（C.militaris）、ウスキサナギタケ（C.takaomontana）等のコルジセプス属は、子嚢果に子嚢胞子を有する子実体を形成するが、子実体よりも菌糸体の方に薬理活性及び／又は有効成分の含有量を豊富に持つ場合も多い。また、活性に遜色がなければ、菌糸体の方が培養日数も短く、培養条件も調節しやすい利点をもつ。そこで、発明者らは、特にコルジセプス属等に属する冬虫夏草を、培地に菌を接種し、３５０〜５５０ｎｍの波長域にピークをもつ光を連続又は間欠的に照射することで、薬理活性及び／又は含有成分を効率よく安定に有する菌糸体を取得する方法を開発した。
【００１７】
このような発明の方法に従えば、１〜２ヶ月間程度の培養により、充分量の冬虫夏草の菌糸体を得ることができる。しかも得られる菌糸体は、神経細胞の分化誘導活性、及び／又は、癌細胞増殖抑制活性及び／又は抗疲労活性を有する。また、抗菌活性を有するコルジセピンの含有量が高くなるという効果を有する。
【００１８】
本発明の上記培養方法により得られる冬虫夏草菌糸体（以下本発明の冬虫夏草菌糸体ともいう。）としては、例えばコルジセプス（Cordyceps）属（例えばサナギタケ（C.militaris）、ウスキサナギタケ（C.takaomontana）等）等の冬虫夏草菌糸体が挙げられ、コルジセプス（Cordyceps）属（例えばサナギタケ（C.militaris）、ウスキサナギタケ（C.takaomontana）等）が好ましい。
本発明の冬虫夏草菌糸体より抽出されるエキスは、上記冬虫夏草菌糸体を常法により有機溶媒（例えば３０％エタノール等）や水等で抽出して得ることができる。
【００１９】
本発明の冬虫夏草菌糸体は、神経細胞の分化誘導活性、及び／又は、癌細胞増殖抑制活性及び／又は抗疲労活性を有する。また、抗菌活性を有するコルジセピンの含有量が高くなっているという特徴を有する。例えば、本発明の冬虫夏草菌糸体１ｇ中のコルジセピンの含有量は、少なくとも１ｍｇ／ｇ−Ｄ．Ｗ．（乾燥菌体）以上、好ましくは２ｍｇ／ｇ−Ｄ．Ｗ．〜２０ｍｇ／ｇ−Ｄ．Ｗ．、さらに好ましくは３ｍｇ／ｇ−Ｄ．Ｗ．〜１２ｍｇ／ｇ−Ｄ．Ｗ．である。コルジセピンの含有量があまり高いと、他の活性（例えば抗癌活性）が失われる傾向もあり、全体のバランスからは上記の範囲であるのが好ましい。
本発明の培養方法により、薬理活性が、上記３つの活性（神経細胞の分化誘導活性、癌細胞増殖抑制活性、抗疲労活性）を同時に有し、かつ、抗菌活性を有するコルジセピン含有量が上記の範囲である菌糸体を得ることが可能となる。
【００２０】
このように、本発明の培養方法によれば、得られる冬虫夏草菌糸体の薬理活性及び／又は有効成分の含有量を増大させることができ、その結果、冬虫夏草菌糸体の薬理作用、例えば神経細胞の分化誘導作用、癌細胞増殖抑制作用、抗疲労作用、抗菌作用、強心、強壮強精作用、免疫調整作用や増強作用等を増強することができる。
例えば、抗菌活性を有するコルジセピンの含有量は、同一の培地を用いて、昼色光を用いた場合に比べ、抽出エキスあたり少なくとも２０重量％以上増加する。
ここで、本明細書において昼色光とは、白色蛍光管による光をいう。
【００２１】
本明細書で使用する冬虫夏草菌糸体、及び／又は菌糸体より抽出されるエキスとは、冬虫夏草菌糸体の乾燥物をそのまま、あるいは粉末化したもの、あるいは、例えば有機溶媒や水等で抽出した抽出エキス、及び抽出エキスを特定の化合物に完全精製に至るまでの任意の純度まで精製したもの（部分精製品）をいう。形態としては、水溶性や、減圧濃縮し乾固させた固体、凍結乾燥品等の液状物や固形物を含む。本発明の方法によって得られた冬虫夏草菌糸体、当該菌糸体より抽出されるエキスは、脳神経細胞の細胞死を伴う疾患及び／又は免疫増強及び／又は心臓・呼吸器系疾患及び／又は癌の予防及び／又は治療用、免疫増強及び疲労回復に有効な医薬若しくは食品として、又は後述する適当な担体等とともに医薬組成物若しくは食品組成物として利用できる。
【００２２】
食品組成物として、本発明の冬虫夏草菌糸体、及び／又は菌糸体より抽出されるエキスを、例えば、ジュース、清涼飲料、茶、スープ、豆乳、豆腐、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキ、パン、クッキー、スナック菓子等に含有させることもできる。（これらも、本明細書では食品組成物に含める）。あるいは、菌子体粉末等又はそのエキスを、デキストリン、乳糖、デンプン、コーンスターチ等の賦形剤や香料、色素等とともに、ペレット、錠剤、顆粒等に加工したり、ゼラチン等で被覆してカプセルに加工して健康食品や栄養補助食品等として利用できる。
本発明の食品組成物の「食品」は、食品全般を意味するが、いわゆる健康食品を含む一般食品の他、栄養補助食品、厚生労働省の保健機能食品制度に規定される特定保健用食品や栄養機能食品をも含むものである。
【００２３】
食品組成物における、本発明の冬虫夏草菌糸体等の配合量は、食品や組成物の種類や状態により一律に規定しがたいが、菌糸体の乾燥物あるいは粉末化したもの、あるいは抽出エキス固形物やその部分精製品として、通常約０．０１〜５０重量％、好ましくは０．１〜３０重量％である。配合量が０．０１重量％未満では経口摂取による効果が期待できず、５０重量％を超えると食品の種類によっては風味を損なったり、当該食品を調製できなくなる場合がある。
【００２４】
医薬組成物として、菌子体粉末やそのエキス、又は、エキスの部分精製品等を用いることができる。更に、食品組成物と同様に、デキストリン、乳糖、デンプン等の賦形剤や香料、色素等とともに、ペレット、錠剤、顆粒等に加工したり、ゼラチン等で被覆してカプセルに加工して利用することもできる。下記の通常、医薬組成物に用いられる担体と共に用いることができ、当業者公知の方法により以下に記載の医薬製剤の形態とすることができる。
【００２５】
医薬組成物の形態としては、例えば、錠剤、顆粒剤、細粒剤、散剤、カプセル剤、丸剤、液剤、乳剤、懸濁剤、シロップ剤、トローチ剤等の経口剤、注射剤、点眼剤、エアゾール剤、経皮吸収剤、坐剤等の非経口剤が挙げられる。
【００２６】
本発明の医薬組成物は、本発明の培養方法で得られた冬虫夏草菌糸体、及び／又は当該菌糸体より抽出されるエキスと、医薬として許容できる担体を含む。本明細書で使用する「医薬として許容できる担体」という用語は添加剤も含む。「医薬として許容できる担体」として、当業者公知のように、例えば、賦形剤（例えば、デンプン、コーンスターチ、ブドウ糖、果糖、ソルビトール、マンニトール、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、乳糖、ショ糖、ヒドロキシプロピルセルロース、炭酸マグネシウム、酸化マグネシウム、リン酸カルシウム等）、結合剤（例えば、アラビアゴム、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、ゼラチン、デキストリン、ヒドロキシプロピルセルロース、ポリビニルピロリドン、ポリエチレングリコール、デンプン、コーンスターチ、ショ糖等）、崩壊剤（例えば、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、デンプン、ヒドロキシプロピルセルロース等）、界面活性剤（例えば、ラウリル硫酸ナトリウム、大豆レシチン、ショ糖脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル等）、滑沢剤（例えば、ケイ酸マグネシウム、ステアリン酸カルシウム、ステアリン酸ケイ酸マグネシウム、タルク等）、希釈剤（例えば、水、食塩水、大豆油、ゴマ油、オリーブ油等のような植物油等）、軟膏基剤（例えば、パラフィン、ラノリン、白色ワセリン、ミツロウ等）、矯味剤（例えば、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピル等のようなパラオキシ安息香酸エステル類、安息香酸ナトリウム等）、等張化剤（例えば、塩化ナトリウム、グリセリン、ブドウ糖、マンニトール等）、保存剤（例えば、パラヒドロキシ安息香酸エステル等）、増粘剤（例えば、グアガム等）、酸化防止剤（例えば、ビタミンＥ等）等が挙げられるが、これらに限定されない。
【００２７】
注射剤の場合は、安定性の点から、バイアル等に充填後冷凍し、通常の凍結乾燥処理により水分を除き、使用直前に凍結乾燥物から液剤を再調製することもできる。
【００２８】
投与量は、患者の年齢、体重、疾患の程度により異なるが、経口の場合、通常成人１日あたり前記部分精製品を、１〜１０００ｍｇを１日１回から数回に分けて服用するのが適当である。非経口の場合、通常、成人で１日あたり前記部分精製品を０．５〜５００ｍｇを１日１回から数回に分けて静脈注射、皮下注射、筋肉注射するのが好ましい。
【００２９】
本発明の培養方法によって得られた冬虫夏草菌糸体、そのエキス、あるいは、エキスの部分精製品は、従来より用いられてきた生薬材料を原料とするもので、有効投与量での毒性は極めて低く、副作用はほとんど認められない。ヒト、ウシ、ウマ、イヌ、ネコ等の哺乳類に対して安全に投与することができる。家畜の飼料用途に適用する場合には、例えば冬虫夏草菌糸体、そのエキス、あるいは、エキスの部分精製品を、通常の飼料に添加して投与することができる。
【００３０】
【実施例】
以下、本発明を詳細に説明するために実施例を記載するが、本発明はこれらの実施例によって何ら限定されるものではない。
【００３１】
実施例１（サナギタケ菌糸体の培養方法）
割麦150 g、乾燥ビール酵母（キリンビール（株）製）30 gと水300 mlを混合し、121℃で15分間、高圧蒸気滅菌器にて殺菌してから室温になるまで放置し、その後無菌状態で、滅菌容器に培地を充填し、試験区とした。サナギタケの菌糸体の直径3 mmの寒天片（寒天培地（ポテトデキストロース）で前培養したコロニーよりコルクボーラーで3 mmΦの寒天片を打ち抜いたもの）を上記培地に接種し、24℃、湿度90％以上、21日間培養した。その間、1日に、400〜500 nmの波長域にピークを持つ青色光（ＮＥＣ製：カラー蛍光ランプ）を6時間照射し、6時間暗所に置くことを繰り返した。菌糸が覆った培地を割りほぐして、同様に光を間欠的に照射しながら、更に14〜21日培養を続け、本発明の菌糸培養物463 gを得た。対照区として、おが屑80 g、米糠64 g、サナギ粉36 gと水300 mlを混合した培地で、昼色光（白色蛍光管）を間欠的にあてて、同様に試験を行い、菌糸体培養物460 ｇを得た。得られた菌糸体（全量）より30％エタノールにて抽出し、エキス各々90 gおよび71 gを得た。得られたエキスを用いて、後述のように神経細胞の分化誘導活性、及び、骨髄性白血病細胞の増殖抑制作用を調べた。また、液体クロマトグラフィーにてコルジセピンを分析した。
【００３２】
神経細胞の分化誘導活性は、以下のように調べた。
1321N1アストロサイトーマ細胞はグリア細胞の一種であるアストロサイトが癌化したものであり、アストロサイトと同様に外部からの刺激により神経栄養因子を分泌する。また、神経細胞のモデル細胞であるPC12細胞は、ラット副腎髄質由来褐色細胞腫より樹立された細胞株（親クロム細胞腫細胞）であり、神経成長因子（NGF）に応答して神経突起を伸展し、神経細胞様に変化する。1321N1アストロサイトーマ細胞に関しては5（v/v）％牛胎児血清を含んだダルベッコ変法イーグル培地中で、37℃、5％二酸化炭素混有空気（水蒸気飽和）中でpH 7.2〜7.4で培養した。PC12細胞に関しては10（v/v）％牛胎児血清及び5（v/v）％馬血清を含んだダルベッコ変法イーグル培地中で、37℃、5％二酸化炭素混有空気（水蒸気飽和）中でpH 7.2〜7.4で培養した。
【００３３】
35 mmシャーレに細胞を約2万個ずつ1321N1アストロサイトーマ細胞を分注し、翌日細胞が容器に付着したことを確認し、前記抽出エキスを添加した。抽出エキスは、ジメチルスルホキシドに溶解し、ミリポアフィルター（0.2 μm）にてろ過滅菌後、最終濃度10 nMになるように添加した。分化誘導の陽対照には、phorbol 12-myristate 13-acetate（PMA）をジメチルスルホキシドに溶解させ、最終濃度100 nMとなるように添加した。2日後その培養上清を取り、あらかじめ培養しておいたPC-12細胞の培養用培地と交換した。PC-12細胞を培養上清で2日間培養後、位相差顕微鏡により形態観察を行い、その分化の程度を評価した。その評価方法は、個々の細胞について、全く変化していないものを0点、細胞体の直径と同程度の突起進展が見られるものを1点、細胞体の2-3倍の突起進展が見られるものを2点、非常に長い突起進展やシナプス形成が見られるものを3点とし、100個の細胞について得られた点数の平均値を細胞分化の指標とした。この結果を表1に示す。この結果から、本発明の方法により得られた冬虫夏草菌糸体が高い神経細胞の分化誘導活性を有していることがわかる。
【００３４】
【表１】
骨髄性白血病細胞に対する増殖抑制活性は、以下のように調べた。
ヒト白血病細胞HL-60は、前骨髄性白血病由来の癌細胞株（原ATCC株CCL-240、浮遊細胞）であり、好中球、マクロファージに分化できる能力を持ち、分化及びアポトーシス研究に多様される。この細胞をシャーレの中で静置培養し、前記抽出エキスを添加して、細胞に対する増殖抑制能について検討した。培養液はRPMI1640培地に10（v/v）％牛胎児血清を含み、37℃、5％二酸化炭素混有空気（水蒸気飽和）中でpH7.2〜7.4に保った。
【００３６】
24穴プレート（浮遊細胞用）を用い、8〜20万cells/mlになるように、1 ml/穴の細胞懸濁液を接種し、細胞培養2〜3日目に、希釈した試料を10 μl/穴で添加した。前記抽出エキスは、ジメチルスルホキシドに溶解・希釈し、ミリポアフィルター（0.2 μm）にてろ過滅菌後、HL-60細胞培養液に最終濃度が100 μg/mlになるように添加した。負対照には、希釈液ジメチルスルホキシド10 μl/穴を添加した。
【００３７】
培養1日後、細胞懸濁液15 μl/穴を取り、培地で10倍に希釈した。この細胞希釈液のATP活性をATPアナライザー（東亜電波工業製）で測定した。また、細胞形態を顕微鏡で観察した。細胞がつぶれて、培養液中に顆粒が散乱しているものを、細胞増殖抑制＋（有り）、細胞形態がきれいで変化しないものを、細胞増殖抑制−（無し）とした。
【００３８】
試料は、n=3穴でATP活性を測定し、平均と標準偏差を求めた。無添加（DMSOのみ添加）の平均値を100として、各試料の平均値と標準偏差値を算出し、T-検定法を用いて、無添加に対する有意差を求めた。p<0.05を有意差有りとした。結果を表2に示す。この結果から、本発明の方法により得られた冬虫夏草菌糸体が高い骨髄性白血病細胞の増殖抑制活性を有していることがわかる。
【００３９】
【表２】
また、抗菌活性を有するコルジセピンの含有量は、以下のように調べた。逆相系液体クロマトグラフィー（カラムTSK-GEL ODS-80TM、東ソー社製）を用いて前記抽出エキス成分を分離し、エキス中のコルジセピン含有量を測定した。検出にはUV波長268 nmを使用し、移動相には、10％メタノールを使用した。含有量を表3に示す。この結果から、本発明の方法により得られた冬虫夏草菌糸体はコルジセピンを高濃度に含有していることがわかる。
【００４１】
【表３】
実施例２（ウスキサナギタケ菌糸体の培養方法）
培地は、No.1（米糠90 g、コーンコブ粉砕物45 g、豆皮45 g）、No.2（おが屑80 g、米糠50 g、コーンコブ粉砕物20 g、サナギ粉30 g）、No.3（割麦70 g、おが屑80 g、サナギ粉30 g）、No.4（割麦120 g、おが屑30 g、サナギ粉30 g）、No.5（割麦150 g、サナギ粉30 g）、No.6（割麦150 g、煮干し30 g）、No.7（割麦140 g、酵母40 g）、No.8（割麦160 g、酵母エキス20 g）、No.9（蕎麦160 g、酵母エキス20 g）、No.10（割麦50 g、サナギ粉100 g、酵母30 g）を使用し、水300 mlを加えて培地を作成した。なお、酵母は乾燥ビール酵母（キリンビール製）を使用した。酵母エキスは乾燥酵母エキス（ナカライラスク（株）製）を使用した。昼色光、又は、400〜500 nmの波長域にピークを持つ青色光を実施例1と同様に間欠的に照射して実施例1と同様に培養を行い、菌糸体培養物を得た。得られた菌糸体（全量）より30％エタノールにてエキスを抽出した。得られたエキスを実施例1記載の方法で、神経細胞の分化誘導活性、骨髄性白血病細胞の増殖抑制活性の有無、及びコルジセピンの含有量を測定した。結果を表4に示す。この結果から、本発明の方法（青色光照射）により得られた冬虫夏草菌糸体は、昼色光を用いた場合に比べてコルジセピンを高濃度に含有していることがわかる。また、昼色光を用いた場合に比べ、本発明の方法を用いると、神経細胞分化誘導活性の指標値の増加がみられる。さらに、No.5、6、10では、昼色光を用いた場合には骨髄性白血病細胞増殖抑制活性が認められなかったのに対し、本発明の方法では当該抑制活性が認められた。
【００４３】
【表４】
実施例３（冬虫夏草株の培養）
実施例1記載（試験区）の本発明の方法で、異なる冬虫夏草株の菌糸体培養を行い、菌糸体培養物を得た。得られた菌糸体（全量）より30％エタノールにて抽出しエキスを得た。得られたエキスを実施例1記載の方法で、神経細胞の分化誘導活性、骨髄性白血病細胞の増殖抑制活性の有無、及びコルジセピンの含有量を測定した。結果を表5に示す。この結果から、本発明の方法により得られたこれらの冬虫夏草菌糸体はコルジセピンを高濃度に含有し、神経細胞の分化誘導活性及び骨髄性白血病細胞の増殖抑制活性を有していることがわかる。
【００４５】
【表５】
実施例４（抽出エキスを含む固形物の製造方法）
実施例1記載（試験区）の本発明の方法により、培養・抽出したエキスに、倍量の重量のコーンスターチを加え均一になるまで混合・練合する。この練合物を乾燥機にて60〜70℃で24時間乾燥する。乾燥物をミキサーにて粉砕して粉末とした。この製剤は医薬組成物又は食品組成物として利用できるものである。
【００４７】
実施例５（抽出エキスを含む固形物の製造方法）
実施例1記載（試験区）の本発明の方法により、培養・抽出したエキスを、デキストリン、グアガム等の混合物に噴霧して顆粒を形成させ、実施例4と同様に利用することができる。
【００４８】
実施例６（抽出エキスを含む液状物の製造方法）
実施例1記載（試験区）の本発明の方法により、培養・抽出したエキス150 mg、精製大豆油125 g、ミツロウ15 mg及びビタミンE10 mgを窒素ガス雰囲気下で約40℃に加温し、十分に混合して均質な液状物とした。これをカプセル充填機に供給して1粒内容量300 mgのゼラチンカプセル製剤を作製した。この製剤は、実施例4と同様に利用することができる。
【００４９】
【発明の効果】
本発明の冬虫夏草菌糸体の培養方法は、350〜550 nmの波長域にピークをもつ光を連続又は間欠的に照射しながら培養することで、薬理活性及び含有成分を有する菌糸体を効率的に、かつ、安定に生産することができる。また、これらの冬虫夏草培養品より抽出を行うことで、前記薬理活性画分及び／又は有効成分を効率よく回収することができる。前記薬理活性及び／又は有効成分を含む固形物及び液状物を調製することで、これらは、脳神経細胞の細胞死を伴う疾患、癌に対する予防及び治療、免疫増強等の医薬品用組成物及び食品用組成物を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for cultivating Cordyceps mycelium, an mycelium obtained by the method, an extract extracted from the mycelium, and a pharmaceutical composition or a food composition containing them.
[0002]
[Prior art]
Cordyceps has long been prized as an antidote for longevity and nourishment, and as a treatment for tuberculosis and jaundice. In recent years, the usefulness has been re-recognized by research on medicinal effects and active ingredients, and the demand for pharmaceuticals and foods is further increasing. For example, recently, anticancer activity, as shown in WO 96/00580 pamphlet (PCT / JP95 / 01298) (Patent Document 1), such as cardiotonic, antihypertensive, antitussive and antifatigue, A tonicity effect as shown in Japanese Patent No. 245340 (Patent Document 2) and a hypoglycemic action as shown in JP-A-10-245341 (Patent Document 3) have been reported. Further, Cordycepin, which has antibacterial action, and N, which has myocardial contraction-inhibiting action, are used as components of cordyceps. 6 -(2-hydroxyethyl) -adenosine (HEA) (Furuya T. et al., Phytochemistry, Vol. 22, No. 11, 1983, p. 2509) (Non-patent Document 1) and immunization Myriocin having an inhibitory action has been isolated and identified.
[0003]
However, cordyceps extract has a very large variation in pharmacological activity, and stable activity cannot be expected. In addition, it is very difficult to obtain a large amount of cordyceps itself from nature.
[0004]
Various studies have been made on the method for cultivating Cordyceps sinensis. For example, as shown in Japanese Patent Application Laid-Open No. 9-327232 (Patent Document 4), Japanese Patent Application Laid-Open No. 10-42691 (Patent Document 5) As shown in Japanese Patent Application Laid-Open No. 10-117770 (Patent Document 6), a method using a medium containing a cocoon cocoon component as a main component, A method for adding an extract of insects to a medium has been reported. However, all of these culture methods only resemble the nutrient composition of natural cordyceps and the fungus state is greatly influenced by environmental factors even if the same medium is used. It is not possible to obtain cordyceps having stable pharmacological activity and contained components.
[0005]
Furthermore, in JP-A-9-62 (Patent Document 7), a medium in which nutrient materials such as rice bran and soybean hulls are mixed with a medium base material such as sawdust and corn cob pulverized material is used. A method for cultivating Cordyceps sinensis (fruit bodies) by controlling temperature, humidity, etc. is described. Furthermore, in Japanese Patent Application Laid-Open No. 2001-269054 (Patent Document 8), yellow or red light is irradiated in the mycelia culture step, and blue or green light is irradiated in the fruit body generation step. A method for cultivating cordyceps to improve yield is described.
However, there have been no studies so far on the effects of light having a peak in a specific wavelength range on the pharmacological activity and contained components of the obtained mycelium culture during the cultivation of Cordyceps mycelium.
[0006]
[Patent Document 1]
WO96 / 00580 pamphlet
[Patent Document 2]
JP-A-10-245340
[Patent Document 3]
JP-A-10-245341
[Patent Document 4]
JP 9-327232 A
[Patent Document 5]
Japanese Patent Laid-Open No. 10-42691
[Patent Document 6]
Japanese Patent Laid-Open No. 10-117770
[Patent Document 7]
JP-A-9-62
[Patent Document 8]
JP 2001-269054 A
[Non-Patent Document 1]
Furuya T., Phytochemistry, Vol. 22, No. 11, 1983, p. 2509
[0007]
[Problems to be solved by the invention]
The present invention can efficiently obtain Cordyceps mycelium having stable pharmacological activity and contained components, a method for cultivating Cordyceps mycelium, a mycelium obtained by the method, an extract extracted from the mycelium, And it aims at providing the pharmaceutical composition or food composition containing them. Another object of the present invention is to provide a method for enhancing the pharmacological action of Cordyceps mycelium.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that Cordyceps is richer in pharmacological activity and active ingredient content in the mycelium than in the fruit body, such as Cordyceps spp. Since there are many cases, the mycelium has a shorter number of culture days and easier to adjust the culture conditions than the fruit body if the activity is not inferior. Furthermore, the inventors of the present invention, after the research, efficiently inoculated Cordyceps mycelium having stable pharmacological activity and contained components by continuously or intermittently irradiating the inoculated bacteria with light having a peak in the wavelength range of 350 to 550 nm. It has been found that it can be obtained well, and the Cordyceps mycelium obtained by this culturing method has been found to have increased pharmacological activity and / or content of active ingredients, and has thus completed the present invention.
[0009]
That is, the present invention is as follows.
(1) A method for cultivating Cordyceps mycorrhizal mycelium characterized by inoculating bacteria in a medium and continuously or intermittently irradiating light having a peak in a wavelength range of 350 to 550 nm,
(2) The culture method according to (1) above, wherein the medium contains cereals, or cereals and yeast or an extract thereof,
(3) The culture method according to (1) above, wherein Cordyceps belongs to the genus cordyceps,
(4) Cordyceps mycelium obtained by the culture method according to (1) above,
(5) An extract extracted from the cordyceps mycelium of (4) above,
(6) A pharmaceutical composition comprising Cordyceps mycelium obtained by the culture method according to (1) above, and / or an extract extracted from the mycelium, and a pharmaceutically acceptable carrier,
(7) A food composition comprising Cordyceps mycelium obtained by the culture method according to (1) above and / or an extract extracted from the mycelium,
(8) A method for enhancing the pharmacological action of Cordyceps mycelium, characterized by inoculating bacteria in a medium and continuously or intermittently irradiating light having a peak in a wavelength range of 350 to 550 nm, and
(9) The enhancing method according to the above (8), wherein the medium contains cereals, cereals and yeasts or extracts thereof.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
Cordyceps, which is the subject of the present invention, belongs to Ascomycota, Pyrenomycetes, Clavicipitales, and Ergotaceae (Clavicipitaceae or Hypocreaceae), and has complete and incomplete generations. It is a microorganism. For example, Cordyceps genus (for example, Sanagitake ( C.militaris ), Usukinasagitake ( C.takaomontana ) Etc.). Cordyceps is preferred because the mycelium has more pharmacological activity and / or content of active ingredients than fruiting bodies. These bacteria are distributed in Japan, Taiwan, China, Nepal, etc., and the occurrence time is from March to November. Infested with moth moths, larvae, etc., it ingests nutrients and proliferates, generating fruit bodies from insect carcasses.
[0011]
The method for cultivating Cordyceps mycelium according to the present invention is preferably cultivated in a medium containing cereals or cereals and yeast or an extract thereof. Cereals generally include crops that are human staple foods, and examples include rice, rice bran, straw, wheat (wheat, barley, pigeons, etc.), buckwheat, barnyard millet, millet, corn, amaranth, and beans. Examples of yeast include dry beer yeast, bread yeast, sake yeast, wine yeast and the like. Although yeast can be used as it is, yeast extract (yeast extract) extracted with water, hot water, an organic solvent or the like can also be used. You may add any one or more of animal powders, such as legumes, beans, such as a bean husk, okara, sanagi powder, fish meal, and dried pulverized material, to cereals, or cereals and yeast, or an extract thereof. In addition, a medium substrate such as sawdust and corn cob pulverized material may be used, and cereals, cereals and yeasts or extracts thereof may be added thereto, and the above-mentioned beans, animal powders, and the like may be added. By using cereals, it also serves as a support for mycelium growth and a nutrient source. Moreover, since yeast or its extract contains abundant amino acids and minerals, efficient growth can be obtained in a small amount. Moreover, cereals and yeasts are easily available and have a relatively stable medium composition.
[0012]
The blending amount of the cereal is usually 5.0 to 55.0% by weight, preferably 10.0 to 35.0% by weight, based on the total weight of the medium components (components that do not include water and the like described later). When using yeast, the compounding quantity is 1.0 to 10.0 weight% normally of the total weight of a culture medium component, Preferably it is 4.0 to 7.0 weight%. When using an extract of yeast, the compounding quantity is 0.01 to 3.0 weight% normally of the total weight of a culture medium component, Preferably it is 0.02 to 1.0 weight%.
Furthermore, when adding beans, animal powders, etc., the compounding quantity is 1.0-10.0 weight% normally of the total weight of a culture-medium component, Preferably it is 4.0-7.0 weight%. Moreover, when adding culture medium base materials, such as sawdust and a corn cob ground material, the compounding quantity is 5.0 to 50.0 weight% normally of the total weight of a culture medium component, Preferably it is 6.0 to 20.0 weight %.
Among the above-mentioned culture media, a culture medium composed of wheat (cracked wheat) and dry beer yeast is preferred, and a culture medium blended at a ratio of 140 to 160 g of wheat (brown wheat) and 20 to 40 g of dry beer yeast is more preferred. Specifically, for example, 150 g of wheat (cracked wheat): 30 g of dried beer yeast: 300 ml of water, 140 g of wheat (brown wheat) 140 g: 40 g of dried beer yeast: 300 ml of water, or 160 g of wheat (broken wheat): 20 g of dried beer yeast: A medium mixed with 300 ml of water is preferred.
The medium containing the above components can be prepared by a conventionally known method. For example, water is usually 50.0 to 85.0% by weight, preferably 60% with respect to the total weight of these medium components. It can be prepared by adjusting to 0.0 to 75.0% by weight and sterilizing with a high-pressure steam sterilizer.
[0013]
In the present invention, bacteria are inoculated in the culture medium exemplified above as culture conditions, and light having a peak in a wavelength region of 350 to 550 nm (mainly blue or green light) is continuously or intermittently irradiated. Thus, it is possible to efficiently and stably have pharmacological activity and contained components.
It is known that light effective for fruiting body generation is blue light, and as shown in the above-mentioned Japanese Patent Application Laid-Open No. 2001-269054 (Patent Document 8), In the fruit body generation process, the yield of fruit bodies is improved by using blue or green.
However, as a culture method having an efficient and stable pharmacological activity and components in the mycelium, the peak is in the wavelength range of 350 to 550 nm, preferably 350 to 530 nm, more preferably 400 to 500 nm, immediately after inoculation of the mycelium. It has been found that it is desirable to continuously or intermittently irradiate light having If the wavelength range is less than 350 nm, the tendency to inhibit the growth of mycelium increases, and if the wavelength range exceeds 550 nm, the pharmacological activity and the content components are reduced.
[0014]
The light having a peak in the wavelength range of 350 to 550 nm is the light having the strongest relative light emission intensity among the light in the visible light range among the light irradiated from the light source. It refers to light in the wavelength region of 550 nm. As a light source capable of irradiating light having a peak in a wavelength range of 350 to 550 nm, for example, in a fluorescent tube, there are blue, green, black light, etc. of a color fluorescent lamp, and in a light emitting diode, blue or green light is emitted. Although a diode is mentioned, it is not limited to these, As long as it is a light source with a peak in an equivalent range, all can be used.
The illuminance is usually 10 lux or more, preferably 1200 to 5000 lux. In the case of intermittently irradiating light, it is preferable that the irradiation time and the dark time are 1: 1. For example, if the irradiation time is 30 minutes, the dark place time is 30 minutes, and if the irradiation time is 12 hours, the dark place time is 12 hours.
[0015]
The inoculation of the bacterium is not particularly limited and can be performed by a conventionally known method. For example, the bacterium can be inoculated by inoculating a strain purely isolated from a wild bacterium into an agar medium or a liquid medium.
The culture temperature is usually 15 to 30 ° C., preferably 19 to 23 ° C., and the culture humidity is usually 60 to 95%, preferably 85 to 95%. The culture period is usually 1 to 2 months, preferably 4 to 6 weeks.
[0016]
Japanese bamboo shoot ( I. Japonica ) And the like form a fruiting body having an asexual spore, but the fruiting body often has a pharmacological activity and / or an active ingredient, and Oshima et al. A new terpene component has been found (Japanese Patent Application No. 2002-52140). On the other hand, Sanagitake ( C.militaris ), Usukinasagitake ( C.takaomontana The genus Cordyceps forms a fruiting body having ascospores in the ascomycete, but often has more pharmacological activity and / or active ingredient content in the mycelium than the fruiting body. If the activity is not inferior, the mycelium has the advantage that the culture days are shorter and the culture conditions are easier to adjust. Therefore, the inventors invented Cordyceps spp. Belonging to the genus Cordyceps etc., inoculating the medium with fungus, and continuously or intermittently irradiating light having a peak in the wavelength range of 350 to 550 nm, whereby pharmacological activity and / or We have developed a method for obtaining mycelium containing the components efficiently and stably.
[0017]
According to such a method of the invention, a sufficient amount of cordyceps mycelium can be obtained by culturing for about 1 to 2 months. In addition, the mycelium obtained has nerve cell differentiation-inducing activity and / or cancer cell proliferation inhibitory activity and / or anti-fatigue activity. Moreover, it has the effect that content of cordycepin which has antibacterial activity becomes high.
[0018]
Examples of Cordyceps mycelium (hereinafter also referred to as Cordyceps mycelium of the present invention) obtained by the above-described culture method of the present invention include, for example, the genus Cordyceps (for example, Sanagitake ( C.militaris ), Usukinasagitake ( C.takaomontana ) Etc.), and Cordyceps genus (for example, Sanagitake ( C.militaris ), Usukinasagitake ( C.takaomontana Etc.) is preferred.
The extract extracted from the cordyceps mycelium of the present invention can be obtained by extracting the cordyceps mycorium mycelium with an organic solvent (for example, 30% ethanol, etc.) or water by a conventional method.
[0019]
The Cordyceps mycelium of the present invention has nerve cell differentiation-inducing activity and / or cancer cell proliferation inhibitory activity and / or anti-fatigue activity. Moreover, it has the characteristics that content of the cordycepin which has antibacterial activity is high. For example, the content of cordycepin in 1 g of cordyceps mycelium of the present invention is at least 1 mg / g D.D. W. (Dry cell) or more, preferably 2 mg / g-D. W. ~ 20 mg / g-D. W. And more preferably 3 mg / g-D. W. ~ 12 mg / g-D. W. It is. When the content of cordycepin is too high, other activities (for example, anticancer activity) tend to be lost, and the above range is preferable from the overall balance.
By the culture method of the present invention, the pharmacological activity has the above three activities (neuronal cell differentiation-inducing activity, cancer cell growth inhibitory activity, and anti-fatigue activity) simultaneously, and the cordycepin content having antibacterial activity is as described above. It becomes possible to obtain mycelium in the range.
[0020]
Thus, according to the culture method of the present invention, it is possible to increase the pharmacological activity of the obtained Cordyceps mycelium and / or the content of the active ingredient. Differentiation-inducing action, cancer cell proliferation inhibitory action, anti-fatigue action, antibacterial action, cardiotonic, tonic tonic action, immune regulating action and enhancing action can be enhanced.
For example, the content of cordycepin having antibacterial activity is increased by at least 20% by weight or more per extract compared to when daylight is used using the same medium.
Here, daylight light in this specification refers to light emitted from a white fluorescent tube.
[0021]
As used herein, Cordyceps mycelium and / or extracts extracted from mycelium are dried or powdered Cordyceps mycelium, or extracted with, for example, an organic solvent or water. This refers to a product (partially purified product) obtained by purifying an extract and an extracted extract into a specific compound to any purity up to complete purification. Forms include water-soluble, liquids such as solids obtained by drying under reduced pressure, and lyophilized products, and solids. Cordyceps mycelium obtained by the method of the present invention, and an extract extracted from the mycelium, are used for diseases associated with cell death of brain neurons and / or immune enhancement and / or prevention of heart / respiratory diseases and / or cancer. And / or can be used as a pharmaceutical or food effective for therapeutic use, immune enhancement and recovery from fatigue, or as a pharmaceutical composition or food composition together with an appropriate carrier described below.
[0022]
For example, juice, soft drinks, tea, soup, soy milk, tofu, salad oil, dressing, yogurt, jelly, pudding, sprinkles, and the like are extracted from the Cordyceps mycelium and / or mycelium of the present invention as a food composition. It can also be contained in infant formula, cakes, breads, cookies, snacks and the like. (These are also included in the food composition herein). Alternatively, mycelium powder or its extract is processed into pellets, tablets, granules, etc. together with excipients such as dextrin, lactose, starch, corn starch, fragrances, pigments, etc., or coated with gelatin etc. to process into capsules It can be used as a health food or nutritional supplement.
“Food” in the food composition of the present invention means all foods, but in addition to general foods including so-called health foods, dietary supplements, foods for specified health use and nutrition prescribed in the Health Functional Food System of the Ministry of Health, Labor and Welfare Includes functional foods.
[0023]
The amount of Cordyceps mycelium of the present invention in the food composition is difficult to specify uniformly depending on the type and state of the food or composition, but the dried or powdered mycelium or the solid extract extract As a partially purified product thereof, it is usually about 0.01 to 50% by weight, preferably 0.1 to 30% by weight. If the blending amount is less than 0.01% by weight, the effect of oral ingestion cannot be expected, and if it exceeds 50% by weight, depending on the type of food, the flavor may be impaired or the food may not be prepared.
[0024]
As the pharmaceutical composition, mycelium powder, an extract thereof, a partially purified product of the extract, or the like can be used. Furthermore, as with food compositions, it is processed into pellets, tablets, granules, etc. together with excipients such as dextrin, lactose, starch, flavorings, pigments, etc., or coated with gelatin etc. and used as capsules You can also. Usually, it can be used with the carrier used for a pharmaceutical composition, and can be made into the form of the pharmaceutical preparation described below by a method known to those skilled in the art.
[0025]
Examples of the form of the pharmaceutical composition include tablets, granules, fine granules, powders, capsules, pills, liquids, emulsions, suspensions, syrups, lozenges, and other oral preparations, injections, and eye drops. And parenteral agents such as aerosols, transdermal absorption agents, and suppositories.
[0026]
The pharmaceutical composition of the present invention contains Cordyceps mycelium obtained by the culture method of the present invention, and / or an extract extracted from the mycelium, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes additives. As a “pharmaceutically acceptable carrier”, as known to those skilled in the art, for example, excipients (eg starch, corn starch, glucose, fructose, sorbitol, mannitol, carboxymethylcellulose, carboxymethylcellulose calcium, lactose, sucrose, hydroxypropyl Cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, etc.), binder (for example, gum arabic, carboxymethylcellulose, sodium carboxymethylcellulose, gelatin, dextrin, hydroxypropylcellulose, polyvinylpyrrolidone, polyethylene glycol, starch, corn starch, sucrose, etc.), Disintegrants (eg, carboxymethylcellulose, carboxymethylcellulose calcium, starch, hydroxypropyl cell) Etc.), surfactant (eg, sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester, etc.), lubricant (eg, magnesium silicate, calcium stearate, magnesium stearate silicate) , Talc, etc.), diluents (eg, vegetable oils such as water, saline, soybean oil, sesame oil, olive oil, etc.), ointment bases (eg, paraffin, lanolin, white petrolatum, beeswax, etc.), flavoring agents (eg, , Paraoxybenzoates such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate, etc.), isotonic agents (eg, sodium chloride, glycerin, glucose, mannitol, etc.), storage Agents (eg, parahydroxybenzoic acid esters) ), Thickeners (e.g., guar gum, etc.), antioxidants (e.g., vitamin E, etc.) and the like, without limitation.
[0027]
In the case of an injection, from the viewpoint of stability, it can be frozen after filling into a vial or the like, the water can be removed by a normal freeze-drying process, and the liquid can be re-prepared from the freeze-dried product immediately before use.
[0028]
The dose varies depending on the patient's age, weight, and degree of illness, but in the case of oral administration, the above-mentioned partially purified product is usually taken from 1 to 1000 mg once a day to 1 to several times a day. Is appropriate. In the case of parenteral, it is usually preferable for adults to inject 0.5 to 500 mg of the partially purified product per day into 1 to several times a day by intravenous injection, subcutaneous injection or intramuscular injection.
[0029]
Cordyceps fungus mycelium obtained by the culture method of the present invention, its extract, or a partially purified product of the extract is a raw material made from a herbal medicine that has been used conventionally, and its toxicity at an effective dose is extremely low, There are few side effects. It can be safely administered to mammals such as humans, cows, horses, dogs and cats. When applied to livestock feed applications, for example, Cordyceps mycelium, its extract, or a partially purified product of the extract can be added to a normal feed for administration.
[0030]
【Example】
EXAMPLES Hereinafter, examples will be described to describe the present invention in detail, but the present invention is not limited to these examples.
[0031]
Example 1 (Method for culturing Sanagitake mycelium)
150 g of wheat, 30 g of dry brewer's yeast (Kirin Brewery Co., Ltd.) and 300 ml of water are mixed, sterilized at 121 ° C for 15 minutes in a high-pressure steam sterilizer, and then left to reach room temperature. Under aseptic conditions, the culture medium was filled into a sterilized container to prepare a test group. An agar piece of diameter 3 mm of mycelium of sanagitake mushroom (inoculated with a cork borer from a colony pre-cultured with agar medium (potato dextrose)), inoculated into the above medium, 24 ° C, 90% humidity The culture was continued for 21 days. Meanwhile, on the 1st, blue light having a peak in a wavelength range of 400 to 500 nm (manufactured by NEC: color fluorescent lamp) was irradiated for 6 hours, and was repeatedly placed in a dark place for 6 hours. The medium covered with the mycelium was broken up, and the culture was further continued for 14 to 21 days while intermittently irradiating light similarly to obtain 463 g of the mycelial culture of the present invention. As a control group, in a medium mixed with 80 g of sawdust, 64 g of rice bran, 36 g of willow powder and 300 ml of water, a daylight light (white fluorescent tube) was applied intermittently, the same test was conducted, and the mycelium culture 460 g was obtained. Extraction was performed with 30% ethanol from the obtained mycelium (total amount) to obtain 90 g and 71 g of extract, respectively. Using the resulting extract, the neuronal differentiation inducing activity and the myelocytic leukemia cell proliferation inhibitory activity were examined as described below. Moreover, cordycepin was analyzed by liquid chromatography.
[0032]
The neuronal differentiation-inducing activity was examined as follows.
The 1321N1 astrocytoma cell is a cancerous type of astrocyte, a type of glial cell, and secretes neurotrophic factors by external stimuli in the same way as astrocytes. PC12 cells, which are model cells of neurons, are a cell line (parent chromocytoma cells) established from rat adrenal medulla pheochromocytoma, which extends neurites in response to nerve growth factor (NGF). However, it changes like a neuron. 1321N1 astrocytoma cells cultured in Dulbecco's modified Eagle's medium containing 5 (v / v)% fetal calf serum at 37 ° C in 5% carbon dioxide mixed air (water vapor saturation) at pH 7.2 to 7.4 did. For PC12 cells, in Dulbecco's modified Eagle's medium containing 10 (v / v)% fetal calf serum and 5 (v / v)% horse serum in 37 ° C, 5% carbon dioxide mixed air (water vapor saturation) And cultured at pH 7.2-7.4.
[0033]
1321N1 astrocytoma cells were dispensed in about 20,000 cells in a 35 mm dish and confirmed that the cells adhered to the container the next day, and the extract was added. The extract was dissolved in dimethyl sulfoxide, sterilized by filtration through a Millipore filter (0.2 μm), and added to a final concentration of 10 nM. As a positive control for differentiation induction, phorbol 12-myristate 13-acetate (PMA) was dissolved in dimethyl sulfoxide and added to a final concentration of 100 nM. Two days later, the culture supernatant was taken and replaced with a culture medium for PC-12 cells that had been cultured in advance. After culturing PC-12 cells in the culture supernatant for 2 days, the morphology was observed with a phase contrast microscope to evaluate the degree of differentiation. The evaluation method is as follows: 0 points for individual cells that have not changed at all, 1 point for protrusions that are comparable to the diameter of the cell body, and 2 to 3 times the protrusions of the cell body. 2 points, 3 points with very long protrusions and synapse formation, and the average of the scores obtained for 100 cells was used as an index of cell differentiation. The results are shown in Table 1. From these results, it can be seen that Cordyceps mycelium obtained by the method of the present invention has a high neuronal differentiation-inducing activity.
[0034]
[Table 1]
The growth inhibitory activity against myeloid leukemia cells was examined as follows.
Human leukemia cell line HL-60 is a promyelocytic leukemia-derived cancer cell line (original ATCC line CCL-240, floating cell) that has the ability to differentiate into neutrophils and macrophages and is widely used in differentiation and apoptosis studies. The The cells were statically cultured in a petri dish, and the extract was added to investigate the ability of the cells to inhibit growth. The culture solution contained 10 (v / v)% fetal calf serum in RPMI1640 medium, and maintained at pH 7.2 to 7.4 in 37 ° C. and 5% carbon dioxide mixed air (water vapor saturation).
[0036]
Using a 24-well plate (for floating cells), inoculate 1 ml / well of cell suspension at 80-200,000 cells / ml, and on the 2nd to 3rd day of cell culture, Added in μl / well. The extract was dissolved and diluted in dimethyl sulfoxide, sterilized by filtration through a Millipore filter (0.2 μm), and added to the HL-60 cell culture solution so that the final concentration was 100 μg / ml. As a negative control, 10 μl / well of diluent dimethyl sulfoxide was added.
[0037]
After 1 day of culture, 15 μl / well of cell suspension was taken and diluted 10-fold with medium. The ATP activity of this cell dilution was measured with an ATP analyzer (manufactured by Toa Denpa Kogyo). In addition, the cell morphology was observed with a microscope. The cells that collapsed and the granules were scattered in the culture medium were defined as cell growth inhibition + (present), and the cells whose morphology was clean and unchanged did not represent cell growth inhibition− (absent).
[0038]
Samples were measured for ATP activity in n = 3 wells, and the average and standard deviation were determined. The average value and standard deviation value of each sample were calculated with the average value of no addition (only DMSO added) being 100, and a significant difference with respect to no addition was determined using the T-test method. p <0.05 was considered significant. The results are shown in Table 2. From these results, it can be seen that Cordyceps mycelium obtained by the method of the present invention has high myelocytic leukemia cell growth inhibitory activity.
[0039]
[Table 2]
In addition, the content of cordycepin having antibacterial activity was examined as follows. The extract extract component was separated using reverse phase liquid chromatography (column TSK-GEL ODS-80TM, manufactured by Tosoh Corporation), and the content of cordycepin in the extract was measured. A UV wavelength of 268 nm was used for detection, and 10% methanol was used for the mobile phase. Table 3 shows the content. From this result, it is understood that Cordyceps mycelium obtained by the method of the present invention contains cordycepin at a high concentration.
[0041]
[Table 3]
Example 2 (Method for culturing myxomycetes mycelium)
Medium No.1 (90 g of rice bran, 45 g of ground corn cob, 45 g of bean hull), No.2 (80 g of sawdust, 50 g of rice bran, 20 g of ground corn cob, 30 g of willow powder), No.3 (70 g of cracked wheat, 80 g of sawdust, 30 g of banana flour), No.4 (120 g of cracked wheat, 30 g of sawdust, 30 g of banana flour), No.5 (150 g of cracked wheat, 30 g of banana flour) No.6 (150g cracked wheat, 30g boiled), No.7 (140g cracked wheat, 40g yeast), No.8 (160g cracked wheat, 20g yeast extract), No.9 (160 buckwheat) g, yeast extract 20 g), No. 10 (50% cracked wheat, 100 g banana flour, 30 g yeast), and 300 ml water was added to prepare a medium. In addition, dry brewer's yeast (made by Kirin Beer) was used for yeast. As the yeast extract, a dry yeast extract (manufactured by Nacala Iraq) was used. Daylight light or blue light having a peak in the wavelength range of 400 to 500 nm was intermittently irradiated in the same manner as in Example 1 and cultured in the same manner as in Example 1 to obtain a mycelium culture. Extracts were extracted from the obtained mycelium (total amount) with 30% ethanol. The obtained extract was measured for the differentiation-inducing activity of nerve cells, the presence or absence of growth-inhibiting activity of myeloid leukemia cells, and the content of cordycepin by the method described in Example 1. The results are shown in Table 4. From these results, it can be seen that Cordyceps mycelium obtained by the method of the present invention (irradiation with blue light) contains cordycepin at a higher concentration than when daylight light is used. In addition, when the method of the present invention is used, an increase in the index value of nerve cell differentiation-inducing activity is observed as compared with the case of using daylight light. Further, in Nos. 5, 6, and 10, when daylight light was used, myelocytic leukemia cell proliferation inhibitory activity was not observed, whereas in the method of the present invention, the inhibitory activity was observed.
[0043]
[Table 4]
Example 3 (Cultivation of Cordyceps strain)
By the method of the present invention described in Example 1 (test section), mycelium cultures of different Cordyceps strains were performed to obtain mycelium cultures. Extraction was obtained from the obtained mycelium (total amount) with 30% ethanol. The obtained extract was measured for the differentiation-inducing activity of nerve cells, the presence or absence of growth-inhibiting activity of myeloid leukemia cells, and the content of cordycepin by the method described in Example 1. The results are shown in Table 5. From these results, it can be seen that these Cordyceps mycelium obtained by the method of the present invention contain cordycepin at a high concentration, and have nerve cell differentiation-inducing activity and myeloid leukemia cell growth-inhibiting activity.
[0045]
[Table 5]
Example 4 (Method for producing a solid containing an extract)
According to the method of the present invention described in Example 1 (test group), corn starch having a double weight is added to the cultured and extracted extract and mixed and kneaded until uniform. This kneaded product is dried in a dryer at 60 to 70 ° C. for 24 hours. The dried product was pulverized with a mixer to obtain a powder. This preparation can be used as a pharmaceutical composition or a food composition.
[0047]
Example 5 (Method for producing a solid containing an extract)
According to the method of the present invention described in Example 1 (test section), the cultured / extracted extract is sprayed onto a mixture of dextrin, guar gum and the like to form granules, which can be used in the same manner as in Example 4.
[0048]
Example 6 (Method for producing liquid substance containing extract)
According to the method of the present invention described in Example 1 (test group), 150 mg of the cultured and extracted extract, 125 g of purified soybean oil, 15 mg of beeswax and 10 mg of vitamin E are heated to about 40 ° C. in a nitrogen gas atmosphere, Mix well to obtain a homogeneous liquid. This was supplied to a capsule filling machine to prepare a gelatin capsule preparation having an inner volume of 300 mg. This preparation can be used in the same manner as in Example 4.
[0049]
【The invention's effect】
The method for cultivating Cordyceps mycelium of the present invention efficiently cultivates mycelium having pharmacological activity and contained components by culturing while continuously or intermittently irradiating light having a peak in a wavelength range of 350 to 550 nm. And stable production. Moreover, the said pharmacologically active fraction and / or an active ingredient can be efficiently collect | recovered by extracting from these Cordyceps centennial culture products. By preparing solid substances and liquid substances containing the pharmacological activity and / or active ingredient, these can be used for pharmaceutical compositions and foods such as diseases accompanied by cell death of cranial nerve cells, prevention and treatment for cancer, immunity enhancement, etc. A composition can be provided.
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| CN104686208A (en) * | 2015-03-26 | 2015-06-10 | 北京美拉德中药饮片科技有限公司 | Usage and method for detection, processing and artificial cultivation of tussah cordyceps militaris taken as traditional Chinese medicinal materials |
| CN110547141A (en) * | 2019-09-30 | 2019-12-10 | 江苏康能生物工程股份有限公司 | Cultivation method for increasing polysaccharide content in cordyceps militaris sporocarp |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104686208A (en) * | 2015-03-26 | 2015-06-10 | 北京美拉德中药饮片科技有限公司 | Usage and method for detection, processing and artificial cultivation of tussah cordyceps militaris taken as traditional Chinese medicinal materials |
| CN110547141A (en) * | 2019-09-30 | 2019-12-10 | 江苏康能生物工程股份有限公司 | Cultivation method for increasing polysaccharide content in cordyceps militaris sporocarp |
| CN111226694A (en) * | 2020-03-20 | 2020-06-05 | 大连市现代农业生产发展服务中心 | Method for cultivating and obtaining cordyceps militaris sporocarp by taking tenebrio molitor larvae as raw materials |
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