JP4721128B2 - New Cordyceps strain - Google Patents
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- JP4721128B2 JP4721128B2 JP2005183162A JP2005183162A JP4721128B2 JP 4721128 B2 JP4721128 B2 JP 4721128B2 JP 2005183162 A JP2005183162 A JP 2005183162A JP 2005183162 A JP2005183162 A JP 2005183162A JP 4721128 B2 JP4721128 B2 JP 4721128B2
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Description
本発明は、ウスキサナギタケ（Ｃｏｒｄｙｃｅｐｓ ｔａｋａｏｍｏｎｔａｎａ）の菌株、ならびに当該菌株の培養物およびエキスに関する。 The present invention relates to strains of Cordyceps Takaomontana, and cultures and extracts of such strains.
従来、冬虫夏草と言えば、生薬の中国産コルジセプスシネシンス（Cordyceps sinensis）が最も有名である。その他にも、サナギタケ（コルジセプスミリタリス（Cordyceps militaris）やハナサナギタケ（Isaria japonica）が知られているが、冬虫夏草の種類は多く、世界中に約４００種が、また日本国内でも３００種以上が記録されている。最近の研究では、抗癌作用、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、特許文献２に示されるような強壮強精作用、また、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。化合物では、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（以後ＨＥＡと略す）（非特許文献１）および免疫抑制作用を有するミリオシン（Isaria sinclairii由来）等の成分も単離同定されている。 Traditionally speaking, Cordyceps sinensis, the herbal medicine, is most famous when it comes to cordyceps. In addition, sanagitake (Cordyceps militaris) and hanasanatake (Isaria japonica) are known, but there are many types of cordyceps, about 400 species all over the world, and over 300 species in Japan. In recent studies, anticancer activity, as shown in Patent Document 1, cardiotonic, antihypertensive, antitussive, and anti-fatigue effects, astonishing tonicity as shown in Patent Document 2, and patents Various medicinal effects have been reported in addition to the hypoglycemic action as shown in Reference 3. Among compounds, cordycepin having an antibacterial action, N 6- (2-hydroxyethyl) -adenosine having a myocardial contraction inhibitory action (hereinafter HEA) And other components such as myriocin (derived from Isaria sinclairii) having an immunosuppressive action have been isolated and identified.
しかしながら、採取物の薬理活性のばらつきは非常に大きく、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が非常に困難である。そこで、冬虫夏草の培養方法について、種々検討が行われている。薬理活性および含有成分の低下を防ぎ、大量に培養する方法としては、特許文献４に記載されているような培地中にさなぎ粉を含有する方法、特許文献５に記載されているような蚕の蛹組成成分を主成分とする方法、また、特許文献６に記載されているような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などがあげられる。しかしながら、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、培地組成を天然の冬虫夏草に類似させただけでは、薬理活性および含有成分を安定に維持することはできない。 However, the variation in pharmacological activity of the collected material is very large, and stable activity cannot be expected. Also, it is very difficult to obtain a large amount of cordyceps itself from nature. Accordingly, various studies have been conducted on the method for cultivating cordyceps. As a method for preventing a decrease in pharmacological activity and contained components and culturing in a large amount, a method of containing pupa flour in a medium as described in Patent Document 4, a method of rice cake as described in Patent Document 5 Examples thereof include a method comprising a cocoon composition component as a main component, and a method of adding an extract of an insect of the same species parasitic on cordyceps as described in Patent Document 6 to the medium. However, even if the same medium is used, the state of the fungus is greatly influenced by environmental factors. Therefore, the pharmacological activity and the contained components cannot be stably maintained only by making the medium composition similar to natural Cordyceps.
そこで、本発明者らは特許文献７に示すように、３５０〜５５０ｎｍの波長域にピークをもつ光を照射することで冬虫夏草の薬理活性および含有成分を安定に維持する方法を見出した。この方法は、多くの株で安定な培養方法ではあるが、株本来の特性が失われるものではない。従って、生理活性および含有成分を保持し、安定な株を選ぶことは、培養方法を更に有意なものにする。
本発明は、安定した人工培養ができ、有用な生理活性等を安定維持することが可能なウスキサナギタケ菌株およびその培養物やエキスを提供することを目的とする。 It is an object of the present invention to provide a Usxanagitake strain that can perform stable artificial culture and stably maintain useful physiological activity, and a culture or extract thereof.
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、冬虫夏草菌株を培養し評価することで、生理活性及び有効成分を有するエキス、および、それを安定に培養できるウスキサナギタケ菌株を見出し、本発明を完成させるに至った。 As a result of intensive studies to achieve the above-mentioned object, the present inventors have cultivated and evaluated Cordyceps fungus strains to extract extracts having physiological activity and active ingredients, and Usxanagitake strains that can be stably cultured. As a result, the present invention has been completed.
即ち、本発明は、以下の（１）〜（４）に関する。
（１）寄託番号がＦＥＲＭ Ｐ−１９５０１であることを特徴とするウスキサナギタケの菌株。
（２）上記（１）に記載の菌株の培養物。
（３）上記（１）に記載の菌株培養物由来のエキス。
（４）上記（１）に記載の菌株の培養物又は当該菌株培養物由来のエキスを含有してなる組成物。
That is, the present invention relates to the following (1) to (4).
(1) A strain of Usxanagitake, whose deposit number is FERM P-19501.
(2) A culture of the strain described in (1) above.
(3) An extract derived from the strain culture described in (1) above.
(4) A composition comprising a culture of the strain described in (1) above or an extract derived from the strain culture.
本発明のウスキサナギタケの新菌株ならびに当該菌株の培養物およびエキスは、優れた生理活性及び有効成分を有する。さらに本発明の菌株は安定に培養することが可能である。従って、本発明の菌株を培養した培養物やそれから抽出したエキスを調製することで、有用な生理活性および有効成分を安定、かつ、効率よく回収することができる。 The new strain of Usxanagitake and the culture and extract of the strain of the present invention have excellent physiological activity and active ingredients. Furthermore, the strain of the present invention can be stably cultured. Therefore, by preparing a culture obtained by culturing the strain of the present invention and an extract extracted therefrom, useful physiological activity and active ingredients can be recovered stably and efficiently.
以下、本発明を詳述する。
まず、本発明の対象となる冬虫夏草菌株は、子嚢菌類（Ascomycota）、核菌網（Pyrenomycetes）、麦角菌目（Clavicipitales）、麦角菌科（ClavicipitalesまたはHypocreacea）に属し、完全世代と不完全世代とを有する微生物である。本発明の菌株はコルジセプス（Cordyceps）属の一種であるウスキサナギタケ（C.takaomontana）に属する。コルジセプス属ウスキサナギタケは、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。蛾の蛹、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。
The present invention is described in detail below.
First, Cordyceps fungus strains that are the subject of the present invention belong to Ascomycota, Pyrenomycetes, Clavicipitales, Clavicipitales or Hypocreacea, complete generation and incomplete generation It is a microorganism having The strain of the present invention belongs to C. takaomontana which is a kind of the genus Cordyceps. Cordyceps spp. Is distributed in Japan, Taiwan, China, Nepal, etc., and the occurrence time is from March to November. Infested with moths, larvae, etc., ingested nutrients and proliferated, generating fruit bodies from insect corpses.
ウスキサナギタケは、自然界においては、蛾の蛹に寄生し、高さは１．５〜４ｃｍで淡黄色、棍棒型の子実体を生じる。淡いシトロンイエローであり、結実部は上半部に生じ、わずかにふくらみのある円筒形である。発生時期は７〜９月で、日本、台湾に分布するが、発生はサナギタケより少ない。（「原色冬虫夏草図鑑」、清水大典著、誠文堂新光社、１９９４年）。上記の形態学的特徴に基づいて、採取した菌株を分類後、純粋分離し、評価および培養検討を行った（発明者自ら採取、同定した株も含む）。 In the natural world, Ussusanagitake is parasitic on the cocoon of the cocoon, and has a height of 1.5 to 4 cm and produces a light yellow, stick-shaped fruit body. It is a light citron yellow, and the fruiting part occurs in the upper half, and is a slightly swelled cylindrical shape. Occurrence time is from July to September, and it is distributed in Japan and Taiwan. ("Primary color caterpillar picture book", Daisuke Shimizu, Seikodo Shinkosha, 1994). Based on the above morphological characteristics, the collected strains were classified, then purely isolated, evaluated and cultured (including the strains collected and identified by the inventors themselves).
まず、ウスキサナギタケを固形培養し、抗菌活性を持つコルジセピンを生産する株を見出した。 First, a strain that solid-cultures Usxanagitake and produces cordycepin with antibacterial activity was found.
コルジセピンは液体培養では生産されず、固体培養で生産される。培地は、特に限定されるものではないが、糸状菌によく用いられるポテトデキストロースや麦芽エキス寒天培地などがあげられる。またコルジセピンの含有量は、栄養繁殖の過程において菌糸体に淡黄色から濃橙色の着色を呈する菌株において、含有量が高いことがわかった。 Cordycepin is not produced in liquid culture but in solid culture. The medium is not particularly limited, and examples thereof include potato dextrose and malt extract agar medium often used for filamentous fungi. In addition, it was found that the content of cordycepin is high in strains that display light yellow to dark orange color on mycelium in the process of vegetative propagation.
本発明のウスキサナギタケ菌株の菌学的諸性質を以下に示す。
直径３ｍｍの円盤状種菌をシャーレに接種し、１６日間、白色光６時間照射下で生育状態を観察した。
（１）麦芽エキス寒天培地（２５℃培養）
初期、淡黄色でビロード状の菌糸。気菌糸有り。増殖につれて、中心部はやまぶき色から濃橙色で気菌糸増加。
（２）ポテトデキストロース寒天培地（２５℃培養）
境界線菌糸は白、中心部は淡黄色から濃橙色で気菌糸増加。
（３）ツアペック・ドックス寒天培地（２５℃培養）
境界線菌糸は白、中心部は淡黄色から濃橙色で気菌糸増加。
（４）サブロー培地（２５℃培養）
初期、淡黄色でビロード状の菌糸。気菌糸有り。増殖につれて、境界線菌糸は白、中心部は淡黄色から濃橙色で気菌糸増加。
（５）オートミール寒天培地（２５℃培養）
初期淡黄色のビロード状の菌糸。気菌糸少ない。増殖につれて境界線菌糸は白、中心部は淡黄色から濃橙色で気菌糸増加。
（６）最適生育温度
直径３ｍｍの円盤状種菌をシャーレに接種し、各温度で生育状態を観察した。その結果最適な生育温度は菌糸で２０〜２５℃、子実体で１６℃〜２０℃であった。また、５℃、３５℃ではほとんど生育しなかった。
（７）最適生育ｐＨ
麦芽エキス液体培地（寒天を含まない）５０ｍｌを３００ｍｌ三角フラスコに分注して殺菌し、酸またはアルカリで各ｐＨに調整後、前培養した種菌を接種して２５℃、１週間培養した後、菌体の生育状態を観察した。その結果、最適生育はｐＨ４〜８で差はみられなかった。また、生育可能なｐＨ範囲は３〜１０であった。
The mycological properties of the Usxanagitake strain of the present invention are shown below.
A petri dish having a diameter of 3 mm was inoculated into a petri dish, and the growth state was observed for 16 days under irradiation with white light for 6 hours.
(1) Malt extract agar medium (cultured at 25 ° C)
Early, pale yellow, velvety mycelium. There is aerial hyphae. As it grows, the center increases from a bright yellow to a deep orange color.
(2) Potato dextrose agar medium (25 ° C culture)
The boundary mycelium is white, and the center is light yellow to dark orange, increasing the aerial hyphae.
(3) Tuapeck Docs agar medium (cultured at 25 ° C)
The boundary mycelium is white, and the center is light yellow to dark orange, increasing the aerial hyphae.
(4) Sabouraud medium (cultured at 25 ° C)
Early, pale yellow, velvety mycelium. There is aerial hyphae. As it grows, the boundary mycelium is white and the center is light yellow to dark orange, increasing in aerial hyphae.
(5) Oatmeal agar medium (cultured at 25 ° C)
Early pale yellow velvety mycelium. Less aerial hyphae. As it grows, the mycelium of the border line is white, and the central part is light yellow to dark orange, increasing in aerial hyphae.
(6) Optimal growth temperature A discoid inoculum having a diameter of 3 mm was inoculated into a petri dish, and the growth state was observed at each temperature. As a result, the optimum growth temperature was 20 to 25 ° C. for mycelia and 16 to 20 ° C. for fruit bodies. Moreover, it hardly grew at 5 degreeC and 35 degreeC.
(7) Optimum growth pH
Disperse 50 ml of malt extract liquid medium (without agar) into 300 ml Erlenmeyer flask and sterilize it, adjust to each pH with acid or alkali, inoculate precultured inoculum and culture at 25 ° C for 1 week, The growth state of the cells was observed. As a result, optimum growth was not observed at pH 4-8. Moreover, the pH range which can be grown was 3-10.
本発明のウスキサナギタケ菌株は、上記の形態学的特徴に基いて採取、分類後、純粋分離したものである。すなわち、本発明のウスキサナギタケ菌株は、栄養繁殖の過程において菌糸体に淡黄色から濃橙色の着色を呈することを特徴としている。 The Ususanagitake strain of the present invention is purely isolated after being collected, classified based on the above morphological characteristics. That is, the usxanagitake strain of the present invention is characterized in that the mycelium is colored from pale yellow to deep orange in the process of vegetative propagation.
更に、これらウスキサナギタケの菌株の遺伝子レベルでの特定を行うため、種の同定に用いられるＩＴＳ領域（核リボソームＤＮＡの内部転写スペーサー領域をさす）の塩基配列を、以下のようにして調べた。 Furthermore, in order to identify these strains of Usxanagitake at the gene level, the base sequence of the ITS region (pointing to the internal transcription spacer region of nuclear ribosomal DNA) used for species identification was examined as follows.
ＤＮＡは、凍結乾燥子実体、または、集菌した凍結乾燥菌糸体を使用して、酵素ＰｒｏｔｅｉｎａｓｅＫとセチルトリメチルアンモニウムブロミド（ＣＴＡＢ）を使用して抽出をおこなった。次に、真菌類用の配列をもつＰｒｉｍｅｒを用いて、ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）法により、ＩＴＳ領域の増幅を行い、クローニングを行った。増幅には、シークエンス増幅機サーマルサイクラーを使用した。アガロース電気泳動により、増幅バンドを確認し、シークエンスサンプルとした。
シークエンス用反応液は、精製されたＩＴＳ領域増幅産物を用いて作成し、シークエンス反応を行った。反応させたサンプルは、エタノール沈殿法やスピンカラム法等で精製し、シークエンスサンプルとした。
シークエンサーにより塩基配列を決定した。
最後に、データベースを利用して、調べたＩＴＳ領域の塩基配列と相同性をもつ菌株の検索を行った。
DNA was extracted using freeze-dried fruit bodies or collected lyophilized mycelium using the enzymes Proteinase K and cetyltrimethylammonium bromide (CTAB). Next, using a primer having a fungal sequence, the ITS region was amplified and cloned by PCR (Polymerase Chain Reaction) method. For amplification, a sequence amplifier thermal cycler was used. The amplification band was confirmed by agarose electrophoresis, and used as a sequence sample.
A sequencing reaction solution was prepared using the purified ITS region amplification product and subjected to a sequencing reaction. The reacted sample was purified by an ethanol precipitation method, a spin column method, or the like, and used as a sequence sample.
The base sequence was determined with a sequencer.
Finally, using a database, strains having homology with the nucleotide sequence of the examined ITS region were searched.
以上のようにして、調べた塩基配列をデータベースにて検索した結果、データベース上の菌株とは、遺伝子の相同性が低く、本発明のウスキサナギタケは新株であることが判明した。 As described above, as a result of searching the examined nucleotide sequence in the database, it was found that the gene homology with the strains on the database is low, and the mussel from the present invention is a new strain.
したがって、本発明のウスキサナギタケ菌株は、染色体ＤＮＡ上の核リボゾームＲＮＡをコードする領域（核リボゾームＤＮＡ）のＩＴＳ領域に、配列番号１の塩基配列が含有されることを特徴とする。 Therefore, the Usxanagitake strain of the present invention is characterized in that the nucleotide sequence of SEQ ID NO: 1 is contained in the ITS region of the region (nuclear ribosomal DNA) encoding nuclear ribosomal RNA on the chromosomal DNA.
これらの株の中から、コルジセピン含量が高く、培養が安定しているＣｔ１−２株について下記に示す薬効評価を行った。その結果、ｉｎ ｖｉｔｒｏ系による癌細胞の増殖抑制作用、神経細胞の分化誘導活性および抗疲労活性を有することがわかった。 Among these strains, Ct1-2 strain having a high cordycepin content and stable culture was evaluated for the efficacy shown below. As a result, it was found that they have cancer cell proliferation inhibitory action, neuronal differentiation-inducing activity and anti-fatigue activity by the in vitro system.
本発明のウスキサナギタケＣｔ１−２菌株は、上記形態学的特徴と核リボゾームＤＮＡ中のＩＴＳ領域の塩基配列を指標に自然界から採取することが可能である。なお、本発明のウスキサナギタケＣｔ１−２菌株は、２００３年８月２７日に日本国茨城県つくば市東１丁目１番地１ 中央第６の独立行政法人産業技術総合研究所特許生物寄託センター（ＩＰＯＤ）に受託番号ＦＥＲＭ Ｐ−１９５０１として寄託されている。 The Usxanagitake Ct1-2 strain of the present invention can be collected from nature using the above morphological characteristics and the base sequence of the ITS region in the nuclear ribosomal DNA as an index. In addition, Usuxanagitake Ct1-2 strain of the present invention was established on August 27, 2003 at 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan. 6th National Institute of Advanced Industrial Science and Technology (IPOD) Is deposited under the accession number FERM P-19501.
本発明のウスキサナギタケ菌株は有用な生理活性及び有効成分を有する。
「有用な生理活性」としては、特に限定されないが、例えば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、抗菌活性、強心活性、強壮強精活性、免疫調整活性、免疫増強活性等が挙げられる。その中でも神経細胞の分化誘導活性、癌細胞増殖抑制活性、抗疲労活性及び抗菌活性が優れている。
ここで、「神経細胞の分化誘導活性」とは、未分化な神経細胞の機能的成熟を誘導する活性、又は分化した神経細胞の機能的亢進を誘導する活性をいう。
The Usxanagitake strain of the present invention has useful physiological activity and active ingredients.
“Useful physiological activity” is not particularly limited, but for example, nerve cell differentiation-inducing activity, cancer cell growth-inhibiting activity, anti-fatigue activity, antibacterial activity, cardiotonic activity, tonic tonic activity, immune modulating activity, immune enhancing activity Etc. Among them, nerve cell differentiation-inducing activity, cancer cell proliferation inhibitory activity, anti-fatigue activity and antibacterial activity are excellent.
Here, “neuronal cell differentiation-inducing activity” refers to an activity that induces functional maturation of undifferentiated neurons, or an activity that induces functional enhancement of differentiated neurons.
「有効成分」としては、特に限定されないが、例えば抗菌作用や抗癌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（以後ＨＥＡと略すことがある）、免疫抑制作用を有するミリオシン等が挙げられる。好ましくはコルジセピンである。 The “active ingredient” is not particularly limited. For example, cordycepin having an antibacterial action and an anticancer action, N 6- (2-hydroxyethyl) -adenosine having a myocardial contraction inhibitory action (hereinafter sometimes abbreviated as HEA), Examples include myriocin having an immunosuppressive action. Cordycepin is preferable.
本発明のウスキサナギタケ菌株は、上記有用な生理活性及び有効成分を安定して維持することが可能な菌株である。
「有用な生理活性及び有効成分を安定して維持することが可能」とは、本発明の菌株を当業者が用いる通常の培養条件で複数回数継代培養、又は長期間保存等しても当該菌株中の上記有用な生理活性及び有効成分の含有量が実質的に同等であることをいう。
The Usxanagitake strain of the present invention is a strain capable of stably maintaining the useful physiological activity and active ingredient.
“A useful physiological activity and active ingredient can be stably maintained” means that the strain of the present invention can be subcultured several times under ordinary culture conditions used by those skilled in the art or stored for a long period of time. It means that the content of the useful physiological activity and active ingredient in the strain is substantially equivalent.
「複数回数」とは、少なくとも２回以上、好ましくは５回以上、より好ましくは１０以上をいう。
「長期間」とは、少なくとも半年以上をいう。
「実質的に同等」とは、前記継代培養・保存後の当該生理活性等が前記継代培養・保存前と比較して少なくとも５０％以上、好ましくは６０％以上、より好ましくは７０％以上、更に好ましくは８０％以上、最も好ましくは９０％以上であることをいう。
“Multiple times” means at least 2 times, preferably 5 times or more, more preferably 10 times or more.
“Long term” means at least half a year.
“Substantially equivalent” means that the physiological activity after the subculture / storage is at least 50% or more, preferably 60% or more, more preferably 70% or more, compared to before the subculture / storage. More preferably, it means 80% or more, most preferably 90% or more.
以下、本発明で用いた生理活性および薬効評価の方法を示す。
まず、ウスキサナギタケ菌株の生理活性を評価するにあたり、以下の人工培養およびエキス抽出を行った。
ジャガイモ浸出液にグルコースを加えた液体培地で１０〜１４日間、静置培養し、得られた菌糸体を、遠心分離にて回収した。表面の培地成分を洗浄後、培養物を乾燥させた。乾燥培養物をエタノール水溶液に浸漬後、得られた抽出液を濃縮・乾燥後、以下の評価に使用した。
Hereinafter, the method of evaluating physiological activity and drug efficacy used in the present invention will be shown.
First, the following artificial culture and extract extraction were performed in evaluating the physiological activity of the Usxanagitake strain.
Static culture was performed for 10 to 14 days in a liquid medium in which glucose was added to potato exudate, and the mycelium obtained was collected by centrifugation. After washing the surface medium components, the culture was dried. After immersing the dried culture in an aqueous ethanol solution, the obtained extract was concentrated and dried, and then used for the following evaluation.
生理活性の評価は、抗菌活性を持つコルジセピンの含有量を中心に行い、ｉｎ ｖｉｔｒｏ系による癌細胞の増殖抑制作用および神経細胞の分化誘導活性、動物実験による抗疲労作用でも調べた。 The physiological activity was evaluated mainly by the content of cordycepin having antibacterial activity, and the in vitro system was also examined for cancer cell proliferation inhibitory activity, nerve cell differentiation-inducing activity, and anti-fatigue activity by animal experiments.
コルジセピンの含有量は、逆相系液体クロマトグラフィーを用いて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。 For the content of cordycepin, the extract component was separated using reverse phase liquid chromatography, and the content of cordycepin in the extract was measured.
また、ｉｎ ｖｉｔｒｏ系による癌細胞の増殖抑制作用は、ヒト白血病細胞を用いた。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、分化およびアポトーシス研究に多用される。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。 Further, human leukemia cells were used for the cancer cell growth inhibitory action by the in vitro system. Human leukemia cell HL-60 is a promyelocytic leukemia-derived cancer cell line (original ATCC line CCL-240, floating cell), has the ability to differentiate into neutrophils and macrophages, and is frequently used for 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.
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ＰＣ−１２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をシャーレに分注し、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。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。
The neuronal differentiation-inducing activity was examined as follows.
The 1321N1 astrocytoma cell is a cancerous type of astrocyte, which is a type of glial cell, and secretes neurotrophic factors by external stimuli in the same manner as astrocytes. PC-12 cells, which are model cells of nerve cells, are cell lines (parent chromocytoma cells) established from rat adrenal medulla-derived pheochromocytoma, and respond to nerve growth factor (NGF) to neurites. Extends and changes like a neuron. 1321N1 astrocytoma cells were dispensed into a petri dish, and the next day, it was confirmed that the cells adhered to the container, and the extract was added. 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, the morphology was observed with a phase contrast microscope to evaluate the degree of differentiation.
抗疲労活性は、マウスをロコモーターにて３時間運動させた後、前記抽出エキスを投与し、さらに９０分自由運動させた時の運動量を、投与しない対照群と比較することにより調べた。 The anti-fatigue activity was examined by comparing the amount of exercise when the mouse was exercised with a locomotor for 3 hours, then administered with the extract, and then allowed to move freely for 90 minutes with a control group that was not administered.
また、本発明のウスキサナギタケ菌株は、安定して人工培養することが可能な菌株である。ここで、「安定して人工培養することが可能」とは、本発明の菌株を当業者が用いる通常の培養条件で複数回数継代培養し、又は長期間保存等しても当該菌株の形質が変化せず、良好に増殖することをいう。 In addition, the Usxanagitake strain of the present invention is a strain that can be stably artificially cultured. Here, “stable artificial culture is possible” means that the strain of the present invention can be obtained even if it is subcultured several times under ordinary culture conditions used by those skilled in the art or stored for a long period of time. Means that it grows well without changing.
本発明のウスキサナギタケ菌株は、自体公知の培地を用いて培養することが可能であるが、穀類、又は穀類及び酵母若しくはその抽出物を含む培地が好ましい。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。 The Usxanagitake strain of the present invention can be cultured using a medium known per se, but a medium containing cereals or cereals and yeast or an extract thereof is preferred. Cereals generally include crops that are human staples, such as rice, rice bran, straw, wheat (wheat, barley, pigeons, etc.), buckwheat, barnyard millet, millet, corn, amaranth, beans and the like. 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, peas, such as okara, banana powder, fish meal, and dried pulverized material, to cereals, or cereals and yeast, or an extract thereof. Further, a medium substrate such as sawdust or pulverized corn cob 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 yeast are easily available and have a relatively stable medium composition.
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、又は麦（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。
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.
培養に適する湿度は、通常６０〜９５％、好ましくは８５〜９５％である。培養期間は、培地の種類、ｐＨ、培養温度、湿度等により適宜選択されるが、通常１〜２ヶ月、好ましくは４〜６週間培養することにより充分量の菌体を得ることができる。 The humidity suitable for culturing is usually 60 to 95%, preferably 85 to 95%. The culture period is appropriately selected depending on the type of medium, pH, culture temperature, humidity, and the like. Usually, a sufficient amount of cells can be obtained by culturing for 1 to 2 months, preferably 4 to 6 weeks.
本発明のウスキサナギタケ菌株は、上述の培地、温度、ｐＨ、湿度等の条件において安定した人工培養が可能であり、有用な生理活性等を安定して維持することが可能な菌株であるが、培地に当該菌を接種し、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、有用な生理活性及び有効成分を更に効率的且つ安定的に菌体中、好ましくは菌糸体中に含有させることが可能である。 The Usxanagitake strain of the present invention is a strain capable of stable artificial culture under the above-mentioned conditions of medium, temperature, pH, humidity and the like, and can stably maintain useful physiological activity, etc. Inoculate the medium with the bacteria and continuously or intermittently irradiate light with a peak in the wavelength range of 350 to 550 nm (mainly blue or green light) to further improve useful physiological activity and active ingredients. In addition, it can be stably contained in the mycelium, preferably in the mycelium.
即ち、菌糸を培地に接種した直後から、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。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。 That is, it is desirable to irradiate light having a peak in a wavelength region of 350 to 550 nm, preferably 350 to 530 nm, more preferably 400 to 500 nm, immediately or intermittently, immediately after inoculating the mycelium into the medium. If the wavelength range is less than 350 nm, the tendency to inhibit the growth of mycelium is increased.
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。 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.
照度は、白色蛍光管の場合、通常１０ｌｕｘ以上、好ましくは１２００〜５０００ｌｕｘである。光の照射は連続的であっても間欠的であってもよいが、好ましくは間欠的である。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の比は１：３０〜１０：３の範囲であればよく、好ましくは１：１５〜５：３の範囲、より好ましくは１：６〜２：３の範囲、更に好ましくは約１：３である。１回の照射時間は１０分〜２４時間であり、好ましくは１〜１２時間、より好ましくは３〜１０時間、更に好ましくは４〜８時間、最も好ましくは約６時間である。 In the case of a white fluorescent tube, the illuminance is usually 10 lux or more, preferably 1200 to 5000 lux. The light irradiation may be continuous or intermittent, but is preferably intermittent. In the case of intermittent light irradiation, the ratio of the irradiation time to the dark time may be in the range of 1:30 to 10: 3, preferably in the range of 1:15 to 5: 3. Preferably it is in the range of 1: 6 to 2: 3, more preferably about 1: 3. One irradiation time is 10 minutes to 24 hours, preferably 1 to 12 hours, more preferably 3 to 10 hours, still more preferably 4 to 8 hours, and most preferably about 6 hours.
本発明のウスキサナギタケ菌株は、スラント等の適切な状態で１〜７℃の温度、好ましくは２〜６℃、より好ましくは３〜５．５℃、もっとも好ましくは５℃にて長期間生存状態にて維持することが可能である。保存可能な期間は少なくとも１年以上である。
保存された当該菌株は、安定した人工培養ができ、有用な生理活性や有効成分を安定して維持できるという当該菌株特有の形質を良好に維持するために、定期的に継代することが好ましい。継代の頻度は少なくとも１０年以内に１回、好ましくは５年以内に１回、より好ましくは２年以内に１回、更に好ましくは６ヶ月以内に１回である。
The Usxanagitake strain of the present invention is in a long-term survival state at a temperature of 1 to 7 ° C, preferably 2 to 6 ° C, more preferably 3 to 5.5 ° C, and most preferably 5 ° C in an appropriate state such as a slant. It is possible to maintain at. The storable period is at least one year.
The preserved strain is preferably subcultured regularly in order to maintain stable characteristics unique to the strain such that stable artificial culture can be performed and useful physiological activities and active ingredients can be stably maintained. . The frequency of passage is at least once within 10 years, preferably once within 5 years, more preferably once within 2 years, and even more preferably once within 6 months.
また、本発明はウスキサナギタケ菌株の培養物に関する。
「培養物」には、ウスキサナギタケ菌株自体の子実体、菌糸体、胞子、およびそれらを培養する培地等が含まれる。本発明では、子実体培養物よりも菌糸体培養物の方が有用な生理活性及び／又は有効成分を豊富に含有する場合が多い。菌糸体の方が、培養日数が短い、菌糸体の方が培養条件を調節しやすい等の利点を有するからである。
当該「培養物」には、培養物の乾燥物、それを粉末化したもの等も含まれる。
The present invention also relates to a culture of the Usxanagitake strain.
“Culture” includes fruit bodies, mycelium, spores, and a culture medium for culturing them. In the present invention, mycelium cultures often contain more useful physiological activities and / or active ingredients than fruit body cultures. This is because the mycelium has advantages such as a shorter number of culture days, and the mycelium is easier to adjust the culture conditions.
The “culture” includes a dried product of the culture, a powdered product thereof, and the like.
更に本発明はウスキサナギタケ菌株培養物由来のエキスに関する。
「エキス」には、当該菌株の培養物の浸出液、その濃縮液、それらの乾燥物（乾燥エキス）に加え、それらの部分精製品（当該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）等が含まれる。形態としては、液状物、油状物、固形物等が含まれる。
The present invention further relates to an extract derived from a culture of Usxanagitake strain.
The “extract” includes a leachate of a culture of the strain, a concentrated solution thereof, and a dried product (dried extract) thereof, as well as a partially purified product thereof (arbitrary product obtained by completely purifying the extract into a specific compound). Purified to purity). Forms include liquids, oils, solids and the like.
当該エキスには、特に限定されないが、少なくともコルジセピンが含有されることが好ましく、より好ましくはコルジセピン、神経細胞の分化誘導活性、癌細胞増殖抑制活性、抗疲労活性及び抗菌活性が含有され、更に好ましくは上述の有用な生理活性及び有効成分の全てが含有される。 The extract is not particularly limited, but preferably contains at least cordycepin, more preferably cordycepin, nerve cell differentiation-inducing activity, cancer cell proliferation inhibitory activity, anti-fatigue activity and antibacterial activity, and more preferably Contains all of the above-mentioned useful physiological activities and active ingredients.
当該エキス中のコルジセピンの含有量は、エキスの調製方法等によって変動するため、一律に設定することは困難であるが、当該エキスの乾燥重量１ｇにつき、通常１〜２００ｍｇ、好ましくは１０〜１５０ｍｇの範囲である。 The content of cordycepin in the extract varies depending on the preparation method of the extract, and therefore it is difficult to set uniformly. However, the dry weight of the extract is usually 1 to 200 mg, preferably 10 to 150 mg. It is a range.
本発明のウスキサナギタケ新菌株培養物由来のエキスを調製する方法としては、自体公知の方法を用いることができる。例えば、日本薬局方（第１２改正）等に記載されている冷浸法、パーコレーション法等により、当該菌株の培養物を浸出用溶媒に浸すことにより、当該菌株の培養物の浸出液を得る。 A method known per se can be used as a method for preparing an extract derived from a culture of a new strain of Usxanagitake of the present invention. For example, by immersing a culture of the strain in a leaching solvent by a cold immersion method, a percolation method, or the like described in the Japanese Pharmacopoeia (12th revision), a leachate of the culture of the strain is obtained.
浸出用溶媒は、適宜選択することができるが、有機溶媒（例えばエタノール等のアルコール類等）、無機溶媒（例えば水、緩衝液等）あるいはそれらの混合溶媒が挙げられる。好ましくはエタノール、水（熱水を含む）又はエタノールと水の混合溶媒であり、より好ましくは水（熱水を含む）又はエタノールと水の混合溶媒である。
浸出用溶媒がエタノールと水の混合溶媒である場合のエタノール濃度は、適宜選択することができるが、通常５〜９０％の範囲、好ましくは１０〜７０％、より好ましくは１５〜５０％、更に好ましくは２０〜４０％である。
The leaching solvent can be appropriately selected, and examples thereof include organic solvents (for example, alcohols such as ethanol), inorganic solvents (for example, water, buffer solutions, and the like), and mixed solvents thereof. Preferred is ethanol, water (including hot water) or a mixed solvent of ethanol and water, and more preferred is water (including hot water) or a mixed solvent of ethanol and water.
The ethanol concentration when the leaching solvent is a mixed solvent of ethanol and water can be appropriately selected, but is usually in the range of 5 to 90%, preferably 10 to 70%, more preferably 15 to 50%, and further Preferably it is 20 to 40%.
浸出時の温度は、浸出用溶媒の種類等により適宜選択することができるが、例えば浸出にエタノールと水の混合溶媒を用いる場合は、浸出時の温度（浸出用溶媒の温度）として、通常５〜６０℃、好ましくは１０〜５０℃、より好ましくは１５〜４０℃、の範囲内である。
また、浸出用溶媒に水を用いる場合は、浸出時の温度は、通常１０〜１３０℃、好ましくは２０〜１２０℃、より好ましくは２０〜８０℃、の範囲内である。
浸出時の温度が低すぎると抽出効率が低下し、浸出時の温度が高すぎると浸出液中の有効成分等が分解されてしまう。
The temperature at the time of leaching can be appropriately selected depending on the type of the leaching solvent. For example, when a mixed solvent of ethanol and water is used for leaching, the leaching temperature (temperature of the leaching solvent) is usually 5 It is in the range of -60C, preferably 10-50C, more preferably 15-40C.
When water is used as the leaching solvent, the temperature during leaching is usually within the range of 10 to 130 ° C, preferably 20 to 120 ° C, more preferably 20 to 80 ° C.
If the temperature at the time of leaching is too low, the extraction efficiency is lowered, and if the temperature at the time of leaching is too high, the active ingredients in the leachate are decomposed.
浸出の時間は、浸出用溶媒の種類等により適宜選択することができるが、例えば浸出にエタノールと水の混合溶媒を用いる場合は、通常１〜１２０時間、好ましくは５〜９６時間、より好ましくは１５〜８０時間の範囲内である。
また、浸出用溶媒に水を用いる場合は、通常０．１〜１２０時間、好ましくは０．２〜９６時間、より好ましくは０．２５〜８０時間の範囲内である。
浸出時間が短すぎると有効成分等の回収効率が低くなり、浸出時間が長すぎると浸出液中の有効成分等が分解されてしまう。
Although the leaching time can be appropriately selected depending on the type of the leaching solvent, etc., for example, when a mixed solvent of ethanol and water is used for leaching, usually 1 to 120 hours, preferably 5 to 96 hours, more preferably Within 15-80 hours.
Further, when water is used as the leaching solvent, it is usually in the range of 0.1 to 120 hours, preferably 0.2 to 96 hours, more preferably 0.25 to 80 hours.
If the leaching time is too short, the recovery efficiency of the active ingredients and the like is lowered, and if the leaching time is too long, the active ingredients and the like in the leachate are decomposed.
また、本発明は、ウスキサナギタケ菌株の培養物あるいは当該菌株培養物由来のエキスを含有してなる組成物に関する。
当該組成物には、特に限定されないが、例えば、医薬組成物、食品組成物、化粧用組成物等が挙げられる。
The present invention also relates to a composition comprising a culture of Usxanagitake strain or an extract derived from the strain culture.
Although it does not specifically limit to the said composition, For example, a pharmaceutical composition, a food composition, a cosmetic composition etc. are mentioned.
組成物が医薬組成物である場合には、当該医薬組成物には、本発明のウスキサナギタケ菌株の培養物又は当該菌株培養物由来のエキス以外に、医薬として許容できる担体を配合することができる。「医薬として許容できる担体」としては、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。 When the composition is a pharmaceutical composition, the pharmaceutical composition can contain a pharmaceutically acceptable carrier in addition to the culture of the Usxanagitake strain of the present invention or an extract derived from the strain culture. . “Pharmaceutically acceptable carrier” includes, for example, excipients (eg, starch, corn starch, glucose, fructose, sorbitol, mannitol, carboxymethylcellulose, carboxymethylcellulose calcium, lactose, sucrose, hydroxy, as known in the art. Propyl cellulose, magnesium carbonate, magnesium oxide, calcium phosphate, etc.), binder (eg, gum arabic, carboxymethyl cellulose, sodium carboxymethyl cellulose, gelatin, dextrin, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, starch, corn starch, sucrose, etc.) Disintegrating agents (eg, carboxymethylcellulose, carboxymethylcellulose calcium, starch, hydroxypropyl cellulose) Loin), surfactants (eg, sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester, etc.), lubricants (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 (for example, sodium chloride, glycerin, glucose, mannitol, etc.), preservatives (For example, parahydroxybenzoic acid ester Etc.), thickeners (e.g., guar gum, etc.), antioxidants (e.g., vitamin E, etc.) and the like, without limitation.
医薬組成物の剤形としては、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、湯剤、膏剤、酒剤、チンキ剤等の経口剤、注射剤、点眼剤、エアゾール剤、経皮吸収剤、坐剤、膏剤、丹剤等の非経口剤が挙げられるが、これらに限定されない。 Examples of the dosage form of the pharmaceutical composition include tablets, granules, fine granules, powders, capsules, pills, solutions, emulsions, suspensions, syrups, troches, hot water, salves, liquors. , Oral preparations such as tinctures, parenteral preparations such as injections, eye drops, aerosols, transdermal absorption agents, suppositories, salves, and tans, but are not limited thereto.
医薬組成物の投与量は、投与対象、患者の年齢、体重、疾患の程度により異なるが、例えば経口投与の場合、通常成人１日あたり培養物の乾燥重量に換算して、１〜１０００ｍｇ相当を１日１回から数回に分けて服用するのが適当である。非経口投与の場合、通常、成人で１日あたり培養物の乾燥重量に換算して、０．５〜５００ｍｇ相当を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。 The dose of the pharmaceutical composition varies depending on the subject of administration, the age, body weight, and degree of disease of the patient. For example, in the case of oral administration, it is usually equivalent to 1-1000 mg in terms of the dry weight of the culture per day for an adult. It is appropriate to take it once to several times a day. In the case of parenteral administration, the equivalent of 0.5 to 500 mg is usually divided into one to several times a day by intravenous, subcutaneous, or intramuscular injection in terms of the dry weight of the culture per day for adults. Is preferred.
本発明の本発明のウスキサナギタケ菌株の培養物又は当該菌株培養物由来のエキスは、従来用いられてきた生薬材料を原料とするものであるので、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。 Since the culture or the extract derived from the strain culture of the present invention of the present invention is based on a herbal material conventionally used, its toxicity at an effective dose is extremely low, and side effects are Almost not recognized. It can be safely administered to mammals such as humans, cows, horses, dogs and cats.
また、組成物が食品組成物である場合は、当該食品組成物としては、本発明のウスキサナギタケ菌株の培養物又は当該菌株培養物由来のエキスを、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、スナック菓子等の一般の食品に含有させたものが挙げられる。あるいは、当該培養物又はそのエキスを、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たりして健康食品や栄養補助食品等として利用できる。その他「食品」としては、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、家畜の飼料等が挙げられるが、これらに限定されない。 Further, when the composition is a food composition, the food composition includes, for example, a culture of the Usxanagitake strain of the present invention or an extract derived from the strain culture, such as juice, soft drink, tea, soup, Examples include soy milk, tofu, salad oil, dressing, yogurt, jelly, pudding, sprinkles, infant formula, cakes, bread, cookies, snacks, and other general foods. Alternatively, the culture or extract thereof can be 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 dietary supplement. Examples of other “food” include, but are not limited to, foods for specified health use, functional nutritional foods, livestock feed, and the like as defined in the Health Functional Food System of the Ministry of Health, Labor and Welfare.
上記食品組成物における、本発明のウスキサナギタケ菌株の培養物又は当該菌株培養物由来のエキスの配合量は、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、乾燥培養物の乾燥重量として、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。 In the above food composition, the amount of the extract of the Usxanagitake strain of the present invention or the extract derived from the strain culture is difficult to define uniformly depending on the type and state of the food or composition, but the dried culture is dried. The weight 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.
本発明のウスキサナギタケ菌株、当該菌株の培養物または当該菌株培養物由来のエキスを含有してなる組成物は、脳神経細胞の細胞死を伴う疾患（例えば、虚血性脳疾患、アルツハイマー病等の痴呆症、パーキンソン病、筋萎縮性側索硬化症等）、腫瘍（例えば骨髄性白血病等）、感染症、心臓・呼吸器系疾患（例えば心筋梗塞、狭心症、高血圧等）等の疾患の予防・治療や、免疫増強、強壮強精、疲労回復に用いることができる。 A composition comprising the Usxanagitake strain of the present invention, a culture of the strain or an extract derived from the strain culture is used for diseases associated with cell death of cranial nerve cells (for example, dementia such as ischemic brain disease and Alzheimer's disease). Disease, Parkinson's disease, amyotrophic lateral sclerosis, etc., tumors (eg myeloid leukemia, etc.), infections, heart / respiratory diseases (eg myocardial infarction, angina, hypertension, etc.)・ It can be used for treatment, immune enhancement, tonic, and fatigue recovery.
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、本発明はこれら実施例によって何ら限定されるものではない。 EXAMPLES Hereinafter, examples will be described to describe the present invention in detail, but the present invention is not limited to these examples.
〔実施例１〕
（菌糸体の培養およびエキス抽出）
表１に示すそれぞれの菌株を、ジャガイモ３５０ｇの浸出液にグルコース２０ｇを加えて蒸留水で１Ｌとした液体培地で、２８〜３５日間静置培養した。培養条件は、２５℃、湿度９０％以上、白色光を６時間／日照射して行った。得られた菌糸体を、遠心分離にて回収した。表面の培地成分を洗浄後、培養物を乾燥させた。乾燥培養物を３０％エタノール水溶液に浸漬後、得られた抽出液を濃縮・乾固してエキスを得た。
ついで抗菌活性を有するコルジセピンの含有量を、以下のように調べた。逆相系液体クロマトグラフィーを用いてエキス成分を分離した。検出にはＵＶ波長２６８ｎｍを使用し、移動相には、１０％メタノールを使用した。菌糸の色とコルジセピン含有量を表１に示す。比較としてサナギタケを同様に培養・エキス抽出を行った。
[Example 1]
(Mycelium culture and extract extraction)
Each strain shown in Table 1 was statically cultured for 28 to 35 days in a liquid medium made by adding 20 g of glucose to a leachate of 350 g of potato and making it 1 L with distilled water. The culture conditions were 25 ° C., 90% humidity and irradiation with white light for 6 hours / day. The obtained mycelium was collected by centrifugation. After washing the surface medium components, the culture was dried. After the dried culture was immersed in a 30% aqueous ethanol solution, the resulting extract was concentrated and dried to obtain an extract.
Subsequently, the content of cordycepin having antibacterial activity was examined as follows. The extract component was separated using reverse phase liquid chromatography. A UV wavelength of 268 nm was used for detection, and 10% methanol was used for the mobile phase. Table 1 shows the color of mycelium and the content of cordycepin. As a comparison, sanagitake was similarly cultured and extracted.
〔実施例２〕
（菌糸体の培養）
割麦１５０ｇ、乾燥ビール酵母３０ｇと水３００ｍｌ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た。ウスキサナギタケ株の菌糸を接種し、２５℃、湿度９０％以上、２１日間培養する。その間、１日に、４００〜５００ｎｍの波長域にピークを持つ青色光を６時間照射し、１８時間暗所に置くことを繰返した。菌糸が覆った培地を割りほぐして、同様に光を照射しながら、更に１４日培養を続け、菌糸培養物を得た。
培養物より、実施例１記載の方法で、エキス抽出を行い、得られたエキスを前記方法にて、評価した。結果を表２に示す。
[Example 2]
(Culture of mycelium)
150 g of wheat, 30 g of dry brewer's yeast and 300 ml of water are mixed, sterilized at 121 ° C. for 15 minutes in a high-pressure steam sterilizer and left to reach room temperature, and then filled in a sterile container in a sterile condition. . Inoculate with mycelia of Usxanagitake strain and culture for 21 days at 25 ° C. and humidity of 90% or more. In the meantime, the blue light having a peak in the wavelength range of 400 to 500 nm was irradiated for 6 hours and placed in a dark place for 18 hours. The medium covered with the mycelium was broken up, and the culture was further continued for 14 days while similarly irradiating light to obtain a mycelial culture.
Extracts were extracted from the cultures by the method described in Example 1, and the obtained extracts were evaluated by the above methods. The results are shown in Table 2.
〔実施例３〕
（菌糸体の培養）
蕎麦１６０ｇ、酵母エキス２０ｇと水３００ｍｌ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た。培養条件は実施例２記載の方法で同様に行った。培養物より、実施例１記載の方法で、エキス抽出を行い、得られたエキスを実施例１記載の方法にて、評価した。結果を表３に示す。
Example 3
(Culture of mycelium)
160 g of buckwheat, 20 g of yeast extract, and 300 ml of water were mixed, sterilized at 121 ° C. for 15 minutes in a high-pressure steam sterilizer, and allowed to reach room temperature, and then the sterilized container was filled with the medium in an aseptic condition. The culture conditions were the same as in Example 2. The extract was extracted from the culture by the method described in Example 1, and the obtained extract was evaluated by the method described in Example 1. The results are shown in Table 3.
〔実施例４〕
（ＩＴＳ領域遺伝子配列）
ウスキサナギタケ１−７株のＩＴＳ領域塩基配列を以下の方法で調べた。
（ＤＮＡの抽出）
酵母エキス０．２％、麦芽エキス２％、および、グルコース２％の液体培地を用いて、Ｃｔ１−２を接種し、２１日間、２５℃にて静置培養を行った。
得られた菌糸体をＬｙｓｉｓ ｂｕｆｆｅｒ ５ｍｇ／ｍｌに懸濁し、４℃で３０分間静置して、１０％ＳＤＳを１／１０量加え軽く懸濁し、６５℃で３０分静置し、さらに氷中に１５分間静置した。この溶液にＰｒｏｔｅｉｎａｓｅＫ（プロメガ社製、特異的活性３０単位／ｍｇ以上）を１ｍｇ加え、３７℃で４時間以上静置した。次に、７０００ｒｐｍにて、４０℃で２０分間遠心分離し、上清を別の遠心管にとり、５Ｍ ＮａＣｌを１／５量加え、さらに１０％ＣＴＡＢを１／１０量加えて、６５℃で１０分間静置した。同量のクロロホルム／イソアミルアルコール（２４：１）を加え、７０００ｒｐｍにて、４℃で１０分間遠心分離し、上清を別の遠心管に移し、これを３回繰り返した。上清に対して、７．５Ｍ酢酸アンモニウムを１／１０量加え、さらに冷エタノールを２．５倍量加え、−２０℃で２時間以上静置した。
これを７０００ｒｐｍにて、４℃で２０分間遠心分離し、得られた沈殿を７０％冷エタノールでリンスし、更に７０００ｒｐｍにて、４℃で１０分間遠心分離で沈殿物を得た。沈殿物を風乾後、ＴＥ ｂｕｆｆｅｒに懸濁し、ＲＮａｓｅ １ｍｇを加えて３７℃で２時間以上静置した。同量のクロロホルム／イソアミルアルコール（２４：１）を加え、１００００ｒｐｍ、４℃、１０分間遠心分離し、上清を別の遠心管に移し、これを３回繰り返した。上清に対して、７．５Ｍ酢酸アンモニウムを１／１０量加え、さらに冷エタノールを２．５倍量加え、−２０℃で２時間以上静置した。これを１００００ｒｐｍにて、４℃で２０分間遠心分離し、得られた沈殿を７０％冷エタノールでリンスし、更に１００００ｒｐｍ、４℃で１０分間遠心分離して沈殿物を得た。沈殿物を風乾後、ＴＥ ｂｕｆｆｅｒに懸濁し、以下の実験に使用した。
（ＰＣＲ増幅）
ＩＴＳ領域の増幅は、ＰＣＲ増幅機サーマルサイクラーを使用した。Ｐｒｉｍｅｒは、真菌類特有のＩＴＳ−１（５’−ＴＣＣＧＴＡＧＧＴＧＡＡＣＣＴＧＣＧＧ−３’）（配列番号２）およびＩＴＳ−４（５’−ＴＣＣＴＣＣＧＣＴＴＡＴＴＧＡＴＡＴＧＣ−３’）（配列番号３）の２種類を使用した。
ＰＣＲ用反応溶液は、Ｐｒｉｍｅｒ各１０ｐｍｏｌ、抽出したゲノムＤＮＡ１０ｎｇ、ｄＮＴＰｓ（タカラバイオ製）各２０ｎｍｏｌ、Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ製）２．５Ｕ、ＰＣＲ ｂｕｆｆｅｒ（タカラバイオ製）を加え、全量２５μｌとした。ＰＣＲ条件は、９２℃、５分間の熱処理の後、９２℃、３０秒間、６０℃、３０秒間、７２℃、１分間の反応を３０回繰り返し、最後に７２℃、１０分間の処理を行った。
（増幅産物の確認）
アガロース電気泳動は、５０Ｖ、１時間、ゲル濃度１．５％（Ｓｉｇｍａ製、Ｔｙｐｅ−ＩＩ−Ａ）で行った。染色は、エチジウムブロマイドを使用し、紫外線を照射して増幅バンドを確認した。
（ＩＴＳ領域増幅産物の精製）
ＩＴＳ領域増幅産物の精製は、ＳＵＰＲＥＣ−０２（タカラバイオ製）を使用してＰｒｉｍｅｒの除去および精製・濃縮を行い、シークエンスサンプルとした。
（シークエンス反応）
シークエンス用反応液は、精製されたＩＴＳ領域増幅産物をＤｙｅ−ｔｅｒｍｉｎａｔｅｒ Ｋｉｔ（Ａｐｐｌｉｅｄ ｂｉｏｓｙｓｔｅｍｓ製）を使用して作成した。ＰＣＲ反応は、９６℃、７分間の熱処理の後、９６℃、３０秒間、５０℃、１５秒間、６０℃、４分間の反応を２５回繰り返し、最後に４℃の処理を行った。
反応させたサンプルは、エタノール沈殿法やスピンカラム法等で精製し、シークエンスサンプルとした。反応液２０μｌに７．５Ｍ酢酸アンモニウム２μｌ、９５％エタノール５０μｌを加え、室温にて１５分放置した。この溶液を１５０００ｒｐｍ、室温で２０分間遠心分離後、上清を除去した。７０％エタノール２００μｌを加え、軽く攪拌後、１５０００ｒｐｍ、室温で５分間遠心分離して、上清を除去した。沈殿物を乾燥後、−２０℃で保存した。
ＡＢＩ Ｐｒｉｓｍ Ｍｏｄｅｌ ３１０シークエンサー（Ａｐｐｌｉｅｄ ｂｉｏｓｙｓｔｅｍｓ製）を利用し、塩基配列を調べた。
（塩基配列相同性の確認）
データベース検索は、Ｗｅｂサイトにおいて、米国立医学図書館生物工学情報センター（ＮＣＢＩ）データベースＢＬＡＳＴ（ｈｔｔｐ：／／ｂｌａｓｔ．ｇｅｎｏｍｅ．ａｄ．ｊｐ／）を利用して、調べたサンプルのＩＴＳ領域の塩基配列と相同性をもつ菌株の検索を行った。
ウスキサナギタケ１−７株のＩＴＳ領域は、同じ塩基配列（配列番号１）を示した。ＢＬＡＳＴでの遺伝子配列相同性検索結果を表４に、菌株系統樹を図１に示す。
Example 4
(ITS region gene sequence)
The ITS region nucleotide sequence of Usxanagitake 1-7 strain was examined by the following method.
(DNA extraction)
Ct1-2 was inoculated using a liquid medium of yeast extract 0.2%, malt extract 2%, and glucose 2%, and static culture was performed at 25 ° C. for 21 days.
The obtained mycelium was suspended in Lysis buffer 5 mg / ml, allowed to stand at 4 ° C. for 30 minutes, 1/10 volume of 10% SDS was added and suspended briefly, then allowed to stand at 65 ° C. for 30 minutes, and further in ice For 15 minutes. 1 mg of Proteinase K (manufactured by Promega, specific activity 30 units / mg or more) was added to this solution, and the mixture was allowed to stand at 37 ° C. for 4 hours or more. Next, the mixture is centrifuged at 7000 rpm for 20 minutes at 40 ° C., the supernatant is put in another centrifuge tube, 1/5 amount of 5M NaCl is added, and 1/10 amount of 10% CTAB is further added. Let stand for a minute. The same amount of chloroform / isoamyl alcohol (24: 1) was added, the mixture was centrifuged at 7000 rpm at 4 ° C. for 10 minutes, the supernatant was transferred to another centrifuge tube, and this was repeated three times. To the supernatant, 1/10 amount of 7.5M ammonium acetate was added, 2.5 times the amount of cold ethanol was further added, and the mixture was allowed to stand at −20 ° C. for 2 hours or more.
This was centrifuged at 7000 rpm at 4 ° C. for 20 minutes, and the resulting precipitate was rinsed with 70% cold ethanol, and further centrifuged at 7000 rpm at 4 ° C. for 10 minutes to obtain a precipitate. The precipitate was air-dried, suspended in TE buffer, 1 mg of RNase was added, and the mixture was allowed to stand at 37 ° C. for 2 hours or more. The same amount of chloroform / isoamyl alcohol (24: 1) was added, and the mixture was centrifuged at 10,000 rpm, 4 ° C., 10 minutes, and the supernatant was transferred to another centrifuge tube, which was repeated three times. To the supernatant, 1/10 amount of 7.5M ammonium acetate was added, 2.5 times the amount of cold ethanol was further added, and the mixture was allowed to stand at −20 ° C. for 2 hours or more. This was centrifuged at 10,000 rpm for 20 minutes at 4 ° C., and the resulting precipitate was rinsed with 70% cold ethanol, and further centrifuged at 10,000 rpm at 4 ° C. for 10 minutes to obtain a precipitate. The precipitate was air-dried and then suspended in TE buffer and used for the following experiments.
(PCR amplification)
For amplification of the ITS region, a PCR amplifier thermal cycler was used. Primer used two types of fungi-specific ITS-1 (5′-TCCGTAGGTGAACCTGCGGG-3 ′) (SEQ ID NO: 2) and ITS-4 (5′-TCCTCCGCTTATTGATAGC-3 ′) (SEQ ID NO: 3).
The PCR reaction solution was 10 pmol each of Primer, 10 ng of extracted genomic DNA, 20 nmol each of dNTPs (Takara Bio), 2.5 U Taq DNA Polymerase (Takara Bio), and PCR buffer (Takara Bio) to a total volume of 25 μl. . PCR conditions were as follows: 92 ° C., 5 minutes heat treatment, 92 ° C., 30 seconds, 60 ° C., 30 seconds, 72 ° C., 1 minute reaction repeated 30 times, finally 72 ° C., 10 minutes treatment .
(Confirmation of amplification products)
Agarose electrophoresis was performed at 50 V, 1 hour, with a gel concentration of 1.5% (manufactured by Sigma, Type-II-A). For the staining, ethidium bromide was used and irradiated with ultraviolet rays to confirm the amplification band.
(Purification of ITS region amplification product)
For purification of the ITS region amplification product, Primer was removed, purified and concentrated using SUPREC-02 (manufactured by Takara Bio Inc.) to obtain a sequence sample.
(Sequence reaction)
A sequencing reaction solution was prepared by using a purified ITS region amplification product using Dye-terminator Kit (manufactured by Applied biosystems). In the PCR reaction, after heat treatment at 96 ° C. for 7 minutes, the reaction of 96 ° C., 30 seconds, 50 ° C., 15 seconds, 60 ° C. and 4 minutes was repeated 25 times, and finally the treatment at 4 ° C. was performed.
The reacted sample was purified by an ethanol precipitation method, a spin column method, or the like, and used as a sequence sample. To 20 μl of the reaction solution, 2 μl of 7.5M ammonium acetate and 50 μl of 95% ethanol were added and left at room temperature for 15 minutes. After centrifuging this solution at 15000 rpm and room temperature for 20 minutes, the supernatant was removed. After adding 200 μl of 70% ethanol and stirring gently, the supernatant was removed by centrifugation at 15000 rpm for 5 minutes at room temperature. The precipitate was dried and stored at -20 ° C.
The base sequence was examined using an ABI Prism Model 310 sequencer (manufactured by Applied biosystems).
(Confirmation of base sequence homology)
The database search is performed by using the National Medical Library Biotechnology Information Center (NCBI) database BLAST (http://blast.genome.ad.jp/) on the website and the base sequence of the ITS region of the sample examined. We searched for strains with homology.
The ITS region of Usxanagitake 1-7 strain showed the same base sequence (SEQ ID NO: 1). The results of gene sequence homology search with BLAST are shown in Table 4, and the strain tree is shown in FIG.
〔実施例５〕
（Ｃｔ１−２株の生理活性評価）
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を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ｐＨ７．２〜７．４で培養した。ＰＣ−１２細胞に関しては１０（ｖ／ｖ）％牛胎児血清および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ｐＨ７．２〜７．４で培養した。
３５ｍｍ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（０．２μｍ）にてろ過滅菌後、最終濃度１０ｎＭ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１００ｎＭ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５に示した。
Example 5
(Evaluation of physiological activity of Ct1-2 strain)
The neuronal differentiation-inducing activity was examined as follows.
1321N1 astrocytoma cells in Dulbecco's modified Eagle's medium containing 5 (v / v)% fetal calf serum, pH 7.2-7.4 in 37 ° C., 5% carbon dioxide mixed air (water vapor saturation) In culture. For PC-12 cells in Dulbecco's modified Eagle's medium containing 10 (v / v)% fetal calf serum and 5 (v / v)% horse serum, 37 ° C., 5% carbon dioxide mixed air (water vapor saturation) ) At pH 7.2 to 7.4.
1321N1 astrocytoma cells were dispensed in about 35 thousand cells in a 35 mm petri dish, and it was confirmed that the cells adhered to the container the next day, and the extract was added. The extract was dissolved in dimethyl sulfoxide, filtered and sterilized with 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. PC-12 cells were cultured in the culture supernatant for 2 days, followed by morphological observation 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 where very long protrusions and synapse formation were observed, and the average of the scores obtained for 100 cells was used as an index of cell differentiation. The results are shown in Table 5.
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、１０（ｖ／ｖ）％牛胎児血清を含む、ＲＰＭＩ１６４０培地で、３７℃、５％二酸化炭素混有空気（水蒸気飽和）、ｐＨ７．２〜７．４で培養した。
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、１ｍｌ／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１０μｌ／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（０．２μｍ）にてろ過滅菌後、ＨＬ−６０細胞培養液に最終濃度が１００μｇ／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１０μｌ／穴を添加した。
培養１日後、細胞懸濁液１５μｌ／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、−（無）とした。
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６に示す。
The growth inhibitory activity against myeloid leukemia cells was examined as follows.
Human leukemia cells HL-60 were cultured in RPMI 1640 medium containing 10 (v / v)% fetal bovine serum at 37 ° C., 5% carbon dioxide mixed air (water vapor saturation), pH 7.2 to 7.4. .
Using a 24-well plate (for floating cells), inoculate 1 ml / well of the cell suspension at 80 to 200,000 cells / ml, and at 2 to 3 days of cell culture, dilute the sample at 10 μl / well. Added in the hole. The extract was dissolved and diluted in dimethyl sulfoxide, sterilized by filtration with 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.
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. Cells whose cells were crushed and granules were scattered in the culture medium were defined as cell growth inhibition + (present), and cells whose cell morphology was clean and unchanged did not represent − (none).
Samples were measured for ATP activity at n = 3 holes, 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 6.
抗疲労活性は以下のようにして調べた。
マウスをロコモーターにて３時間強制歩行させた後、前記抽出エキスを１００μｇ／mouse脳室内投与し、さらに９０分自由運動させた時のロコモーター回転数を、投与しない対照群と比較した。結果を表７に示す。
Anti-fatigue activity was investigated as follows.
After the mice were forcibly walked for 3 hours with a locomotor, the extract was administered into the ventricle at 100 μg / mouse and allowed to move freely for 90 minutes, and the rotational speed of the locomotor was compared with the control group not administered. The results are shown in Table 7.
〔実施例６〕
（繰り返し培養）
実施例２と同様の培養方法にて、Ｃｔ１−２株の培養を１０回繰り返した時の生理活性を表８に示す。
Example 6
(Repeated culture)
Table 8 shows the physiological activity when Ct1-2 strain culture was repeated 10 times in the same culture method as in Example 2.
〔実施例７〕
（保存後の培養）
Ｃｔ１−２株をポテトデキストロース寒天培地（日水製薬製）に接種し、１４日間、２５℃にて培養後、このスラントを５℃にて１年保存した。１年後、スラントよりシャーレに接種し、培養した後、実施例２記載の方法で培養し、実施例１および５記載の方法で、生理活性を評価した。結果を表９に示す。
Example 7
(Culture after storage)
The Ct1-2 strain was inoculated into potato dextrose agar medium (manufactured by Nissui Pharmaceutical), cultured for 14 days at 25 ° C., and the slant was stored at 5 ° C. for 1 year. One year later, a petri dish was inoculated from the slant, cultured, and cultured by the method described in Example 2. The physiological activity was evaluated by the method described in Examples 1 and 5. The results are shown in Table 9.
配列番号１：ｒＤＮＡ ＩＴＳ
配列番号２：真菌類のｒＤＮＡのＩＴＳ領域を検出するためのプライマーとして機能するよう設計されたオリゴヌクレオチド
配列番号３：真菌類のｒＤＮＡのＩＴＳ領域を検出するためのプライマーとして機能するよう設計されたオリゴヌクレオチド
Sequence number 1: rDNA ITS
SEQ ID NO: 2: oligonucleotide designed to function as a primer for detecting the ITS region of fungal rDNA SEQ ID NO: 3: designed to function as a primer to detect the ITS region of fungal rDNA Oligonucleotide
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