JP2004344133A - Pleurotus nebrodensis strain, cultivation method thererof and disease prophylactic and ameliorative agent containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Pleurotus nebrodensis strain that has excellent disease prophylactic and ameliorative effects, a diseases prophylactic and ameliorative agents containing the same and a cultivation method for Pleurotus nebrodensis that can harvest a sufficient amount of fruit bodies in a short period of time. <P>SOLUTION: This Pleurotus nebrodensis strain is deposited to Kogyou Gijutuin Seimei Kogaku Kogyou Gijutiin Kenkyuusho as Pleurotus nebrodensis No. 17 FERM-22-5-2003. A disease prophylactic and ameliorative agent including mainly Pleurotus nebrodensis as a main component is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００３０】
表１より、電気インパルスを与えると、子実体の発生日が早まり、収量が増加することが確認された。子実体の発生日は、電圧が強いほど早まり、２０〜３０ｋＶでは１４日目から、１０〜１５ｋＶでは１６日目から、５ｋＶでは１８日目から子実体が発生し、１５ｋＶでは２６日目までに、２０ｋＶでは２４日目までに、２５ｋＶでは２２日目までに、３０ｋＶでは２０日目までにすべての培養瓶で子実体が発生した。
【００３１】
また収量は、２０ｋＶにおいて最も多く、２０〜３０ｋＶでは電気インパルスを与えない時の２倍以上の収量があった。さらに高い電圧を与えて試験したところ、６０ｋＶにおいても、子実体発生日が早まり、収量が増加する効果が確認された。
よって、Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓを短期間で多量に収穫するためには、培養終了時に５〜６０ｋＶ、特に１５〜３０ｋＶの電気インパルスを与えることが好ましく、収量の点では２０ｋＶが最適であることが確認された。
【００３２】
さらに、温度条件を以下のようにしたところ、さらに短期間でより多くの子実体を収穫できた。よって、電気インパルスに加えて、低温刺激を与えることが好ましいことが確認された。
温度条件：室温−１℃／相対湿度９０％で５日間維持し、室温を５℃に上昇させ５日間維持し、その後、室温を１８℃に上昇させた。
【００３３】
１．高血圧予防改善剤
（１）試験ラットの飼育、及び被験薬の投与方法
▲１▼予備飼育（６〜７週齢）
日本チャールスリバー（株）より購入した６週齢の雄性自然発症高血圧ラット（ＳＨＲ）、及び正常血圧のウィスター京都ラット（ＷＫＹ）を、室温２２±１℃、湿度６０±１０％に調節された飼育室において、白色蛍光灯で１日１２時間（７〜１９時明期）の光調節を行い、飼料及び水道水を自由摂取させ１週間予備飼育した。
予備飼育後、各個体の体重及び血圧の測定を行い、１群８頭とし、血圧値の平均値がほぼ等しくなるようにＳＨＲラットをＡ〜Ｈ群に、ＷＫＹラットをＩ〜Ｊ群に分類した。
【００３４】
▲２▼Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ抽出物の調製方法
Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ（平成１５年５月２２日付で工業技術院生命工学工業技術研究所に寄託。識別のための表示：Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ Ｎｏ．１７）子実体の乾燥物を粉砕し、１６メッシュのふるいにかけて粉末とした。また、この粉末３、６、９，１２ｇをそれぞれ６００ｍｌの熱水（８０℃）中で２時間抽出し、熱水抽出液を得た。以下、それぞれを３ｇ抽出物、６ｇ抽出物、９ｇ抽出物、１２ｇ抽出物という。
【００３５】
▲３▼被験薬投与方法（８〜１８週齢）
下記の要領で、各群のラットに被験薬投与を行い、８週齢からさらに１２週間、２０週齢まで飼育した。飼料と水道水を各群とも自由摂取させた。飼育室の環境は、温度２２±１℃、湿度６０±１０％、白色蛍光灯で１日１２時間の採光下（７〜１９時明期）とした。
各抽出物はすべて経口投与とし、毎日午前１０時より施行し、抽出物投与後は滅菌水を自由摂取させた。
【００３６】
Ａ群：ＳＨＲラット（無投与）
Ｂ群：ＳＨＲラット（３ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記３ｇ抽出物１０ｍｌを投与する。
Ｃ群：ＳＨＲラット（６ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記６ｇ抽出物１０ｍｌを投与する。
Ｄ群：ＳＨＲラット（９ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記９ｇ抽出物１０ｍｌを投与する。
Ｅ群：ＳＨＲラット（１２ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記１２ｇ抽出物１０ｍｌを投与する。
Ｆ群：ＳＨＲラット（乾燥粉末３％）・・・飼料に３％のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ乾燥粉末を混合し、自由摂取させる。
Ｇ群：ＳＨＲラット（乾燥粉末６％）・・・飼料に６％のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ乾燥粉末を混合し、自由摂取させる。
Ｈ群：ＳＨＲラット（乾燥粉末９％）・・・飼料に９％のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ乾燥粉末を混合し、自由摂取させる。
Ｉ群：ＷＫＹラット（無投与）
Ｊ群：ＷＫＹラット（乾燥粉末６％）・・・飼料に６％のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ乾燥粉末を混合し、自由摂取させる。
【００３７】
（２）結果
▲１▼血圧検査
血圧検査は２週間に１回、被験薬投与前に行い、各群ラットの平均値を求めた。
結果を表２に示す。
【表２】

【００３８】
自然発症高血圧ラットであるＳＨＲラット（Ａ〜Ｈ群）はＷＫＹラット（Ｉ〜Ｊ群）と比較して、血圧値が高かった。しかしながら、ＳＨＲラットにおいて、無投与群（Ａ群）と比較して、６，９，１２ｇ投与群（Ｃ〜Ｅ群）、６、９％群（Ｇ，Ｈ群）では１４週齢当たりから、血圧上昇の抑制が確認された。さらに驚くべきことに、６％群（Ｇ群）では１４週齢から、９、１２ｇ投与群（Ｄ，Ｅ群）及び９％群（Ｈ群）では１６週齢から、６ｇ投与群（Ｃ群）では１８週齢から、血圧の降下が確認された。
血圧上昇抑制及び血圧降下の作用は、用量が多い群ほど高い傾向にあった。１２ｇ投与群（Ｅ群）において血圧の降下作用が、９ｇ投与群（Ｄ群）よりも高くなかったのは、薬効用量に伴う生物曲線の発現と考えられる。また、元々正常血圧であるＷＫＹラットにおいては、血圧の降下（異常）は起こらなかった。
【００３９】
以上より、本発明のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓには、用量依存的に高血圧症予防改善効果があることがわかった。また、正常血圧である場合には血圧降下作用を示さないことから、従来の高血圧治療薬のように単に血圧を下げるのではなく、血圧を正常値に近づける効果があり、より安全に服用できることがわかる。また、乾燥粉末でも、その抽出物でも効果があることがわかった。
【００４０】
▲２▼血液検査
実験最終日の前日に全ラットを絶食させ、翌日に深麻酔（ネンブタール，４５ｍｇ／ｋｇ，ｉ．ｐ．）し、左心室から２０Ｇ採血針で可能な限り採血を行った。
採取した血液を用いて、下記の項目において生化学的検査（自動化学分析装置：Ａｕｔｏ Ｌａｂ， Ｒａｄｉｏ ｌｍｍｕｎｏ Ａｓｓａｙ 法）を行い、平均値を求めた。
結果を表３に示す。
【００４１】
【表３】

【００４２】
ＳＨＲラット（Ａ〜Ｈ群）はＷＫＹラット（Ｉ〜Ｊ群）と比較して、ＬＤＬコレステロール、βリポタンパク、中性脂肪、アルブミン、尿素窒素、クレアチニン、尿酸、アンジオテンシンの値が高く、総コレステロール、ＨＤＬコレステロール、総タンパク、Ａ／Ｇ比、白血球数の値が低いことがわかった。しかしながら、ＳＨＲラットにおいて、無投与群（Ａ群）と比較して、投与群（Ｂ〜Ｈ群）では各項目において著明な改善が確認された。
【００４３】
アンジオテンシン
アンジオテンシンは、肝臓で産生されるレニン基質に、腎糸球体より分泌されるレニンが作用し生成される活性ペプチドである。高値を示す疾患としては、腎血管性高血圧症、悪性高血圧症等が挙げられる。よって、アンジオテンシン値は、高血圧症の指標に用いられる。
Ａ群と比較して、Ｂ〜Ｈ群ではアンジオテンシンが低下し、正常なＩ群の値に近づき、特にＢ〜Ｅ、Ｇ，Ｈ群ではほぼＩ群の値に等しくなった。このようにアンジオテンシン値においても、高血圧予防改善効果が確認された。
【００４４】
コレステロール
コレステロールは、生体細胞膜の構成成分等として必須の成分であり、善玉コレステロール（ＨＤＬ）と、悪玉コレステロール（ＬＤＬ）とに分けられる。ＬＤＬは動脈硬化を促進し、ＨＤＬは血管壁に付着したＬＤＬを取り除く働きをする。よって、これらのコレステロール値を測定することにより、脂質代謝異常をきたす疾患の推測ができる。
【００４５】
Ａ群と比較してＢ〜Ｈ群では、ＨＤＬの上昇、及びＬＤＬの低下が見られ、Ｉ群の値に近づき、特にＤ〜Ｅ、Ｇ，Ｈ群ではほぼＩ群の値に等しくなった。また総コレステロール値も正常なＩ群の値に近づいた。よって、高血圧予防改善効果に伴い、脂質代謝異常の予防改善効果を有することが確認された。
【００４６】
βリポタンパク、中性脂肪
βリポタンパクの主な機能は、コレステロールを肝臓から各臓器に移送することである。よってβリポタンパク値は脂質代謝異常疾患の指標とされる。
Ａ群と比較して、Ｂ〜Ｈ群では用量依存的にβリポタンパク値の低下が見られ、正常値に近づいた。
さらに、Ａ群と比較してＢ〜Ｈ群では、用量依存的に中性脂肪の低下も見られた。よって、βリポタンパク、中性脂肪値においても、脂質代謝異常の予防改善が確認された。
【００４７】
尿素窒素、クレアチニン、尿酸
尿素窒素、クレアチニン、尿酸の測定は、肝・腎機能の指標に用いられる。
Ａ群と比較して、Ｄ、Ｅ、Ｇ，Ｈ群では尿素窒素、クレアチニン、尿酸値が低下し、正常なＩ群の値に近づいた。よって、高血圧予防改善効果に伴い、肝・腎機能の改善効果も有することが確認された。
【００４８】
総タンパク、アルブミン、Ａ／Ｇ比、白血球数
さらに、Ａ群と比較してＢ〜Ｈ群では、用量依存的に総タンパク、Ａ／Ｇ比、白血球数の値の上昇、及びアルブミン値の低下が見られ、正常なＩ群の値に近づいた。
【００４９】
以上のことから、Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓの投与により、高血圧予防改善作用に伴い、脂質代謝異常の改善効果、肝・腎機能の改善効果が発揮されることが確認された。また、正常ラットにおいては、投与によりこれらの値には目立った変化（異常）を示さないことから、安全性が高いことがわかる。
【００５０】
２．高脂血症予防改善剤・肥満予防改善剤
Ｚｕｃｋｅｒラットの遺伝形式は常染色体性の単純劣性遺伝様式を取り、病因遺伝子（ｆａ遺伝子）をホモに持つ個体（ｆａ／ｆａ）のみが肥満を呈し（Ｚｕｃｋｅｒ−ｆａｔｔｙラット：（ＺＵＣ）−ｆａ／ｆａ）、ヘテロ接合体（ｆａ／＋）及び野生型（＋／＋）は肥満を呈さない（Ｚｕｃｋｅｒ−ｌｅａｎラット：（ＺＵＣ）−ｌｅａｎ）。Ｚｕｃｋｅｒ−ｆａｔｔｙラットは、生後４週齢頃より体重増加及び外観によって肥満状態が認められ、１０週齢頃までに急速に肥満が進行し、その後も徐々に進行する。さらに高脂血症、高インシュリン血症、高レプチン血症及び耐糖性の異常を示すことが知られている。このＺｕｃｋｅｒ−ｆａｔｔｙラット及び比較としてＺｕｃｋｅｒ−ｌｅａｎラットを使用し、高脂血症予防改善効果及び肥満予防改善効果について試験を行った。
【００５１】
（１）試験ラットの飼育、及び被験薬の投与方法
▲１▼予備飼育（５〜６週齢）
日本チャールスリバー（株）より購入した５週齢の雄性Ｚｕｃｋｅｒ−ｆａｔｔｙラット、及び雄性Ｚｕｃｋｅｒ−ｌｅａｎラットを、室温２２±１℃、湿度６０±１０％に調節された飼育室において、白色蛍光灯で１日１２時間（７〜１９時明期）の光調節を行い、飼料及び水道水を自由摂取させ１週間予備飼育した。
予備飼育後、各個体の体重測定及び糖負荷試験を行い、１群５頭とし、体重の平均値がほぼ等しくなるようにＺｕｃｋｅｒ ｆａｔｔｙラットをＫ〜Ｏ群に、Ｚｕｃｋｅｒ−ｌｅａｎラットをＰ〜Ｑ群に分類した。
【００５２】
▲２▼Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ抽出物の調製方法
前述の通りとする。
▲３▼被験薬投与方法（６〜１６週齢）
下記の要領で、各群のラットに被験薬投与を行い、さらに１０週間、１６週齢まで飼育した。飼料と水道水を各群とも自由摂取させた。飼育室の環境は、温度２２±１℃、湿度６０±１０％、白色蛍光灯で１日１２時間の採光下（７〜１９時明期）とした。
各被験薬はすべて経口投与とし、毎日午前１０時より施行し、投与後は滅菌水を自由摂取させた。
【００５３】
Ｋ群：Ｚｕｃｋｅｒ−ｆａｔｔｙラット（無投与）
Ｌ群：Ｚｕｃｋｅｒ−ｆａｔｔｙラット（３ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記３ｇ抽出物１０ｍｌを投与する。
Ｍ群：Ｚｕｃｋｅｒ−ｆａｔｔｙラット（６ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記６ｇ抽出物１０ｍｌを投与する。
Ｎ群：Ｚｕｃｋｅｒ−ｆａｔｔｙラット（９ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記９ｇ抽出物１０ｍｌを投与する。
Ｏ群：Ｚｕｃｋｅｒ−ｆａｔｔｙラット（１２ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記１２ｇ抽出物１０ｍｌを投与する。
Ｐ群：Ｚｕｃｋｅｒ−ｌｅａｎラット（無投与）
Ｑ群：Ｚｕｃｋｅｒ−ｌｅａｎラット（９ｇ抽出物投与）・・・１日に体重１ｋｇ当たり前記９ｇ抽出物１０ｍｌを投与する。
【００５４】
（２）結果
▲１▼体重変動
体重測定は週１回、被験薬投与前に行い、各群ラットの平均値を求めた。結果を表４に示す。
【表４】

【００５５】
Ｚｕｃｋｅｒ−ｆａｔｔｙラット（Ｋ〜Ｏ群）は、Ｚｕｃｋｅｒ−ｌｅａｎラット（Ｐ〜Ｑ群）と比較して、体重の増加が激しかった。しかしながら、Ｚｕｃｋｅｒ−ｆａｔｔｙラットにおいて、無投与群（Ｋ群）と比較して、投与群（Ｌ〜Ｏ群）では１０週齢当たりから体重増加の抑制が見られた。体重増加の抑制効果は、用量が多い群ほど高かった。
また、正常体重であるＺｕｃｋｅｒ−ｌｅａｎラットにおいても、正常な範囲内での体重増加の抑制が確認された。
以上より、本発明のＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓには、用量依存的に肥満を抑制する効果が見られ、正常な場合に対しても、適度な脂肪沈着抑制効果があることがわかった。
【００５６】
▲２▼血液検査
実験最終日の前日に全ラットを絶食させ、翌日に深麻酔（ネンブタール，４５ｍｇ／ｋｇ，ｉ．ｐ．）し、左心室から２０Ｇ採血針で可能な限り採血を行った。
採取した血液を用いて、下記の項目において生化学的検査（自動化学分析装置：Ａｕｔｏ Ｌａｂ， Ｒａｄｉｏ ｌｍｍｕｎｏ Ａｓｓａｙ 法）を行い、平均値を求めた。
結果を表５に示す。
【００５７】
【表５】

【００５８】
表５より、Ｚｕｃｋｅｒ−ｆａｔｔｙラット（Ｋ〜Ｏ群）はＺｕｃｋｅｒ−ｌｅａｎラット（Ｐ〜Ｑ群）と比較して、総コレステロール、遊離コレステロール、βリポタンパク、中性脂肪、リン脂質、総脂質、尿素窒素、ＡＳＴ（ＧＯＴ）、ＡＳＴ（ＧＰＴ）、インスリンの値が高く、ＨＤＬコレステロール、遊離脂肪酸、クレアチニン、尿酸の値が低いことがわかった。しかしながら、Ｚｕｃｋｅｒ−ｆａｔｔｙラットにおいて、無投与群（Ｋ群）と比較して、投与群（Ｌ〜Ｏ群）では各項目において、著明な予防改善効果が確認された。
【００５９】
中性脂肪、遊離脂肪酸、リン脂質、総脂質
Ｋ群と比較してＬ〜Ｏ群では、用量依存的に中性脂肪、リン脂質、総脂質が低下し、遊離脂肪酸が上昇する傾向が見られた。遊離脂肪酸は、大部分が脂肪組織に蓄積された中性脂肪が分解され血中に放出されたもので、心筋、骨格筋、腎等の主要臓器で燃焼され、末梢組織でエネルギー源として利用される。よってこの結果は、肥満の改善を示す。また、中性脂肪、総脂質の著しい低下に比べて、細胞膜の構成成分として重要であるリン脂質は低下が少なく、正常なＰ群の値以下になることはなかった。また上記の作用は、正常ラットにおいても、正常な範囲内で確認された。以上より、高脂血症、肥満の予防改善作用が確認された。
【００６０】
コレステロール
前述のようにコレステロール値は、脂質代謝異常の指標となる。
Ｋ群と比較してＬ〜Ｏ群では、善玉コレステロールであるＨＤＬが上昇するのに対し、総コレステロール、遊離コレステロールは用量依存的に低下する傾向があった。以上より、コレステロール値において、脂質代謝の改善効果が確認された。
【００６１】
βリポタンパク
前述のように、βリポタンパク値は脂質代謝異常疾患の指標とされる。
Ｋ群と比較してＬ〜Ｏ群では、用量依存的にβリポタンパク値が低下し、正常なＰ群の値に近づいた。よって、βリポタンパク値においても、脂質代謝の改善が確認された。
【００６２】
インスリン
インスリンは、膵臓のβ細胞から分泌されるホルモンで、筋肉・脂肪組織でのグルコースの取り込み促進、及び肝での糖放出抑制等を介して血糖を降下させる。またグリコーゲン蓄積、脂肪蓄積、ケトン体低下、蛋白合成等にも関与している。よってインスリンの測定は、糖代謝異常の指標となる。
Ｋ群と比較して、Ｌ〜Ｏ群では用量依存的にインスリン値の低下が見られ、正常なＰ群の値に近づいた。よって、肥満予防改善効果に伴い、糖代謝の改善も確認された。
【００６３】
尿素窒素、クレアチニン、尿酸、ＡＳＴ
尿素窒素、クレアチニン、尿酸、ＡＳＴは、肝・腎機能の指標に用いられる。
Ｋ群と比較してＬ〜Ｏ群では、用量依存的に尿酸窒素、ＡＳＴ値が低下し、クレアチニン、尿酸値が上昇する傾向が見られ、Ｏ群においては、正常なＰ群とほぼ同じ値となった。よって、肥満予防改善効果に伴い、肝・腎機能の改善も確認された。
【００６４】
また、抽出液投与によって、元々正常であった総タンパク、アルブミン、Ａ／Ｇ比等には異常をきたすことはなかった。
以上のことから、Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓの投与により、高脂血症予防改善効果、肥満予防改善効果、及びそれに伴う脂質代謝異常の改善効果、糖代謝の改善効果、肝・腎機能の改善効果が発揮されることが確認された。
【００６５】
３．ヒト臨床検査
次に本発明の疾患予防改善剤におけるヒト臨床検査について説明する。
被検試薬の調製（図１）
Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ（平成１５年５月２２日付で工業技術院生命工学工業技術研究所に寄託。識別のための表示：Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ Ｎｏ．１７）乾燥粉末１００ｇと滅菌水１Ｌを三角フラスコに加え、９５℃の湯浴中で１時間抽出後、室温まで放冷した。減圧濾過により第一濾液と残渣とに分離する。残渣に滅菌水５００ｍＬを加え、９５℃の湯浴中で１時間抽出後、室温まで放冷し、減圧濾過により第二濾液と残渣とに分離する。第一濾液と第二濾液を混合し、６０℃にて減圧濃縮する。さらに、９５℃で１０分殺菌後、固形分５０％となるまで６０℃にて濃縮する。固形分に対して、５０％の賦形剤を添加し、加熱溶解、凍結乾燥にて粉末化する。６０メッシュのふるいにかけ、押出し造粒法にて顆粒化する。
【００６６】
３３歳、４２歳、５０歳の３人の男性に、前記方法にて調製した被検試薬を１日６ｇずつ毎日服用してもらい、服用前、服用１ヶ月目、服用３ヶ月目、服用６ヶ月目にそれぞれ血液検査を行った。結果を表６に示す。
【００６７】

【００６８】
なお、正常値は、総コレステロール：１２０〜２２０ｍｇ／ｄｌ、ＨＤＬコレステロール：４１〜８４ｍｇ／ｄｌ、中性脂肪：３０〜１８０ｍｇ／ｄｌ、β−リポタンパク：１５０〜５００ｍｇ／ｄｌとされている。服用前はどの男性も、総コレステロール、中性脂肪、β−リポタンパクの値が正常値を超えており、１人を除いてＨＤＬが正常値以下であった。
しかしながら、Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ乾燥粉末を服用することにより、これらの値に改善が見られ、服用６ヶ月目には３人ともすべての値が正常範囲内に改善された。よって、Ｐｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓはヒトに対しても疾患予防改善作用を持つことが確認された。また服用によって、下痢、嘔吐、排尿異常等の副作用は一切起こらなかった。
【００６９】
【発明の効果】
本発明にかかるＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ、特に平成１５年５月２２日付で工業技術院生命工学工業技術研究所に寄託し、識別のための表示がＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓ Ｎｏ．１７であるＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓは、優れた疾患予防改善効果を有する。
また、本発明にかかるＰｌｅｕｒｏｔｕｓ ｎｅｂｒｏｄｅｎｓｉｓの栽培方法によれば、培養終了直後に電気インパルスを与えることでより、短期間で充分な量の子実体を収穫することができる。
【図面の簡単な説明】
【図１】本発明にかかる披検試薬の調製方法を示すフローチャートである。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a Pleurotus nebrodensis strain, a cultivation method thereof, and a disease prevention / improvement agent containing the same, particularly a Pleurotus nebrodensis strain having a disease prevention / improvement effect, and improvement of the yield.
[0002]
[Prior art]
In recent years, there has been a tendency for young people to become obese due to a variety of eating habits and a gastronomic boom. In addition, it is predicted that diseases related to aging and lifestyle will increase with the entry into a super-aging society and a stressed society. In particular, hypertension tends to increase due to obesity and lack of exercise. Furthermore, hypertension is known to be easily accompanied by adult diseases such as diabetes, impaired glucose tolerance, hyperlipidemia, abnormal lipid metabolism, and obesity.
[0003]
Conventionally, as a treatment method for hypertension, a method using a Ca antagonist, a β receptor agonist, and an angiotensin converting enzyme inhibitor is effective and widely used. However, side effects are often a problem with long-term administration of these drugs. Exercise therapy and diet therapy are the main methods for improving obesity, but it is difficult to continue them. For this reason, interest in natural foods and health foods (including functional foods), which are unlikely to cause side effects and can be taken continuously, is increasing.
[0004]
On the other hand, edible mushrooms are in strong demand due to diversification of food culture and recognition as health foods, and their production is increasing. Therefore, the production technology has been developed corresponding to each mushroom. At present, most mushrooms, such as shiitake mushroom, enokitake mushroom, bunashimeji, maitake, oyster mushroom, nameko, eryngii, etc., can be artificially cultivated, except for some mushrooms having special ecology.
Methods for cultivating edible mushrooms include cultivation of raw wood such as shiitake mushrooms and cultivation of container fungi using a mixture of sawdust, rice bran, bran, water and the like as a medium, such as enokitake and oyster mushrooms.
[0005]
In the container cultivation, since the composition of the medium has a great influence on its growth, various materials have conventionally been used as the medium to increase the yield of high-quality mushrooms.
For example, there are a method of adding red algae powder to a medium (Japanese Patent Application Laid-Open No. 06-113670) and a method of adding a crushed egg shell to the medium (Japanese Patent Application Laid-Open No. 06-253677).
[0006]
[Patent Document 1] JP-A-06-113670
[Patent Document 2] JP-A-06-253677
[0007]
[Problems to be solved by the invention]
However, in the case of Pleurotus nebrodensis, a conventional cultivation method cannot obtain a sufficient harvest amount, and a cultivation method capable of further increasing the harvest amount has been desired.
The present invention has been made in view of the above-mentioned problems of the prior art, and aims to provide a Pleurotus nebrodensis strain having an excellent disease prevention / ameliorating effect, and a disease prevention / improvement agent containing the same, and a sufficient amount in a short period of time. An object of the present invention is to provide a method for cultivating Pleurotus nebrodensis capable of harvesting fruiting bodies of Pleurotus nebrodensis.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the present inventors have conducted intensive studies, and as a result, deposited Pleurotus nebrodensis, particularly on May 22, 2003, with the Institute of Biotechnology and Industrial Technology and Research Institute of Life Science and Technology, and displayed it for identification. Is Pleurotus nebrodensis No. It has been found that Pleurotus nebrodensis 17 has an excellent disease prevention and improvement effect. Furthermore, it has been found that the application of an electric impulse promotes the generation of Pleurotus nebrodensis fruiting bodies, and has completed the present invention.
That is, the first subject of the present invention was deposited on May 22, 2003, with the Institute of Biotechnology and Industrial Technology, and the display for identification was Pleurotus nebrodensis No. 1. 17 Pleurotus nebrodensis strain.
[0009]
A second subject of the present invention is a method for cultivating Pleurotus nebrodensis, comprising the following steps (a) to (d).
(A) a step of inoculating a medium with a seed of Pleurotus nebrodensis;
(B) a step of, after the step (a), culturing at a temperature of 20 to 30 ° C. to spread mycelia in the medium;
(C) a step of applying an electrical impulse of 5 to 60 kV after the step (b).
(D) a step of generating fruiting bodies at a temperature of 10 to 20 ° C. after the step (c).
[0010]
In the cultivation method described above, it is preferable to set the temperature to 10 to 20 ° C. after applying a low-temperature stimulus of −1 to 2 ° C. in the step (d).
In the cultivation method, in the step (a), it was deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Technology, and the display for identification was Pleurotus nebrodensis No. It is preferable to inoculate the inoculum of Pleurotus nebrodensis which is No. 17.
[0011]
A third subject of the present invention is a disease prevention / amelioration agent comprising Pleurotus nebrodensis as a main component.
The agent for preventing or improving disease preferably contains a dry powder of Pleurotus nebrodensis and / or a hot water extract thereof.
Pleurotus nebrodensis strain was deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Science and Technology, and the label for identification was Pleurotus nebrodensis No. 1 on May 22, 2003. Preferably, the strain is Pleurotus nebrodensis strain No. 17.
In the agent for preventing or improving disease, the disease is preferably one or more selected from hypertension, hyperlipidemia, and obesity.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in detail.
First, a method for cultivating Pleurotus nebrodensis according to the present invention will be described.
(1) Preparation of medium
In the present invention, the material used as the medium is not particularly limited as long as the effects of the present invention are not hindered, and those conventionally used can be used. For example, rice bran, sawdust and the like prepared by adding water are used. Further, a hyphal activator and the like may be mixed.
The container for filling the medium is not particularly limited as long as the effects of the present invention are not hindered, and may be filled in an appropriate container. Usually, a bottle-shaped container (culture bottle) or a bag-shaped container (culture bag) having an upper opening is used.
[0013]
(2) Sterilization of medium
The prepared medium is sterilized by sterilization means such as steam sterilization. The sterilization conditions are the same as before, and are not particularly limited. For example, the sterilization may be performed at 100 ° C. for about 4 hours.
(3) Inoculation of inoculum
Then, after cooling to about 20 ° C., an appropriate hole is made substantially in the center of the container filled with the culture medium, and the inoculum of Pleurotus nebrodensis is inoculated. The inoculum of the inoculum is not particularly limited as long as the effects of the present invention are not hindered, and can be performed by a usual method. Although the depth of the hole is not particularly limited, it is preferable to set the depth until reaching the vicinity of the bottom of the container in consideration of the promotion around the bacteria.
[0014]
(4) Culture
After inoculation of the inoculum, the cells are cultured to spread the mycelium in the medium. The room temperature during the culturing is preferably 20 to 30 ° C. and the humidity is preferably 70 to 100%, and the culturing is preferably performed until the hyphae are sufficiently spread.
The culturing conditions are not particularly limited as long as they are in the above-mentioned range. However, in order to perform culturing quickly, it is preferable to temporarily lower the room temperature at a later stage of the culturing and then raise it again.
[0015]
Specifically, it is preferable that the room temperature in the early stage of culture is 18 to 22 ° C., the temperature once lowered in the second stage of culture is 8 to 12 ° C., and the temperature subsequently raised is 23 to 32 ° C.
The period of the first half of the culture is preferably about 40 days, and the period of the second half of the culture is preferably about 20 days. The timing of the rise of the room temperature in the latter stage of the culture is preferably to keep the room temperature of 8 to 12 ° C. for about 10 days, then to raise the temperature rapidly to 23 to 32 ° C., and it is better to keep that state for about 10 days.
[0016]
(5) Generation of fruiting body
By applying an electrical impulse after the mycelium has spread in the medium, the fruiting body is sped up and the yield is increased. The electrical impulse is preferably between 5 and 60 kV, in particular between 20 and 30 kV, even more preferably between 20 kV. If it is less than 5 kV, the effects of the present invention may not be sufficiently exhibited, and if it is more than 60 kV, it may be difficult to generate fruit bodies, which is not preferable. It is preferable that the timing of applying the electric impulse is the same as the timing of entering the fruiting body development stage after the completion of the culture.
[0017]
(6) Generation of fruiting body
In order to generate fruiting bodies, it is preferable that the room temperature be lower than that during the culture, that the room temperature be 10 to 20 ° C and the humidity be 80 to 100%.
The temperature and humidity conditions suitable for the fruiting body generation are as described above. However, in order to further accelerate the fruiting body generation and increase the yield, it is preferable to maintain the room temperature at a low temperature and raise it halfway. Specifically, after maintaining the state of -1 to 2 ° C for about 5 days and applying a low-temperature stimulation to the hypha, the temperature is raised to 3 to 7 ° C and maintained for about 5 days, and then further raised to 15 to 20 ° C. It is preferable to maintain it. This period may be appropriately adjusted depending on the state of the hypha.
[0018]
Further, it is particularly preferable to increase the carbon dioxide concentration and / or the illuminance at almost the same time as the second-stage temperature increase.
Specifically, the carbon dioxide concentration is preferably increased from about 400 ppm to about 2000 ppm, and the illumination luminous intensity is preferably increased from about 100 Lux to about 400 Lux.
[0019]
The Pleurotus nebrodensis strain according to the present invention was deposited on May 22, 2003 by the present applicant, Fumiyo Eguchi, with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and displayed for identification. Of Pleurotus nebrodensis No. Seventeen. The scientific properties are as follows.
1. Scientific properties: Characteristics of the bacterium: Form a white colony in a nutrient medium containing a carbon source and a nitrogen source. Under an optical microscope, a clamp connection is observed.
[0020]
2. Taxonomic position ... Basidiomycete
3. Culture conditions
(1) Medium name: SMYA medium (S: saccharose, M: malt extract, Y: yeast extract, A: agar)
S, A → Commercial product M, Y → Difco (Difco)
{Circle around (2)} Composition of medium: per 1000 ml of medium
1% Saccharose 10g
10% 1% malt extract
4g 0.4% yeast extract
2g agar 20g
[0021]
{Circle around (3)} pH of the culture medium: 5.0 to 7.0 (optimum pH 5.5)
(4) Sterilization conditions for culture medium: 121 ° C, 20 minutes
(5) Medium temperature: 28 ° C
(6) Culture period: 10 days
(7) Oxygen demand: aerobic
[0022]
4. Storage conditions
Can be stored by the freezing method.
(1) Freezing conditions: -80 ° C
(2) Protecting agent: 10-20% glycerin aqueous solution (optimal 20%)
(3) Restoration rate after freezing: 100% in one year, 99% in three years
[0023]
5. Survival conditions
(1) Restoration of microorganisms: 40 ° C
{Circle around (2)} Inoculation, culture and confirmation methods: Under the same conditions as the culture conditions.
[0024]
The Pleurotus nebrodensis strain of the present invention can be used to produce a disease prevention / amelioration agent. The agent for preventing or improving disease of the present invention is orally administered, and is applied for preventing / ameliorating diseases, particularly hypertension, hyperlipidemia and obesity. The intake amount when used as a disease prevention / improvement agent varies depending on the symptoms and the like, and is not particularly limited. However, a sufficient effect can be expected if 1 to 15 g, particularly 6 to 9 g, of Pleurotus nebrodensis dry powder per adult is taken per day. it can.
[0025]
Pleurotus nebrodensis can also be taken as an extract of a dry powder. As the extract, it is preferable to extract a Pleurotus nebrodensis fruit body dry powder with hot water. The extract may be an extract or a dried product thereof.
[0026]
The agent for preventing or improving disease of the present invention is usually administered as an internal medicine.
In the case of internal medicine, powders, tablets, capsules, granules, teas, suspending agents, fluid extracts, liquids, syrups and the like can be prepared in a usual manner. In the case of formulation, usual formulation carriers, for example, excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, flavoring agents, and the like may be used as necessary. it can. In addition, if necessary, the coating can be applied with an appropriate coating agent or the like.
[0027]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
Effect of electric impulse
1. A culture medium comprising 150 parts by weight of sawdust, 100 parts by weight of bran, 10 parts by weight of corn meal, and 15 parts by weight of mycelial activator was prepared, and the water content was adjusted to about 65%. A cultivation bottle made of polypropylene (diameter: 52 mm, capacity: 850 ml) was filled with the prepared medium in an amount of about 650 g, and capped.
2. The solution was sterilized by a steam sterilizer at 121 ° C. for 4 hours, and cooled until the temperature of the medium became 20 ° C. or less.
3. An inoculation hole having a diameter of about 20 mm was provided at the center of the culture medium to the bottom, and was inoculated to Pleurotus nebrodensis (deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on May 22, 2003. Indication for identification: Pleurotus nebrodensis No. 17). 15 g were inoculated.
4. The bottle inoculated with the inoculum was placed in a culture room set at room temperature 25 ° C./70% relative humidity for 60 days. As a result, the mycelium spread sufficiently in the medium, and the mycelium was ripe.
5. An electric impulse of a predetermined voltage is applied to the bottle in which the bacteria have been cultured.
6. The plants were transferred to a growth room set at a room temperature of 10 ° C. and a relative humidity of 90%, and the occurrence of fruiting bodies was observed every other day.
[0028]
100 culture bottles were prepared for each voltage. Table 1 shows the number of culture bottles in which fruiting bodies were generated and the average yield on each day. The average yield indicates the average of the fruiting body yield per culture bottle in which fruiting bodies occurred at each voltage. The number of days was calculated from the day when the electric impulse was given (the day when the cells were transferred from the culture room to the growth room).
[0029]

[0030]
From Table 1, it was confirmed that when the electric impulse was given, the date of occurrence of the fruiting body was earlier and the yield was increased. The date of occurrence of the fruiting body is earlier as the voltage is stronger, and the fruiting body occurs from the 14th day at 20 to 30 kV, the 16th day at 10 to 15 kV, the 18th day at 5 kV, and the 26th day at 15 kV. , 20 kV by day 24, 25 kV by day 22, and 30 kV by day 20.
[0031]
Further, the yield was the highest at 20 kV, and at 20 to 30 kV, the yield was more than twice that when no electric impulse was given. When a test was conducted by applying a higher voltage, it was confirmed that even at 60 kV, the fruiting body emerged earlier and the yield increased.
Therefore, in order to harvest a large amount of Pleurotus nebrodensis in a short period of time, it is preferable to apply an electric impulse of 5 to 60 kV, particularly 15 to 30 kV at the end of culture, and it has been confirmed that 20 kV is optimal in terms of yield. .
[0032]
Furthermore, when the temperature conditions were set as follows, more fruiting bodies could be harvested in a shorter period of time. Therefore, it was confirmed that it is preferable to apply a low-temperature stimulus in addition to the electric impulse.
Temperature conditions: Maintained at room temperature-1 ° C./90% relative humidity for 5 days, increased room temperature to 5 ° C. and maintained for 5 days, then increased room temperature to 18 ° C.
[0033]
1. Antihypertensive agent
(1) Breeding of test rats and administration of test drugs
(1) Preliminary rearing (6-7 weeks old)
Six-week-old male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) purchased from Japan Charles River Co., Ltd. were bred at room temperature of 22 ± 1 ° C. and humidity of 60 ± 10%. In the room, light control was performed with a white fluorescent lamp for 12 hours a day (light period from 7 to 19 o'clock), feed and tap water were freely taken, and the animals were preliminarily reared for one week.
After preliminary breeding, the body weight and blood pressure of each individual were measured, and one group was divided into eight animals, and SHR rats were classified into groups A to H, and WKY rats were classified into groups I to J so that the average values of blood pressure values were almost equal. did.
[0034]
(2) Preparation method of Pleurotus nebrodensis extract
Pleurotus nebrodensis (deposited with the Institute of Biotechnology, Institute of Biotechnology, Japan, on May 22, 2003. Identification for identification: Pleurotus nebrodensis No. 17) A dried substance of the fruit body was pulverized and sifted through a 16 mesh sieve. And The powders 3, 6, 9, and 12 g were each extracted in 600 ml of hot water (80 ° C.) for 2 hours to obtain a hot water extract. Hereinafter, these are referred to as 3 g extract, 6 g extract, 9 g extract, and 12 g extract, respectively.
[0035]
(3) Test drug administration method (8-18 weeks old)
The test drug was administered to each group of rats according to the following procedure, and the rats were bred from 8 weeks of age to 20 weeks of age for further 12 weeks. Each group had free access to feed and tap water. The environment of the breeding room was a temperature of 22 ± 1 ° C., a humidity of 60 ± 10%, and a white fluorescent lamp under lighting for 12 hours a day (light period from 7 to 19).
Each extract was orally administered, and the test was performed daily at 10 am. After administration of the extract, sterile water was allowed to be freely ingested.
[0036]
Group A: SHR rat (no administration)
Group B: SHR rats (administration of 3 g extract): 10 ml of the 3 g extract is administered per kg of body weight per day.
Group C: SHR rats (administration of 6 g extract): 10 ml of the 6 g extract is administered per kg of body weight per day.
Group D: SHR rats (administration of 9 g extract) ... 10 ml of the 9 g extract is administered per kg of body weight per day.
Group E: SHR rats (administration of 12 g extract): 10 ml of the 12 g extract is administered per kg of body weight per day.
Group F: SHR rat (dry powder 3%): 3% of Pleurotus nebrodensis dry powder is mixed with the feed and allowed to freely ingest.
Group G: SHR rats (dry powder 6%): 6% of Pleurotus nebrodensis dry powder is mixed with the feed and allowed to freely ingest.
Group H: SHR rat (dry powder 9%): 9% of Pleurotus nebrodensis dry powder is mixed with the feed and allowed to freely ingest.
Group I: WKY rats (no administration)
Group J: WKY rats (dry powder 6%): 6% of Pleurotus nebrodensis dry powder is mixed with the feed and fed freely.
[0037]
(2) Result
▲ 1 ▼ Blood pressure test
The blood pressure test was performed once every two weeks before administration of the test drug, and the average value of rats in each group was determined.
Table 2 shows the results.
[Table 2]

[0038]
SHR rats (groups A to H), which are spontaneously hypertensive rats, had higher blood pressure values than WKY rats (groups I to J). However, in the SHR rats, compared with the non-administration group (group A), the 6,9,12 g administration group (C to E group) and the 6,9% group (G, H group) started from around 14 weeks of age. Suppression of blood pressure elevation was confirmed. Even more surprisingly, the 6% group (Group G) from the age of 14 weeks and the 9,12 g group (D and E groups) and the 9% group (H group) from the age of 16 weeks (Group C). ), A drop in blood pressure was confirmed from the age of 18 weeks.
The effects of increasing blood pressure and lowering blood pressure tended to be higher in the higher dose group. The reason why the blood pressure lowering effect in the 12 g administration group (group E) was not higher than that in the 9 g administration group (group D) is considered to be the development of a biological curve associated with the efficacious dose. Also, in WKY rats that were originally normotensive, no drop in blood pressure (abnormality) occurred.
[0039]
From the above, it was found that the Pleurotus nebrodensis of the present invention had a dose-dependent effect of preventing and improving hypertension. In addition, since it does not show a blood pressure lowering effect when it is normal blood pressure, it has the effect of bringing blood pressure closer to the normal value rather than simply lowering blood pressure like conventional antihypertensive drugs, so that it can be taken more safely. Understand. In addition, it was found that both dry powders and extracts thereof were effective.
[0040]
▲ 2 ▼ Blood test
All rats were fasted the day before the last day of the experiment, deep anesthetized (Nembutal, 45 mg / kg, ip) the next day, and blood was collected from the left ventricle with a 20G blood collection needle as much as possible.
Using the collected blood, the following items were subjected to a biochemical test (automatic chemical analyzer: Auto Lab, Radio Immunoassay method), and the average value was determined.
Table 3 shows the results.
[0041]
[Table 3]

[0042]
SHR rats (groups A to H) have higher levels of LDL cholesterol, β-lipoprotein, neutral fat, albumin, urea nitrogen, creatinine, uric acid, angiotensin, and total cholesterol than WKY rats (groups I to J). , HDL cholesterol, total protein, A / G ratio, and white blood cell count were low. However, in the SHR rats, marked improvement was confirmed in each item in the administration group (BH group) as compared with the non-administration group (group A).
[0043]
Angiotensin
Angiotensin is an active peptide produced by the action of renin secreted from the glomerulus of the kidney on a renin substrate produced in the liver. Diseases showing high levels include renal vascular hypertension, malignant hypertension and the like. Therefore, the angiotensin value is used as an indicator of hypertension.
Compared with the group A, the angiotensin was decreased in the groups B to H and approached the normal value of the group I, and in particular, was substantially equal to the value of the group I in the groups B to E, G and H. As described above, the effect of preventing and improving hypertension was confirmed also with respect to the angiotensin value.
[0044]
cholesterol
Cholesterol is an essential component as a constituent of the living cell membrane and the like, and is classified into good cholesterol (HDL) and bad cholesterol (LDL). LDL promotes arteriosclerosis, and HDL serves to remove LDL adhering to the vessel wall. Therefore, by measuring these cholesterol levels, it is possible to infer a disease that causes abnormal lipid metabolism.
[0045]
In groups B to H, an increase in HDL and a decrease in LDL were observed in comparison with group A, approaching the values in group I, and in particular, in groups D to E, G, and H, they were almost equal to the values in group I. . The total cholesterol level also approached that of the normal group I. Therefore, it was confirmed that the anti-hypertensive effect has the effect of preventing and improving abnormalities of lipid metabolism.
[0046]
β lipoprotein, neutral fat
The main function of β-lipoprotein is to transport cholesterol from the liver to each organ. Therefore, the β-lipoprotein value is used as an index for a disorder of lipid metabolism.
Compared with the group A, the β-lipoprotein values were seen in the B-H groups in a dose-dependent manner, approaching normal values.
Furthermore, in the BH group as compared with the A group, a decrease in neutral fat was also observed in a dose-dependent manner. Therefore, prevention and improvement of abnormal lipid metabolism were also confirmed in β-lipoprotein and neutral fat levels.
[0047]
Urea nitrogen, creatinine, uric acid
Measurements of urea nitrogen, creatinine, and uric acid are used as indicators of liver and kidney function.
Compared with the group A, the urea nitrogen, creatinine, and uric acid levels decreased in the groups D, E, G, and H and approached the normal values in the group I. Therefore, it was confirmed that the hepatic and renal functions were also improved together with the effect of preventing and improving hypertension.
[0048]
Total protein, albumin, A / G ratio, white blood cell count
Furthermore, in the groups B to H, as compared with the group A, the total protein, the A / G ratio, the leukocyte count, and the albumin level decreased in a dose-dependent manner, and approached the normal group I value. Was.
[0049]
From the above, it was confirmed that the administration of Pleurotus nebrodensis exerts the effect of preventing and improving hypertension, the effect of improving lipid metabolism abnormality, and the effect of improving liver and kidney function. In normal rats, these values do not show any noticeable change (abnormality) upon administration, indicating high safety.
[0050]
2. Hyperlipidemia prevention / improvement agent
The inheritance pattern of Zucker rats adopts an autosomal simple recessive inheritance pattern, and only individuals (fa / fa) having a homologous pathogenic gene (fa gene) are obese (Zucker-fatty rats: (ZUC) -fa / fa), heterozygotes (fa / +) and wild type (+ / +) do not exhibit obesity (Zucker-lean rats: (ZUC) -lean). Zucker-fatty rats become obese due to weight gain and appearance from around 4 weeks of age, and obesity progresses rapidly by around 10 weeks of age, and then progresses gradually thereafter. In addition, it is known to show hyperlipidemia, hyperinsulinemia, hyperleptinemia and abnormal glucose tolerance. Using this Zucker-fatty rat and a Zucker-lean rat as a comparison, tests were carried out on the effects of preventing and improving hyperlipidemia and obesity.
[0051]
(1) Breeding of test rats and administration of test drugs
(1) Preliminary rearing (5-6 weeks old)
Five-week-old male Zucker-fatty rats and male Zucker-lean rats purchased from Japan Charles River Co., Ltd. were bred with white fluorescent lamps in a breeding room adjusted to a room temperature of 22 ± 1 ° C. and a humidity of 60 ± 10%. Light control was performed for 12 hours per day (light period from 7 to 19 o'clock), feed and tap water were freely available, and the animals were preliminarily reared for one week.
After preliminary breeding, the weight of each individual was measured and a glucose tolerance test was carried out. Five rats were taken per group, Zucker fatty rats were placed in groups K to O, and Zucker-lean rats were placed in groups P to Q so that the average values of body weights were almost equal. Classified into groups.
[0052]
(2) Preparation method of Pleurotus nebrodensis extract
As described above.
(3) Test drug administration method (6 to 16 weeks of age)
The test drug was administered to each group of rats in the following manner, and the rats were further bred for 10 weeks until the age of 16 weeks. Each group had free access to feed and tap water. The environment of the breeding room was a temperature of 22 ± 1 ° C., a humidity of 60 ± 10%, and a white fluorescent lamp under lighting for 12 hours a day (light period from 7 to 19).
Each test drug was orally administered, and the test was carried out every day at 10 am, and after the administration, sterile water was freely taken.
[0053]
Group K: Zucker-fatty rat (no administration)
Group L: Zucker-fatty rat (administration of 3 g extract) ... 10 ml of the 3 g extract is administered per kg of body weight per day.
Group M: Zucker-fatty rat (administration of 6 g extract) ... 10 ml of the 6 g extract is administered per kg of body weight per day.
Group N: Zucker-fatty rat (administration of 9 g extract) ... 10 ml of the 9 g extract is administered per kg of body weight per day.
Group O: Zucker-fatty rats (administration of 12 g extract) ... 10 ml of the 12 g extract is administered per kg of body weight per day.
Group P: Zucker-lean rat (no administration)
Group Q: Zucker-lean rats (administration of 9 g extract) ... 10 ml of the 9 g extract is administered per kg of body weight per day.
[0054]
(2) Result
▲ 1 ▼ Weight fluctuation
The body weight was measured once a week before administration of the test drug, and the average value of rats in each group was determined. Table 4 shows the results.
[Table 4]

[0055]
The weight of the Zucker-fatty rats (KO group) increased more than that of the Zucker-lean rats (PQ group). However, in the Zucker-fatty rats, suppression of body weight gain was observed at about 10 weeks of age in the administration group (groups L to O) as compared to the non-administration group (group K). The inhibitory effect on weight gain was higher in the higher dose group.
In addition, suppression of body weight gain within a normal range was also confirmed in normal-weight Zucker-lean rats.
From the above, it was found that Pleurotus nebrodensis of the present invention has an effect of suppressing obesity in a dose-dependent manner, and has a moderate fat deposition suppressing effect even in a normal case.
[0056]
▲ 2 ▼ Blood test
All rats were fasted the day before the last day of the experiment, deep anesthetized (Nembutal, 45 mg / kg, ip) the next day, and blood was collected from the left ventricle with a 20G blood collection needle as much as possible.
Using the collected blood, the following items were subjected to a biochemical test (automatic chemical analyzer: Auto Lab, Radio Immunoassay method), and the average value was determined.
Table 5 shows the results.
[0057]
[Table 5]

[0058]
From Table 5, Zucker-fatty rats (Groups K to O) were compared with Zucker-lean rats (Groups P to Q) in total cholesterol, free cholesterol, β-lipoprotein, neutral fat, phospholipid, total lipid, It was found that the values of urea nitrogen, AST (GOT), AST (GPT), and insulin were high, and the values of HDL cholesterol, free fatty acids, creatinine, and uric acid were low. However, in the Zucker-fatty rat, a remarkable preventive improvement effect was confirmed in each item in the administration group (LO group) as compared with the non-administration group (group K).
[0059]
Neutral fats, free fatty acids, phospholipids, total lipids
Compared with the K group, in the L to O groups, the neutral fat, phospholipid, and total lipid tended to decrease and the free fatty acids tended to increase in a dose-dependent manner. Free fatty acids are mostly neutral fats accumulated in adipose tissue and released into the blood after being decomposed.They are burned in major organs such as heart muscle, skeletal muscle, and kidneys, and used as energy sources in peripheral tissues. You. Thus, this result indicates an improvement in obesity. In addition, compared with the remarkable decrease in neutral fat and total lipid, the phospholipid which is important as a component of the cell membrane did not decrease much and did not fall below the normal P group value. The above-mentioned effects were also confirmed in normal rats within a normal range. From the above, the effect of preventing and improving hyperlipidemia and obesity was confirmed.
[0060]
cholesterol
As described above, the cholesterol level is an index of lipid metabolism abnormality.
Compared with the K group, in the L to O groups, HDL, which is a good cholesterol, increased, while total cholesterol and free cholesterol tended to decrease in a dose-dependent manner. As described above, the effect of improving lipid metabolism in cholesterol levels was confirmed.
[0061]
β lipoprotein
As described above, the β-lipoprotein value is used as an index of a disorder of lipid metabolism.
In the L to O groups, the β lipoprotein value decreased in a dose-dependent manner as compared with the K group, approaching the normal P group value. Therefore, the improvement of lipid metabolism was confirmed also in the β lipoprotein value.
[0062]
Insulin
Insulin is a hormone secreted by β cells of the pancreas, and lowers blood glucose through promotion of glucose uptake in muscle and adipose tissue and suppression of glucose release in the liver. It is also involved in glycogen accumulation, fat accumulation, ketone body lowering, protein synthesis and the like. Therefore, the measurement of insulin serves as an index of abnormal glucose metabolism.
Compared with the K group, the L to O groups showed a dose-dependent decrease in insulin level, approaching the normal P group. Therefore, improvement of glucose metabolism was also confirmed along with the effect of preventing and improving obesity.
[0063]
Urea nitrogen, creatinine, uric acid, AST
Urea nitrogen, creatinine, uric acid, and AST are used as indicators of liver and kidney function.
Compared with the K group, in the L to O groups, the urinary nitrate and AST values tend to decrease and the creatinine and uric acid values tend to increase in a dose-dependent manner. In the O group, the values are almost the same as those in the normal P group. It became. Therefore, improvement of liver and kidney function was confirmed along with the effect of preventing and improving obesity.
[0064]
The administration of the extract did not cause any abnormality in the originally normal total protein, albumin, A / G ratio, and the like.
From the above, by the administration of Pleurotus nebrodensis, the effect of preventing and improving hyperlipidemia, the effect of preventing and improving obesity, the effect of improving lipid metabolism disorder, the effect of improving glucose metabolism, and the effect of improving liver and kidney function are exhibited. Was confirmed.
[0065]
3. Human clinical test
Next, a human clinical test using the agent for preventing or improving disease of the present invention will be described.
Preparation of test reagent (Fig. 1)
Pleurotus nebrodensis (deposited with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology on May 22, 2003. Indication for identification: Pleurotus nebrodensis No. 17) 100 g of dry powder and 1 L of sterilized water were added to an Erlenmeyer flask, and 95 ° C. After extraction in a hot water bath for 1 hour, the mixture was allowed to cool to room temperature. Separate into a first filtrate and a residue by vacuum filtration. 500 mL of sterilized water is added to the residue, and the mixture is extracted in a hot water bath at 95 ° C. for 1 hour, cooled to room temperature, and separated into a second filtrate and a residue by filtration under reduced pressure. The first filtrate and the second filtrate are mixed and concentrated at 60 ° C. under reduced pressure. Further, after sterilizing at 95 ° C for 10 minutes, the mixture is concentrated at 60 ° C until the solid content becomes 50%. An excipient of 50% with respect to the solid content is added, heat-dissolved, and lyophilized to powder. The mixture is sieved through a 60 mesh sieve and granulated by extrusion granulation.
[0066]
Three men, 33, 42 and 50 years old, took the test reagent prepared by the above method daily at 6 g / day, before taking, 1 month after taking, 3 months after taking, 6 months after taking. Monthly blood tests were performed each month. Table 6 shows the results.
[0067]

[0068]
The normal values are 120-220 mg / dl for total cholesterol, 41-84 mg / dl for HDL cholesterol, 30-180 mg / dl for neutral fat, and 150-500 mg / dl for β-lipoprotein. Before taking the drug, all men had total cholesterol, triglyceride, and β-lipoprotein values exceeding normal values, and HDL was below the normal value except for one.
However, taking Pleurotus nebrodensis dry powder improved these values, and at 6 months of taking all three were within normal limits. Therefore, it was confirmed that Pleurotus nebrodensis has a preventive and ameliorating effect on humans as well. Also, no side effects such as diarrhea, vomiting and urination abnormalities occurred at all by taking the drug.
[0069]
【The invention's effect】
The Pleurotus nebrodensis according to the present invention, in particular, was deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and the indication for identification is Pleurotus nebrodisis No. Pleurotus nebrodensis 17 has an excellent disease prevention and improvement effect.
Further, according to the method for cultivating Pleurotus nebrodensis according to the present invention, a sufficient amount of fruiting bodies can be harvested in a short period of time by applying an electric impulse immediately after the completion of the culture.
[Brief description of the drawings]
FIG. 1 is a flowchart showing a method for preparing a test reagent according to the present invention.

Claims (8)

Deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, and displayed for identification in Pleurotus nebrodensis No. 17. Pleurotus nebrodensis strain. A method for cultivating Pleurotus nebrodensis, comprising the following steps (a) to (d).
(A) a step of inoculating a medium with a seed of Pleurotus nebrodensis;
(B) a step of, after the step (a), culturing under conditions of a temperature of 20 to 30 ° C. to spread mycelia in the medium;
(C) a step of applying an electrical impulse of 5 to 60 kV after the step (b).
(D) a step of generating fruiting bodies at a temperature of 10 to 20 ° C. after the step (c). 3. The method for cultivating Pleurotus nebrodensis according to claim 2, wherein in the step (d), after applying a low-temperature stimulus of -1 to 2 ° C, the temperature is set to 10 to 20 ° C. In the cultivation method according to claim 2 or 3, in the step (a), the composition was deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Technology, and the display for identification is Pleurotus nebrodensis No. 1. 17. Pleurotus characterized by inoculating a seed of Pleurotus nebrodensis 17
Cultivation method of nebrodensis. A disease prevention / improvement agent containing Pleurotus nebrodensis as a main component. The disease prevention / improvement agent according to claim 5, comprising Pleurotus nebrodensis dry powder and / or a hot water extract thereof. 7. The agent for preventing or ameliorating a disease according to claim 5 or 6, wherein the Pleurotus nebrodensis strain was deposited on May 22, 2003 with the Institute of Biotechnology and Industrial Technology, and the indication for identification is Pleurotus nebrodensis No. 17. A disease prevention / amelioration agent, which is Pleurotus nebrodensis strain No. 17. The disease prevention / amelioration agent according to any one of claims 5 to 7, wherein the disease is one or more selected from hypertension, hyperlipidemia, and obesity.

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