JP6728344B2 - 桔梗抽出物を含有する筋肉疾患の予防及び治療用又は筋機能改善用組成物 - Google Patents
桔梗抽出物を含有する筋肉疾患の予防及び治療用又は筋機能改善用組成物 Download PDFInfo
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- JP6728344B2 JP6728344B2 JP2018514758A JP2018514758A JP6728344B2 JP 6728344 B2 JP6728344 B2 JP 6728344B2 JP 2018514758 A JP2018514758 A JP 2018514758A JP 2018514758 A JP2018514758 A JP 2018514758A JP 6728344 B2 JP6728344 B2 JP 6728344B2
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- muscular dystrophy
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Description
本発明は、桔梗抽出物を有効成分として含む筋肉疾患の予防又は治療用薬学組成物を提供する。
但し、下記の実施例は本発明を例示するのみであり、本発明の内容が下記の実施例に限定されるものではない。
桔梗抽出物の製造
<1-1>桔梗メタノール抽出物の製造
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料100gを100%メタノール1Lに入れて48時間常温で抽出した。抽出した試料はワットマン(Whatman)2番濾紙で濾過して、濾過された抽出液を真空回転濃縮機で濃縮して溶媒成分を除去した後、桔梗メタノール抽出物を得た。
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料100gを100%エタノール1Lに入れて48時間常温で抽出した。抽出した試料はワットマン(Whatman)2番濾紙で濾過して、濾過された抽出液を真空回転濃縮機で濃縮して溶媒成分を除去した後、桔梗エタノール抽出物を得た。
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料100gを100%酢酸エチル1Lに入れて48時間常温で抽出した。抽出した試料はワットマン(Whatman)2番濾紙で濾過して、濾過された抽出液を真空回転濃縮機で濃縮して溶媒成分を除去した後、桔梗酢酸エチル抽出物を得た。
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料100gを100%ヘキサン1Lに入れて48時間常温で抽出した。抽出した試料はワットマン(Whatman)2番濾紙で濾過して、濾過した抽出液を真空回転濃縮機で濃縮して溶媒成分を除去した後、桔梗ヘキサン抽出物を得た。
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料100gを水1Lに入れて100℃で2時間攪拌しながら抽出した。抽出された試料はワットマン(Whatman)2番濾紙で濾過して、濾過した抽出液を真空回転濃縮機で濃縮して溶媒成分を除去した後、桔梗熱水抽出物を得た。
乾燥した桔梗地下部をミキサーで粉砕し、粉砕した桔梗試料1gと18%エタノール76mLをポリエチレン(polyethylene)パックに入れて密封した後、超高圧抽出装置(Frescal MFP-7000; Mitsubishi Heavy Industries、Tokyo、Japan)を用いて抽出した。超高圧抽出条件は抽出圧力が320MPa、抽出時間は5minであった。抽出された試料はワットマン(Whatman)2番濾紙で濾過して、濾過された抽出液を真空回転濃縮機で濃縮して溶媒成分を除去することにより、桔梗超高圧抽出物を得た。
乾燥した桔梗をミキサーで粉砕し、粉砕した桔梗試料1gを試料カートリッジに充填して超臨界流体抽出装置(SFX 3560、Isco Inc.、Lincoln、NE、USA)を用いて抽出した。超臨界流体抽出条件は、抽出圧力が40MPa、抽出温度は50℃、超臨界二酸化炭素の流速は60mL/min、抽出時間は60minであった。超臨界流体抽出が完了すると、抽出装置の圧力を下げて超臨界流体状態を解除して、桔梗超臨界流体抽出物を得た。
乾燥した桔梗をミキサーで粉砕し、粉砕した桔梗試料1gを蒸留水10mLに入れて亜臨界流体抽出装置(DIONEX Accelerated Solvent Extractor 100、DIONEX co.,USA)を用いて抽出した。亜臨界流体抽出条件は2.5MPa、抽出温度150℃、抽出時間15分の条件下で亜臨界抽出を行った。抽出された試料はワットマン2番濾紙で濾過して濾過された抽出液を−40℃で凍結乾燥して、桔梗亜臨界流体抽出物を得た。
桔梗抽出物の筋タンパク質合成の増加効果
筋肉細胞であるL6 myoblast(ATCC、Manassas、VA、USA)を10%fetal bovine serum(FBS; Hyclone、Logan、UT、USA)が含有されたDulbecco's modified Eagle's Media(DMEM; Hyclone)と共に6ウェルプレートに1X105cell/mlになるように入れた。細胞密度が約80〜85%になったとき、ウェルの培地を除去して2% horse serum(HS; Hyclone)が含有されたDMEMに実施例1-1乃至1-5の桔梗抽出物をそれぞれ10μg/mLの濃度で溶解して細胞に処理してmyotube分化を誘導した。このとき、試料の代わりに0.01%DMSOを処理した群を対照群とした。この過程を2日ずつ6日間進行して分化させた後、proteinase inhibitor cocktailが含まれたNP-40緩衝溶液(ELPIS-Biotech、Daejeon、Korea)で溶解させた。緩衝溶液に溶解された細胞を1.5mlチューブ(tube)に移して13,000rpmで10分間遠心分離して上澄み液だけを採った。上澄み液をブラッドフォード(Bradford、Bio-Rad Laboratories Inc.,Hercules、CA、USA)法を用いて定量した。定量されたタンパク質を5分間煮沸後、10%SDS-PAGEで電気泳動して分離し、分離されたタンパク質をニトロセルロース膜にトランスファーした。p-mTOR 1次抗体(Cell signaling technology、Beverly、MA、USA)を2.5%bovine serum albumin(BSA)に1:1000の割合で希釈して、ニトロセルロース膜にトランスファーされたタンパク質と20時間常温で反応させた。1次抗体を反応させた後Tris-buffer Saline Tween20(TBST)を用いて、ニトロセルロース膜を10分間3回洗浄した。洗浄後、1次抗体を認知するhorseradish peroxidaseが接合されたanti-rabbit 2次抗体(Bethyl Laboratories、Inc.,Montgomery、TA、USA)を2.5%BSAに1:5000になるように希釈して、ニトロセルロース膜と2時間常温で反応させ、TBSTを用いて10分ずつ3回洗浄した。Protein bandはECL western blotting detection reagents(Amersham、Tokyo、Japan)を用いて発色し、G; BOX EF imaging system(Syngene、Cambridge、UK)を用いて発色されたProtein bandを確認した。その結果を図1に示した。
桔梗メタノール抽出物の筋タンパク質合成の増加効果
前記実施例1-1の桔梗メタノール抽出物を1及び10μg/mLの濃度で前記実施例2と同じ方法で実験を行った。p-mTOR 1次抗体(Cell signaling technology)とp-p70S6K抗体(Santa Cruz Biotechnology、Santa Cruz、CA、USA)を処理してprotein bandを確認した。
その結果、図2に示した通り、桔梗メタノール抽出物を処理することにより、L6筋肉細胞においてp-mTORとp-p70S6Kの発現量が増加したことを確認できた。これは、本発明の桔梗メタノール抽出物が筋肉細胞内で筋肉の生成を増加させる能力が優れていることを意味する。
桔梗抽出物の筋肉分化活性
筋肉細胞であるL6 myoblast(ATCC)を、10%FBS(Hyclone)が含まれたDMEM(Hyclone)と共に6ウェルプレートに2X105cell/mlになるように入れた。細胞密度が約80〜85%になったとき、ウェルの培地を除去し、2%HS(Hyclone)が含まれたDMEM(Hyclone)に前記実施例1-2乃至実施例1-5で製造した桔梗ヘキサン抽出物、酢酸エチル抽出物、エタノール抽出物、熱水抽出物を10μg/mLの濃度で溶解した後、細胞に処理してmyotube分化を誘導した。このとき、試料の代わりに0.01%DMSOを処理した群を対照群とした。この過程を2日ずつ6日間進行して分化させた後、TRIzol試薬(Invitrogen、Carlsbad、CA、USA)を用いて総RNAを分離した。分離した総RNAはナノドロップ(NanoDrop 1000; Thermo Fisher Scientific Inc.,MA,USA)を用いて定量した。定量された16μLのRNAをReverse Transcriptase Premix(ELPIS-Biotech)とPCR装置(Gene Amp PCR System 2700; Applied Biosystems、MA、USA)を用いて、42℃で55分、70℃で15分の条件でcDNAに合成した。16μLの生成されたcDNAのうち4μLのcDNA、下記の特定のプライマー(Bioneer、Daejeon、Korea)及びPCR premix(ELPIS-Biotech)で、95℃で30秒、60℃で1分、72℃で1分を30回繰り返してPCRを行った。
Forward primer:5'-TTTCGACTCACCAGACCTGC-3'(配列番号1)
Reverse primer:5'-CAGAGCCTGCAGACCTTCAA-3'(配列番号2)
Forward primer:5'-TTTCGCACCTGATGGACCTG-3'(配列番号3)
Reverse primer:5'-CTTTCTTGAGCCTGCGCTTC-3'(配列番号4)
Forward primer:5'-AGCCATGTACGTAGCCATCC-3'(配列番号5)
Reverse primer:5'-CTCTCAGCTGTGGTGCTGAA-3'(配列番号6)
その結果、図3に示した通り、桔梗ヘキサン抽出物、酢酸エチル抽出物、エタノール抽出物、熱水抽出物を処理することにより、L6筋肉細胞からMyoD及びmyogeninのmRNA発現が増加することが分かった。これは、本発明の桔梗抽出物が筋肉細胞内で筋肉分化を促進する能力が優れていることを意味する。
桔梗メタノール抽出物による筋肉分化活性
前記実施例4と同じ方法で筋肉細胞のL6 myoblast(ATCC)を培養した後、2%HS(Hyclone)が含まれたDMEM(Hyclone)に前記実施例1-1で製造した桔梗メタノール抽出物を1と10μg/mLの濃度で溶解した後、細胞に処理してmyotube分化を誘導した。このとき、試料の代わりに0.01%DMSOを処理した群を対照群とした。この過程を2日ずつ6日間進行して分化させた後、実施例4と同じ方法でRT-PCRを行った。
桔梗の筋タンパク質分解抑制活性
筋肉細胞であるL6 myoblast(ATCC)を、10%FBS(Hyclone)が含まれたDMEM(Hyclone)と共に6ウェルプレートに2X105cell/mlになるように入れた。細胞密度が約80〜85%になったとき、ウェルの培地を除去して2%HS(Hyclone)が含まれたDMEM(Hyclone)を細胞に処理してmyotube分化を誘導した。2日に1度ずつ新たな培地と交換して合計6日間分化を行った。分化した後、50ng/mL tumor necrosis factor alpha(TNF-α; PeproTech、Rocky Hills、NJ、USA)が含まれたDMEM(Hyclone)に前記実施例1-2乃至実施例1-5で製造した桔梗ヘキサン抽出物、酢酸エチル抽出物、エタノール抽出物、熱水抽出物を10μg/mLの濃度で溶解した後、細胞に処理した。6時間後、TRIzol試薬(Invitrogen)を使用して総RNAを分離した。分離した総RNAはナノドロップ(NanoDrop 1000; Thermo Fisher Scientific Inc)を用いて定量した。定量された16μLのRNAをReverse Transcriptase Premix(ELPIS-Biotech)とPCR装置(Gene Amp PCR System 2700; Applied Biosystems)を用いて42℃で55分、70℃で15分の条件でcDNAで合成した。16μLの生成されたcDNAのうち4μLのcDNA、下記特定のプライマー(Bioneer)とPCR premix(ELPIS-Biotech)で、95℃で30秒、60℃で1分、72℃で1分を30回繰り返してPCRを行った。
Forward primer:5'-CCCTGAGTGGCATCGCCCAA-3'(配列番号7)
Reverse primer:5'-AGGTCCCGCCCATCGCTCA-3'(配列番号8)
Forward primer:5'-GAAATGCTATGCAGAACCTG-3'(配列番号9)
Reverse primer:5'-ATTCCTGCTTGTAGATGTCG-3'(配列番号10)
Forward primer:5'-AGCCATGTACGTAGCCATCC-3'(配列番号11)
Reverse primer:5'-CTCTCAGCTGTGGTGCTGAA-3'(配列番号12)
その結果、図5に示した通り、桔梗を処理することにより、L6筋肉細胞からgin-1及びMuRF-1のmRNA発現が減少することが分かった。これは、本発明の桔梗が筋肉細胞内で筋タンパク質分解を抑制する能力が優れていることを意味する。
桔梗高圧抽出物による筋肉生成活性
前記実施例1-6で製造した桔梗超高圧抽出物、実施例1-7で製造した桔梗超臨界流体抽出物、実施例1-8で製造した桔梗亜臨界流体抽出物を20ppmの濃度で前記実施例2と同じ方法で筋肉細胞に処理した。ECLウェスタンブロット検出試薬(western blotting detection reagents; Amersham、Tokyo、Japan)を使用してp-mTORタンパク質バンドを発色し、G; BOX EF imaging system(Syngene、Cambridge、UK)を用いて発色されたタンパク質バンドのdensityを測定した。このとき、対照群タンパク質バンドのdensityを100%にして、試料を処理した実験群の相対的なタンパク質バンドのdensityをパーセント(%)で示した。
動物モデルから筋肉量増加効果を確認
5週齢のWistar ratを1週間適応させ、TNF-α 100ng/gを2週間供給して筋萎縮を誘導させた後、体重を基に無作為的に群を配置してそれぞれ8匹ずつ合計3群に分けて実験に利用した。実験群には、実施例1-2で製造した桔梗エタノール抽出物500mg/kg体重、実施例1-5で製造した桔梗熱水抽出物500mg/kg体重の濃度で0.25%カルボキシメチルセルロース(carboxymethylcellulose)に懸濁して1日1回ずつ8週間一定の時間に投与した。このとき、対照群には実験群が摂取するのと同じ量の0.25%カルボキシメチルセルロースにTNF-αを投与した群を使用した。
<1−1>健康食品の製造
前記実施例1の桔梗抽出物 1000mg、ビタミンA酢酸塩 70μg、ビタミンE 1.0mg、ビタミンB1 0.13mg、ビタミンB2 0.15mg、ビタミンB6 0.5mg、ビタミンB12 0.2μg、ビタミンC 10mg、ビオチン10μg、ナイアシンアミド 1.7mg、葉酸 50μg、パントテン酸カルシウム 0.5mg、硫酸第一鉄 1.75mg、酸化亜鉛 0.82mg、炭酸マグネシウム 25.3mg、第1リン酸カリウム 15mg、第2リン酸カルシウム 55mg、クエン酸カリウム 90mg、炭酸カルシウム 100mg、塩化マグネシウム 24.8mgを混合して製造することができ、その配合比を任意に変更して実施してもよく、通常の健康食品の製造方法により前記の成分を混合した後、顆粒を製造して通常の方法により健康食品組成物の製造に使用することができる。
前記実施例1の桔梗抽出物 1000mg、クエン酸 1000mg、オリゴ糖 100g、梅濃縮液 2g、タウリン 1gに精製水を加えて全体 900mlとし、通常の健康飲料製造方法により前記の成分を混合した後、約1時間の間85℃で攪拌加熱した後、作製された溶液を濾過して滅菌された2Lの容器に取得して密封滅菌した後、冷蔵保管して健康飲料組成物の製造に使用することができる。
ガムベース 20重量%、砂糖 76.9重量%、香料 1重量%及び水 2重量%と、実施例1の桔梗抽出物 0.1重量%を配合して通常の方法でチューインガムを製造した。
砂糖 60重量%、水飴 39.8重量%及び香料 0.1重量%と実施例1の桔梗抽出物 0.1重量%を配合して通常の方法でキャンディーを製造した。
小麦粉(薄力1級) 25.59重量%、小麦粉(中力1級) 22.22重量%、精白糖 4.80重量%、食塩 0.73重量%、グルコース 0.78重量%、パームショートニング 11.78重量%、アンモニウム 1.54重量%、重曹 0.17重量%、亜硫酸水素ナトリウム 0.16重量%、米粉 1.45重量%、ビタミンB 0.0001重量%、ミルク香料 0.04重量%、水 20.6998重量%、全脂粉乳 1.16重量%、代用粉乳 0.29重量%、第1リン酸カルシウム 0.03重量%、散布塩 0.29重量%及び噴霧油 7.27重量%と、前記実施例1の桔梗抽出物 1.0重量%を配合して通常の方法でビスケットを製造した。
<2−1>散剤
前記実施例1の桔梗抽出物を50mg、結晶セルロース 2gを混合した後、通常の散剤の製造方法により機密包に充填して散剤を製造した。
前記実施例1の桔梗抽出物を50mg、結晶セルロース 400mg、ステアリン酸マグネシウム 5mgを混合した後、通常の錠剤の製造方法により打錠して錠剤を製造した。
前記実施例1の桔梗抽出物を30mg、乳清タンパク質 100mg、結晶セルロース 400mg、ステアリン酸マグネシウム 6mgを混合した後、通常のカプセル剤の製造方法によりゼラチンカプセルに充填してカプセル剤を製造した。
<3−1>栄養化粧水(ミルクローション)
前記実施例1の桔梗抽出物を下記表3の栄養化粧水剤形の割合の通り通常の方法により栄養化粧水を製造した。
前記実施例1の桔梗抽出物を下記の表4の柔軟化粧水製剤の比率の通り通常の方法により柔軟化粧水を製造した。
前記実施例1の桔梗抽出物を下記表5の栄養クリーム剤形比率の通り通常の方法により栄養クリームを製造した。
前記実施例1の桔梗抽出物を下記の表6のマッサージクリーム剤形比率の通り通常の方法によりマッサージクリームを製造した。
桔梗抽出物を下記表5のパック剤形比率の通り通常の方法によりパックを製造した。
前記実施例1の桔梗抽出物を下記の表8のゲル剤形比率の通り通常の方法によりジェルを製造した。
Claims (8)
- 桔梗抽出物を有効成分として単独で含む筋肉疾患の予防又は治療用薬学組成物であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である薬学組成物。 - 水、炭素数1乃至6の有機溶媒、亜臨界流体、超臨界流体及びこれらの混合物からなる群より選ばれた一つ以上の溶媒による桔梗抽出物を有効成分として単独で含む筋肉疾患の予防又は治療用薬学組成物の製造方法であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である方法。 - 前記炭素数1乃至6の有機溶媒は、炭素数1乃至6のアルコール(alcohol)、アセトン(acetone)、エーテル(ether)、ベンゼン(benzene)、クロロホルム(chloroform)、酢酸エチル(ethyl acetate)、塩化メチレン(methylene chloride)、ヘキサン(hexane)、シクロヘキサン(cyclohexane)及び石油エーテル(petroleum ether)からなる群より選ばれたことを特徴とする請求項2記載の方法。
- 100MPa乃至1000MPaの超高圧条件下で桔梗を抽出して収得した桔梗抽出物を有効成分として単独で含む筋肉疾患の予防又は治療用薬学組成物の製造方法であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である方法。 - 桔梗抽出物を有効成分として単独で含む筋肉疾患の予防又は筋機能改善用食品組成物であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である食品組成物。 - 桔梗抽出物を有効成分として単独で含む筋機能改善用化粧料組成物であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である化粧料組成物。 - 桔梗抽出物の筋肉疾患の予防又は治療剤製造のための使用であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である使用。 - 桔梗抽出物を、これを必要とする動物(ヒトを除く)に有効量で投与することを特徴とする筋肉疾患の予防又は治療方法であって、
前記筋肉疾患は、緊張減退症(atony)、筋萎縮症(muscular atrophy)、筋異栄養症(muscular dystrophy)、筋肉退化、筋硬直症、筋ジストロフィー、筋萎縮性軸索硬化症、筋無力症、悪液質(cachexia)及び筋肉減少症(sarcopenia)からなる群より選ばれる1つ以上である方法。
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