JP5753542B2 - 虎杖根ブタノール分画物またはエチルアセテート分画物を用いた肥満治療及び予防用組成物と機能性食品 - Google Patents
虎杖根ブタノール分画物またはエチルアセテート分画物を用いた肥満治療及び予防用組成物と機能性食品 Download PDFInfo
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Description
(発明を実施するための形態)
<実施例1>虎杖根水抽出物製造
実験に使用した虎杖[Polygonum cuspidatum Sieb.et Zucc.=Reynotria japonica Houtt.;Polygonaceae]の根茎(虎杖根)は、大田に位置した百済堂漢方薬局で購入して(にちへんに景)園大生命科学科キム・ズファン教授が同定した。証拠標本(no.KIOM−POCU1)は、韓国漢方医学研究院糖尿合併症研究センターの標本室に保管中である。
<実施例2>虎杖根エチルアルコール抽出物製造
陰干して細切した虎杖根6.8kgをエチルアルコール36lに入れて、抽出容器で常温状態で24時間3回抽出した後、40℃の減圧下で濃縮させてエチルアルコール抽出物を得た。前記抽出液をろ過した後、減圧状態で濃縮した。ここで、構成成分の分解及び加水分解を防止できるように濃縮時の温度を40〜45℃以下に維持した。その結果、エチルアルコール抽出物580gを得た。
<実施例3>虎杖根ヘキサン分画物製造
実施例2で得た虎杖根エチルアルコール抽出物を蒸留水2lに懸濁させた後、図1に示されたようにノルマルヘキサン2lを用いて3回反復抽出してノルマルヘキサン層46gを得た。
<実施例4>虎杖根エチルアセテート分画物製造
実施例2で得た虎杖根エチルアルコール抽出物を蒸留水2lに懸濁させた後、図1に示されたようにノルマルヘキサン2lを用いてノルマルヘキサン層と水層を分離した。このようにして得た水層にエチルアセテート2lを用いて3回反復抽出してエチルアセテート層250gを得た。
<実施例5>虎杖根ブタノール分画物製造
実施例2で得た虎杖根エチルアルコール抽出物を蒸留水に懸濁させた後、図1に示されたようにノルマルヘキサン、エチルアセテート(EtOAc)、ノルマルブタノール(n−BuOH)で順次に系統分離をして減圧下で濃縮して凍結乾燥してノルマルブタノール分画物150gを得た。
<実施例6>虎杖根エチルアルコール抽出物のエチルアセテート分画物からレスベラトロ−ルの製造
虎杖根エチルアセテート分画物(250g)をシリカゲルカラムクロマトグラフィー(70−230mesh、φ12×60cm、メチレンクロライド/メタノール=100:0→0:100)を実施して総10個の1次小分画に分けた(F1〜F10)。その中の分画物F5(4.9g)をクロロホルムとメタノール混合溶媒で中圧液体クロマトグラフィー(クロロホルム100%、40分;98%、30分;95%、40分;90%、60分;Hi−Flash 2Lシリカゲルカラム、φ12×60cm、Yamazen YFLC AI−580、日本)を実施して、トランスレスベラトロ−ルを純粋分離して構造を同定した。
1H-NMR (300 MHz, acetone-d6) δ: 7.36 (2H, d, J = 8.4 Hz, H-2'/H-6'), 7.02 (1H, d, J = 16.4 Hz, H-8), 6.78 (2H, d, J = 8.4 Hz, H-3'/H-5'), 6.74 (1H, d, J = 16.4 Hz, H-7), 6.51 (2H, d, J = 2.0 Hz, H-2/H-6), 6.25 (1H, t, J = 2.0 Hz, H-4). 13C-NMR (75 MHz, acetone-d6) δ: 159.6 (C-3'/C-5'), 158.2 (C-4), 141.0 (C-1'), 130.1 (C-1), 129.2 (C-8), 128.8 (C-2/C-6), 126.9 (C-7), 116.5 (C-3/C-5), 105.8 (C-2'/C-6'), 102.8 (C-4')
虎杖根エチルアルコール抽出物とその抽出物のヘキサン分画物、エチルアセテート分画物、ブタノール分画物と水抽出物及びレスベラトロ−ルを用いて、試験管内でブタ膵臓脂肪分解酵素活性抑制実験を実施した。
<実験例2>動物での脂肪エマルジョン短期間吸収能力分析
虎杖根エチルアルコール抽出物とその抽出物のヘキサン分画物、エチルアセテート分画物、ブタノール分画物と水抽出物及びレスベラトロ−ルが、ネズミの腸管で脂肪が分解されて吸収される程度を抑制する能力を測定した。
<実験例3>抗肥満効能指標である脂肪生成(Anti−lipogenesis)抑制効能
虎杖根ブタノール分画物(以下POCU−1bとする。)の体重増加抑制効能及び脂肪生成、脂肪肝、高脂血症、インスリン分泌などに及ぼす効能を検証した。
<3−1>体重変化観察
高脂肪飼料のみを投与した群は持続的に体重が増加したが、虎杖根ブタノール分画物 1.0%混合群は有意に体重増加が抑制された(*p<0.01)(図2参照)。体重増加程度は、高脂肪飼料投与群が10.9gで、ゼニカル投与群は6.1gであるが、虎杖根ブタノール分画物混合群は3.6gでゼニカルよりも優れた体重増加予防効果が示された($,*p<0.01)(図3参照)。
<3−2>各部位脂肪増加予防効能観察
脂肪組織の増加有無を分析するために、生殖器周囲脂肪(左/右)、腎臓周囲脂肪(左/右)、皮下脂肪(左/右)及び褐色脂肪を注意深く摘出して生理食塩水で洗浄した後、ろ過紙で水分を除去した後、脂肪組織の重量を測定した。
<3−3>脂肪細胞の大きさ抑制効能観察
摘出した脂肪組織を10%ホルマリンに固定した後、パラフィン包埋してスライド組織を準備した後、H&E染色をして光学顕微鏡下で脂肪細胞の大きさを分析した。図7に見られるように、高脂肪食餌群は脂肪細胞の大きさが顕著に増加したが(125μm)、虎杖根ブタノール分画物投与群は正常な水準で(61μm)維持され、ゼニカル(75μm)より効能がさらに優れていた(#p<0.01)(図7参照)。
<3−4>抗高脂血症効能観察
試験終了後、実験動物を16時間絶食させて、剖検時に腹大動脈から血液を採取して 3,000rpmで10分間遠心分離して血清及び血漿を分離した。分離した血清を用いて脂肪代謝関連指標である中性脂肪、高密度脂質タンパク質(HDL)、低密度脂質タンパク質(LDL)、総コレステロールを血清自動分析機(Hitachi 7060,日本)を用いて測定した。
<3−5>脂肪肝に予防効能
脂肪が増加するにつれて脂肪にも脂肪が蓄積して脂肪肝に進展する。脂肪肝に進展すると蓄積された脂肪によって肝臓本来の色相である鮮紅色が消える。
<3−6>インスリン耐性予防効能観察
肥満は、インスリン耐性を誘発して自動的に糖尿病に進展する。虎杖根ブタノール分画物(1%)投与で、インスリン耐性が有意に(#p<0.01)予防されたことが確認された(図17参照)。
<3−7>脂肪生成マーカー(aP2)発現変化
肝組織からmRNAを抽出した後、脂肪生成(adipogenesis)マーカーであるaP2プライマーを用いて、RT−PCRを実施した。図19のように脂肪生成(lipogenesis)マーカーであるaP2 mRNAをRT−PCRを通じて分析した結果、高脂肪食餌群でaP2マーカーが1.4倍以上増加し、虎杖根ブタノール分画物1%給餌群では有意に正常水準に減少した(*p<0.01)。これは、高脂質飼料による脂肪生成が虎杖根ブタノール分画物によって抑制されたことを示す。
<実験例4>虎杖根ブタノール分画物の脂肪分解効能(肥満治療効能)
<4−1>体重減少変化観察
虎杖根ブタノール分画物(POCU−1b)の高脂肪食餌で誘発された肥満ネズミの体重減少及び脂肪分解に及ぼす影響を調べるために、高脂肪飼料を薬物供給前に10週間供給して高度の肥満を誘導した後、虎杖根ブタノール分画物(0.1%、1.0%)を投与して体重の変化を観察した。虎杖根ブタノール分画物(1%)給餌によって、ゼニカルよりも効能が優れ有意な体重減少治療効能を示した(*,**p<0.01)(図20参照)。
<4−2>各部位の脂肪増加予防効能観察
図21〜図24に示したように、各部位別の脂肪重さを測定した結果、生殖器周囲脂肪は高脂肪食餌群で約11gで正常食餌群6.2gに比べて約2倍増加した一方、虎杖根ブタノール分画物(1%)給餌群では6.3gで、1%給餌群では有意に脂肪重さが減少した(p<0.01)。腎臓周囲脂肪及び皮下脂肪の場合にもこのような同一な結果が示され、褐色脂肪の場合虎杖根ブタノール分画物0.1%、1.0%給餌群ですべて有意な脂肪重さの減少が観察された(p<0.01)。
<4−3> 脂肪細胞の大きさ抑制効能観察
剖検時に摘出した脂肪組織を10%中性ホルマリンに固定した後、パラフィン包埋してスライド組織を準備した後、H&E染色をして光学顕微鏡下で脂肪細胞の大きさを分析した。
<4−4>抗高脂血症効能
試験終了後に実験動物を16時間絶食させて、剖検時に腹大動脈から血液を採取して3,000rpmで10分間遠心分離して血清及び血漿を分離した。分離した血清を用いて脂肪代謝関連指標である中性脂肪、HDL、LDL、総コレステロールを血清自動分析機(Hitachi7060,日本)を用いて測定した。
<4−5>脂肪肝治療効能
脂肪が増加するにつれて脂肪にも脂肪が蓄積して脂肪肝に進展する。脂肪肝に進展すると蓄積された脂肪によって肝本来の色相である鮮紅色が消える。
<4−6>肥満性インスリン抵抗性(Obesity−induced insulin resistance)治療効能
肝及び血清(Serum)で虎杖根ブタノール分画物によるインスリン代謝調節の核心酵素であるIL−6、TNF−α、SOSC−3(suppressor of cytokine signalling−3)の発現変化を分析した。肥満は、インスリン抵抗性を誘発して深刻な糖尿(合併症)を誘発することはすでに広く知られている。肥満によって、IL−6、TNF−a、アディポネクチンなどが非正常的に作用して肝臓や筋肉でSOCS経路とTSC/mTOR経路を通じてインスリン耐性を引き起こす。
<4−7>筋肉及び肝組織で脂肪代謝に及ぼす影響
脂肪代謝は、脂肪生成と脂肪分解の均衡が適切に維持されることが理想的であるが、飲食物を通じた過度な営養分の摂取はこのような脂肪代謝の均衡を破って体内筋肉及び肝組織に過度な脂肪の蓄積を引き起こすようになる。このような脂肪代謝の不均衡に虎杖根ブタノール抽出物が及ぼす影響を詳しく調べた。
Claims (11)
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、肥満予防及び治療用薬学的組成物。
- 虎杖根ブタノール分画物が、虎杖根抽出物を水に溶解した後、ヘキサン、エチルアセテート、ブタノールを順に用いて分画したブタノール分画物であることを特徴とする、請求項1に記載の肥満予防及び治療用薬学的組成物。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、肥満予防及び改善用機能性食品。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、高脂血症予防及び治療用薬学的組成物。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、高脂血症予防及び改善用機能性食品。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、代謝症侯群予防及び治療用薬学的組成物。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出した虎杖根ブタノール分画物を有効成分として含む、代謝症侯群予防及び改善用機能性食品。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にエチルアセテートで抽出した虎杖根エチルアセテート分画物を有効成分として含む、肥満予防及び治療用薬学的組成物。
- 虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にエチルアセテートで抽出した虎杖根エチルアセテート分画物を有効成分として含む、肥満予防及び改善用機能性食品。
- 薬学的に有効な量の虎杖根ブタノール分画物または虎杖根エチルアセテート分画物をヒトを除く、肥満である個体に投与する工程を含み、前記虎杖根ブタノール分画物が虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出したものであり、前記虎杖根エチルアセテート分画物が虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にエチルアセテートで抽出したものである、肥満治療方法。
- 薬学的に有効な量の虎杖根ブタノール分画物または虎杖根エチルアセテート分画物をヒトを除く個体に投与する工程を含み、前記虎杖根ブタノール分画物が虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にブタノールで抽出したものであり、前記虎杖根エチルアセテート分画物が虎杖根の水、アルコール、またはそれらの混合溶媒の抽出物を追加的にエチルアセテートで抽出したものである、肥満予防方法。
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