JP2022014440A - 大麦若葉由来の膵リパーゼ阻害剤、脂質吸収抑制用剤、及びそれらを含む飲食品 - Google Patents
大麦若葉由来の膵リパーゼ阻害剤、脂質吸収抑制用剤、及びそれらを含む飲食品 Download PDFInfo
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- Coloring Foods And Improving Nutritive Qualities (AREA)
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Abstract
Description
〔2〕大麦若葉処理物が、大麦若葉の乾燥粉末、搾汁液又は該搾汁液を乾燥させた搾汁末であることを特徴とする上記〔1〕記載の膵リパーゼ阻害剤。
〔3〕タンパク質が、以下の(a)~(f)よりなる群から選ばれる何れかのタンパク質であることを特徴とする上記〔1〕又は〔2〕記載の膵リパーゼ阻害剤。
(a)配列番号2に記載のアミノ酸配列からなるタンパク質
(b)配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換、挿入若しくは付加されたアミノ酸配列からなり、かつ膵リパーゼ阻害活性を有するタンパク質
(c)配列番号3に記載の塩基配列からなるDNAによりコードされるタンパク質
(d)配列番号3に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
(e)配列番号4に記載の塩基配列からなるDNAによりコードされるタンパク質
(f)配列番号4に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
〔4〕以下の(a)~(f)よりなる群から選ばれる何れかのタンパク質を有効成分として含むことを特徴とする膵リパーゼ阻害剤。
(a)配列番号2に記載のアミノ酸配列からなるタンパク質
(b)配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換、挿入若しくは付加されたアミノ酸配列からなり、かつ膵リパーゼ阻害活性を有するタンパク質
(c)配列番号3に記載の塩基配列からなるDNAによりコードされるタンパク質
(d)配列番号3に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
(e)配列番号4に記載の塩基配列からなるDNAによりコードされるタンパク質
(f)配列番号4に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
〔5〕上記〔1〕~〔4〕のいずれか記載の膵リパーゼ阻害剤を有効成分として含むことを特徴とする脂質吸収抑制用剤。
〔6〕上記〔1〕~〔4〕のいずれか記載の膵リパーゼ阻害剤、又は上記〔5〕記載の脂質吸収抑制用剤を有効成分として含んでなる膵リパーゼ阻害用又は脂質吸収抑制用の飲食品。
もできる。
。
選択マーカーとしては、通常使用されるものを常法により用いることができる。例えばテトラサイクリン、アンピシリン、又はカナマイシン若しくはネオマイシン、ハイグロマイシン又はスペクチノマイシン等の抗生物質耐性遺伝子等が例示される。
すなわち、本発明のタンパク質が宿主細胞内に蓄積する場合には、遠心分離やろ過等の操作により宿主細胞を集め、これを適当な緩衝液(例えば濃度が10M~100mM程度のトリス緩衝液、リン酸緩衝液、HEPES緩衝液、MES緩衝液等の緩衝液。pHは用いる緩衝液によって異なるが、pH5.0~9.0の範囲が望ましい)に懸濁した後、用いる宿主細胞に適した方法で細胞を破壊し、遠心分離により宿主細胞の内容物を得る。
特開2009-148213号公報に記載の方法に準じて次のとおり調製した。刈り取った大麦の若葉(茎葉)10kgを圧搾し、得られた搾汁液8kgを乾燥し、大麦若葉エキス末0.5kgを得た。
大麦若葉エキス末4.5gをイオン交換水45mLに懸濁し、24時間室温で振盪した。懸濁液を10,000×g、室温で10分間遠心分離して得られた上清を冷凍保存した。凍結した上清を解凍し、再度10,000×g、室温で10分間遠心分離して得られた上清に飽和硫酸アンモニウム水溶液を滴下することで20%硫酸アンモニウムに調整後、4℃で10時間静置した。溶液を10,000×g、室温で10分間遠心分離して、20%硫酸アンモニウム沈殿画分と上清を得た。上清には再度飽和硫酸アンモニウム水溶液を加えて同様にして30%硫酸アンモニウム沈殿画分、40%硫酸アンモニウム沈殿画分、50%硫酸アンモニウム沈殿画分を得た。
(1)測定法
トリオレインミセル溶液300μL(トリオレイン、レシチン、タウロコール酸を緩衝液に懸濁して超音波処理によりミセル化した溶液)と被験物質溶液100μL(50%ジメチルスルホキシド(DMSO)水溶液)を1.5mLマイクロチューブ内で混合し、37℃で5分間のプレインキュベートを行った。その後ブタ膵臓リパーゼ酵素溶液100μLを加え、37℃で10分間反応させた後、1M塩酸50μLを加えて反応を停止させた。
結果を図2に示す。精製タンパク質0.015625μg/mlの低濃度より、コントロールと比較して有意な阻害活性が確認された。図2中、PC(陽性対照)は10μMセチリスタットである。有意差の検定結果(t-test)は、mean±SD *p<0.05である。
精製物の約34kDaバンドのnano-LC MS/MS解析を株式会社日本プロテオミクスにて実施した。その概要を次に示す。
(1)SDS-PAGEバンドの切り出し
(2)脱色、還元処理
(3)ゲル内タンパク質のトリプシン消化
(4)ペプチド断片の抽出、脱塩
(5)nano-LC MS/MS解析
(6)Mascot searchによりタンパク質の同定
(1)プラスミドベクターの構築
配列番号3の配列(Uniprot A0A287NGA7のcDNA配列:Ensembl Plants Transcript: HORVU4Hr1G019570.5より)を大腸菌での発現用に最適化した配列(配列番号4)をpET-19bベクターに導入したプラスミドベクターを構築した。なお、発現用に最適化した配列(配列番号4)の合成とプラスミドベクターの構築は、GenScript社へ委託した。
上記(1)で精製したプラスミドベクターをBL21(DE3) Competent E. coli(New England Bio Labs社製)に導入して形質転換体を得た。この形質転換体を100μg/mLのアンピシリンを添加したLB培地50mLにてOD600が0.4-0.6程度となるまで培養後、1mMとなるようにイソプロピル-β-チオガラクトピラノシド(IPTG)を加えて37℃で二時間培養した。
溶液を遠心分離(10,000×g、10分)して得られた上清をバイオデザイン透析チューブ(#D102、BioDesign Inc. of New York)に入れ、4℃で10日間トリス緩衝液(50mMトリス、0.1M塩化ナトリウム、pH8.0)に対して透析後、さらにトリス緩衝液を交換して4℃で1日間透析した。
測定は上述の実施例3と同様の方法で行った。ただし、陽性対照のみ5μMのセチリスタットに濃度を変更している。その結果を図4に示す。図4中、PC(陽性対照)はセチリスタットである。有意差の検定結果(t-test)は、mean±SD *p<0.05である。
図2、図4から、組換えタンパク質は、大麦若葉エキス末から精製したタンパク質と同等の膵リパーゼ阻害活性を有していることが分かる。
以下の2群のマウスを用いて、糞中脂質(トリグリセライド量と遊離脂肪酸量)を解析した。
・HFD群:Research Diet社のD12492飼料を給餌するマウス群
・6%HV群:6%の大麦若葉エキス末を添加したD12492飼料を給餌するマウス群
Claims (6)
- 大麦若葉由来のタンパク質を有効成分として含有する大麦若葉処理物を含むことを特徴とする膵リパーゼ阻害剤。
- 大麦若葉処理物が、大麦若葉の乾燥粉末、搾汁液又は該搾汁液を乾燥させた搾汁末であることを特徴とする請求項1記載の膵リパーゼ阻害剤。
- タンパク質が、以下の(a)~(f)よりなる群から選ばれる何れかのタンパク質であることを特徴とする請求項1又は2記載の膵リパーゼ阻害剤。
(a)配列番号2に記載のアミノ酸配列からなるタンパク質
(b)配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換、挿入若しくは付加されたアミノ酸配列からなり、かつ膵リパーゼ阻害活性を有するタンパク質
(c)配列番号3に記載の塩基配列からなるDNAによりコードされるタンパク質
(d)配列番号3に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
(e)配列番号4に記載の塩基配列からなるDNAによりコードされるタンパク質
(f)配列番号4に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質 - 以下の(a)~(f)よりなる群から選ばれる何れかのタンパク質を有効成分として含むことを特徴とする膵リパーゼ阻害剤。
(a)配列番号2に記載のアミノ酸配列からなるタンパク質
(b)配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換、挿入若しくは付加されたアミノ酸配列からなり、かつ膵リパーゼ阻害活性を有するタンパク質
(c)配列番号3に記載の塩基配列からなるDNAによりコードされるタンパク質
(d)配列番号3に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質
(e)配列番号4に記載の塩基配列からなるDNAによりコードされるタンパク質
(f)配列番号4に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるタンパク質であって、かつ該タンパク質が膵リパーゼ阻害活性を有するタンパク質 - 請求項1~4のいずれか記載の膵リパーゼ阻害剤を有効成分として含むことを特徴とする脂質吸収抑制用剤。
- 請求項1~4のいずれか記載の膵リパーゼ阻害剤、又は請求項5記載の脂質吸収抑制用剤を有効成分として含んでなる膵リパーゼ阻害用又は脂質吸収抑制用の飲食品。
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