JP7510695B2 - 植物組織由来ナノファイバー - Google Patents
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- JP7510695B2 JP7510695B2 JP2021527166A JP2021527166A JP7510695B2 JP 7510695 B2 JP7510695 B2 JP 7510695B2 JP 2021527166 A JP2021527166 A JP 2021527166A JP 2021527166 A JP2021527166 A JP 2021527166A JP 7510695 B2 JP7510695 B2 JP 7510695B2
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Description
少なくとも1つの構造炭水化物を含む、或いは少なくとも1つの構造炭水化物から構成されている1又は2以上の鎖(strands)を含む伸長した繊維を含んでもよい。
植物組織から遊離される細胞成分を含む、低ポリフェノール含有量の溶液中で均質化した植物組織を調製すること;
存在する場合、細片を均質化した植物組織から除去すること;及び
任意で、均質化した植物組織を透析して、複合体を形成していない化合物を除去すること、又はサイズ排除によって複合体を形成していない化合物を除去すること、
を含み、それによって、細胞成分の自己組織化によって形成される、ナノファイバーを含む溶液を提供する。
植物組織から遊離される細胞成分を含む、低ポリフェノール含有量の溶液中で均質化した植物組織を調製すること;
存在する場合、細片を均質化した植物組織から除去すること;
細胞成分の自己組織化によるナノファイバーの形成を可能にすること;及び
フリーズドライ、スプレードライ、又はナノスプレードライして、ナノファイバーを含む粉末を形成することを含む。
生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、上記ナノファイバー又は複数のナノファイバーのいずれかを、対象に投与することを含む。
上記ナノファイバー又は複数のナノファイバーのいずれかを対象に投与することを含む。
上記ナノファイバー又は複数のナノファイバーのいずれかを対象に投与することを含む。
上記ナノファイバー又は複数のナノファイバーのいずれかを対象に投与することを含む。
上記ナノファイバー又は複数のナノファイバーのいずれかを対象の皮膚に塗布することを含む。
細胞又は組織又は器官を、上記ナノファイバー又は複数のナノファイバーのいずれかで処置することを含む。
上記ナノファイバー又は複数のナノファイバーのいずれかを対象に投与することを含む。
ペクチンベースの内側コア;
内側コアの周囲の中間層であって、ペクチン及びヘミセルロースの高分子及び/又はその切断産物、ポリフェノール、及び有機酸を含む中間層;及び
熟成の間及び/又は植物組織の均質化の間の異化作用から形成されていてもよい高分子の炭水化物及びタンパク質を含むフィブリル化外層を含んでもよい。
ペクチンベースの内側コア;
内側コアの周囲の中間層であって、ペクチン又はその誘導体、及び1又は2以上のヘミセルロース由来分子又はその誘導体を含む、1又は2以上のらせん状のフィブリル構造を含む中間層;並びに
ヘミセルロース又はその切断産物、及び細胞タンパク質に由来する1又は2以上のペプチドを含むフィブリル化外層を含んでもよい。
植物組織から遊離される細胞成分を含む、溶液中で均質化した植物組織を調製すること;
存在する場合、細片を均質化した植物組織から除去すること;及び
任意で、均質化した植物組織を透析して、複合体を形成していない化合物を除去すること、又はサイズ排除によって複合体を形成していない化合物を除去すること、
それによって、細胞成分の自己組織化によって形成される、ナノ粒子を含む溶液を提供することを含む。
植物組織から遊離される細胞成分を含む、溶液中で均質化した植物組織を調製すること;
細胞成分の自己組織化によるナノ粒子の形成を可能にすること;
存在する場合、細片を均質化した植物組織から除去すること;及び
フリーズドライ、スプレードライ、又はナノスプレードライして、ナノ粒子を含む粉末を形成することを含む。
化学的又は物理的な方法の使用により生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、いずれかの上記ナノ粒子又は複数のナノ粒子を、対象、細胞、又は生物に投与することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
別の実施形態では、それを必要とする対象において肥満を治療する又は減少させるための方法が本明細書に提供され、前記方法が、
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象の皮膚に塗布することを含む。
細胞又は組織又は器官を、上記ナノ粒子又は複数のナノ粒子のいずれかで処置することを含む。
上記ナノ粒子又は複数のナノ粒子のいずれかを対象に投与することを含む。
ナノスフェア様構造を形成するための、互いに巻きつく(又は巻き上がる)繊維構造を含んでもよい。
スミノミザクラ、又はその水性抽出物;
アーモンドミルク又はアーモンドの別のホモジネート;
豆乳、又はダイズの別のホモジネート;
ブロッコリー、又はその水性抽出物;及び
ウコン、又はその粉末を含んでもよい。
約25~30v/v%スミノミザクラ抽出物(少なくとも約0.1mg/mlのポリフェノール当量);
約25~30v/v%アーモンドミルク又はアーモンドの他のホモジネート;
約10~18v/v%豆乳又はダイズの他のホモジネート;
約25~30v/v%ブロッコリー抽出物;及び
約0.5~2.5w/v%ウコン又はその粉末を含んでもよい。
植物組織から遊離される細胞成分を含み、細胞成分が、1又は2以上の構造炭水化物又はその切断産物を含み、溶液中で均質化した植物組織が、少なくとも1つの栄養剤をさらに含む、溶液中で均質化した植物組織を調製すること;
任意で、存在する場合、細片を溶液中で均質化した植物組織から除去すること;及び
溶液中で均質化した植物組織をフリーズドライ、凍結乾燥、スプレードライ、又はナノスプレードライして、食物粉末を形成することを含む。
スミノミザクラ、又はその水性抽出物;
アーモンドミルク又はアーモンドの別のホモジネート;
豆乳、又はダイズの別のホモジネート;
ブロッコリー、又はその水性抽出物;及び
ウコン、又はその粉末を含んでもよい。
約25~30v/v%スミノミザクラ抽出物(少なくとも約0.1mg/mlのポリフェノール当量);
約25~30v/v%アーモンドミルク又はアーモンドの他のホモジネート;
約10~18v/v%豆乳又はダイズの他のホモジネート;
約25~30v/v%ブロッコリー抽出物;及び
約0.5~2.5w/v%ウコン又はその粉末を含んでもよい。
化学的又は物理的な方法の使用により生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、いずれかの上記食物粉末又は複数の食物粉末を、対象又は細胞又は生物に投与することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む方法が、本明細書に提供される。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
細胞又は組織又は器官を、上記食物粉末又は複数の食物粉末のいずれかで処置することを含む。
上記食物粉末又は複数の食物粉末のいずれかを対象に投与することを含む。
ナノ粒子の脱水、凍結乾燥、又はフリーズドライしたサンプルを提供すること;
生物学的に活性な薬剤又はカーゴをナノ粒子のサンプルと混合して混合物を提供すること;及び
水又は水性ベースの溶液を混合物に添加してナノ粒子を縮小して、その中に生物学的に活性な薬剤又はカーゴをトラップすることを含む。
ナノ粒子、
一実施形態では、均質化した植物組織に由来する自己組織化した細胞成分を含むナノ粒子であって、細胞成分が1又は2以上の細胞壁及び/又は他の細胞成分を含む、ナノ粒子が本明細書に提供される。特定の実施形態では、細胞成分が、均質化によって植物組織から遊離される成分を含んでもよく、これが、例えば、ナノ粒子に自己組織化する。特定の実施形態では、1又は2以上の細胞壁成分が、ペクチン及び/又はその誘導体を含んでもよい。
a.カロテノイド(ベータカロチン、リコペン、ルテイン及び/又は他のキサントフィル、アスタキサンチンなど);
b.アンノナシン(抗がん特性を有するアンノナ果実に由来するポリケチド);
c.ボスウェリア(Boswellia)(クルクミンと協同して作用することが可能であり、追加の抗炎症性機能等を提供する);
d.アシュワガンダ(抗ストレス、及びがん予防特性を有するウィタノライド(withanoliides)を含有するハーブ成分)、主にステロイド構造を有する;
e.食用ショウガ(ショウガ(Zingiber officinalis))、マンゴージンジャー(クルクマアマダ(Curcuma amada))及びジンゲロール及びショウガオールを含む、いくつかの生理活性成分を含有するその他のものを含むショウガ科のメンバー;
f.クルクマロンガ(Curcuma longa)(クルクミンを含有するウコン);
g.幻覚誘発活性のないアサ属種(Cannabis sp.)の医薬活性成分であるカンナビノジオール;
h.プレバイオティック含有量を増強するための、フラクトオリゴ糖及びガラクトオリゴ糖並びにエレサレムアーティチョークからのイヌリン;
i.コショウ科のメンバー、例えば、コショウ及びピペリンを含有するその野生の類縁体及びいくつかの誘導体;及び/又は
j.上に列挙されていない植物からの任意の他の適切な成分が含まれ得る。
一実施形態では、均質化した植物組織からナノ粒子(例えば、本明細書に記載されるもの、等々)を調製するための方法が本明細書に提供され、前記方法が、
植物組織から遊離される細胞成分を含む、溶液中で均質化した植物組織を提供すること;
存在する場合、細片を均質化した植物組織から除去すること;及び
任意で、均質化した植物組織を透析して、複合体を形成していない化合物を除去すること、又はサイズ排除によって複合体を形成していない化合物を除去すること、
それによって、細胞成分の自己組織化によって形成される、ナノ粒子を含む溶液を提供することを含む。
植物組織から遊離される細胞成分を含む、溶液中で均質化した植物組織を調製すること;
細胞成分の自己組織化によるナノ粒子の形成を可能にすること;
存在する場合、細片を均質化した植物組織から除去すること;及び
フリーズドライ、スプレードライ、又はナノスプレードライして、ナノ粒子を含む粉末を形成することを含む。
ナノファイバー
ナノファイバーも開発されており、本明細書に記載される。ナノ粒子及びナノファイバーは両方とも植物組織から調製され得るが、これらの2つのナノ構造は著しく異なる方法を使用して本明細書で調製されており、これらの2つのナノ構造が著しく異なる構造を採ること、組成の点で有意に異なることを特色とすること、機能及び/又は生物学的効果の点で興味深い差(及び類似性)を特色とすることが本明細書で見出されている。
別の実施形態では、均質化した植物組織からナノファイバー(例えば、本明細書に記載されるもの)を調製するための方法が、
植物組織から遊離される細胞成分を含む、低ポリフェノール含有量の溶液中で均質化した植物組織を調製すること;
存在する場合、細片を均質化した植物組織から除去すること;及び
任意で、均質化した植物組織を透析して、複合体を形成していない化合物を除去すること、又はサイズ排除によって複合体を形成していない化合物を除去すること、
それによって、細胞成分の自己組織化によって形成される、ナノファイバーを含む溶液を提供することを含む。
植物組織から遊離される細胞成分を含む、低ポリフェノール含有量の溶液中で均質化した植物組織を調製すること;
存在する場合、細片を均質化した植物組織から除去すること;
細胞成分の自己組織化によってナノファイバーの形成を可能にすること;及び
フリーズドライ、スプレードライ、又はナノスプレードライして、ナノファイバーを含む粉末を形成することを含む。
食物粉末及び添加物
別の実施形態では、本明細書に記載のナノ粒子及び/若しくはナノファイバー、又はその一部又は成分、又はそれに関連する構造を含む食物粉末又は食品添加物が本明細書に提供される。特定の実施形態では、食物粉末が、例えば、ミクロンスケールの微細な粉末形態等を形成するナノ粒子及び/又はナノファイバーの基礎の構造成分を含んでもよい。
スミノミザクラ、又はその水性抽出物;
アーモンドミルク又はアーモンドの別のホモジネート;
豆乳、又はダイズの別のホモジネート;
ブロッコリー、又はその水性抽出物;及び
ウコン、又はその粉末を含んでもよい。
約25~30v/v%スミノミザクラ抽出物(少なくとも約0.1mg/mlのポリフェノール当量);
約25~30v/v%アーモンドミルク又はアーモンドの他のホモジネート;
約10~18v/v%豆乳又はダイズの他のホモジネート;
約25~30v/v%ブロッコリー抽出物;及び
約0.5~2.5w/v%ウコン又はその粉末を含んでもよい。
1.本明細書に記載される炭水化物ベースのナノ粒子又はナノファイバーを形成することができるスミノミザクラ(又はスミノミザクラ抽出物)、又は別の果実又は野菜(又はその抽出物);
2.疎水性分子を追加の物質から組み込むことができる疎水性成分(例えば、限定されないが、アーモンドミルク)(他の例はココナツミルク、又は食用ナッツに由来するミルク等);及び
3.例えば、食物粉末の所望の適用のために調整される1又は2以上の生理活性の成分/複数の成分を含有する栄養含有物質(例えば、機能的な食物抽出物等)。
カロテノイド(すなわち、ベータカロチン、リコペン、ルテイン及び他のキサントフィル、アスタキサンチンなど);
アンノナシン(すなわち、抗がん特性を有するアンノナ果実に由来するポリケチド);
ボスウェリア(クルクミンと協同して作用することが可能であり、追加の抗炎症性機能を提供する);
アシュワガンダ(抗ストレス、及びがん予防特性を有するウィタノライドを含有するハーブ成分、主にステロイド構造を有する);
例えば、食用ショウガ、マンゴージンジャー(クルクマアマダ)及びジンゲロール及びショウガオールを含むいくつかの生理活性成分を含有するその他のものを含み得るショウガ科のメンバー;クルクマロンガ(すなわち、クルクミンを含有するウコン);
カンナビノジオール(幻覚誘発活性のないアサ属種の医薬活性成分である);
プレバイオティック含有量を増強するための、フラクトオリゴ糖及びガラクトオリゴ糖並びにエレサレムアーティチョークからのイヌリン;
コショウ科のメンバー、例えば、コショウ及びピペリンを含有するその野生の類縁体及びいくつかの誘導体;及び/又は
これまで上に列挙されていない、任意の他の適切な成分(例えば、限定されないが、植物に由来するもの);
及び/又は、それから単離された任意の抽出物、誘導体、産物、又はそのような成分を含有する物質又は植物組織。
別の実施形態では、均質化した植物組織から食物粉末を調製するための方法が本明細書に提供され、前記方法が、
植物組織から遊離される細胞成分を含み、細胞成分が、1又は2以上の構造炭水化物又はその切断産物を含み、溶液中で均質化した植物組織が、少なくとも1つの栄養剤をさらに含む、溶液中で均質化した植物組織を調製すること;
任意で、存在する場合、細片を溶液中で均質化した植物組織から除去すること;及び
溶液中で均質化した植物組織をフリーズドライ、凍結乾燥、スプレードライ、又はナノスプレードライして、食物粉末を形成することを含む。
スミノミザクラ、又はその水性抽出物;
アーモンドミルク又はアーモンドの別のホモジネート;
豆乳、又はダイズの別のホモジネート;
ブロッコリー、又はその水性抽出物;及び
ウコン、又はその粉末を含んでもよい。
約25~30v/v%スミノミザクラ抽出物(少なくとも約0.1mg/mlのポリフェノール当量);
約25~30v/v%アーモンドミルク又はアーモンドの他のホモジネート;
約10~18v/v%豆乳又はダイズの他のホモジネート;
約25~30v/v%ブロッコリー抽出物;及び
約0.5~2.5w/v%ウコン又はその粉末を含んでもよい。
本明細書に記載のナノ粒子、ナノファイバー、及び食物粉末の企図される使用の例は、以下に記される。これらの例は、限定される対象とならないが、代わりに当業者用として意図された例示の例を提供する。以下の実施例のセクションに記される実験研究が、例示的な方法及び使用に関するさらなる詳細を提供する。
一実施形態では、生物学的に活性な薬剤をそれを必要とする対象に送達する方法が本明細書に提供され、前記方法が、
生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、本明細書に記載のナノ粒子を、対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を対象の皮膚に塗布することを含む。
細胞又は組織又は器官を、本明細書に記載のナノ粒子と接触させることを含む。
本明細書に記載のナノ粒子を対象に投与することを含む。
本明細書に記載のナノ粒子を投与することを含む。
本明細書に記載のナノ粒子を投与することを含む。
一実施形態では、生物学的に活性な薬剤をそれを必要とする対象に送達する方法が本明細書に提供され、前記方法が、
生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、本明細書に記載のナノファイバーを、対象に投与することを含む。
本明細書に記載のナノファイバーを対象に投与することを含む。
本明細書に記載のナノファイバーを対象に投与することを含む。
本明細書に記載のナノファイバーを対象に投与することを含む。
本明細書に記載のナノファイバーを対象の皮膚に塗布することを含む。
任意で細胞傷害剤と複合体を形成している又はコンジュゲートされているナノファイバーを、細胞又は組織又は器官に導入することを含む。
本明細書に記載のナノファイバーを対象に投与することを含む。
一実施形態では、生物学的に活性な薬剤を、それを必要とする対象又は生物に送達する方法が本明細書に提供され、前記方法が、
化学的又は物理的な方法の使用により生物学的に活性な薬剤と複合体を形成している又はコンジュゲートされている、本明細書に記載の食物粉末を、対象に投与することを含む。
本明細書に記載の食物粉末を対象に投与することを含む方法が、本明細書に提供される。
本明細書に記載の食物粉末を対象に投与することを含む。
本明細書に記載の食物粉末を対象に投与することを含む。
本明細書に記載の食物粉末を対象に投与することを含む。
本明細書に記載の食物粉末を対象に投与することを含む。
本明細書に記載の食物粉末を対象に投与することを含む。
細胞又は組織又は器官を、本明細書に記載の食物粉末で処置することを含む。
本明細書に記載の食物粉末を対象に投与することを含む。
細胞破壊条件下でのスミノミザクラ(酸果桜桃(Prunus cerasus L.))果実における高分子のナノ粒子及びナノファイバーへの自然組織化並びにそれらの比較
本発明者らは、細胞破壊が行われ、分子相互作用が生じ得る環境を提供し、明瞭なナノ構造の形成を潜在的にもたらす特定の果実加工方法によってナノ構造が生成され得ると仮定した。本実施例は、例えば食物機能、慢性疾患予防、並びに/又は細胞への薬物及び/若しくは生理活性送達の改善における有益な役割を有し得る、異なる方法を使用した生物学的供給源(この実施例ではスミノミザクラ果実)に由来するナノ粒子及びナノファイバーの調製、単離、物理化学特性、及び構造的特徴を記載する。
スミノミザクラ:この実施例において使用したスミノミザクラ果実は、これらを生殖質として維持するVineland Research Station、Vineland、Ontarioのものであった。全ての品種は、ポリフェノール含量が生体重100g当たり300~500mgの範囲に及ぶ高ポリフェノール含有品種である。本研究のために使用した主要な品種はV 70151であり、その他の品種、例えばV 71261、Hymann Conserva、及びHymann Rubisnもまた高レベルのナノ粒子形成を示した。この実施例において使用したスミノミザクラは酸果桜桃であった。
ナノ粒子に関して、抽出方法は、PTA 10プローブを備えるポリトロンホモジナイザーを使用して、1:1w/wの割合(水又はアルコール1mlに対して組織1g)の媒体(この実施例において使用する場合、好ましくは水だが、例えば無水又は希釈メタノール又はエタノールを代替的に使用してもよい)中、中間(4~5)設定で、果実組織が完全に均質化するまでスミノミザクラ果実を均質化することを含んだ。ホモジネートを、4層のチーズクロスを介して濾過して、細片を除去した。結果として得たホモジネートをSorvall RC 6 Plus遠心分離機において20分間遠心分離した(18,000×g)。上清をデカントした。5mlの上清を水(1リットル)に対して4℃で12時間透析した(spectra-Por、6~8kDのカットオフ)。透析バッグ内の透析抽出物を上清(粗抽出物と呼ぶ)と同じように-20℃で凍結して保存した。抽出物は、水性溶液中に存在する場合、ナノ粒子を分解し得る酵素を含有する場合があるため、透析抽出物は低温で維持した。アルコール媒体又は実質的アルコール媒体を使用する複数の実施形態では、そのような酵素は変性している及び/又は低下した活性を有する可能性が高いが、依然として低温は一般に好ましい。本明細書に記載されるように、(例えば)凍結乾燥又はスプレードライすると、ナノ粒子は安定である。
ナノ粒子の特徴付け:これらの研究では、水性媒体中での果実の均質化は、細胞の高分子及び単一分子の天然の構成を破壊し、その結果、別個の物理化学及び構造的特徴を有する構造の生成を引き起こす、それらの無作為だが構造化した組織化をもたらす。ポリフェノールと、ポリフェノールと複合体を形成したナノ構造との間のサイズの差を使用して、サイズ排除分離を可能にする低分子質量カットオフ膜又はPD-10カラムを使用する透析によりこれらの構造体を分離した。スミノミザクラ粗抽出物、ナノ粒子を含有する透析抽出物、及び透析物(膜によって排除された約6kD以下の分子質量の分子を含有する)のポリフェノール含量を表1に示す。抽出物におけるほぼ80%のポリフェノールは透析膜内に保持され、ポリフェノールが複合体を形成した状態で存在することを示唆した。ブドウ及びブルーベリー等の他の果実との比較によると、複合体形成はスミノミザクラにおいてはるかに高かった(が、ナノ粒子はブドウ及びブルーベリーにおいても形成された)。ポリフェノール性成分のHPLC-MS分離は、主要なアントシアニンとしてのシアニジン-3ルチノシドとより少ない存在量のペオニジン3-ルチノシドとの存在を明らかにした。クロロゲン酸及びパラ-クマロイルキナ酸等のフェノール酸ははるかに小さいレベルで存在した(表2を参照のこと)。水性抽出物とメタノール抽出物の両方からの粗抽出物、透析抽出物(ナノ粒子;NP,nanoparticle)、及び透析物の抗酸化活性を表3に示す。全ての抽出物は高レベルのスーパーオキシド、ヒドロキシル、及びDPPHラジカル捕捉活性を示した。粗抽出物は両方の抽出条件下で最も高い抗酸化能を示した。透析物は最も低い抗酸化活性を有した。有意な一部の抗酸化能はナノ粒子(すなわち透析抽出物)に関連した。
ナノ粒子の酸性性質:ナノ粒子は水性溶液中でも乾燥粉末としても高度に安定であった。ナノ粒子のサイズ排除クロマトグラフィーの間、ナノ粒子に結合したポリフェノールは空隙容量に溶出し、ポリフェノールに関して特徴的な520nmにおける極大吸収、及び3のpHを示した。溶液に存在する遊離ポリフェノールの溶出は遅延し、溶出液は、青灰色に見え、アントシアニンのベンゾピラン環開裂が関連する赤色の喪失と共に起こる7に近いpHを示した。ジュース分子中の遊離有機酸を除去したと思われるスミノミザクラ抽出物の徹底した透析後、透析抽出物は依然として3に近いpHを示し、ホモガラクツロナンのオリゴガラクツロン酸部分(pH3)の他に構造成分としてナノ粒子に結合した酸性成分(有機酸)が存在し得ることを示唆した。そのような成分を同定するために、ナノ粒子及びナノファイバーを乾燥させ、トリメチルシリル誘導体を調製し、その誘導体をGC-MS分析に供した。結果を図9に示す。上段の(A)のパネルは標準的な有機酸の溶出パターンを示す。(B)及び(C)はそれぞれ、ナノ粒子及びナノファイバーからの化合物の溶出プロファイルを示す。ナノ粒子とナノファイバーの両方は、13.7分で溶出したリンゴ酸の明確な存在を示した(質量スペクトル、トリメチルシリル誘導体)。リンゴ酸はスミノミザクラ果実に存在する主要な有機酸である。ヒドロキシル酸であるリンゴ酸は水素結合を形成して、ナノ粒子及びナノファイバーの成分間の分子会合を安定化し得る。20~25分の間に溶出するピークは誘導糖又はオリゴ糖由来である。図9の下側のパネルは参照リンゴ酸トリメチルシリル誘導体の質量スペクトルを示す。
野生型及びETKOマウスにおける体重に対するナノ粒子処置の効果
材料及び方法:
インビボ分析 - 動物及び遺伝子型同定 - Pcyt2+/-マウス(本明細書においてETKOマウスとも称す)をこれまでに記載されたように生成し、遺伝子型を同定した(Fullerton MD, et al. 2007)。Pcyt2とは、細胞膜及び細胞小器官の重要な成分であるリン脂質、すなわちホスファチジルエタノールアミン(PE,phosphatidylethanolamine)の産生の制御を担う遺伝子である。Pcyt2遺伝子を完全にノックアウトすること(ヌル動物)は、早期胚性致死(子が誕生前に死亡する)をもたらすが、ヘテロ接合型動物(1コピーの遺伝子のみを有する)は誕生時及び誕生後に正常である。しかしながら、ヘテロ接合型マウスはリン脂質代謝の変化の結果として慢性的な体重増加を経験する。リン脂質を構成する成分のリダイレクションがあり、細胞機構がPEの産生からトリグリセリド(脂肪)の産生へと変化する。このことは、マウスが体重の増加、肝臓及び血漿におけるより多くの脂肪、並びに最終的にインスリンに対する感応性の減少(インスリン抵抗性)を有することを意味する。
材料及び方法:
組織学的分析。肝臓組織学を検査するために、組織を10%中性緩衝ホルマリン+リン酸緩衝生理食塩水中で固定し、パラフィンに包埋した。10μm肝臓切片をヘマトキシリン及びエオジン(H&E,hematoxylin and eosin)で染色し、光学顕微鏡法によって可視化した。組織脂質を、これまでに記載されたように、イメージングデンシトメーター(Image J)で分析した(Fullerton et al. 2007)。
野生型及びETKOマウスの肝臓におけるトリグリセリドレベルに対するナノ粒子処置の効果を調査する研究を実施した。通常の生理的状態では、肝臓が脂肪の貯蔵に関与していないため、肝トリグリセリドレベルは比較的低い。非アルコール性脂肪性肝臓のトリグリセリドは、血漿から細胞への脂肪酸取込みの速度及び肝細胞の貯蔵容量に依存する(Bradbury MW. 2006、Kawano Y 2013)。肥満マウス肝臓組織におけるトリグリセリドの蓄積のため、トリグリセリドのレベルを、ナノ粒子(NP)処置を伴う又は伴わないWT及びETKOマウスにおいて調査した。図19は、マウス肝臓におけるトリグリセリドレベルの変化に対するナノ粒子(NP)処置の効果を示す。肝臓組織のトリグリセリドレベルを、Wako社製L-type TG Mアッセイキット(Wako Life Sciences社、CA、USA)を使用して、記載されている手順によって推定した。NP処置に供したETKOマウスにおいてトリグリセリドレベルの有意な減少が存在した。上段のパネルは、雄マウスと雌マウスの両方から取得したデータを示す。下段のパネルは、雄マウスから取得したデータを示す。ナノ粒子(NP)処置による肝臓トリグリセリドの減少は、肥満マウス肝臓、又はそれを必要とする別の動物若しくは対象の肝臓におけるトリグリセリドレベルを減少させるためのNPの使用を支持する。
結果及び考察:
様々な関連する生物学的経路及び/又は状態に関与する様々な肝要な遺伝子の発現レベルに対するナノ粒子処置の効果を調査する研究を実施した。マウス(野生型、WT、及びETKOノックアウト、KO)をナノ粒子で処置し、遺伝子発現レベルの変化を分析した。
送達ビヒクルとしてのナノファイバー(及びナノ粒子)、並びに結腸直腸がん細胞に対するスミノミザクラ(酸果桜桃)果実からのナノ粒子及びナノファイバーの細胞毒性
材料及び方法:
スミノミザクラ:この実施例において使用した果実は、これらを生殖質として維持するVineland Research Station、Vineland、Ontarioのものであった。全ての品種は、ポリフェノール含量が生体重100g当たり300~500mgの範囲に及ぶ高ポリフェノール含有品種である。本研究のために使用した主要な品種はV 70151であり、その他の品種、例えばV 71261、Hymann Conserva、及びHymann Rubisnもまた高レベルのナノ粒子形成を示した。
抗酸化特性(活性酸素種又は活性化ラジカルを捕捉する能力)を、スーパーオキシドアニオンラジカル、ヒドロキシルアニオンラジカル、及びDPPHラジカルの捕捉における効率をインビトロアッセイ条件下で評価することによって実行した(Jacob, J.K. and Paliyath, G. (2008) Physico-chemical characteristics of nanovesicle-carbohydrate complexes in grape juice, J. Agr. Food Chem., 56, 1305-1315)。これらのラジカルを捕捉することにおける効率を具体的な消去率によって示す。
ナノ粒子及びナノファイバーの形態学:起源であるスミノミザクラ果実及びエタノール脱色したスミノミザクラ果実から単離したナノ粒子及びナノファイバーは別個の特徴を示した(実施例1及び3における考察、並びに図1及び17(ナノ粒子)及び図37(ナノファイバー)を参照のこと)。起源であるサクランボ抽出物からのナノ粒子は形態学の点で球状であった。詳細な分析は、ある特定の実施形態では球状複合体がペクチン成分、ヘミセルロース由来分子、細胞壁タンパク質に由来するペプチド、アントシアニン、及びリンゴ酸を含む繊維様構造によって囲まれるペクチンコアによって形成され得ることを示す。これらの複合体は溶液中で直径50~250nmであった。アントシアニン及び他の低分子(すなわち、ある特定の実施形態ではリンゴ酸)が除去されるスミノミザクラ果実のエタノール脱色後、結果として得た果実は均質化の間に長さ数マイクロメートル及び直径5~10nmの繊維様構造を形成した(図37を参照のこと)。ナノ粒子とナノファイバーの両方を使用してそれらの機能特性を評価した。
スミノミザクラ果実の均質化の間に、細胞壁を起源とする細胞成分(セルロース、ペクチン、タンパク質)、並びにアントシアニン及びリンゴ酸等の液胞成分を含む細胞成分の自己組織化を介して形成されるナノ粒子及びナノファイバーの組成的及び構造的特色を評価する。ナノ粒子は、らせん状の繊維形状構造が周りに巻きついているペクチンコアを含む直径約50~約250nmの球状の複合体である。対照的に、エタノール脱色したサクランボ果実から形成したナノファイバーは、繊維様(横幅約5~約10nm)であり、長さマイクロメートルであった。アントシアニン及びリンゴ酸を果実から除去して、これらの研究におけるナノファイバー形成を容易にした。したがって、アントシアニンが少ない果実は、ここに記載される方法に従ってナノファイバーを優先的に形成する傾向を有し得る。ナノ粒子におけるアントシアニンの存在のため、これらは高抗酸化活性を示し、したがって栄養に関連する構造的複合体であり得る。細胞毒性試験において、ナノファイバーは、抗がん薬に対して複合体を形成した場合、はるかに高い活性を示した。本実施例は、哺乳動物細胞におけるナノ粒子及びナノファイバーの潜在的な機能的効果を記載する。
食物粉末調製及び構造調査
この実施例では、ブロッコリー、スミノミザクラ、アーモンド、ダイズ、及び/若しくはウコン、又はその任意の組合せ(並びに特に、これらの成分の全て)の抽出物から高剪断混合及びナノスプレードライにより作製した機能的食物粉末を記載する。粉末の各粒子は、各未処理成分を起源とする個々の乾燥粉末の混和物と比較して実質的に一様な、原材料(スルフォラファン、インドール-3-カルビノール、ポリフェノール、フィトステロール、イソフラボン、クルクミン等)を起源とする栄養補助食品の混和物を含有した。
ブロッコリー-栄養補助食品成分(すなわち栄養剤)、例えばスルフォラファン及びインドール-3-カルビノールの前駆体を含有する。両方の成分は、自然抗酸化系(KEAP-1/NRF2/ARE経路)及び生体異物の排出に関与する第2相酵素を増強する。
1.本明細書に記載される炭水化物ベースのナノ粒子又はナノファイバーを形成することができるスミノミザクラ(若しくはスミノミザクラ抽出物)又は別の果実若しくは野菜(若しくはその抽出物);
2.添加された物質からの疎水性分子を組み込むことができる疎水性成分(例えば、限定されないが、アーモンドミルク)(他の例は、例えばココナツミルク又は食用ナッツに由来するミルクである);及び
3.例えば食物粉末の所望の適用のために調整された1又は2以上の生理活性成分を含有する栄養含有物質(例えば機能的食物抽出物等)
を含み得る。
カロテノイド(すなわちベータカロテン、リコペン、ルテイン及び他のキサントフィル、アスタキサンチン等);
アンノナシン(すなわち抗がん特性を有するバンレイシ属果実に由来するポリケチド);
ボスウェリア(追加された抗炎症性機能を提供するクルクミンと共に作用し得る);
アシュワガンダ(抗ストレス及びがん予防特性を有するウィタノリドを含有するハーブ成分、主にステロイド構造を有する);
例えば、食用ショウガ(ショウガ)、マンゴージンジャー(クルクマアマダ)、又はジンゲロール及びショウガオールを含むいくつかの生理活性成分のいずれかを含有するその他のものを含み得るショウガ科のメンバー;クルクマロンガ(すなわちクルクミンを含有するウコン);
カンナビノジオール(幻覚誘発活性を有しないアサ種の医薬的に活性な成分である);
プレバイオティクス含量を増強するフラクトオリゴ糖及びガラクトオリゴ糖、及びエルサレムアーティチョークからのイヌリン;
コショウ科のメンバー、例えばコショウ、及びピペリン及びいくつかの誘導体を含有するその野生の類縁体;並びに/或いは
まだ上に列挙されていない任意の他の適切な成分(例えば、限定されないが、植物に由来する成分);
並びに/或いはそれから単離された任意の抽出物、誘導体、産物、又はそのような成分を含有する材料若しくは植物組織。
1.スミノミザクラの水性抽出物;
2.アーモンドミルクとして市販されているアーモンドのホモジネート;
3.豆乳として市販されているダイズのホモジネート;
4.ブロッコリーの水性抽出物;及び
5.ウコン粉末。
約8~10%w/vの100mlのアーモンドミルク;
約8~10%w/vの50mlの豆乳;
100mlのブロッコリー抽出物;及び
5gのウコン粉末
を含んだ。
材料及び方法:
発現分析-化学物質及び試薬。L-typeトリグリセリドMキットをWako Diagnostics社(日本、大阪、及びNeuss、Germany)から購入した。RNA単離のためのRNeasy PlusキットはQiagen社(Valencia、CA、USA)からであった。cDNA調製のためのHigh-Capacity cDNA Reverse Transcription KitはApplied Biosystems社(Foster city、CA、USA)からであった。RT2Profiler(商標)PCR Array Mouse Inflammatory Cytokines & ReceptorsキットはQiagen社(Valencia、CA、USA)からであった。iQ SYBR Green SupermixはBio-rad社(California、USA)からであった。
水性条件下での有機分子のナノファイバー及びナノ粒子との結合能を評価する実験を実施した。ナノファイバーとナノ粒子の両方の主要な成分は、キシラン及びアラビナン等の他の主要ではない炭水化物ポリマーと一緒になったポリガラクツロン酸(PG,polygalacturonic acid)ポリマーである(以下を参照のこと)。PGのカルボン酸部分は、4に近いpKaを有する水性溶液中のPGに高度に酸性の特性を与える。このpHでは、大半のカルボン酸は解離した形態(COO-)において存在することになる。これはマルチストランド構造における個々のストランドと反発する傾向を有することがあり、場合によりリガンド分子の結合の増加を容易にする。
肥満のマウスモデルにおける食物粉末処置の効果を評価する研究をマウスにおいて行った。実験の条件は上に記載した通りである。切片を、エオジン/ヘマトキシリンで染色した。
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Claims (26)
- サクランボ又はスミノミザクラに由来する均質化した植物組織に由来する自己組織化した細胞成分を含むナノファイバーであって、前記細胞成分が1又は2以上の構造炭水化物又はその切断産物を含み、脂質及びポリフェノールが前記ナノファイバーの構造成分ではなく、前記ナノファイバーが少なくとも1つの構造炭水化物を含む、或いは少なくとも1つの構造炭水化物から構成されている1又は2以上の鎖を含む伸長した繊維を含む、前記ナノファイバー。
- 脂質を含まないか、ポリフェノールを含まないか、又はその両方である、請求項1に記載のナノファイバー。
- ナノファイバーがペクチン、ヘミセルロース、ペプチド及び/又はタンパク質、有機酸、その切断産物、又はその任意の組合せを含み、前記有機酸が、リンゴ酸、アスコルビン酸、又はその両方を含む、請求項1に記載のナノファイバー。
- 細胞成分が、ナノファイバーに自己組織化することができる、熟成の間又は熟成した果実の均質化の間の植物組織から遊離される細胞成分を含む、請求項1に記載のナノファイバー。
- 1又は2以上の構造炭水化物が、ペクチン、ペクチン酸、ペクチンのメチルエステル、ペクチン誘導体、ポリガラクツロン酸、ラムノガラクツロナン、キシログルカン、ヘミセルロース;グルコース、マンノース、若しくはキシロースのβ-(1→4)-連結骨格を保持するキシログルカン;及び/又はアラビノガラクタン、及び/又はその切断産物のうちの1又は2以上を含む、請求項1に記載のナノファイバー。
- 前記ナノファイバーが非結晶性であり、直径5~10nmを有する繊維形状を有する、請求項1に記載のナノファイバー。
- ナノファイバーが、水素結合相互作用によって安定化され、植物組織の細胞成分の異化作用に由来する高分子の間で形成され、ヒドロキシル基及び/又はアミノ基及び/又は有
機酸基を保持している、請求項1に記載のナノファイバー。 - 生物学的に活性な薬剤をさらに含み、前記生物学的に活性な薬剤が、薬学的な活性薬、タンパク質、酵素、栄養補助食品、栄養分、金属、又は化学物質である、請求項1に記載のナノファイバー。
- 水溶液中にあり、粉末形態であり、脱水、凍結乾燥、フリーズドライ、スプレードライ、又はナノスプレードライ形態である、請求項1に記載のナノファイバー。
- ポリフェノールが均質化前に前記植物組織から除去されている、請求項1に記載のナノファイバー。
- サクランボ又はスミノミザクラに由来する均質化した植物組織からナノファイバーを調製するための方法であって、
植物組織から遊離される細胞成分を含む溶液中で均質化した植物組織を調製すること;
存在する場合、細片を前記均質化した植物組織から除去すること;及び
任意で、前記均質化した植物組織を透析して、複合体を形成していない化合物を除去すること、又はサイズ排除によって前記複合体を形成していない化合物を除去すること
を含み、それによって、前記細胞成分の自己組織化によって形成される前記ナノファイバーを含む溶液を提供し、前記ナノファイバーが少なくとも1つの構造炭水化物を含む、或いは少なくとも1つの構造炭水化物から構成されている1又は2以上の鎖を含む伸長した繊維を含む、前記方法。 - ナノファイバーを含む溶液を脱水、凍結乾燥、フリーズドライ、スプレードライ、又はナノスプレードライするステップをさらに含む、請求項11に記載の方法。
- ナノファイバーが、水性の媒体中での自己組織化によって形成される、請求項11に記載の方法。
- 溶液中で均質化した植物組織を調製するステップが、植物組織を水性、有機性、又は混合された水性-有機性媒体中で均質化すること;及び
任意で、前記植物組織を、高剪断均質化及び/又は水、エタノール、メタノール、又はアセトンのいずれか1又は2以上を含む水性、有機性、又は混合された水性-有機性媒体中でのソノレーションに供すること;及び
任意で、前記植物組織の均質化前に、前記植物組織を脱色して、それからポリフェノールを除去することを含む、請求項11に記載の方法。 - 脱色することが、抽出溶媒での植物組織からのポリフェノールの抽出を行うことを含み、前記抽出溶媒がエタノールを含む、請求項14に記載の方法。
- 細片を除去するステップが、透析、均質化した植物組織を濾過すること、前記均質化した植物組織を遠心分離すること、又は前記均質化した植物組織にタンジェンシャルフロー濾過又は連続フロー濾過を行うこと、又はその任意の組合せを含む、請求項13に記載の方法。
- 請求項1~10のいずれかに定義されるナノファイバーを調製するための、請求項11に記載の方法。
- サクランボ又はスミノミザクラに由来する均質化した植物組織からナノファイバーを調製するための方法であって、
植物組織から遊離される細胞成分を含む溶液中で均質化した植物組織を調製すること;
存在する場合、細片を前記均質化した植物組織から除去すること;
前記細胞成分の自己組織化によるナノファイバーの形成を可能にすること;及び
フリーズドライ、スプレードライ、又はナノスプレードライして、前記ナノファイバーを含む粉末を形成すること
を含み、前記ナノファイバーが少なくとも1つの構造炭水化物を含む、或いは少なくとも1つの構造炭水化物から構成されている1又は2以上の鎖を含む伸長した繊維を含む、前記方法。 - 自己組織化が、水性の媒体中で生じる、請求項18に記載の方法。
- 溶液中の均質化した植物組織を調製するステップが、植物組織を水性、有機性、又は混合された水性-有機性媒体中で均質化すること;及び
任意で、前記植物組織を、高剪断均質化及び/又は水、エタノール、メタノール、又はアセトンのいずれか1又は2以上を含む水性、有機性、又は混合された水性-有機性媒体中でのソノレーションに供すること;及び
任意で、前記植物組織の均質化前に、前記植物組織を脱色して、それからポリフェノールを除去することを含む、請求項18に記載の方法。 - 脱色することが、抽出溶媒での植物組織からのポリフェノールの抽出を行うことを含み、前記抽出溶媒がエタノールを含む、請求項20に記載の方法。
- 細片を除去するステップが、透析、均質化した植物組織を濾過すること、前記均質化した植物組織を遠心分離すること、又は前記均質化した植物組織にタンジェンシャルフロー濾過又は連続フロー濾過を行うこと、又はその任意の組合せを含む、請求項18に記載の方法。
- 請求項1に定義されるナノファイバーを調製するための、請求項18に記載の方法。
- 薬剤として使用するための請求項1~10のいずれかに記載のナノファイバーであって、前記ナノファイバーが、1又は2以上の生物学的に活性な薬剤をさらに含む、前記ナノファイバー。
- 薬剤として使用するための請求項24に記載のナノファイバーであって、前記薬剤が、がん細胞、又は多剤耐性がん細胞において細胞毒性を誘導するためのものである、前記ナノファイバー。
- 薬剤として使用するための請求項24に記載のナノファイバーであって、前記薬剤が、多剤耐性細菌感染を治療する又は予防するためのものである、前記ナノファイバー。
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