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Studies on the Physiological Functions of Kaki Fruit Extracts against Oxidative Stress in Skeletal Muscle

Nayla Majeda Alfarafisa 岐阜大学

2020.09.18

概要

高齢化社会を迎え,健康長寿を目指した食品科学によるアプローチの重要性が増している。しかし,老化とはさまざまな生物学的反応が関与する非常に複雑な現象であることから,単純化したモデル系での研究は困難であり,より対象を明確にした分子レベルでの研究の重要性が今後ますます増してくると考えられる。本学位論文では,老化現象を説明する一つのメカニズムとして,酸化ストレス状態で蓄積されたフリーラジカル種が細胞内の分子に障害を与え,老化という悪影響をもたらすことに注目している。具体的には,老化によってもたらされるサルコペニア(骨格筋の質量と強度の変性損失)の発症に寄与する病態生理学的要因の一つである酸化ストレスに焦点を当て,骨格筋を構成する細胞の損傷を防ぐために,酸化ストレス状態にある筋芽細胞(C2C12 細胞)に対して,柿(Diospyros kaki)の抽出物が有する効果の検証を行うことを目的に,従来の単一培養系に替えて,ヒト腸管細胞(Caco-2 細胞)を用いた共培養系を構築して研究を行った。

本研究では,まず柿果実を 25%,50%,75%の 3 種類のエタノールを溶媒としてホットエタノール法で抽出し,柿の粗抽出液を分画したところ,6 種類の画分が得られた。各画分の機能解析を行うため,生理活性化合物の活性を評価するため,フリーラジカル消去剤である DPPH を用い,DPPH アッセイから IC50 値を算出した。その結果,柿画分のラジカル消去活性は,画分 F>画分 B と D と E>画分 C>画分 A の順に効果を発揮した。また,ORAC 法を用いて,ペルオキシルラジカル誘導酸化に対する抗酸化剤介在性保護を測定した。その結果,柿画分の ORAC値は,画分F(8.0 mmol TE/100 g)>画分B(6.2 mmol TE/100 g)とD(7.2 mmol TE/100 g)と E(7.1 mmol TE/100 g)>画分A(2.7 mmol TE/100 g)とC(3.5 mmol TE/100 g)の順に効果を発揮した。さらに,全フェノール含有量は,画分F(3.87 g CE/100 g)>画分 D(1.10 g CE/100 g)>画分B(0.75 g CE/100 g)と E(0.84 g CE/100 g)>画分A(0.22 g CE/100 g)と C(0.39 g CE/100 g)の順に高かった。以上より,他の疎水性画分と同等の値を示した E 画分を除いて,柿由来の疎水性画分(B, D, F)は親水性画分(A, C)と比較して,より強い抗酸化力を示す傾向が示された。そこで,さらなる分析のために,画分 B, D, E および F を選択した。

直接的細胞毒性は,WST-8 アッセイおよび TEER 値を用いて評価した。0.01–1 mg/mL の濃度範囲で B, D, E, F 画分を添加したところ,Caco-2 もしくは C2C12 細胞培養において,24 時間培養後の細胞生存率の有意な低下は認められなかった。また,TEER 値は,Caco-2 細胞が完全に分化したことを示すだけでなく,腸管細胞における被験物質の亜致死毒性も示すことができることを利用して調査したところ,柿画分の添加直後に観測された TEER 値の低下は,24 時間培養後には 0.1 mg/mL および 0.5 mg/mL ともに初期値を回復することが確認された。このことは,用いた柿画分が腸管細胞に有害な毒性を有さないことを意味している。

さらに,共培養系として,1 mM の H2O2 を添加して 6 時間培養すると,C2C12 細胞の生存率が著しく低下することを利用し,低濃度(0.1 mg/mL)と高濃度(0.5 mg/mL)の柿画分をCaco- 2 細胞に作用させた後,回収した基底膜側培地を用いて C2C12 筋芽細胞を 24 時間培養して,細胞生存率に対する影響を検討した。その結果,低濃度では,細胞生存率を回復させなかった一方,高濃度の柿画分で前処理すると,H2O2 に対してより顕著な細胞保護効果を示した(B,D,および F)。このことから,柿画分の 0.5 mg/mL 画分 B, D, F によって誘導されるCaco-2 細胞の分泌物は,H2O2 による酸化ストレスに対する細胞保護効果を有していることが示された。そして,このような細胞保護効果を説明するために,H2O2 による酸化ストレスで誘導された C2C12細胞の細胞内活性酸素量を測定した結果,柿画分を低濃度で添加して得られた基底膜側培地での培養では,C2C12 細胞内の活性酸素レベルの有意な上昇を回復させることはできなかったが,高濃度の B 画分を添加して得られた基底膜側培地での培養では,酸化ストレス誘導された C2C12 細胞内の活性酸素レベルを有意に減少させることができた。このことから,酸化ストレスが誘発する細胞毒性からの保護効果は,細胞内の活性酸素の発生を抑制する効果に依存している可能性が高いことが示唆された。

以上の結果より,本学位論文では,柿果実に由来する抽出物は,in vitro においてフリーラジカル消去活性と抗酸化物質を介したペルオキシルラジカル誘導酸化に対する保護作用を示し,また,筋芽細胞における酸化ストレスに対する細胞保護作用を有する代謝物の分泌をヒト腸管細胞に対して誘導するという,筋芽細胞内の活性酸素消去効果を間接的に有していることを発見したことを報告している。これらの知見は,骨格筋細胞のように食品成分と直接接触できない組織への食品成分の作用機序の一端を示唆するものであり,共培養系を用いた新しい食品成分の機能性評価に寄与するものである。

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参考文献

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