五倍子(Galla chinensis) の抗アミロイド凝集作用とその有効成分

Abstract

室蘭工業大学博士（工学）アルツハイマー病（AD）は，加齢に伴い発症する慢性の神経変性疾患であり，認知機能の低下や記憶障害，さらに行動や精神機能の変化を特徴とし，時間の経過とともに進行する．本疾患の主な原因は，脳内の酸化ストレスとともにアミロイドβ（Aβ）が蓄積するためであると考えられている．植物由来の低分子は，初期の病理学的事象，特にAβ凝集を標的とした治療法への適用の可能性がある．五倍子(Galla chinensis) は，抗酸化作用，抗炎症作用，神経保護作用が報告されているポリフェノール成分を豊富に含む伝統的な生薬である．また，最近の研究では，認知機能低下に対する有効性が報告されており，これに含まれるAβ凝集を阻害する化合物が注目されている． 本研究は，Aβ凝集を効果的に阻害する五倍子のエタノール抽出物から活性成分を単離することを目的とした．Aβ凝集阻害活性は，チオフラビンT (ThT) 法により評価した．五倍子EtOH抽出物のEC50値は1.65 mg/mLであり，抽出物から液―液分配により得られたEtOAc 画分はその42%に相当する活性を示した．活性化合物は，Aβ凝集阻害活性を指標に，種々のクロマトグラフィーにより分画・精製し，2種の活性化合物を得た．これらの化合物の1H-および13C-NMRスペクトル,質量スペクトルの詳細な解析により，その構造をpentagalloyl glucose (PGG) およびmethyl Gallate (MG) と同定した． PGGとMGのAβ凝集阻害活性は，それぞれEC50 = 46.7μMおよび111.9μMであり，MGよりもPGGは強い活性を示した．また，PGGおよびMGは五倍子エタノール抽出物の活性に対して，それぞれ1.5および0.7％貢献することが明らかとなった．さらに，DPPHラジカル消去アッセイにより抗酸化活性を評価した．その結果，PGGおよびMGのEC50値はそれぞれ1.16および6.44μMであり，PGGはより強い抗酸化力を示した．これらの化合物のSH-SY5Y細胞株を対象としたAβ誘発細胞毒性試験を実施した．その結果，30μMの化合物濃度で，PGG (p < 0.0005) はMG (p < 0.05)と比較して細胞毒性を有意に軽減した． 以上，本研究により五倍子とPGGは，AD発症過程で重要な因子と考えられているAβ凝集の抑制と酸化ストレスの消去活性に対して有効性を示し，五倍子の生物活性成分に関する知見を得た．今後，五倍子からさらなる生物活性化合物の同定とADの潜在的治療薬の開発を目指すAlzheimer's disease (AD) is an age-related chronic neurodegenerative disorder that progresses with time, characterized by cognitive decline, memory impairment, and alterations in behavioral and mental functions. The accumulation of amyloid- beta (Aβ) plaques along with oxidative stress in the brain are considered as the key contributors to AD pathogenesis. Recently, plant-derived small molecules have gained attention for their therapeutic potential, particularly in targeting early pathological events such as Aβ aggregation, offering promising strategies for AD prevention and treatment. Gobaishi (Galla chinensis) is a traditional herbal medicine that is composed of a rich source of polyphenolic constituents that have been reported to possess antioxidant, anti-inflammatory, and neuroprotective effects. In addition, recent studies have reported its effectiveness in improving cognitive functions, which has garnered the attention to seek the compounds capable of inhibiting Aβ aggregation. In this study, the aim was to isolate the active constituents of Gobaishi EtOH extract which can effectively prevent the Aβ aggregation. The Aβ aggregation inhibitory activity was evaluated by the thioflavin T (ThT) method. EC50 value of Gobaishi EtOH extract is 1.65 mg/mL and the liquid–liquid partitioned EtOAc fractions contributes to 42% of the extract. The active compounds were fractionated and purified by various chromatography under the guidance of Aβ aggregation inhibitory activity, and two active compounds were obtained. Detailed analysis of 1H- and 13C-NMR spectra and mass spectra of these compounds identified their structures as pentagalloyl glucose (PGG) and methyl gallate (MG). The Aβ aggregation inhibitory activity of PGG and MG was EC50 = 46.7 and 111.9 μM, respectively, indicating that PGG was more active than MG, and PGG and MG contributed 1.5 and 0.7%, respectively, to the activity of EtOH extract of Gobaishi. Furthermore, antioxidant activity was evaluated by DPPH radical scavenging assay. The EC50 values of PGG and MG were 1.16 and 6.44 μM, respectively, indicating that PGG showed stronger antioxidant activity. In addition, Aβ-induced cytotoxicity studies of these compounds in the SH-SY5Y neuroblastoma cell line were conducted. The results showed that PGG significantly reduced cytotoxicity (p < 0.0005) compared to MG (p < 0.05) at a compound concentration of 30 μM. In conclusion, this study demonstrated the efficacy of Gobaishi and PGG in inhibiting Aβ aggregation and radical scavenging activity, which are considered to be important factors in the pathogenesis of AD and provided insight into the bioactive components of Gobaishi.doctoral thesi