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研究生:馬浩庭
研究生(外文):Ma, Hao-Ting
論文名稱:金錢薄荷乙酸乙酯萃取物之抗氧化、乙醯膽鹼酯酶活性抑制及神經保護功效探討
論文名稱(外文):Studies on the Antioxidant, Acetylcholinesterase Inhibitory Activities and Neuroprotective Effect of Glechoma hederacea Ethyl Acetate Extracts
指導教授:周淑姿周淑姿引用關係
指導教授(外文):Chou, Su-Tze
口試委員:張珍田陳淑茹
口試委員(外文):Chang, Chen-TienChen, Shu-Ju
口試日期:2016-07-21
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:138
中文關鍵詞:金錢薄荷PC12 cells分配萃取神經保護
外文關鍵詞:Glechoma hederaceaPC12 cellspartitioned fractional extractsneuroprotection
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阿茲海默症 (Alzheimer’s disease, AD) 為最常見的神經退化症,有許多研究指出阿茲海默症與神經傳導物乙醯膽鹼含量具有相關性,而從天然中藥草提取有效活性成分,減少乙醯膽鹼被乙醯膽鹼酯酶(Acetylcholinesterase, AChE) 分解代謝,進而保護神經細胞及改善神經退化性疾病為一研究趨勢。金錢薄荷(Glechoma hederacea) 為唇形科(Lamiaceae) 植物,為一傳統中藥材。本實驗首先以金錢薄荷經熱水萃取後所得之粗萃物 (hot water of Glechoma hederacea, HWG),再經分配萃取得乙酸乙酯層 (ethyl acetate, EA)、丁醇層 (n-butanol) 及水層 (H2O) 之萃取物,分析其乙醯膽鹼酯酶活性抑制試驗評估及酵素抑制動力學以確定其抑制型態,並以體外抗氧化試驗測其抗氧化效力,再從細胞模式探討分配萃取物對經H2O2誘發氧化損傷之大鼠腎上腺髓質嗜鉻細胞瘤 (PC12細胞) 之保護功效,進而以高效液相層析 (High-performance Liquid Chromatography, HPLC) 分析其組成分。
從體外試驗結果顯示,三種分配萃取物隨著濃度之上升皆有良好抑制乙醯膽鹼酯酶活性、清除DPPH及ABTS+自由基、並具良好之還原力及抑制脂質過氧化能力,其中EA extract的效果最佳。在細胞試驗結果顯示,三種分配萃取物於12.5~50 μg/ml 之添加對經H2O2誘發氧化損傷之PC12細胞之影響,因濃度增加其細胞存活率也隨之增加,有劑量依賴效應,其中仍以EA extract的效果最佳,故選EA extract做進一步實驗。EA extract之添加可有效改善經H2O2誘發氧化損傷之PC12細胞其氧化/抗氧化系統恆定狀態,降低脂質過氧化物質MDA生成量及glutathione (GSH) 含量,並調節glutathione peroxidase (GSH-Px)、superoxide dismutase (SOD) 與 catalase (CAT) 之酵素活性,進而減少細胞的DNA損傷及凋亡。於高效液相層析分析萃取物可得知,其中主要含量的化合物為迷迭香酸 (rosmarinic acid),推測可能為生理活性來源。
綜合上述,EA extract具良好之抗氧化及乙醯膽鹼酯酶活性抑制效力,且能調控氧化/抗氧化系統恆定,減少細胞凋亡,進而降低經H2O2誘發PC12細胞之氧化損傷,具有良好神經保護之潛力。

Alzheimer’s disease is the most common neurodegenerative disease. Many studies have indicated that Alzheimer’s disease was related to neurotransmitter acetylcholine content. Recently, there is an increasing interest in the study how to protect nerve cells and improve the neurodegenerative disease. Glechoma hederacea belongs to the Lamiaceae family, is used for medication purpose widely and possesses several health benefits. This study was aimed to evaluate the effects of the crude extract (HWG, GH50, GH95) and partitioned fractional extract (EA, n-butanol, H2O) of G. hederacea on H2O2-induced toxicity and oxidative damage in rat pheochromocytoma line ( PC12 cells). Furthermore, we also identified the phenolic compounds of the G. hederacea extracts by using reverse-phase high performance liquid chromatography (HPLC) coupled with UV/vis detector.
The results demonstrated that three partitioned fractional extracts from G. hederacea possessed good inhibition of acetylcholinesterase, DPPH and ABTS+ radical, and have well reducing power and inhibit lipid peroxidation activities. It was obvious that EA extract was more effective protection ability. In the cell model, the EA extract didn’t possess the cytotoxicity in PC12 cells and can recover the cell morphology and the cell viability of H2O2-treated PC12 cells. In addition, EA extract can improve the glutathione (GSH) levels, decrease the malondialdehyde (MDA) levels and regulate the glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase activities. Moreover, the EA extract can prevents H2O2-induced DNA damage and apoptosis in PC12 cells. The rosmarinic acid content in G. hederacea extracts was the highest.
In conclusion, EA extract possessed the AchE inhibitory activity and balanced the oxidant-antioxidant defense in H2O2-treated PC12 cells. EA extract inhibited the cell apoptosis and DNA damage. The EA extract may have the antioxidant activity and neuroprotective potential.

目錄 I
表目錄 IV
圖目錄 V
中文摘要 IX
英文摘要 XI
第一章 前言 1
第二章 文獻回顧 3
第一節 植物介紹 3
一、 唇形科 (Lamiaceae) 植物簡介 3
二、 金錢薄荷簡介 3
第二節 萃取 6
1.固、液萃取法 7
2.液、液萃取法 7
第三節 氧化壓力與抗氧化防禦系統 8
一、自由基與活性氧物質之簡介 11
二、體內抗氧化防禦系統 12
第四節 阿茲海默症 (Alzheimer’s disease, AD) 17
第五節 氧化傷害與神經退化性疾病之相關性 18
第六節 乙醯膽鹼酯酶作用及其抑制 20
第七節 H2O2誘發PC12細胞氧化損傷 22
一、PC12細胞之簡介 22
二、誘發細胞氧化損傷之簡介 22
第三章 研究目的 25
第四章 實驗流程 26
第五章 材料方法 28
第一節 儀器設備、器材 28
第二節 藥品 30
第三節 實驗方法 33
一、 金錢薄荷不同溶劑萃取物及熱水萃取物之分配萃取物之製備 33
二、 乙醯膽鹼酯酶活性測定試驗 33
三、 體外抗氧化試驗 36
四、 細胞試驗 43
五、 高效液相層析分析 (High-performance Liquid Chromatography, HPLC) 52
第六章 統計分析 54
第七章 結果 55
一、 金錢薄荷不同溶劑萃取物及熱水萃取物之分配萃取物之萃取率 55
二、 金錢薄荷不同溶劑萃取物及熱水萃取物之分配萃取物對乙醯膽鹼酯酶活性及其動力學型態之影響 56
三、 金錢薄荷熱水萃取物之分配萃取物之體外抗氧化活性 66
(一) 自由基清除能力 66
(二) 金錢薄荷熱水萃取物之分配萃取物之還原力 68
(三) 抑制脂質過氧化測定 68
(四) 金錢薄荷熱水萃取物之分配萃取物之總多酚及類黃酮含量 69
四、 金錢薄荷不同溶劑萃取物及熱水萃取物之分配萃取物對PC12細胞存活率之影響 73
五、 金錢薄荷不同溶劑萃取物及熱水萃取物之分配萃取物對經H2O2誘發氧化損傷之PC12細胞存活率之影響 73
六、 金錢薄荷熱水萃取物與乙酸乙酯萃取物 (EA extract) 對H2O2誘發氧化損傷之PC12細胞DNA損傷之影響 79
七、 金錢薄荷熱水萃取物與乙酸乙酯萃取物 (EA extract) 對H2O2誘發氧化損傷之PC12細胞氧化/抗氧化系統恆定之影響 85
八、 金錢薄荷分配萃取物之高效液相層析 (High-performance Liquid Chromatography, HPLC) 及相關性分析 98
第八章 討論 107
第九章 結論 117
第十章 參考文獻 119

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