孫璐西Hwang, Lucy Sun臺灣大學:食品科技研究所羅喬文Luo, Chiau-WenChiau-WenLuo2010-05-112018-06-292010-05-112018-06-292008U0001-1808200823495300http://ntur.lib.ntu.edu.tw//handle/246246/182163阿滋海默症 (Alzheimer''s disease, AD) 是一種神經退化性疾病，病理上最大特徵是大量的老化斑塊 (senile plaques) 堆積於腦部神經細胞外，而細胞內有神經元纖維糾結 (neurofibrillary tangle) 沉積。β-amyloid peptide (Aβ )為老化斑塊的主要組成，已知Aβ 在阿滋海默症的致病過程中扮演著重要的角色。許多研究指出過多的 Aβ 聚集會導致氧化壓力 (oxidative stress)、細胞內鈣離子失衡，而促使細胞走向凋亡 (apoptosis)。本研究以「Aβ聚集導致PC-12 細胞死亡之抑制」作為阿滋海默症之細胞模式 ，篩選一些具有抗氧化功效以及預防或延緩阿滋海默症之功效的食藥材。將桑椹水萃物、洛神花水萃物和蓮子心 70% 甲醇粗萃物以及源自於何首烏 (Polygonum multiflorum Thunb.) 的 2,3,5,4''-tetrahydroxystilbene-2-O-β-gluco pyranoside (PM-SG) 和 許多漿果中存在的 pterostilbene 進行體外抗氧化能力測定與其抑制 Aβ25-35 毒性作用之功效，結果顯示蓮子心 70% 甲醇粗萃物的抗氧化能力和保護 PC-12 細胞免於 Aβ25-35 毒性的效果最好。因此將蓮子心 70% 甲醇粗萃物進一步以不同溶劑區分，得到正己烷區分物 (Nn-M-H)、乙酸乙酯區分物 (Nn-M-EA)、正丁醇區分物 (Nn-M-B) 和水區分物 (Nn-M-W)，將此四個區分物進行體外抗氧化能力測定和抗 Aβ1-40Alzheimer''s disease (AD), the major cause of dementia, is a neurodegenerative disorder associated with impairment in memory and cognitive function. AD is characterized pathologically by the presence of senile plaques (amyloid plaques), and neurofibrillary tangles (NFT). β-amyloid peptide (Aβ) has been proposed to play an important role in the pathogenesis of AD. Deposits of Aβ are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. Many studies have shown that aggregated Aβ can induce caspase-dependent apoptosis and death by oxidative stress and increase intracellular calcium levels in cultured neurons. The aim of my study is to investigate the antioxidative activities and neuroprotective effects of some edible plant extracts and stilbenoids.he antioxidative potency was evaluated by DPPH radical scavenging activity and trolox equivalent antioxidant capacity (TEAC). The neuroprotective activity was evaluated in vitro in the “Aβ-induced cell death of rat pheochromocytoma (PC-12) cells” model system. Among the different samples (mulberry, roselle and the embryo of Nelumbo nucifera Gaertn. seed extracts), methanol extract of the embryo of Nelumbo nucifera GAERTN. seed (Nn-M) showed better antioxidative activity and also higher inhibitory effect against Aβ25-35 –induced cytotoxicity. Therefore, Nn-M was fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fractions. The n-butanol fraction of Nn-M was found to have the best inhibitory effect against Aβ1-40 –induced cytotoxicity. Among stilbenoids, stilbene glycoside purified from Polygonum multiflorum Thunb. (PM-SG) has better inhibitory effect against Aβ25-35 –induced and Aβ1-40 –induced cytotoxicity than pterostilbene. he mechanism of the possible protective action of PM-SG and n-butanol fraction of Nn-M was further investigated. Results showed that Aβ1-40 signficantly increased ROS level, intracellular [Ca2+] and caspase-3 activity, and also signficantly decreased mitochondrial membrane potential and glutathione content in PC-12 cells. PM-SG and n-butanol fraction of Nn-M showed a significant reduction of the elevated ROS, intracellular [Ca2+] and caspase-3 activity and also replenished the loss of mitochondrial membrane potential and glutathione content induced by Aβ1-40. This study concludes that PM-SG and n-butanol fraction of Nn-M have potential neuroprotective ability against Aβ-mediated cell damage.致謝.......................................................I寫表 (Abbreviateions)..................................III文摘要..................................................IVbstract..................................................VI錄....................................................VIII次.....................................................XII次....................................................XIII一章 前言................................................1二章 文獻整理............................................2.1 老化與神經退化........................................2.2 阿滋海默症 (Alzheimer''s disease, AD)..................2.3 阿滋海默症的病理特徵..................................5.4 阿滋海默症的分類......................................8.5 阿滋海默症的病理機制..................................8.5.1 β-類澱粉胜肽 (β-amyloid peptide; Aβ)...............8.5.2 脂蛋白元E (Apolipoprotein E; ApoE).................14.5.3 Tau 蛋白...........................................14.6 Amyloid cascade 假說.................................15.7 氧化壓力與阿滋海默症.................................17.8 阿滋海默症的治療方法.................................20.9 PC-12 細胞模式與阿滋海默症的研究.....................22.10 實驗材料介紹.........................................23.10.1 銀杏葉萃出物 (EGb 761)............................23.10.2 桑椹..............................................25.10.3 洛神花............................................25.10.4 蓮子心............................................26.10.5 Stilbenoids.......................................27三章 研究目的與實驗架構.................................30.1 研究目的.............................................30.2 實驗架構.............................................31四章 材料與方法.........................................35.1 實驗材料.............................................35.2 實驗細胞株...........................................35.3 實驗用試藥與耗材.....................................36.4 裝置與儀器設備.......................................39.5 細胞實驗溶液配方.....................................41.6 實驗方法.............................................42. 測試樣品製備..........................................42. 體外抗氧化測定........................................43.6.1 DPPH 自由基清除能力測定............................43.6.2 總抗氧化力測定 (Trolox equivalent antioxidant capacity, TEAC)...........................................43. Aβ 聚集導致 PC-12 細胞死亡模式......................44.6.3 PC-12 細胞之培養與保存.............................44.6.4 細胞存活率測定.....................................46.6.5 細胞數與 MTT 或 WST-1 吸光值之標準曲線.............47.6.6 Aβ25-35 與 Aβ1-40 之聚集與其對 PC-12 細胞之毒性測試........................................................47.6.7 試驗樣品抗 agg-Aβ 細胞毒殺作用之活性篩選..........48. 保護作用之機制評估...................................49.6.8 細胞內 ROS 含量.....................................49.6.9 流式細胞儀測定粒線體膜電位..........................49.6.10 流式細胞儀測定細胞內鈣離子.........................50.6.11 細胞內 glutathione 含量測定........................51.6.12 流式細胞儀測定 caspase-3 活性......................52.7 統計分析..............................................53五章 結果與討論.........................................54.1 粗萃物樣品及 stilbenoids 之體外抗氧化活性測定.........54.1.1 DPPH 自由基清除能力測定.............................54.1.2總抗氧化力測定 (Trolox equivalent antioxidant capacity, TEAC)...........................................56.2 Aβ25-35 對 PC-12 細胞之毒性測試.....................65.2.1 細胞數與 MTT 或 WST-1 吸光值之標準曲線..............66.2.2 Aβ25-35 對 PC-12 細胞之毒性.......................67.2.3 粗萃物樣品與 stilbenoids 抗 Aβ25-35 細胞毒性作用之活性篩選....................................................69.3 蓮子心 70% 甲醇粗萃物之區分...........................71.4 蓮子心區分物之體外抗氧化活性測定......................71.4.1 DPPH 自由基清除能力測定.............................71.4.2總抗氧化力測定 (Trolox equivalent antioxidant capacity, TEAC)...........................................72.5 Aβ1-40 對 PC-12 細胞之毒性測試......................77.5.1 Aβ1-40 對 PC-12 細胞之毒性........................77.5.2 蓮子心70% 甲醇粗萃物的四種區分物與 stilbenoids 抗 Aβ 1-40 細胞毒性作用之活性篩選...............................77.6 保護作用之機制評估....................................81.6.1 細胞內 ROS 含量.....................................81.6.2 粒線體膜電位測定....................................85.6.3 細胞內鈣離子測定....................................91.6.4 細胞內 glutathione 含量.............................94.6.5 Caspase-3 活性測定..................................98六章 結論..............................................102七章 參考文獻..........................................104application/pdf2243408 bytesapplication/pdfen-US阿滋海默症類澱粉蛋白PC-12氧化壓力蓮子心stilbenoidsAlzheimer’s diseaseβ-Amyloid peptideOxidative stressEmbryo of the seed of Nelumbo nucifera GAERTN.Stilbenoidsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182163/1/ntu-97-R95641005-1.pdf