孫璐西臺灣大學：食品科技研究所林家凱Lin, Jia - KaiJia - KaiLin2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/56316阿滋海默症為神經退化性疾病，主要因為老化斑塊（senile plaque）的沉積、神經纖維糾結（neurofibrillary tangles）與神經退化，造成患者記憶與認知的衰退。β-類澱粉胜Alzheimer’s disease (AD) is a neurodegenerative disorder. The decline of memory and cognition in AD is underlied by the progressive deposition of senile plaques, neurofibrillary tangles and neuronal degeneration. The principal component in senile plaques is the β-amyloid peptide (Aβ). Aβ has been shown to lead to oxidative stress and apoptosis of neurons. Many of studies have demonstrated that antioxidants may have the potential to protect Aβ-induced cytotoxicity. The theory of traditional Chinese medicine (TCM) postulated that kidney functions is a key factor in AD development and progression. We screened the dried edible plant materials which have remarkable antioxidation activities and based on the theory of TCM, we chose TCMs which can improve liver and kidney functions to screen for the anti-Alzheimer effect. Scientific preparations of TCMs were extracted with water or methanol at room temperature. The dried edible plant materials were extracted with boiling water or methanol at room temperature. The neuroprotective effect of Ginkgo biloba extract (EGb 761) has been demonstrated by several in vitro and in vivo studies. In vitro, EGb 761 showed dose-dependent effect to protect cultured PC-12 cells against death induced by Aβ. In vivo, daily oral administration of EGb 761 improved cognitive function in patients with Alzheimer’s disease. Therefore, we utilized EGb 761 as the positive control in this study. In this study, we used the “inhibition of aggregated Aβ-induced PC-12 cells death” as the screening model for anti-Alzheimer’s disease activity, and EGb 761 was used as the positive control. PC-12 cells were incubated with samples and 15 μM Aβ25-35 for 48h. The inhibition of Aβ-induced cytotoxicity were observed with methyl thiazolyl tetrazolium (MTT) assay and were represented by cell viability and the percentage of inhibition (inhibition ratio). Among the water extracts, superior inhibition effects were exhibited by Panax ginseng（Pg-W）, Dioscorea opposit（Do-W）, Schisandra chinensis, Polygonum multiflorum（Pm-W）, Lycium barbarum（Lb-W）, Poncirus trifolata（Pt-W）, Psoralea coryhoflia（Pc-W）, green tea（GT-W）, oolong tea（OT-W）, pu-erh tea（PT-W）, GABA tea（GT-W）, Sesamum indicum（Si-W）, nelumbinis folium（Nf-W）, Perilla frutescens（Pf-W）, nelumbinis embryo（Ne-W） and Dimoracarpus longan（Dl-W） at concentration range of 50 ~200 μg/mL. Superior inhibition effects of the ethanol extracts of TCMs were shown by Cornus officinalis（Co-E）, Dioscorea opposite（Do-E）, Acorus tatarinowii（At-E）, Cistanche deserticola（Cd-E）, Lycium barbarum（Lb-E）, Astragalus membranaceus（Am-E）, Angelica sinensis（As-E）, Psoralea coryhoflia（Pc-E） and Polygala Tenuifolia（Pt-E） at concentration range of 50~200 μg/mL. Methanol extract of Sesamum indicum（Si-M）gave the greatest inhibition effects among all the edible plant materials. It also indicated that Si-W, Ne-W and Pt-W were the three most effective extracts, and all of them did not exhibit any cytotoxicity. Microscopic examination of the morphology of PC-12 cells, showed that 200 μg/mL Si-W and Ne-W could induce neurite outgrowth in PC-12 cells even when Aβ25-35 was present. In the cell cycle analysis by flow cytometer, Si-W, Ne-W and Pt-W were demonostrated to significantly inhibit Aβ25-35-induced apoptosis. Si-W, Ne-W and Pt-W were fractionated into ethyl acetate fraction, n-butanol fraction and aqueous fraction. The aqueous fractions of these three extracts were found to have better inhibition effects against Aβ-induced cytotoxicity than the other two fractions. In the cell cycle analysis, the aqueous fractions of Si-W, Ne-W and Pt-W could all significantly inhibit Aβ25-35-induced apoptosis when the concentration was 200 μg/mL. We presumed that Sesamum indicum, nelumbinis embryo and Poncirus trifolata have anti-Alzheimer’s disease capacity could probably be related to compounds having higher polarity.摘要 I 英文摘要 III 目錄 VI 表次 XI 圖次 XII 壹、前言 1 貳、文獻整理 3 一、阿滋海默症 (Alzheimer，s disease, AD) 3 二、阿滋海默症的病理特徵（Pathological features） 4 三、阿滋海默症的診斷 6 四、阿滋海默症之分類 7 五、阿滋海默症之致病機轉 7 六、阿滋海默症之治療 13 七、現代中醫與阿滋海默症 19 八、實驗材料介紹 20 （一）藥材部份 20 1.人參 20 2.山茱肉 22 3.山藥 22 4.五味子 23 5.巴戟天 23 6.牛膝 23 7.石菖蒲 24 8.肉蓯蓉 24 9.何首烏 25 10.杜仲 25 11.刺五加 26 12.枸杞子 26 13.枳實 26 14.茴香 27 15.茯苓 27 16.淫羊藿 28 17.菟絲子 28 18.黃耆 28 19.當歸 29 20.補骨脂 30 21.遠志 30 22.熟地黃 30 （二）食材部份 31 1.綠茶 31 2.烏龍茶 32 3.普洱茶 32 4.佳葉龍茶 32 5.芝麻 33 6.荷葉 33 7.紫蘇 34 8.蓮子心 34 9.龍眼花 34 九、大鼠的腎上腺髓質嗜鉻細胞株（pheochromocytoma, PC-12） 35 參、研究目的與實驗設計 37 一、研究目的 37 二、實驗設計 38 肆、材料與方法 39 一、實驗材料 39 （一）藥材部份 39 （二）食材部份 39 （三）正控制組 40 二、實驗細胞株 40 三、化學藥品、溶劑與耗材 40 （一）化學藥品 40 （二）溶劑 41 （三）細胞耗材 42 （四）細胞實驗各種溶液配方 43 四、儀器設備 44 （一）樣品前處理與萃取相關儀器設備 44 （二）樣品分析相關儀器設備 44 （三）細胞培養相關儀器設備 45 五、實驗方法 46 （一）樣品的製備 46 （二）萃出率之測定 47 （三）大鼠的腎上腺髓質嗜鉻細胞瘤（pheochromocytoma, PC-12）之培養與保存 48 （四）細胞存活率分析-MTT assay 49 （五）β-amyloid peptide（Aβ25-35）聚集 50 （六）Aβ25-35對PC-12細胞之細胞毒性 50 （七）樣品對Aβ25-35聚集導致PC-12細胞死亡之抑制 50 （八）以流式細胞儀偵測實驗樣品對PC-12細胞週期之影響 51 六、統計分析 52 伍、結果與討論 53 一、不同溶劑之萃出率 53 二、Aβ25-35對PC-12細胞之毒性 55 三、EGb 761對PC-12細胞存活率之影響與抗Aβ25-35聚集導致PC-12細胞死亡 56 四、樣品萃出物對Aβ25-35聚集導致PC-12細胞死亡之抑制與對PC-12細胞存活率之影響 57 （一）科學中藥 57 （二）食材 67 五、三種樣品水萃物各區分層之萃出率及其所佔有之百分比 75 六、三種樣品水萃出之區分物對Aβ25-35聚集導致PC-12細胞死亡之抑制 76 （一）芝麻水萃物 76 （二）蓮子心水萃物 77 （三）枳實水萃物 78 七、細胞週期分析 79 （一）抗Aβ25-35細胞毒性功效較佳之三種水萃物對PC-12 細胞週期之影響 81 （二）抗Aβ25-35細胞毒性功效較佳之三種水萃物對PC-12 細胞週期之影響 82 陸、結論 85 柒、參考文獻 111 捌、附錄 1235379591 bytesapplication/pdfen-US阿滋海默症食藥材PC-12 細胞β-類澱粉胜Alzheimer’s diseaseplant materialsPC-12 cellAβthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56316/1/ntu-95-R93641011-1.pdf