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研究生:徐雯敏
研究生(外文):Wen-Min Hsu
論文名稱:香椿對老化促進小鼠學習記憶能力、抗氧化狀態與精子品質之影響
論文名稱(外文):The effects of Toona sinensis Roemor on learning and memory ability, antioxidant status and sperm quality in senescence accelerated mice
指導教授:詹吟菁詹吟菁引用關係
指導教授(外文):Yin-Ching Chan
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文出版年:2005
畢業學年度:92
語文別:中文
論文頁數:198
中文關鍵詞:抗氧化狀態香椿學習記憶能力類澱粉蛋白精子品質
外文關鍵詞:learning and memoryß-amyloid proteinantioxidative statusToona sinensis Roemorsperm quality
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摘要
香椿含有大量的酚類化合物,可能具有抗氧化之功效,而能降低活性氧物質對生物體的傷害。本研究之目的在探討不同部位之香椿萃取物清除DPPH自由基的效力及對老化促進小鼠(SAMP8)學習記憶能力、抗氧化狀態與精子品質之影響。體外試驗測試香椿葉、根皮及樹皮萃取物清除DPPH自由基之效力。體內試驗則選用3月齡雄性老化促進小鼠,分別餵食1%香椿葉、1%香椿根皮及1%香椿樹皮萃取物進行12週;另以老化正常小鼠(SAMR1)為正對照組。實驗期間記錄體重及攝食量,並進行行為活動量試驗(open field activity test)、單次被動迴避試驗(single-trial passive avoidance test)及主動迴避試驗(active shuttle avoidance test)。實驗後將動物犧牲,取肝臟和腦部海馬迴、紋狀體及皮質檢測其丙二醛(MDA)含量、超氧歧化酶(SOD)、觸酶(CAT)及麩胱光肽過氧化酶(GSH-Px)之活性,並觀察腦部類澱粉蛋白(Aß)堆積情形。此外,評估精子活動力、濃度及畸形率,另進行血液生化值檢查。體外試驗顯示,香椿葉、根皮及樹皮萃取物於濃度0.625mg/ml時,其清除DPPH自由基效力均達80%以上。體內試驗結果發現攝食量、體重及活動量於五組間並無顯著差異。單次被動迴避試驗顯示以香椿葉萃取物組有較佳學習記憶能力;而主動迴避試驗結果則顯示香椿三部位萃取物均較對照組與正對照組有較佳學習記憶能力,但香椿萃取物組間並無顯著差異。另,給予香椿萃取物的三組(葉、根皮及樹皮組)均能顯著較對照組與正對照組降低腦中類澱粉蛋白的堆積與肝臟及腦部丙二醛之含量,但三組間未達顯著差異。餵食香椿萃取物之三組較對照組與正對照組皆能顯著增加其腦部及肝臟之超氧歧化酶、觸酶及麩胱甘肽過氧化酶活性。三組萃取物中以根皮具最佳促進海馬迴及肝臟中超氧歧化酶之活性與肝臟中觸酶活性之功效,葉萃取物組則有最高的紋狀體及皮質超氧歧化酶活性與肝臟中麩胱甘肽過氧化酶之活性;此外樹皮萃取物組則最能有效增加海馬迴,紋狀體及皮質中麩胱甘肽過氧化酶之活性。餵食香椿三部分萃取物皆能顯著增加精子活動力、精子濃度,並能顯著降低其血清中膽固醇與三酸甘油酯之濃度,但三組間無顯著差異。此外,以香椿葉組有最低的血糖值。相關性分析結果則顯示Aß與腦部SOD、CAT與GSH-Px之活性成負性相關,另與腦部MDA含量成顯著正性相關(P<0.01)。綜合以上的結果顯示,飲食中添加香椿葉、根皮及樹皮萃取物皆能有效促進學習記憶能力,降低脂質過氧化物濃度及腦部組織病變,提升抗氧化防禦系統及精子之品質,至於三種不同萃取物所造成影響之可能機轉則有待進一步的探討。


關鍵詞: 香椿、學習記憶能力、類澱粉蛋白、抗氧化狀態、精子品質
Abstract

Toona sinensis Roemor (TS) may decrease the damage of reaction oxygen species (ROS) because contain a lot of polyphenol compounds which have the antioxidant capacity. The purpose of this study was to examine the DPPH scavenging ability of different parts of TS extracts, and also evaluated the effects on the learning and memory ability, antioxidant status and sperm quality in senescence-accelerated-prone mice (SAMP8). In vitro experiment, the antioxidant capacity of Toona sinensis Roemor leave (TSL), Toona sinensis Roemor root (TSR) and Toona sinensis Roemor bark (TSB) extracts were evaluated by the DPPH radical scavenging test. In vivo experiment, 3-month-old male SAMP8 mice were fed with four different diets for 12 weeks: casein diet group (the control group), casein diet supplemented with 1% TSL, 1% TSR and 1% TSB extracts. Senescence-accelerated-resistant mice (SAMR1) was used for the normal control group and fed with casein diet. Body weight, food intake, open field activity test, single-trial passive avoidance and active shuttle avoidance test were performed. The malondiadehyde (MDA) concentration, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities of liver, hippocampus, striatum and cortex, and the ß-amyloid protein (Aß) deposition of brain were examined after sacarificed. The motility, concentration and abnormality of spermatozoa, and serum biological values were also evaluated. The results of vitro study showed that the DPPH scavenging ability of different TS extracts were over 80% in the concentration of 0.625 mg/ml. In vivo study, the results showed that were no significant differences in the food intake, body weight and locomotion among five groups. The mice fed with TSL extracts had significantly better single-trial passive avoidance results, while three TS diet groups showed better active shuttle avoidance results than the control and normal control groups but no differences were found among three TS diet groups. The Aß deposition of brain and the MDA concentration of liver and brain in three TS extracts groups were significantly lower than the control and normal control groups, whereas no differences were found among three TS diet groups. The SOD, CAT and GSH-Px activity of the brain and liver in the TS extracts groups were significantly higher than the control and normal control groups. It was noted that the TSR diet group had the best SOD activity in the hippocampus and liver, and the best CAT activity in liver; the TSL extract group showed higher SOD activity in striatum and cortex, and GSH-Px activity in liver; and the TSB extracts group had the best GSH-Px activity in the hippocampus, striatum and cortex. The motility and concentration of spermatozoa in mice fed with three TS extracts were significantly higher than the control and normal control groups. The serum triglyceride and total cholesterol concentration of TS extracts groups were significantly lower than the control group but no differences were found among three TS diet groups. The mice fed with TSL extracts were found to have the lowest serum glucose concentration. Pearson correlation analysis showed that the ß-amyloid protein deposition were negatively associated with the SOD, CAT and GSH-Px activity of brain, and also significantly positively associated with the MDA concentration of brain (p<0.01). In summary, we conclude that the supplements of TSL, TSR and TSB extracts could improve the learning and memory ability, reduce the lipid peroxidation and brain pathological changes, and also promote the antioxidative defense system and sperm quality. However, the mechanisms of the effects of different TS extracts are still unknown and need further study.

Keywords: Toona sinensis Roemor, learning and memory, ß-amyloid protein, antioxidative status, sperm quality
目錄
頁次
中文摘要………………………………………………………….........................I
英文摘要………………………………………………………………...................III
目錄…………………………………………………………………….....................V
表目錄………………………………………………………………......................IX
圖目錄………………………………………………………………….....................X

第一章 緒論……………………………………………………………....................1
第二章 文獻回顧………………………………………………………....................4
第一節 香椿………………………………………………………...................4
壹、香椿之介紹……………………………………………...................4
貳、香椿之營養成分及藥理作用…………………………...................4
參、香椿生理功效之研究…………………………………...................6
一、香椿之抗癌作用……………………………………....................6
二、香椿之鎮痛作用……………………………………....................6
三、香椿對血小板凝集作用……………………………....................7
四、香椿之降血糖作用…………………………………....................7
五、香椿對人類精子生理功能之作用…………………....................8
六、香椿對血壓之作用…………………………………....................8
七、香椿之抗氧化作用…………………………………....................8
第二節 自由基及活性氧物質對生物體之探討………………....................10
壹、自由基與活性氧物質之介紹………………………....................10
一、自由基與活性氧物質………………………………...................10
二、自由基與活性氧物質之來源………………………...................10
貳、自由基與活性氧物質對生物體之氧化性傷害……....................12
一、氧化壓力之形成……………………………………...................12
二、氧化傷害之原因……………………………………...................13
三、氧化傷害對生物體之影響…………………………...................13
參、氧化性傷害對疾病及衰老之影響…………………....................16
一、氧化傷害與疾病之相關性…………………………...................16
二、氧化傷害與衰老之相關性…………………………...................16
肆、抗氧化防禦系統與氧化傷害之關係…………………..................22
一、生物體之抗氧化防禦系統…………………………...................22
二、抗氧化劑之作用機制………………………………...................24
第三節 脂質過氧化物產物-丙二醛(MDA)之介紹……………....................26
第四節 超氧歧化酶Superoxide dismutase(SOD)之介紹……….................29
壹、SOD生物學特性及分類…………………………….....................29
貳、SOD之特異性……………………………………….....................30
參、SOD之生理作用…………………………………….....................31
肆、SOD測量之方法…………………………………….....................32
伍、SOD對生物體之影響……………………………….....................32
第五節 觸酶Catalase(CAT)之介紹……………………………...................34
壹、CAT之生理作用…………………………………….....................34
貳、CAT對生物體之影響……………………………….....................35
第六節 麩胱甘肽過氧化酶Glutathione peroxidase (GSH-Px)之介紹...........36
壹、GSH-Px之分類………………………………………....................36
貳、GSH-Px之生理作用…………………………………....................37
參、GSH-Px對生物體之影響……………………………....................38
第七節 類澱粉蛋白質β-amyloid protein (Aβ)之探討………..................40
壹、類澱粉前驅蛋白(Amyloid-ß precursor protein; APP)..............41
貳、類澱粉蛋白(ß-amyloid protein)…………………….................42
第八節 學習記憶之探討………………………………………....................47
壹、學習記憶之簡介……………………………………....................47
一、學習之定義…………………………………………...................47
二、記憶之定義…………………………………………...................48
貳、學習和記憶之類型…………………………………....................48
一、學習的類型…………………………………………...................48
二、記憶的類型…………………………………………...................49
參、學習記憶的實驗模式………………………………....................52
一、古典制約條件反射…………………………………...................52
二、操作性條件反射……………………………………...................52
三、逃避或迴避性條件反射……………………………...................52
四、辨別性學習…………………………………………...................53
肆、學習記憶的生理機制…………………………………..................53
一、學習記憶與神經傳導之關係………………………...................53
二、海馬迴與學習記憶之相關性………………………...................54
三、紋狀體與學習記憶之相關性………………………...................55
第九節 精子品質之探討……………………………………......................57
壹、精子的構造及生成…………………………………....................57
貳、活性氧化物質對精子品質之影響…………………....................58
參、抗氧化劑與精子品質之相關研究…………………....................60
第十節 老化促進小鼠…………………………………………....................64
壹、老化促進小鼠簡介…………………………………...................64
貳、老化促進小鼠開發過程……………………………...................65
參、SAMP8系小鼠………………………………………....................65
肆、SAMR1系小鼠………………………………………....................68
第三章 材料與方法……………………………………………………...................72
第一節 香椿之萃取流程………………………………………....................72
第二節 香椿萃取物清除α,α-diphenyl-ß–picrylhydrazyl(DPPH)自由基效力測定.73
第三節 實驗動物與飼料………………………………………....................74
第四節 實驗流程與方法………………………………………....................76
壹、活動量測試…………………………………………...................78
貳、學習記憶能力測試…………………………………...................78
一、單次被動迴避試驗………………………………....................78
二、主動迴避試驗………………………………………..................79
參、器官均質液之製備及分析流程……………………...................84
肆、氧化狀態之分析……………………………………...................86
一、脂質過氧化物丙二醛之分析……………………....................86
伍、抗氧化狀態之分析…………………………………...................89
一、藥品…………………………………………………..................89
二、儀器設備……………………………………………..................90
三、超氧歧化酶之分析………………………………....................90
四、觸酶之分析………………………………………....................91
五、麩胱甘肽過氧化酶之分析………………………....................92
六、總蛋白質含量之測定……………………………....................94
陸、腦部組織病理切片觀察……………………………...................96
一、儀器設備……………………………………………..................96
二、試藥配製……………………………………………..................96
三、組織切片過程………………………………………..................98
四、ß-類澱粉蛋白染色法……………………………...................100
五、腦部組織病理評估標準…………………………...................102
柒、精子品質之觀測…………………………………....................106
一、儀器設備…………………………………………...................106
二、試藥配製…………………………………………...................106
三、精子活動力之評估………………………………...................106
四、精子濃度之評估…………………………………...................107
五、精子畸形率之評估………………………………...................107
第五節 統計分析…………………………………………….....................108
第四章 結果與討論…………………………………………………....................109
第一節 DPPH自由基效力測定……………………………….....................109
第二節 體重及飼料攝取量……………………………………...................112
第三節 活動量…………………………………………………...................115
第四節 學習與記憶能力………………………………………...................117
壹、單次被動迴避試驗…………………………………..................117
貳、主動迴避試驗………………………………………..................120
第五節 器官重量………………………………………………...................123
第六節 脂質過氧化物丙二醛含量……………………………...................125
第七節 超氧歧化酶之活性……………………………………...................130
第八節 觸酶之活性……………………………………………...................135
第九節 麩胱甘肽過氧化酶之活性……………………………...................140
第十節 腦部ß-類澱粉蛋白沉積………………………………...................145
第十一節 精子品質評估………………………………………...................149
壹、睪丸及副睪重量……………………………………....................149
貳、精子活動力…………………………………………....................151
參、精子濃度……………………………………………....................154
肆、精子畸形率…………………………………………....................157
第十二節 血液生化質分析……………………………………...................159
第十三節 相關性分析...................................................163
第五章 總論…………………………………………………………....................170
第六章 參考文獻……………………………………………………....................173
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