臺灣博碩士論文加值系統

於生物體代謝過程中所形成的活性氧物質 ( reactive oxygen species ) 會與生物體內之DNA、蛋白質或生物膜上之不飽和脂肪酸反應，使細胞受損進而導致生物體之氧化性傷害，此傷害為造成老化或與老化有關疾病的主因之一；而體內主要之抗氧化酵素如catalase、superoxide dismutase ( SOD ) 與glutathione peroxidase ( GSH-Px ) 能有效清除活性氧物質以降低細胞所受到之氧化性傷害，其含量可作為體內抗氧化防禦系統能力的指標之一。傳統的中草藥含有許多豐富的化學物質，其中富含anthraquinone及stilbene類的何首烏 ( Polygonum multiflorum Thunb. ) 為蓼科草本植物，據研究指出其塊根具有調節生物體免疫功能、降低血脂及脂質過氧化產物含量等功能，長久以來一直作為抗衰老之主要用藥，因此本實驗主要探討不同溶劑之何首烏萃取物對小白鼠體內抗氧化酵素活性之影響。
何首烏經鑑定後分別以水、50 %乙醇及95 %乙醇為溶劑萃取後，先以體外實驗之方式探討其清除α, α-diphenyl-β-picrylhydrazyl ( DPPH ) 及超氧陰離子自由基之效力與還原力；而後以不同月齡 ( 1月齡、7月齡 ) 之雄性老化促進小白鼠 ( senescence accelerated mice, SAM ) 進行體內試驗，實驗為期18週，以評估不同溶劑何首烏萃取物 ( 0.4g / kg‧BW ) 之給予對其肝臟、心臟及腦中抗氧化酵素活性之影響。並以最具有抗氧化效力之萃取物，經不同極性溶劑進行分配萃取，找尋最具抗氧化活性之成分層。
實驗結果發現，於體外實驗中三種何首烏萃取物其清除DPPH 之效力均可達67 %，且50 %乙醇萃取物所需之半清除濃度 ( IC50 ) 最低；於超氧陰離子自由基之清除效力，水萃取物其清除效力可達73.84 %，且50 %乙醇萃取物具有較低之半清除濃度 ( IC50 ) ；何首烏不同溶劑之萃取物其還原力皆隨樣品添加劑量增加而增加，並於10 mg / ml時達到平衡。在動物實驗方面，何首烏之乙醇類萃取物均可顯著提高1月齡小白鼠之肝臟、心臟及腦中GSH-Px及SOD活性，而50 %乙醇萃取物亦能顯著提高三器官中catalase活性。在7月齡小白鼠方面，何首烏乙醇類萃取物顯著增加肝臟中GSH-Px、catalase活性以及心臟與腦中GSH-Px與SOD活性；何首烏萃取物對於1月齡小白鼠體內抗氧化酵素活性之影響較7月齡小白鼠明顯。同時以何首烏50 %乙醇萃取物進行分配萃取後，其乙酸乙酯成份層具有最顯著之抗氧化活性。
綜合以上結果，不同溶劑之何首烏萃取物具有不同之抗氧化效果，於體外實驗中何首烏萃取物不僅擁有良好之還原力與供氫能力同時亦具有類SOD活性，且在動物實驗中可顯著性增加小白鼠體內抗氧化酵素活性，因此何首烏萃取物可能經由其抗氧化之特性而達到抗衰老之作用。

Reactive oxygen species are species that may cause a wide spectrum of cell damage and aging progress. The primary antioxidant enzymes include superoxide dismutase (SOD), which catalyzes the dismutation of superoxide anions (•O2-) to hydrogen peroxide (H2O2), and catalase as well as glutathione peroxidase (GSH-Px), which catalase the degradation of H2O2 to H2O and O2. The root of Polygonum multiflorum Thunb. (Hoshouwu, PM) has been used as an anti-aging herb in traditional Chinese medicine.
This study aim to evaluate the antioxidative activity of Polygonum multiflorum Thunb. extracted by different solvents (H2O, 50% ethanol, 95% ethanol). The radicals scavenging effects and reducing power were studies in vitro and the antioxidative enzymes activities in tissues for 1 and 7 months old senescence-accelerated mice (SAM) supplemented with PM extracts (0.4 g / kg‧BW) for 18 weeks were studied in vivo.
At a dose of 10 mg / ml, the scavenging effect of PM extracted by H2O, 95% ethanol on the α, α-diphenyl-β-picryl-hydrazyl (DPPH) radical was 72.09 %, while H2O extract showed 73.84 % on the superoxide anion radical. The reducing power of different PM extracts increased with the concentration of all solvents up to 10 mg/ml. SAM were fed with 20 % casein diet supplemented with different PM extracts at 0.4 g / kg‧BW for 18 weeks. The results showed that all PM extracts increased the activities of GSH-Px in liver, heart and brain; on the other hand, the 50 % ethanolic extracts increased both the SOD and catalase activities in three tissues at 1-month old SAM. The ethanolic PM extracts increased the GSH-Px activities in three tissues and the activities of SOD in heart and brain; on the other hand, the 95 % ethanolic extracts increased the catalase activities in three tissues at 7-month old SAM. By the administration of PM extracts, the 1-month old mice showed greater improvement on their antioxidative enzymes activity when in compared with the 7-month old. The 50 % ethanolic PM is extracted partitionary with different solvents (ethyl acetate, n-butanol) and the ethyl acetate layer showed greater antioxidative activity according to in vitro and in vivo experiments.
The results of this study implied that Polygonum multiflorum Thunb. had strong antioxidative ability, which may contribute to the anti-aging effects. Further research on the isolation of the antioxidative components from Polygonum multiflorum Thunb. is demanded.

目錄……………………………………………………………………..Ⅰ
表目錄…………………………………………………………………..Ⅶ
圖目錄…………………………………………………………………..Ⅷ
中文摘要………………………………………………………………..Ⅹ
英文摘要………………………………………………………………..Ⅹ
第一章 前言……………………………………………………..1
第二章 文獻回顧
第一節 抗氧化酵素之重要性
一. 活性氧物質…………………………………………………3
(一) 活性氧物質來源……………………………………..3
(二) 活性氧物質特性……………………………………..5
1. 超氧陰離子自由基…………………………………5
2. 過氧化氫……………………………………………7
3. 氫氧自由基…………………………………………8
(三) 氧化性傷害之相關性探討…………………………..9
1. 活性氧物質對蛋白質之傷害……………………..10
2. 活性氧物質對脂質之傷害………………………..10
3. 活性氧物質對DNA之傷害………………………11
4. 氧化性傷害與疾病之相關性……………………..12
5. 氧化性傷害與老化之相關性……………………..13
二. 抗氧化防禦系統…………………………………………..13
(一) 非酵素性防禦系統………………………………..14
1. 維生素C…………………………………………...15
2. 維生素E…………………………………………...15
3. Glutathione………………………………………...15
(二) 酵素性防禦系統…………………………………..17
1. Superoxide dismutase ( SOD )…………………….17
2. Glutathione peroxidase ( GSH-Px )..……….……..19 3. Catalase……………………………………………21
4. 抗氧化酵素與老化之相關性……………………..22
三. 脂質過氧化產物-丙二醛之介紹………………….……..23
第二節 何首烏
一. 何首烏之主要成分………………………………………..25
二. 何首烏之藥理作用………………………………………..28
(一) 調節血脂作用…………………………………….28
(二) 增強免疫功能…………………………………….29
(三) 延緩衰老作用…………………………………….30
1. 減少脂質過氧化物之含量………………………..30
2. 增加SOD活性……………………………………31
3. 降低單胺氧化-B活性………………………….31
4. 抑制脂褐質之形成……………………….……….32
第三節 老化促進小白鼠
一. SAM系小白鼠之開發歷程……………………………….34
二. SAMP8系小白鼠之老化特徵…………………………….35
第四節 研究目的……………………………………….………….39
第三章 材料與方法
第一節 實驗材料
一. 樣品……………………………………………….………40
二. 試藥……………………………………………………….40
第二節 實驗方法
一. 一般之製備……………………………………….……..42
二. 何首烏不同溶劑萃取物之體外試驗…………….……..43
(一) 何首烏不同溶劑萃取物之產率計算……………44
(二) 何首烏不同溶劑萃取物之總酚類化合物含量
測定……………………………………….……….44
(三) 何首烏不同溶劑萃取物之清除DPPH自
由基效力測定…………………………………..45
(四) 何首烏不同溶劑萃取物之清除超氧陰離
子自由基效力測定……………………………….46
(五) 何首烏不同溶劑萃取物之還原力測定………...48
(六) 何首烏不同溶劑之清除自由基及還原力反應
模擬.……………………………………………...49
1. 抑制常數………………………………….……...49
2. 半清除濃度……………………………………...49
三. 動物試驗…………………………………………………...50
(一) 動物飼養條件………………………………….50
(二) 何首烏萃取物對小白鼠體內抗氧化酵素活性
影響……………………………………………..51
1. 實驗分組………………………………………….51
2. 實驗飼料之製備………………………………….51
(三) 器官均質液之製備.……………………………..53
(四) Glutathione peroxidase活性之測定……………..55
(五) Superoxide dismutase 活性之測定……………..57
(六) Catalase活性之測定…………………….………..58
(七) 總蛋白質含量之測定…………………….………59
(八) 丙二醛含量之測定……………………………….61
四. 分配萃取…………………………………………………...63
(一) 極性分層………………………………………….64
(二) 體外試驗………………………………………….64
1. 何首烏50 %乙醇萃取物其不同極性成分層
產率之計算……………………………………….64
2. 何首烏50 %乙醇萃取物其不同極性成分層
總酚類化合物含量之測定……………………….66
3. 何首烏50 %乙醇萃取物其不同極性成分層
清除DPPH自由基之效力測定..………………...66
4. 何首烏50 %乙醇萃取物其不同極性成分層
清除超氧陰離子自由基之效力測定…………….66
5. 何首烏50 %乙醇萃取物其不同極性成分層
還原力之測定……………………………………67
6. 何首烏50 %乙醇萃取物其不同極性成分層
清除自由基及還原力反應模擬………………67
第三節 統計分析………………………………………………...68
第四章 結果與討論
第一節 何首烏不同溶劑萃取物之體外試驗
一. 何首烏不同溶劑萃取物之產率…………………………..69
二. 何首烏不同溶劑萃取物之總酚類化合物含量…………..69
三. 何首烏不同溶劑萃取物對DPPH自由基之清除效力…...70
四. 何首烏不同溶劑萃取物對超氧陰離子自由基之清除
效力………………………………………………………..74
五. 何首烏不同溶劑萃取物之還原力………………………..77
六. 何首烏不同溶劑萃取物清除自由基及還原力反應模擬..78
(一) 何首烏不同溶劑萃取物之抑制常數………………..78
(二) 何首烏不同溶劑萃取物之半清除濃度……………..83
第二節 動物試驗
一. 何首烏萃取物對小白鼠其攝食效率與體重變化之影
響…………………………………………………………86
二. 何首烏萃取物對小白鼠體內GSH-Px活性之影響……..89
三. 何首烏萃取物對小白鼠體內SOD活性之影響………...91
四. 何首烏萃取物對小白鼠體內Catalase活性之影響……..93
五. 何首烏萃取物對小白鼠體內MDA含量之影響………..95
第三節 分配萃取
一. 何首烏50 %乙醇萃取物其不同極性成份層之產率……99
二. 何首烏50 %乙醇萃取物其不同極性成份層之總酚
類化合物含量……………………………………….…...99
三. 何首烏50 %乙醇萃取物其不同極性成份層對DPPH
自由基之清除效力..……………………………………100
四. 何首烏50 %乙醇萃取物其不同極性成份層對超氧陰
離子自由基之清除效力………………………………..102
五. 何首烏50 %乙醇萃取物其不同極性成份層之還原…..105
六. 何首烏50 %乙醇萃取物其不同極性成份層之清除自由
基或還原力反應模擬…………………………………..105
(一) 何首烏50 %乙醇萃取物其不同極性成份層之抑制
常數…………………………………………………..105
(二) 何首烏50 %乙醇萃取物其不同極性成份層之半清
除濃度………………………………………………..107
第五章 結論………………………………………………………...111
第六章 參考文獻…………………………………………………..112

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