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研究生:吳采柔
研究生(外文):Tsai-Jou Wu
論文名稱:刺蔥萃取物對缺血再灌流損傷大鼠腦組織氧化傷害效用之研究
論文名稱(外文):Effect of Zanthoxylum ailanthoides Sieb & zucc. on oxidative damage in ischemia-reperfusion injury rat brain
指導教授:周淑姿周淑姿引用關係
指導教授(外文):Su-Tze Chou
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
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:79
中文關鍵詞:體內試驗缺血/再灌流刺蔥抗氧化性體外試驗
外文關鍵詞:antioxidantischemia/reperfusionin vitroin vivoZanthoxylum ailanthoides Sieb. &ampZucc.
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近年來從草本植物中尋找良好的天然抗氧化劑,用以對抗生物體內之氧化性傷害日漸受到各界矚目。刺蔥(Zanthoxylum ailanthoides Sieb. &; Zucc.)為芸香科花椒屬植物,別名為食茱萸、鳥不踏、刺江某等,自古以來即為入藥植物,於民間傳用具有治療風寒感冒、跌打腫痛、風濕關節痛等功效;經先前體外研究指出刺蔥水相及50%乙醇相萃取物具有清除自由基、抑制脂質氧化及還原力等抗氧化特性。本研究將進一步以動物試驗探討刺蔥萃取物之給予是否會改變活體組織中氧化―抗氧化狀態之恆定,特別是腦組織,另一方面也分析具抗氧化性之刺蔥萃取物是否具有保護動物體經缺血/再灌流後傷害之作用,實驗乃是將刺蔥萃取物(0.2 g/100 g BW/ day)經由管灌方式給予Sprauge-Dawley品系大鼠連續14天,再施予缺血/再灌流之手術,分析動物體器官內抗氧化物質-維生素C、E、glutathione與抗氧化酵素-superoxide dismutase、glutathione peroxidase、catalase及脂質過氧化物-丙二醛含量之含量,並利用組織切片染色技術觀察腦部梗塞面積之變化。
結果顯示,體外清除ABTS自由基的總抗氧化力測定實驗中,刺蔥葉50%乙醇萃取物(LE)之清除能力與水相萃取物(LW)於測試濃度(0.3125~20 mg/ml)下具有相當的抗氧化力,且抗氧化能力隨著測試濃度增加而有增加的趨勢。而在總類黃酮含量測試上,刺蔥葉50%乙醇萃取物(LE)(93.41 μM, as quercetin/g)含量顯著高於水相萃取物(LW)(38.75 μM, as quercetin/g)。體內試驗結果顯示,給予刺蔥葉萃取物可顯著提升大鼠肝臟及腦組織中的Vit E與腦中GSH含量,並可顯著降低心、肝及腦組織中脂質過氧化物MDA的含量;另外於刺蔥葉萃取物之給予並誘發中腦動脈缺血模式之結果顯示,二種萃取物之給予皆可顯著降低大鼠經缺血再灌流所誘發之腦部缺血面積及腦組織中MDA之含量,然而對腦中抗氧化物質含量或酵素活性並無顯著之影響,因此刺蔥葉萃取物可能藉由其良好之抗氧化活性進而可減少動物體經缺血/再灌流後腦部傷害之發生。
In recent years, there is an increasing interest in finding natural antioxidants from plants, which can protect human body against free radical insult and ameliorate the progress of many chronic diseases. Zanthoxylum ailanthoides Sieb. & Zucc., also called Chhi-chhang or Tana, is a traditional Chinese herbal medicine, belonging to the Rutaceae family. In folk medicine, Z. ailanthoides have several health benefits, such as myocardium disorder attenuation, bone-injury alleviation, and cold resistance. In our previous studies, the water (LW) or 50% ethanolic extracts (LE) of leaves (LE) from Z. ailanthoides had showed antioxidative activities including radical-scavenging effect, reducing power, and lipid peroxidation inhibition. In this study, we aimed to assess whether the Z. ailanthoides extracts will modulate cellular antioxidative capacity in animal and whether Z. ailanthoides extracts will protect brain tissue from ischemic injury. The animals were pretreated or none with LW or LE by gavage for 14 consecutive days prior to the brain ischemia reperfusion (I/R) surgery. We determined the effects of the two extracts on the antioxidants (Vit C, Vit E and GSH), the antioxidant enzymes activities (SOD, catalase and GSH peroxidase) and the lipid peroxidation in the brain of the rats. The evaluation approaches will also include determination of infarct size. The results showed that LW and LE had 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical-scavenger effect. It was found that the radical-scavenging activity of the tested extracts increased with increasing concentration. The total flavonoid contents of LW and LE were 39.41 and 38.75 μM quercetin equivalent/g, respectively. It was noted that 50% ethanolic extract had significant higher total flavonoid contents than did water extracts (P<0.05). Supplement of both the extracts increased the Vit E levels in the liver and brain tissues, while it only increased the GSH level in the brain tissue. Supplement of the extracts could decrease the malondialdehyde (MDA) levels in the three tested tissues. LW or LE pretreatment markedly attenuated cerebral I/R-induced cortical infarction. Furthermore, cerebral I/R increased the levels of MDA, an oxidative stress marker. The reduction of MDA levels was also significantly suppressed by LW or LE pretreatment. Nevertheless, there was no significant difference in brain antioxidants, and antioxidant enzymes activities between the extracts-pretreatment group and I/R group. These results suggest that Z. ailanthoides extracts, including LW and LE exerts a beneficial effect in focal cerebral ischemia, which may be attributed to its antioxidant property.
目錄.................................................. Ⅰ
表目錄................................................ Ⅴ
圖目錄................................................ Ⅵ
中文摘要.............................................. Ⅷ
英文摘要.............................................. Ⅸ
第一章 前言........................................... 1
第二章 文獻回顧....................................... 3
一、刺蔥簡介....................................... 3
二、自由基與活性氧之定義........................... 5
(一) 超氧自由基................................... 6
(二) 過氧化氫..................................... 6
(三) 氫氧自由基................................... 7
(四) 單重態氧..................................... 7
三、自由基與活性氧對生物體之氧化傷害............... 8
四、自由基與疾病之相關性........................... 9
(一) 老化與氧化壓力............................... 9
(二) 動脈粥狀硬化與氧化壓力....................... 9
(三) 癌症與氧化壓力............................... 11
(四) 腦缺血與氧化壓力............................. 11
1. 自由基與腦缺血............................... 11
(1)出血性腦中風................................. 11
(2)缺血性腦中風................................. 12
2. 腦缺血再灌流與氧化損傷之關係................. 12
(1)自由基增多原因............................... 12


五、生物體內抗氧化防禦系統......................... 14
(一) 酵素性防禦系統............................... 14
1. superoxide dismutase (SOD)......................... 14
2. catalase........................................... 15
3. glutathione peroxidase(GSH-Px)..................... 15
(二) 非酵素性抗氧化防禦系統....................... 17
1. 維生素E (α-tocopherol)............................. 17
2. 維生素C (ascorbic acid)...................... 17
3. 麩胱甘(glutathione, GSH)................... 17
六、天然之抗氧化劑................................. 17
第三章 研究目的....................................... 19
第四章 材料與方法..................................... 20
一、試藥........................................... 20
二、儀器設備....................................... 22
三、刺蔥製備....................................... 22
四、實驗架構.......................................... 23
五、刺蔥抗氧化活性分析................................ 24
(一) 體外抗氧化活性測試之樣品液製備................... 24
(二) 總抗氧化力測定................................... 24
(三) 類黃酮含量測定................................... 24
六、動物試驗.......................................... 25
(一) 實驗動物來源及飼養............................. 25
(二) 實驗設計....................................... 25
(三) 中大腦動脈缺血再灌流手術模式................... 25
(四) 生物檢體製備................................... 26
(五) 生物檢體中維生素C之分析方法.................... 26
(六) 生物檢體中維生素E(α-Toc)之分析方法........... 27
(七) 生物檢體中麩胱甘(GSH)之分析方法............ 28
(八) 抗氧化酵素活性測定............................. 30
1. 器官均質液製備................................. 30
2. Glutathione peroxidase(GPx)活性之測定........ 30
3. Superoxide dismutase(SOD)活性之測定.......... 30
4. Catalase活性之測定............................. 31
(九) 丙二醛(MDA)含量之測定........................ 31
(十) 總蛋白質含量之測定............................. 31
(十一) 大腦梗塞面積測量............................. 32
七、統計分析.......................................... 32
第五章 結果與討論..................................... 33
一、體外試驗.......................................... 33
(一) 總抗氧化能力................................... 33
(二) 類黃酮含量..................................... 33
二、動物實驗.......................................... 35
(一) 刺蔥葉萃取物對動物器官中抗氧化物質、酵素及脂質過氧化物含量之影響........................................ 35
(二) 刺蔥葉萃取物對於缺血再灌流大鼠腦組織抗氧化酵素、物質及脂質過氧化物之影響................................ 37
1. 刺蔥葉萃取物對於缺血再灌流大鼠腦部缺血面積之探討37
2. 刺蔥葉萃取物對腦部缺血再灌流大鼠腦組織中抗氧化酵素 活性之影響............................................ 37
3. 刺蔥葉萃取物對經由腦部缺血再灌流大鼠腦組織中抗氧化 物質含量之影響........................................ 38
4. 刺蔥葉萃取物對經由腦部缺血再灌流大鼠腦組織中脂質過 氧化之影響......................................... 38
(三) 刺蔥葉萃取物之給予對於缺血再灌流大鼠心臟及肝臟氧化-抗氧化恆定之影響.................................... 39

1. 刺蔥葉萃取物對於腦部缺血再灌流大鼠心臟及肝臟中 catalase活性之影響.................................... 39
2. 刺蔥葉萃取物對於腦部缺血再灌流大鼠心臟及肝臟中SOD活性之影響............................................ 40
3. 刺蔥葉萃取物對於經由腦部缺血再灌流大鼠心臟及肝臟中GPx活性之影響......................................... 40
4. 刺蔥葉萃取物對於經由腦部缺血再灌流大鼠心臟及肝臟中 Vit C含量之影響.................................... 41
5. 刺蔥葉萃取物對於大鼠經由腦部缺血再灌流心臟及肝臟中 Vit E含量之影響.................................... 41
6. 刺蔥葉萃取物對於經由腦部缺血再灌流大鼠心臟及肝臟中 GSH含量之影響...................................... 41
7. 刺蔥葉萃取物對於經由腦部缺血再灌流大鼠心臟及肝臟中脂 質過氧化含量之影響................................. 42
第六章 結論........................................... 44
附錄A、刺蔥參考圖鑑................................... 66
附錄B、自由基與活性氧的來源........................... 67
附錄C、數種自由基及活性氧之種類及其半衰期............. 68
附錄D、自由基對細胞的傷害............................. 69
附錄E、與自由基相關的臨床疾病......................... 70
附錄F、人體血漿中之抗氧化劑........................... 71
第七章 參考文獻....................................... 72
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