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研究生:劉建志
研究生(外文):Chien-Chih Liu
論文名稱:香蘭葉萃取物抗氧化與輻射效應影響之研究
論文名稱(外文):Studies on the antioxidative activity and the influence of radiation effects of Pandanus amaryllifolius Roxb. extracts
指導教授:王愛義王愛義引用關係
指導教授(外文):Ai-Yih Wang
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學影像暨放射技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:101
語文別:中文
論文頁數:53
中文關鍵詞:抗氧化香蘭葉輻射效應
外文關鍵詞:antioxidantsPandanus amaryllifoliusradiation effect
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本研究旨在進行香蘭葉 (Pandanus amaryllifolius Roxb.) 萃取物抗氧化與輻射效應之影響。實驗中利用超音波萃取技術進行香蘭葉的萃取,再以總多酚、總類黃酮、DPPH 自由基清除率、亞鐵離子螯合等非酵素系統進行香蘭葉萃取物抗氧化能力的評估。另外再以過氧化氫對細胞傷害測定香蘭葉萃取物於細胞內抗氧化能力,香蘭葉萃取物之輻射效應影響則是以 5.5 戈雷 (Gy) 的輻射劑量照射,以 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 分析方法測定細胞的存活,評估香蘭葉萃取物對輻射效應之影響。研究結果顯示香蘭葉乾燥粉末總多酚當量為 139.89±0.43 GAE mg/g,總類黃酮當量為 44.24±0.61 RE mg/g,香蘭葉萃取物之 DPPH 半清除濃度為 156.43±0.52g/mL,而香蘭葉萃取物亞鐵離子螯合的 IC50 為 6.12±0.03mg/mL。在 H2O2 對細胞傷害的實驗中,香蘭葉萃取物不論是對於 Clone 9 細胞還是 Hep G2 細胞都不具有保護的能力。香蘭葉萃取物對於 Hep G2 細胞隨著萃取物濃度增加具顯著 (p <0.05) 的毒殺作用,在萃取物濃度為 25g/mL 經過 72 小時培養後其存活率為 64.72%,當萃取物濃度增加為 1500g/mL 同樣經過 72 小時培養其存活率降低為 30.62%。低濃度香蘭葉萃取物對於 Clone 9 細胞有輻射增敏效果,高濃度則具有輻射保護作用,單純以輻射照射其存活率為 40.98%,在照射時使用 25g/mL 的萃取物時其存活率會降為 23.69%。濃度增為 1000g/mL 時其存活率會增加至 66.20%。綜合以上的結果,香蘭葉萃取物具有抗氧化和輻射效應影響的能力,但其真正的作用機制需要做進一步的實驗探討。
The aims of this study were to evaluate the antioxidative and the influence of radiation effects of extracts of Pandanus amaryllifolius Roxb. (P. amaryllifolius) by ultrasound extraction. For non-enzymatic antioxidants, several methods were used, they were the total polyphenol, total flavonoid, α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging assay, ferrous ion chelation. The cytotoxic effect of oxidative stress induced by hydrogen peroxide (H2O2) was used to evaluate the intracellular antioxidative ability of the extracts. Cells were exposed to 5.5 Gy of X-ray, the surviving fraction of cells were determined by MTT method to evaluate the radiation effect of the extracts of P. amaryllifolius. The results revealed that extracts of amaryllifolius has a total phenolic content of 139.89±0.43 gallic acid equivalents (GAE) mg/g, total flavonoid content of 44.24±0.61 rutin equivalents (RE) mg/g. The half-inhibition concentration (IC50) of DPPH radical scavenging and ferrous metal ion chelating of the extracts of extracts of P. amaryllifolius were 156.43±0.52g/mL and 6.12±0.03 mg/mL, respectively. There was no significant protective effect on oxidative stress induced by hydrogen peroxide (H2O2). There was significant (p<0.05) cytotoxic effect of extracts of P. amaryllifolius on liver hepatocellular carcinoma (HepG2), and the cell viability was decreased with the concentration of extracts of P. amaryllifolius. The cell viability was 64.72 % at 25 g/mL, and it decreased to 30.62% at 1500 g/mL of extracts of P. amaryllifolius incubated for 72 hr. There was radiosensitive effect on low concentration of extracts of P. amaryllifolius, and radioprotective effect on high concentration of extracts of P. amaryllifolius in clone 9, the survival fraction was 40.98% in irradiation control group, it decrased to 23.69% at 25 g/mL, and it increased to 66.20% at 1000 g/mL. The results show the extracts of P. amaryllifolius had strong antioxidative activity and the ability of influence of radiation damage, but the influence mechanism required further investigation.
致謝 I
中文摘要 II
英文摘要 III
目錄 V
表目錄 VIII
圖目錄 IX
第一章 前言 1
第二章 研究背景 3
2.1 植物介紹 3
2.2 自由基與活性氧物質來源 4
2.2.1 超氧陰離子(Superoxide anion radical, ⋅O2-) 6
2.2.2 過氧化氫(Hydrogen peroxide, H2O2) 6
2.2.3 氫氧自由基(Hydroxyl radical, ⋅OH) 7
2.2.4 脂質過氧化自由基(Lipid peroxide, ROO⋅) 7
2.2.5 單重態氧(Singlet oxygen, 1O2) 7
2.3 自由基的產生來源 8
2.3.1 自由基的傷害 8
2.4 天然抗氧化物質 10
2.4.1 多酚類化合物(Polyphenol) 10
2.4.2 類黃酮化合物(Flavonoid) 10
2.5 輻射的生物效應 13
2.5.1 輻射效應化學影響因子 13
2.5.2 輻射保護劑 14
2.5.3 輻射增敏 18
第三章 材料與方法 20
3.1 實驗材料 20
3.2 實驗流程與架構 23
3.3 超音波萃取 24
3.3.1 樣品前處理 24
3.3.2 超音波萃取原理 24
3.3.3 萃取條件測試之萃取物處理 24
3.3.4 條件確定後之萃取物處理 25
3.4 抗氧化能力測定 25
3.4.1 總多酚測量 25
3.4.2 總類黃酮測量 25
3.4.3 清除 DPPH 自由基測試 26
3.4.4 亞鐵離子螯合能力測試 26
3.5 細胞培養與藥品配製 27
3.5.1 細胞來源 27
3.5.2 細胞培養液配製 27
3.5.3 杜氏磷酸鹽緩衝液(Dulbecco's phosphate-buffered saline, DPBS)配製 28
3.5.4 胰蛋白酶(Trypsin-EDTA)配製 28
3.5.5 細胞解凍程序 28
3.5.6 細胞繼代培養 28
3.5.7 細胞數目計數 29
3.5.8 細胞冷凍保存程序 30
3.6 細胞試驗 30
3.6.1 細胞存活率試驗 30
3.6.2 過氧化氫造成氧化壓力的保護作用試驗 31
3.6.3 細胞毒殺測試 31
3.6.4 輻射效應影響之試驗-細胞存活率測試 32
3.7 統計分析方法 32
第四章 結果與討論 33
4.1 萃取條件比較 33
4.2 細胞外抗氧化能力 37
4.2.1 總多酚當量和總類黃酮當量測定 37
4.2.2 DPPH自由基清除測試 37
4.2.3 亞鐵離子螯合能力測試 38
4.3 香蘭葉萃取物之細胞毒殺作用 40
4.4 香蘭葉萃取物對過氧化氫造成氧化壓力的保護作用 43
4.5 香蘭葉萃取物對輻射效應的影響 45
第五章 結論 48
參考文獻 49

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