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研究生:陸毅航
研究生(外文):Lu, Yihang
論文名稱:蔓荊子之抗氧化活性研究
論文名稱(外文):Studies on the Antioxidative Activities of Vitex trifolia
指導教授:凃瑞澤凃瑞澤引用關係吳淑姿吳淑姿引用關係
指導教授(外文):Too, JuirzeWu, Shutzu
口試委員:鄭啟清張基郁凃瑞澤吳淑姿
口試委員(外文):Jeng, KeechingChang, ChiyueToo, JuirzeWu, Shutzu
口試日期:2012-06-22
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:67
中文關鍵詞:蔓荊子熱迴流萃取多酚類化合物類黃酮化合物抗氧化活性HPLCPC12MTT
外文關鍵詞:Vitex trifoliareflux extractionpolyphenolicsflavonoidsantioxidant activityHPLCPC12MTT
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  本研究利用不同溶劑(水、50%乙醇、甲醇、95%乙醇、乙酸乙酯與正己烷)熱迴流萃取中藥材蔓荊子(Vitex trifolia L.),並對萃取物進行總多酚化合物與類黃酮化合物含量測定及抗氧化能力分析。抗氧化能力分析包括:DPPH自由基的清除能力、還原力、亞鐵離子螯合能力、清除超氧陰離子能力及清除ABTS陽離子能力,並與標準品BHA、EDTA、Trolox、維他命C及沒食子酸比較其抗氧化能力。
  以不同溶劑萃取蔓荊子,其中以50%乙醇之萃取率最高,達9.49%;總多酚與總類黃酮含量以正己烷萃取時最高,分別為53.13 ± 0.10 mg/g與1.12 ± 0.10 mg/g。清除DPPH自由基能力試驗,以乙酸乙酯萃取物最佳,在濃度0.8 mg/mL時,達100%,IC50 (半數清除濃度)以50%乙醇萃取物較佳,為0.02 ± 0.00 mg/mL。還原力以水萃取物最佳,濃度0.8 mg/mL時,還原力較標準品BHA為佳。螯合亞鐵離子試驗以水萃取物在濃度4 mg/mL時最佳,達98.8%,與EDTA之螯合力相當 (99.9%),IC50以水萃取物的0.29 ± 0.01 mg/mL較佳。清除ABTS陽離子自由基能力之試驗,以水萃取物最高,達96.3%,IC50為0.18 ± 0.01 mg/mL。清除超氧陰離子能力試驗,以水萃取物活性最高,達63.2%, IC50為0.19 ± 0.00 mg/mL。經HPLC分析各萃取物中之化合物,除50%乙醇萃取物外,各萃取物均有發現槲皮素 (3,3',4',5'-7-penta-hydroxy flavone, quercetin)之存在,化合物以極性較小之溶劑萃取物含量較高,可達202.93 mg/g。
  蔓荊子水萃取物經試驗證明,對PC12細胞沒有太大的負面影響,但隨著萃取物濃度提高,細胞會受到滲透壓的影響而死亡。最後在H2O2誘導損傷保護試驗中,利用長時間(24 h)及短時間(1 h),兩種標準品對照蔓荊子水萃取物保護活性。其保護力優良,在高濃度時略低標準品槲皮素保護力,但高於羥苯醇。實驗結果顯示,蔓荊子萃取物具有高抗氧化能力,值得進一步深入研究並開發應用。

  In this study, traditional Chinese herb medicine, Vitex trifolia, was extracted by using various solvents (H2O, 50% ethanol, methanol, 95% ethanol, ethyl acetate, and n-hexane) with reflux. The contents of polyphenolics and flavonoids in the extracts were measured, and their antioxidant activities were analyzed. The analyses of antioxidant activities included DPPH scavenging activity, reducing power, superoxide anion and ABTS scavenging activities, and Fe2+ chelating ability, and the above antioxidative activities of the extracts were compared with those of BHA, EDTA, Trolox, Vitamin C and Gallic acid.
  In comparison of extracts by various solvents, the highest extraction yield (9.49%) was obtained by using 50% ethanol; the extraction by n-hexane has the highest total polyphenolic and total flavonoid compounds, and they were 53.13 ± 0.10 mg/g and 1.12 ± 0.10 mg/g, respectively. In the DPPH free radical scavenging activity experiment, the extract by ethyl acetate reached 100% scavenging activity at an extract concentration of 0.8 mg/mL. The smallest DPPH IC50 is 0.02 ± 0.00 mg/mL of the extract by 50% ethanol. The extract by H2O at a concentration of 0.8 mg/mL has reducing power even better than that of BHA; In the Fe2+ chelating test, the extract by H2O has the greatest reaction activity, up to 98.8%, which is equivalent to that of EDTA (about 99.9%), and the IC50 of the extract was 0.29 ± 0.01 mg/mL. Finally, the extract by H2O had the highest superoxide anion scavenging activity (96.3%) and ABTS scavenging activity (63.2%) and their IC50’s were 0.18 ± 0.01 mg/mL and 0.19 ± 0.00 mg/mL, respectively. By using HPLC analysis, quercetin (3,3',4',5'-7-penta-hydroxy flavone) was found in all the extracts, In addition to 50% ethanol extracts.. The extracts contained higher amount of this compounds when non-polar solvents were used during extraction. In an in vitro experiment, results indicated that the extract by water had good protection to PC12 cells when they were damaged by H2O2. Furthermore, higher concentrations of the extract made cell protection in a dose-dependent manner.
  Based on the findings above, the extracts of Vitex trifolia L. have high antioxidant activities there is showing that the extract (by water) could correct PC12 cell damage induced by H2O2. However, Vitex trifolia L. is still worth for further study and other applications.
目錄

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vii
目錄 viii
圖目錄 xi
表目錄 xii
1. 緒論 1
2. 文獻回顧 2
2.1 氧化壓力 2
2.1.1 自由基與氧化傷害 2
2.1.2 氧化壓力與疾病 2
2.2 抗氧化 3
2.2.1 抗氧化物質 3
2.2.2 人工合成之抗氧化劑 3
2.2.3 天然抗氧化劑 4
2.2.4 食物中的抗氧化物 4
2.3 蔓荊子 7
2.3.1 蔓荊子簡介 7
2.3.2 蔓荊植物型態 7
2.3.3 蔓荊子之傳統療效 7
2.3.4 蔓荊子臨床療效 9
2.3.5 蔓荊子主要成分 9
2.4 槲皮素 9
2.4.1 槲皮素簡介 9
2.4.2 槲皮素的生物活性 11
2.5 木樨草素 11
2.5.1 木樨草素簡介 11
2.5.2 木樨草素的生物活性 11
2.6 紫花牡荊素 15
2.6.1 紫花牡荊素簡介 15
2.6.2 紫花牡荊素的生物活性 15
2.7 PC12 15
3. 材料與方法 19
3.1 植物材料 19
3.2 藥品 19
3.3 儀器 22
3.4 實驗方法 23
3.4.1 實驗架構 23
3.4.2 蔓荊子萃取物之製備 23
3.4.3 抗氧化成分分析 25
3.4.4 抗氧化能力分析 27
3.4.5 HPLC分析 29
3.4.6 細胞試驗 29
4. 結果與討論 31
4.1 蔓荊子以不同溶劑熱迴流萃取 31
4.1.1 萃取率 31
4.2 抗氧化成分含量分析 31
4.2.1 總多酚化合物含量 31
4.2.2 總類黃酮化合物含量 34
4.3抗氧化能力分析 34
4.3.1 清除DPPH自由基能力 34
4.3.2 鐵離子還原抗氧化力 35
4.3.3 螯合亞鐵離子能力 39
4.3.4 清除超氧陰離子能力 39
4.3.5 清除ABTS自由基能力 42
4.4 HPLC分析 42
4.5 PC12細胞試驗 45
4.5.1 細胞存活度試驗 45
4.5.2 H2O2誘導氧化壓力保護試驗 48
5. 結論 54
5.1 結論 54
5.2 未來展望 54
參考文獻 56
附錄 62

圖目錄 xi

圖2.1 TBHQ、BHA、TBMP及PG之化學結構 5
圖2.2 飲食中可攝取到的類黃酮基本結構及其衍生物 6
圖2.3 蔓荊 8
圖2.4 槲皮素的結構式 10
圖2.5 木樨草素的結構式 12
圖2.6 木樨草素誘導A549肺癌細胞株之可能途徑 13
圖2.7 木樨草素的生物活性與可能的作用機制 14
圖2.8 紫花牡荊素的結構式 16
圖3.1 實驗流程圖 24
圖3.2 抗氧化活性分析 26
圖4.1 蔓荊子不同溶劑萃取物之清除DPPH自由基能力 37
圖4.2 蔓荊子不同溶劑萃取物之三價鐵還原力 38
圖4.3 蔓荊子不同溶劑萃取物之螯合亞鐵離子能力 40
圖4.4 蔓荊子萃取物之清除超氧陰離子能力 41
圖4.5 蔓荊子萃取物清除ABTS自由基能力 43
圖4.6 不同濃度蔓荊子水萃取物處理PC12細胞1小時 46
圖4.7 不同濃度蔓荊子水萃取物處理PC12細胞24小時 47
圖4.8 不同濃度H2O2處理PC12細胞1小時 49
圖4.9 不同濃度H2O2處理PC12細胞24小時 50
圖4.10 抗氧化劑保護PC12細胞H2O2氧化損傷1小時 51
圖4.11 抗氧化劑保護PC12細胞H2O2氧化損傷24小時 52

表目錄 xii

表2.1 紫花牡荊素對腫瘤細胞的抑制活性 17
表4.1 不同溶劑萃取蔓荊子之萃取率 32
表4.2 不同溶劑萃取蔓荊子其總酚及總類黃酮含量 33
表4.3 蔓荊子不同溶劑萃取物之半數清除或螯合力(IC50) 36
表4.4 HPLC分析蔓荊子萃取物之槲皮素含量 44
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