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研究生:莊鼎彬
研究生(外文):Chuang, Tingpin
論文名稱:彩葉草萃取物之抗氧化能力與細胞試驗
論文名稱(外文):Antioxidative Activity and Cell Assay of Coleus blumei Extracts
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):Tu, JuitseWu, Shutzu
口試委員:陳錦坤胡淳怡凃耀國吳淑姿
口試委員(外文):Chen, ChinkunHu, ChunyiTu, YaokuoWu, Shutzu
口試日期:2011-06-17
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:2011
畢業學年度:99
語文別:中文
論文頁數:103
中文關鍵詞:彩葉草抗氧化迷迭香酸人類肝癌細胞大鼠腎上腺嗜鉻瘤細胞細胞存活率分析
外文關鍵詞:Coleus blumeiantioxidativerosmarinic acidHepG2 cellPC-12 cellcell viability
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本研究以熱迴流方式利用不同溶劑(水、甲醇、乙醇、乙酸乙酯和正己烷)萃取、不同前置處理之彩葉草(新鮮彩葉草、日曬、40℃、60℃、80℃和100℃)。探討不同萃取條件對抗氧化能力之影響。抗氧化能力分析包括:總多酚類與類黃酮含量、對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、清除ABTS 陽離子能力及抑制微脂粒氧化作用能力,並與BHA、EDTA及沒食子酸之抗氧化能力做比較。同時對彩葉草萃取物抗氧化能力高的部份進行成分分析與細胞試驗。
  實驗結果顯示,新鮮彩葉草以不同溶劑萃取,萃取物之萃取率和總多酚含量皆是水萃取物最高,而類黃酮含量則是乙酸乙酯萃取物最高。在抗氧化能力分析方面,皆是新鮮彩葉草萃取物表現較佳,清除DPPH 自由基能力試驗,以水和乙酸乙酯萃取物最佳,其IC50皆小於0.01 mg/mL;相對還原力試驗,以水萃取物最佳,其IC50為0.7 ± 0.00 mg/mL;清除ABTS 陽離子試驗,以乙酸乙酯萃取物最佳,其IC50小於0.01 mg/mL;螯合亞鐵離子能力試驗,以水萃取物最佳,其IC50為0.13 ± 0.02 mg/mL;清除超氧陰離子能力試驗,以水萃取物最佳,其IC50為0.35 ± 0.00 mg/mL;抑制微脂粒氧化作用,以正己烷萃取物最佳,其IC50為3.89 ± 0.14 mg/mL。萃取物成分分析,以新鮮彩葉草水和乙酸乙酯萃取物進行HPLC 分析,與標準品迷迭香酸比對後,確認皆含有迷迭香酸成分,滯留時間為2-3 min 之間,以水萃取物含量較高,為232.09 mg/g。長期貯存萃取物,其主要成分迷迭香酸含量會降低。於細胞實驗,發現新鮮彩葉草水萃取物對HepG2細胞生存率之影響,濃度為400 μg/mL時,其細胞存活率為47.1%,而對PC-12細胞生存率之影響並不顯著,濃度為400 μg/mL時,其存活率為76.6%。另外,新鮮彩葉草水萃取物具有保護PC-12細胞免受氧化壓力之傷害,於缺血再灌流之模式中,未添加保護劑之細胞其存活率為35.8%,而添加濃度為400 μg/mL時,其存活率為48.8%。而以H2O2誘導PC-12細胞凋亡之保護效果方面,未添加保護劑之細胞其存活率為46.0%,而添加濃度為100 μg/mL時,其存活率為66.1%。
  綜合研究結果顯示,新鮮彩葉草萃取物之抗氧化能力效果最佳,總酚和類黃酮含量較多,亦可抑制肝癌細胞生長並可保護神經不受氧化壓力之傷害。此結果將有助於機能性食品之開發,與後續更深入研究之基礎。

Fresh leaves of Coleus blumei were first dried under various temperatures (40, 60, 80 and 100℃) and then were extracted using a hot water reflux. Also some fresh leaves of Coleus blumei were first sun-dried and then extracted by using a hot reflux with one solvent (water, methanol, ethanol, ethyl acetate or n-hexane). The purpose of this study is to examine the effect of extraction method on antioxidative activities of extracts. The assays of antioxidative activities included DPPH (α,α-diphenyl- β-picrylhydrazyl) radical scavenging ability, Fe2+ chelating ability, relative reducing power, superoxide anion scavenging ability, the inhibition of Fe/ascorbate-induced lipid peroxidation, and ABTS cation scavenging ability. These antioxidative activities of Coleus blumei extracts were measured and compared with those of butylated hydroxyanisole (BHA), ethylene diamine tetracetic acid (EDTA) or gallic acid. The component analysis and cell assay were also carried out for Coleus blumei extracts with the highest antioxidative activities.
The results have showed that both of the extraction yield and the content of total phenols reached the highest when fresh leaves of Coleus blumei were extracted by hot water. The fresh leaves of Coleus blumei extracted by ethyl acetate had the highest content of total flavonoids, and however, most of total phenols and total flavonoids lost during drying. For the antioxidative activities, the extracts obtained by water and ethyl acetate had a higher DPPH radical scavenging ability (IC50<0.01 mg/mL); the aqueous extract had the highest relative reducing power (IC50=0.7 ± 0.00 mg/mL), the highest Fe2+ chelating ability (IC50=0.13 ± 0.02 mg/mL), and the highest superoxide anion scavenging ability (IC50=0.35 ± 0.00 mg/mL); the extract by ethyl acetate had the highest ABTS cation scavenging ability (IC50<0.01 mg/mL); the extract by n-hexane had the highest inhibition ability of lipid peroxidation (IC50=3.89 ± 0.14 mg/mL). In addition, the components of the extracts by water and ethyl acetate were analyzed by using an HPLC. The result showed that rosmarinic acid was the major component, and its content in the aqueous extract (232.09 mg/g) is higher than that in the ethyl acetate extract. In cell assays, the results show that the aqueous extract affected the HepG2 cell viability but didn’t affect the PC-12 cell viability. The aqueous extract showed the ability to protect PC-12 cells against hypoxia and H2O2-induced oxidative stress.
In summary, the contents of total phenols and total flavonoids, and the antioxidant activities were highest for the extract from fresh leaves of Coleus blumei obtained by a hot water reflux. The extract could inhibit the growth of HepG2 cells and protect nerve cells against oxidative stress. The results obtained in this study are useful for future research and development of functional foods.

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xiv

1. 緒論1
2. 文獻回顧2
2.1 彩葉草簡介2
2.2 彩葉草之種類2
2.3 自由基之介紹4
2.3.1 自由基的定義4
2.3.2 自由基在生物上的作用5
2.3.3 自由基的產生和清除6
2.4 抗氧化劑介绍及其分類6
2.5 迷迭香酸9
2.5.1 簡介9
2.5.2 迷迭香酸生合成12
2.5.3 迷迭香酸的生物活性12
3. 材料與方法15
3.1 植物材料15
3.2 藥品15
3.3 儀器16
3.4 實驗方法17
3.4.1 實驗架構17
3.4.2 植物萃取之製備20
3.4.3 抗氧化分析23
3.4.4 迷迭香酸分析26
3.4.5 細胞株27
3.4.6 培養基與試劑配製27
3.4.7 細胞分析31
4. 結果與討論37
4.1 新鮮彩葉草之不同溶劑熱迴流萃取37
4.1.1 萃取率37
4.1.2 抗氧化成分含量分析34
4.1.3 抗氧化能力分析40
4.2 不同乾燥處理彩葉草之熱迴流萃取50
4.2.1 萃取率50
4.2.2 抗氧化成分含量分析52
4.2.3 抗氧化能力分析54
4.3 日曬乾燥處理彩葉草之不同溶劑熱迴流萃取64
4.3.1 萃取率64
4.3.2 抗氧化成分含量分析64
4.3.3 抗氧化能力分析67
4.4 彩葉草之成分分析78
4.4.1 新鮮彩葉草水與乙酸乙酯萃取物之HPLC分析78
4.4.2 貯存期間之迷迭香酸含量變化83
4.5 細胞試驗86
4.5.1 細胞存活率分析86
4.5.2 細胞保護能力試驗85
5. 結論94
5.1 結論94
5.2 未來展望95
參考文獻96
附錄101

圖目錄
圖2.1 彩葉草(a)葉子、(b)花3
圖2.2 細胞中含氧和含氮自由基之生成7
圖2.3 細胞中含氧和含氮自由基之去除8
圖2.4 迷迭香酸的結構式10
圖2.5 迷迭香酸生合成路徑13
圖3.1 實驗流程圖18
圖3.2 抗氧化活性分析19
圖3.3 萃取流程21
圖3.4 熱迴流萃取裝置22
圖3.5 細胞實驗流程33
圖3.6 細胞存活率分析34
圖4.1 不同溶劑萃取彩葉草對清除DPPH自由基能力之影響41
圖4.2 不同溶劑萃取彩葉草之相對還原力之影響43
圖4.3 不同溶劑萃取彩葉草對螯合亞鐵離子能力之影響45
圖4.4 不同溶劑萃取彩葉草對清除ABTS陽離子能力之影響46
圖4.5 不同溶劑萃取彩葉草對清除超氧陰離子能力之影響48
圖4.6 不同溶劑萃取彩葉草對抑制微脂粒氧化作用之影響49
圖4.7 不同乾燥處理之彩葉草萃取物對清除DPPH自由基能力之影響55
圖4.8 不同乾燥處理之彩葉草萃取物之相對還原力之影響57
圖4.9 不同乾燥處理之彩葉草萃取物對螯合亞鐵離子能力之影響59
圖4.10不同乾燥處理之彩葉草萃取物對清除ABTS陽離子能力之影響60
圖4.11不同乾燥處理之彩葉草萃取物對清除超氧陰離子能力之影響62
圖4.12不同乾燥處理之彩葉草萃取物對抑制微脂粒氧化作用之影響63
圖4.13日曬乾燥彩葉草不同溶劑萃取之萃取物對清除DPPH自由基能力之影響68
圖4.14日曬乾燥彩葉草不同溶劑萃取之萃取物對相對還原力之影響70
圖4.15日曬乾燥彩葉草不同溶劑萃取之萃取物對螯合亞鐵離子能力之影響72
圖4.16日曬乾燥彩葉草不同溶劑萃取之萃取物對清除ABTS陽離子能力之影響73
圖4.17日曬乾燥彩葉草不同溶劑萃取之萃取物對清除超氧陰離子能力之影響75
圖4.18日曬乾燥彩葉草不同溶劑萃取之萃取物對抑制微脂粒氧化作用之影響76
圖4.19新鮮彩葉草水熱迴流萃取萃取物之HPLC分析圖79
圖4.20迷迭香酸之HPLC層析圖80
圖4.21新鮮彩葉草水熱迴流萃取萃取物之HPLC分析圖82
圖4.22貯存期間新鮮彩葉草水萃取物對清除DPPH自由基能力之影響85
圖4.23新鮮彩葉草水萃取物之存活率分析87
圖4.24標準品迷迭香酸之存活率分析88
圖4.25缺血再灌流模式下新鮮彩葉草萃取物對PC-12之保護效果90
圖4.26H2O2對PC-12細胞之存活率分析91
圖4.27新鮮彩葉草萃取物對H2O2誘導PC-12細胞凋亡之保護效果93

表目錄
表2.1 植物界中含迷迭香酸之分布11
表3.1 HepG2細胞之培養基28
表3.2 PC-12細胞之培養基29
表3.3 磷酸鹽緩衝液溶液配方30
表4.1 不同溶劑萃取彩葉草之萃取率38
表4.2 不同溶劑萃取之彩葉草總酚及總類黃酮含量39
表4.3 不同溶劑萃取彩葉草之半數效應濃度(IC50)42
表4.4 不同乾燥處理之彩葉草萃取率51
表4.5 不同乾燥處理之彩葉草總酚與總類黃酮含量53
表4.6 不同乾燥處理彩葉草之半數清除濃度(IC50)56
表4.7 日曬乾燥彩葉草不同溶劑萃取之萃取率65
表4.8 日曬乾燥彩葉草不同溶劑萃取之總酚及總類黃酮含量66
表4.9 日曬乾燥彩葉草不同溶劑萃取之半數清除濃度(IC50)69
表4.10新鮮彩葉草萃取物之迷迭香酸含量81
表4.11貯存期間之迷迭香酸含量變化84


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