Antioxidative activity and cell assay of coleus blumei extracts 莊鼎彬、.瑞澤
E-mail: 343845@mail.dyu.edu.tw
ABSTRACT
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.
Keywords : Coleus blumei、antioxidative、rosmarinic acid、HepG2 cell、PC-12 cell、cell viability Table of Contents
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?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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