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研究生:張竣淵
論文名稱:利用化學顯色與冷光法以及紅血球螢光檢測方法對於不同類胡蘿蔔素之抗氧化能力比較
論文名稱(外文):Comparison of the anti-oxidative capacity of carotenoids by colorimetry, chemiluminescence, and RBC-based fluorescent assay
指導教授:黎耀基黎耀基引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
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:2014
畢業學年度:102
語文別:英文
論文頁數:87
中文關鍵詞:類胡蘿蔔素抗氧化紅血球過氧化氫活性氧
外文關鍵詞:Carotenoidsanti-oxidative capacityerythrocyte (red blood cell)hydrogen peroxide (H2O2)reactive oxygen specie (ROS)
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類胡蘿蔔素為廣泛分佈的紅黃色色素,具有結構多樣性和許多的功能特性,且可被植物和微生物所合成。在許多研究中指出,攝取類胡蘿蔔素的多寡與退化性疾病之間存在著對比關係,且它們也有具有良好的抗氧化效果。從日常生活所攝取的類胡蘿蔔素預防退化性疾病的結果被認為是因為其抗氧化能力,保護細胞及組織免於氧化的損害。好的抗氧化劑可以有效地去除或是捕捉自由基。而在人體裡最主要的自由基為活性氧,如羥自由基及過氧化自由基。人體裡的活性氧的產生來自於細胞進行呼吸作用所產生的有害副產物,過多的活性氧會造成人體的氧化壓力,導致許多退化性疾病,甚至是癌症。因此人們開始重視抗氧化,來預防許多疾病的產生。
許多類胡蘿蔔素都已被不同方法來檢測其抗氧化的能力,然而這些方法所使用的自由基、反應條件及細胞等都不盡相同,導致難以一起比較其結果差異。在這篇研究中,我們建立了同樣以過氧化氫誘發的氧化但在不同環境中反應的實驗方法。利用LPSC方法以過氧化氫誘發化學冷光及CPA-e方法在紅血球模式下以過氧化氫所產生的螢光來比較出類胡蘿蔔素在不同環境中所表現出的抗氧化結果差異。
我們從特定微生物及雨生紅球藻得到不同種類的類胡蘿蔔素,藉由重力管柱層析來純化出要進一步分析抗氧化能力的類胡蘿蔔素。此篇研究的目的在利用我們所建立出的抗氧化方法(LPSC and CAP-e assay)與化學顯色法(ABTS and FRAP assay)將純化出的類胡蘿蔔素與市售的類胡蘿蔔素做抗氧化能力的比較。在這些不同方法的結果中,不同類胡蘿蔔素顯示出對於不同自由基的去除能力有所差異。分析並比較其不同方法結果發現,我們純化出的類胡蘿蔔素Z和H表現出有效的抗氧化活性。在ABTS方法中,ZEA、Z及H有高達50 %以上,甚至88 %的抑制率。而-類胡蘿蔔素、茄紅素及我們所純化出新的類胡蘿蔔素P也有33 %以上的抑制率。H及玉米黃素對於三價鐵的還原能力比其他的類胡蘿蔔素高6倍以上。在LPSC方法中,類胡蘿蔔素對於過氧化氫所產生的氧化都有不錯的抑制能力,抑制率都可達到52 %以上,甚至到達97 %。-類胡蘿蔔素與茄紅素相較於其他xantophylls抑制率在同樣濃度下只達到52 %左右。蝦紅素與我們純化出的類胡蘿蔔素F3其在胞外實驗表現的抗氧化能力相近,然而在細胞實驗中,蝦紅素與F3雖為同一類胡蘿蔔素卻表現出不同的抗氧化活性差異。角黃素與我們純化出的類胡蘿蔔素F1也有相同的情形。在CAP-e方法,我們所純化出的相較於市售的相同類胡蘿蔔素,對於過氧化氫所產生的氧化壓力,抑制率明顯有所差異。我們所純化出的天然類胡蘿蔔素抑制率高達64 %,而市售的的抑制率在32 %左右。推測其差異可能是因立體構型不同導致細胞攝取及在細胞膜分布不相同。

Carotenoids are yellow to red pigments that are notable for their wide distribution, structural diversity, and various functions. They are synthesized in plants and in some microorganisms.
The epidemiologic studies have revealed that there is an inverse relationship between the presence of degenerative disorders and dietary carotenoids. Moreover, anti-oxidative activities of carotenoid pigments have been notified in a number of studies. The prevention of these degenerative disorders by dietary carotenoids has been associated with capacity of carotenoids to protect cells and tissues from oxidative damages.
A powerful antioxidant can scavenge or trap free radical effectively. The common free radicals in human body are reactive oxygen species (ROS) such as hydroxyl radical and peroxyl radical that may cause oxidative stress. ROS are generated during normal cell aerobic respiration, which are harmful byproducts. Thus, people are beginning to attach importance to antioxidant for preventing free-radical-involved diseases. To date, the anti-oxidative activity of carotenoids has been measured by different methods; however, it is difficult to compare the results directly because of variation in free radicals, reaction conditions, and the types of cells used for analysis.
The aim of this research was to set up ABTS, FRAP, LPSC, and CAP-e assays to compare the anti-oxidative performance for a variety of carotenoids obtained from the specific microorganism and Haematococcus pluvialis (F1, F2, F3, P, Z and H, respectively) and commercial caroteinoids (astaxanthin, canthaxanthin, zeaxanthin, -carotene and lycopene) as well as from commonly used non-pigment antioxidants (BHA and Trolox). Carotenoids obtained from recombinant bacteria and algae were purified via Open Column Chromatography (OCC) followed by reprecipitation method. Among the four assays which were all modified for application in this study, LPSC and CAP-e were particularly adopted because they both use hydrogen peroxide (H2O2) as the source of oxidative stress.
In all four assays, Z and H (the carotenoids we purified) presented strong anti-oxidative activity in general. Zeaxanthin, Z and H had inhibition from 50 to 88%; and-carotene, lycopene and P also had over 33 percent inhibition in ABTS assay. The sample H and zeaxanthin had 6 times higher ferric reducing ability than other carotenoids in the FRAP analysis.
In LPSC and CAP-e assays, all analyzed carotenoids showed > 52% inhibition of H2O2-induced oxidation, indicating good anti-oxidative activity of carotenoids. Xantophylls presented significantly better antioxidative activities as compared with -carotene and lycopene at the same concentration.
We further compared natural carotenoids purified in this research with commercial carotenoids which were supposed to be in all-trans form, and found that their anti-oxidative activities had significant difference in CAP-e assays. Although commercial astaxanthin standard and the isolated F3 showed similar anti-oxidative activity in ABTS, FRAP and LPSC methods, F3 presented significantly higher anti-oxidative activities as compared with astaxanthin standard (P<0.05). Likewise, the commercial canthaxanthin showed significantly lower activities in comparison with F1 (P<0.001). At concentration of 5 μg/ml, natural carotenoids (F1 and F3) purified in this study showed 64% inhibition in average, while commercial carotenoids (astaxanthin and canthaxanthin) reached 32% only.
The possible reasons of the variation in anti-oxidative activities might be addressed to difference of chemical structures between stereoisoforms that probably affect the solubility and cellular uptake as well as distribution in the cell membrane.

Chapter 1 Introduction
1.1 Carotenoids
1.1.1 Astaxanthin
1.1.2 Cantaxanthin
1.1.3 Zeaxanthin
1.1.4 b-carotene
1.1.5 Lycopene
1.2 The properties of antioxidant
1.2.1 The free radical and ROS
1.2.1.1 Superoxide (O2-)
1.2.1.2 Hydrogen peroxide (H2O2)
1.2.1.3 Peroxy radical (LOO.)
1.2.1.4 Hydroxyl radical (HO.)
1.2.1.5 Singlet oxygen (1O2)
1.2.2 The natural antioxidant and synthetic antioxidant
1.3 Open column chromatography
1.4 Colorimetry assay
ABTS assay
FRAP assay
1.5 Chemiluminescence assay
LPSC assay
1.6 Fluorescent assay
Cell-based antioxidant protection in erythrocytes (CAP-e) assay
1.7 The Aim
Chapter 2 Materials and Methods
2.1 Chemical reagents
2.2TLC Method
2.3 OCC Method
2.4 Reprecipitation of primarily purified carotenoids
2.5 Sample preparation
2.6 Analysis of the absorption spectrum of the carotenoids
2.7 ABTS assay
2.8 FRAP assay
2.9 LPSC assay
2.10 Cell-based antioxidant protection
in erythrocytes (CAP-e) assay
2.11 Flow Cytometry Analysis
Chapter 3 Results
3.1 Separating single carotenoid from crude extracts
3.1.1 Sample F
3.1.2 Sample H
3.1.3 Sample P
3.1.4 Sample Z
3.2 The absorption spectrum and optical density (OD) value of different carotenoids
3.3 The ability of different compounds to scavenge ABTS•+
3.4 The reducing ability of different compounds
in FRAP assay
3.5 The ability of scavenging H2O2-induced oxidation in different compounds in LPSC assay
3.6 The anti-oxidative ability of different compounds to reduce the H2O2-induced oxidative stress in RBCs
Chapter 4 Conclusions and Discussions
4.1 The separation of different carotenoids
4.2 Anti-oxidative activity of compounds assessed with the different methods
4.2.1 ABTS assay
4.2.2 FRAP assay
4.2.3 LPSC assay and CAP-e assay
4.3 Summary
References
Figures
Tables
Appendixes


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