臺灣博碩士論文加值系統

近年來，自由基、氧化壓力與抗氧化物間的關係已成為疾病作用機制中備受重視的一環。自由基能氧化許多重要的生物分子，如蛋白質、脂肪、去氧核糖核酸 而誘發一些氧化病變的產生。聚球藻（Synechococcus sp.）屬藍綠藻，其富含藻藍素（phycocyanin）為一光吸收色素-蛋白質複合體，目前已知具有抗氧化能力、抗發炎、神經保護及肝臟保護作用而呈現多樣化的生理活性。本研究目的為評估聚球藻中其藻藍素特性探討及安全性，以評估聚球藻開發之潛能。結果顯示，在萃取條件上以凍結-解凍破碎細胞且以水萃為佳。將萃取液以超濃縮過濾濃縮約20倍後，經疏水性交換層析及陰離子交換層析純化，得一分子量約為106.5 kDa的三聚體分子（Trimer molecule），經變性後裂解成兩個次單元，其分子量分別約為18.4 kDa及17.1 kDa。當藻藍素純化液濃度為90 μg/ml時，其清除DPPH自由基能力、超氧離子、螯合亞鐵離子能力與過氧化氫清除效應分別為88.2％、69.4％、47.6％及61.8％。在進行毒性試驗與致突變性試驗，數據顯示在測定濃度0.1-1 mg/plate之藻藍素純化液均無具毒性及致突變性。而在抗致突變試驗中，其抗致突變率皆隨著藻藍素純化液濃度提高而增加。藻藍素純化液於4℃、避光及pH=7的環境下安定性較佳。在金屬離子的影響方面，以添加濃度100mM的HgCl2與CuSO4影響最大，其殘存率不到30％。在化學添加物方面，以添加濃度100 mM的SDS影響最大，其損失率約78.1％。

Free radical, oxidative stress and antioxidants have become commonly used terms in modern discussions of disease mechanisms. Free radicals may induce some oxidative damage to biomolecules such as proteins, lipids and DNA. Synechococcus sp., a member of cyanobacteria family, containing phycocyanin is one of the major light harvesting pigment-protein which exhibiting diverse antioxidation capacities, anti-inflammatory, neuroprotective and hepatoprotective effect. The main purpose of the study is Synechococcus sp was subjected as an example to investigate its potential for nutracieutical by characterizing and safety assessment of C-phycocyanin from Synechococcus sp. U2. Freezing -thawing in combination with distilled water was the best method for extracting phycocyanin from Synechococcus sp.U2. The crude extracts was concentrated up to 20 fold by ultrafiltration and consecutively purified by butyl- sepharose and DEAE- sepharose resin. It migrated in native polyacrylamide gel as a singal protein（106.5 kDa）, which split under
denaturing conditions into two bands having an apparent molecular weight of 18.4 and 17.1 kDa. The purity extracts of 90 μg/ml from Synechococcus sp.U2 scavenged DPPH 、superoxide and H202 by 88.2％ 、69.4％、61.8％, respectively. In addition to, 47.6％ chelating ability on ferrous ions was observed in the present study. Three different dosage of purified phycocyanin extracts were used, 0.1, 0.5 and 1.0 mg/plate, showed no toxicity and mutagenicity. The purified phycocyanin extracts from Synechococcus sp.U2 indicated the best antimutagenic effect when the concentration of 1 mg/plate phycocyanin was used. The purified phycocyanin extracts was stable over pH=7 at 4℃without light. In the presence of metal ions, the content of phycocyanin was reduced by adding 100 mM HgCl2 and CuSO4 and only less than 30％ residual left. In related to chemical additives study, the content of phycocyanin was reduced 78.1％ by adding 100 mM SDS.

目錄

摘要 I
Abstract II
誌謝 IV
目錄 V
圖表目錄 X
第1章 前言 1
第2章 文獻回顧 3
2.1 聚球藻簡介 3
2.2 藻膽蛋白 4
2.3 藻藍素生理活性簡介 6
2.4 自由基與活性氧之形成 15
2.5 自由基對生物體所造成之危害 15
2.6 抗氧化酶與天然抗氧化物 17
2.6.1 抗氧化酶 17
2.6.1.1 超氧歧化酶 17
2.6.1.2 觸酶 19
2.6.1.3 麩胱甘肽過氧化酶 19
2.6.2 天然抗氧化物 20
2.6.2.1 花青素 20
2.6.2.2 抗壞血酸 22
2.6.2.3 維生素E 22
2.6.2.4 類胡蘿蔔素 24
2.6.2.5 酚類 28
2.6.2.6 藻藍素 28
2.7癌症與飲食 30
2.7.1 食物中常見的致突變物及致癌物 30
2.7.1.1 異環胺類化合物 30
2.7.1.2 黴菌毒素 31
2.7.1.3 亞硝基化合物 31
2.7.1.4 多環芳香烴類 32
2.7.2 食品中之抗致突變物及抗致癌物 32
2.7.3 抗致突變物及抗致癌物之作用機制 32
2.7.4 安氏試驗法 33
2.7.5 鼠傷寒沙門氏桿菌組織胺酸突變位置及檢測特性 33
2.7.5.1 Histidine requirement 35
2.7.5.2 rfa mutation 35
2.7.5.3 uvrB mutation 35
2.7.5.4 R-factor 36
2.7.5.5 pAQ1 plasmid 36
2.7.6 安氏試驗法常用各株鼠傷寒沙門氏菌特性 36
2.7.6.1 TA97 36
2.7.6.2 TA98 36
2.7.6.3 TA100 36
2.7.6.4 TA102 37
第3章 材料與方法 39
3.1 實驗設計 39
3.2 聚球藻來源 40
3.3 安氏試驗試驗菌株 40
3.4 實驗試藥與設備 40
3.4.1 抗氧化分析試藥 40
3.4.2 電泳分析試藥 40
3.4.3 安氏試驗分析試藥 41
3.4.4 藻藍素純化材料 41
3.4.5 儀器設備 41
3.5 實驗方法 42
3.5.1 聚球藻培養 42
3.5.2 聚球藻前處理 43
3.5.3 最適萃取條件之探討 43
3.5.3.1 最適萃取方法 43
3.5.3.2 最適萃取溶劑 43
3.5.4 藻藍素含量測定 43
3.5.5 藻藍素之分離與純化 44
3.5.5.1 藻藍素粗萃液之製備 44
3.5.5.2 疏水性交換層析 44
3.5.5.3 陰離子交換層析 44
3.5.5.4 蛋白質含量測定 45
3.5.5.5 純化總表之製作 45
3.5.6 分子量之測定 45
3.5.6.1 樣品之處理 45
3.5.6.2 電泳分析試劑之配製 46
3.5.6.3 電泳膠體之配製 48
3.5.6.4 電泳之進行 48
3.5.6.5 膠體之染色 49
3.5.7 抗氧化活性測定 49
3.5.7.1 清除DPPH自由基試驗 49
3.5.7.2 清除超氧陰離子能力試驗 49
3.5.7.3 螯合亞鐵離子能力之測定 50
3.5.7.4 還原力測定 50
3.5.7.5 過氧化氫清除能力之測定 50
3.5.8 藻藍素之安全性評估 51
3.5.8.1 安氏試驗之萃取液之製備 51
3.5.8.2 試驗菌株之活化 51
3.5.8.3 基因型態之確認 51
3.5.9 安氏試驗所需溶液之配製 52
3.5.10 安氏試驗 55
3.5.10.1 毒性試驗 55
3.5.10.2 致突變性分析 56
3.5.10.3 抗致突變分析 56
3.5.11 藻藍素穩定性之測定 57
3.5.11.1 溫度穩定性之測定 57
3.5.11.2 光穩定性之測定 57
3.5.11.3 pH穩定性之測定 57
3.5.11.4 金屬離子對藻藍素穩定性之影響 58
3.5.11.5 化學試劑對藻藍素穩定性之影響 58
3.5.12 統計分析 58
第4章 結果與討論 59
4.1 藻種培養及前處理 59
4.2 最適萃取條件之探討 59
4.2.1 最適萃取方法 59
4.2.2 最適萃取溶劑 63
4.3 粗萃液之製備 63
4.4 藻藍素之純化 65
4.4.1 疏水性交換層析 65
4.4.2 陰離子交換樹脂層析 65
4.4.3 純化總表 68
4.5 分子量之測定 68
4.5.1 Native-PAGE 68
4.5.2 SDS-PAGE 73
4.6 抗氧化活性測定 73
4.6.1 清除DPPH自由基試驗 73
4.6.2 清除超氧陰離子能力試驗 75
4.6.3 螯合亞鐵離子能力之測定 79
4.6.4 還原力測定 81
4.6.5 過氧化氫清除能力之測定 81
4.7 藻藍素之安全性評估 83
4.7.1 試驗菌株基因型態之確認 83
4.7.1.1 組胺酸需求性之確認 85
4.7.1.2 rfa突變之測試 85
4.7.1.3 uvrB突變之測試 85
4.7.1.4 R-factor之測試 87
4.7.2 毒性試驗 87
4.7.3 致突變性試驗 87
4.7.4 抗致突變性試驗 92
4.8 藻藍素之穩定性測定 92
4.8.1 溫度穩定性之測定 92
4.8.2 光穩定性之測定 93
4.8.3 pH穩定性之測定 93
4.8.4 金屬離子對藻藍素穩定性之影響 99
4.8.5 化學試劑對藻藍素穩定性之影響 99
第5章 結論 103
參考文獻 105
作者簡介 124

















圖表目錄

圖 1、 海洋微生物食物網與有機碳循環關係...…………………..…... 5
圖 2、 藻膽色體其概要構造圖……………………….………………... 7
圖 3、 藻藍素之bilin發色基團與膽紅素之化學結構………………… 8
圖 4、 膽紅素與麩胱甘肽於細胞中之抗氧化效果…………………… 12
圖 5、 ROS之形成過程.……………………….……………………….. 16
圖 6、 釋放的自由基對細胞分子的影響…………..………………….. 18
圖 7、 麩胱甘肽-抗壞血酸之相關關係...………..……………………. 23
圖 8、 不同結構之生育醇及生育三烯醇...……………………………. 25
圖 9、 在未提供6-OH基團氫離子給脂質自由基前α-tocopherol之結構………………………………………………………………… 26
圖10、 類胡蘿蔔素之構造……………………………………………… 27
圖11、 類黃酮的基本結構..…………………………………...……….. 29
圖12、 抗致突變劑之作用機制分類…………………………………… 34
圖13、 聚球藻之菌種觀察(a) 聚球藻培養液 (b)聚球藻粉末外觀.... 60
圖14、 Synechococcus sp. U2中藻藍素之butyl-sepharose管柱層析圖... 66
圖15、 Synechococcus sp. U2中藻藍素之DEAE-sepharose管柱層析圖. 67
圖16、 經純化後所得之藻藍素純化液……………................................ 70
圖17、 經純化後之藻藍素吸收波長曲線圖....………………………… 71
圖18、 Synechococcus sp. U2中藻藍素之Native-PAGE電泳圖............. 72
圖19、 Synechococcus sp. U2中藻藍素之SDS-PAGE電泳圖......…….. 74
圖20、 Synechococcus sp. U2中藻藍素清除DPPH能力....…………… 76
圖21、 Synechococcus sp. U2中藻藍素清除超氧陰離子能力....….….. 78
圖22、 Synechococcus sp. U2中藻藍素螯合亞鐵離子能力....………… 80
圖23、 Synechococcus sp. U2中藻藍素之還原力....…………………… 82
圖24、 Synechococcus sp. U2中藻藍素清除過氧化氫能力....………… 84
圖25、 聚球藻中藻藍素於不同溫度下其穩定性試驗....……….…… 96
圖26、 聚球藻中藻藍素光穩定性試驗....…………………….……… 97
圖27、 聚球藻中藻藍素於不同pH下其穩定性試驗....……………..… 98
圖28、 化學試劑對聚球藻中藻藍素之影響....….......................…..… 102
表1、 藻藍素抗氧化之相關體外試驗……….……………………… 13
表2、 藻藍素抗氧化之相關體內試驗…………………….......………. 14
表3、 主要的ROS及其代謝……………………................................... 21
表4、 致突變性試驗中所使用之沙門氏菌試驗菌株………………… 38
表5、 不同的聚球藻其藻藍素含量之比較….……………………… 61
表6、 不同萃取方法對於聚球藻中藻藍素萃取效果之比較………… 62
表7、 不同萃取溶劑對於聚球藻中藻藍素萃取效果之比較………… 64
表8、 Synechococcus sp.U2藻藍素之純化總表………………………. 69
表9、 鼠傷寒沙門氏桿菌TA98、TA100基因型態測定結果.................. 86
表10、 聚球藻中藻藍素對S. typhimurium TA98菌株之毒性試驗(有無添加S9混和物)………..………….…………………………… 88
表11、 聚球藻中藻藍素對S. typhimurium TA100菌株之毒性試驗(有無添加S9混和物)………………………………………………. 89
表12、 聚球藻中藻藍素對S. typhimurium TA98菌株之致突變性試驗(有無添加S9混和物)..................................................................... 90
表13、 聚球藻中藻藍素對S. typhimurium TA100菌株之致突變試驗(有無添加S9混和物).................................................................... 91
表14、 聚球藻中藻藍素對S. typhimurium TA98菌株之抗致突變性試驗.................................................................................................... 94
表15、 聚球藻中藻藍素對S. typhimurium TA100菌株之抗致突變性試驗................................................................................................ 95
表16、 金屬離子對聚球藻中藻藍素之影響............................................ 101

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