臺灣博碩士論文加值系統

落葵（Basella alba）俗稱皇宮菜，屬蔓藤類蔬菜，其成熟漿果呈深紫色，所含色素值得探討。將成熟新鮮漿果去籽，分別利用水、80%甲醇（含0.2%甲酸）萃取（1:5, w/v）及整顆漿果直接壓榨取得新鮮汁液，再經真空凍結乾燥後秤重，三種粗萃物之收率分別為1.79、1.77及3.7 g/100 g漿果。經比較三種粗萃物TEAC（trolox equivalent antioxidant capacity）抗氧化能力，以甲醇萃取者最高，後續檢測均以甲醇進行萃取。將甲醇粗萃物回溶於不同pH緩衝溶液（250 μg/mL）後，分析其於pH值2.12~ 8.9之呈色及於pH值3、5及7之溶液並置於不同溫度（4、30、60、90及120 oC）之安定性，結果顯示，於pH 2.12~ 8時，均呈現紫紅色，其可見光最大吸收波長為540 nm，於pH 8.9時，其可見光最大吸收波長位移至570 nm，顏色轉為藍色。在不同pH值下（pH 3、5及7）溶液中，以4 oC最安定，溫度愈高，色素降解愈快。另外，將甲醇粗萃物（A）、甲醇粗萃液加熱（90 oC，2 h）再凍結乾燥（B）、甲醇萃取液經SPE（solid phase extraction）分離再凍結乾燥（C）及甲醇萃取液經SPE分離經加熱（90 oC，2 h）後再凍結乾燥（D）等固形物回溶於水，其可見光最大吸收波長分別為540、510、540及510 nm，各溶液經加熱處理後其顏色均變為紅色。經定量A、B、C及D固形物之總酚含量，分別為0.23、0.26、0.45及0.37 mg/g；當回溶水溶液濃度為500 μg/mL時，其TEAC相當於483、372、1378及1359 μM Trolox；濃度為50 μg/mL時，其DPPH（α,α-diphenyl-β-picrylhydrazyl）自由基清除能力分別為22.9、27.7、83.5及88.4%，相當於4.7、6.0、22.1及23.5 μg/mL之BHT；濃度均為100 μg/mL時，其還原力分別相當於8.2、5.8、33.1及30.1 μg/mL Vit C，其亞麻油酸抗氧化能力分別相當於9.13、18.9、33.1及35.9 μg/mL之BHT。另外，將SPE萃取物進行HPLC分離純化及回收鑑定，可得三個主要色素成分，經UV-Visible光譜及ESI-MS分析，顯示三成分均具為betacyanin（甜菜�ㄕ滽嚏^主要化學特性，其中兩個色素成分主峰m/z值均為 713，其碎片離子m/z值為551與389；另一色素成分主峰m/z值為551，其碎片離子m/z值389，故可鑑定其化合物分別為betanidin-dihexose、Iso-betanidin-dihexose及betanidin-hexose。此外，利用HPLC分析比較Basella rubra之主要色素gomphrenin I及甜菜根（Beta vulgaris）中的betanin及其主要色素之UV-Visible光譜，以推斷Basella alba漿果之主要色素成分（betanidin-hexose）之完整結構。結果顯示Basella alba與Basella rubra之主要波峰滯留時間一致，且其UV-Visible光譜一樣，故主要色素成分可鑑定為gomphrenin I。測定所分離純化之主要色素gomphrenin I之抗氧化能力時，濃度為181.82 μM時，其TEAC相當於534 μM Trolox；濃度為22.73 μM 時，其DPPH清除能力為84%，相當於103 μM BHT；濃度為45.45 μM時，其還原力相當於129 μM Vit C；濃度為181.82 μM時，其亞麻油酸抗氧化能力為20%，相當於68.39 μM BHT。進一步以漿果之水及甲醇粗萃物以及所純化之gomphrenin I為材料，進行小鼠巨噬細胞（RAW264.7）經0.5 μg/mL lipopolysaccharide處理之抗發炎活性測定時。當兩種粗萃物濃度為200 μg/mL或gomphrenin I為100 μM時，均具有降低細胞一氧化氮含量（nitrite）之生成量，抑制率分別為47.8、38.2及100%；對細胞prostaglandin（E2）之抑制率分別為8.1、3.2及8.2%；對細胞激素IL-1β及TNF-α之抑制率分別為18.1、0、17.8% 及9.3、6.2、5.1%。整體而言，落葵漿果富含水溶性色素-甜菜�ㄕ滽嚏A並具良好的抗氧化能力。經分離純化得到之gomphrenin I，具有相當強之抗氧化及抑制NO之生成能力，顯示特定成熟度之落葵漿果具有開發高附加價值產品之潛力。

目 錄
頁次
中文摘要 I
英文摘要 II
目錄 V
表目錄 XII
圖目錄 XIII
附錄 XVI
壹、前言 1
貳、文獻回顧 2
一、落葵簡介 2
二、食用色素 3
三、Betalains簡介 4
（一）一般性質 4
（二）Betalains基本結構與特性 5
（三）Betalains生合成 5
（四）Betalains來源 7
（五）Betalains之生物活性 7
1. 抗氧化作用 7
2. 抗腫瘤 10
四、抗氧化與自由基 10
（一）自由基簡介 10
（二）自由基種類 10
（三）體內的自由基來源 11
（四）自由基對生物體的傷害 12
（五）人體抗氧化防禦系統 12
（六）抗氧化劑作用原理 13
五、免疫系統 14
六、發炎反應 15
（一）巨噬細胞角色與功能 16
（二）脂多醣（lipopolysaccharide, LPS） 16
（三）一氧化氮及RNS與發炎反應之相關性 17
（四）前列腺素（prostaglandin, PG） 18
（五）TNF-α與IL-1β之細胞激素 19
參、實驗材料與方法 21
一、實驗架構 21
二、實驗材料 23
三、化學試劑 23
四、實驗用細胞株 26
五、儀器及相關設備 26
六、實驗方法 28
（一）樣品製備 28
1. 落葵漿果甲醇粗萃物及落葵漿果甲醇粗萃物之加熱處理 28
2. 落葵漿果SPE萃取物及落葵漿果SPE萃取物之加熱處理 28
3. 落葵漿果水萃物製備 29
4. 新鮮落葵漿果直接壓搾汁液製備 29
（二）落葵漿果萃取物固形物回收率 29
（三）落葵漿果色素安定性 29
1. 在不同pH值下的紫外線-可見光吸收光譜分析 29
2. 落葵漿果甲醇粗萃物之熱安定性 30
3. 比較甲醇粗萃物、甲醇萃取物之加熱處理、SPE萃取物及SPE萃取物之加熱處理 30
（四）抗氧化分析 31
1. 總酚類化合物含量測定 31
2. TEAC（trolox equivalent antioxidant capacity） 31
3. DPPH（α,α-diphenyl-β-picrylhydrazyl）自由基清除能力測定 32
4. 還原力測定 32
5. 亞麻油酸抗氧化能力測定 33
（五）落葵漿果色素成分之分離純化 34
1. Solid phase extraction（SPE）前處理 34
2. 半製備型（semi-preparative）HPLC分離回收 34
3. 分析型（analytical）HPLC-DAD分析 35
4. 質譜分析（MS analysis） 35
5. Basella alba、Basella rubra、甜菜根（Beta vulgaris）主要色素分析 36
（六）小鼠巨噬細胞（RAW 264.7）之前處理與培養 36
（七）落葵漿果水萃物、落葵漿果甲醇粗萃物及所純化之gomphrenin I對小鼠巨噬細胞細胞（RAW 264.7）存活率分析 38
（八）落葵漿果水萃物、落葵漿果甲醇粗萃物及所純化之gomphrenin I抑制經由LPS（lipopolysaccharide）誘導小鼠巨噬細胞細胞（RAW 264.7）一氧化氮（NO）之生成 38
（九）落葵漿果水萃物、落葵漿果甲醇粗萃物及所純化之gomphrenin I抑制經由LPS誘導小鼠巨噬細胞細胞（RAW 264.7）PGE2（prostaglandin E2）之生成 39
（十） 落葵漿果水萃物、落葵漿果甲醇粗萃物及所純化之gomphrenin I抑制經由LPS誘導小鼠巨噬細胞細胞（RAW 264.7）之TNF-α（tumor necrosis factor-alpha）及IL-1β（interleukin-1β）之生成 40
（十一）落葵漿果分離純化色素gomphrenin I熱安定性 41
（十二）統計分析 42
肆、結果與討論 43
一、落葵漿果以水、80%甲醇（含0.2%甲酸）萃取及漿果直接壓榨之新鮮汁液粗萃物回收率及其TEAC（trolox equivalent antioxidant capacity）抗氧化活性 43
二、落葵漿果色素安定性 44
（一）落葵漿果甲醇粗萃物在不同pH值之紫外線-可見光吸收光譜 44
（二）落葵漿果甲醇粗萃物之熱安定性 44
（三）落葵漿果甲醇粗萃物、落葵漿果甲醇粗萃物之加熱處理、落葵漿果SPE萃取物及落葵漿果SPE萃取物之加熱處理之紫外線-可見光吸收光譜圖 45
三、比較落葵漿果甲醇粗萃物、落葵漿果甲醇粗萃物之加熱處理、落葵漿果SPE萃取物及落葵漿果SPE萃取物之加熱處理之抗氧化活性 46
（一）總酚類化合物含量比較 46
（二）TEAC抗氧化活性比較 46
（三）DPPH（α,α-diphenyl-β-picrylhydrazyl）自由基清除活性比較 47
（四）還原力之比較 48
（五）亞麻油酸抗氧化活性比較 48
四、落葵漿果主要色素成分之分離純化 49
五、由落葵漿果分離純化之gomphrenin I抗氧化活性 53
（一）TEAC抗氧化活性測定 53
（二）DPPH自由基清除能力測定 53
（三）還原力測定 54
（四）亞麻油酸抗氧化能力測定 54
六、落葵漿果水萃物、落葵漿果甲醇粗萃物及gomphrenin I對小鼠巨噬細胞（RAW 264.7 ）存活率（cell viability）及誘發小鼠巨噬細胞一氧化氮（nitric oxide, NO）生成之影響 54
七、落葵漿果水萃物、落葵漿果甲醇粗萃物及gomphrenin I對LPS誘發小鼠巨噬細胞PGE2生成之影響 56
八、落葵漿果水萃物、落葵漿果甲醇粗萃物及gomphrenin I對LPS誘發小鼠巨噬細胞TNF-α生成之影響 56
九、落葵漿果水萃物、落葵漿果甲醇粗萃物及gomphrenin I對LPS誘發小鼠巨噬細胞IL-1β生成之影響 57
十、由落葵漿果分離純化之gomphrenin I熱安定性 58
伍、結論 59
陸、參考文獻 92
柒、個人資料 108

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