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| 研究生： |
蔡依蓉 Cai, Yi-Rong |
|---|---|
| 論文名稱： |
不同加工條件製成之蘿蔔乾生物活性成分分析與其對抗腫瘤及抗發炎細胞之影響 Analysis of biologically active ingredient from different processing conditions of dried radish on anti-tumor and anti-inflammatory in vitro |
| 指導教授： |
邱秋霞
Chiu, Chiu-Hsia 黃卓治 Huang, Tzou-Chi 林麗雲 Lin, Li-Yun |
| 學位類別： |
碩士 Master |
| 系所名稱： |
農學院 - 食品科學系所 Department of Food Science |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 138 |
| 中文關鍵詞： | 蘿蔔 、抗氧化能力 、揮發性化合物 、抗發炎 、抗腫瘤 |
| 外文關鍵詞： | Radish, Antioxidant Capacity, Volatile, Anti-inflammatory, Anti-tumor |
| DOI URL： | http://doi.org/10.6346/THE.NPUST.FS.003.2019.E11 |
| 相關次數： | 點閱：82 下載：9 |
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蘿蔔 (Raphanus sativus L.)為十字花科蘿蔔屬，屬於深根性農作物。在台灣傳統文化中，取新鮮蘿蔔以食鹽醃漬後，再日曬製成具有獨特色澤及風味之蘿蔔乾，主要目的為長時間儲藏及有效防止腐敗。本研究擬探討其機能性成分。(1)將新鮮白蘿蔔表面洗淨皮肉分離，兩個部位分別進行生物活性成分分析與細胞體外試驗；(2)將新鮮白蘿蔔表面洗淨切片直接進行熱風乾燥，分別為50℃、70℃及90℃；(3)將新鮮白蘿蔔表面洗淨，整顆蘿蔔以食鹽醃漬，使其脫水，醃漬約二至三天使白蘿蔔水分充分脫水，而後切片進行熱風乾燥，分別以50℃、70℃及90℃；(4)新鮮白蘿蔔表面洗淨，整顆蘿蔔以食鹽醃漬，使其脫水，醃漬約二至三天使白蘿蔔水分充分脫水，進行第一次日曬，而後切片在第二次鹽醃脫水，再進行二次日曬乾燥。熱風乾燥最終判斷決定於水活性<0.6，熱風乾燥部分大約為期2-3天，日曬需約一個月。另外，以沙鹿公有市場購入之3-4年市售老菜脯作為比較。以純水、乙醇、甲醇及乙酸乙酯萃取蘿蔔乾，探討其生物活性成分之變化及巨噬細胞、黑色素瘤細胞等體外試驗。
本實驗結果顯示，三種不同加工方式蘿蔔乾之水活性介於0.4-0.6，使其易於保存。萃取率最高以甲醇>純水>乙醇>乙酸乙酯，其中四種溶劑萃取率皆以SH50為最高，分別甲醇為41.92%、純水為33.55%、乙醇為24.61%、乙酸乙酯為8.29%。總多酚類及總黃酮類含量皆以甲醇萃取最佳，其中總多酚含量以甲醇萃取H70最高為43.71 μg GAE/g；總類黃酮含量以甲醇萃取H90最高為1.60 μg QE/g。另外，抗氧化能力方面，四種溶劑萃取物皆具有良好之抗DPPH及ABTS清除自由基之能力，不論以純水、甲醇或乙醇萃取蘿蔔乾之清除率皆高於新鮮蘿蔔之清除率，其中DPPH之EC50以甲醇萃取H90為20.45 μg TE/g最高；ABTS之EC50以水溶液萃取H70為44.80 μg TE/g最高。蘿蔔硫素部份，僅在新鮮蘿蔔肉中檢測出 8.28 µg/g，因蘿蔔硫素為不穩定中間產物，會因儲藏時間愈久含量逐漸減少。呈味部分，最具貢獻力者為5’-GMP (鳥苷酸)及5’-IMP (肌酐酸)，其中以H70在呈味上最為強烈，兩者核苷酸含量分別為24.42 μg/g及25.07 μg/g，表示熱風乾燥有助於增加呈味強度。脂肪及脂肪酸部份，老菜脯具有較高脂肪含量為6.14%，脂肪酸部份，以老菜脯有較高總脂肪酸含量1.90 mg/g，其不飽和脂肪酸含量佔總脂肪酸含量約65%，而H70之總脂肪酸含量為1.16 mg/g，較老菜脯少0.74 mg/g，其不飽和脂肪酸含量佔總脂肪酸含量約70%，表示H70脂肪酸組成與老菜脯相似。植物固醇部份，以H70總植物固醇含量最高為843.80 μg/100g，其中以γ-sitosterol含量最多，而植物固醇在許多文獻被證實具有預防肥胖及降低慢性疾病發生率之風險，表示H70在此方面具有高潛力。揮發性化合物部份，十字花科蔬菜香氣來源主要為異硫氰酸酯類及硫化物，本實驗總共鑑定出10種硫化物、2種異硫氰酸酯類、3種吲哚類及5種脂肪酸；新鮮白蘿蔔鑑定出4-(methylthio)-3-butenyl isothiocyanate (MTBITC)含量為8.05 μg/g，文獻指出其為白蘿蔔中最具影響力之化合物，亦為異硫氰酸酯類主要指標之一，然而經乾燥後皆發現4-isothicyanato-1-(methylthio)-1-butene，其亦為異硫氰酸酯類化合物，且在H70含量最高為22.66 μg/g；另外，值得探討的是鑑定出5種脂肪酸，此結果與上述脂肪酸結果相符，皆是以老菜脯含量較高，其中又以多元不飽和脂肪酸中的次亞麻油酸含量最高為183.61 μg/g。
體外模式結果顯示，以巨噬細胞作為抗發炎試驗，SH70水萃取物及H70乙醇萃取物須在0.01 μg/mL時才不具細胞毒性，而在抑制TNF-α及IL-6促發炎因子產生方面，H70乙醇萃取物0.01 μg/mL時，就能有效降低TNF-α約183.31 ng/mL，而老菜脯乙醇萃取物在0.1 μg/mL時，亦能降低TNF-α約214.89 ng/mL。B16-F10細胞作為抗腫瘤試驗，並以流式細胞儀作為細胞週期分析，在H70水萃取物濃度為1 mg/mL時，細胞週期滯留於G2/M期為11.59%最高。由於蘿蔔硫素、異硫氰酸酯類及植物固醇皆被證實能夠使細胞週期停滯於G2/M期，以擾亂細胞活性及降低癌細胞形成之潛力，進而誘導細胞壞死，並非細胞凋亡，但細胞凋亡之誘導常伴隨著G2/M期之時間依賴性。
綜合上述，H70具有發展為媲美老菜脯之生理活性成分及風味，亦能縮短老菜脯醃漬過程，因其具有高抗氧化能力，亦富含異硫氰酸酯類、植物固醇及多元不飽和脂肪酸，更能有效抑制TNF-α及IL-6促發炎因子產生，甚至使腫瘤細胞滯留於細胞中的G2/M期，證實70℃熱風乾燥具有工業發展之潛力。
Radish (Raphanus sativus L.) is the Brassicaceae genus and belongs to deep-rooted crops. In traditional, fresh radishes are salted and then sun dried to produce dried radish with unique color and flavor. The main purpose is to store for a long time and prevent corruption. However, rarely literatures discuss the functional ingredients of radish. In this study, fresh radishes were prepared by hot air drying (H50、H 70、H 90) and were dried in the sun and heated-air drying (SH50、SH 70、SH 90) after preserved in 6% salt concentration. Compare with the commercial old dried-radish. The radishes are extracted with water, ethanol, methanol and ethyl acetate. Explore biologically active ingredients such as anti-oxidant, anti-tumor and other activities. The preliminary experimental results show that the water activity of three different processing methods of radish is between 0.4 and 0.6, making it easy to preserve. The extraction rate was methanol > water > ethanol > ethyl acetate. Especially the SH50 is the highest of 41.92%, 33.55%, 24.61% and 8.29%, respectively. the total polyphenols and total flavonoids were extracted with methanol superior to water, ethanol, and ethyl acetate. The content of total polyphenols extracted with methanol was the highest at 43.71 μg GAE/g, which was the H70. The total flavonoid extracted with methanol was the highest at 1.60 μg QE/g, which the H90. In addition, the antioxidant capacity, the scavenging rate of dried radish extracted with water, methanol, and ethanol was higher than the fresh radish. The DPPH of EC50 values extracted with methanol was the highest at 20.45 μg TE/g, which was the H90. The ABTS of EC50 values extracted with ethanol was the highest at 83.13 μg TE/g, which was the H90. The sulforaphane was only found in fresh radish pulp content 8.28 μg/g. Because sulforaphane is an unstable intermediate. The taste, which the most contributors are 5’-GMP and 5’-IMP. The H50, H 70 and H 90°C increased the most significant nucleotide content. The 5’-GMP of fresh radish was 0.22 μg/g increase to 13.42 μg/g, 24.42 μg/g and 17.78 μg/g, respectively. On the other hand, 5’-IMP of fresh radish was increased from 0.39 μg/g to 14.05 μg/g, 25.07 μg/g and 16.80 μg/g, respectively. Among them, the H70 was the most intense in the taste, indicating that the hot air drying process was helpful to increase its taste intensity. Fat and fatty acid was the highest at 6.14%, which is the commercial old dried-radish. In addition, total fatty acid was the highest at1.90 mg/g which is the commercial old dried-radish. The commercial old dried-radish of UFA content accounts for about 65% of TFA content. However, TFA of H70 was 1.16 mg/g, which less than 0.74 mg/g of the commercial old dried-radish. The H70 of UFA content accounts for about 70% of TFA content. Indicates that the H70 fatty acid composition is similar to that of the commercial old dried-radish. The phytosterol were identified the Campesterol and γ-sitosterol. Both had the highest contents at H70, which total phytosterol were 843.80 μg/100g. The phytosterol have been shown in many literatures to prevent obesity and reduce the risk of chronic disease. Indicates that H70 has high potential in this respect. Brassicaceae vegetable aroma sources are mainly isothiocyanates and sulfides. On this experiment a total of 10 kinds of sulfides, 2 kinds of isothiocyanates, 3 kinds of indole and 5 kinds of fatty acids were identified. In addition, found in the preliminary results of volatile compounds, the fresh radish contains 4-(methylthio)-3-butenyl isothiocyanate, which content in fresh radish were 8.04 μg/g. But after processing it is only found the 4-isothicyanato-1-(methylthio)-1-butene in the same retention time, which content the highest in H70 were 22.66 μg/g. In addition, the five fatty acids were identified, which content in commercial old dried-radish were the hightest. And the linolenic acid of PUFA were the hightest, which content in commercial old dried-radish were 183.61μg/g. The in vitro results show that SH70 water extract and H70 ethanol extract must not be cytotoxic at 0.01 μg/mL. When H70 ethanol extract is 0.01 μg/mL, it can effectively inhibit the production of TNF-α. Reduce the TNF-α about 183.31 ng/mL. And the ethanol extract of the commercial old dried-radish at 0.1 μg/mL, it also reduced TNF-α about 214.89 ng/mL. Using flow cytometry as a cell cycle analysis. When the H70 water extract concentration is 1 mg/mL, the cell cycle was retained in the G2/M phase with a maximum of 11.59%. Due to the sulforaphane, isothiocyanates and phytosterols have been shown to arrest the cell cycle in the G2/M phase. Order to disrupt cell activity and reduce the potential for cancer cell formation. Inducing cell necrosis, but not apoptosis. However, induction of apoptos is often accompanied by the time dependence of the G2/M phase.
中文摘要...................................................................I
Abstract..................................................................IV
謝誌.....................................................................VII
目錄....................................................................VIII
表目錄...................................................................XII
圖目錄..................................................................XIII
1. 前言....................................................................1
2. 文獻回顧................................................................2
2.1 蘿蔔簡述..............................................................2
2.1.1 蘿蔔品種介紹.......................................................2
2.1.1.1 東方蘿蔔......................................................2
2.1.1.2 櫻桃蘿蔔 (四季蘿蔔)............................................3
2.1.1.3 葉用蘿蔔......................................................3
2.1.1.4 鼠尾蘿蔔......................................................3
2.1.2 蘿蔔功效及用途.....................................................4
2.1.3 台灣蘿蔔種植面積及生產量............................................4
2.2 蘿蔔乾介紹.............................................................7
2.2.1 蘿蔔乾加工.........................................................7
2.3 蘿蔔活性成分...........................................................8
2.3.1 硫代葡萄糖苷 (Glucosinolates)水解物................................8
2.3.2 蘿蔔硫素 (Sulforaphane)..........................................10
2.3.3 維生素C (Vitamin C)..............................................12
2.3.4 植物固醇 (Phytosterol)...........................................14
2.3.5 多酚化合物 (Polyphenol)...........................................16
2.3.6 黃酮類化合物 (Flavonoids).........................................18
2.4 自由基與抗氧化活性....................................................21
2.4.1 自由基...........................................................21
2.4.2 抗氧化活性........................................................22
2.5 呈味化學介紹..........................................................23
2.5.1 核苷酸 (Nucleotide)..............................................25
2.5.2 游離胺基酸 (Amino acid)...........................................25
2.5.3 有機酸 (Organic acid)............................................29
2.5.4 可溶性糖.........................................................29
2.6 抗發炎試驗............................................................30
2.6.1 抗發炎反應機制....................................................30
2.6.2 巨噬細胞之介紹....................................................30
2.6.3 脂多醣之介紹......................................................31
2.6.4 脂多醣活化巨噬細胞之機制...........................................31
2.7 細胞週期 (Cell cycle)................................................33
3. 材料與方法..............................................................35
3.1 實驗架構.............................................................35
3.2 原料.................................................................36
3.2.1 原料製備流程......................................................37
3.3 實驗材料.............................................................41
3.3.1 試藥.............................................................41
3.3.2 儀器設備.........................................................43
3.4 樣品製備及前處理......................................................44
3.5 分析方法及測定........................................................44
3.5.1 一般成分分析之測定................................................44
3.5.1.1 水分含量之測定................................................44
3.5.1.2 脂肪含量之測定................................................45
3.5.2 游離脂肪酸含量之測定..............................................45
3.5.3 水活性之測定......................................................46
3.5.4 維生素C含量測定...................................................46
3.5.5 植物固醇含量測定..................................................47
3.5.6 抗氧化能力之分析..................................................48
3.5.6.1 DPPH自由基清除能力試驗........................................48
3.5.6.2 ABTS自由基清除能力試驗........................................48
3.5.6.3 總多酚類化合物含量測定.........................................48
3.5.6.4 總類黃酮含量測定..............................................49
3.5.7 呈味成分分析......................................................50
3.5.7.1 核苷酸含量測定................................................50
3.5.7.2 游離胺基酸含量測定............................................50
3.5.7.3 可溶性糖之測定................................................52
3.5.7.4 有機酸含量之測定..............................................52
3.5.8 蘿蔔硫素分析......................................................54
3.5.9 揮發性成分之分析..................................................54
3.5.9.1揮發性成分之鑑定...............................................55
3.6 細胞模式─小鼠巨噬細胞株RAW 264.7.......................................56
3.6.1 溶液配製.........................................................56
3.6.2 RAW 264.7 之活化.................................................56
3.6.3 RAW 264.7 之細胞保存..............................................56
3.6.4 細胞及藥物培養條件................................................57
3.6.5 細胞存活率測定 (MTT assay)........................................57
3.6.6 酵素連結免疫分析法─TNF-α..........................................57
3.6.7 酵素連結免疫分析法─IL-6...........................................58
3.7 細胞模式─小鼠黑色素瘤細胞株B16-F10.....................................58
3.7.1 溶液配製.........................................................58
3.7.2 B16-F10 之活化...................................................59
3.7.3 B16-F10 之細胞保存................................................59
3.7.4 細胞及藥物培養條件................................................59
3.7.5 細胞存活率測定 (MTT assay)........................................60
3.7.6 細胞週期分析 (Cell cycle analysis)................................60
3.7.7 細胞週期定量分析..................................................60
3.8 統計分析.............................................................61
4. 結果與討論..............................................................62
4.1 加工成品之水分及水活性.................................................62
4.2 加工成品之脂肪及脂肪酸含量.............................................64
4.3 維生素C含量測定結果...................................................67
4.4 新鮮白蘿蔔及加工成品之植物固醇含量分析..................................69
4.5 新鮮白蘿蔔及加工成品之機能性成分含量分析................................73
4.5.1 多酚類化合物含量測定之結果.........................................73
4.5.2 總黃酮類含量測定之結果.............................................76
4.5.3 新鮮蘿蔔及蘿蔔乾之蘿蔔硫素測定結果..................................79
4.6 新鮮白蘿蔔及加工成品之抗氧化能力分析....................................81
4.6.1 清除DPPH自由基之能力結果..........................................81
4.6.2 總抗氧化能力測定結果..............................................86
4.7 新鮮蘿蔔及加工成品呈味含量測定之結果....................................89
4.7.1 新鮮蘿蔔及蘿蔔乾之核苷酸含量測定結果................................89
4.7.2 新鮮蘿蔔及蘿蔔乾之游離胺基酸含量測定結果............................91
4.7.3 新鮮蘿蔔及蘿蔔乾之可溶性糖含量測定結果..............................93
4.7.4 新鮮蘿蔔及蘿蔔乾之有機酸含量測定結果................................93
4.8 新鮮蘿蔔及加工成品揮發性化合物含量測定之結果.............................96
4.9 以體外模式評估抑制發炎效果之結果─RAW264.7..............................101
4.9.1 新鮮蘿蔔及蘿蔔乾萃取物對巨噬細胞生長之影響.........................101
4.9.2 新鮮蘿蔔及蘿蔔乾萃取物抑制發炎效果評估.............................104
4.10 以體外模式評估抑制腫瘤效果之結果─B16-F10..............................113
4.10.1 新鮮蘿蔔及蘿蔔乾萃取物對小鼠黑色素瘤細胞生長之影響.................113
4.10.2 新鮮蘿蔔及蘿蔔乾萃取物抑制腫瘤效果評估............................116
5. 結論..................................................................123
6. 參考文獻..............................................................126
8. 作者介紹..............................................................138
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