在保健微藻食品行銷上，產品之保健效果的學術研究，可提升其商業價值。Nannochloropsis oculata 大多用於幼魚餌料，而它在保健應用較缺乏研究，因此本論文從 N. oculata中分離抗發炎的成份。結果顯示在震盪 110 rpm、溫度 25 oC 、光照 15000 lux、鹽度 2.8% 可得最佳藻量，額外添加營養及 CaCO3 也可略為提高產量。在分析 N. oculata 抗發炎成分時，是以萃取物對 RAW 264.7 之 iNOS、COX-2蛋白質表現之抑制的程度來判定其抗發炎活性。在 TLC 測試時，發現高極性分子可同時抑制 iNOS 及 COX-2 蛋白質表現，抑制率分別為 83% 及 81%。從丙酮層分離出 AWP2-11-1 及 AWP2-11-3 純物質。以 RP-C18 分離得到 A3-19-21 化合物對於 iNOS 及 COX-2 抑制率各別為 99% 和 95%。AWP3-11-13及AWP6-11-12 對於 iNOS 的抑制率為 99% (蛋白質表現為 1%)。正己烷分層中 HP2-69-70 化合物對於 iNOS 的抑制率為 64%。分離純化所得純物質是以 1H-NMR、13C-NMR、2 D NMR及質譜儀來鑑定結構。

New bioactive compounds in microalgae increase their commercial values for healthy food. Nannochloropsis oculata is mostly used for juvenile feed, while application for health is unavailable so this research isolated the compositions of the anti-inflammatory ingredients from N. oculata. The results showed growth of N. oculata yielded well in 110 rpm, 25 oC, 15000 lux, and 2.8% salinity; additional nutrients and CaCO3 slightly increased production. The analysis of N. oculata anti-inflammatory ingredients was based on the inhibition ratios of iNOS and COX-2 protein expression in RAW 264.7 under LPS induction. TLC separation showed that highly polar molecules inhibited iNOS and COX-2 expressions. AWP2-11-1 and AWP2-11-3 compounds were from acetone partition. In RP-C18 separation of the extracts, A3-19-21 compound inhibited iNOS and COX-2 expressions by 99% and 95%, respectively. HP2-69-70 compound from n-hexane partition inhibited iNOS expression by 64%. The resulting compounds were identified for their structures via 1H-NMR, 13C-NMR, 2 D NMR, and mass spectrometries.

目錄
壹、前言
發炎現象 1
巨噬細胞 3
一氧化氮合成酶 3
環氧化酶 6
微藻抗發炎應用 8
研究目的 9
貳、材料與方法
培養方法 10
微藻之生長曲線 11
細胞解凍步驟 11
細胞繼代培養 12
細胞培養 12
SDS-PAGE 13
西方點墨法 14
分離步驟 16
NMR 與質譜數據收集 17
参、結果
物理環境對N. oculata 生長影響 18
營養環境對N. oculata生長影響 20
EtOAc粗萃物分離之抗發炎活性 21
Hexane Parition (HP) 分離層之抗發炎活性分析 21
AWP 分離層之抗發炎活性分析 22
RP-C18 分離之抗發炎活性分析 23
Chlorella、Spirulina、N. oculat萃取物抗發炎活性分析 24
N. oculata 藻粉 (DM) 萃取結果 24
AWP8 (1) 結構解析 25
AWP3-20-4 (2)結構判定 30
AWP2-11 -1 (3) 解析 34
AWP2-11-3 (4)結構解析 37
肆、討論
環境生長條件 43
DMSO及萃取物濃度條件選擇 45
不同微藻粗萃物對抗發炎活性比較 46
N. oculata 萃取物抗炎效能 46
Mannitol對iNOS 抑制表現 48
伍、參考文獻 50
陸、圖表 64

表目錄
Table 1. Extract yield of N. oculata 65
Table.2 AWP-8 66
Table.3 AWP320-4 67
Table.4 AWP211-1 68
Table. 5 AWP-211-3 1H-NMR 69
Table. 6 AWP-211-3 13C-NMR 70

圖目錄
Fig. 1. Growth of N. oculata culture in the different environments 71
Fig. 2. Effects of nutrition and CaCO3 on the growth of N. oculata 72
Fig. 3. Effects of ethyl acetate (EtOAc) extracts from N. oculata on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 73
Fig. 4. Effects of ethyl acetate (EtOAc) extracts from N. oculata on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 75
Fig. 5. Effects of n-hexane partitions (HP) on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 77
Fig. 6. Effects of acetone-water partitions (AWP) on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 79
Fig. 7. Effects of acetone-water partitions (AWP) on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 81
Fig. 8. Effects of the fractions from the second acetone-water partitions on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 83
Fig. 9. Effects of the fractions from the second acetone-water partitions on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 85
Fig. 10. Effects of RP-C18 separation fractions from AWP partitions on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 87
Fig. 11. Effects of RP-C18 separation fractions from DM extracts and HP2 on the expressions of iNOS and COX-2 in murine macrophage cell line RAW 264.7 89
Fig. 12 Effects of microalgae extract on the expression on iNOS and COX-2 murine macrophage cell line RAW 264.7 91
Fig. 13 From low to high polar extraction microalgae pure (DM) extractions 93
Fig. 14 From high to low polar extraction microalgae pure (DM) extractions 94
Fig. 15. The 1H-NMR spectrum of AWP8 (400 MHz, in MeOD) 95
Fig. 16. The 13C-NMR spectrum of AWP8 (400 MHz, in MeOD) 96
Fig. 17. The qCOSY of AWP8 (400 MHz, in MeOD) 97
Fig. 18. The qHSQC of AWP8 (400 MHz, in MeOD) 98
Fig. 19.The qHMBC of AWP8 (400 MHz, in MeOD) 99
Fig. 20. The ESI-MS spectrum of AWP8 100
Fig. 21. The DEPT spectrum of AWP8 101
Fig. 22. The IR spectrum of AWP8 102
Fig. 23. The UV spectrum of AWP8 103
Fig. 24. The 1H-NMR spectrum of AWP3-20-4 (400 MHz, in D2O) 104
Fig. 25. The qCOSY spectrum of AWP3-20-4 (400 MHz, in D2O) 105
Fig. 26. The 1H-NMR spectrum of AWP2-11-1 (500 MHz, in D2O) 106
Fig. 27. The 13C-NMR spectrum of AWP2-11-1 (500 MHz, in D2O) 107
Fig. 28. The qCOSY spectrum of AWP2-11-1 (500 MHz, in D2O) 108
Fig. 29. The qHSQC spectrum of AWP2-11-1 (500 MHz, in D2O) 109
Fig. 30. The qHMBC spectrum of AWP2-11-1 (500 MHz, in D2O) 110
Fig. 31. The DEPT spectrum of AWP2-11-1 (500 MHz, in D2O) 111
Fig. 32. The ESI-MS spectrum of AWP2-11-1 112
Fig. 33. The 1H-NMR spectrum of AWP2-11-3 (500 MHz, in D2O) 113
Fig. 34. The 13C-NMR spectrum of AWP2-11-3 (500 MHz, in D2O) 114
Fig. 35. The qCOSY spectrum of AWP2-11-3 (500 MHz, in D2O) 115
Fig. 36. The qHSQC spectrum of AWP2-11-3 (500 MHz, in D2O) 116
Fig. 37. The qHMBC spectrum of AWP2-11-3 (500 MHz, in D2O) 117
Fig. 38. The DEPT spectrum of AWP2-11-3 (500 MHz, in D2O) 118
Fig. 39. The ESI-MS spectrum of AWP2-11-3 119

附錄
Fig.. S1 Pathway of COX 120
Fig. S2 N. oculata separation plan flowchart 121
Fig. S3 Separation flow chart of AWP2-11-1, AWP2-11-3, AWP3-20-4 and AWP8 122
Fig. S4 Separation flow chart of AWP1 ~ AWP14 123
Fig. S5 Separation flow chart of HP2 124
Fig. S6 Separation flow chart of microalgae powder (DM) extraction 125
Fig. S7 Operation Flowchart of 300 MHz NMR Fig. S8. 126
Fig. S8 Effects of DMSO on the expression of iNOS and COX-2 in murine macrophage cell line RAW 264.7 127
Fig. S9 iNOS and COX-2 pathway 129

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