臺灣博碩士論文加值系統

本研究自台灣原生特有種的二葉松與五葉松之松針與松樹皮為料中，萃取純化具有生物活性的酚酸類物質，以提供台灣林業資源次生代謝產物的合理化開發評估。
第一部分實驗，以台灣五葉松針(PM)為原料，使用L18直交表實驗設計法進行，利用酵素水解與熱壓流體(HPFE)萃取酚酸類生物活性成分。實驗結果顯示，纖維酵素、果膠酵素、半纖維素及蛋白水解酵素在連續或批式製程中皆有助於植物細胞結構組織的鬆解。酵素浸漬處理後，以連續熱壓流體萃取製程的最適當條件為使用混合酵素1.5％wt於70%乙醇水溶液（pH 6）中，在689 kPa壓力與343 K 溫度下，以10 mL/min流量萃取進行180分鐘，每克萃出物中酚酸含量可達39.80mg，而批式製程可得31.34 mg，萃提率可提升27%。五葉松(PM)與二葉松(PT)針萃出物在乙酸乙酯可溶部份，皆具有最強的抗氧化能力。PT萃出物在清除DPPH自由基、抑制過氧化自由基、清除一氧化氮自由基、螯合二價鐵離子與還原力能力等五項皆優於PM；在抑制脂質過氧化能力上，則是PM優於PT。使用化學發光法就萃出物的水溶性與油溶性部分進行抗氧化能力檢測，顯示每克PM萃出物的抗氧化能力分別相當於0.78mmol VC、1.63 mmol VE 優於PT萃出物 0.29mmol VC 、1.48 mmol VE。兩種萃出物在抑制人類癌細胞株增殖能力方面，對AGS胃腺癌與A549肺癌兩種細胞隨劑量的增加具有增生趨勢，其中AGS細胞特別顯著；對A431皮膚表皮癌細胞、DU145前列腺癌及DBTRG腦膠質母細胞瘤等三種，抑制細胞增殖能力皆隨劑量呈正相關性，在不影響對照組胚胎細胞下，分別對於人類腦膠質母細胞瘤DBTRG細胞株有10% 與 16% 的抑制增殖作用。
第二部分以台灣五葉松樹皮生物活性成分之分離與抗氧化能力評估為主；不同樹齡樹皮之甲醇萃取物，以乙酸乙酯可溶部份活性最高。化學抗氧化評估，顯示清除DPPH自由基以低樹齡樹皮（1∼3年）為佳，其餘在抑制過氧化自由基釋出、抑制脂質過氧化自由基、清除一氧化氮自由基、螯合二價鐵離子與還原力等五種抗氧化效能皆隨樹齡（15∼20年）成正相關性。高樹齡甲醇萃取物經正相層析分離純化後，以榭皮素（Quercetin）為，分別以化學法與化學發光法進行篩選，發現EF-18(7.13mmol VC/g、5.07 mmol VE/g)，EF-14 (0.61 mmol VC/g、9.57 mmol VE/g) 等兩份，總抗氧化能力高於榭皮素（Quercetin）(5.57 mmol VC/g、3.88 mmol VE/g)，分別達1.29、1.08倍。酚酸化合物影響總抗氧化能力之強弱，依序為結構型態、羥基取代位置有關；構型以羥肉桂酸型＞黃烷醇型＞羥苯甲酸型＞花青素型、黃酮醇型＞二羧酸型；黃烷醇型取代位置以B環上具有鄰位二羥基者為佳。
五葉松針與樹皮抽提物中的酚酸類化合物，因種類與結構形式的不同，是造成生物活性差異的主要原因；在清除DPPH自由基、過氧化陰離子自由基、一氧化氮自由基與抑制脂質過氧化作用能力上，以樹皮部優於葉部，可取代市面合成抗氧化劑Butyled hydroxyanisole。台灣二葉松針在清除NO自由基能力明顯勝於五葉松針；整體而言，台灣五葉松資源的開發潛力優於台灣二葉松。

Pine species are rich in polyphenols and are important for regenerating biomass that have increasingly attracted attention in the fields of nutrition, health and medicine. In this study, two parts of experiments have been carried out. we are interested in using pine needles and pine barks of wood residue from the agricultural and forestry industries in Taiwan.
Firstly, Enzymatic hydrolysis on the hot pressurized fluid extraction (HPFE) of polyphenols from the needles of Pinus taiwanensis (PT) and Pinus morrisonicola (PM) were carried out. A mixture of cellulose, hemicellulase, pectinase and protease is able to enhance plant cell breakage. Hence, these enzymatic catalyzed HPLE process dramatically reduce the extraction pressures and temperature. The results showed that the optimum conditions of two step extraction process were as follows: 1.5 % wt of mixed enzyme in 70% ethanol (pH 6) at a flow rate of 10 mL/min, under pressure 689 kPa and 343 K for 180 min. The maximum contents of polyphenols obtained was 39.8 mg per gram of extract, and the continuous hot pressurized ethanol aqueous solution was superior to batch 31.34 mg. The extracts from PT needles possessed higher antioxidant activities against DPPH radical, superoxide anion radical (O2−) and nitrite oxide radical (NO), and higher reducing ability of chelating ferrous cations (Fe2+) than those from PM needles. Particularly, the ability of scavenging NO by the PT needle extracts is more than 1.8 times better than that of scavenging by the PM needle extracts. However, the PM needle extracts inhibited peroxide radical (O22−) much better than PT needle extracts did. In addition, the total anti-oxidative capacity determined by photochemiluminescence method, the PM needles extracts demonstrated higher than that from the PT needles extracts did. In the bioassay tests, the extracts of PM and PT needles exhibited 10% and 16% of growth inhibition effect on DBTRG-glioblastoma cell of the human brain, respectively.
Secondly, Expermental results showed that the ethyl acetate partition of the shoot, offshoot and trunk of PM bark of the methanol extracts was superior to the n-hexane, n-butanol and water partitions on antioxidant efficacy. The shoot extract showed excellent ability for screening DPPH free radical. But the extract of trunk showed the most obvious abilities for inhibiting lipoid peroxide, screening nitrite oxide radical and superoxide anion radical, and these abilities were all positively dependent on the growth period of tree. Furthermore, the methanol extract of trunk bark was subject to silicon chromatographic separation, and the anti-oxidative ability of the eluent was examined. The total anti-oxidative capacity of EF-14 and EF-18 fractions was superior to quercetin. Furthermore, comparing with antioxidation of the needle, the truck bark presents better anti-oxidation ability in the extracts of Pinus morrisonicola. Anti-oxidative ability is mainly related to the contents of phenols in the extracts. The bark primarily contained condensed tannins and the needle segment mainly hydroxycinnamates and flavonoids.

摘 要 ...............................................ii
ABSTRACT ...............................................iv
目 錄...................................................vi
表 目 錄 ..............................................ix
圖 目 錄 ................................................x
符號說明 ...............................................xii
第一章、緒論.......................................... ..13
1.1 研究動機.............................................13
1.2 研究目的............................................ 14
第二章、文獻回顧.........................................16
2.1 台灣原生種松樹簡介...................................17
2.2 松針化學成分與生化活性...............................18
2.3 自由基與植物抗氧化劑.................................21
2.4 壓力流體萃取技術.....................................24
2.5 酵素水解技術.........................................27
第三章、酵素水解對壓力流體萃取台灣五葉松針活性成分之影響.33
3.1 台灣五葉松針簡介.....................................33
3.2 實驗材料與設備 .......................................33
3.2.1 松針原料...........................................33
3.2.2 藥品與試劑.........................................34
3.2.3 設備與儀器.........................................37
3.3 實驗方法與步驟.......................................39
3.3.1 松針酵素水解與壓力提取............................ 39
3.3.2 化學成份分析.......................................40
3.3.3 化學抗氧化能力分析.................................41
3.3.4 抑制癌細胞增殖能力.................................47
3.4 數據分析............................................49
3.4.1 單一酵素水解之探討.................................50
3.4.2 混合酵素水解之探討.................................52
3.4.3 酵素水解-壓力流體萃取..............................53
3.4.4 松針萃出物的抗氧化能力評估.........................55
3.4.5 癌細胞生長抑制試驗.................................56
第四章、台灣五葉松樹皮生物活性成分之分離與抗氧化能力比較.57
4.1 法國海岸松簡介.......................................57
4.2 低聚原花青素 (OPC) 簡介..............................58
4.3 實驗材料與設備.......................................67
4.3.1 台灣五葉松樹皮原料.................................67
4.3.2 藥品與試劑.........................................67
4.3.3 設備與儀器 .......................................69
4.4 實驗方法與步驟 .......................................70
4.4.1 萃取與分離處理.....................................70
4.4.2 原花青素定性定量分析...............................71
4.5 數據分析............................................73
4.5.1不同部位松樹皮抗氧化能力比較........................73
4.5.2 高齡樹皮不同極性萃取物抗氧化能力比較...............74
4.5.3 乙酸乙酯可溶性部份分離物之抗氧化能力比較...........75
4.5.4 酚酸結構對總抗氧化能力之影響.......................76
第五章、結論.............................................80
參考文獻.................................................82
表 目 錄
表3-1台灣原生種松針五大成份分析..........................92
表3-2酵素水解-熱壓流體萃取台灣五葉松針製程 L18 直交表法實驗設計表...................................................93
表3-3 酵素-壓力流體連續萃取製程對台灣五葉松與二葉松萃出物酚類成份與抗氧化能力分析...................................94
表4-1酚酸類化合物的顯色反應特徵..........................95
表4-2 五葉松高齡樹皮常溫25℃甲醇萃出物不同可溶性劃份之抗氧化能力比較.................................................96
表4-3 五葉松高齡樹皮乙酸乙酯可溶部分正相層析分離物之總抗氧化能力比較.................................................97
表 4-4 酚酸構型與總抗氧化能力之關連性....................98
附表2-1 活性氧與活性氮的種類............................116
附表2-2 自由基與非自由基的類別..........................117
附表2-3 酚酸化合物二次代謝產物..........................118
附表2-4 酚類化合物的抗氧化性質..........................120
附表4-1 典型的松樹皮萃出物組成(HPLC)....................121
附表4-2 葡萄籽/皮與松樹皮萃出物中多元酚化合物組成之比較.122
圖 目 錄
圖3-1 溫度效應對酵素水解五葉松針之影響..................102
圖3-2 pH效應對酵素水解五葉松針之影響....................103
圖3-3 纖維酵素劑量對水解五葉松針處理時間之影響..........104
圖3-4 酵素組成對水解效率之影響..........................105
圖3-5 混合酵素劑量對水解效率之影響......................106
圖3-6 台灣二葉松針EtOAc可溶性萃出物對抑制人類腫瘤細胞株之影響......................................................107
圖3-7 台灣五葉松針EtOAc可溶性萃出物對抑制人類腫瘤細胞株之影響......................................................108
圖4-1 五葉松樹皮(PM)粗萃物的抗氧化能力比較..............111
圖4-2 臺灣松樹幼枝皮部粗萃物的抗氧化能力比較............112
圖4-3 五葉松高齡樹幹皮EtOAc可溶性部分正相層析分離物之清除DPPH自由基能力比較......................................113
圖4-4 五葉松高齡樹幹皮EtOAc可溶性部分正相層析分離物之清除NO•自由基能力比較......................................114
圖4-5 五葉松高齡樹幹皮EtOAc可溶性部分正相層析分離物之清除LOO•
自由基能力比較..........................................115
附圖2-1 活性氧的種類、來源及其對人體之影響..............123
附圖2-2 酚類化合物的分類與其分子結構....................124
附圖2-3 類黃酮衍生物之結構..............................125
附圖2-4 類黃酮抗氧化機制................................126
附圖2-5 植物細胞壁的木質纖維素及纖維素的分子結構........127
附圖2-6 纖維分解酵素之作用機制假說......................127
附圖2-7 纖維素分解酵素協同作用機制......................128
附圖2-8 纖維分解酵複合體的作用模式......................128
附圖2-9 果酸酯酵素的作用位置............................129
附圖3-1 壓力流體萃取實驗裝置............................130
附圖4-1 抗氧化性複合物(低聚原花青素群)的化學結構........131
附圖4-2 各類單寧結構示意圖..............................132
附圖4-3 原花青素結構通式................................133
附圖4-4 原花青素單體結構................................134
附圖4-5 原花青素二聚體構型..............................134
附圖4-6 原花青素B型二聚體(B1~B8)結構式..................135

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