臺灣博碩士論文加值系統

Pinus morrisonicola Hay (PM)，即台灣五葉松”生長在台灣海拔
300-2300公尺處。傳統被用來當作降血壓功能的飲料，已被證實可以
降低血漿膽固醇、低密度脂蛋白膽固醇含量和預防高血壓。超臨界流
體萃取技術 (supercritical fluid extraction; SCFE)是一種環保、快速和
安全的萃取方法，SCFE 技術已經成為傳統食品、醫藥及化工等方面
更好的選擇。我們在溫度（40，50，60℃），壓力（15，20，25 MPa），
時間（5，10，15 分鐘）和輔助溶劑（1，2，3 毫升乙醇）之不同條
件下萃取PM，共獲得PME 9 種萃取物，分別編號為1- 9，萃取率
大約在1.0〜3.9％左右。本研究的目的是探討PME 之抗氧化潛力及
其活性成分。研究顯示PME 1，3，4和6的細胞存活率高於PME 2，
5，7，8，9，且有較低的細胞毒性，並也發現PME 3（萃取溫度40℃，
壓力25 MPa，時間15分鐘和輔助溶劑3 ml）有最佳的收率（3.9％）
及抗氧化潛力。PME 3 清除DPPH自由基的IC50為20 mg/ml，在100
μg/ml 之濃度下對Cu+2（50 μM）誘導LDL (Low-Density Lipoprotein)
的氧化（100 μg/ml）所形成之TBARS和共軛二烯分別為22％和85
％。此外，PME 3在10 μg/ml 之低濃度下對RAW 264.7巨噬細胞具
有抑制泡沫細胞形成的效果。 PME3 的總多酚和類黃酮的含量分別
為14500和9600 μg/g，以HPLC進行分析，發現所含成分為沒食子
酸(gallic acid)、表兒茶素 (epicatechin)及香豆酸 (p-coumaric acid)。此
外，我們以GC / MS 對PME3進行分析，發現有效的活性化合物分
別1-Docosene（1.6％）、Neophytadiene （7.7％）、 Methyl abietate
（3.4％）及Octadecane 1-(ethenyloxy)（55％），綜合上述，PME 3應
具有開發為抗氧化保健產品潛力。
關鍵詞：臺灣五葉松、心血管疾病、超臨界萃取

Pinus morrisonicola Hay (PM), also known as “five leaves pine” is grown at 300-2300 m altitude in Taiwan. Traditionally, it has been used as beverages for lowing blood pressure. PM has been shown to reduce plasma cholesterol and LDL levels and to prevent hypertension. Supercritical fluid extraction (SCFE) technology has emerged as a superior alternative to the conventional techniques for extraction of natural products in food, pharmaceutical and chemical industries. SCFE technology is an environment, quick and safety extraction method. We used different conditions, temperature (40, 50, 60℃), pressure (15, 20, 25 MPa), time (5, 10, 15 min) and adjuvant solvents (1, 2, 3 ml ethanol) to extract PM and obtained PME 9 were obtained extracts or materials, respectively numbered 1 - 9, and the extraction yield were about 1.0~3.9 %. The aim of this study was to explore the antioxidant effects of PME and their active components. The cell viability of PME 1, 3, 4 and 6 had lower toxicity than that of PME 2, 5, 7, 8, 9. The results also shows that PME 3 (40℃、25 Mpa, 15 min and 3 ml ethanol) had the better extraction yield (3.9%) and potent antioxidant effects. The IC50 of scavenging DPPH radical is 20 mg/ml. The inhibition of Cu+2 (50 M)–induced oxidative LDL (100 g/ml) on TBARS and conjugated diene formation in the of PME 3 were 22% and 85% at 100 g/ml , respectively. In addition, PME 3 (100 g/ml) possessed effect and foam cells formation in Raw264.7 macrophage. The content of total polyphenol and flavonoids in PME3 was 14500 and 9600 g/g, and only gallic acid, epicatechin and p-coumaric acid were detected and confirmed by HPLC. Furthermore, we analyzed PME3 by GC/MS and found that the potent active compounds were Octadecane 1-(ethenyloxy) (55%), Neophytadiene (7.7%), Methyl abietate (3.4%) and 1-docosene (1.6%), respectively. Overall, PME3 has the potential for development as a antioxidant agent.

Keywords：Pinus Morrisonicola Hay, Cardiovoscular disease, Supercritical fluid extraction

中文摘要…………………………………………………………………i
英文摘要 ……………………………………………………………… iii
目錄 …………………………………………………………………… iv
表次 …………………………………………………………………… vi
圖次 ……………………………………………………………………vii
壹、前言…………………………………………………………………1
貳、文獻回顧……………………………………………………………4
一、五葉松的植物特性………………………………………………4
二、國人的食用習慣…………………………………………………4
三、五葉松的功能性…………………………………………………4
四、五葉松抗氧化的相關研究……………………………10
五、五葉松抗氧化成分……………………………………11
六、國人高血脂疾病…………………………………………………14
七、超臨界萃取的定義及應用………………………………………22
八、精油類抗氧化研究………………………………………………25
參、實驗架構……………………………………………………………29
肆、材料與方法…………………………………………………………30
一、實驗器材與藥劑…………………………………………………30
二、實驗方法…………………………………………………………32
(一)五葉松超臨界萃取物（PME）之制備………………………32
(二)細胞存活率測定………………………………………………35
(三)五葉松針超臨界萃取物 (PME)之抗氧化能力評估 ……… 35
(四)成分分析..…………………………………………………..…38
三、統計分析……………….………………………………………...41
伍、結果與討論 ……………………………………………………….42
一、五葉松針超臨界萃取條件的建立…………………………… 42
二、五葉松針超臨界萃取物對RAW 264.7巨噬細胞存活率的
影響……………………………………………………………45
三、五葉松針超臨界萃取物清除DPPH自由基能力……………47
四、五葉松針超臨界萃取物抑制Cu+2 (50 μM)誘導LDL
（100 μg/ml）的脂質過氧化的能力…………………………49
五、五葉松針超臨界萃取物對ox-LDL誘導巨噬細胞泡沫
細胞形成的影響………………………………………………53
六、成分分析………………………………………………………57
陸、結論 ………………………………………………………………62
柒、參考文獻 …………………………………………………………63
表一、臺灣地區主要死亡原因…………………………………………3
表二、台灣五葉松相關之有效成分與功效……………………………5
表三、不同年齡之國人血清總膽固醇含量料…………………………14
表四、血脂異常的分類標準……………………………………………18
表五、人體血脂正常與異常值…………………………………………19
表六、松樹精油之主要成分與功效……………………………………27
表七、控制因子及其水準表……………………………………………33
表八、Level 9 L 9 ( 34)直交表 ……………………………………33
表九、五葉松超臨界萃取條件L9 (34)直交表及相對產率……………44
表十、不同濃度之五葉松針超臨界萃取物 (PME 1-9)對Cu+2
誘導LDL氧化產生TBARS的能力………………………………50
表十一、五葉松針超臨界萃取物PME1-9抗氧化潛力綜合評估 ……56
表十二、PME3的總多酚類含量及組成…………………………………58
表十三、五葉松超臨界萃取物 (PME 3)中揮發性成分之百分比 (%)59
圖一、類黃酮的化學結構式……………………………………………7
圖二、物體超臨界流體相變化…………………………………………23
圖三、本論文之研究大綱………………………………………………29
圖四、超臨界CO2萃取簡圖……………………………………………34
圖五、田口實驗法之實驗設計…………………………………………43
圖六、五葉松針超臨界萃取物(PME 1-9)對RAW 264.7巨噬細胞
存活率的影響……………………………………………………46
圖七、DPPH自由基清除能力的影響 …………………………………48
圖八、五葉松針超臨界萃取物PME 1，3，4，6 (10 μg/ml)
的抑制 Cu+2 (50 μM)誘導LDL (100 μg/ml) 氧化
產生共軛雙烯的能力……………………………………………51
圖九、PME 3 (10~100 μg/ml)抑制在3小時的反應時間下對
Cu+2誘導LDL氧化產生共軛雙烯………………………………52
圖十、五葉松針超臨界萃取物PME 1,3, 4, 6 (10 μg/ml)對
ox-LDL (50 μg/ml)誘導巨噬細胞泡沫細胞形成的影響...55
圖十一、PME 3之總多酚類HPLC圖譜 …………………………………58
圖十二、PME 3之GC-MS 圖譜 ……………………………………… 61

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