臺灣博碩士論文加值系統

黃蓮蕉 (Canna indica) 為美人蕉科 (Cannaceae)、美人蕉屬 (Canna) 植物，近年來已有許多研究證實，黃蓮蕉具有許多生理活性，包括調控血糖、抗 HIV 反轉錄酶活性、抗氧化能力、治療淋病、婦女併發症等功效，然而目前仍無詳細文獻報告針對美人蕉屬植物根部之抗氧化力和抗發炎特性及相關機轉作研究。因此本研究主要探討以不同溶劑 (熱水、80% 乙醇) 萃取黃蓮蕉萃取物，以評估其抗氧化特性及抗氧化成分，並採用 LPS 誘導巨噬細胞 RAW 264.7 為發炎細胞模式，進一步測試黃蓮蕉萃取物抗發炎之能力。
首先探討黃蓮蕉萃取物之抗氧化性及其活性成分，結果顯示，黃蓮蕉熱水萃取物抗氧化活性在DPPH 自由基清除能力、總抗氧化能力 (TEAC) 及還原力較黃蓮蕉 80% 乙醇萃取物佳，總酚含量也以黃蓮蕉熱水萃取物顯著高於黃蓮蕉 80% 乙醇萃取物 (p< 0.001)；在類黃酮 (p< 0.007) 及花青素 (p< 0.033) 含量方面則以 80% 乙醇萃取物顯著較高。另外黃蓮蕉 80% 乙醇萃取物在高劑量下其抑制血管收縮素轉化酶 (ACE) 活性能力較佳，約有 74 % (p< 0.05)。
另外在 LPS 誘導 RAW 264.7 巨噬細胞發炎反應模式下，結果發現在 10-500 μg/mL 之黃蓮蕉熱水萃取物和 80% 乙醇萃取物均可顯著抑制 LPS 誘導 RAW 264.7 巨噬細胞生成 Nitric oxide (NO)、Soluble intercellular adhesion molecule-1 (sICAM-1)、Prostaglandin E2 (PGE2) 與 Interleukin-1β (IL-1β) 之能力，但對於 Tumor necrosis factor alpha (TNF-α) 分泌量並無顯著影響。
綜合以上結果推測，黃蓮蕉萃取物含有豐富之抗氧化成分 (總酚及類黃酮)，因此具有良好之抗氧化能力，此外，研究也進一步證實黃蓮蕉萃取物確實能減少經由 LPS 誘發巨噬細胞的發炎反應。

The biological activities of Canna species have caused most attentions, but limited scientifical evidence is available on their health-related activities. The objective of this study was to identify antioxidant and anti-inflammation properties of Canna indica (Cannaceae). Hot water extract (CI-W) and 80% ethanol extract (CI-E) from Canna indica (Cannaceae) were investigated for their total phenolic, total flavonoid and anthocyanin contents, antioxidant and anti-inflammatory activities.
The results showed that CI-W has higher DPPH free radical scavenging activity, trolox equivalent antioxidant capacity, reducing power and total phenolic contents and antioxidant activities than CI-E. However, CI-E has higher total flavonoids and anthocyanin contents than CI-W. A higher inhibition of angiotensin converting enzyme activity of CI-E at high doses was about 74%.
With respect to anti-inflammatory properties, both CI-W and CI-E showed the inhibitory effects on production of LPS-induced nitric oxide, soluble intercellular adhesion molecule-1, prostaglandin E2 and interleukin-1β by RAW 264.7 macrophages. However, there was no significant change in tumor necrosis factor alpha.
In conclusion, the results indicate that Canna indica extracts were rich in antioxidant ingredients (total phenolics and total flavonoids), and have antioxidant capacity and anti-inflammatory properties.

中文摘要……………………………………………………………………… I
Abstract……………………………………………………………………… III
目錄…………………………………………………………………………… IV
圖目錄………………………………………………………………………… VII
表目錄………………………………………………………………………… VIII
縮寫總表……………………………………………………………………… IX
壹、前言……………………………………………………………………… 1
1.1 研究動機 ……………………………………………………………1
1.2 研究目的 ……………………………………………………………2
貳、 文獻整理………………………………………………………………… 4
2.1 黃蓮蕉文獻回顧…………………………………………………… 4
2.1.1 黃蓮蕉簡介………………………………………………………… 4
2.1.2 黃蓮蕉成分分析…………………………………………………… 5
2.1.3 黃蓮蕉功能性分析………………………………………………… 5
2.1.4 塊根類植物之功能性 ………………………………………………8
2.2 血管收縮素轉化酶 (Angiotensin Converting Enzyme, ACE) ……………………………………………………………10
2.2.1 ACE 之生化特性…………………………………………………… 10
2.2.2 ACE 抑制劑作用機制……………………………………………… 11
2.3 發炎反應 (inflammatory response) 與發炎調控路徑…… 11
2.3.1 發炎 (inflammation) 之簡介………………………………… 11
2.3.2 脂多醣 (Lipopolysaccharide) 與巨噬細胞……………… 12
2.3.3 發炎反應相關因子………………………………………………… 14
2.3.3.1 Proinflammatory cytokines 促發炎反應細胞激素……… 14
2.3.3.2 NO 及 iNOS……………………………………………………… 15
2.3.3.3 PGE2 及 COX-2 前列腺素E2 與環氧化酶-2………………… 17
2.3.3.4 細胞間黏著分子-1………………………………………………… 18
2.3.3.5 胞核因子-κB 發炎調節路徑………………………………………19
參、實驗材料與方法……………………………………………………………21
3.1 實驗架構…………………………………………………………… 21
3.2 實驗材料…………………………………………………………… 22
3.2.1 材料………………………………………………………………… 22
3.2.1.1 黃蓮蕉來源………………………………………………………… 22
3.2.1.2 實驗細胞株………………………………………………………… 22
3.2.1.3 樣品製備…………………………………………………………… 22
3.2.2 試驗藥品…………………………………………………………… 22
3.2.2.1 抗氧化實驗與成分分析…………………………………………… 22
3.2.2.2 抑制血管收縮素轉化酶活性……………………………………… 23
3.2.2.3 細胞培養…………………………………………………………… 23
3.2.2.4 細胞存活率測定…………………………………………………… 23
3.2.2.5 一氧化氮釋放量分析 ……………………………………………… 24
3.2.2.6 蛋白質定量………………………………………………………… 24
3.2.2.7 蛋白質膠體電泳 (SDS-PAGE) 分析…………………………… 24
3.2.2.8 Western Blot…………………………………………………… 24
3.2.2.9 Cytokine ELISA kit…………………………………………… 25
3.2.3 儀器設備…………………………………………………………… 25
3.3 實驗方法…………………………………………………………… 26
3.3.1 抗氧化成分分析…………………………………………………… 26
3.3.1.1 總酚類化合物含量測定…………………………………………… 26
3.3.1.2 類黃酮含量測定…………………………………………………… 26
3.3.1.3 總花青素含量測定………………………………………………… 26
3.3.2 抗氧化活性之測定………………………………………………… 27
3.3.2.1 DPPH自由基清除能力測定………………………………………… 27
3.3.2.2 總抗氧化能力分析………………………………………………… 28
3.3.2.3 亞鐵螯合能力……………………………………………………… 28
3.3.2.4 還原力測定………………………………………………………… 29
3.3.3 血管收縮素轉化酶抑制活性之檢測……………………………… 29
3.3.4 細胞試驗…………………………………………………………… 30
3.3.4.1 RAW264.7 巨噬細胞株之培養…………………………………… 30
3.3.4.2 MTT 細胞存活率試驗……………………………………………… 31
3.3.4.3 亞硝酸鹽含量分析………………………………………………… 31
3.3.4.4 前列腺素E2 (Prostaglandin E2, PGE2) 含量分析……… 32
3.3.4.5 腫瘤壞死因子-α (TNF-α) 含量分析…………………………… 32
3.3.4.6 介白素-1β (Interleukin-1β, IL-1β) 含量分析………… 33
3.3.4.7 細胞激素sICAM-1測定…………………………………………… 33
3.3.4.8 細胞蛋白質抽取…………………………………………………… 34
3.3.4.9 蛋白定量 (Bio-Rad Protein Assay)……………………… 34
3.3.4.10 聚丙烯醯胺膠體電泳法……………………………… 35
3.3.4.11 西方轉漬法 (Western blot assay)…………… 35
3.3.5 統計分析…………………………………………………………… 36
肆、 結果……………………………………………………………………… 37
伍、 討論 ……………………………………………………………………… 56
陸、 結論 ……………………………………………………………………… 61
柒、 參考文獻………………………………………………………………… 62

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