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研究生:蘇嘉淇
研究生(外文):Su, Chia-Chi
論文名稱:綠花白千層精油抗氧化及抗黑色素生成之機制探討
論文名稱(外文):Study on the Anti-oxidation and Anti-melanogenesis of Essential Oil from Melaleuca Quinquenervia
指導教授:周淑姿周淑姿引用關係
指導教授(外文):Chou, Su-Tze
口試委員:陳淑茹張珍田
口試委員(外文):Chen, Shu-JuChang, Chen-Tien
口試日期:2015-07-28
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:95
中文關鍵詞:綠花白千層精油抗氧化黑色素酪胺酸酶B16小鼠黑色素瘤細胞
外文關鍵詞:Melaleuca quinquenerviaessential oilantioxidantmelanintyrosinaseB16 melanoma cells
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精油是由植物提煉之芳香物質,為脂溶性且分子小,具可穿透皮膚及黏膜等特性,已知植物精油大多具抗菌、抗感染等效用,且其具特殊香氣故廣泛應用於化粧品工業與調香業,近年來逐漸有文獻探討精油對於黑色素生成之影響,酪胺酸酶為黑色素生成之關鍵酵素,而自由基與黑色素生成有密切相關性,因此本研究利用體外試驗探討綠花白千層 (Melaleuca quinquenervia) 精油及其主要組化物之抗氧化能力,並以B16 小鼠黑色素瘤細胞 (B16 melanoma cell line) 評估其對於黑色素生成之影響。
結果顯示,以GC-MS分析其主要組化物,可發現1,8-cineole、α-pinene及α-terpineol為綠花白千層精油之主要成分。綠花白千層精油可有效清除ABTS+自由基並具有良好之還原能力,而綠花白千層精油及其三種主要組化物皆可有效延緩脂質過氧化之發生﹔另外綠花白千層精油及其主要組化物1,8-cineole也能有效抑制酪胺酸酶活性。於B16細胞模式中,綠花白千層精油及其主要組化物隨著劑量上升皆能降低α-MSH誘發後黑色素之生成量及降低酪胺酸酶活性,並調節細胞經α-MSH誘發之氧化-抗氧化恆定、降低脂質過氧化之程度、增加glutathione (GSH) 濃度、提升superoxide dismutase (SOD)、gluathione peroxidase (GPx) 及catalase (CAT) 活性。此外,綠花白千層精油能調控酪胺酸酶蛋白表現,且經由DAPI染色和彗星試驗法得知綠花白千層精油也能緩解經α-MSH誘發B16細胞氧化損傷後之細胞凋亡及DNA損傷。綜合上述,綠花白千層精油及其主要組化物在體外試驗具有良好抗氧化能力,並能改善細胞氧化-抗氧化狀態,進而降低黑色素之生成,並可推估1,8-cineole、α-pinene和α-terpineol為綠花白千層精油具有抑制酪胺酸酶活性與黑色素生成之主要組化物。

Natural essential oils are extracted from different plants of aromatic crops, which have anti-bacterial and anti-infective effects. The characteristics of plant essential oil are fat-soluble and small molecules that can easily penetrate the skin and mucous membranes. Essential oils have been widely used for medicinal and cosmetic applications. Recently many studies are focusing on the anti-melanogenesis and major active biomolecules in plant essential oils. However, no scientific study demonstrates that Melaleuca quinquenervia essential oil (MQ-EO) on tryosinase activity and melanogenesis. Because tyrosinase plays an important role in melanogenesis, the purpose of this study is to investigate the effects of MQ-EO and the major compounds on antioxidant, tyrosinase activity and melanogenesis.
The results of in vitro study had shown that MQ-EO with antioxidant activities. From GC-MS analysis results, the major compounds of MQ-EO are 1,8-cineole, α-pinene, and α-terpineol. Furthermore, our results indicated that treatment of B16 melanoma cells with MQ-EO and the major compounds along with the dose increases would significantly decrease α-MSH-induced melanin contents, cellular tyrosinase activity, reduced lipid peroxidation levels and increased the GSH level, superoxide dismutase (SOD), gluathione peroxidase (GPx) and catalase (CAT) activities. In addition, MQ-EO can regulate the expression of tyrosinase protein ,and rescue the cells from α-MSH-induced apoptosis and DNA damage which was confirmed by DAPI stain and comet assay. In conclusion, our study has established the information of anti-tyrosinase activity and anti-melanogenesis potential of MQ-EO and the major compounds, and we supposed the major compounds is the major contributor to the biological activities of MQ-EO.


目錄 I
表目錄 IV
圖目錄 V
中文摘要 VII
英文摘要 VIII
第一章 前言 1
第二章 文獻回顧 3
第一節 精油之簡介 3
一、 精油之化學組成 3
二、 精油之功效 6
第二節 綠花白千層 (Melaleuca quinquenervia) 之簡介 9
第三節 皮膚之結構 11
一、表皮層 11
二、真皮層 11
三、皮下組織 11
第四節 黑色素細胞 (Melanocyte)、黑色素小體 (Melanosomes) 與黑色素 (Melanin) 13
1. 第一階段 13
2. 第二階段 13
3. 第三階段 13
4. 第四階段 13
第五節 黑色素相關酵素 16
一、 酪胺酸酶 (Tyrosinase) 16
二、 酪胺酸酶相關蛋白 (tyrosinase related proteins ﹔TRP1和TRP2) 16
第六節 黑色素生成 (Melanogenesis) 機轉 17
一、 UV 17
二、 α-MSH (α-melanocyte stimulating hormone) 19
第七節 黑色素生成與氧化/抗氧化恆定 22
第三章 研究目的與架構 23
一、 動機 23
二、 目的 23
三、 研究架構 24
第四章 材料與方法 25
第一節 儀器設備及器材 25
第二節 藥品與試劑 26
第三節 實驗方法 29
一、 精油來源與GC / MS分析 29
二、 體外抗氧化試驗 29
三、 細胞培養與保存 33
四、 細胞存活率測定 (MTT assay) 34
五、 黑色素細胞誘發培養 35
六、 細胞內黑色素測量 35
七、 細胞內酪胺酸酶活性測定 36
八、 DNA損傷測定 36
九、 抗氧化物質與酵素之活性測定 38
十、 西方墨點法 (Western blotting) 42
十一、 統計分析 45
第五章 結果 46
一、 綠花白千層精油經GC-Mass 之成分鑑定 46
二、 體外抗氧化能力評估 48
1. 綠花白千層精油 (MQ-EO) 及其主要組化物ABTS自由基清除能力分析 48
2. 綠花白千層精油 (MQ-EO) 及其主要組化物還原力分析 50
3. 綠花白千層精油 (MQ-EO) 及其主要組化物β-carotene/linoleic acid試驗法 52
4. 綠花白千層精油 (MQ-EO) 及其主要組化物對酪胺酸酶活性抑制能力 55
三、 綠花白千層精油 (MQ-EO) 及其主要組化物經α-MSH刺激B16細胞其 細胞存活率之影響 57
四、 綠花白千層精油 (MQ-EO) 對α-MSH刺激B16細胞其黑色素含量、酪胺酸酶活性及酪胺酸酶蛋白表現量之影響 60
五、 綠花白千層精油之主要組化物對α-MSH刺激B16細胞其黑色素含量及酪胺酸酶活性之影響 62
六、 綠花白千層精油 (MQ-EO) 對α-MSH刺激B16細胞DNA損傷之影響 64
七、 綠花白千層精油 (MQ-EO) 及其主要組化物對α-MSH刺激B16細胞其氧化 / 抗氧化酵素恆定之影響 68
第六章 討論 76
第七章 結論 81
第八章 參考文獻 82


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