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研究生:翁月君
研究生(外文):Weng, Yuehchun
論文名稱:桉樹葉精油抗發炎生物活性之探討
論文名稱(外文):Eucalyptus essential oils inhibit inflammatory responses in LPS-activated macrophages
指導教授:花國鋒花國鋒引用關係
指導教授(外文):Hua, Kuofeng
口試委員:吳世雄陳安趙國評何振隆花國鋒
口試委員(外文):Wu, ShihhsiungChen, AnnChao, LouiskuopingHo, ChenlungHua, Kuofeng
口試日期:2012-06-13
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物資源學院碩士在職專班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:92
中文關鍵詞:抗發炎桉樹葉精油檸檬桉葉精油
外文關鍵詞:anti-inflammatoryEucalyptus essential oilEucalyptus citriodora
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桉樹(Eucalyptus)為世界主要之紙漿用材,分布廣泛,引進台灣已有百年歷史。已知其生物活性有抗氧化(antioxidative)與抗微生物(antimicrobial)(細菌、黴菌及木材腐朽菌)等活性,本研究以四種不同種類之桉樹葉片,即垂尾桉(E. urophylla) 、玫瑰桉(E. grandis)、赤桉(E. camaldulensis)及檸檬桉(E. citriodora),以水蒸餾法萃取桉樹葉精油,並進一步評估其抗發炎活性(anti-inflammatory activity)。結果顯示四種桉樹葉精油中以檸檬桉葉精油降低巨噬細胞中由細菌內毒素(LPS)所誘導產生一氧化氮(NO)的效果最好,故進一步將檸檬桉葉精油進行管柱層析 (column chromatography),得到Fraction A~H,在研究中證實Fraction F可以抑制LPS所誘導產生的腫瘤壞死因子(TNF-alpha)、IL-6、NO、一氧化氮合成酶(iNOS) 與環氧化酵素二型(COX-2)的表現。進一步探討Fraction F對細胞訊息傳遞的影響,結果顯示Fraction F可以抑制ERK1/2、p38、PKC-alpha、PKC-epsilon與 PKC-delta的磷酸化,然而無法抑制JNK1/2的磷酸化。另外Fraction F也可以抑制NF-kappaB的活性。綜合以上研究結果顯示,檸檬桉葉精油分離純化後的產物Fraction F具有很好的抗發炎效果。


Eucalyptus is an important pulpwood genus distribution widely and was introduced to Taiwan over a century ago. The reported bio-activities of the leaves of eucalypts were antioxidative and antimicrobial (including bacteria, mildews and wood decaying fungi). In this study, essential oils from the leaves of 4 species, i.e., E. urophylla, E. grandis, E. camaldulensis, and E. citriodora were extracted using water distillation, and were evaluated for their anti-inflammatory activity in murine macrophages cell lines RAW 264.7. Experimental results showed that essential oils of E. citriodora had the best efficacy in NO inhibition. Essential oils of E. citriodora were fractioned into A~H fractions using column chromatography. We found that Fraction F inhibited TNF-alpha, IL-6, NO, iNOS and COX-2 expression in LPS-stimulated macrophages. Furthermore, the phosphorylation of ERK1/2, p38, PKC-alpha, PKC-epsilon and PKC-delta induced by LPS were inhibited by Fraction F, while the phosphorylation of JNK1/2 was not reduced by Fraction F. Fraction F also inhibited NF-kappaB activation in LPS-stimulated macrophages. Our current results indicated that Fraction F is the major anti-inflammatory ingredient of E. citriodora.
總目錄
中文摘要  Ⅱ
英文摘要  Ⅲ
致謝  Ⅳ
總目錄  Ⅴ
表目錄  Ⅷ
圖目錄  Ⅸ
縮寫檢索表  XI
第一章、緒論  1
第一節、精油  2
第一項、精油之介紹  2
第二項、精油之萃取及應用  4
第三項、桉樹葉精油之應用  6
第二節、免疫與發炎反應相關因子  9
第一項、發炎反應之調節機制  9
第二項、巨噬細胞及其功能  10
第三項、脂多醣  11
第四項、發炎機制相關調控物質  12
第三節、研究方向與動機  17
第二章、研究材料與方法  18
第一節、實驗研究材料  19
第一項、試驗材料  19
第二項、實驗細胞  20
第三項、藥品及化學試劑  21
第四項、抗體  23
第五項、分析試劑組  24
第六項、儀器設備  25
第七項、實驗用耗材  26
第八項、分析軟體  27
第二節、研究方法  28
第一項、細胞解凍  28
第二項、細胞繼代培養  28
第三項、細胞數目測定  30
第四項、細胞保存  31
第五項、細胞存活率偵測分析  31
第六項、一氧化氮之定量與分析  33
第七項、西方墨點法  35
第八項、IL-6、TNF-alpha 濃度測定  44
第九項、NF-kappaB 訊息傳遞分析  46
第十項、統計分析  46
第三章、研究結果  47
第一節、四種桉樹葉精油對 RAW 264.7 存活率之影響  48
第二節、四種桉樹葉精油對 LPS 刺激 RAW 264.7 所產生 NO 的影響  48
第三節、Fraction A~H 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  49
第四節、Fraction D~G 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  50
第五節、Fraction F 與 fraction G 對 RAW 264.7 存活率之影響  50
第六節、Fraction F 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  51
第七節、Fraction F 對 LPS 刺激 RAW 264.7 所產生 IL-6、TNF-alpha 的影響  51
第八節、Fraction F 對 LPS 刺激 RAW 264.7 所活化 NF-kappaB 的影響  52
第九節、Fraction F 對 LPS 刺激 RAW 264.7 所產生的 iNOS 及 COX-2的影響  52
第十節、Fraction F 對 LPS 刺激 RAW 264.7 所產生的 MAPK 磷酸化的影響  53
第十一節、Fraction F 對 LPS 刺激 RAW 264.7 所產生的 PKC 磷酸化的影響  53
第十二節、Fraction F 對 LPS 刺激 RAW 264.7 所產生的 IKK-alpha 及IkappaB-alpha 磷酸化的影響  54
第十三節、Fraction F 成份組成  54
第四章、結果與討論  55
第一節、Fraction F 於巨噬細胞存活率之影響  56
第二節、Fraction F 於發炎相關物質之影響  57
第三節、Fraction F 所含的成分  59
第五章、結論與展望  60
第六章、實驗結果圖表  62
參考文獻  83
表目錄
表 1.Fraction F 與 fraction G 組成  63
Table 1.Ingredients of fraction F and fraction G  63
圖目錄
圖 1.巨噬細胞之功能  64
Fig. 1.Macrophage function  64
圖 2.脂多醣 (Lipopolysaccharide, LPS) 之結構圖  65
Fig. 2.Structure of lipopolysaccharide  65
圖 3.MAPK 及 NF-kappaB 訊息傳遞路徑  66
Fig. 3.Signaling pathways of MAPK and NF-kappaB  66
圖 4.NO 合成途徑及功能  67
Fig. 4.Synthetic pathway of NO and its function  67
圖 5.NF-kappaB 訊息傳遞路徑  68
Fig. 5.Signaling pathway of NF-kappaB  68
圖 6.LPS 刺激單核球活化 MAPK 及 NF-kappaB 訊息傳遞路徑  69
Fig. 6.MAPK and NF-kappaB signaling pathways in LPS-activated monocytes  69
圖 7.四種桉樹葉精油對 RAW 264.7 存活率之影響  70
Fig. 7.Effect of leaf essential oils from four Eucalyptus species on RAW 264.7 viability  70
圖 8.四種桉樹葉精油對 LPS 刺激 RAW 264.7 所產生 NO 的影響  71
Fig. 8.Effect of leaf essential oils from four Eucalyptus species on NO generation in LPS-activated RAW 264.7  71
圖 9.Fraction A~H 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  72
Fig. 9.Effect of fractions A~H on NO generation in LPS-activated RAW 264.7  72
圖 10.Fraction D~G 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  73
Fig.10.Effect of fractions D~G on NO generation in LPS-activated RAW 264.7  73
圖 11.Fraction F 與 fraction G 對 RAW 264.7 存活率之影響  74
Fig.11.Effect of fraction F and fraction G on RAW 264.7 viability  74
圖 12.Fraction F 對 LPS 刺激 RAW 264.7 所產生 NO 的影響  75
Fig.12.Effect of fraction F on NO generation in LPS-activated RAW 264.7  75
圖 13.Fraction F 對 LPS 刺激 RAW 264.7 所產生 IL-6 的影響  76
Fig.13.Effect of fraction F on IL-6 secretion in LPS-activated RAW 264.7  76
圖 14.Fraction F 對 LPS 刺激 RAW 264.7 所產生 TNF-alpha 的影響  77
Fig.14.Effect of fraction F on TNF-alpha secretion in LPS-activated RAW 264.7  77
圖 15.Fraction F 對 LPS 刺激 RAW 264.7 所活化 NF-kappaB 的影響  78
Fig.15.Effect of fraction F on NF-kappaB activation in LPS-activated RAW 264.7  78
圖 16.Fraction F 對 LPS 刺激 RAW 264.7 所產生的 iNOS 及 COX-2 的影響  79
Fig.16.Effect of fraction F on the expression of iNOS and COX-2 in LPS-activated RAW 264.7  79
圖 17.Fraction F 對 LPS 刺激 RAW 264.7 所產生的 MAPK 磷酸化的影響  80
Fig.17.Effect of fraction F on MAPK phosphorylation in LPS-activated RAW 264.7  80
圖 18.Fraction F 對 LPS 刺激 RAW 264.7 所產生的 PKC 磷酸化的影響  81
Fig.18.Effect of fraction F on PKC phosphorylation in LPS-activated RAW 264.7  81
圖 19.Fraction F 對 LPS 刺激 RAW 264.7 所產生的 IKK-alpha 及 IkappaB-alpha 磷酸化的影響  82
Fig.19.Effect of fraction F on the phosphorylation of IKK-alpha and IkappaB-alpha in LPS -activated RAW 264.7  82

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