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研究生:魏吟潔
研究生(外文):Yin-Jie Wei
論文名稱:道手香水蒸餾精油吸入對正常及氣喘模式小鼠免疫調節之影響
論文名稱(外文):Immunomodulatory effects of steam distillation essential oil of Coleus amboinicus inhalation in normal and asthmatic mice
指導教授:林金源林金源引用關係
口試委員:林璧鳳陳炳宏
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
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:151
中文關鍵詞:過敏性氣喘道手香免疫細胞吸入OVA 致敏及刺激水蒸餾精油Th1/Th2 細胞激素
外文關鍵詞:allergic asthmaColeus amboinicusimmune cellsinhalationOVA-sensitization and challengesteam distillation essential oilsTh1/Th2 cytokines
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植物精油為植物二級代謝產物的複雜混合物,可能具有抗過敏、抗發炎及抗癌功效。目前有關植物水蒸餾精油之免疫調節作用尚無完整研究,因此本研究選取金錢薄荷(Glechoma hederacea) 、金盞花(Calendula officinalis) 、香茅(Cymbopogon nardus )、土肉桂(Cinnamomum osmophloeum Kanehira.) 葉、迷迭香(Rosmarinus officinalis) 及道手香(Coleus amboinicus Lour.) 六種具潛力植物為材料,製備其水蒸餾精油進行免疫調節之研究。
先於體外(in vitro)試驗進行免疫調節功能評估,以調節小鼠初代脾臟細胞Th1/Th2 細胞激素分泌傾向、初代腹腔巨噬細胞促/抗發炎細胞激素分泌變化及初代肺臟肥大細胞之安定性試驗進行評估,結果發現道手香水蒸餾精油添加於脾臟細胞使其細胞激素分泌傾向於抗發炎之Th2 型免疫反應、於腹腔巨噬細胞可抑制發炎反應且對於肥大細胞去顆粒化之安定性較佳,顯示道手香水蒸餾精油具抗發炎及抗過敏之潛力,因此篩選出道手香水蒸餾精油,進行動物實驗。
於動物體內試驗,以吸入介入方式探討其對正常小鼠及OVA 致敏及刺激的氣喘小鼠免疫調節之影響。結果顯示,短暫吸入道手香水蒸餾精油,對正常小鼠於肺氣管內變化,於脾臟細胞Th1/Th2 細胞激素分泌及增生,於血清中非特異抗體變化,於肺臟肥大細胞去顆粒化作用,皆無顯著影響,但於腹腔巨噬細胞,則可降低IL-1β/IL-10 分泌比值,具抗發炎之效果。然而吸入高劑量(6 mg/l)道手香水蒸餾精油雖會減少小鼠肺氣管內蛋白質含量,卻降低腎臟臟器重量,為避免副作用,因此選用低劑量(0.3 mg/l)及中劑量(1.2 mg/l)道手香水蒸餾精油,進行氣喘小鼠體內試驗。顯示短暫吸入道手香水蒸餾精油後,對OVA 致敏及刺激的氣喘小鼠,於肺氣管內可降低嗜伊紅細胞浸潤、減少發炎媒介物產生,於腹腔巨噬巨細胞可抑制發炎反應,於血清內可增加OVA 特異性IgG2a/IgE 抗體比值分泌,及增加肺臟肥大細胞安定性等。綜合本研究結果證實吸入道手香水蒸餾精油噴霧具有改善過敏性氣喘之作用。
Essential oils are a complex mixture of plant secondary metabolites that may have anti-allergic, anti-inflammatory and anti-cancer effects. However, immunodualtory functions of steam distillation essential oils from plants remain unclear. Therefore, six potential plants including Glechoma hederacea, Calendula officinalis,Cymbopogon nardus, leaves of Cinnamomum osmophloeum Kanehira., Rosmarinus officinali and Coleus amboinicus Lour. were selected to extract their steam distillation essential oils for immunomodulatory study.
First, effects of these essential oils on immunodualtory functions in vitro were evaluated by observing Th1/Th2 cytokines produced by primary splenocytes, pro- and
anti-inflammatory cytokine secretions by peritoneal macrophages and the stability test to the degranulation of mast cells from BALB/c mice. The results showed that steam distillation essential oil of Coleus amboinicus among six essential oils had Th2-skewed but anti-immunodualtory responses to splenocytes, anti-immunodualtory effects on peritoneal macrophages and the better stability to the degranulation of mast cells. Thus,the essential oil was selected to perform animal study for its potential antiimmunodualtory and anti-allergic effects in vitro.
Next, the potential essential oil with anti-immunodualtory and anti-allergic functions was further selected to administer to normal and ovalbumin (OVA)-sensitized and challenged BALB/c mice using inhalation intervention in vivo. In normal mice, the results showed that there were no significant differences among Th1/Th2 cytokine secretions and the profileration of splenocytes, no-specific antibodies in the serum and the degranulation of mast cells after inhalation intervention. However, IL-1β/IL-10 secretion ratios by peritoneal macrophages decreased, exhibiting anti- immunodualtory effects. Although high dose (6 mg/l) inhalation of Coleus amboinicus essential oil reduced protein contents in the trachea, the weight of the kidney also decreased. To avoid side effects, low (0.3 mg/l) and medium (1.2 mg/l) doses of Coleus amboinicus essential oil were further selected to treat OVA-sensitized and challenged mice. The results showed that inhalation intervention using Coleus amboinicus essential oil reduced eosinophilia infiltration and inflammatory mediators in the trachea, inhibited inflammatory reactions by macrophages, increased OVA-specific IgG2a/IgE antibody ratio secretions in the serum and stabilized mast cells degranulation in the lung. Overall,our results evidenced that inhalation intervention using Coleus amboinicus essential oil improved allergic asthma.
摘要 ................................................................................................................................... i
Abstract............................................................................................................................. ii
縮寫對照表................................................................................................................... xiv
緒言.................................................................................................................................. 1
第一章文獻回顧 ............................................................................................................ 2
第一節免疫反應................................................................................................... 2
一、免疫系統............................................................................................... 2
二、輔助型T細胞第一型與第二型之免疫平衡......................................... 3
三、Th1/Th2 及促/抗發炎細胞激素........................................................... 4
四、免疫與發炎........................................................................................... 5
五、免疫球蛋白........................................................................................... 9
第二節過敏性氣喘介紹..................................................................................... 12
一、過敏性氣喘......................................................................................... 12
二、過敏反應機制與類型......................................................................... 14
三、肥大細胞去顆粒化............................................................................. 15
四、氣喘治療目標..................................................................................... 17
第三節傳統中草藥或香料物質的潛在功用..................................................... 18
第四節精油與免疫調節..................................................................................... 20
第五節研究動機與目的..................................................................................... 21
第二章六種植物水蒸餾精油在體外試驗對小鼠初代脾臟細胞Th1/Th2 平衡之影響
........................................................................................................................................ 24
第一節前言......................................................................................................... 24
第二節 材料與方法........................................................................................... 24
一、六種水蒸餾精油之製備 ................................................................... 24
二、六種植物樣品粉末之水份測定 ....................................................... 28
三、六種水蒸餾精油中總多酚、總類黃酮及總皂苷含量測定............. 28
四、六種水蒸餾精油對BALB/c 雌鼠脾臟細胞細胞毒性之影響.......... 30
五、六種水蒸餾精油對BALB/c雌鼠脾臟細胞細胞激素分泌之影響.... 33
六、水蒸餾精油中總多酚、總類黃酮及總皂苷含量與脾臟細胞細胞激素
分泌之相關性...................................................................................... 36
七、統計分析............................................................................................. 36
第三節 結果....................................................................................................... 37
一、六種植物樣品粉末之水分含量分析................................................. 37
二、六種水蒸餾精油之萃取率................................................................. 37
三、六種水蒸餾精油之總多酚、總類黃酮、總皂苷成分分析............. 37
四、六種水蒸餾精油對BALB/c雌鼠脾臟細胞細胞生長之影響............ 40
五、六種水蒸餾精油對BALB/c雌鼠脾臟細胞細胞激素分泌之影響.... 43
六、六種水蒸餾精油水蒸餾精油中總多酚、總類黃酮及總皂苷含量與脾
臟細胞細胞激素分泌之相關性.......................................................... 51
第四節討論......................................................................................................... 57
第五節結論......................................................................................................... 59
第三章三種水蒸餾精油對小鼠初代巨噬細胞發炎及肥大細胞安定性之影響 ...... 60
第一節前言......................................................................................................... 60
第二節 材料與方法........................................................................................... 60
一、實驗架構................................................................................................ 60
二、金盞花、迷迭香及道手香水蒸餾精油對BALB/c雌鼠初代巨噬細胞促
發炎與抗發炎細胞激素分泌之影響.................................................. 61
三、金盞花、迷迭香及道手香水蒸餾精油對BALB/c雌鼠初代肥大細胞
β-hexosaminidase 釋放之影響.............................................................. 62
四、統計分析................................................................................................ 66
第三節 結果....................................................................................................... 67
一、金盞花、迷迭香及道手香水蒸餾精油對BALB/c雌鼠初代巨噬細胞促
發炎及抗發炎細胞激素分泌之影響..................................................... 67
二、金盞花、迷迭香及道手香水蒸餾精油對BALB/c雌鼠初代肥大細胞
β-hexosaminidase 釋放之影響.............................................................. 75
第四節 討論....................................................................................................... 77
第五節 結論....................................................................................................... 79
第四章道手香水蒸餾精油吸入對正常及過敏性氣喘BALB/c小鼠之影響............. 80
第一節 前言....................................................................................................... 80
第二節 材料與方法........................................................................................... 81
第一部分 道手香水蒸餾精油吸入對正常BALB/c小鼠之影響..................... 81
一、實驗大綱................................................................................................ 81
二、實驗動物飼養與分組............................................................................ 82
三、小鼠犧牲後測定項目............................................................................ 82
第二部分道手香水蒸餾精油吸入對以卵蛋白(ovalbumin) OVA致敏及吸入刺
激之BALB/c小鼠之影響.................................................................. 84
一、實驗大綱................................................................................................ 84
二、實驗動物飼養與分組............................................................................ 85
三、小鼠犧牲後測定項目............................................................................ 86
四、脾臟及腹腔細胞激素分泌量之測定.................................................... 86
五、肺氣管沖洗液(bronchoalveolar lavage fluid, BALF) 取得與分析... 88
六、血液取得與分析.................................................................................... 92
七、肺臟肥大細胞去顆粒化之測定............................................................ 95
八、統計分析................................................................................................ 95
第三節 結果....................................................................................................... 96
第一部分 道手香水蒸餾精油吸入對正常BALB/c小鼠之影響..................... 96
一、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠體重變化之影
響 .......................................................................................................... 96
二、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠臟器絕對與相
對重量量變化之影響.......................................................................... 96
三、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠肺氣管沖洗液
細胞總數與細胞族群比率變化之影響.............................................. 99
四、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠肺氣管沖洗液
中細胞激素分泌量之影響.................................................................. 99
五、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠肺氣管沖洗液
發炎媒介物與趨化素含量之影響.................................................... 102
六、不同劑量道手香水蒸餾精油吸入後對正常BALB/c小鼠脾臟細胞生
長之影響............................................................................................ 102
七、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠脾臟細胞分泌
細胞激素之影響................................................................................ 105
八、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠腹腔細胞分泌
促/抗發炎細胞激素之影響............................................................... 105
九、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠血清中非特異
性IgE、IgA、IgM及IgG抗體分泌之影響........................................110
十、不同劑量道手香水蒸餾精油吸入對正常BALB/c小鼠肺部肥大細胞
去顆粒作用之影響.............................................................................110
第二部分 道手香水蒸餾精油吸入對以OVA致敏及吸入刺激之BALB/c小鼠
之影響..................................................................................................................113
一、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠體重變化之影響.........................................................................113
二、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠臟器絕對與相對重量量變化之影響.........................................113
三、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠肺氣管沖洗液細胞總數與細胞族群比率變化之影響 .............116
四、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠肺氣管沖洗液之發炎媒介物與趨化素含量之影響.................116
五、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠肺氣管沖洗液中細胞激素分泌量之影響.................................119
六、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠脾臟細胞生長之影響................................................................ 121
七、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠脾臟細胞分泌細胞激素之影響................................................ 121
八、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠脾臟細胞分泌促及抗發炎細胞激素之影響............................ 125
九、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠腹腔細胞分泌促及抗發炎細胞激素之影響............................ 125
十、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之BALB/c
小鼠血清中非特異性IgE、IgA、IgM及IgG抗體分泌之影響....... 129
十一、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之
BALB/c小鼠血清中OVA特異性IgG1、IgG2a及IgE抗體分泌之影響
............................................................................................................ 129
十二、不同劑量道手香水蒸餾精油吸入對以卵蛋白致敏及吸入之
BALB/c小鼠肺臟肥大細胞去顆粒作用之影響............................... 134
第四節 討論..................................................................................................... 136
第五節 結論..................................................................................................... 138
第五章總討論與總結 ................................................................................................ 139
第六章參考文獻 ........................................................................................................ 140
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