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研究生: 李宥苡
You-Yi Li
論文名稱: 山苦瓜萃取物暨其區分物對於痤瘡桿菌誘導發炎反應的影響
Effects of wild bitter melon extracts on Propionibacterium acnes-induced inflammation
指導教授: 蔡帛蓉
Tsai, Po-Jung
學位類別: 碩士
Master
系所名稱: 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 138
中文關鍵詞: 痤瘡痤瘡桿菌山苦瓜發炎
英文關鍵詞: acne, Propionibacterium acne, Momordica Charantia, inflammation
論文種類: 學術論文
相關次數: 點閱:211下載:11
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  • 痤瘡(acne)俗稱青春痘,其致病因子複雜。過度皮脂生成(sebaceous hyperproduction)、毛囊過度角質化(follicular hyperkeratinization)、痤瘡桿菌增生(Propionibacterium acnes colonization)和發炎反應(periglandular dermal inflammation)是重要的致病因子。其中痤瘡桿菌(P. acnes)被認為在痤瘡發展過程中扮演重要的角色,P. acnes藉由活化單核球細胞(monocytes)與角質細胞(keratinocytes)的toll-like receptor,啟動下游的訊息傳遞路徑產生發炎介質誘導痤瘡發炎情形;另外P. acnes誘發產生活性氧分子(reactive oxygen species, ROS) 與基質金屬蛋白酶(matrix metalloproteinases, MMP)亦參與發炎反應。苦瓜(Momordica charantia)萃取物具有抗發炎、抗氧化能力。因此,本研究欲探討台灣山苦瓜(Momordica charantia Linn. var. abbreviata Ser.; wild bitter melon)萃取物是否具有抑制P. acnes生長與其誘發之發炎反應的效用。
    製備數種山苦瓜萃取物,收集山苦瓜樣品(育種之花蓮1~4號,67-11品系與野生品種)之不同部位(果實、葉、藤),果實以溶劑(乙醇、正己烷、乙酸乙酯和水)進行萃取,而葉以甲醇進行萃取。以broth dilution method實驗的結果發現67-11與野生品種葉、藤甲醇萃物能抑制痤瘡桿菌生長。以P. acnes刺激THP-1 monocytes的模式發現山苦瓜果實乙酸乙酯和乙醇萃取物與葉子甲醇萃物具in vitro抗發炎作用。為進一步探討山苦瓜果實萃取物之區分物(fractions)的抗發炎作用,取得皂化物、非皂化物、oleic acid, linoleic acid, α-linolenic acid, conjugated-linolenic acid, phytol和lutein,結果顯示不皂化物和lutein抑制cytokine和MMP-9表現量,皆具有in vitro抗發炎作用,而長鏈脂肪酸(oleic acid, linoleic acid, α-linolenic acid, CLN)僅降低IL-1β濃度。另發現中鏈脂肪酸(capric acid和lauric acid)能夠減少P. acnes刺激THP-1 monocytes的cytokines產生。在in vivo抗發炎實驗,將P. acnes注射至ICR小鼠耳朵測試樣品的抗發炎能力,結果顯示山苦瓜果實EA萃物、capric acid、山苦瓜葉子甲醇萃物與總多酚萃取物能夠抑制P. acnes誘發的發炎反應與耳朵腫脹情形。
    綜合論述,山苦瓜果實EA萃物與葉子甲醇萃物在in vitro and in vivo實驗中顯示具有抑制P. acnes誘發的促發炎介質與小鼠耳朵腫脹情形,而果實EA萃物中含的lutein與葉子中含的總多酚可能是抗發炎的有效成份。

    Acne vulgaris is a common skin disease involving pilosebaceous follicle. The pathogenesis of acne vulgaris is multifactorial, including increased sebum production, comedogenesis and Propionibacterium acnes proliferation. P. acnes plays an important role not only in the process of inflammation but in the formation of comedones. Previous studies have shown P. acnes would activate monocytes to secrete pro-inflammatory cytokines, some studies have found that matrix metalloproteinases (MMPs) will participate in the progression of acne. Wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) possesses numerous pharmacological actions such as antibacterial, antioxidant, anti-diabetic, and anti-inflammatory activities. The aim of this study was to evaluate the anti-microbial activity as well as in vitro and in vivo inhibitory effect of wild bitter melons extracts and carpic acid on P. acnes-induced inflammation.
    Our results showed that leaves and vine extracts from #67-11 and wild varirties of wild bitter melons effectively inhibited the growth of P. acnes. The ethyl acetate and ethanol extract from WBM fruit significantly reduced IL-8, TNF-α, IL-1β and MMP-9 levels by P. acnes-stimulated THP-1 cells. We further evaluated the fractions and composition of WBM fruit EA extract, the results showed that saponification and unsaponification fractions of WBM fruit, phytol, lutein, capric acid, and lauric acid can reduce cytokine levels. Unsaponification fraction of WBM fruit, lutein, and lauric acid can reduce MMP-9 protein levels. Fatty acids including oleic acid, linoleic acid, α-linolenic acid and conjugated linolenic acid reduced IL-1β production. In addition, ethyl acetate extract of WBM fruit, capric acid, methanolic extract and total phenol extract of WBM leaves reduced P. acnes-induced inflammation of mice ear and then showed in vivo anti-inflammatory activity.
    Our results suggested that wild bitter melon extracts have anti-inflammatory effects against P. acnes and may be useful in the adjuvant treatment of acne.

    第一章 緒論 1 壹、研究動機 1 貳、研究目的 2 第二章 文獻探討 3 第一節 皮膚結構 3 第二節 痤瘡的致病機轉 5 壹、痤瘡的成因 5 貳、痤瘡桿菌與發炎反應 7 (一) 細胞外基質(Extracellular matrix, ECM) 10 (二) 基質金屬蛋白酶(Matrix metalloproteinases, MMPs) 10 參、 誘發發炎的訊息傳遞路徑 12 肆、 痤瘡與基質重塑(Matrix remodeling) 16 第三節 痤瘡的治療 18 第四節 實驗材料 19 一、山苦瓜 19 二、脂肪酸 23 第三章 材料與方法 30 第一節 實驗架構 30 (一) 抗菌與抗發炎評估 30 (二) 萃物暨其區分物抗發炎評估 30 第二節 樣品製備 31 第三節 研究材料 32 一、藥品與試劑 32 二、儀器設備與耗材 34 三、細胞培養 35 四、細菌培養 36 第四節 實驗方法 38 一、抑菌實驗 38 二、細胞存活率測定 (MTT) 38 三、抗發炎活性評估 39 四、轉錄因子nuclear factor-kappa B (NFκB)活化分析 40 五、MMP-9蛋白質表現量評估 42 六、動物實驗 (in vivo抗發炎能力評估) 46 七、脂肪酸成分分析 50 八、總多酚含量測定 51 九、Maximum inhibition(%)計算 52 十、統計分析 52 第四章 結果與討論 53 第一節 山苦瓜萃取物之抑菌與抗發炎作用 54 壹、山苦瓜萃取物之萃取率 54 貳、抑菌實驗 54 參、細胞存活率測定 54 肆、In vitro抗發炎活性評估 55 伍、討論 63 第二節 評估山苦瓜果實萃物暨其區分物之抗發炎作用 66 壹、In vivo anti-inflammatory effect of WBM EA extract 66 貳、皂化物與不皂化物之成分 67 參、細胞存活率測定 67 肆、In vitro抗發炎活性評估 68 伍、討論 87 第三節 評估山苦瓜葉甲醇萃物與其多酚萃物之抗發炎作用 94 壹、總多酚含量測定 94 貳、細胞存活率測定 94 參、In vitro抗發炎活性評估 95 肆、轉錄因子nuclear factor-kappa B (NFκB)活化分析 95 伍、In vivo anti-inflammatory effect 96 陸、討論 105 第四節 評估capric acid之抗菌、抗發炎作用 107 壹、抑菌實驗 107 貳、細胞存活率測定 107 參、In vitro抗發炎活性評估 107 肆、In vivo anti-inflammatory effect 108 伍、討論 115 第五章 綜合討論 118 第六章 參考文獻 127 第七章 附錄(動物實驗同意書) 137

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