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研究生: 石雅欣
Shih, Ya-Hsin
論文名稱: 以痤瘡桿菌刺激單核球細胞為模式探討山苦瓜葉萃取物之區分物抑制IL-8生成
Subfractions of wild bitter melon leaf extracts inhibit interleukin-8 induction by P. acnes in THP-1 cells
指導教授: 蔡帛蓉
Tsai, Po-Jung
林麗純
Lin, Lie-Chwen
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: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 139
中文關鍵詞: 痤瘡桿菌山苦瓜葉介白素8抗發炎
英文關鍵詞: P. acnes, wild bitter melon leaf, interleukin 8, anti-inflammation
DOI URL: https://doi.org/10.6345/NTNU202204448
論文種類: 學術論文
相關次數: 點閱:184下載:3
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  • 痤瘡桿菌(P. acnes),屬於革蘭氏陽性厭氧菌(gram-positive anaerobic bacterium),是存在人類皮膚毛囊、口腔、大腸、結膜的正常菌群之一,為尋常性痤瘡皮膚毛囊中的主要致病菌,也與眼內炎、心內膜炎、角膜炎、前列腺發炎癌化等發炎性疾病相關。痤瘡桿菌活化細胞膜上類鐸受體(TLR),啟動下游MyD88與MAPK等訊息傳遞,增加NF-κB與AP-1等轉錄因子活性,造成促發炎介質的生成。
    苦瓜是常見的蔬菜,文獻指出苦瓜(Momordica charantia L.)具有降血糖、降血脂、抗氧化、抗腫瘤、抗菌、抗發炎等作用,其活性成份也陸續被分離出來,像是類黃酮、酚酸、三萜、共軛次亞麻油酸等。先前研究指出,山苦瓜葉甲醇萃取物能夠抑制THP-1人類單核球細胞受痤瘡桿菌刺激之IL-8 (CXCL8)、TNF-α、IL-1β產生,並能降低細胞核內轉錄因子NF-κB,在動物實驗中亦能減緩ICR小鼠耳朵受痤瘡桿菌刺激所引起之紅腫發炎現象。
    本篇研究分析山苦瓜葉之營養成分並接續分離山苦瓜葉甲醇萃取物,以抑制化學趨化激素IL-8為指標,希望找出具有抗發炎活性之山苦瓜葉區分物與成份。 實驗結果顯示,花蓮一號山苦瓜葉含有水份81.22%、粗蛋白6.26%、粗脂肪1.22%、粗纖維1.92%、灰份2.85%、維生素C 1920.14 μg/g drybasis。另外,山苦瓜葉甲醇萃取物之正已烷層區分物Hex-2m能夠顯著抑制痤瘡桿菌誘導之IL-8生成,抑制ERK、JNK、p38蛋白質磷酸化,並且能降低小鼠耳朵受痤瘡桿菌刺激所引起之紅腫發炎現象。透過化學呈色法、NMR、GC-FID、LC-MS分析出山苦瓜葉活性區分物Hex-2m中含有三萜類化合物,脂肪酸部分以α-次亞麻油酸(α-linolenic acid, C18:3 n-3)為主要成份。除此之外,GC-MS資料庫比對到squalene、phytol、維生素K1、維生素E (α-tocopherol、γ-tocopherol)、β-ionone與dihydroactinidiolide等化合物亦是活性區分物Hex-2m中之成份。

    Propionibacterium acnes (P. acnes) is a gram-positive bacterium that forms part of the normal flora of the human skin, oral cavity, large intestine, and conjunctiva. P. acnes plays an important role in acne vulgaris and other diseases, including endophthalmitis, endocarditis, keratitis, and inflammation of the prostate leading to cancer. P. acnes can stimulate toll-like receptor (TLR) on the cell membrance, active MyD88 and MAPK signaling, and increase the activaction of transcription factors NF-κB and AP-1. The signal transduction of TLR leads to the expression of pro-inflammatory mediators.
    Bitter melon (Momordica charantia L.) is a common vegetable and has been reported to possess hypoglycemic, antihyperlipidemic, antioxidant, anticancer, antibacterial, and anti-inflammatory properties. The bioactive compounds in bitter melon, such as flavonoids, phenolic acids, triterpenoids, and conjugated linolenic acids, have been identified. Previous study showed that methanolic extracts from wild bitter melon leaf inhibited P. acnes-induced IL-8 (also called CXCL8), TNF-α, IL-1β, and NF-κB in THP-1 cells and suppressed the inflammatory response in vivo.
    In this study, we analyzed the nutrient contents of Hualien-1 wild btiier melon leaf (H1L) and tried to find the active subfractions and compounds followd by the inhibitory effects on P. acnes-induced IL-8. Our resultes showed the nutrient contents of H1L were 81.22% moisture, 6.26% crude protein, 1.23% crude fat, 1.92% crude fiber, 2.85% ash, and vitamin C 1920.14 μg/g dry basis, respectively. The subfraction, Hex-2m, inhibited the level of P. acnes-induced IL-8 and suppressed ERK, JNK, p38 protein phosphorylation in THP-1 cells. This subfraction also showed an anti-inflammatory activity in P. acnes-stimulated ears of ICR mice. Triterpenoids, fatty acids, including α-linolenic acid, were measured in this subfraction by using colorimetric method, NMR, GC-FID and LC-MS. Squalene, phytol, vitamin K1, α-tocopherol, γ-tocopherol, β-ionone, and dihydroactinidiolide were identified by GC-MS with the library data.

    目錄 i 圖目錄 v 表目錄 ix 中文摘要 x Abstract xi 縮寫表 xii 第一章 緒論 1 第一節、研究動機 1 第二節、研究目的 2 第二章 文獻探討 3 第一節 痤瘡桿菌與發炎 3 壹、前言 3 貳、發炎反應 4 參、TLR singnaling pathway 5 肆、痤瘡桿菌與尋常性痤瘡(Acne vulgaris) 7 一、痤瘡的流行病學 7 二、痤瘡的成因 8 三、痤瘡的治療 11 第二節 實驗材料 13 壹、山苦瓜 13 貳、抗發炎藥物 17 第三章 材料與方法 20 第一節 實驗架構 20 第二節 研究材料 21 壹、藥品與試劑 21 貳、儀器設備 30 第三節 實驗方法 32 壹、樣品萃取與分離純化 32 一、山苦瓜葉甲醇粗萃物製備 32 二、山苦瓜葉區分物製備 32 貳、山苦瓜葉成份分析 38 一、水份含量測定 38 二、粗蛋白含量測定 38 三、粗脂肪含量測定 39 四、灰份含量測定 39 五、粗纖維含量測定 40 六、維生素C含量測定 40 七、總多酚含量測定 41 八、脂肪酸組成分析 41 九、三萜類含量測定 43 十、LC-MS分析 43 十一、GC-MS分析 44 參、細胞培養 45 肆、細菌培養 46 伍、抑菌實驗 48 陸、細胞存活率測定-MTT assay 48 柒、in vitro抑制IL-8生成之活性評估-ELISA assay 49 捌、山苦瓜葉區分物對MAPK蛋白質磷酸化之影響 50 玖、in vivo抗發炎活性評估 54 拾、統計分析 58 第四章 結果 59 第一節 山苦瓜葉萃取物萃取率 59 第二節 山苦瓜葉成份分析 60 壹、山苦瓜葉的營養成份 60 貳、山苦瓜葉甲醇萃取物總多酚含量 61 參、山苦瓜葉區分物成份分析 62 第三節 In vitro study-抗發炎藥物 70 一、細胞存活率測定 70 二、抗發炎藥物抑制IL-8生成之活性評估 70 三、抑菌實驗-luteolin 73 第四節 In vitro study-山苦瓜葉區分物 74 壹、山苦瓜葉partition後各層區分物 74 一、細胞存活率測定 74 二、Partition後各層區分物抑制IL-8生成之活性評估 74 貳、正丁醇層區分物 77 一、細胞存活率測定 77 二、正丁醇層區分物抑制IL-8生成之活性評估 77 參、乙酸乙酯層區分物 80 一、細胞存活率測定 80 二、乙酸乙酯層區分物抑制IL-8生成之活性評估 80 肆、正已烷層區分物 84 一、細胞存活率測定 84 二、正己烷層區分物抑制IL-8生成之活性評估 84 三、抑菌實驗-Hex-2m 84 四、Hex-2m對MAPK訊息傳遞蛋白磷酸化之影響 90 第五節 In vivo study 94 第五章 討論 96 第一節 山苦瓜葉區分物in vitro及in vivo抗發炎活性評估 96 第二節 山苦瓜葉成份探討 99 第三節 總結 106 第六章 參考文獻 108 第七章 附錄 120

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