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研究生: 洪雅菱
Hung, Ya-Ling
論文名稱: 生物活性導向分離鑑定山苦瓜葉抑制牙齦卟啉單胞菌誘導發炎反應的活性物質
Bioassay-guided isolation and identification of anti-inflammatory compounds from wild bitter melon leaf against Porphyromonas gingivalis
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: 2015
畢業學年度: 103
語文別: 中文
論文頁數: 99
中文關鍵詞: 牙周致病菌山苦瓜葉介白素8抗發炎
英文關鍵詞: P. gingivalis, anti-inflammation, wild bitter melon leaf, interleukin-8
論文種類: 學術論文
相關次數: 點閱:158下載:0
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  • 牙齦卟啉單胞菌Porphyromonas gingivalis (P. gingivalis),為厭氧革蘭氏陰性菌,黑色、短桿狀,引起慢性牙周炎的致病菌之一。當宿主受到P. gingivalis感染,入侵口腔表皮細胞後,宿主將啟動免疫反應,刺激細胞激素、化學趨化素分泌,例如:介白素-8 (Interukin-8 ; IL-8),誘導嗜中性白血球移動至發炎處浸潤,而產生了紅、腫的發炎現象。
    山苦瓜(Momordica charantia L. var. abbreviate Seinge) 俱有多種功效,降血糖、降血脂、抗菌、抗發炎等功效。本研究室先前研究自苦瓜葉萃取物分離鑑定數種俱有抗發炎的活性物質,例如:beta-ionone, momordicoside aglycone等。
    本研究以牙齦卟啉單胞菌(P. gingivalis)刺激人類單核球細胞(THP-1)細胞模式,以生成細胞激素介白素-8 (Interukin-8 ; IL-8)作為篩選指標。自山苦瓜葉萃取物分離並鑑定抗發炎活性的組成分:Charantadiol A ( 5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol )—苦瓜二醇。細胞實驗結果顯示能夠抑制P. gingivalis 誘導THP-1細胞分泌IL-6、IL-8;而在動物實驗發現可抑制P. gingivalis 誘導小鼠牙齦組織IL-6和TNF-α mRNA表現。
    本研究結果證實Charantadiol A俱有抗發炎的活性,故推測Charantadiol A俱有抑制牙周病炎和其他P. gingivalis 誘導發炎之相關疾病。

    Porphyromonas gingivalis, a gram-negative anaerobic rod bacterium, is a major periodonto-pathogen that plays a role in the pathogenesis of periodontal disease, which is considered a typical chronic inflammatory disease. Wild bitter melon (WBM; Momordica charantia L. var. abbreviate Seinge) possesses hypoglycemic, hypolipidemic, antibacterial, and anti-inflammatory properties. The aim of this study was to identify the anti-inflammatory active compounds from WBM leaf using P. gingivalis-stimulated human monocytic THP-1 cells in vitro.
    WBM leaves were extracted with ethanol and separated into five fractions by elution of n-hexane/acetone. Among them, the fra5 significantly inhibited P. gingivalis-induced interleukin (IL)-8 by THP-1 cells. Hence, fra5 was subjected to separation and purification by using various chromatographic techniques. Charantadiol A (5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol) was identified by comparing spectral data. Treatment of charantadiol A effectively reduced IL-6 and IL-8 production by P. gingivalis-stimulated human monocytic THP-1 cells in vitro. In addition, charantadiol A also significantly inhibited heat-killed P. gingivalis-induced IL-6 and TNF-a mRNA levels in gingival tissue of mice. In summary, these findings suggest that charantadiol A P. gingivalis-induced cytokine production or expression, suggesting its therapeutic potential in P. gingivalis-related inflammatory diseases.

    中文摘要 i Abstract ii 目錄 iii 圖目錄 v 表目錄 vii 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 Porphyromonas gingivalis與牙周病 3 壹、 前言 3 貳、 牙周病 4 參、 P. gingivalis致病因子 10 肆、 P. gingivalis對宿主誘導之免疫反應 12 伍、 P. gingivalis誘導發炎反應 14 第二節 山苦瓜 18 壹、 前言 18 貳、 成分及功效 19 參、 苦瓜中所含的化合物 21 第三章 材料與方法 24 第一節 實驗架構 24 第二節 研究材料 25 壹、 藥品與試劑 25 貳、 儀器設備 33 第三節 實驗方法 34 壹、 細胞培養 34 貳、 細菌培養 36 中文摘要 i Abstract ii 目錄 iii 圖目錄 v 表目錄 vii 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 Porphyromonas gingivalis與牙周病 3 壹、 前言 3 貳、 牙周病 4 參、 P. gingivalis致病因子 10 肆、 P. gingivalis對宿主誘導之免疫反應 12 伍、 P. gingivalis誘導發炎反應 14 第二節 山苦瓜 18 壹、 前言 18 貳、 成分及功效 19 參、 苦瓜中所含的化合物 21 第三章 材料與方法 24 第一節 實驗架構 24 第二節 研究材料 25 壹、 藥品與試劑 25 貳、 儀器設備 33 第三節 實驗方法 34 壹、 細胞培養 34 貳、 細菌培養 36 參、 樣品萃取與純化分離 38 肆、 細胞存活率測定-MTT assay 41 伍、 in vitro抗發炎活性評估 42 陸、 in vivo抗發炎活性評估 49 柒、 統計分析 52 第四章 結果 53 壹、 山苦瓜葉萃取物之萃取率 53 貳、 山苦瓜花蓮一號葉區分粗萃物對THP-1存活率影 55 響 55 參、 山苦瓜花蓮一號葉區分粗萃物抑制IL-8生成之活性評估 56 肆、 山苦瓜花蓮一號葉區分物抑制IL-8生成之活性評估 57 伍、 Fra 5-3對THP-1細胞存活率影響 58 陸、 Fra 5-3抑制P. gingivalis生成細胞激素之活性 59 柒、 Fra 531、532抑制P. gingivalis誘導生成IL-8的能 60 力 60 捌、 Charantadiol A對於P. gingivalis-induced MAPK 63 訊息傳遞蛋白磷酸化之影響 63 玖、 P. gingivalis-induced NOD1、RIPK2及TREM-1之mRNA表現 65 壹拾、 Charantadiol A 苦瓜二醇A 68 壹拾壹、 In vivo 抗發炎活性評估 71 第五章 討論與結論 72 第六章 參考文獻 80 第七章 附錄 96

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