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研究生:張育甄
研究生(外文):Yu-chen Chang
論文名稱:南嶺前胡莖部促進U937細胞株吞噬作用之研究
論文名稱(外文):Studies on enhancing U937 cells phagocytosis from the stems of Peucedanum longshengense
指導教授:黃秀琴黃秀琴引用關係
指導教授(外文):Shiow-chyn Huang
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:藥物科技研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:U937 細胞株毒性試驗南嶺前胡標示綠色螢光蛋白之克雷白氏肺炎桿菌有效成分分離促進 U937 細胞吞噬作用
外文關鍵詞:cytotoxcity testPeucedanum lonshengenseU937 cell lineenhancing phagocytosis of U937 cellsisolation and identification of active constitue
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本研究利用本實驗室所建立之細胞毒性試驗及促進類人類巨噬細胞 U937 細胞株吞噬作用篩選平台,分離台灣草藥中有效成分。選用台灣產的厚葉捕魚木莖部及葉部 (GBS, GBL)、山胡椒莖部及葉部 (LCS, LCL) 以及南嶺前胡莖部 (PLS),進行水及 95 % 乙醇萃取,分別得到這些草藥之水萃取物及乙醇萃取物。再將萃取物利用 cell counting kit-8 方法進行細胞毒性試驗,及促進 U937 細胞株吞噬標示綠色螢光蛋白之克雷白氏肺炎桿菌 (Klebsiella pneumoniae-gfp) 試驗進行篩選,選擇對 U937 細胞株無毒性及具有最佳促進 U937 細胞株吞噬作用之草藥萃取物。結果顯示南嶺前胡乙醇萃取物 (PLSE) 在 200 �慊/ml 濃度下,對 U937 細胞具有 104.2 % 存活率,及具有最佳 (44.67 %) 之促進吞噬能力。進而利用乙醇進行南嶺前胡之大量 (965g) 萃取,所得萃取物再以 CH2Cl2、EtOAc、n-BuOH 及 H2O 等溶媒進行分配萃取。共得到五個萃取層:二氯甲烷層 (PLSEC)、乳化層 (PLSEE) 、乙酸乙酯層 (PLSEA)、正丁醇層 (PLSEB) 及水層 (PLSEW)。分別以篩選平台進行篩選,結果顯示其中無毒性及最佳促進吞噬能力之萃取層為 PLSEA,並將其利用矽膠管柱層析分離,可獲得六個分層 (PLSEA 1∼6)。其中 PLSEA 5 對 U937 細胞不具細胞毒性,且促進 U937 細胞吞噬能力最佳 (40.27%)。再次利用矽膠管柱層析法分離 PLSEA 5 之成分,共獲得六個分層 (PLSEA 5-1 ∼ PLSEA 5-6) 。六個分層中 PLSEA 5-2 及 PLSEA 5-5 對 U937 細胞不具細胞毒性,且促進 U937 細胞吞噬能力,分別為 53.46 % 及 51.57 %。其有效成分有待進一步分離鑑定。
The purpose of this study is to isolate the active constituents from Taiwanese herbal medicines (THMs) by using a screening platform for immunity-enhancing properties. Three THMs, namely, the leaves and stems of Grewia biloba (GBL, GBS), the leaves and stems of Litsea cubeba (LCL, LCS), the stems of Peucedanum longshengense (PLS), were collected in Taiwan, and H2O and 95% EtOH were used to produce two different extracts of each THM.
U937 cells, a kind of macrophage-like cells, were used for the cytotoxicity test by the cell counting kit-eight method. The number of Klebsiella pneumoniae-gfp phagocytosed by U937 cells was used as a phagocytosis-enhancing assay by THMs. The results showed that EtOH extract of PLS (PLSE) at 200�n�慊/ml gave 104.2% viability of U937 cells, and was the most effective (44.67%) in enhancing phagocytosis of U937 cells. A large amount (965g) of PLS was extracted with EtOH.
The large scale extract was partitioned with CH2Cl2, EtOAc, n-BuOH and H2O. Five layer of extracts, namely, CH2Cl2 layer (PLSEC), emulsion layer (PLSEE), EtOAc layer (PLSEA), n-BuOH layer (PLSEB) and H2O layer (PLSEW), were obtained, and then they were examined by cytotoxicity and phagocytosis tests. PLSEA had the lowest cytotoxicity and the most effective, and it was subjected on a silica gel column chromatography and eluted with CH2Cl2: MeOH (25 : 1). Six fractions, PLSEA 1~6, were collected. PLSEA 5 had non-cytotoxicity and the strongest enhancing phagocytosis (40.27 %) of U937 cells among the six fractions.
PLSEA 5 was further subjected on a silica gel column chromatography and eluted with CHCl3: MeOH: EtOAc (5:1:1). Six fractions, PLSEA 5-1 ~ 5-6, were collected and were evaluated by cytotoxicity and phagocytosis tests. The results demonstrated that PLSEA 5-2 and 5-5 exhibited non-cytotoxicity and effective enhancing phagocytosis of U937 cell, 53.46 % and 51.57 %, respectively. The active constituents of enhancing phagocytosis of U937 cell of PLSEA 5-2 and 5-5 will further need to be isolated and identified.
中 文 摘 要 I
Abstract III
謝 誌 V
目 錄 VI
圖 目 錄 VIII
表 目 錄 XI
第一章 緒論 1
第一節 中草藥與免疫 1
第二節 免疫與疾病 2
第三節 免疫活性物質之評估方法 5
第四節 類人類白血病細胞株 U937 細胞株特性 9
第五節 流式細胞技術 10
第六節 研究動機及目的 10
第七節 厚葉捕魚木、南嶺前胡及山胡椒簡介與其相關之研究 11
第二章 材料與方法 15
第一節 厚葉捕魚木、山胡椒及南嶺前胡之萃取方法 15
第二節 GB,LC 及 PL 之水及 95 % 乙醇萃取物對 U937細胞株之
細胞毒性試驗 17
第三節 GB,LC 及 PL 之水及 95 % 乙醇萃取物促進 U937 細胞株
之吞噬能力試驗 23
第四節 南嶺前胡莖部有效成分大量萃取與分離方法 29
第三章 結果與討論 32
第一節 厚葉捕魚木、山胡椒、南嶺前胡萃取結果 32
第二節 GB,LC 及 PL 之水及 95 % 乙醇萃取物對 U937 細胞株之
細胞毒性試驗結果 33
第三節 GB,LC 及 PL 之水及 95 % 乙醇萃取物促進 U937 細胞株
吞噬能力試驗結果 41
第四節 大量之南嶺前胡莖部乙醇萃取及分離結果 43
第四章 結論 56
參 考 文 獻 58
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