| 摘要: | 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 g/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 200g/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. |
