利用本研究室所建立之細胞毒性試驗及促進 U937 細胞吞噬作用篩選平台，進行台灣草藥有效成分之分離。本研究選擇台灣產之白英 (SL)、山煙草 (SV)、蛇莓 (DI)、石上柏 (SD) 及南嶺前胡 (PL) 等五種台灣草藥進行 H2O 及 EtOH 萃取，可得到台灣草藥之水萃取物及乙醇萃取物。
本研究使用 U937 細胞株;為一種類人類的巨噬細胞，以 cell counting kit-8 方法進行U937 細胞株之細胞毒性試驗，及促進 U937 細胞株吞噬標示有綠色螢光蛋白之克雷白氏肺炎桿菌 (Klebsiella pneumoniae-gfp) 試驗之方法篩選五種台灣草藥的水萃取物及乙醇萃取物。結果顯示蛇莓乙醇萃取物在 100 μg/ml 濃度下，對 U937 細胞有 114.9% 的存活率及最佳促進吞噬能力 (63.52%)，繼而進行蛇莓乙醇之大量 (1.7 kg) 萃取，並使用 H2O 及 CH2Cl2 兩種溶媒進行分配萃取，可得二氯甲烷層 (50.00 g)、乳化層 (40.00 g) 及水層 (109.27 g) 等萃取層，並將此三層萃取物進行細胞毒性試驗及促進 U937 細胞株吞噬作用，發現二氯甲烷層 (DIEC) 在 100 μg/ml 濃度下，對 U937 細胞具有最高存活率 (96.7%) 及最佳之促進吞噬作用 (72.74%)。繼而將 DIEC 進行矽膠管柱層析分離並以 CH2Cl2 及MeOH (5：1) 進行沖提，共得到 DIEC1 ~ DIEC5 五個分層，再將這五個分層分別進行細胞毒性試驗及促進 U937 細胞株之吞噬作用，結果顯示 DIEC3 分層在 50 μg/ml 濃度下，有 102.8% 的 U937 細胞存活率及最佳促進 U937 細胞株吞噬效果 (81.59%)，因此選擇DIEC3 繼續進行矽膠管柱層析並以 C6H6 及 CO(CH3)2 (25：1) 進行沖提分離，將所得 DIEC3-1 ~ DIEC3-6 等六個分層進行細胞毒性試驗及促進 U937 細胞株之吞噬作用，由試驗結果得到 DIEC3-4 及 DIEC3-5 分層對 U937 細胞的毒性較低且分別具有 58.72% 及 42.67%較高之促進 U937 細胞株之吞噬效果，其有效成分有待進一步分離鑑定。 The purpose of this study is to use a screening platform for immunity-enhancing function and to isolate the active constituents from Taiwanese herbal medicines (THMs). Five Taiwanese herbal medicines, including Solanum lyratum (SL), Solanum verbascifolium (SV), Duchesnea indica (DI), Selaginella doederleinii (SD) and Peucedanum longshengense (PL), were collected in Taiwan. They were respectively extracted with H2O and EtOH, for corresponding extracts.
U937 cells, kind of macrophage-like cells, were used for the cytotoxicity test by cell counting kit-8 method. The number of Klebsiella pneumoniae-gfp phagocytosed by U937 cells was used as phagocytosis-enhancing assay by THMs. These two methods were applied to screen H2O and EtOH extracts of five THMs, respectively. The results showed that EtOH extract of DI (DIE) at 100 μg/ml gave 114.9% viability of U937 cells and was the most effective (63.52%) in enhancing phagocytosis of U937 cells. A larger amount (1.7 kg) of DI was extracted with EtOH, partitioned, and extracted with CH2Cl2 and H2O solvents. Three layers extracts, namely, CH2Cl2 layer (DIEC), emulsion layer (DIEE) and H2O layer (DIEW) extracts were respectively obtained. The three layers extracts were examined by cytotoxicity and phagocytosis tests. We found DIEC at 100 μg/ml had 96.7% viability of U937 cells and the most effective (72.74%) in enhancing phagocytosis of U937 cells. The DIEC extract was subjected to chromatography on a silica gel column and eluted with CH2Cl2-MeOH (5:1) to give five fractions. The five fractions were again screened by cytotoxicity and phagocytosis tests. The results show that DIEC3 at 50 μg/ml had 102.8% viability of U937 cells and the 81.59% in enhancing phagocytosis of U937 cells. DIEC3 was subjected to chromatography on a silica gel column and eluted with benzene-acetone (25:1) to give six fractions. The six fractions were again screened by cytotoxicity and phagocytosis tests, and the results show that DIEC3-4 and DIEC3-5 had lower cytotoxicity and more effective in enhancing phagocytosis of U937 cells. The active constituents to enhance immune system in DIEC3-4 and DIEC3-5 remain to be isolated.
