目的:建立高效逆流色谱法(HPCCC)从白益母草中分离制备4 个抗氧化成分。方法:白益母草95% 乙醇水提取物经D101 大孔树脂柱色谱,先用水和45% 乙醇水各1 000 mL 洗脱,然后用70% 乙醇水(5 000 mL)洗脱。70% 乙醇水洗脱液减压回收至干,用HPCCC 法以正己烷-正丁醇-水-冰醋酸 (1∶2∶1∶0.1) 为两相溶剂系统,在主机转速 1 000 r·min-1,检测波长 261 nm 条件下进行分离制备,得到化合物1~4,经谱学数据分析,确定其结构式。同时,利用1,1-二苯基-2-三硝基苯肼(DPPH)法测定化合物1~4 的抗氧化活性。结果:从白益母草70% 乙醇水洗脱液中分离得到4 个化合物,分别为5,7,4′-三羟基黄酮-7-O-(6′′-O-反式-咖啡酰基)-β-葡萄糖苷(1)、5,7,4′-三羟基黄酮-7-O-2′′,6′′-O-反式-二香豆酰基)-β-葡萄糖苷(2)、5,7,4′-三羟基黄酮-7-O-6′′-O-反式-香豆酰 基)-葡萄糖苷(3)和毛蕊花苷(4)。化合物1~4 表现出显著的抗氧化活性,其有效半数浓度(EC50)平均值分别为(22.9±2.7)、(26.2±3.4)、(24.4±3.1)和(19.5±1.8)μg·mL-1。结论:建立的方法可高效分离纯化白益母草中可作为天然抗氧化剂的4 个化合物。
Objective: To develop an high performance counter current chromatography(HPCCC) method combined with macroporous resin chromatography for separation and preparation of antioxidant compounds from Panzeria alashanica. Methods: The 95% ethanol extract of Panzeria alashanica was subjected to D101 macroporous resin column and was eluted with water(1 000 mL),45% ethanol(1 000 mL) and 70% ethanol (5 000 mL). The 70% ethanol eluate was recovered to dry under reduced pressure,which was isolated by HPCCC with n-hexane-n-butanol-water-glacial acetic acid(1∶2∶1∶0.1) as a two-phase solvent system with a 1 000 r·min-1 of rotation rate. The antioxidant activities of compounds 1-4 isolated from the 70% ethanol fraction of Panzeria alashanica were tested using 1,1-diphenyl-2-picrylhydrazyl(DPPH). Results: Four compounds 1-4, namely 5, 7, 4′-trihydroxyflavone-7-O-(6′′-O-[E]-caffeyl)-β-glucopyranoside (1), 5, 7, 4′-trihydroxyflavone-7-O-(2′′, 6′′-O-[E]-dicoumaroyl)-β-glucopyranoside(2), 5, 7, 4′-trihydroxyflavone-7-O-(6′′-O-[E]-coumaroyl)-β-glucopyranoside(3) and verbascoside(4), were isolated from the 70% ethanol fraction of Panzeria alashanica. Compounds 1-4 demonstrated significant antioxidant activity. The mean±SD values of EC50 were (22.9±2.7), (26.2±3.4), (24.4±3.1) and (19.5±1.8)μg·mL-1 for compounds 1-4 ,respectively. Conclusion: The established method can effectively separate and purify four compounds which can be used as natural antioxidants.
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