In the present study, an efficient preparative procedure was developed for the preparation of three main acylated pentasaccharides from Polygalae Radix using integrated extraction-adsorption method followed by semi-preparative high performance liquid chromatography. Firstly, the acylated pentasaccharides were simultaneously extracted and purified by column chromatographic extraction integrated with macroporous resin purification. Comparing this integrated process to conventional one, some obvious advantages were observed, namely more than 38.9% enhancement of the recovery of acylated pentasaccharides, about 49.4% and 50.4% reduction in water and ethanol consumption, respectively. And then, the obtained extracts were further purified by semi-preparative high performance liquid chromatography. Finally, three known acylated pentasaccharides were purified from 100 g of Polygalae Radix, including tenuifoliose I (13 mg), tenuifoliose H (15 mg), and tenuifoliose A (21 mg). The purities of the compounds were determined by high performance liquid chromatography and the chemical structures were confirmed by mass spectrometric and NMR analysis. (C) 2015 Elsevier B.V. All rights reserved.