For comprehensive utilization of Polygonum cuspidatum Sieb. et Zucc., a systematic, environmental friendly preparative process was developed for purification of polydatin, resveratrol and anthraglycoside B simultaneously from the herb. The process was simple, consisting of macroporous resin adsorption and reversed-phase liquid chromatography. Macroporous resin adsorption separated the solvent extract into fraction I containing polydatin and fraction II containing resveratrol as well as anthraglycoside B. The fractions I and II were further purified by reversed-phase chromatography to get the fractions corresponding to polydatin, resveratrol and anthraglycoside B, respectively. Two polydivinylbenzene microspheres (PDVB-SPG and PDVB-Swelling) were prepared by SPG membrane emulsification and by two-step activated swelling polymerization, respectively. These new media were compared with a commercial C-18 bonded silica medium in the reversed-phase liquid chromatography step. The PDVB-SPG column demonstrated a better separation compared with C-18 and PDVB-Swelling column. The pH value of the mobile phase showed little effect on the separation of polydatin and resveratrol, and large effect on the separation of anthraglycoside B for both HPLC analysis and preparative reversed-phase liquid chromatography. After the chromatographic process and a further crystallization, polydatin, resveratrol and anthraglycoside B were purified from 7.5%, 1.3% and 2.4% to 98.8%, 98.2% and 98.6%, respectively, with total recoveries of 81.3%,77.4% and 80.2%. (C) 2009 Elsevier B.V. All rights reserved.