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An efficient and convenient method, three-dimensional (3-D) cell bioreactor coupled with high performance liquid chromatography-mass spectrometry was developed for affinity screening and analysis of multiple bioactive components from herbal medicines. Cancer cells were cultured on a porous scaffold to form a 3-D cell bioreactor. After interacting with live and fixed cells, the HPLC fingerprinting chromatograms of herbal medicine extract were compared to evaluate the binding properties of herbal components on cells. Model anticancer drugs (paclitaxel and resveratrol) and non-anticancer drugs (ketoprofen and penicillin G) were chosen to investigate the feasibility. When cell-drug interaction time was 30 min, the binding degrees of paclitaxel and resveratrol (each 15 μg/ml) were 82.2±7.2% and 66.1±4.1%, and for ketoprofen and penicillin G (each 15 μg/ml) were less than 3%. This method was used to screen bioactive components from Polygonum cillinerve (Nakai) Ohwi (PCO) extract, and the binding degrees of two main components in PCO extract (10 μg/ml), aristolochic acid A and aristolochic acid B, were 63.0±5.1% and 18.8±0.9%, respectively. These results demonstrated that this method was highly specific, efficient and convenient for affinity screening and analysis of bioactive components interacted with cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)