5b). complex mechanism of herbal medicines. Traditional Chinese Medicine (TCM), a complete system of healing developed in ancient China, is receiving more and more attention in China and throughout the world in recent decades1,2. However, the essential elements in TCM natural herbs have not been clearly recognized and their exact mechanisms and focuses on have yet to be discovered, which seriously delays its integration into the modern health-care system3,4. How to characterize the active ingredients in TCM and their molecular focuses on is still probably the most demanding task at present5,6. Therefore, the development of a new strategy is extremely essential to display active ingredients and elucidate drug-target relationships7. Traditionally, the PEG6-(CH2CO2H)2 parts in TCM formulae were separated and recognized by phytochemical methods, and evaluated by pharmacological assays for his or her molecular focuses on and mechanism, but the processes were incomprehensive, time-consuming and also inefficient8. In recent years, cell membrane chromatography (CMC) has been extensively utilized for active PEG6-(CH2CO2H)2 component testing and identification based on the relationships between membrane receptors and active elements9,10,11. It combines the advantages of both biomaterials and classic chromatography and realizes online and high-throughput screening of potential active ingredients from complicated biological samples12. In our earlier study, a comprehensive two-dimensional (2D) high throughput testing system was firstly founded which have successfully screened several active parts from TCM natural herbs8,13. This biological chromatographic technique offers been proven to be a powerful approach to screening active parts from TCM. target identification, also known as reverse testing, is a technique that can be used to fast determine potential focuses on of small molecules and construct drug-target networks14. This approach has been successfully used to identify fresh potential biological focuses on for known compounds, and focuses on for compounds among a family of related receptors15,16,17. In recent years, a large number of computational target fishing methods and data bases have been developed18,19. In light of the existing huge amounts of parts in TCM and their complex targets, this method may be a desired strategy to explore the ingredient-target connection and the practical mechanism underlying the multi-component mixtures in the molecular level. In this study, a new strategy that combines the comprehensive 2D K562/CMC system and target identification has been developed to characterize active parts and their focuses on in (Qingdai), an important TCM herb that has been used in several effective formulae for leukemia20,21. As demonstrated in Fig. 1, a comprehensive 2D K562/CMC system was first founded for screening potential active parts from Qingdai. Then, the anti-leukemia effects of the screened parts were verified by cell viability and apoptosis assays. Next, target identification methods were employed for target screening. Compound-receptor relationships were further confirmed by molecular docking, CMC competitive displacement assays, kinase inhibition assays and surface plasmon resonance (SPR) analysis. Furthermore, receptor mediated molecular mechanism of K562 cell cycle PEG6-(CH2CO2H)2 regulation was analyzed. This novel strategy and strategy may provide a new way for characterizing active parts from TCM and additional complex systems and their focuses on. Open in a separate window Number 1 The circulation diagram of characterizing anti-leukemia parts and their focuses on from from the combination of comprehensive 2D K562/cell membrane chromatographic system and target identification. Results Recognition of active parts by comprehensive 2D K562/CMC system A comprehensive two-dimensional K562/CMC system was first founded based on our CT19 earlier study8,13 (Fig. 2a,b). Two standard medicines, dexamethasone (binding to intracellular glucocorticoid receptor) and imatinib (acting on membrane receptor c-Kit and PDGFR) were selected to demonstrate the feasibility and selectivity of the proposed 2D K562/CMC system. The three-dimensional (3D) PEG6-(CH2CO2H)2 PEG6-(CH2CO2H)2 storyline of mixed requirements was demonstrated in Fig. 2c. As expected, dexamethasone has small retention behavior on K562/CMC system, while imatinib has a significant retention characteristic within the K562/CMC model. Open in a separate window Number 2 Building and software of the proposed comprehensive 2D K562/CMC system Scheme of the 2D K562/CMC system.(a) K562/CMC column was equilibrated, and the 1st fraction was collected in pre-column 1. (b) The 1st portion was analyzed by a C18 column coupled with TOFMS while the 2nd portion was collected in pre-column 2, then into the C18 column and TOFMS for analysis, alternately. (c) 3D storyline of mixed requirements acquired by 2D K562/CMC system. Dexamethasone was used as bad control, and showed no retention behavior on K562/CMC. Imatinib was used as positive control, and showed a significant retention characteristic within the K562/CMC. (d) 3D.