Characterization Of Anti-leukemiacomponents From Indigo Naturalisusing Cell Membranechromatography And Target Identification

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. TCMhasthe "multi-component-multi-target" and its peculiar constituents synergy character, however, active component and its mechanism of TCM hard to explain, which highly limits the progress of the modernizationof TCM. Thus, to clarify the active ingredient and its mechanism is a great challenge for themodernization of TCM.Cell Membrane Chromatography (CMC) is a biological chromatographic technique, this affinity analysis technology is based on the concept of "multi-component-multi-target". It enables rapid identification and separation of the active ingredient of any complex system, such as TCM herbs. In silico target identification couldscreen potential targets for a specific drug or active small molecule compounds through calculational methods.A new strategy based on cell membrane chromatography and in silico target identification technology was established for the rapid active ingredient screening and target identification. The efficient and convenient technology presents new ideas for rapid targetscreening.As a kind of TCM herbs, indigo naturalis has been applied for thousands of years in China. In recent decades, indigo naturaliswas found to have a certain effect for the treatment of leukemia, which makes it become a research focus. Indirubin was an acknowledgedanti-leukemia ingredient in indigo naturalis, but one component can’t well replace the effect of indigo naturalis.This suggests that other anti-leukemia ingredients might exist in indigo naturalis. Therefore, indigo naturaliswas firstly selected to verify the feasibility of the neotype strategy.In this study, a new strategy that combines the CMC system and in silico target identification has been developed.To verify the feasibility of this strategy, indigo naturaliswas selected as an example for active ingredient screening and target identification. Firstly,2D K562/CMC system wasestablished, and three potential active ingredient:indirubin, tryptanthrin and isorhmnetin were successfully screened from indigo naturalis. Secondly, their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Cancer-related targets of indirubin and tryptanthrin have been reported before, however, cancer-related targets of isorhamnetin are still undefined.Next, using in silico target identification, proto-oncogene tyrosine-protein kinase (Src)was screened as a potential anti-tumor target of isorhmnetin. Then, molecular docking, CMC competitive displacement assays, kinase inhibition assays and surface plasmon resonance (SPR) analysis were conducted,and Src was verifiedas a direct target of isorhmnetin. Finally, cell cycle analysis,western-blotting assay and gene interference technique were selected to explore the molecular mechanism, and the cell cycle regulation effect of isorhamnetin was verified through Src/ATR/Wee 1/Cyclin B1/CDK1 pathway.In conclusion, three active components were successfully screened from indigo naturalis, and the target of isorhamnetin was identified. To shed light the anti-leukemiamode of isorhamnetin,molecular mechanism study was conducted.This paper provides a new strategy of active components screening and target identification, and the feasibility of this strategywas verified through indigo naturalis. Based on "multi-component-multi-target" character of TCM,and the advantage of high efficiency, high throughput and easy operation, the strategy will provide new ideas and methods for the modernization of TCM.