A few methods have been developed to separate and

A few methods have been developed to separate and prepare compounds from L. chinensis, such as silica gel column chromatography [5,6] and high-performance liquid chromatography (HPLC) [7]. Silica gel column chromatography is still used for its simple equipment and low cost. However, this method is often tedious, time-consuming and poor reproducibility, which makes the comprehensive survey of chemical substances in L. chinensis a difficult job. HPLC is the most powerful separation tool nowadays and it has been widely used for isolation of compounds from TCMs [8,9]. Based on chiral HPLC, four novel lobechinenoids A-D were separated from L. chinensis [7]. However, one-dimensional liquid chromatography fails to provide sufficient resolving power for the separation of various compounds with huge differences in category, polarity and concentration. Therefore, a two-dimensional liquid chromatography (2D-LC) method based on CN and ODS columns has been developed to comprehensively separate components in L. chinensis. Using this method, at least 536 components were detected and 6 of them were identified as apigenin 7-O-rutinoside, luteolin, lobetyolinin, lobetyolin, diosmin, and linarin, demonstrating the powerful resolution and high peak capacity of the 2D-LC method in the analysis of complex samples [10]. Reversed-phase liquid chromatography (RPLC) and Fusaric Acid mg interaction liquid chromatography (HILIC) are the most widely used in sample separation, take advantage of their different retention mechanisms favorable for constructing highly orthogonal 2D-LC systems. Based on XTerra C18 and Click β-CD, a 2D-RPLC × HILIC system was developed for the separation of polar and medium-polar components in TCMs [11]. This 2D-RPLC × HILIC system was also used for the isolation of flavonoids from licorice extract [12]. These 2D-LC methods provide a powerful means for the separation and purification of components from TCMs. To the best of our knowledge, it has not yet been reported to isolate new compounds via prepared 2D-LC methods from L. chinensis. Based on chemical constituents obtained from L. chinensis, studying their mechanisms of action is important for lead discovery, as well as elucidation of its clinical feature. Choi et al. evaluated the anti-mycobacterium tuberculosis activity of L. chinensis extracts using resazurin microtiter assay and mycobacteria growth indicator tube 960 system assay and found that L. chinensis extracts could block the growth of mycobacterium tuberculosis through a new pharmacological action [13]. The flavonoid compound luteolin could cause many kinds of tumor cell to die by activation of TNF-α [14], and inhibit dose-dependently HO-8910PM cell proliferation in vitro via inhibition of MMP- 9 secretion and ERK2 expression [15]. Although these studies have provided some understanding about the functions of L. chinensis, the exact molecular mechanisms are still unknown. Label-free cell phenotypic assay, as one of the new generation phenotype-based assay techniques, has become an attractive approach for determining target engagement of drugs [[16], [17], [18]]. This assay uses label-free resonant waveguide grating (RWG) biosensor to convert drug-induced dynamic redistribution of cellular constituents in cells into an integrated and kinetic response, termed dynamic mass redistribution (DMR). The DMR is recorded as a shift in resonant wavelength in picometer (pm) and represents a cell phenotypic response covering a wide range of targets/pathways [[19], [20], [21]]. Furthermore, due to its non-invasiveness, DMR assay can be performed in multiple formats and permits intervention with probe molecules, thus enabling mechanistic elucidation of drug pharmacology [22,23]. In the present work, we will first develop a solid phase extraction method to selectively enrich the flavonoids from the total extract after removing most of the pigment and interfering components. Thus, many minor flavonoid compounds could be also selectively enriched. Considering hydrophilic and hydrophobic properties of the flavonoid chemical structure, a structure-guided method development strategy will be employed to design a 2D-HILIC × RPLC system. The chromatographic conditions will be systematically optimized in the analytical level and the preparative level to improve separation selectivity, peak capacity and peak shape. This method has the advantage of separation and preparation of series of flavonoid compounds with similar structures even at the trace level. Label-free cell phenotypic assays, as one of new generation pharmacological assays, will be used to evaluate bio-activity of flavonoids to discovery their new targets. As far as we know, the combination of 2D HILIC × RPLC method and label-free cell phenotypic assays is first used to isolate and bioactive-evaluate of flavonoid glycosides from L. chinensis. This study will not only provide an effective method for the interpretation of the material basis of TCMs, but also shed light on the mechanisms of action of novel flavonoids..