Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography.

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, high-performance liquid chromatography (HPLC) was used for fast screening of G-quadruplex ligands from the crude extract of Kalopanax septemlobus (Thunb.) Koidz, a traditional Chinese medicine. Four potent G-quadruplex ligands were firstly selected through HPLC by comparing the peak profiles and absorption intensity of the crude sample before and after interaction with G-quadruplex DNA. Then the target compounds were isolated and purified by high-speed countercurrent chromatography (HSCCC) for further confirmation of their stabilities of G-quadruplex by temperature-dependent circular dichroism (CD). Four compounds were isolated and identified as 2,4-dihydroxybenzoic acid (I), chlorogenic acid (II), caffeic acid (III) and 5-feruloylquinic acid (IV) each by MS and NMR. Finally, compound I, II, III were each proved to be potent G-quadruplex ligands by decreasing the peak intensity in HPLC chromatogram after complexation with G-quadruplex, which stabilize G-quadruplex by 7±2 °C, 10±2 °C, and 3±2 °C respectively, based on CD analyses. However, compound IV showed no G-quadruplex stability. The decrease of peak absorption intensity in HPLC chromatogram is the most important signal to find G-quadruplex ligands. This provides a very promising strategy for fast screening G-quadruplex ligands from natural plant extracts.