An on-line high-performance liquid chromatography−diode-array detector−multi-stage mass spectrometry−deoxyribonucleic acid−4′,6-diamidino-2-phenylindole−fluorescence detector system for screening the DNA-binding active compounds in Fufang Banbianlian Injection

Abstract

Fufang Banbianlian Injection (FBI), a well-known traditional Chinese medicine formula, has been recently approved and extensively used as a newly anti-inflammatory and anti-tumor drug. This prescription comprises an equal ratio of three traditional Chinese herbs, Lobelia chinensis Lour, Scutellaria barbata D. Don and Hedyotis diffusa Willd. The relationships between its chemical compositions and activities have not been understood well yet. To investigate the ingredients and their DNA-binding activities in FBI, an on-line high-performance liquid chromatography−diode-array detector−multi-stage mass spectrometry−deoxyribonucleic acid−4′,6-diamidino-2-phenylindole−fluorescence detector (HPLC−DAD−MSn−DNA−DAPI−FLD) system was developed using a combination of chromatographic, mass spectrometric and fluorescent detection techniques. 4′,6-Diamidino-2-phenylindole (DAPI) specifically binds to three ATT base pairs on the DNA minor groove, and thus can be used as a fluorescent probe for screening active compounds that compete ATT sequences with DAPI. Using this system, 21 of 58 identified or tentatively characterized compounds in FBI showed DNA-binding activities, with most of the active compounds being flavone glycosides. In addition, the structure–activity relationships of these active compounds suggested that conjugated planar structures are favorable for DNA-binding activities, and adjacent hydroxyl groups in flavonoids can significantly improve their activities. This is, to the best of our knowledge, the first application of DAPI as a fluorescent probe for the screening of DNA-binding active compounds in complex samples.