On-line study of flavonoids of Trollius chinensis Bunge binding to DNA with ethidium bromide using a novel combination of chromatographic, mass spectrometric and fluorescence techniques

Abstract

The study of the interaction between drugs and DNA is an important way to understand the role of drug molecules. A novel online analytical method for this purpose combining high-performance liquid chromatography-diode array detector-electrospray ionization-ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS(n)) and DNA-ethidium bromide detection with a fluorescence detector (DNA-EB-FLD) was firstly developed, which could rapidly identify the chemical constituents and obtain the profile related to DNA binding activity. This method has been applied for a precise or probable identification of the chemical constituents by ultraviolet (UV) absorption and MS(n) data analysis, while the DNA binding profile has been characterized by directly measuring the fluorescence intensity of compound-DNA-EB. Using this method, Trollius chinensis Bunge was studied and 18 constituents were identified by MS(n) data; six of them (4'-methoxy-2″-O-(2‴-methylbutyryl)vitexin,2″-O-(3‴-methoxycaffeoyl)vitexin) and 4'-methoxy-2″-O-(2‴-methylbutyryl)orientin,acacetin-7-O-rutinoside,quercetin-3-O-xylosylglucoside,quercetin-3-O-arabinosylglucoside) were identified for the first time in T. chinensis Bunge, and 16 constituents accounted for its activity of binding to DNA. The established (HPLC-DAD-ESI-IT-TOF-MS(n) DNA-EB-FLD) system has proved to offer a useful strategy for correlating the chemical profile with the binding to DNA activities of the components without their isolation and purification, and may be used for multicomponent analysis of active substances in other herbs.