采用电喷雾质谱(ESI-MS)、紫外吸收光谱(UV)以及荧光光谱结合溴化乙锭荧光探针研究黄酮类化合物木犀草 素-7-O-葡萄糖苷与双链DNA的相互作用. 质谱研究结果表明木犀草素-7-O-葡萄糖苷可与双链DNA形成复合物, 且Lug与DNA之间存在氢键作用, 通过串联质谱数据推断出其结合模式为插入型. 紫外吸收光谱研究结果表明DNA的加入使木犀草素-7-O-葡萄糖苷产生明显的减色红移效应, 说明该化合物可能插入DNA双螺旋碱基对间. 荧光光谱研究结果表明木犀草素-7-O-葡萄糖苷能引起溴化乙锭-DNA体系荧光强度明显减弱和波长轻微蓝移, 主要是单一的静态猝灭, 猝灭常数K_q=8.61×10¹¹ L·mol^-1·s^-1, 进一步说明Lug与DNA的主要作用方式为插入型.

The interaction between luteolin-7-O-glucoside and duplex DNA was investigated by ESI-MS, ultraviolet and fluorescence spectroscopy in conjunction with fluorescent probe ethidium bromide (EB). We synthetized a critical sequence of GC-rich human survivin promoter DNA for ESI-MS investigation. The stock solutions of DNA and the drug for MS analysis were diluted with methanol/20 mmol/L ammonium acetate (20:80, V/V) solvent, giving a final concentration of DNA of 5 and 20 μmol/L for luteolin-7-O-glucoside. In negative ion mode, we found the duplex DNA/drug complex as well as complex of single-strand oligodeoxynucleotide/luteolin-7-O-glucoside in ESI-MS spectrum. At the same time, it can be seen that the flavonoid shows 1:1, 1:2 and 1:3 binding stoichiometries. In positive ion mode, only 1:1 complex was observed from the spectrum. This suggests that the complex between the drug and the duplex has hydrogen bond interaction. The spectra of tandem mass spectrometry reveal that the 5- charged 1:1 complex underwent the predominant loss G base and a little of losing a neutral drug. Moreover, for 6- charged 1:1 complex of duplex and luteolin-7-O-glucoside, its fragmentation pathway is loss of a 1-charged drug yielding a 5- charged DNA ion. The results of MS/MS data demonstrate that luteolin-7-O-glucoside is an intercalator. UV data were measured by addition of ct-DNA (80 μmol/L) to Lug (20 μmol/L)-20 mmol/L ammonium acetate solution. The result shows obvious hypochromic and red shift effect, indicating that the compound may be inserted between the pairs of the DNA helix bases. The fluorescence experiments were obtained by the drug (final concentrations: 0—66.66 μmol/L) titrating certain EB-DNA system (c_(EB)=3 μmol/L, c_(ct-DNA)=9.78 μmol/L) in 20 mmol/L ammonium acetate solvent. The emission spectra were recorded at an excitation wavelength of 520 nm. The results show that luteolin-7-O-glucoside can quench the fluorescence of EB-DNA system, and the wavelength of EB-DNA hypsochromic shift. The quenching of luteolin-7-O-glucoside on the fluorescence of EB-DNA is a static quenching process, quenching constant K_q=8.61×10¹¹ L·mol^-1·s^-1. This reveals that luteolin-7-O-glucoside has only one mode to interact with duplex DNA. Based on the above mentioned experimental results, we can conclude that luteolin-7-O-glucoside is an intercalator. These results are expected to provide a basis to understand the DNA-binding properties of flavonoids and useful information for development of new efficient duplex DNA ligands.