Analysis of Xanthine Oxidase Inhibitors from Clerodendranthus spicatus with Xanthine Oxidase Immobilized Silica Coated Fe3O4 Nanoparticles

In this study, xanthine oxidase immobilized silica coated Fe3O4 nanoparticles (Fe3O4@SiO2-XO) were successfully prepared and characterized by transmission electron microscope, X-ray powder diffraction, Fourier transform infrared spectroscopy and vibrating sample magnetometer. The average diameter of the Fe3O4 nanoparticles was about 300 nm to 350 nm with a shell thickness of 60 nm. The maximum saturation magnetization of the Fe3O4@SiO2-XO nanoparticles was 44.9 emu/g, which ensured the separation from the medium within one minute by using an ordinary magnet. A xanthine oxidase (XO) inhibitor screening method using Fe3O4@SiO2-XO nanoparticles was established and utilized in the extract of Clerodendranthus spicatus. Under the optimized conditions, two compounds were screened out and identified as gardenin B and eupatorin. The half maximal inhibitory concentration (IC50) values of these two compounds were 1.488 μg/mL and 11.197 μg/mL, respectively. The interactions between these two compounds and XO were investigated by the fluorescence spectroscopic method. The results suggested that the quenching effects of gardenin B and eupatorin were due to a static quenching mechanism. Furthermore, gardenin B showed stronger binding capacity than that of eupatorin. In conclusion, this screening method exhibited efficiency and reusability in screening, identification and analysis of enzyme inhibitors from complex mixtures.