A novel method for simultaneously screening of superoxide anion scavenger and xanthine oxidase inhibitor using fluorescent probe hydroethidine coupled with high-performance liquid chromatography-mass spectrometry
Excess reactive oxygen species can cause cellular damage, and involve in many pathological progresses such as inflammation, atherosclerosis and cancer. Reactive oxygen species can be generated by several mechanisms, one of which involves the reaction of xanthine oxidase with xanthine to generate superoxide anion and uric acid. In this study, a new method using hydroethidine as a fluorescent probe coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) was developed for simultaneously screening of xanthine oxidase inhibitor and superoxide anion scavenger. The method was validated using the known superoxide anion radical scavenger L-ascorbic acid and xanthine oxidase inhibitor febuxostat. As a result, six compounds, including salvianolic acid C, quercetin, apigenin, α-tocopherol, formononetin and aloin, were successfully screened by this method. Compared with the traditional colorimetric methods, the developed method was sensitive, accurate and fast. This work demonstrated that the developed fluorescent probe method coupled with HPLC-MS was effective for simultaneously discovering xanthine oxidase inhibitors and superoxide anion radical scavenger from natural product libraries.