Discovery and characterization of the flavonoids in Cortex Mori Radicis as naturally occurring inhibitors against intestinal nitroreductases

Gut bacterial nitroreductases are found to be heavily related with the intestinal toxicity of nitroaromatic compounds in food or medicine, which can be converted into mutagenic and enterotoxic nitroso or N-hydroxyl intermediates. Thus, inhibiting the gut microbe-encoded nitroreductases has become an attractive method to reduce the mutagen metabolites in colon and prevent intestinal diseases. In this study, the inhibitory effects of sixteen constituents in Cortex Mori Radicis on two kinds of gut bacterial nitroreductases (EcNfsA and EcNfsB) were evaluated with nitrofurazone (NFZ) as substrate and NADPH as electron donor. The results clearly demonstrated that four flavonoids including kuwanon G, kuwanon A, sanggenol A and kuwanon C showed dual inhibition on both EcNfsA and EcNfsB mediated NFZ reduction; morusin, morin, and sanggenone C were strong inhibitors towards EcNfsA; kuwanon H and kuwanon E exhibited effective inhibition on EcNfsB. Further inhibition kinetic analysis and molecular docking simulations displayed that all inhibitors above suppressed both EcNfsA and EcNfsB activities in competitive manners, except non-competitive inhibition of morin on EcNfsA and non-competitive inhibition of kuwanon C on EcNfsB, respectively. Taking together, these findings revealed that most flavonoids in Cortex Mori Radicis presented effective inhibition on gut microbial nitroreductases, suggesting that Cortex Mori Radicis might be a promising candidate for ameliorating nitroreductases mediated intestinal mutagenicity.