Discovery And Mechanism Of Natural Products In Cortex Mori Radicis As Inhibitors Against Intestinal Nitroreductases

Background:The intestinal microbiota is the largest microbiota in the human body.A normal person microbiota is estimated to contain 3.8×10¹³ bacteria,more than human somatic cells.At the genetic level,the intestinal microbiota contains about 30 times more genes than the human genome.As a rich transformation pool comparable to human metabolic enzyme pool,intestinal enzymes have huge metabolic potential.The intake of xenobiotics can affect the composition of intestinal microbiota by inhibiting or promoting the growth of bacteria.More importantly,a variety of enzymes produced by intestinal bacteria will metabolize endogenous and exogenous substances,thus changing their biological activities in the host.The interaction between intestinal microbiota and host will also be affected by various metabolites generated from enzyme metabolisms of intestinal bacteria.Nitro-aromatic compounds are contained in foods,drugs and environmental pollutants.Intestinal bacteria nitroreductases play an important role in the metabolism of nitro-aromatic compounds.Intestinal bacterial nitroreductases metabolize nitro-aromatic compounds,converting their original nitro groups into corresponding hydroxylamines or amino groups,and generate reactive nitro-hydroxy intermediates and aromatic amines.These products can interact with DNA to produce toxic and mutagenic effects.Inhibition of intestinal bacterial nitroreductase is an effective approach to prevent colon DNA damage.Objective:The purpose of this research is to study the inhibitory activity and mechanism of the natural products of Cortex Mori Radicis on the intestinal nitroreductase of,and further explore the inhibitory effect of the natural products on the toxic metabolites of CB1954 at the molecular and cellular levels.Overall,this study is to explore the modulation of intestinal enzyme metabolisms by natural products,and the potential ability of natural inhibitors to alleviate intestinal injury.Methods:Two kinds of intestinal bacteria nitroreductase（Ec Nfs A and Ec Nfs B）were recombined and expressed.By screening the effective inhibitors from Cortex Mori Radicis,the half inhibition concentration,inhibition kinetic parameters,synergistic and antagonistic effects were determined.The interactions between enzymes and inhibitors were further studied through molecular docking simulation.With a system that mimic the process of the intestinal toxicity induced by nitroreductase metabolism of chemotherapy drug CB1954,SOS reactions were performed to evaluate the DNA damage in bacteria strains,which confirmed the effect of the natural product of Cortex Mori Radicis on reducing intestinal damage.Results:The results showed that the flavonoids in Cortex Mori Radicis could effectively inhibit the activities of nitroreductases in different ways.Kuwanon G（1）,kuwanon A（2）,sanggenol A（3）,kuwanon C（4）exhibited dual inhibition on both Ec Nfs A and Ec Nfs B;morusin（5）,morin（6）,sanggenone C（7）were Ec Nfs A inhibitor;kuwanon H（8）and kuwanon E（9）only had inhibition on Ec Nfs B.The inhibition ability of all flavonoids exceeded that of the positive inhibitor dicoumarol.The inhibition kinetics analysis showed that most of the flavonoids blocked the reduction of nitrofurazone catalyzed by nitroreductase in a competitive manner,while morin and kuwanon C were used as non-competitive inhibitors of Ec Nfs A and Ec Nfs B,respectively.Molecular docking simulations showed that these naturally occurring nitroreductase inhibitors were tightly bound to the target enzyme mainly through hydrogen bonds and hydrophobic interactions,and then hinder the interaction of substrate and enzyme.HPLC analysis showed that flavonoids in Cortex Mori Radicis could also inhibit the reduction of CB1954 to toxic metabolites catalyzed by nitroreductase,and the SOS reaction confirmed that natural flavonoids could reduce the drug toxicity produced by CB1954 through inhibiting the activity of nitroreductase.Conclusion:In summary,the inhibitory effects and mechanisms of the major constituents in Cortex Mori Radicis on gut bacterial nitroreductases（Ec Nfs A&Ec Nfs B）were carefully investigated for the first time.Cortex Mori Radicis can reduce the activation of nitro compounds in the intestine by inhibiting the activity of intestinal bacterial nitroreductase.In the study of nitroreductase metabolism CB1954,it was proved that flavonoids from Cortex Mori Radicis can effectively inhibit the production of toxic metabolites and reduce bacterial SOS reaction caused by these metabolites.In summary,the flavonoids in Cortex Mori Radicis can be used as natural inhibitors of intestinal bacteria nitroreductase,and can reduce the intestinal DNA damage caused by drug metabolism,which provides new ideas and effective drugs for reducing intestinal toxicity caused by intestinal bacteria enzyme metabolism.