Screening Inhibitors Against Intestinal Bacterial β-Glucuronidase From Homology Of Medicine And Food

Background:Irinotecan（CPT-11）is a first-line chemotherapy for advanced or metastatic colorectal cancer.Irinotecan and its powerful metabolite SN-38（7-ethyl-10-hydroxycamptothecin）are topoisomerase I inhibitors,which can prevent the formation of lysates and cause DNA strand breaks,thereby killing cancer cell.After that,SN38 is metabolized by UDP-glucuronidase（UGT）in the liver to inactive substance SN38G,which enters the gastrointestinal tract with bile.The commensal bacteriaβ-glucuronidase in the intestine will reactivate SN38G as a toxic product SN38 accumulating in the gastrointestinal tract,cause intestinal epithelial cell damage and delay diarrhea and the dose limitation of CPT-11 will affect the therapeutic effect.It can be seen that the activity ofβ-glucuronidase is particularly important for the degree of secondary activation of SN38G.Therefore,screening of high-efficiency and broad-spectrum inhibitors provides an effective way to relieve the clinical side effect of chemotherapy-induced diarrhea.Objective:This study screensβ-glucuronidase inhibitors from natural Chinese herbal medicines and compound preparations,developing its structural properties in vitro and preliminary inhibition mechanisms in vivo,to expore the auxiliary preparations for alleviating chemotherapy-induced diarrhea.Methods:（1）The activity detection system ofβ-glucuronidase was constructed with p-nitrophenol-β-D-glucuronic acid（PNPG）as a substrate,and the active components of Mulberry bark,Caryophylli flos,and Ginkgo biloba were detected to analyse its inhibitory effect,and explore its inhibitory mechanism from both practical and theoretical aspects through molecular dynamics and molecular simulation,respectively.（2）The mouse model of preventive diarrhea was induced by intraperitoneal injection of irinotecan,and then treated with different concentrations of compound Chinese herbal medicine（Huangqin Tang）.During the experiment,the mice’s diet,water intake,body weight,colon and abdominal cavity were observed,then detecting the changes inβ-glucuronidase activity in the colon and feces.Results:（1）Some active components in Mulberry bark,Caryophylli flos,and Ginkgo biloba all showed strong inhibitory effect onβ-glucuronidase.Among them,the amentoflavone from Ginkgo biloba had a strong effect on allβ-glucuronidase from three kinds of bacteria,with IC₅₀ values of 2.36μM,2.88μM and 3.43μM,respectively.The strong inhibitory compounds selected above are all non-competitive inhibitors ofβ-glucuronidase.The results of enzyme-inhibitor docking showed that morin,sanggenon C,3,4,8,9,10-Pentahydroxy Urolithin and amentoflavone all can form strong interactions withβ-glucuronidase through the form of hydrogen bonds.In addition,the amentoflavone can be linked to the non-catalytically active sites ofβ-glucuronidase from three bacteria sources through the form of hydrogen bonds,thereby exerting a role.（3）The results of animal experiments showed that the compound Chinese herbal medicine（Huangqin Decoction）can obviously alleviate the damage of the colon tissue of mice with diarrhea induced by irinotecan.In addition,theβ-glucuronidase activities of the CPT-11 group in the colon and feces were significantly higher than that of the NC group,and the GUS activity of the H+CPT-11 group in the treatment group was significantly lower than that of the CPT-11 group approached the normal group.It was also found that the fecal GUS activity and the therapeutic effect were dose-dependent with Huangqin Decoction.The results showed that medium and high doses of Huangqin Decoction can inhibit the activity of GUS in the intestine,while the inhibitory effect of low doses may need to be further explored.Conclusion:In this study,inhibitory effects on bacterialβ-glucuronidase of natural products in Mulberry bark,Caryophylli flos and Ginkgo biloba were carefully investigated.It was discovered for the first time that the amentoflanone exhibited strong inhibitory effects onβ-glucuronidase with broad potency.The Morin,Sanggenon C and3,4,8,9,10-Pentahydroxy Urolithin were all non-competitive inhibitors ofβ-glucuronidase via strong interactions with Ec GUS.In addition,the compound preparation（Huangqin Decoction）can effectively alleviate the colon injury in mice with diarrhea induced by irinotecan,In addition,the results of in vivo experiments in mice showed that the enzyme activity level of the high-dose treatment group was significantly lower than that of the CPT-11 group,suggesting that the compound preparation（Huangqin Decoction）can effectively alleviate the colon injury in mice with diarrhea induced by irinotecan.These results indicated that natural Chinese herbal medicines are precious source for development of broad-spectrum inhibitors ofβ-glucuronidase,which will provide a great expectation for application as an adjuvant drug to relieve chemotherapy-induced diarrhea.