Establishment Of Human Microbiota-associated Rat Model And Its Application In The Study Of Flavonols

Gut microbiota can participate in drug metabolism and thus have an important effect on drug exposure in vivo,thereby altering the pharmacokinetics and pharmacodynamic properties of drugs and related metabolites.However,there are significant species differences in these metabolisms mediated by gut microbiota.Flavonols are the active components of many kinds of traditional Chinese medicines and plant medicines,which have the pharmacological activity of protecting cardiovascular health,and mainly exist in the form of glycosides.The systemic exposure levels of the unchanged and its metabolites were significantly lower than the concentrations required to exhibit their biological activities,which challenged the elucidation of the pharmacodynamic material basis and mechanism of flavonols.Flavonols can interact with gut microbiota,there are two steps in the metabolism of flavonol glycosides under the human gut microbiota: the flavonol glycosides are deglycosylated to form aglycone,and the aglycone through ring fission to form phenolic acids.However,the rats’ gut microbiota can only mediate the first-step metabolism of flavonol glycosides(the second-step metabolism is very weak),which is not conducive to the pharmacokinetic and pharmacodynamic studies of flavonols in rats.To solve the problem of species differences in the metabolism of gut microbiota,we constructed a human microbiota-associated rat model by fecal microbiota transplantation.Quercetin,Isoquercitrin(Isoquercitrin,quercetin-3-O-glucoside),Rutin(Rutin,quercetin-3-O-rutinoside)were used as research compounds.Human feces were collected in an anaerobic preservation solution to prepare suspension(in vitro metabolism experiments showed that the gut microbiota of human feces could mediate the deglycosylation of Rutin to produce quercetin and its ring fission to produce 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid).The metabolic function and bacterial diversity of human microbiota-associated rats after fecal microbiota transplantation were more similar to donors,which were applied to the study of gut-luminal exposure of flavonols,to reveal the dynamic process and material basis of the interaction with intestinal microbiota in the gut lumen.After the model was successfully established,the gut-luminal exposures of quercetin,isoquercitrin,and rutin were compared by oral(16 μmol/kg)and intravenous(2.4 μmol/kg)routes of administration.The contents of the gastrointestinal tract(stomach,duodenum,jejunum,ileum,cecum,colon),blood,and fecal samples were collected at different time points.The results of oral administration showed that quercetin and rutin were present in the upper digestive tract as unchanged components,and isoquercitrin was present as quercetin II conjugated products.When the unchanged compound and metabolites entered the large intestine(cecum and colon),they were rapidly metabolized into 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid by gut microbiota,and only trace amounts of quercetin were detected.Results of intravenous administration showed that the unchanged compound and metabolites of quercetin,isoquercitrin,and rutin were excreted into the intestinal lumen via bile,but compared with oral administration,its gut-luminal exposure was relatively low(related to dose and in vivo disposition).Based on the study of gut-luminal exposure in human microbiotaassociated rats after oral administration of quercetin and rutin,a large number of phenolic acids were produced by gut microbiota,but no significant exposure of phenolic acids in systemic circulation was observed.The results suggest that gut microbiota may be a potential target for quercetin and rutin to exert beneficial effects.This research aimed to study the process of flavonols and the target of its effective substance in vivo,to establish a human microbiota-associated rat model by fecal microbiota transplantation,and to study the characteristics of gut-luminal exposure of flavonol and its metabolite,this study will provide support for the research on the gut-luminal exposure of flavonols by microbiota and the target arrival of flavonols exposure in vivo.