Study On The Metabolism And Transport Of Flavonoids By UGTs And OATP

Flavonoids are an important class of natural organic compounds widely distributed in nature.Flavonoids have a fused ring system consisting of an aromatic ring and a benzopyran ring with a phenyl substituent.The flavonoids are found in plants as glycosides.Before oral absorption,flavonoids undergo deglycosylation via intestinal bacteria,and generate flavonoid aglycones.After the flavonoid aglycone enters the liver through the portal vein after absorption,extensive hepatic first-pass effects occur.The hepatic first-pass effect of flavonoids is mainly attributed to the presence of phenolic hydroxyl groups in the structure,which may lead to extensive phase II metabolism via glucuronidation and sulfonation.Glucuronidation of flavonoids can affect drug effect to a considerable extent and the investigation of glucuronidation metabolism at initial stages of drug development is very imperative.In the metabolism part of this study we mainly studied the UGTs-mediated glucuronidation of flavonoids,and enzyme kinetics of flavonoids in different human recombinant UDP-glucuronosyltransferase(UGT)enzymes.Organic anion-transporting polypeptide OATP1B1 is a membrane transport protein,which is predominantly expressed on the sinusoidal(basolateral)membrane of human hepatocytes.Recent studies have demonstrated OATP1B1 participates in the transport of glucuronide for many drugs and endogenous substances.Thus,this study further investigated the transport of flavonoids and their glucuronic acid metabolites in OATP1B1 cells.Through studies on the metabolism and transport of luteolin,it was found that luteolin is metabolized into three monoglucuronides in human liver microsomes,namely luteolin-3'-O-glucuronide,luteolin-7-O-glucuronide,and luteolin-4'-O-glucuronide,respectively,among them,luteolin-3'-O-glucuronide is the main metabolite.Different UGT enzyme phenotypes exhibited different activity towards catalyzing luteolin glucuronidation.UGT1A1,1A9 and 2B7 mainly catalyze the production of 3'-O-glucuronide,with UGTl A9 exhibiting the strongest activity.UGT1A1,1A6 and 1A9 mainly catalyze the formation of 7-O-glucuronide.The production of 4'-O-glucuronide is also mainly mediated by UGT1A1,1A9 and 1A10.Enzymatic kinetic studies showed that the intrinsic clearance of Luteolin-3'-O-glucuronide catalyzed by UGT1A1 was more than double of UGT1A9.The intrinsic clearances of UGT1A1 and UGT1A9 were 1.01 ± 0.08 and 0.44 ± 0.01 m L/min/mg protein,respectively.For the catalyzing of luteolin-7-O-glucuronide formation,UGT1A1 and UGT1A9 showed similar clearance rates of 0.34 ± 0.00 and 0.46 ± 0.03 m L/min/mg protein,respectively.Further transport studies revealed that OATP1B1 selectively uptake luteolin-3'-O-glucuronide but not parent luteolin and luteolin-7-O-glucuronide.Studies on the metabolism and transport of baicalein showed that baicalein was mainly metabolized into two monoglucuronides in human liver microsomes,which are baicalein-7-O-glucuronide(main metabolite)and baicalein-6-O-glucuronide.The metabolism of baicalein-7-O-glucuronide is mainly mediated by UGT1A1,1A3 and 1A9,among which UGT1A9 exhibited the strongest affinity(Km 2.68 ± 0.40 ?M)and UGT1A1 exhibited the highest capacity(9.10 ± 1.40 nmol/min/mg protein).OTAP1B1 uptake studies revealed that baicalein was not a substrate for OATP1B1,whereas baicalin was obviously a substrate of OATP1B1,and baicalein glucuronide methyl ester is also a substrate of OATP1B1.However,the baicalein glucoside is not a substrate of OATP1B1,indicating that small changes in the structure of the substituents on the hydroxyl group at the 7-position of baicalein can also affect the transport of OATP1B1 to them.Finally,the relationship between the glucuronidation activity of flavonoids by different UGTs and the structure of flavonoids was studied.It was found that the glucuronidation of flavonoids occurs predominantly at the 7-hydroxyl group and 3-hydroxyl group.The glucuronidation of the 7-hydroxyl group is mainly mediated by UGT1A1,1A3,1A9,and 2B7,and the glucuronidation of the 3-hydroxyl group is mainly mediated by 1A9.UGT1A1,1A9 and 2B7 mainly catalyze the glucuronidation of 3?-hydroxyl group and 4?-hydroxyl group.But the 5-hydroxyl group is less prone to glucuronidation due to larger steric hindrance,and the glucuronidation of the 5-hydroxyl group is mainly mediated by by UGT 1A3,1A6,1A8,and 1A9.From the UGTs subtype identification results,it can also be seen that UGT1A1 and UGT1A9 have the strongest activity towards flavonoid glucuronidation among the 12 UGTs tested.In addition,UGT 1A7,1A8 and 1A10 were also involved in the glucuronidation of flavonoids,nidicating that glucuronidation of flavonoids occured not only in the liver but also in the gastrointestinal tract.This study systematically studied the glucuronidation of flavonoids by UGTs and transport of flavonoids by OATP1B1,clarifying the metabolism and transport mechanism of flavonoids in the liver.Out study provides scientific basis for clinical rational and safe usage of flavonoids.