Studies On Flavonoids Glucuronidation Using Recombinant UGTs And Their Structure-Metabolism Relationships

Flavonoids are a broad class of low molecular weight,widely distributed in fruits, vegetables,nuts,seeds,herbs,spices,stems,flowers,as well as tea and red wine. Flavonoids are benzo-γ-pyrone derivatives with phenolic and pyrane rings and are classified according to substitutions.The chemical structure of flavonoids has three phenolic rings referred to as A,B,and C rings.The biochemical activities and metabolism of flavonoids depend on their chemical structures and the relative orientation of various moieties on the molecule.Flavonoids are classified according to their chemical structures.The major flavonoid classes include flavonols,flavones, flavanones,flavanols,anthocyanidins,isoflavones,dihydroflavonols,and chalcones. Over 8000 flavonoids have been identified to 2002.And flavonoids are an integral part of the human diet.The amount of daily flavonoids consumed would be about 50-1000 mg per day according to different region and living habits.The flavonoid natural products exert a wide range of biochemical and pharmacological properties,in particular cardiovascular disease and cancer.As the further research,we know that flavonoids in our daily diet play an important role in human health and disease protection.As a class of plant phenols phenolics,flavonoids have more than one free hydroxyl group,are easy to be conjugated with UDPGA.Conjugation reactions with glucuronic acid seem to be the most common type of metabolic pathways for the flavonoids.In particular,there are now strong evidences for extensive phaseⅡmetabolism of the aglycones such as genistein,apigenin,quercetin,kaempferol and baicalein to glucuronides in rats or human urine.Glucuronidation of flavonoids via UGTs may be the most common metabolic pathways,and studies on flavonoids glucuronidation and structure-metabolism relationships are meaningful,do a great favor to bioflavonoids development and rational drug design.For the importance of UGTs in flavonoids glucuronidation,we try to elucidate the UGTs role in flavonoids metabolism and structure-metabolism relationships between UGTs and flavonoids using human recombinant UGTs.1.In vitro glucuronidation of apigenin by human liver microsomes and its main metabolite bioactivityIn the previous study,potential metabolic pathways of apigenin in rats were found. Incubation in the presence of human recombinant UGTs demonstrated that M1 is almost exclusively catalyzed by UGT1A6,M2 is formed by UGT1A3,UGT1A9 and only to a minor extent by UGT1A6 and UGT2B7,whereas M3 is mainly produced by UGT2B7,while UGT1A4 have no contribution to apigenin glucuronidation. UGT1A3.1,UGT1A3.4 And UGT1A3.5 had much higher glucuronidation efficiency to the 7-hydroxyl group of apigenin than other two UGT1A3 variants(UGT1A3.2 and UGT1A3.3).Apigenin glucuronides were isolated from a reaction mixture consisting of apigenin and UGTs fortified with UDPGA and identified by hydrolysis reaction withβ-glucuronidase,HPLC-MS and UV analysis.On the basis of these results,M1 was identified as 5-glucuronide apigenin,M2 as 7-glucuronide apigenin and M3 as 4'-glucuronide apigenin.There were species differences in glucuronidation of apigenin with liver microsomes obtained from human,rabbit,rat and dog.Kinetic analysis indicated apigenin had the highest affinity for the rabbit liver microsomal enzyme(Km=36.31μmol·L^-1)and the lowest affinity for the dog liver mcirosomal enzyme(Km=120.85μmol·L^-1)in four species.The glucuronidation metabolic activity (Vmax/Km)of apigenin was in the following order:rabbit＞dog＞rat＞human.The results showed that 7-glucuronide apigenin had a stronger antioxidant bioactivity than apigenin assayed by MDA method.The recombinant UGTs expressed by Bac-to-Bac system showed different organic solvent inhibitions compared with UGTS expressed other cell lines.According to flavonoids UGT1A6 substrate reported,we concluded that 5-,7-,4'-position hydroxyl groups,without 3-hydroxyl group may be the essential chemical elements.2.Glucuronidation of flavonoids by human UGT1A3,UGT1A9 and structure-metabolism relationshipsA recent study has suggested that only UGT1A1,1A3,1A4,1A6 and 1A9 are expressed in human liver of the UGT1 gene complex.Previously,we consider that UGT1A1,1A3,and 1A9 are the main enzymes catalyzing the glucuronidation of flavonoids in human liver while human UGT1A4 mainly catalyze amines and UGT1A6 preferentially the glucuronidation of planar phenols.This study proposes features of the flavonoid structure necessary to confer it as a substrate for human UGT1A3 and UGT1A9.The preferred substrates for UGT1A3 and UGT1A9 contain the hydroxyl group at C7-position.The glycon and the position of B ring have conspicuous influences on the glucuronidation activity,and other chemical structures of flavonoids have minor affects.Generally speaking,the substrates for UGT1A3 and UGT1A9 are different,the result shows that the two UGTs have mostly overlapped flavonoids substrates,except quercetin-3-O-arabinoside.From the quantitative study, UGT1A9 in general has higher glucuronidation efficiency than UGT1A3.The high glucuronidation efficiency toward many flavonoids observed suggests that the contribution of UGTs in the metabolism of these flavonoids may be significant.3.QSMR studies of flavonoids and glucuronidation involved in human UGT1A3QSAR(quantitative structure-activity relationships)is wildly used in pharmacology and pharmaceutical chemistry.And in drug metabolism,QSMR is mainly focus on CYP450,such as,pharmacophore and 3D-QSMR models have been developed that can be used to infer the active site binding requirements of substrates and inhibitors for numerous CYP450 isoforms and that can predict apparent Km for substrates and apparent Ki for inhibitors.However,the development of models for predicting metabolism and for characterizing structural features of substrates for UGT isoforms is less advanced relative to CYP450.In order to explore the 2D-QSMR model for flavonoids substrates of human UGT1A3,the parameters which are closely related with the flavonoids activity.The relationship between flavonoid structures and values of Km for human UGT1A3 was studied for 11 flavonoids compounds by the calculation of quantum chemistry at MOPAC-AM1.Combining their calculated results with experimental data,a QSMR (pKm=-2.20707+0.00256HOF+3.21290 Q_C5)was obtained with stepwise regress. The results show the heat of formation(HOF)and Q_C5have conspicuous contributions to the values of Km for UGT1A3.