| ObjectiveThe aim of the present study is to evaluate the inhibitory effects of liverUDP-glucuronosyltransferases (UGTs) by glycyrrhizic acid and glycyrrhetinicacid, which are the bioactive ingredients isolated from licorice. The resultsshowed that glycyrrhetinic acid exhibited stronger inhibition towards all thetested UGT isoforms, indicating that the deglycosylation process played animportant role in the inhibitory potential towards UGT isoforms. Further-more,the inhibition kinetic type and parameters were determined for the inhibition ofglycyrrhetinic acid towards UGT1A3and UGT2B7. Data fitting using Dixon andLineweaver-Burk plots demonstrated that the inhibition of UGT1A3andUGT2B7by glycyrrhetinic acid was best fit to competitive and noncompetitivetype, respectively. The second plot using the slopes from Lineweaver-Burk plotsversus glycyrrhetinic acid concentrations was employed to calculate theinhibition kinetic parameters (Ki), and the values were calculated to be0.2and1.7mM for UGT1A3and UGT2B7, respectively. All these results remind us thepossibility of UGT inhibition-based herb-drug interaction. However, theexplanation of these in vitro parameters should be paid more caution due tocomplicated factors, including the probe substrate-dependent UGT inhibitionbehaviour, environmental factors affecting the abundance of herbs’ ingredients,and individual difference of pharmacokinetic factors. Copyright?2012JohnWiley&Sons, Ltd.Materials and methods1. Chemicals. Glycyrrhetinic acid and glycyrrhizic acid (purity≥ 98%) were purchased from Sichuan Weikeqi Bio-technology Co. Ltd(SichuanChina).4-methylumbelliferone(4-MU),4-methylumbelliferone-b-D-glucuronide (4-MUG), Tris-HCl,7-hydroxycoumarin and uridine-5’-diphosphoglucuronic acid.5’-diphosphoglucuronic acid (UDPGA)(trisodiumsalt) were purchased from Sigma-Aldrich (St Louis, MO). Recombinanthuman UGT supersomes (UGT1A1, UGT1A3, UGT1A9, and UGT2B7)expressed in baculovirus-infected insect cells were obtained from BD GentestCorp.(Woburn, MA, USA). All other reagents were of HPLC grade orof the highest grade commercially available.2. Investigation of inhibition of UGT isoforms by glycyrrhetinic acidand glycyrrhizic acid.4-MU, the nonspecific probe for UGT isoforms, wasused to investigate the inhibition of UGT isoforms by glycyrrhetinic acid andglycyrrhizic acid. Incubation system and condition were carried out as previouslydescribed (Dong et al.,2012). The mixture (200ml) contained recombinantUGTs (final concentrations:0.25,0.05,0.05, and0.05mg/ml forUGT1A1, UGT1A3, UGT1A9, and UGT2B7, respect-ively),5mM UDPGA,5mM MgCl2,50mM Tris-HCl buffer (pH=7.4), and4-MU in the absence orpresence of different concentrations of glycyrrhetinic acid orglycyrrhizic acid. The concentrations of4-MU were as follows:110mM forUGT1A1,1200mM for UGT1A3,30mM for UGT1A9, and350mM forUGT2B7. After5-min pre-incubation, UDPGA was added into the mix-ture toinitiate the reaction. The incubation time was120min for UGT1A1and UGT2B7,75min for UGT1A3, and30min for UGT1A9. The reactions were quenched byadding100ml acetonitrile with7-hydroxycoumarin (100mM) as internalstandard. The mixture was centri-fuged at20000g for10min, and an aliquotof supernatant was transferred to an auto-injector vial for HPLC analysis. TheHPLC system.(Shimadzu, Kyoto, Japan) contained a SCL-10A system controller,two LC-10AT pumps, a SIL-10A auto injector, and a SPD-10AVP UV detector.Chromatographic separ-ation was carried out using a C18column (4.6200mm,5mm, Kromasil) at a flow rate of1ml/min and UV detector at316nm. Themobile phase consisted of acetonitrile (A) and H2O containing0.5%(v/v)formic acid (B). The following gradient condition was used:0-15min,95-40%B; 5-20min,10%B;20-30min,95%B.Determination of inhibition kinetic type and parameters. Inhibition kineticarameters (Ki) were determined utiliz-ing various concentrations of4-MU in theresence of different concentrations of glycyrrhetinic acid. Dixon andineweaver plots were adapted to determine the inhibition type, andecond plot of slopes from Lineweaver-Burk plot versus glycyrrhetinic acidoncentrations was utilized to calculate the Ki value.ResultsAs shown in Fig.1,100mM of glycyrrhizic acid inhibited the activity ofGT1A1, UGT1A3, UGT1A9, and UGT2B7by17.8%,21.5%,33.6%, and8%, respectively. Glycyrrhetinic acid showed stronger inhibition towards theseour UGT isoforms, with the activity of UGT1A1, UGT1A3, UGT1A9, andGT2B7inhibited by78.4%,93.6%,72.5%, and99.7, respectively.Furthermore, the inhibition kinetic type and para-meters ofGT1A3and UGT2B7by glycyrrhetinic acid were evaluated. Data fittingsing Dixon plot and Lineweaver-Burk plot were carried out, and theecond plot using the slopes from Lineweaver-Burk plot versus theoncentrations of glycyrrhetinic acid was used to calculate the Ki values. Bothixon plot (Figs.2B and3B) and Lineweaver-Burk (Figs.2C and3C) showedhat the inhibition of UGT1A3and UGT2B7by glycyrrhetinic acid was bestit to competitive and noncompetitive type, respectively. The inhibitionarameters (Ki) were determined to be0.2and1.7mM for UGT1A3nd UGT2B7, respectively.ConclusionsThe glycyrrhetinic acid exhibits inhibition towards UGT subtypesUGT1A3UGT2B7). This inhibition is most suitable for competitive andon-competitive type. |
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