Effects Of C-18Epimers Of Glycyrrhizic Acid And Their Hydrolysis Products On Ⅱ Phase Detoxification Enzymes And Its Mechanism Research

Motherland medicine have long recognized that licorice had a role on reconcile the various drugs and detoxification, which considered licorice to be alleviated the toxicity and strong of other drugs. Compatibility of Chinese Herbs and Licorice Root are used to ease their toxicity. The scientific traditional understanding of "Cabernet drug toxicity" have been demonstrated by a large number of clinical practice and experimental studies. Glycyrrhizic acid (GL) is the main active component of licorice (Radix Glycyrrhiza), one molecule of GL can be transformed to two molecule of glucuronic acid and one molecule of glycyrrhetinic acid (GA) after hydrolyzation. Base on the different configuration of C18-H bond of triterpene saponin mother nucleus, GL has two different epimers:18a-GL and18(3-GL and they can be hydrolyzed to corresponding18a-GA and18β-GA. They were not distinguished at most previous researches. But the difference of C-18epimers of glycyrrhizic acid has attracted more and more attention after magnesium isoglycyrrhetate was marketed in2006. Preliminary studies of our research group had found that licorice can induce the expression of P-gp, The effects of C-18epimers of Licorice extract, glycyrrhizic acid and their hydrolysis products on II phase detoxification enzymes were introduced in this paper.OBJECTIVEStudy the effects of liquorice extract, C-18epimers of glycyrrhizic acid and their hydrolysis products on protein transcription and expression of UGT1A, UGT2B, and its isozyme UGT1A1and UGT1A6and GSTπ in vitro and in vivo studies and investigate that whether there are stereoselectivity on them; and intended confirmation that the induced effect and mechanism of Liquorice extract, C-18epimers glycyrrhizic acid and their hydrolysis products on the II phase detoxification enzyme; Study the effects of glycyrrhizin preparations on the pharmacokinetics of paracetamol, initially investigate possible drug interaction between glycyrrhizin preparations and substrate of II phase detoxification enzyme UGT that may occur in clinical practice.METHODS 1.Liquorice extract and glycyrrhizic acid18H epimer from different concentra-tions and at different times of the two-phase detoxification enzyme of ratsLicorice extract from different concentrations (90,180,360mg.kg-1) and18a-GL and18β-GL (12.5,25,50mg.kg-1) and at different times (3,6,12consecutive days) in rats by induced Experimental (n=5), By Immunohistochem-ical techniques with chip array experiments investigated the impact of the test drugs on the two-phase detoxification enzyme UGT1A and UGT2B and GSTπ.2. Licorice extract, glycyrrhizic acid18H epimer and their hydrolysis products of Ⅱ-phase detoxification enzymes in ratsThe42rats were randomly divided into control group (saline), licorice extract (concluding14.3%glycyrrhizic acid) group,18a-GL,18β-GL,18a-GA18P-GA,positive control group (phenobarbital)(n=6), respectively, which were induced by7consecutive days. Through the liver microsomal method, the Western blot and the array of tissue microarray methods, and the "trinity"of experimental methods from subcellular level and tissue morphology of the liver tissue level investigated the effect on the licorice extract and its main components18α, β-GL and18α, β-GA on UGT1A, the UGT1A1, UGT1A6,UGT2B and GSTπ.3. HepG2cell culture and MTT toxicity testHepG2cell was cultured with MEM (Modified Eagle's Medium) and was applied to study the effects of licorice extract, GL and GA epimers on the Ⅱ-phase detixification enzyme of UGT and GST. MTT (Methyl thiazolyl tetrazolium) assay was applied to determine the maximum non-cytotoxic dose of each test drugs the activity of cells during the experiment, and ensure maximum test concentration of each drugs.4. The effect licorice extract, glycyrrhizic acid18H epimer and hydrolysis products on the two phase detoxification enzyme protein expression and transcription in HepG2cellsRifampicin was used to positive control, the HepG2cells of blank treatment as a negative control. By Western-Blot and Q-PCR method to study the effect Licorice extract and its main components18a,β-GL and18a, β-GA of protein expression levels and mRNA levels on the Ⅱ phase detoxification enzyme UGT1A, UGT2B isoenzyme UGT1A1and UGT1A6and GSTπ.5. The effect of licorice extract, glycyrrhizic acid18H epimer and their hydrolysis products on UGT1A1and UGT1A6transcriptional activity by the nuclear receptors PXR and CARBuild hPXR, hCAR and the RXRa Eukaryotic expression plasmid and UGT1A1and UGT1A6luciferase reporter plasmid, which were transiently cotransfected into HepG2cells, DMSO was used to a blank control group, rifampicin was used to PXR positive control group, PCN was used to a PXR-negative control group, CITCO was used to CAR positive control group, TCPOBOP was used to CAR negative control group. Drug treatment groups:licorice extract (2,20,200mg. mL-1) and18a-glycyrrhizic acid,18β-glycyrrhizic acid,18α-glycyrrhetinic acid,18β-glycyrrhetinic acid (1,10,100μM). Detection of licorice extract and its main component of18α, β-GL and of18a,(3-GA on the transcriptional activity by the reporter gene luciferase reporter gene method.6. Effects of glycyrrhizin preparation on the pharmacokinetics of paracetamolThe study were assigned to open-label, randomized-sequence, latin square design which consisted of three1-day treatment periods and two7-day washout periods.12subjects were randomly divided into three groups, Blood samples (4mL each) were collected at0,10,20,30and45min; then1.0,1.5,2.0,3.0,4.0,6.0,8.0,10.0,12.0and24.0hours after dosing. Urine samples were collected prior to the administration and0-2,2-4,4-6,6-8,8-12,12-24hours after administration. The blood and urine samples obtained were frozen at-75℃until analysis. The HPLC-MS/MS method was used to determinated the plasma concentration of paracetamol and its conjugates. The DAS2.1.1software was used to calculate the pharmacokinetic parameters.The SPSS version13.0(SPSS Inc., Chicago, Illinois) was used for calculation of the pharmacokinetic parameters.RESULTS AND CONCLUSION1. The results of the liver tissue array immunohistochemistry chip by statistical analysis found that the induction effect of licorice extract and180-GL on Ⅱ-phase detoxification enzyme UGT1A and UGT2B in a dose-dependent and time-dependent. The glutathione transferase GSTπ also was induced, but the time-dependent and dose-dependent manner is not obvious.2. The results showed that licorice extract induced UGT1A, UGT1A1,UGT1A6, UGT2B significantly after the rats were induced by the experimental drug for seven consecutive days, but the effect is not obvious for GSTπ. The18a-GL and18β-GL have an effect on the UGT and its isozymes UGTIA1, UGT1A6, meantime,18β-GL have an more pronounced induced effect on Ⅱ-phase enzyme.The18a-GA have a significant induction effect on UGT2B, by compared,the18(3-GA have a more obviously effect on UGT1A and its isozyme UGT1A1and UGT1A6. Glycyrrhizic acid and glycyrrhetinic acid as well as their isomers have some differences in a variety of studies, both the difference in metabolism should be taken attention seriously.3. Licorice extract enhanced significantly the level of protein expression and the transcriptional level of mRNA expression of UGT1A, UGT2B, the UGT1A1, UGT1A6and GSTπ in HepG2cells.18a, β-GL enhanced UGT1A, UGT2B, UGTIA1, UGT1A6and GSTπ protein expression and mRNA expression;18β-GL had a significantly enhanced UGTIA1and UGT1A6protein expression and transcriptional levels, which consistent with reported that18(3-GL had a more induction significantly than18a-GL in rat liver tissue;18a-GA and18β-GA at the cellular level have a more obviously induction impact on protein expression and mRNA expression of UGT1A,UGT2B, UGTIAI, UGT1A6and GSTπ than that in the liver tissue of rats, especially the18β-GA have a significant induction at the transcriptional level of UGT1A, UGT1A1,UGT1A6, UGT2B, and GSTπ mRNA. In the protein expression level,18β-GA had also a significant induction role for UGT1A and UGT1A1.4. Licorice extract,18α,β-GL and of18a, β-GA have an impact on UGTIA1and UGT1A6transcriptional activity through the nuclear receptor CAR or PXR, and this effect is more obvious at high doses. The effect of18a, β-GL on this transcriptional activity shows some differences, especially18a-GL have not significant impact at low dose on UGTIA1and UGT1A6transcriptional activity by the nuclear receptor CAR or PXR except for a obvious role at high doses, however,18β-GL have a significant induction at low, medium and high dose through the nuclear receptor CAR or PXR on UGT1A1and UGT1A6transcriptional activity, which also may be the real reason for the mechanism that18β-GL induce significantly Ⅱ-phase detoxification enzyme UGT.18α, β-GA have an impact on the UGT1A1and UGT1A6transcriptional activity by the nuclear receptor CAR or PXR, but only at high doses, this effect is more obvious. In short, licorice extract,18α, β-GL and18a,(3-GA have an induction of through the nuclear receptor CAR or PXR, and further induce downstream target gene UGT1A1and UGT1A6expression, which may be licorice detoxification one of the mechanisms, the next step study can be carried out from the joint mechanism of PXR/CAR-mediated detoxification enzymes and efflux pump (such as P-gp-UGT pumps or MRP-UGT cotransporter system), to proven licorice detoxification of real joint mechanism.5. The pharmacokinetic experiments of paracematol show that the acetamin-ophen pharmacokinetic parameters (AUC0.24, AUC0-∞, Cmax) between the experiment-tal and control groups are large different, and AUC0-∞had a statistically significance (P<0.05), compared with the placebo group, paracetamol total elimination half life has been accelerated that also been observed in experiment group. We also compared changes in plasma concentrations of PG in the three experimental groups, which found PG main pharmacokinetic parameters of diammonium glycyrrhininate capsules had a significant changes compared with the placebo group, especially in the AUC0-24and AUC0-∞has a significant difference (P<0.05). Compared with the placebo group, glucuronolactone experimental group have an impact on the main pharmacokinetic parameter of paracetamol and its conjugates, but not significantly.Therefore, we speculated that the diammonium glycyrrhininate capsules group may induce two-phase enzyme UGT,thus speeding up the metabolism and excretion of the substrate acetaminophen, so that the PG increased accordingly. Glucuronolactone group showed that increase of glucuronic acid can speed up the UGT substrate metabolism and excretion to a certain extent, but it play a major role that may be induced two phase conjugating enzyme UGT. In our research, it is also observed that some main pharmacokinetic parameters of APAP and PG did not change significantly, possibly induction time is too short, or there may be the aspolymorphism of UGT isozyme UGT1A6, the UGT1A1and UGT1A9in vivo influence of substrate on the metabolism of acetaminophen, the next step experiment can be developmented from the expansion of the sample size, genotyping and further study of glycyrrhizic acid preparations which affect on the Ⅱ-phase detoxification enzyme.In this study, induction effect of licorice extract and of18α-GL and18β-GL on two-phase detoxification enzyme UGT1A and UGT2B in a dose-dependent and time-dependent. The glutathione transferase GSTπ also was induced, but the time-dependent and dose-dependent manner is not obvious. Licorice extract and18β-GL significantly induced phase Ⅱ detoxifying enzymes, and validation of experimental drugs by up-regulating the upstream factor of PXR and CAR signaling pathway, thereby increasing a series of downstream UGT1A1and UGT1A6expression of detoxification genes, which this signal pathway is one of the important mechanism of detoxification of licorice, Compared with the isomers the effect on phase-conjugating enzyme UGT and GST found that there were some differences between them,but there is no significant impact. In this study, it is the provide a new theoretical support that licorice used in detoxification in clinical application, and also provide new ideas for the detoxification of traditional Chinese medicine research, furthermore, it is provide scientific and theoretical basis for licorice clinical drug interaction studies.