The Influence Of Daphne Genkwa And Its Main Components On UGT Enzyme Activity

Genkwa Flos,the dried flower bud from Daphne genkwa Sieb.et Zucc which has a long history of clinical application.It has been used for the purposes of antitussive,expectorant,diuretic,abortifacient,anti-inflammatory,antibacterial,anti-convulsants and antitumor for centuries.it is expected to become commonly used adjuvant therapy because of its wide range of biological activity and potential antitumor activity,and it may be more and more common used in combination with other drugs in the future.However,there is evidence that excessive and chronic use of Genkwa Flos will finally result in serious damage to liver,kidney,brain and other organs,and it was publicly known to have irritation to mucous and skin.Some studies have shown that Daphnane diterpene esters is not only bioactive ingredient but also the main toxic substances.Thus,for a long time due to the toxicity of Genkwa Flos which make it in clinical applications has been greatly restricted.At present,the study on the toxicity of Genkwa Flos is mostly the toxic substances findings,but the study of its toxic mechanism is still rare.UDP-glucuronosyltransferases(UGTs)enzymes is one of the important phase II metabolic enzymes in the body,it is mainly distribute in the liver,gut,kidneys and other organs.Its main function is catalyze the conjugation of various endogenous substances and exogenous compounds and phase I metaboletes,which made the polar of them increased and then excreted with the urine and the feces by the kidneys.Therefore,the number and activity of the enzyme UGTs will direct influence the metabolic activation and efflux of drugs and endogenous substance in vivo,to maintain the body's healthy and drug occurred efficacy is essential.Some studies have reported that the effect of Genkwa Flos on cytochrome CYP450 enzyme activity,but there is no report about its influence on the metabolic enzyme activity of UGTs.In view of this,this paper studied the influence of the main components of Genkwa Flos on UGT enzyme activity.This research can not only predict the effects of Genkwa Flos on endogenous metabolites,also from the perspective of metabolic enzyme speculate about the toxic mechanism of Genkwa Flos.The possible toxic mechanism is that some compounds of Genkwa Flos inhibited UGT enzymes activity,UGT enzymes which can catalyze the metabolic transformation of the diterpenoid who is the toxic components of Genkwa Flos,this inhibition will resulting in the phase II detoxification process of toxic components is blocked,leading to toxicity reaction,this study can provide a new ideas for the further investigate on the specific of toxicity to Daphne genkwa.In this paper,4-nitrophenol(the probe drug of UGTs),estradiol and bilirubin(the probe drug of UGT1A1)were incubated with or without main compounds of Genkwa Flos in liver microsomes,respectively.After the incubation,the consumption of the substrates were quantified by UV and the metabolites of the substrates were quantified HPLC or UPLC/MS to Investigate the effect of the main components of Genkwa Flos on UGTs and UGT1A1 enzyme activity.The experimental studies are as follows:1 Optimizing the conditions for determination of UGT1A1 enzyme activity in vitroThis part selects estradiol as the probe substrate of UGT1A1,the activity of the enzyme can be expressed by the concentration of the metabolites produced in the unit time per unit of protein.This article established the method of estradiol and its metabolites in the liver microsomes by high performance liquid detection at first.The method has good specificity,which precision,accuracy and stability of them are in line with methodology requirements,and the method can well meet the quantitative requirements for the following study.Based on the method,the proper incubation condition were selected as 30 min,0.5mg·mL-1 protein in RLM,HLM and rhUGT1A1.2 Effects of Genkwa Flos and its main ingredients on enzyme acticity of UGTs in liver microsomes2.1 Quantification of the main component of Genkwa FlosEthanol extract of Genkwa Flos was prepared by ethanol heating reflux extraction method,and the chloroform extract was extracted from ethanol extract.And then,we determined the contant of apigenin,genkwanin and hydroxygenkwanin in the two kinds of extracts.The result was that the contents of apigenin,hydroxygenkwanin and genkwanin in the ethanol extract were 6.34%,8.72%and 6.06%,respectively.The contents of apigenin,hydroxygenkwanin and genkwanin in the chloroform extract were 1.00%,6.40%and 18.38%,respectively;Colorimetric method has been applied to the determination of total diterpene contents were 30.72%and 36.33%in the both extracts,respectively.The purpose of this experiment is provided the basis for the next study.2.2 In vitro effects of Genkwa Flos and its main ingredients on UGTs activities of liver microsomesIn this experiment,the effects of main components of Genkwa Flos on UGTs activities of rat,mice and human liver microsomes were investigated in vitro.4-Nitrophenol was selected as probe substrate to determine the activities of UGTs by UV method.The results show that,ethanol extract of Genkwa Flos,chloroform extract of Genkwa Flos,apigenin,genkwanin and hydroxygenkwanin significantly inhibited rat,mice and liver microsome UGTs activity,among them ethanol extract of Genkwa Flos showed the strongest inhibition,In addittion,effects of main components of Genkwa Flos on UGTs activity showed significant difference between different species.2.3 In vitro effects of Genkwa Flos and its main ingredients on UGT1A1 activities of liver microsomes2.3.1 Effects of Genkwa Flos and its main ingredients on UGT1A1 activities of rat liver microsomesIn this experiment,in order to evaluate the effect of Genkwa Flos ethanol extract,Genkwa Flos chloroform extract and hydroxygenkwanin on UGT1A1 activity in rat liver microsomes,estradiol was selected as probe substrate to UGT1A1 and HPLC method was used to measure the contents of metabolites.The results showed that,ethanol extract of Genkwa Flos,chloroform extract of Genkwa Flos and hydroxygenkwanin significantly inhibited rat liver microsome UGT1A1 activity.The inhibit strong-to-weak sequence of Genkwa Flos on UGT1A1 activity was chloroform extract(IC50=8.76?M)>hydroxygenkwanin(IC50=10.93?M)>ethanol extract(IC50=46.32?M);Ethanol extract and chloroform extract and hydroxygenkwanin all showed moderate inhibitory effect on UGT1A1,the inhibition types were competitive inhibition(Ki=4.62?M)and noncompetitive inhibition(Ki=28.3 ?M)and competitive inhibition(Ki=32.3 ?M),respectively.2.3.2 Effects of Genkwa Flos and its main ingredients on UGT1A1 activities of human liver microsomesIn this experiment,in order to evaluate the effect of Genkwa Flos ethanol extract,Genkwa Flos chloroform extract and hydroxygenkwanin on UGT1A1 activity in human liver microsomes,estradiol was selected as probe substrate to UGT1A1 and HPLC method was used to measure the contents of metabolites.In addition,billirubin was selected as probe substrate to UGT1A1 and HPLC-MS method was used to measure the contents of billirubin metabolites,to evaluating the effect of genkwanin and apigenin on UGT1A1 activity in human liver microsomes.Results showed that Genkwa Flos ethanol extract and Genkwa Flos chloroform extract all showed moderate inhibitory effect on UGT1A1 activity.The order of inhibition strength was Genkwa Flos chloroform extract(IC50=10.36?M)>Genkwa Flos ethanol extract(IC50=32.49?M).The inhibition types of Genkwa Flos ethanol extract and chloroform extract were uncompetitive inhibition with Ki about 1.464?M,46.90?M,respectively.Genkwanin,hydroxygenkwanin and apigenin are three major flavonoids in Genkwa Flos.Hydroxygenkwanin showed weak inhibitory effect on UGT1A1,but genkwanin and apigenin all showed moderate inhibitory effect on UGT1A1 activity.The intensity of inhibition function was apigenin(IC50=76.3 ?M)>genkwanin(IC50=23.21?M)>hydroxygenkwanin(IC50=12.40?M).The inhibition types of them were competitive inhibition.2.3.3 Effects of Genkwa Flos and its main ingredients on UGT1A1 activities of recombinant human UGT1A1 enzymeIn this experiment,in order to evaluate the effect of Genkwa Flos ethanol extract and hydroxygenkwanin on UGT1A1 activity in recombinant human UGT1A1 enzyme,estradiol was selected as probe substrate to UGT1A1 and HPLC method was used to measure the contents of metabolites.Results showed that Genkwa Flos ethanol extract and hydroxygenkwanin all showed moderate inhibitory effect on UGT1A1 activity.The order of inhibition strength was Genkwa Flos chloroform extract(IC50=10.36?M)>hydroxygenkwanin(IC50=20.07?M).The inhibition type of Genkwa Flos ethanol extract and was competitive inhibition with Ki about 5.59?M and hydroxygenkwanin was mixed inhibition with Ki about 3.44?M,respectively.3 In vivo effects of Genkwa Flos and its main ingredients on UGTs and UGT1A1 activities of rat liver microsomesIn this experiment,rats were administrated Genkwa Flos ethanol extract(0.25g·kg-1·d-1,0.05g·kg-1·d-1)and chloroform extract(0.25g·kg-l-d-1,0.05g·kg-1·d-1)and blank reagent by intragastric administration,respectively.Rats were administrated hydroxygenkwanin(100 mg·kg-1·d-1)by intraperitoneal injection and blank reagent,respectively.After induced 7 days,liver microsomes were prepared,to determine the effects of various components on the UGTs and UGT1A1 activity.Results showed that ethanol extract,chloroform extract and hydroxygenkwanin can significantly inhibited rat liver microsome UGTs activity in vivo,the effects of them on UGTs activity were consistent with the results previously obtained in vitro.After compared with the blank group,there is no significant induction of the extract of Genkwa Flos on the activity of UGT1A1 in vivo.This result was contrary to the previously obtained in vitro.In addition,hydroxygenkwanin can slightly inhibit the rat liver UGT1A1 enzyme activity in vivo,This inhibition,however,was not statistically significant compared with the blank control group.In summary,the main component of Genkwa Flos can be significantly inhibited the UGTs activity in vitro and in vivo,such as ethanol extract,chloroform extract,genkwanin,hydroxygenkwanin and apigenin.There are some reseaches repoted that apigenin and yuanhuapine were metabolized and eliminated via glucuronic acid of binding reaction.So,the activity of UGTs was inhibited by constituents of Genkwa Flos(flavonoids)which may hinder the phase ? metabolism of toxic components of Genkwa Flos,resulting in toxic components and secondary toxic metabolites accumulation in vivo and caused toxic reaction.This may be the mechanism of Genkwa Flos induced toxicity.Besides,the inhibit effect of Genkwa Flos on UGT1A1 activity,suggesting that Genkwa Flos may disrupt the metabolism of bilirubin,which can cause hyperbilirubinemia,even severe liver cell damage.On the other hand,the effect of Genkwa Flos on UGTs and UGT1A1 activity also indicated that we should be on guard against the drug-drug interactions happened when we applicate Genkwa Flos in clinical.In addation,the effects of Genkwa Flos and its main ingredients on UGT1A1 activities were different in vivo and in virto,the causes of different might be:after a series of metabolise in vivo,there is a big difference between the components of drug effect on UGT1A1 enzyme and in vitro incubation system,which eventually lead to different results.