Studies On The Characters Of Liquiritigenin Sulfonation And The Mechianism Involved In The Sulfate Efflux

Background:Flavonoids are widely found in fruits,nuts,vegetables and herbs and have a variety of pharmacological activities.Currently,a variety of flavonoids have been clinically applied,but none of the drugs are designated for systemic delivery.One of the important reasons is that such compounds are prone to phase II metabolism?such as glucuronidation,sulfonation?,resulting in lower oral bioavailability.Liquiritigenin is one of the main active ingredients of traditional Chinese medicine liquorice.It belongs to flavonoids and has many pharmacological effects such as anti-inflammatory,anti-oxidation,anti-diabetes,anti-cancer and liver protection.However,the poor bioavailability has limited its application.In addition,studies have reported that phase II metabolites of liquiritigenin?glucuronidates and sulfates?can be detected in rats,mice,rabbits and dogs.Therefore,we speculate that the occurrence of phase II metabolism may be an important factor leading to low bioavailability of liquiritigenin.At present,the glucuronidation metabolism of liquiritigenin has been reported,and the SULT metabolism is not clear.Objective:The purpose of this paper is to investigate the kinetics of liquiritigenin sufonation and to evaluate the role of efflux transporters?such as BCRP and MRPs?in the efflux of liquiritigenin sulfate.Defining the characteristics of liquiritigenin sulfonation and its influencing factors may have certain significance for guiding the clinical use of liquiritigenin.Methods:Firstly,the sulfonation of liquiritigenin was characterized using recombinant human sulfotransferases?SULTs?.Then human embryonic kidney?HEK?293 cells overexpressing SULT1A3?named as HEK-SULT1A3 cells?were conducted using transfection technique.In order to confirm whether the constructed HEK-SULT1A3 cell line has the function of the SULT1A3 enzyme,liquiritigenin sulfonation was detected in HEK-SULT1A3 cell lysate.Furthermore,the correlations of liquiritigenin sulfonation characters?7-OH?in HEK-SULT1A3 cell lysate ans recombinant SULT1A3 enzyme were evaluated.Then biological inhibition and chemical inhibition method were conducted to investigate the effect of efflux transporters on the efflux of liquiritigenin sulfate.Finally,the influences of efflux transporter protein knockdown on liquiritigenin sulfonation were evaluated using HEK-SULT1A3 cells.Results:1.When liquiritigenin was incubated with recombinant human SULT1A1,SULT1A2,SULT1A3,SULT1B1,SULT1C2,SULT1C4,SULT1E1 and SULT2A1 enzymes,7-hydroxyl sulfate of liquiritigenin was detected.The experimental results show that liquiritigenin sulfonation in all of these enzymes displayed the classical Michaelis-Menten profile.According to the intrinsic clearance(CL_int)value,the sulfonation rates of liquiritigenin-7-OH by expressed SULT enzymes followed the following rank order:SULT1C4>SULT1A3>SULT1E1>SULT1A1>SULT1A2>SULT1B1>SULT1C2>SULT2A1.However,the Michaelis constant values of liquiritigenin sulfonation in SULT1A3 was lower than that in SULT1C4,which indicated that SULT1A3 has shown a better affinity with liquiritigenin and plays an more important role in liquiritigenin sulfonation.2.Liquiritigenin generated one mono-sulfate metabolite?7-O-sulfate?in HEK-SULT1A3 cell lysate and SULT1A3,and the sulfonation kinetics followed the Michaelis-Menten model.The derived V_max value was18.50 nmol/min/mg and the K_m was 6.58?M in SULT1A3 enzyme.The V_max was 0.83 nmol/min/mg and the K_m was 7.12?M in HEK-SULT1A3 cell lysate.There was no difference in the K_m value between the two groups?p>0.05?.And the sulfonation characters of liquiritigenin?7-OH?in HEK-SULT1A3 cell lysate were strongly correlated with that in SULT1A3 enzyme.3.When the BCRP inhibitor Ko143 was used,the efflux rate and clearance of liquiritigenin sulfate were significantly decreased.When the inhibitor concentration was 20?M,the efflux rate and clearance rate were significantly reduced by 42.5%?p<0.001?and 60.0%?p<0.001?.When the pan-MRPs inhibitor MK-571was used,the efflux rate and clearance of liquiritigenin sulfate were also decreased significantly.At the concentration of 20?M,MK-571 reduced the efflux rate and clearance rate of liquiritigenin sulfonated by 97.4%?p<0.05?and 99.3%?p<0.001?,respectively.4.Knockdown of BCRP in HEK-SULT1A3 cells,the efflux rate and clearance rate were respectively reduced by 15.9%-16.9%and 26.5%-29.5%.Then,we knocked down the expression of MRP4 protein by the same method.The efflux rate and clearance rate of liquiritigenin sulfonated metabolites decreased by20.2%-32.5%and 44.8%-47.1%,respectively.In addition,knockdown of the expression of the above two efflux transporters resulted in a decrease in the total metabolic level(f_met)of liquiritigenin in HEK-SULT1A3 cells by 20.6%and 22.2%.It indicates that the efflux transporter regulates the intracellular sulfonation of liquiritigenin.Conclusions:BCRP and MRP4 have played important roles in the efflux of liquiritigenin sulfate.At the same time,the efflux transporter affected the extent of liquiritigenin sulfonation.