Study On The Metabolism Properties Of The Active Constituents Of Polygonum Multiflorum In Vitro And In Vivo

Polygonum multiflorum,a natural product originated from the root of polygonum multiflorum Thunb,has been shown to display multiple pharmacological activities,including anti-aging,improving memory,hyperlipidemia,anti-atherosclerosis and premature graying.The clinical use prefers polygonum multiflorum praeparata.Polygonum multiflorum praeparata has beenwidely used in Chinese medicine prescriptionand health care products.However,a seriesof cases of polygonum multiflorum praeparatainduced hepatotoxicity were reported in recent years,the exact mechanism of polygonum multiflorum praeparatainduced hepatotoxicity and the basis of toxic substances is unknown.Recently,the liver toxicity of polygonum multiflorum praeparata related to the metabolic activation of its active constituents in vivo had been demonstrated,but the metabolic properties of the active constituents of polygonum multiflorum praeparatais not systematic.A rapid LC-MS/MS method for the simultaneous analysis of emodin(EM),aloe-emodin(AE)and 2,3,5,4-Tetrahydroxystilbene-2-O--D-glucoside(TSG)was developed and validated.The method was successfully used to determine the active constituents of polygonum multiflorum praeparata.In addition,the metabolic stabilities,reaction phenotyping,enzyme inhibition and efflux transport properties were also studied in the incubation system of liver microsomes,human recombinantenzyme and Caco-2 cell.The research could provide experimental date and scientificbase for the in-depth study of liver toxicity and clinical use of polygonum multiflorum praeparata.In this study,a LC-MS/MS method was developed and validated for the simultaneous analysis of the three active constituents of polygonum multiflorum praeparata.Through enzymatic hydrolysis,the corresponding conjugates could be quantitatively determined.The method was smart and validated to meet the quantitative requirement.The absorbance and metabolism of EM,AE and TSG were rapid after a single oral administration of the extract of polygonum multiflorum praeparata to rats.The peak time of EM and AE was both at 1 h,and the elimination half life was 0.5 h and 0.4 h,respectively.In addition,a large number of glucuronidated conjugates produced by EM and AE were detected.The plasma concentration of TSG was low,and the peak time of parent drug and glucuronidated conjugates was at 1 h and 0.25 h,respectively.The Cmax was(39.1±23.3)ng/mL and(61±34.4)ng/mL,respectively.The absorbance of parent drug and glucuronidated conjugates of EM and AE was rapid after a single oral administration of EM or AE,and a large number of glucuronidated conjugates produced by EM and AE were also detected.But compared to the extract polygonum multiflorum praeparata,the elimination was significantly slower andthe half time and MRT were longer.Also,the concentration of glucuronidated conjugates is lower.After oral administration of the extract of polygonum multiflorum praeparata,the accumulative excretion of bile of EM,AE and TSG were 49.5%,48.7% and 1.6%,respectively,and the glucuronide conjugates were the main metabolites.The bile recovery rate of EM and AE within 72 h was 37.7% and 7.3% after oral administration of EM or AE.Compared to the extract,the bile excretion recovery of parent drug of EM and AE was significantly reduced,and the glucuronide conjugates of AE were significantly reduced,too.ATPase assay illustrated that EM and AE may be the substrates of BCRP.After oral administration of the extract of polygonum multiflorum praeparata,BCRP specific inhibitor KO143 significantly reduced the recovery of EM and AE,which further confirmed that EM and AE were BCRP substrates.The EM,AE and TSG could distribute to the tissues rapidly.The concentration of EM,AE and TSG in the most tissues reached the peak level at 0.25 h,and the exposure concentration of liver and kidney were the highest.The urine excretion of EM and AE was 13.1% and 9.4%,respectively.The fecal excretion was 14.0% and 14.3%,respectively.And the parent drug was the main excretory form.TSG was widely metabolized in rats and was not detected in urine and feces.Both EM and TSG performed phase I and phase II metabolism elimination in HLM and RLM,but there was a difference in species.AE was metabolically eliminated in the presence of NADPH in HLM and performed phase I and phase II metabolism elimination in RLM.The results of enzyme kinetics showed that EM,AE and TSG were metabolically eliminated rapidly in HLM and RLM,the metabolic elimination in RLM was quicker than HLM.EM and AE underwent multi-enzyme mediated phase I metabolism in liver.CYP1A2,2B6,2C19 and 3A4 were the major CYP enzymes of EM metabolism,and CYP1A2,2C9 and 3A4 contributed to more than 20%.AE was mainly mediated by CYP1A2,2B6,2C19 and 3A4,and the contribution rate of metabolism mediated by CYP1A2 and 3A4 to AE was both more than 20%.The phase II metabolism of EM was mainly mediated by UGT1A1,1A8,1A10,2B7.EM and AE had inhibitory effect on CYP enzyme,EM was a strong inhibitor of CYP1A2,and AE was a moderate inhibitor of CYP1A2.Therefore,EM and AE and CYP1A2 substrates in combination should pay attention to the possible drug-drug interaction.In summary,EM,AE and TSG were absorbed rapidly in rat after oral administration of the extract of polygonum multiflorum praeparata,and the drug exposure in liver and kidney were significantly higher than in other tissues.The EM and AE were metabolically eliminated quickly by CYP and UGT,and then transported by BCRP to the bile.EM and AE were both the substrates of CYP1A2 and also could significantly inhibit the enzyme activity of CYP1A2,which causes a risk of drug-drug interactions.