In Vivo Study On The Isoflavone-7-O-?-D-glucopyranoside And Aglycone In Rats

Our research group found that calycosin-7-O-?-D-glucopyranoside was able to fight against coxsackie virus B3?CVB₃?an viral myocarditis.We also found its aglycone?calycosin?has better effects than the prototype drug.Additionally,calycosin elicited anti-human immunodeficiency virus?HIV?activity.Therefore,it is of great significance to research the in vivo process of calycosin-7-O-?-D-glucopyranoside and calycosin,in order to explain the efficacy and research on new drug.1.Metabolism of calycosin-7-O-?-D-glucopyranoside in vivo and in vitroWith the aid of high performance liquid chromatography-quadrupole time-of-flight mass spectrometer series?HPLC-Q-TOF?,the accurate mass of molecules and fragment ions can be obtained.According to the fragmentation patterns and characteristic fragment ions of the reference substances,the metabolites in the portal vein plasma,systemic plasma,liver,heart,kidney,urine and feces were identified after oral administration of calycosin-7-O-?-D-glucopyranoside in Sprague-Dawley rats.Meanwhile,the influence factors regarding the metabolism of calycosin-7-O-?-D-glucopyranoside were investigated by using the intestinal flora and the primary hepatocytes.A total of 20 metabolites were identified from the biological samples after rats orally taking calycosin-7-?-D-glucopyranoside.The prototype drug was observed in the portal vein plasma,systemic plasma and feces,and 18,14 and 8 metabolites were observed in the portal vein plasma and systemic plasma,respectively.Morever,there were 11,13,7 and 18 metabolites identified in the liver,kidney,heart and urine,respectively.According to the in vitro results,calycosin-7-O-?-D-glucopyranoside was metabolized into calycosin in the intestinal bacteria incubation system,and then calycosin was transformed into daidzein,formononetin and other metabolites through demethylation,dehydroxylation and other pathways.In addition,calycosin-7-?-D-glucopyranoside can be metabolized into calycosin glucuronides by hydrolysis and glucuronidation in the primary hepatocytes.Summarily,the orally administratedcalycosin-7-O-?-D-glucopyranoside underwent the main metabolic pathways of demethylation,dehydroxylation,glycosylation,methylation,sulfate and glucuronidation in rats.2.Pharmacokineticandtissuedistributionofcalycosin-7-O-?-D-glucopyranosideA liquid chromatography–tandem mass spectrometry method was established and validated for simultaneous determination of calycosin-7-O-?-D-glucopyranoside and calycosin in the plasma and different organs,including of liver,kidney,and heart.This method was applied to the pharmacokinetics and tissue distribution of calycosin-7-O-?-D-glucopyranoside and calycosin,following oral administration of calycosin-7-O-?-D-glucopyranoside at 120 mg/kg in rats.Consequently,the exposure of calycosin-7-O-?-D-glucopyranoside and calycosin were both similarly low in the systemic plasma,but the levels of calycosin were 53.5 folds higher than that of calycosin-7-O-?-D-glucopyranoside in the portal vein plasma,corresponding to the liver extraction ratio?ER?of calycosin-7-O-?-D-glucopyranoside and calycosin at 0.3%and 98.5%,respectively.Therefore,our results revealed that the intestinal first-pass effect was predominant for the poor circulating levels of calycosin-7-O-?-D-glucopyranoside,whereas liver first-pass effect played the predominant role in the poor circulating levels of calycosin on calycosin-7-O-?-D-glucopyranoside treatments.In contrast to no observation of calycosin-7-O-?-D-glucopyranoside,the calycosin levels were 212.1,30.5 and 4.7 folds higher in the liver,kidney and heart than its circulating levels,respectively.The high tissue distribution of calycosin can explain the discrepancy of calycosin-7-O-?-D-glucopyranoside between the low bioavailability and the pharmacological activity in vivo.3.Metabolism of calycosin in vivo and in vitroThe metabolites in portal vein plasma,systemic plasma,liver,heart,kidney,urine and feces were analyzed utlizing HPLC-Q-TOF-MS,after oral administration of calycosin in rats.There were 21 metabolites,including 4 phase I metabolites and 17phase II metabolites,identified in those biological samples.Besides the prototype,there were 19,18,13,15,5,5,and 10 metabolites were observed in the portal vein plasma,systemic plasma,liver,kidney,heart,feces and urine,respectively.The metabolic pathways of calycosin included of dehydroxylation,demethylation,reduction,methylation,sulfation,and glucuronidation.In addition,calycosin could be metabolized in the intestinal bacteria and primary hepatocytes,where the glucuronidation is predominant metabolic pathway.4.Pharmacokinetic and tissue distribution of calycosinThe ER of calycosin was as high as 99.6%after oral administration of calycosin in rats.This manifested that the severe liver first-pass effect prevented the majority of calycosin inaccessible to the circulating system.On one hand,this was in accordance with the previous in vitro results that calycosin underwent obvious hepatic metabolism.On the other hand,the considerable hepatic distribution of calycosin also made certain contributions to the liver first-pass effect of calycosin.In addition to the liver,the high exposure of calycosin were detected in the kidney and heart.The high tissue distribution can partly explain the discrepancy between low circulating levels of calycosin and its substantial pharmacological effects by oral administration.Furthermore,compared to those on calycosin treatments?76.4 mg/kg?,there were higher levels of calycosin in the plasma and tissues on calycosin-7-O-?-D-glucopyranoside treatments with equimolar amount?120 mg/kg?,indicating that calycosin-7-O-?-D-glucopyranoside can be served as the prodrug of calycosin.The AUCs of calycosin glucuronides were 4.6 and 1552.8times higher than those of calycosin in the portal vein plasma and the systemic plasma,respectively,after oral administration of calycosin.The results showed that both the intestinal and liver played a critical role in the glucuronidation of calycosin.Calycosin glucuronides were also predominant in the liver,kidney and heart.calycosin glucuronides may make significant contributions to the health improvement and disease management of calycosin in vivo,awaiting further research.The metabolites were identified from the biological samples,and the prototype and main metabolite were determined after rats orally taking calycosin-7-?-D-glucopyranoside and calycosin.The metabolism,pharmacokinetics,liver first-pass effect and tissue distribution of calycosin-7-O-?-D-glucopyranoside and calycosin were researched,and the pharmacodynamic material basis of calycosin-7-?-D-glucopyranoside and calycosin were illuminated.