Study On The Factors Regarding Biotransformation And Bioavailability Of Astragaloside ?

Astragaloside IV(ASIV)is a typical bioactive constituent of Radix Astragali.The study aimed to investigate the major factors of low bioavailability of ASIV based on disposition of drug in organism first.The influence of the gastrointestinal digest,intestinal and liver metabolic enzymes,and intestinal bacteria are the keys for ASIV to reach the systemic circulation in vivo.The contribution of enterohepatic circulation of ASIV and the impact of activity of intestinal microbiota on the deposition of ASIV and its metabolites were evaluated.Secondly,effect of the solubility of ASIV on the bioavailability was also investigated from itself chemical properties.The absorption and metabolic mechanism of ASIV was studied according to the dynamic change of ASIV and metabolites from administration to getting systemic circulation.Furthermore illustrate the main factors of the hard process of absorption,and the radical factor of low bioavailability of ASIV.Descriptions are as follows:1.Biotransformation of ASIV in ratsLC-MSn method was employed to investigate the metabolic profiles of ASIV in several biosamples,such as feces,urine,bile and plasma of rats after oral administration of ASIV.Seven metabolites were detected,including deglycosylation of ASIV and cycloastragenol(CA),which was sapogenin of ASIV,and metabolites of CA.The amount of metabolites in bile and feces were higher than urine,in which little of glucuronidation of CA was detected besides the unchanged ASIV.In addition of ASIV,CA and its isomer,iso-CA was found in plasma.2.Pharmacokinetics of ASIV in ratsThe amounts of ASIV and its metabolites were quantified by an LC-MS/MS method.The Pharmacokinetic study of ASIV and its metabolites was investigated in rats after single or multiple oral and intravenous administration of ASIV.After rats were intragastrically administrated with single 10 mg/kg ASIV,ASIV was predominant in plasma,the AUC0-t values of ASIV,CA and iso-CA were 109±55,26.8±17.9 and 77.9±35.1 nM·h,respectively.The contribution of ASIV,CA and iso-CA was 51.0%,12.5%and 36.5%.After rats were intragastrically administrated with multiple 10 mg/kg ASIV,the plasma concentration of ASIV was stable,but CA and iso-CA were changed greatly.The AUC0-t values of CA and iso-CA were increased 10 and 8 times,respectively.The amount of iso-CA was higher than ASIV in plasma,consequently iso-CA was predominant in plasma.Pharmacokinetics of ASIV and its metabolites in rats were studied after an i.v.injection of 1.5 mg/kg ASIV.When rats received an i.v.injection of ASIV,ASIV traveled in rat systemic circulation mainly as itself.Very tiny amounts of CA and iso-CA were detected in rat plasma from 4 h after administration.The contribution of ASIV,CA and iso-CA to the total exposure were 99.7%,0.05%and 0.3%,respectively3.Stability of ASIV and CA in biological fluids in vitroThe stability of ASIV and CA in vitro was studied by using LC-MSn method.ASIV and CA were stable at different pH solution,and in gastrointestinal digestive juice.ASIV was comparatively stable to rat liver and intestinal S9 fraction.CA underwent moderate metabolism in rat liver S9 fraction but was hardly biotransformed by intestinal S9 fraction.When CA was incubated with rat liver S9 fraction at 37? for 1 h,approximate 25%of CA was metabolized,among which 6%was converted to iso-CA.ASIV was slowly metabolized by intestinal microbiome.After 4 h incubation,ASIV began to be converted to brachyoside B(Bra B),cyclogaleginoside B(Cyc B),cycloastragenol(CA),and dehydrogenated metabolite of CA(CA-2H)by intestinal bacteria.After 24 h incubation,about 72%of ASIV was metabolized by gut bacteria,among which 36%was converted to CA,35%to CA-2H,and 1%to iso-CA%.When 1 ?M CA reacted with intestinal bacteria,No other metabolites except CA-2H and iso-CA were found in the intestinal bacteria incubation samples of CA.4.Effect of enterohepatic circulation and the activity of intestinal bacteria on the bioavailable degree of ASIVThe plasma distribution of ASIV and its metabolites between rats with and without bile duct-cannulation(control)at 2,8 and 10 h after administration of ASIV from duodenum were investigated to evaluate the effect of enterohepatic circulation on the disposition of ASIV.After rats received bile duct drainage surgery,the plasma amount of ASIV at 2 h was significantly lower than that in control rats at the same point.Moreover,the amounts of CA and iso-CA in bile duct-cannulated rats became immeasurable at 8 and 10 h.These data declared that enterohepatic circulation played an important role in the disposition of ASIV after oral administration.We also compared the plasma distribution of ASIV and its metabolites between bile duct-cannulated rats and control rats after i.v.injection.Neither CA nor iso-CA was found in control and bile duct-cannulated rats at 2,8 and 10 h after i.v.injection.Hydrolysis of ASIV by gut microbiota is a key factor in the disposition of ASIV after oral administration.The influence of activity of intestine microbiota on the deglycosylation of ASIV,in vitro gut microbial conversion of ASIV were compared between the intestinal microbiota obtained from antibiotic-pretreated rats and that from control rats.No metabolites of ASIV were observed in antibiotic-pretreated gut microbiota samples over the same time period.The metabolism of ASIV was obviously inhibited by inactivated intestinal microbiome.Pharmacokinetic profile of ASIV in antibiotic-pretreated rats was similar to that in control rats.Nevertheless,the plasma concentrations of CA in antibiotic-pretreated rats were immeasurable.The result indicated that the disposition of ASIV was obviously affected by the activity of intestinal bacteria after oral administration.5.Effect of Solubility on the bioavailable degree of ASIVThe effect of the solubility of ASIV on the bioavailability was also investigated.Tea phenol(TP)could modify the solubility of ASIV.Although the bioavailability of ASIV was increased 10 times,it was still very low.In summary,ASIV was metabolized by intestinal bacteria to form Bra B,Cyc B,CA,iso-CA,and CA-2H.CA and iso-CA circulated in blood besides ASIV when rats received ASIV intragastrically or intravenously.The plasma distribution of ASIV was significantly affected by bile duct drainage when ASIV was administrated through duodenum.ASIV,Bra B and Cyc B were secreted from bile after duodenal administration of ASIV.Antibiotics markedly inhibited the metabolism of ASIV in intestinal microbiota.After rats were pretreated with antibiotics,the AUC0-t of iso-CA was 4.8 times less than that in control rats and the concentration of CA became undetectable.Variations in intestinal microbiota may change the disposition of ASIV and subsequently influent its potential health benefits.Modified solubility could enhance the absorption of ASIV,but it was not the main reason for the low bioavailability of ASIV.