Studies On The Absorption, Metabolism And Drug-drug Interactions Of Effective Components Of Chrysanthemum Morifolium Extracts

Flos Chrysanthemi is the flower of Chrysanthemum morifolium Ramat..It has many beneficial effects such as dispelling wind and heat,improving eyesight,calm the liver and suppress yang,heat-clearing and detoxicating.The pharmacological investigations indicated that CME has antioxidant activity and can be used in the treatment of cardiovascular diseases.And the further researches shows that the flavonoids in CME play an important role in these benefit.The main flavonoids in CM were luteolin-7-O-β-D-glucoside and apigenin-7-O-β-D-glucoside,which can be deglycosidated to their effective aglycone——luteolin and apigenin during absorption.The pharmacokinetic,absorption,distribution and excretion of main CM flavonoids have been systematically studied in our lab.After oral administration of CME,the main effective components were luteolin and apigenin.AUC of apigenin was higher than luteolin in pharmacokinetic study,and they can excrete by urine,stool and bile.They can distribute in various tissues and their distribution was different between normal and myocardial ischemia rats.While,the hydrolysis and absorption site of glucosides in CME,the effect of conditions on the hydrolysis and absorption process as well as the reason of the higher AUC of apigenin were still not well elucidated.In addition,CM can be use as food and drug,so there was more chance of drug-drug interactions,which was not reported too.In this paper,we will investigate the absorption,metabolism and drug-drug interactions of CME in vivo,in situ and in vitro level to explore new ideas and methods for seeking new drugs,and providing scientifical evidence to scientifically evaluate animal pharmacodynamics,pharmacokinetics and toxicology.1.Deglyeosidation and absorption of active flavonoids of CME in rats.Objective:To investigate the deglycosidation and absorption of active flavonoids of CME in rat.Methods:Established HPLC methods for analyzing luteolin and apigenin in rat gastrointestinal contents and tissues,and then evaluated the distribution of luteolin and apigenin in gastrointestine.Results:1 ) The hydrolysis and distribution of CME components in gastrointestine were very fast.Luteolin and apigenin emerged in plasma,the contents and tissues of stomach,duodenum and jejunum at 5min after administration.2) The highest concentration of contents were 5min,5min,2h,5h and 8h for stomach,duodenum, jejunum,ileum,cecum and colon,respectively.The total residence time of luteolin and apigenin in gastrointestinal tract exceeded 12h,and contained high concentration level in large intestine contents for long time.3 ) Luteolin and apigenin were absorbed fast,while the elimination process of them in plasma were relatively slow,Tmax of luteolin and apigenin in plasma were about 30min and 5h,respectively,which implied that luteolin was mainly absorbed in the upper instine while apigenin may be absorbed in wider scope,and the plasma concentration of apigenin was still high at 12h after administration.3 )The ratio of luteolin and apigenin in plasma,tissue and contents was different,such as the concentration of apigenin was far beyond luteolin in plasma,but concentration of luteolin was higher than apigenin in cecum and colon.Conclusion:CME components can be hydrolyzed fast in the whole gastrointestine and producted effective components——luteolin and apigenin.This process was site and substrate dependent.2.Deglycosylation and absorption of active components of CME in situ perfusing iigated intestinal segment.Objective:To assess the hydrolysis and absorption ability of different gastrointestinal parts independently and impact of factors on hydrolysis and absorption.Methods:An in situ perfusing ligated intestinal segment model was used.After incubated CME in various gastrointestinal segments,the plasma,tissues and contents sample were collected and evaluated to assess the discrepancy of different parts.In addition,jejunum was selected to investigate the pharmacokinetics of deglycosidation and absorption.Factors affecting hydrolysis and absorption(contents,bacteria,long term administration,gender and acid condition of stomach ) were also evaluated.Results:1 ) All parts of gastraintestine could hydrolyze CME,and luteolin and apigenin could be detected after 10min incubation with various parts.The hydrolysis ability of cecum on luteolin glucoside was higher than apigenin glucosides,while other parts had the fair ability of hydrolyzing luteolin and apigenin glucosides,and the hydrolysis rate were all beyond 50%.Luteolin and apigenin could be uptaked by various gastrointestinal tissues,but only small intestine could transport them to blood. 2 ) CME was hydrolyzed and absorpted fast in jejunum,and the highest concentration emerged was 5min after administration.The absorption of apigenin was higher than luteolin with Cmax value 8.43 and 5.41μg/mL,respectively,while AUC_0-∞were 999±100 and 339.4±49.9mg/L*min,respectively.The hydrolysis and absorption potential depended on the dosage.3 ) Without contents,using antibiotics as well as long-time administration were all decreased the concentration of luteolin and apigenin in jejunum contents,while gender had no significant effect on it.Without contents,the tissue uptake and plasma concentration of luteolin and apigenin were increased,but other factors have no significant effect on their tissue and plasma concentration.Conclusion:CME components can be hydrolyzed fast in the whole gastrointestine and producted effective components——luteolin and apigenin.They can be absorbed in small intestine.This process was site and substrate dependent,and can be influenced by various factors.3.Deglycosidation of CME in cell free fractions of different tissues.Objective:To investigate the hydroysis ability of cell-free fractions of various tissues in vitro.Methods:Determinated the concentration of luteolin and apigenin after incubated CME with various cell free fractions.Investigated the best concentration of substrates, incubation time and protein contents,and compare the hydrolysis ability of various cell free fractions.LC-MS was also used to identify the metabolites.Results:The incubation condition selected was:18.75mg/mL of substrates,0.5mg protein/mL,and incubate for 30min.CME can deglycosidated in all investigated cell-free fractions,and produced effective luteolin and apigenin.Kidney cell free fraction can hydrolyze more CME than other tissues during the same time.Jejunum can hydrolyze more CME than other gastrointrestinal tissues,while stomach was the weakest one.Conclusion:CME can be hydrolyzed in many tissues,and produce effective lutelin and apigenin.Kidney and jejunum cell free fractions can hydrolyze CME more efficently. 4.Metabolism of luteolin and apigenin in primary cultured hepatocytes.Objective:Investigated the conjugation metabolism of luteolin and apigenin in liver after absorption.Methods:Primary cultured rat hepatocytes model was selected.The elimination process of them was evaluated and the effect of other flavonoids on the elimination rate of luteolin and apigenin was also studied.Results:1 ) The eliminaton of luteolin and apigenin was very fast.91.9%of luteolin and 86.7%of apigenin disappeared after 120min incubation.The percentage of phaseâ…¡metabolites reached 54%and 73%of dose for luteolin and apigenin,respectively. 2 ) Gender and pH have no significant effect on the elimination rate of luteolin and apigenin.3 ) Luteolin can transformed to apigenin to mimor extent(＜5%).4 ) When monolayers were exposed to increasing amounts of luteolin and apigenin,a concentration-dependent inhibition were observed;They can inhibit the elimination of each other with the IC₅₀ value of 17.3μmol/L and 38.2μmol/L,respectively. Elimination of luteolin and apigenin were also inhibited in HCME.5 ) Other flavonoids glycosides didn't make any difference in elimination rate of luteolin and apigenin.As to flavonoids aglycone,flavonol and flavone can inhibit their elimination effectively,while isoflavone and catechin have no significant effect.Conclusion:Metabolism of luteolin and apigenin in hepatocytes was very fast.Drug interactions occured in this process.5.Modulation effect of CM flavonoids on metabolizing enzymes of hepatocytes.Objective:To investigate the Modulation effect of CM flavonoids on metabolizing enzymes.Methods:A rat primary cultured hepatocytes was used.The modulation effect was valued by the elimination of probes.The probes of CYP3A were testosterone and nifedipine and probe of CYP2E was chlorzoxazone.Results:Metabolism of testosterone after 1h incubation were inhibited to 44.0±8.2%, 48.5±4.7%,20.1±0.4%and 70.5±0.8%by luteolin,apignein,CME and HCME, respectively.Metabolism of nifedipine after 2h incubation were inhibited to 79.1±1.3%,80.1±0.2%,60.4±2.6%and 92.2±0.4%,respectively.Metabolism of chlorzoxazone after 2h incubation were inhibited to 54.3±0.2%,42.6±0.8%, 41.9±2.7%and 69.9±1.8%,respectively.The inhibition of elimination of probes was HCME＞luteolin≈apignein＞CME.Conclusion:CM fiavonids can inhibit CYP3A and CYP2E enzymes of hepatocytes. Hydrolysis process may bioactivate this inhibition effect.6.Modulation effect of CM flavonoids on human P-gp.Objective:Investigated the modulation effect of CM flavonoids on human P-gp.Methods:Overexpressed MDR1 cell line——MDR1-Bcap and MDR1-MDCK were selected to investigate the inhibition effect of CM flavonoids.The accumulation of rodamine 123 in above cell lines was used to value this inhibition effect.The inhibition of rodamine accumulation was HCME＞luteolin＞apignein＞CME,and the results from different models was equol.Results:Luteolin,apignein,CME and HCME can inhibit human P-gp significantly, and this inhibition was dose dependent.The accumulation of rodamine in Bcap-MDR1 can reach 2.79±0.063,1.68±0.078,1.15±0.051 and 5.73±0.073 fold of control for the highest dose of luteolin,apignein,CME and HCME,respectively;The accumulation of R123 in MDCK-MDR1 can reach 2.07±0.039,1.44±0.065, 1.67±0.091 and 7.23±0.65 fold of control for the highest dose of luteolin,apignein, CME and HCME,respectively.Conclusion:CM flavonoids can mudulate human P-gp.Hydrolysis process may bioactivate this inhibition effect.