Distribution And Excretion Studies On The Effective Components Of Chrysanthemum Morifolium Extracts In Rats

The flower of Chrysanthemum morifolium Ramat.(CM)has been used as Chinese traditional healthy food and medicine for hundreds of years.It contains flavonoids,chlorogenic acid and aetherolea.Studies showed that the main flavonoids in CM were luteolin-7-O-β-D-glucoside and apigenin-7-O-β-D- glucoside,which also were the main components effecting anti-oxidant values and preventing cardiovascular diseases.Both of the main flavonoids in CM would be bio-transformed into their aglycones—luteolin and apigenin in the intestine by microorganism and hydrolase in epithelial cell after an oral administration of the extract of CM to animals.The previous studies of our laboratory have proved that luteolin and apigenin are the main bioactive components of Chrysanthemum morifolium extract(CME)in vivo.Few references are available on the tissue distribution and excretion of the effective components of CME.Therefore,the present study evaluates the distribution rule of luteolin and apigenin in rats and elucidates their distribution difference under the normal and myocardial ischemia conditions.Moreover,this study investigates the excretion of luteolin and apigenin in urine,feces and bile,to observe the information of excretion routes and forms of CME in rats,to perfect the fate of CME in animal models,and provide powerful evidence for compound foundation of the effect of CME anti-myocardial ischemia. Tissue distribution studies on the effective components of Chrysanthemum morifolium extracts in rats1.Tissue distribution studies on the effective components of CME in healthy rats after single dose administrationObjective:To establish an HPLC method for analyzing luteolin and apigenin in rat tissue,and evaluates the distribution rule of luteolin and apigenin in rats. Methods:The tissue samples were collected at different times after rats were orally administrated of a single dose(200mg/kg)of CME.The concentration of luteolin and apigenin in tissue were determined by reversed-phase high performance liquid chromatography(RP-HPLC)method after samples were homogenated and hydrolyzed.Results:Luteolin and apigenin were distributed in most of all tissues collected,such as heart,liver,spleen,lung,kidney,brain,muscle,fat,testes,stomach and intestine, especially in stomach and intestine,the concentration of luteolin and apigenin were the highest,and that in liver,kidney and lung were higher than that in brain and testes.In most tissue,luteolin and apigenin reached the highest peak at 0.5h after administration,which was similar with that in plasma.Conclusion:Luteolin and apigenin both have a quick and extensive tissue distribution.2.Tissue distribution studies on the effective components of CME in rats of myocardial ischemiaObjective:To study the distribution difference of luteolin and apigenin in rats under the normal and myocardial ischemia conditions,and to provide powerful evidence for the effect of anti-myocardial ischemia of CME.Methods:Male SD rats were grouped randomly into steady-state, pre-administrated and post-administrated groups.The dose of CME was 200mg/kg. All groups were administrated continuously for 7 days.The post-administrated group was operated to myocardial ischemia 24h before the first day administrated.The pre-administrated group was operated to myocardial ischemia after the sixth day administrated.All rats were executed at 0.5h after the last administration,and main tissues were collected immediately.The concentration of luteolin and apigenin in tissue were detected by HPLC,and t-test was applied to clarify the difference among three groups.Results:In pre-administrated group,the distributions of luteolin and apigenin were both lower than steady-state group.There were no noticeable differences of luteolin's concentration in most tissues between post-administrated group and steady-state group,while,in post-administrated group,the concentration of apigenin in heart is higher than steady-state group.Take the comparsion of pre-administrated group with post-administrated group,the former's concentration of luteolin and apigenin in most tissues were higher than the latter's.In plasma and heart,the concentration of luteolin and apigenin in post-administrated group is higher than that in pre-administrated group,whereas,that in brain and testes.Conclusion:There are noticeable differences of luteolin's and apigenin's concentration among three groups.In large blood-flow tissues,both of luteolin and apigenin have differences between two administrated patterns of myocardial ischemia rats.Excretion studies on the effective components of Chrysanthemum morifolium extracts in ratsObjective:To investigate the main three courses' excretion of metabolites of CME (luteolin and apigenin)in rats after oral administration,and to evaluate the information of main excretion routes and forms of CME in rats.Methods:The concentration of luteolin and apigenin in urinary,fecal and biliary were measured by RP-HPLC.Prior to HPLC analysis,urine and bile was hydrolyzed byβ-glucuronidase and sulfatase for the degradation of conjugates to aglycones.And feces samples were homogenized in methanol and sonicated.Then,the accumulated excretion rate of luteolin and apigenin in the three courses were calculated.Results:The total recovery of the dose of luteolin was 37.8%(6.54%in urine; 31.3%in feces)and that of apigenin was 45.2%(16.6%in urine;28.6%in feces). Cumulative luteolin and apigenin excreted in the bile were 2.05%and 6.34%of the dose,respectively.Conclusion:Both of the total recovery of luteolin and apigenin didn't reached at 50%,of dosage,which suggested that luteolin and apigenin may be metabolized to simple compounds or other metabolites.The existence of biliary excretion appears the conjugates of luteolin and apigenin undergo enterohepatic cycling resulting in deconjugation and reabsorption of luteolin and apigenin.It may be associated with double peaks in the blood concentration-time curves and prolongs the plasma half-life of luteolin and apigenin in rats following oral administration.Studies on metabolites of CME in ratsObjective:Study metabolites from urine sample of CME(M1,M2),to find its metabolism route.Methods:Compare the urine sample with diosmetin standard sample using HPLC-MS.Results:The two metabolites had the same molecule weight with diosmetin,but only one metabolite's(M2)abundance was the same with diosmetin.And M2 had the same retention time and UV-absoption with diosmetin.Conclusion:It is hypothesized that the two metabolites are isomers,and one metabolite(M2)is diosmetin.