Metabolism And Kinetic Studies On The Effective Components Of Chrysanthemum Morifolium Extracts

Flos Chrysanthemi, the flower of Chrysanthemum morifolium Ramat. was harvested in October-December generally. According to 《Ben Cao Gang Mu》, 《Ben Cao Shu Jing》 , Flos Chrysanthemi has many beneficial effects of dispelling wind and heat, improving eyesight, calm the liver and suppress yang, heat-clearing and detoxicating. The pharmacological investigations indicated that Chrysanthemum morifolium extracts (CME) had some pharmacological effects and was widely used in the treatment of cardiovascular diseases. And the further researches attributed these beneficial effects to some flavonoids contained in CME. Therefore, systematic studies about metabolic information in vivo, especially for the pharmacokinetics and absorption kinetics characteristics in vivo or in vitro of flavonoids, play the key role in flavonoids research and may be the foundation for elevating preparations quality, optimizing administration program and new drug design of CME.Studies showed that luteolin and apigenin were the bioactive components of CME when it was orally administrated. In this paper, HPLC methods are established for analyzing biological samples containing the objective components (luteolin and apigenin), then applied the methods to investigate the pharmacokinetics differences and observe the informations of enteric absorption, intestinal flora metabolism and liver microsome metabolism. Moreover, the paper evaluates initially the interindividual difference characteristics of in absorption, metabolic and excretion in different species. For further, to explore new ideas and methods for seeking new drugs, and provide scientifical evidence in order to direct safe and reasonableappalication on clinic, scientifically evaluate animal pharmacodynamics, pharmacokinetics and toxicology.Pharmacokinetics studies on the effective component of Chrysanthemum morifolium extracts1. Studies on the effective components pharmacokinetics of CME and its acid hydrolysate in rabbitsAims: To establish an HPLC method for analyzing luteolin and apigenin in rabbit plasma, and apply the method to characterize and compare the metabolic pharmacokinetics of the effective components (luteolm and apigenin) in rabbit after oral administration of acid hydrolysate of CME or intact CME, and clarify whether the bioavailability and pharmacokinetics of flavoloids from Chrysanthemum morifolium Ramat. would modified due to the flavonoids forms orally administrated. Methods: The blood samples were collected from ear edge vein in a tube containing heparin at different times after rabbits were orally administrated of a single dose (200 mg/kg, n=3) of intact CME and hydrolysate of CME, and the plasma was isolated by centrifugation. The concentration of luteolin and apigenin in plasma were determined by reversed-phase high performance liquid chromatography (RP-HPLC) method in order to elucidate whether are there some differences in pharmacokinetics of luteolin and apigenin of hydrolysate of CME and intact CME. The pharmacokinetic parameters were calculated by software 3P87, and statistical assay was performed on software Microsoft Excel. Results: The plasma concentration of luteolin could be determined accurately and the pharmacokinetics process was best fitted to one-compartment model. However, the present study showed that the concentration of apigenin was too low to be measured accurately. There were some extent differences in pharmacokinetics of effective component (luteolin) of hydrolysate of CME and intact CME in apparent. Furthermore, oral hydrolysate of CME resulted in latter Tmax, lower Cmax and longer MRT (mean residence time) for luteolin than those after oral intact CME. By Mest statistics, oral administration of acid hydrolysate of CME or intact CME to rabbit, the plasma concentration and AUC of luteolin reveals no significant difference (P>0.05). Conclusions: Whether hydrolysate of CME or intact CME were orally administered to rabbits, no statistical difference was observed inpharmacokinetics characteristics of luteolin.2. Linear pharmacokinetics of effective components of CME following single oral administration to ratsAims: To study the linear pharmacokinetics and metabolism characteristics of effective components of CME after oral administration to rats and investigate the dosage range of CME for linear pharmacokinetics. Methods: 20 male SD rats received single oral doses of CME (100, 200, 400 mg/kg and 12g/kg as the maximal tolerated dose, n=5), in a randomized, single-blind study. The blood samples were collected from tail vein in a tube containing heparin at different times after rats received single oral doses of CME. The concentration of the total luteolin and apigenin in plasma were determined by HPLC after plasma hydrolyzed for studying linear pharmacokinetics of effective components of CME. Results: The results indicated that the plasma concentration-time profiles of luteolin and apigenin fitted well one-compartment models after single oral dose of 100 mg/kg, while two-compartment kinetics for 200 and 400 mg/kg groups. The areas under the concentration versus time curves (AUCo-oo) and the peak concentrations (Cmax) of luteolin and apigenin increased linearly with the dose of CME. CME was well tolerated by all subjects with a low incidence of adverse effects after oral adminisration of 12g/kg, as the maximal tolerated dose. Conclusions: CME was well tolerated by all subjects. The Cmax and AUCo-oo of luteolin and apigenin increased in a dose dependent manner. Overall, luteolin and apigenin exhibited linear kinetics in the dose range of 100-400 mg/kg CME.3. Pharmacokinetics of effective components in dogs after successive oral administration of CMEAims: To investigate pharmacokinetics characteristics of the active components in dogs after continuous 9 months oral administration of high and low doses of CME. Methods: The blood samples were collected from saphenous vein at different times after Beagle dogs were given orally CME at dosages of 918 mg/(kg-day), 102 mg/(kg-day) for successive 9 months. The concentration of the total luteolin and apigenin were determined by HPLC and pharmacokinetic parameters were calculated. Results: After Beagle dogs were administered orally CME for successive 9 months, the concentration of luteolin in plasma was much higher than that of apigenin. The results simulated by 3P87 software indicated the plasma concentration-time profiles of luteolin and apigenin fitted one-compartment models well after oral dose of 918mg/(kg-day), while two-compartment kinetics for 102mg/(kg-day) groups. For luteolin and apigenin, the maximum plasma concentrations (Cmax) and the areas under the concentration-time curve (AUCo-oo) increased with dosage, but a under-proportionality was detected in exposure with increasing doses of CME. No accumulation of luteolin and apigenin were observed after 9 months of repeated daily administration. Conclusions: There were no remarkable changes in behavior or demeanor recorded during the daily animal observations for all dogs after administration of CME to dogs at doses of 102 and 918 mg/(kg-day) for up to 9 months. Moreover, no significant difference was observed roughly in the 102 mg/(kg-day) group when compared with the 918 mg/(kg-day) treated group.4. Studies on pharmacokinetics and excretion of the effective components of CME in humanAims: To characterize the pharmacokinetics of effective components in human plasma and urine after oral administration of CME tablets, know the respective urinal excretion of luteolin and apigenin, and provide scientific bases for rational administration in clinic. Methods: The blood and urine samples were collected at different times after eight healthy volunteers administrated CME tablets orally (20mg/kg). The concentration of the total luteolin and apigenin were determined for studying the pharmacokinetics of effective components of CME, and the urine samples were incubated with |3-glucuronidase/sulfatase for obtaining the cumulative amount excreted in urine of total luteolin and apigenin (free + sulpho or/and glucuronide conjugates) within 12h. Results: The described method was applied to assay luteolin and apigenin in plasma samples of eight healthy volunteers after oral dosing of CME tablets, moreover, their Tmax were both <1.0h. The data demonstrated that the plasma concentration-time profiles of luteolin and apigenin both fitted one-compartment models well. After urine samples were incubated with P-glucuronidase /sulfatase, the mean concentrations of luteolin in urine was high than that of apigenin. The cumulative amount excreted in urine of total luteolin and apigenin (free + sulpho or/and glucuronide conjugates) within 12h were only 2.30% and 6.09%, respectively. Conclusions: Luteolin and apigenin were absorpted rapidly into plasma after volunteers administered of CME tablets, and eliminated slowly. The fraction of oral dose excreted in urine was little for luteolin and apigenin within 12h, respectively. The results suggested that sulpho or glucuronide conjugates of luteolin and apigenin only represent a part of metabolites, or the urinary excretion was not animportant route.Studies on disposition in intestinal tract of the effective components of Chrysanthemum morifolium extracts1. Studies on CME metabolism in intestinal flora in vitroAims: To study and compare metabolism of CME in vitro intestinal flora from human, rat, Beagle dog and rabbit. Methods: CME in intestinal flora of human, rat, Beagle dog and rabbit was anaerobic culture at 37°C in vitro. After extracted by acetic ether, the main metabolites of CME were separated and quantified by HPLC/LC-MS. Results: In vitro, CME was metabolized easily by the intestinal flora and the main metabolites in incubation medium were determined in high concentration. Identified by LC-MS, luteolin and aipigenin were identified, respectively;both metabolites were degraded with prolongation of incubation time and the concentration of luteolin and apigenin was below the LOQ after 24h. Moreover, CME was metabolized quickly by human, rat, beagle dog intestinal flora, and gently by rabbit intestinal flora. Conclusions: In vitro, CME is metabolized easily by intestinal flora of human, rat, Beagle dog and rabbit, and the main metabolites are luteolin and apigenin.2. Study on intestinal absorption of the effective components of hydrolyzed CME in animals with the everted sac techniqueAims: To investigate and compare the dynamics of intestinal absorption by the method of everted gut sac of different animals for profound on the process in vivo. Methods: Using the reverted gut sac experiments of rat and rabbit in vitro to observe absorption of the effective components hydrolyzed CME via intestine. The luteolin and apigenin in gut sac were determined by HPLC, and the absorption rate constants (Ka) and apparent permeability coefficients (Papp) were calculated. Results: It was found that in rat and rabbit everted gut sac, the Ka of luteolin and apigenin were 0.0185, 0.0253mm"1 and 0.0186, 0.0091mm'1, while the Papp were 3.941*10"5> 10.15xl0'5cm/min and 1.051 xlO'5, 2.443x 10'5 cm/min, respectively. Conclusions: Absorpion of luteolin and apigenin in rat gut sac was relatively better than in rabbit gut, moreover, apigenin was absorpted easily more than luteolin by gut sac. It showed that there was correspondance with the results of in vivo tests which Cmax of apigenin was higher than that of luteolin in rat. By Hest statistics, a significant difference wasfound in the absorbing capacity of two species animals' gut sacs for apigenin (PO.01).Study on metabolism of the effective components of Chrysanthemum morifolium extracts in microsomes in vitro1. Studies on metabolism of hydrolyzed CME in liver microsomes Aims: To study and compare the metabolism of apigenin, one of the effective components of hydrolyzed CME, as well as enzyme kinetics in Beagle dog, rabbit, and rat liver microsomes. Methods: Liver microsomes of Beagle dog, rat and rabbit were prepared by using calcium salts precipitation method. 10.0 ug/mL hydrolysate of CME was incubated with some concentrations (0.3-0.8mg/mL) of liver microsomes at 37°C for different time (5-15min). The concentration of apigenin in the supernatant was determined by HPLC at proper time intervals to select the optimal concentration of liver microsomes and incubation time. The different concentration (10.0-200 Hg/mL) of hydrolysate of CME were incubated with different animals microsomal protein at 37°C for the optimal incubation time, respectively, and HPLC was used to determine the concentration of apigenin in the incubation mixture. The Michaelis-Menten parameters (Michaelis constant [Km] and maximal velocity [1/F]) in liver microsomes were determined. The values of Km and XIV were initially estimated by analysing Line weave-Brurk plot. Results: With the microsomal protein of Beagle dog (0.5mg/mL), rabbit (0.3mg/mL), and rat (0.3mg/mL) in incubation systems spiked with 10.0 ng/mL hydrolysate of CME, the optimal incubation times at 37°C for apigenin in the three microsomes were lOmin, 5min, and 5min, respectively. The Km of apigenin in the microsomes of Beagle dog, rabbit, and rat were 3.608i 0.62,4.363±0.46 and 3.599±0.62 ng/mL, respectively. The Vm were 0.1970±0.010, 0.8924±0.051 and 0.3274±0.061ug/mg/min, respectively. Conclusions: With the concentration range (10.0-200 ug/mL) of hydrolysate of CME, apigenin incubated in liver microsomes of Beagle dog, rabbit and rat was metabolized into the main metabolite, luteolin. By r-test statistics, no significant difference was observed in Km of apigenin among three different microsomes (P>0.05). Metabolism of apigenin in Vm and C/jnt in Beagle dog liver microsome was similar to that in rat liver microsome,while, there was a significant difference between rabbit microsome with the other two CPO.Ol).