Study On Absorption And Metabolism Of The Main Active Constituents In Cortex Daphnes

Cortex Daphnes is the stem cortex and root bark of Daphne giraldii Nitsche,D. tangutica Maxim or D.retusa Hemsl,a folk herb used in west of China.The content of daphnetin and coumarins in raw material of Cortex Daphnes were determined about 0.2%and 3%by HPLC and UV method respectively.The adjuvant-arthritic(AA) rat model was developed to evaluate the anti-arthritic effects of Cortex Daphnes.The results showed that daphnetin significantly reduced paw swelling and erythrocyte sedimentation.The pathological examination demonstrated that articular cartilage degeneration with synovial hyperplasia and inflammatory cells infiltration in AA rats were suppressed by daphnetin.There was significant reduction in production of IL-1, TNF-αand MIF in serum of AA rats treated with daphnetin.In conclusion,daphnetin was the main active constituents in Cortex Daphnes for treating rheumatoid arthritis.The physical and chemical parameters such as apparent solubility and n-octanol-water partition coefficient of daphnetin were measured.Daphnetin belongs to the classⅡdrug of Biopharmaceutics Classification System,which solubility is poor but membrane permeability is fine.Daphnetin concentrations were investigated in blood plasma after ig or iv injection in rats by HPLC and pharmacokinetic parameters were calculated.Pharmacokinetic parameters of daphnetin after intravenous(50mg/kg) and oral administration(250mg/kg) in rats were all fit to the one-compartment model.Daphnetin was distributed and eliminated quickly.The absolute bioavailability was about 24.6%after oral administration. Daphnetin was detected in the urine collected within 2h after oral administration(5,25,125mg/kg) and almost completely excreted within 24h.Cumulative urinary excretion of daphnetin was not more than 6 percent of administration dosage.The mechanism of daphnetin intestinal absorption was first investigated systematically at in vivo and cellular level.The absorption of daphnetin was studied by the rat intestinal perfusion model.The permeability of daphnetin(10μg/mL) in duodenum, jejunum,ileum and colon was 1.52±0.79,1.34±1.09,1.41±0.50,0.54±0.34 respectively; the permeability of daphnetin(20μg/mL) in duodenum,jejunum,ileum and colon was 1.54±0.09,1.48±0.11,1.44±0.36,0.72±0.09 respectively;the permeability of daphnetin (20μg/mL) in duodenum,jejunum,ileum and colon was 2.12±0.81,1.99±0.23,1.59±0.90,1.12±1.05 respectively.The results indicated that the absorption of daphnetin was passive diffusion and no deference in different intestinal segment of rats.The absorption mechanism of daphnetin was investigated by Caco-2 monolayer model.The uptake of daphnetin was positive correlation to time and temperature,but not affected by pH.The ration of P_BA/P_AB was reduced along with the increase of daphnetin concentration.The absorption of daphnetin was passive diffusion at the dominating process.Daphnetin may be a substrate of BCRP and MRP2,but not P-gp substrate.The phaseⅡmetabolic reactions of daphnetin were found in intestine of rats and Caco-2 monolayer model.Four metabolites of daphnetin were detected in the intestine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7--glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation and sulfation.Two metabolites of daphnetin detected in Caco-2 monolayer model were daphnetin-7-glucuronide,daphnetin-7-glucuronide respectively by analyzed LC and MS data.The metabolic pathways of daphnetin in the intestine of rat were mainly glucuronidation.The metabolism of daphnetin in urine,bile and plasma were also investigated.Eight metabolites of daphnetin were detected in urine of rat.These metabolites were daphnetin-7-sulfate,daphnetin-8-sulfate,daphnetin-7-glucuronide,daphnetin-7--glucuronide,daphnetin-7-methoxyl-8-glucuronide,daphnetin-7-methoxyl-8-glucuronide, daphnetin methylate and daphnetin hydroxylate respectively by analyzed LC and MS data. Two metabolites of daphnetin detected in bile of rat were daphnetin-7-glucuronide, daphnetin-7-glucuronide.One metabolite of daphnetin detected in plasma of rat was daphnetin methylate.These metabolites of daphnetin were all found at the first time.