Determination of phillygenin in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies.

The research group has been dedicated to the study of Fructus Forsythiae which was used widely in traditional Chinese medicines. And some research results have been accepted in Chinese Pharmacopeia. In a recent study, phillygenin was found to be a potential "metabolite" of phillyrin and the effective material of phillyrin may be changed according to the in vivo pharmacokinetic process. Therefore, a sensitive, specific, accurate, and reproducible reversed phase HPLC method for the determination of phillygenin in rat plasma was developed. Separation was achieved on a Hypersil ODS C18 column with UV detection at 277 nm. The good linear calibration curves ranged from 0.039 to 20 µg/mL with the limit of quantification estimated as 0.026 µg/mL. The intra- and inter-day precisions were in the range of 98-103 %. The average recoveries of phillygenin were 90.54, 92.47, and 92.15 % for phillygenin of 0.156, 1.25, and 10.0 µg/mL. And the Ruggedness of HPLC method was evaluated. The analytical method was also successfully applied to the pharmacokinetic study of phillygenin in rat for the first time. A rapid distribution was observed from the plasma concentration-time curves, and was followed by a quick elimination for phillygenin. The mean t 1/2z was 6.02, 5.62, and 5.79 min for 1.4, 2.8, and 5.6 mg/kg, respectively. The AUC (0-t) increased linearly from 166.29 to 332.48 mg/L min. All results indicated that, in the range of the doses examined, the pharmacokinetics of phillygenin in rat was based on first-order kinetics.

Assessment and modulation of phillyrin absorption by P-gp using Caco-2 cells and MDR1-MDCKII cells.

The study was to investigate the absorption mechanism and transport modulation of phillyrin by P-gp in Caco-2 cells and MDR1-MDCKII cells. Three concentrations of phillyrin were tested in transport studies. The absorptive transports of phillyrin in the two cell models were not concentration-dependent which indicated passive diffusion as the dominating process in the test concentrations. The absorptive P (app) were 7.15, 6.39 and 10.03 × 10(-6) cm s(-1), respectively, for different concentrations (2.2, 4.8 and 8.4 µg ml(-1)) in Caco-2 cells. And the low absorptive P (app) was consistent with the low oral bioavailability of phillyrin observed in pharmacokinetic experiments. In transport inhibition experiment, the efflux inhibitors, verapamil and GF120918 can increase the absorption of phillyrin in Caco-2 cells which suggested the involvement of efflux transporters. In the further inhibition experiment in MDR1-MDCKII cells, the absorption was greatly increased and the efflux of phillyrin was competitively inhibited by verapamil and GF120918, which confirmed the involvement of P-gp in the efflux of phillyrin.

Investigation on the absorption of phillyrin and forsythiaside in rat digestive tract.

In the present study, an in situ rat model was employed to systemically investigate the absorption of phillyrin and forsythiaisde. Three concentrations of phillyrin (0.2, 0.4 and 1.5 mg) were tested and the results showed that phillyrin cannot be absorbed in the digestive tract. The absorption rates of forsythiaside in stomach were 7.773, 7.228 and 6.751% h(-1) for 0.5, 1 and 2.5 mg, and no significant difference was found in different concentrations. The absorptions of forsythiaside in intestine were investigated in different concentrations and different absorption sites. The mean P% were 6.618, 7.199, 9.210 and 9.747% h(-1) of forsythiaside in intestine for 0.25, 0.5, 1, 2.5 mg dosage, and the statistical analysis showed that the absorption had no relation with concentration. In addition, in different digestive segments, the mean P% were 7.528, 8.382, 8.191, 9.109 and 6.908% h(-1) for the gastric, duodenum, jejunum, ileum and colon, respectively. No statistical differences of absorption were found for forsythiaside among different digestive segments indicated no specific absorption site was found.