| Objective:To develop a specific and rapid high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) method, utilized to investigate the plasma pharmacokinetics of resveratrol and polydatin in rats. Resveratrol (RES) and Polydatin (POL) are the main bioactive constituents in Rhizoma Polygoni Cuspidati (Huzhang), the dried root and rhizome of Polygonum cuspidatum Sieb. et Zucc. The metabolism of POL was researched in vitro to verify the pharmacokinetics results.Method:Rhizoma Polygoni Cuspidati (RPC) extract was obtained through extraction by ethanol. The contents of RES and POL, active constituents, were determined by HPLC method. A Shimadzu LC-2010AHT liquid chromatography system was used, equipped with a Shimadzu VP-ODS C18column (250×4.6mm ID). The column temperature was set to30℃. A gradient elution was selected:phase A was0.1%formic acid, phase B was acetonitrile. The elution procedure was:0min B5%,15min B20%,28min B100%,30min B100%. The flow rate was set to1.0ml/min. The sample size was10μL. RES was detected through channel one with the wave length of307nm, while POL was detected through Channel two with290nm wave length.In vitro study, POL was incubated with liver homogenate or intestinal bacteria liquid for4or24hours at37℃, respectively, to invest the metabolism of plydatin preliminary.In vivo study in rat, four intragastric administration solutions were prepared, such as RES intragastric solution, POL intragastric solution, RES+POL mixed intragastric solution and RPC extract intragastric solution. The pharmacokinetics of RES and POL were determined after rat stomach-lavaging with the above solutions, respectively. Genistein was used as internal standard (IS). A HPLC-MS method was utilized in the study. Separations were performed on a Shim-pack VP-ODS C18column (250×4.6mm,5μm) with column temperature set to30℃, the flow rate was set to1.0ml/min. A linear gradient elution was selected:phase A was0.05%formic acid, while phase B was acetonitrile. The elution procedure:0min B20%,5min B100%, maintaining phase B100%for10minutes. A quadrupole mass spectrometer operated under selected ion monitoring (SIM) mode was used. Through a solvent splitter, a portion of0.2ml/min was delivered into the ion source. The selected ions monitoring (SIM) were:m/z229for RES, m/z391for POL and m/z271for internal standard genistein. A LCMS-solution software package (Shimadzu, Japan) was used for data acquisition.Results:The active constituents RES and POL in RPC extract were determined by HPLC method. The retention time for RES and POL was16.78min and13.68min, respectively. The linear range was1.80-180μg/ml for RES (R2=0.9999),3.50-350μg/ml for POL (R2=0.9999) with a good linear relationship. The quantitation limit and detection limit for RES was0.18μg/ml and0.10μg/ml, which for POL was0.35μg/ml and0.15μg/ml. Both the RSD for within day precision and between-day precision were less than2%by assay of RES and POL reference solutions in three concentration levels. Three batches of PRC extract lyophilized powder were determined. The contents of RES and POL were obtained,17.8,16.5,18.3mg/g for RES and72.1,73.7,69.6mg/g for POL, respectively, with the ratio of1:4between RES and POL contents.In vitro metabolism study, after incubated with rat liver homogenates or intestinal bacteria the content of POL gradually decreased and the aglycone-RES gradually increased, suggesting that POL could be converted into its aglycone-RES under the role of intestinal tract flora and the liver enzymes.In vivo study, the RES and POL levels in rat plasma were determined by HPLC-MS method after rats intragastric administration of RES, POL, RES+POL and RPC extract solutions. The linear range was0.0018-3.6μg/ml for RES,0.0035-7.0μg/ml for POL, R2was0.9986and0.9988, respectively, indicating a good linear relationship. The quantitation low limit and detection limit for RES was0.0018μg/ml and0.0008μg/ml, which for POL was0.0035μg/ml and0.0015μg/ml. The RSD of within day precision were all below5.5%, which of between-day precision were all below6.1%, obtained through assay of RES and POL reference plasma solutions in three concentration levels. The freeze thaw stability test was performed with reference plasma solutions at three levels, consisted of3short-freeze thawing periods (24hours) and1long-freeze thawing period (20days). The obtained RSD values were all below7%, indicating the store environment (-20℃) showed no influence on the contents of RES and POL in rat plasma.The pharmacokinetic parameters of RES and POL in rat plasma were calculated using the pharmacokinetic software. The results showed that the pharmacokinetic of RES and POL in rats fit for a two-compartment open model. The findings obtained were: the delay time (Lag time), peak concentration (Cmax) and peak time (Tpeak) in each group showed no statistically significant change (P>0.05).Compared the pharmacokinetics parameters of POL among the three groups, it can be found:the AUC value and CL in extract group and mixture group showed no significant difference compared with the single intragastric. The T1/2alpha and T1/2beta of POL in both groups increased, indicating the residence time in vivo prolonged. The Ka (0.14vs0.07, P<0.05) and T1/2Ka (4.83vs10.34, P<0.05) of POL between the extract group and mixture group had significant differences. Compared the extract group with the POL signal group, there was no significant difference of Ka and T1/2Ka in both groups. These above results suggested that the dynamic process of POL in rats were mainly impacted by RES. Factors affecting the absorption process are from the other ingredients in Polygonum cuspidatum extract.Compared the pharmacokinetics parameters of RES among the three groups, it can be found:the AUC value (187.56,178.35vs98.18, P<0.05) and clearance (CL,0.54,0.56vs1.02, P<0.05) in RPC extract group and RES+POL group showed significant differences compared with the RES single group, Vd showed no difference, while T1/2values in all groups significantly increased. Between the extract group and mixture group, the T1/2alpha and beta showed significant differences, the AUC, CL, Vd and Ka values no significant differences. These above results suggested that the dynamic process of RES in rats were mainly impacted by POL’s metabolism, RES. And it should be pay attention to the impactation of some ingredients in Polygonum cuspidatum extract. They may affect the distribution and metabolism of RES.Conclusion:High specificity and sensitivity of analytical methods are necessary for a pharmacokinetic study. The in vitro metabolism study confirmed POL may rapidly metabolize to its aglycone (RES) by enzymes in liver and/or intestinal flora in vitro, according to the analysis of the obtained pharmacokinetic parameters. In vivo pharmacokinetic study, the metabolism kinetics of RES was found mainly impacted by some ingredients in Polygonum cuspidatum extract. The increased AUC value and prolonged action duration of RES in vivo was speculated influenced by POL partial converted into RES. Clinical drug metabolism should pay attention because the active ingredient dose resulted in a change impact. It should be pay attention to the changed impact of the active ingredient resulting by metabolism in vivo. |
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