Pharmacokinetic Study Of Anthraquinones From Rhubarb In Rat

Objective:(1) To develop a new high performance liquid chromatographic method (HPLC) for determining the concentration of chrysophan in rat plasma following intravenous injection or intragastric administration of chrysophan solution, and apply to the sutdy of its pharmacokinetics in rat.(2) To determine chrysophan in rat plasma and study its pahrmacokinetics after intragastric administration of the extract of Rhubarb.(3) To develop a method to determine the quantities of aloe-emodin、rhein、emodin、chrysophanol in rat plasma, and apply to the sutdy of its pharmacokinetics in different composition for investigating the effect of different compatibility of Da-Chen-Qi-Tang on the Main Componets’s pharmacokinetics of Rhubarb and discussing principle of Da-Chen-Qi-Tang compatibility.(4) To investigate the effects of Liuwei Dihuang pill (LWDHP) on the activity of CYP1A2 and P-gp in rat intestines.Methods:(1) The HPLC assay was carried out using a Cosmosil C18 column. Methanol -1% phosphoric acid buffer (45:55, v/v) was used as mobile phase The flow rate was 1.0 ml/min. The detection wavelength was set at 254 nm. Internal standard was bendroflumethiazide. After tail vein injection or oral administration of chrysophan solution to rat, blood samples were collected from the femoral artery. Acetonitrile solution contained 4μg/mL benzylfluoridehydrochlorothiazide (internal standard) was used to precipitate plasma protein. A RP-HPLC was adopted to determine the plasma concentration of chrysophan. Moreover, pharmacokinetics parameters were estimated by DAS 2.0.(2) The assay was conducted on a VP-ODS C18 column (4.6 mm×250 mm), and the mobile phase was consisted of methanol and 0.1% phosphoric acid buffer in a volume ratio of 50:50. The flow rate was 1.0 ml/min. The detection wavelength was set at 254 nm. Bendroflumethiazide was used as internal standard. Column temperature was 40℃. Rats were given the extract of rhubarb by intragastric administration. At different times, blood samples were collected through the femoral artery. A HPLC methord was used for determining plasma concentrations of chrysophan and calculating the pharmacokinetic parameters.(3) The HPLC assay was performed on a Kromasil C18 column (4.6 mm×150 mm, 5.0μm) A gradient elution of A (methanol) and B (0.1% phosphoric acid buffer) was used. The gradient was as follows:0-25 min 60% A,25-30 min 60-79% A,30-55 min 79%A The flow rate was set at 1 mL/min. The detector was operated at 254 nm. Internal standard was 17a-deoxy-corticosterone. Rats were given different compatibility groups of Da-Cheng-Qi decoction by intragastric administration. Plasma concentrations of aloe emodin, rhein, chrysophanol and emodin content of rhubarb was determined using the above gradient elution methord. A Dispersed liquid-liquid microextraction methord was used to pre-treat plasma samples.(4) After oral administration LWDHP for 7 days and Sodium Chloride, caffeine was given intravenously at a dose of 10 mg/kg. A developed HPLC method was used to determine the concentrations of caffeine in rat plasma. The difference of caffeine t1/2 was used to evaluate LWDHP on the activity of rat CYP1A2. The absorption kinetics of digoxin at intestine in rats was studied by in situ recirculation and the absorption parameters were used to evaluat the effect of LWDHP on P-gp.Results:(1) The concentration of chrysophan in the standard curve range (0.195～12.5μg/mL) had a good linear relationship. The equation was as follow:Y=0.4478X-0.0167, with correlation coefficient r2=0.9999. Intra-day and inter-day RSD were less than 3.8%, which was used to reflect precision of the assay. Relative recovery was more than 99.69%. An open two-compartment model was used to descripe the pharmacokinetic characteristics of chrysophan by intragastric and intravenous administration.(2) The linear range of chrysophan in Rhubarb extract was 0.24～15.5μg/mL. Intra-day and inter-day RSD values of precision were less than 10.2% and accuracy were greater than 95.8%. The recovery was more than 97.5%. Pharmacokinetic parameters showed that pharmacokinetic characteristics of chrysophan were consistent with a one compartment model, and the speed of distribution is more quickly after administeated the ethanol extract of rhubarb. (3) The linear ranges of Aloe-emodin, rhein, chrysophanol, emodin and physcion were as follows:0.15-20μg/mL,0.31-40μg/mL,0.19-12.7μg/mL,0.54-17.2μg/mL and 0.65-10.5μg/mL respectively. Correlation coefficients r2 were higher than 0.9982. inter-day RSD of five ingredients in Rhubarb were less than 6.83%(n=5) and the intra-day RSD were less than 9.06%(n=5). The recovery of five components was more than 91.8%. The main pharmacokinetic parameters (Cmax, Tmax, T1/2 or AUC) were significantly different with different compatibility of Da-Cheng-Qi-Tang.(4) The t1/2 values of caffeine in groups of LWDHP and control group were (2.61±0.98) h、(5.14±0.21) h, respectively. The absorption rate constants (ka) in LWDHP and control group were 0.030±0.0057、0.024±0.0039, respectively. The absorption percentage in one hour of intestines was 6.54±0.71% and 5.42±0.23% for LWDHP and control group, respectively.Conclusion:(1) The established high performance liquid chromatography method is accurate, reliable and simple, which can be used for determining chrysophan in biological samples and appling for its pharmacokinetic study. Pharmacokinetic parameters of chrysophan are reported for the first time.(2) A new HPLC methord was developed for determining of chrysophan in the ethanol extract of rhubarb in rat plasma, which was sensitive, reliable and can be used for the pharmacokinetic study of chrysophan in ethanol extract of rhubarb.(3) The different compatability of Minister-drug, adjuvant-drug, etc. in Da-Chen-Qi decoction have some certain extent effect to the pharmacokinetical parameters of the dominant drug (Rhubarb).(4) Liuwei Dihuang pill has an inhibitive effect to rats CYP1A2 while it can improve the activity of P-gp in rat intestines.