Pharmacokinetics Of Total Glucosi'des Of Paeony In Immunological Liver Injuried Animals

Objective: White peony root, the dried radix of ranunculaceae peony, can nourish blood and liver and relieve pain. Total glucosides of paeony (TGP) are the bioactive components extracted from white peony root, chiefly including paeoniflorin(Pae), albiflorin(Alb), hydroxypaeoniflorin, peonin, benzoylpaeoniflorin and etc. The content of Pae and Alb is higher than the others. Modern studies indicate that TGP has apparente preventive and therapeutic effect to acute, chronic chemical and immunological hepatic fibrosis, but the physiological disposition under liver injuried pathological state has not been so clear. Pae and Alb as indexs, the study adopt HPLC to explore pharmacokinetics of TGP in immunological liver injuried and normal animals and research tissue distribution features of TGP in normal animals to provide reliable theory evidence for rational use of drug in clinic.Methods:1 Pharmacokinetics of TGP in liver injuried rats1.1 Chromatographic conditionsThe HPLC assay was established by phenomenex C18 column (4.6×250 mm, 5μm) with acetonitrile-0.05% formic acid (15:85) as mobile phase at a flow rate of 1 mL/min. The detection wavelength was UV 230 nm and the temperature 25℃.1.2 Sample pretreatment100μL plasma was precisely taken to a blank centrifuge tube, then added 10μL gentiopicrin internal standard (IS) solution, vortexed 1 min, added 50μL 20% perchloric acid, vortexed 2 min, centrifugated, then the supernatant was gotten out and 10μL was injected into the column.1.3 PharmacokineticsPig serum intraperitoneal injection was used to establish immunological liver injuried rats. The model group were abdominally injected pig serum 0.5 mL each one, twice every week and lasted for 10 weeks. Detect ALT and AST. After succeeding in making the model, three dosages 0.47 g/kg, 1.41 g/kg, 2.82 g/kg aqueous solutions of TGP, were orally administered to normal and model animals (10 mL/kg body weight). Blood samples were collected from each rat through vena orbitalis posterior according to the specific schedule (at times of 15 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 240 min, 360 min, 480 min and 720 min after dosing). With HPLC, determine the concentration of Pae and Alb. According to concentration-time curve the parameters were calculated. Carry out data analysis between groups with SPSS 11.5.2 Pharmacokinetics of TGP in liver injuried miceChromatographic conditions were the same with 1.1. Use BCG and LPS caudal intravenous injection to make mouse immunological liver injuried model. The model group were caudal intravenously injected BCG 2 mg/0.2mL each one and 10 days later LPS 7.5μg/0.2mL. Three dosages 0.47 g/kg, 1.41 g/kg, 2.82 g/kg aqueous solutions of TGP, were orally administered to normal and model animals (10 mL/kg body weight). Blood samples were collected from each mouse through picking eyeball according to the specific schedule (at times of 15 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 240 min, 360 min, 480 min and 720 min after dosing). With HPLC, determine the concentration of Pae and Alb. According to concentration-time curve the parameters were calculated. Carry out data analysis between groups with SPSS 11.5.3 Tissue distributionThe HPLC assay was the same with"1.1", but with different proportional mobile phase. Homogenate all tissues. Make homogenate into freeze-dry powder, added in rational IS, extracted with methanol and 10μL was injected in column. The concentration of Pae and Alb in heart, liver, spleen, lung, kidney, stomach, small intestine and large intestine from normal rats after an oral administration of 2.82 g/kg TGP 1 h, 3 h and 6 h. Results:1 Pharmacokinetics of TGP in liver injuried rats1.1 Methodology studyPae, Alb and IS had good specificity in rat plasma. The regression equation of Pae was y=0.0724x-0.037 (n=7, r²=0.9996, 0.848μg/mL-106μg/mL), Alb y=0.0656x-0.0092 (n=7, r²=0.9998, 0.4375μg/mL-35μg/mL). Intra-day and inter-day precision RSD were less than 10%, accuracy 100%±10%. The absolute recovery of Pae, Alb and IS were all more than 70%. The samples after pretreatment could keep steady after putting at room temperature and freeze-thaw cycle.1.2 Pharmacokinetics parameters After an oral administration of TGP in three dosages, the main PK parameters of Pae in low, medium and high dosage were as follows: Compared with normal group, model group Pae C_max and AUC_0-∞ significantly increased, T_max advanced (p<0.05or p<0.01), Alb T_max advanced, t_1/2 prolonged, AUC_0-∞ increased (p<0.05or p<0.01) in low dosage; Pae T_max advanced, t_1/2 prolonged, AUC_0-t and AUC_0-∞ increased (p<0.05or p<0.01), Alb T_max advanced, t_1/2 prolonged, AUC_0-∞ increased (p<0.05or p<0.01) in medium dosage; Pae T_max advanced, t_1/2 prolonged (p<0.01), Alb T_max advanced, t_1/2 prolonged, C_max increased (p<0.05or p<0.01) in high dosage. Comparing C_max, T_max, t_1/2, AUC_0-t and AUC_0-∞ between three dosages in normal and model group respectively, the results indicated, C_max, AUC_0-t and AUC_0-∞ had significant difference between all two dosages (p<0.01), but T_max and t_1/2 hadn't difference (p>0.05), and dosage with C_max, AUC_0-t and AUC_0-∞ had determinate linear correlation (p<0.05or p<0.01).2 Pharmacokinetics of TGP in liver injuried miceAfter an oral administration of TGP in three dosages, the main PK parameters of Pae in low, medium and high dosage were as follows: Alb's concentration in low dosage can't be measured because of too small content, then the main PK parameters of Alb in medium and high dosage:Compared with normal group, model group Pae Tmax advanced, AUC_0-t and AUC_0-∞ increased (p<0.05or p<0.01) in low dosage; Pae Tmax advanced, C_max, AUC_0-t and AUC_0-∞ increased (p<0.05or p<0.01), Alb Tmax advanced, C_max, AUC_0-t and AUC_0-∞ increased, t_1/2 prolonged (p<0.05or p<0.01) in medium dosage; Pae and Alb Tmax advanced, C_max, AUC_0-t and AUC_0-∞ increased, t_1/2 prolonged (p<0.05or p<0.01) in high dosage. Comparing C_max, Tmax, t_1/2, AUC_0-t and AUC_0-∞ between three dosages in normal and model group respectively, the results indicated, C_max,AUC_0-t and AUC_0-∞ had significant difference between all two dosages (p<0.01), but Tmax and t_1/2 hadn't difference (p>0.05), and dosage with C_max, AUC_0-t and AUC_0-∞ had determinate linear correlation (p<0.05or p<0.01). 3 Tissue distribution3.1 Methodology studyPae, Alb and IS had good specificity in all tissues. Intra-day and inter-day precision RSD were less than 10%, accuracy 100%±10%. The absolute recovery of Pae, Alb and IS were all about 70%. The samples after pretreatment could keep steady after putting at room temperature. The regression equations were as follows:3.2 Tissue distributionThe concentration sequence of Pae and Alb from large to small in all tissues were as follows:1 h Pae: small intestine, stomach, large intestine, kidney, liver, lung, spleen, heart Alb: small intestine, stomach, large intestine, liver, kidney, lung, spleen, heart3 h Pae: small intestine, stomach, large intestine, kidney, spleen, liver, lung, heart Alb: small intestine, stomach, large intestine, kidney, spleen, liver, lung, heart6 h Pae: small intestine, large intestine, stomach, kidney, liver, spleen, lung, heart Alb: small intestine, large intestine, stomach, kidney, liver, lung, spleen, heart Except stomach and small intestine, the concentration of Pae and Alb in other tissues arrived the maximum at 3 h. At 6 h the concentration of Pae in small intestine, large intestine and kidney decreased to 84.1%, 68.1% and 22.1% of that at 3 h, Alb 86.7%, 73.9% and 19.2%. The concentration of Pae and Alb in other tissues decreased to less than 20%.Conclusions:1 Through pharmacokinetics study, we found that the physiological disposition of normal animals and immunological liver injuried model animals were different. Compared with the normal's, in the modle TGP Tmax advanced, Cmax, AUC_0-t and AUC_0-∞ increased, t_1/2 prolonged, which indicated that TGP was absorbed more rapidly, absorbed dose larger, eliminated more slowly and stayed in the body for longer time, suggesting that the dose for the liver injuried patients needs be adjusted to avoid accumulation and toxicity caused by overdose.2 Studying tissue distribution, we found after oral administration of TGP, the drug was distributed rapidly and widely in tissues. Small intestine, stomach, large intestine and kidney, spleen, liver were the chief distribution tissues and TGP readily accumulated in stomach and intestines, less in other tissues. The tissue distribution features have some connection with modern pharmacological actions, adverse reaction and channel entry theory, which provides guidance for further study of the pharmacological actions and mechanism of TGP, and meanwhile provides some scientific evidences for channel entry theory of white peony root.