Pharmacokinetics And Tissue Distribution Studies Of Butulin In Mice With Liver Injury

Objective:Betulin(BT) is a herbal product for the treatment of liver injury, but its pharmacokinetics and tissue distribution had not been investigated. In this study, the liver injury model was induced with acetaminophen (APAP) given orally. A simply and sensitive HPLC assay was developed and validate for rapid determination of Betulin in liver injury mice. It will provide a reference for drug development and clinical reasonable. Methods:All biological samples were prepared by protein precipitation method (used liquid-liquid extraction) using oleanolic acid as an internal standard(IS). Betulin in serum and tissue was extracted by ethyl acetate in a certain percentage. Bio-samples were detected quantificationally in peak area ratio internal standard by RP-HPLC:The chromatographic separation was achieved on SHIMADZU ODS C18 column(150×4.6mm,5μm) maintain at 30℃ temperature at a flow rate of 0.8ml/min.℃A gradient mobile phase consisting of methanol(A) and water(B) was eluted as A to B(85:15, v/v). Set UV wave length at 210nm. The injection volume was 10OμL. Mice were divided into two groups, the normal group and the model group. Betulin was administered at a dose of 75mg/kg to mice both two groups. Collect blood samples at different time points to determine the concentration of betulin. Then remove out the tissues. The concentration of plasma was measured with DAS 2.0 software to find a fitable compartment model, calculating pharmacokinetic parameter and analyzing metabolic law of betulin in mice with time changing. With the same condition, determine concentration of betulin in tissues(heart, liver, spleen, lung and kidney), finding out the distribution characteristics. Results:1. Betulin concentration in serum:Linear regressions of the peak area ratios versus concentration were fitted over the range of 1.0-50.0μg·mL-1 for betulin(R2= 0.9994). The intra-day precision and inter-day precision(RSD,%) were less than 8.07% and 7.01%. Serum samples kept stable concentration within 24 hours, RSD values were less than 7.01%. The average of recoveries was 107.37%. Determination of pharmacokinetic parameters:Plasma concentration were fitted processing at different time points, concentration-time(C-t) curves in mice were fitted by two-compartment model. Measured major pharmacokinetic parameters with α, β，T1/2α, T1/2β，K10, K12, K21, AUC, CL, V, V2, Tmax, Cmax.2. Betulin concentration in tissues:Linear regressions of the peak area ratios versus cincentrations were fitted over the range of 2.0-100.0 μg·mL-1 for betulin in heart(R2=0.9991). Linear regressions of the peak area ratios versus cincentrations were fitted over the range of 1.0-250.0μg-mL-1 for betulin in liver(R2=0.9987). Linear regressions of the peak area ratios versus cincentrations were fitted over the range of 1.0-150.0 μ g-mL-1for betulin in spleen and lung(R2=0.9994 and 0.9992). Linear regressions of the peak area ratios versus concentrations were fitted over the range of 1.0-200.0μg·mL-1 for betulin in kidney(R2=0.9991). Linear regressions of the peak area ratios versus cincentrations were fitted over the range of 1.0-100.0 μ g·mL-1for betulin in mixture tissues(R2=0.9980). The intra-day precision and inter-day precision(RSD,%) were less than 5.67% and 5.02%. Tissues samples kept stable concentration within 24 hours, RSD values were less than 6.37%. Betulin distribution in mice tissues:mice were injected betulin, which rapidly distributed in various tissues. Betulin concentration decreased significantly in each tissue, and the concentration in liver is higher than other tissues. Conclusion:The selectivity, linearity, precision, recovery and stability of the newly developed HPLC method in this study have been validated. Furthermore, the method was successfully applied in pharmacokinetics and tissue distribution study of betulin. Betulin is rapidly absorbed and widely distributed in various tissues. The level of betulin in liver is highest, and followed by kidney, spleen, lung and heart. There was no significant change in betulin absorption and distribution comparing model group with normal group. This showed that the liver injury did not weaken the absorption of betulin. With the preliminary knowledge of in vivo pharmacokinetics and tissues distribution, this study will provide helpful information for further development of betulin in future studies.