Pharmacokinetics And Tissue Distribution Study Of Bfadienolides From Shexiang Baoxin Pill In Mice

Shexiang Baoxin Pill (SBP), a well-known composite formula of traditional Chinese medicine, consists of seven materia medicas, such as Moschus, Radix Ginseng, Calculus Bovis, Cortex Cinnamomi, Styrax, Venenum Bufonis and Borneolum Syntheticum. Being one of the most commonly used traditional Chinese medicine formula for coronary heart disease in clinic, it has been widely used to treat angina, pleura and myocardial infarction resulting from ischemia in cardiac muscles. Bufadienolides, with the characteristic of narrow therapeutic window, are the major active and toxic compounds of Venenum Bufonisand, one of the seven materia medicas comprising Shexiang Baoxin Pill. Previous pharmacokinetics studies just investigated the pharmacokinetic character of bufadienolides from Venenum Bufonisandthe after oral administration to rats. In this study, the mouse, which can more accurately reflect the metabolic behavior of this kind of compounds in human than in rat, were chosen as animal model to investigate their in vivo pharmacokinetics and tissue distribution from the Shexiang Baoxin Pill. The main contents are as follows:Firstly, a specific and sensitive LC-MS/MS method was developed for the simultaneous determination of resibufogenin, bufalin, gamabufotalin, arenobufagin and bufotalin in mouse plasma and tissues. The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3column by gradient elution using water (containing0.1%formic acid) and acetonitrile as the mobile phase. The detection was carried out by a triple-quadrupole tandem mass spectrometer in positive ion multiple-reaction monitoring mode. All analytes showed good linearity over a wide concentration range (r2>0.9950). The lower limit of quantification of resibufogenin, bufalin, gamabufotalin, arenobufagin and bufotalin were2.5ng/mL,2.0ng/mL,2.5ng/mL,2.0ng/mL and0.1ng/mL in mouse plasma, respectively;5.00ng/mL,5.00ng/mL,5.00ng/mL,5.00ng/mL and0.20ng/mL in mouse tissues, respectively. The precisions of all analytes in mouse plasma and tissue (liver) were in the range of0.68%-7.10%; the accuracy were in the range of-7.79%-7.40%; the stability were in the range of1.18%-9.95%; the matrix effects were in the range of86.78%-102.54%; and the extraction recovery were over85.72%. All the results were well within the acceptable limit according to the FDA (USA Food and Drug Administration) bioanalytical method validation guidance. This validated method was successfully applied to the pharmacokinetic and tissue distribution study of five bufadienolides in mice plasma and tissues after oral administration of SBP.Secondly, in the pharmacokinetic and tissues distribution study, the plasma drug-time curves of the five bufadienolides were described and the distribution of all analytes in vivo were investigated. The pharmacokinetic parameters were determined using non-compartmental analysis. The pharmacokinetic results showed that the five bufadienolides were rapidly absorbed into the systemic circulation and were widely distributed throughout the body, followed by a rapid elimination phase. The pharmacokinetic curve showed double peaks after oral administration. The tissue distribution results indicated that all of the analytes underwent a rapid and wide distribution into tissues within the time course examined, and no long-term accumulation of the compounds in tissues was observed. The major tissue depots for resibufogenin, bufalin and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. All of the analytes were observed in the small intestine which may be mainly attributed to the oral mode of administration, and the relatively high distribution in kidney demonstrated that kidney might be the primary excretion organ of bufadienolides.The result of this research can not only provide a scientific basis for the availability and security of the clinical applications of SBP, but also provide theoretical guidance for further exploring the active mechanism and scientific connotation of SBP.