| 1. Determination of a-mangostin across MDCK monolayers with ultraviolet detection:application to a bioaccessibility, biotransformation, and transport studyObjective:The goal of this work was to develop and validate an HPLC method for the determination of a-mangostin in MDCK monolayers, and to predict the oral pharmacokinetic behavior and intestinal transport nature of a-mangostin.Methods:An HPLC method for the determination of a-mangostin was developed via selected chromatographic conditions, specificity study, the recoveries and precision tests. MDCK monolayer was established to simulate the gastrointestinal absorption process of a-mangostin. The samples from Apical, Cell and Basolateral parts were collected at indicated time points with the magnolol as an internal standard and analyzed via HPLC method on RP zobax SB-C18(150mm×4.60mm,5μm) using a methanol-water gradient elution at312nm. The drug concentrations of above three parts were investigated. To evaluate whether the P-gp efflux action works in the transport of a-mangostin across MDCK cell monolayers, the transport assay of a-mangostin was performed with the addition of P-gp inhibitor verapamil. To determine the presence of phase Ⅱ conjugates of a-mangostin, the samples were incubated with β-glucuronidase before extractionResults:The retention times of a-mangostin and magnolol were19.4and15.1min, respectively. The a-mangostin range of linearity in culture medium was between0.08and10μg/mL with the intra-day precision of RSD<5%and inter-day precision of RSD<7%, the average recoveries of98～102%and the LLOQ for40ng/mL. In this study, cell uptake of xanthones from apical chamber was dose dependent and intracellular concentrations peaked by3h. MDCK cells remained about20%of total alpha-mangostin, which was significantly greater than that in the basolateral compartment (3.2%) after6h. Treatment of medium with glucuronidase from H. pomatia revealed that a remarkable portion of a-mangostin was conjugated to glucuronide or sulfate derivatives in both the apical and basolateral compartments in a time-dependent manner. Moreover, the absorption of a-mangostin was hardly influenced by verapamil, which means that the P-glycoprotein (P-gp) efflux may not participate in the absorption process.Conclusion:a-mangostin exhibited the characteristics of low bioavailability, high tissue binding and notable phase Ⅱ metabolic.2. Investigation of pharmacokinetics and plasma protein binding of a-mangostin in vivo after intravenousObjective:An HPLC method for the determination of a-mangostin was developed to study the pharmacokinetics and plasma protein binding in vivo after intravenous.Methods:Rat blood samples were collected at indicated time points after a-mangostin intravenation and analyzed via HPLC method on RP zobax SB-C18(150mm×4.60mm, 5μm) using a methanol-water gradient elution at312nm. The plasma concentration-time data was analyzed by DAS2.0software. The method of equilibrium dialysis was employed to measure plasma protein binding of a-mangostin. The tissue distributions of a-mangostin in mice were tested on istribution, balance and elimination phases in the same location.Results:The a-mangostin range of linearity in plasma or tissue homogenate was between0.10and80.00μg/mL with the intra-and inter-day precision of RSD<10, the average recoveries of98～105%and the LLOQ for40ng/mL. The pharmacokinetic parameters as follows:α=14.10h-1, β=0.24h-1,K10=6.14h-1, K12=7.59h’-1,K21=0.61h-1, AUCo～∞＝5.87μg·h·ml-1, MRT=2.05h, Vc=0.21L/kg and Vp=3.53L/kg. The average rates of plasma protein binding of a-mangostin were62.3%,60.0%and57.4%at the concentrations of1,3and10μg/mL, respectively. The data of tissues distributions indicated a-mangostin mainly in the liver and lungs, but at2h after injection, the rate of the drug in fat was remarkablely accumulated.Conclusion:The pharmacokinetics of a-mangostin fits to two-compartment model, shows a fast distribution and a slow elimination phases in vivo and possesses a moderate binding capacity to plasma protein. |
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