The Bioavailability Of Puerarin Microemulsion For Oral Administration And Its Preliminary Absorption Mechanism

OBJECTIVETo study the absorption mechanism of puerarin microemulsion preliminarily by its bioavailability in rats and uptake in Caco-2 cells.METHODS1 The bioavailability of puerarin microemulsion in ratsSD Rats were orally administrated by puerarin o/w and w/o microemulsion at a dose of 30 mg·kg^-1,respectively,puerarin solution as control group.Blood samples were collected from plexus venosus of fossa orbitalis at different time,the drug concentration was determined by HPLC,and the pharmacokinetic parameters were calculated according to non-compartment model.2 The uptake of puerarin microemulsion in Caco-2 cellsTo carry out toxicity test by MTT assay;To determine the concentration of puerarin in Caco-2 cells by HPLC,and the content of protein in Caco-2 cells by Coomassie brilliant blue method;To study the effect of time,pH and concentration on the uptake of puerarin microemulsion.RESULTS1 The bioavailability of puerarin microemulsion in rats1.1 Determination of puerarin in rat plasma by HPLC The calibration curve of puerarin in rat plasma at concentrations ranging from 0.10 to 10.00μg·mL^-1 was excellent linearity(r=0.9996), with the lower limit of quantification of 0.10μg·mL^-1.The extraction recoveries were from 79.0%to 83.0%,the method recoveries from 97.9% to 106.7%,and the intra-and inter-day RSD less than 6%.1.2 Main pharmacokinetic parameters and relative bioavailabilityMain pharmacokinetic parameters in rats after intragastric administration of puerarin solution,o/w and w/o microemulsion as follow: t_max were 0.27±0.08,1.35±1.03,and 3.67±0.52 h,respectively;C_max 1.315±0.821,1.719±0.475,and 2.154±0.840μg·mL^-1,respectively; AUC_0→t 1.59±0.37,7.02±2.89,and 11.33±2.53μg.h.mL^-1,respectively; AUC0→∞1.83±0.41,7.44±2.89,and 14.96±3.98μg.h.mL^-1,respectively; MRT 1.58±0.62,3.28±0.85,and 7.45±4.11 h.The relative bioavailability of o/w and w/o microemulsion were 407%and 817%.2 The uptake of puerarin microemulsion in Caco-2 cells2.1 Toxicity testThe survival rate of cells were more than 90%when the concentration of puerarin microemulsion was less than 208μg·mL^-1,thus 208μg·mL^-1 was chosed as the maximum concentration in the toxicity test.2.2 Determination of puerarin in Caco-2 cells by HPLCThe calibration curve of puerarin in Caco-2 cells at concentrations ranging from 0.0102 to 10.20μg·mL^-1 was excellent linearity(r=0.9998), with the lower limit of quantification of 0.0102μg·mL^-1.The extraction recoveries were from 95.8%to 97.0%,the method recoveries from 99.8% to 100.6%,and the intra-and inter-day RSD less than 7%.The calibration curve of protein in Caco-2 cells at concentrations ranging from 10 to 100μg was excellent linearity(r=0.992).2.3 The uptake of puerarin microemulsion in Caco-2 cells The uptake amounts of puerarin microemulsion in Caco-2 cells gradually increased with time,when at 120 min the concentration of puerarin in Caco-2 cells reached the maximum,thus 120 min was determined as the experiment time;There was no obvious difference between microemulsion group and solution group in uptake amounts at pH 6.0 and pH 7.4;The uptake amounts of puerarin increased linearly with concentration from 26 to 208μg·mL^-1.At lower concentration there was no obvious difference between microemulsion group and solution group.However,at higher concentration the uptake amounts of puerarin in solution group was much larger than that of microemulsion.CONCLUSION1.The method to determin puerarin concentration in rat plasma and Caco-2 cells is precise,accurate,stable and suitable for the bioavailability of puerarin microemulsion in rats and the uptake of puerarin microemulsion in Caco-2 cells.2.Compared with solution,microemulsion enhanced absorption of puerarin,and improved its bioavailability significantly.Microemulsion can enhance the absorption of puerarin is not only by improving drug solubility according to the traditional theory.Microemulsion has its special absorption mechanism,the paracelluar pathway and lymph transport may contribute to the absorption of microemulsion primarily in integrated form of particle rather than the transcellular pathway.