Study On Andrographolide Self-microemulsifying Drug Delivery System And Its Absorption Mechanism In Caco-2 Cell Model

Andrographolide(AP) has the effect of anti-inflammatory, anti-bacterial,anti-virus and anti-tumor functions, et al. However, the bioavailability of AP oral formulations is very low due to the poor stability and water solubility. In order to expand the clinical application of AP, it is necessary to improve the above deficiencies by means of suitable pharmacy methods. Since the self-emulsifying drug delivery system(SMEDDS) could improve the oral absorption and bioavailability of poorly soluble drugs. The AP self-emulsifying drug delivery system was prepared in this study and its relative performance was evaluated as well. The main contents and results of this study are as follows:A HPLC method was developed to measure the concentration of AP, and the oil-water partition coefficient and the effects of pH/ temperature on the stability and solubility of AP were studied. The results showed that the Papp of AP was 3.90, which indicated AP was a poor absorption drug. Stability test showed that AP was not readily hydrolyzed under acidic condition and below 60℃, indicated that the drug loading process of AP-SMEDDS should be below 60℃, and acidic environment of AP-SMEDDS helped to extend the validity of the drug. Solubility test showed that the solubility of AP varied little while pH or temperature changed, indicated it was difficult to improve the solubility of AP by adjusting the pH or temperature.The optimal formulation of AP-SMEDDS was screened by solubility,compatibility, ternary phase diagram and Box-Behnken design-response surface methodology, with the particle size, zeta electric potential and self-microemulsifying time as parameters. The ability of precipitation inhibition was screened by in vitrodissolution test and optimized by HPMC, and the physic-chemical property and dissolution characters of AP-SMEDDS were also determined.The results showed that the optimum self-microemulsifying drug delivery system was composed of Maisine35-1(14.29%), Tween20(39.68%) and Transcutol P(46.03%). In vitro dissolution tests of optimal formulation showed that the precipitation of AP appeared in about 10 min, and addition of 2% HPMC could effectively sustained a metastable supersaturated state by retarding precipitation. The mean particle diameter of AP-SMEDDS after the emulsification was 19.96 nm, and the zeta electric potential was﹣9.76 mv, and the self-microemulsifying time was less than 20 s. The accumulative dissolution of AP-SMEDDS was 94.63%, which was about 2.14 time as much as that of the marketed pills.Caco-2 cell in vitro absorption model was established and assessed, and the transport mechanism and the ability of improving bioavailability of AP-SMEDDS was studied by using Caco-2 cell model. Caco-2 cells were cultured on the millipore filters fixed in Transwell transport chamber, and the cell morphology, transepithelial electrical resistance, mannitol efflux rate, alkaline phosphatase activities were monitored during culture. The result showed that after 21 days of in vitro culture,formation of tight junction was observed between the cells. The transepithelial electrical resistance reached a relatively stable value of 620 Ωcm2, the mannitol efflux rate was lower than 0.3%·h-1·cm2, and the alkaline phosphatase activity in the apical side was significantly higher than that in the basolateral side. These results indicated that the established Caco-2 cell model showed similar morphology to intestinal epithelial cells with formation of polarity, and could be used as an in vitro model for absorption study.The effects of concentration, temperature and verapamil(P-gp inhibitor) on the transport of AP and AP-SMEDDS in Caco-2 cell monolayer model was studied. The results showed that the Papp(AP-BL) of AP-SMEDDS increased about 5 times compared with AP, and Papp varied little while the concentration or temperature increased. When coupled with p-gp inhibitor(Verapamil), the efflux rate(ER) of AP decreased from1.57 to 1.2, reduced by 23.6%. These results indicated that AP-SMEDDS promotedthe permeation of AP, and the absorption of AP-SMEDDS in Caco-2 cell model was mainly through a passive transcellular transport with active efflux possibly mediated by the P-glycoprotein.