| Paeonol?Pae?is an active component found in peonies such as Paeonia moutan and Radix Cynanchi Paniculati with board range of pharmacological activities in some traditional Chinese medicine remedies.In vitro and in vivo studies showed Pae possessed a board range of pharmacological activities like anti-inflammatory activity,antibacterial activity,anti-diabetic activity,anti-allergic activity,anti-tumer activity,anti-atherogenic activity,as well as the ability to relieve pain.However,the intrinsic properties of Pae with low water solubility,poor stability and low oral bioavailability restrict its clinical application severely.Therefore,it is necessary to develop an appropriate formulation to improve the absorption of Pae under oral administration.Nanoemulsion,a transparent and thermodynamically stable system with nanosized droplets at the range of 1-100 nm.Nanoemulsion has been proved to be an effective carrier for improving the oral bioavailability of drugs through different absorption mechanisms.The improvement of aqueous solubility and stability,the facilitation of lymphatic transport,the enhancement of intestinal permeability,the prevention of P-glycoprotein efflux may contribute to the enhanced oral bioavailability.Nanoemulsion can protect the drugs by encapsulating effect and prevent the recognition of P-gp.The drugs can be easily absorbed into the cell without being squeezed out,which result in the enhanced bioavailability of the drugs under oral administration.In this research,the preparation of Pae nanoemulsion was optimized.The pharmacokinetic parameters of Pae nanoemulsion in rats were investigated in pharmacokinetic studies.Then in situ single-pass intestinal perfusion was carried out to investigate the intestinal absorption of Pae nanoemulsion.In vitro everted gut sacs of rats was performed to investigate the effect of P-gp inhibitor on the absorption of Pae nanoemulsion.Western blot analysis was performed to detect the expression of P-gp in the intestine of Pae nanoemulsion group.From the perspective of P-gp efflux,the mechanism of Pae nanoemulsion enhancing intestinal absorption was initially clarified.Objective:The aim of this work was to optimize a nanoemulsion formulation,increase the oral bioavailability of Pae,and explore the reason why Pae nanoemulsion enhances the oral absorption of Pae.Method:In this research,the solubility of Pae in the vehicles were determined to screen suitable surfactant,co-surfactant and oil phase.The pseudo-ternary phase diagram was used to select the optimal formulation.Then the stable Pae nanoemulsion was prepared and the quality of Pae nanoemulsion was evaluated.Pae suspension and Pae nanoemulsion were orally administrated to each group.About 0.5 mL blood was collected at different time,and the plasma concentration of Pae was detected by HPLC.The pharmacokinetic parameters were calculated by DAS 2.0.In situ single-pass intestinal perfusion model was used to examine the intestinal absorption of Pae solution and Pae nanoemulsion.The absorption mechanism of Pae nanoemulsion was explored by comparing the absorption rate constants?Ka?and apparent permeation coefficients?Papp?of the Pae solution and Pae nanoemulsion groups.The effects of verapamil,a P-gp inhibitor,on the absorption of Pae solution and Pae nanoemulsion were studied using everted gut sacs experiment.The apparent permeability coefficient?Papp?of Pae solution and Pae nanoemulsion was compared after adding verapamil.Western blot analysis was used to evaluate the expression levels of P-gp in rat colon after oral administration with normal saline,Pae suspension,blank nanoemulsion,Pae nanoemulsion and the solution of EL35 with Pae dissolved in?EL35+Pae?.The methods mentioned above provide preliminary elucidation of the absorption mechanism of Pae nanoemulsion.Results:1.The preparation and quality evaluation of Pae nanoemulsionThe optimal Pae formulation was EL35,ethanol,IPM and water?0.5:0.5:0.17:1,w/w/w/w?.Nanoemulsion with the concentration of 10.00 mg/mL Pae was prepared for the following study.The droplet size and zeta potential of Pae nanoemulsion were27.26±1.06 nm,-16.83±1.79 mV,respectively.Pae nanoemulsion showed a uniform and spherical shape from TEM result.2.Pharmacokinetics studyPharmacokinetics result showed that the AUC0?t of Pae nanoemulsion?27.244±8.068 mg?h/L?increased by 4.27-fold than that of Pae suspension?6.378±0.444mg?h/L?.The Cmax of Pae in Pae nanoemulsion group was 5.434±1.254 mg/L,which was significantly improved by 4.02-fold compared to Pae suspension group?1.351±0.129 mg/L?.The Tmax of Pae nanoemulsion was relatively delayed for about 40min compared with Pae suspension.The results showed that nanoemulsion significantly increased oral bioavailability of Pae in rats.3.In situ single-pass intestinal perfusion of Pae nanoemulsion in ratsThe Ka of Pae nanoemulsion in jejunum,ileum and colon had improved by 0.65-,1.35-and 1.64-fold compared with Pae solution group.Meanwhile,the Papp of Pae nanoemulsion in ileum and colon had improved by 0.55-and 0.66-fold compared with Pae solution group.The absorption of Pae nanoemulsion was better than that of Pae solution.The Ka and Papp of Pae nanoemulsion group had the greatest increase in the colon among all bowel segments.4.Effect of Pae nanoemulsion on intestinal P-gpAfter adding verapamil,the Papp of Pae solution group was increased from3.22×10-6cm/s to 7.82×10-6cm/s,which meant that the absorption of Pae in Pae solution significantly increased.However,the Papp of Pae nanoemulsion before and after adding verapamil were 5.94×10-6 cm/s and 6.41×10-6 cm/s,respectively.The absorption of Pae in Pae nanoemulsion was not affected by verapamil.The results of western blot analysis showed a significant reduction of P-gp expression in blank nanoemulsion group compared with normal saline group.Compared to the Pae suspension group,the expression level of P-gp was significantly reduced in Pae nanoemulsion group.Conclusion:1.In this research,an optimiazed Pae nanoemulsion was prepared to improve the oral bioavailability of Pae.2.After oral administration of Pae nanoemulsion,the absorption rate and degree in the small intestine were significantly improved.3.The absorption of Pae was affected by P-gp,which leaded to the intestinal absorption restriction.Pae nanoemulsion could prevent Pae from being recognized by P-gp and being excluded.Moreover,Pae nanoemulsion could inhibit the express level of P-gp in intestinal tract.It may be the main reseaon that nanoemulsion improves the intestinal absorption of Pae. |
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