Preparation And Evaluation Of Nanoemulsion-loaded Gel Based On Paeonol-menthol Eutectic Mixture

Objective In view of the anti-inflammatory and analgesic effect of paenol and the formation of eutectic mixture between menthol and paeonol under certain conditions,the present study simultaneously adopted nano-technology and natural penetration enhancer and prepared nano-emulsion gels of paeonol-menthol eutectic mixture to improve percutaneous penetration performance and stability of paeonol.Methods1.HPLC method for determination of paeonol was established and validated.The saturated solubility of paeonol in different media was determined by shaking flask method.2.The eutectic mixture at different mass ratio of paeonol and menthol were prepared by hot melting method and then analyaed by DSC,XRD,FTIR and NMR methods.3.The saturated solubility of paeonol from different paeonol-menthol eutectic mixtures in water was determined.Using eutectic mixture as the oil phase,Cremophor EL and glycerin as emulsifier and co-emulsifier respectively,a water titration method was adopted to prepare the pseudo-ternary phase diagram of nano-emulsions Then the nanoemulsion formulation was optimized and prepared.The preliminary stability of obtained nano-emulsion was compared on their droplet size,PI value,appearance and viscosity at different temperature.Nano-emulsion gels of paeonol-menthol eutectic mixture were prepared by adding 1%Carbopol 934.Morphology ofparticles in nano-emulsion gel was observed under TEM.Drug content and crystallization of gels were tested in 0,1,2,3 months at room temperature,low temperature（4℃）and high temperature（40℃）respectively.In vitro skin permeation and retention of different gels were investigated by Franz diffusion method.The skin permeation of different gel loaded fluorescein in different time was observed by CLSM.Results1.The established HPLC method for drug content analysis was as follows:methanol-water（70:30,v/v）at 1.0 mL/min as mobile phase,column temperature at 30℃,detection wavelength of 274 nm.This HPLC method showed a good linearity,and specificity,stability and accuracy.Paeonol was poorly soluble in water（<0.001 g/mL）,but with good solubility in organic solvent like alcohol,isopropyl alcohol and so on.2.Eutectic mixture formed at ratio of paeonol and menthol from 1:9 to 9:1（w/w）,the actual and theory melting enthalpy of eutectic mixtures were calculated respectively,which confirmed the interaction between eutectic components.The crystal shape of eutectic mixtures was obviously different under microscope.The results of XRD,NMR and FTIR showed that the interaction between paeonol and menthol might be speculated as hydrogen and Vander Waals force.3.The solubility of paeonol from eutectic mixture（4:6,w/w）eutectic in water was 593μg/mL,which was slightly higher than that of paeonol crude drug（537μg/mL）.The appearance of nano-emulsion containing paeonol-menthol eutectic was homogeneous liquid with a light blue opalescence.The area of pseudo ternary phase diagram for different ratio（w/w）of paeonol and menthol was in the order of 4:6>5:5>6:4.Preliminary stability of nano-emulsions prepared with paeonol-menthol 4:6（w/w）eutectic was better in room temperature than that of nanoeulsions at other ratioes,and showed no crystallization,layering or sedimentation.Nnano-emulsion gel appeared as white and uniform semi-solid,gels and could be easily applied with no greasy feeling.The morphology of gels was uniform droplets under TEM.No crystallization occurred for nano-emulsion gel of paeonol-menthol 4:6（w/w）eutectic after storage at room temperature for 20 days.In vitro study showed that accumulated skin permeation of nano-emulsion gel from paeonol-menthol eutectic was significantly higher than that of gel from physical mixture（4:6,w/w）or paeonol solution（P<0.05）.The accumulated amount of skin peameation at 24 h were 2.882±0.123,1.998±0.125,2.059±0.099（mg/cm²）for nanoemulsion gels of paeonol-menthol（4:6,5:5,6:4,w/w）eutectics,respectively,which were 1.54,1.06 and 1.10 times higher than that of the physical mixture（1.870±0.357 mg/cm²）.Drug retention amount of nano-emulision gel with paeonol-menthol（4:6,w/w）eutectic was 111.81±45.98μg/cm²,which was significantly higher than that of other gels（P<0.05）.The steady state permeation(J_ss)and coefficient of penetration（P）of nano-emulsion gel with paeonol-menthol（4:6,w/w）eutectic were（0.3079±0.0068）and（0.3028±0.007）respectively,which was significantly higher than physical mixture gel and paeonol gel（P<0.05）.It is clear that the eutectic nano-emulsion gel with faster rate and penetrate into skin quickly compare to other gels when investigate by CLSM.Conclusion Both nano-emulsion and its gel containing paeonol-menthol（4:6,w/w）eutectic mixture showed better stability,excellent skin permeation and retention properties.However,the transdermal mechanism,clinical efficacy and interaction of eutectic mixture still need further study.