Preparartion And In Vitro And In Vivo Study Of Asiaicoside-Loaded Nanoemulsions And Nanoemulsions-Based Gels For Transdermal Delivery

Part 1 Preformulation studyObjective:To establish a method of HPLC for determination of asiaticoside,screen the oils,surfactants and surfactants with high solubility for asiatiside and establish a method for determination the entrapment efficiency and solubility of asiaticoside nanoemulsions（ASI-NEs）.Methods:1.The HPLC method was established to determine the concentration of asiaticoside,while its precision,repeatability and stability were also investigated.2.The equilibrium solubility of asiaticoside in different oils,surfactants and cosurfactants was determined by HPLC,and the oil-water partition ratio of asiaticoside was determined by shake-flask method.3.The entrapment efficiency of asiaticoside was observed by the dialysis method.4.The high-speed centrifugation was employed to determine the solution of ASI-NEs.Results:1.The HPLC method for the determination the content of ASI was established,the method accorded with the experimental requirements and could be used for the calculation of encapsulation efficiency.2.The results showed that the higher solubility of asiaticoside in oils was glycerol monolinoleate,castor oil and OA,surfactants with higher solubility was RH-40 and Labrasol,cosurfactants with higher solubility was Transcutol CG,Transcutol P and PEG-400.3.Dialysis method could separate ASI-NEs from free drug and the equilibrium time was determined to be 3 h,which could effectively determine the entrapment efficiency of ASI-NEs.4.The high-speed centrifugation could effectively determine the solution of ASI-NEs.Summary:The determination of ASI content by HPLC,the entrapment efficiency of ASI-NEs by dialysis method and the determination of the solubility of ASI-NEs by high-speed centrifugation were all in accordance with the experimental requirements,which laid the foundation for formulation screening and technological research.Part 2 Preparation of asiaticoside nanoemulsions and asiaticosidenanoemulsions-based gels and properties of asiaticoside nanoemulsionsObjective:To determine the optimal preparation and prescription of ASI-NEs.To determine the preparation method of ASI-NBGs,and investigate the physical and chemical properties of ASI-NEs.Methods:1.The compatibility experiment was used to screen the oil,surfactance and surfactants.In order to determine the species of each phase,the area was used as the standard in the pseudo-termary phase diagrams.2.Single-factor experiments were used to determine the optimal preparation process of ASI-NEs with the encapsulation efficiency as the evaluation index.3.The simplex lattice design（SLD）was utilized to screen and optimize the formulation,the drug solubility and encapsulation efficiency were inspected as evaluation index.In the process,the experimental results were fitted with multivariate model to draw contour map and three-dimensional effect surface map,and to predict the theoretical optimal prescription.The optimized ASI-NEs were determined and prepared to verification the efficiency of regression.4.The physicochemical properties of the optimal prescription of ASI-NEs were investigated.ASI-NBGs were prepared by Carbopol 940.In the process,P940 was expanded in ASI-NEs to swell for 24 h,and triethanolamine was used to regulate pH（7）to produce ASI-NBGs.Results:1.In the process of compatibility experiment and pseudo-termary phase diagrams,the prescription compositions of oil,surfactance and cosurfactance were determined and which were glyceryl monolinoleate,Cremophor RH 40and diethylene glycol monoethyl ether,respectively.2.The optimum preparation technology about rotating speed,temperature and equilibration time through the single-factor investigation of ASI-NEs were determined as follows:700 rpm·min^-1,37°C and 50 min,respectively.3.Following the optimization,the optimal ASI-NEs formulation was comprised of 5%glyceryl monolinoleate,15%Cremophor RH 40,26.3%diethylene glycol monoethyl ether and 53.7%water,respectively.The deviations between the measured value and the predicted value of solubility and entrapment efficiency were 4.55%and 3.69%,respectively.The solubility and encapsulation efficiency of the optimal prescription were 13.97 mg·g^-1and 90.29%,respectively.4.ASI-NEs were O/W nanoemulsion with clear and transparent appearance,pH value of 6.8,average particle size of 132±5.84 nm and PDI of0.2211.ASI-NEs was good stability and without delamination under the high-speed centrifugation.ASI-NBGs had transparent appearance and no caking.Summary:ASI-NEs and ASI-NBGs were successfully prepared and the preparations have good appearance and physicochemical properties.Part 3 Study on the transdermal characteristics of asiaticosidenanoemulsions and asiaticoside nanoemulsions-based gels in vitro.Objective:To study the transdermal characteristics of ASI-NEs and ASI-NBGs in vitro.Methods:The abilities of various formulations to deliver asiaticoside through the skin in vitro were studied using improved Franz diffusion cells.The ASI-NEs and ASI-NBGs were prepared according to the optimal preparation and compared with respect to the marketed cream of asiaticoside（ASI-C）,plain gel and gels added with transdermal enhancers to study the transdermal characteristics.The cumulative release curves were plotted,and the 12 h skin retention was calculated.The transdermal characteristics in vitro were evaluated by the retention time method.Results:Permeation studies in vitro showed that ASI-NEs and ASI-NBGs had faster penetration rate than those of gels added with transdermal enhancers,and the difference between them was statistically significant（P<0.01）.The cumulative amount of ASI permeated from ASI-NEs and ASI-NBGs at 12 h after application were 3504.30±180.93μg·cm^-2 and 1187.40±128.88μg·cm^-2 respectively,which were 6.57 and 2.23 times higher than that in the control group of ASI-C.And the drug deposition in skin of ASI-NEs and ASI-NBGs were 159.48±7.47μg·cm^-2 and 120.53±5.71μg·cm^-2respectively,which were 5.93 and 4.48 times higher than that of ASI-C,the difference was statistically significant（P<0.01）.Summary:ASI-NEs and ASI-NBGs have good characteristics as the vehicle for dermal drug delivery.Part 4 Dermatopharmacokinetic study of asiaticoside nanoemulsions andasiaticoside nanoemulsions-based gelsObjective:To study dermatopharmacokinetics characteristics of asiaticoside nanoemulsions and asiaticoside nanoemulsions-based gels.Methods:To compare the dermatopharmacokinetics of ASI-NEs,ASI-NBG and ASI-C by time-share sampling method in mice.Results:The pharmacokinetic parameters of ASI-NEs,ASI-NBGs and ASI-C were that AUC _0-∞were 6227.85,4994.90 and 2321.36μg·h·g^-1,and which were 2.68 and 2.15 times higher than that in the control group of ASI-C,t_maxax were 6,6 and 8 h,respectively.The study shown that asiaticoside loaded nanoemulsions and nanoemulsions-based gels can quickly reach the peak in the skin,and maintain a stable release in subcutaneous tissue for a long time to remain at a relatively constant level.And ASI-NEs and ASI-NBGs had higher bioavailability.Summary:ASI-NEs and ASI-NBGs have high skin retention in vivo and they are suitable for topical application.Part 5 Study on the skin irritation and transdermal mechanism ofasiaticoside nanoemulsions and asiaticoside nanoemulsions-based gels.Objective:To study the skin irritation and transdermal mechanism of asiaticoside nanoemulsions and asiaticoside nanoemulsions-based gels.Methods:1.New Zealand white rabbits were used to study the skin irritation of ASI-NEs and ASI-NBGs by examining the effects of ASI-NEs and ASI-NBGs on normal and damaged skin with single-dose and multiple-dose administration.2.HE staining method and CLSM were used to study the transdermal mechanism.The topical effect of ASI-NEs and ASI-NBGs on ultrastructure of rabbit skin were evaluated using HE staining method.The localization and the permeation pathway of asiaticoside was provided by CLSM.Results:1.Skin irritation study showed that:after single-dose administration,ASI-NEs and ASI-NBGs showed no irritation to normal skin and damaged skin.After multiple-dose administrations,ASI-NBGs showed no irritation to normal skin and mild ASI-NEs to normal skin,ASI-NEs and ASI-NBGs were mildly irritating to the damaged skin with individual manifestations of barely visible erythema that can be completely self-contained after a day.2.The study of HE staining and CLSM indicated that the transdermal mechanism was related to breaking the ultrastructure of stratum corneum and penetrating by the path of skin adnexa.Summary:The transdermal mechanism of ASI-NEs and ASI-NBGs is related to breaking the ultrastructure of stratum corneum and penetrating by the path of skin adnexa.ASI-NBGs is a safe topical formulation for the skin but should be avoided for damaged skin.Conclusion:In this study,ASI-NEs and ASI-NBGs are successfully prepared and the preparation process is simple and reproducible.ASI-NEs and ASI-NBGs can effectively improve the solubility of asiaticoside.ASI-NEs and ASI-NBGs have good characteristics as the vehicle for dermal drug delivery and higher bioavailability,and ASI-NBGs is a safe topical skin preparation.