| ObjectiveTo prepare and evaluate the ginsenoside Rd microemulsion and microemulsion gel,determinate ginsenoside Rd in rat plasma and brain tissue by liquid chromatography-mass spectrometry after solid-phase extraction and its application in rat pharmacokinetics following intranasal administration,evaluate the pharmacokinetics parameters,bioavailability,drug targeting efficiency,and brain drug direct transport percentage of ginsenoside Rd microemulsion and microemulsion gel after intranasal administration for its better use in brain protection,treatment of acute ischemic stroke.Methods1 An HPLC method was used for measuring pH stability,oil-water partition coefficient,and equilibrium solubility of different oil phase,surfactant and cosurfactant of ginsenoside Rd.2 The pseudo ternary phase diagram method was adopted to optimize the proportion of surfactant and cosurfactants in the prescription.According to the particle size,polydispersity index and amount of drug loading,the prescription of microemulsion was screened and the optimized one was got to prepare ginsenoside Rd microemulsion.For further study of ginsenoside Rd microemulsion,particle size,polydispersity index,transmission electron microscopy,transmittance,conductivity,refractive index,pH,type,viscosity,stability,and the nasal mucous membrane irritation were evaluated.3 The preparation of ginsenoside Rd microemulsion gel was based on the prescription of its microemulsion.After selecting different kinds of gel,the ginsenoside Rd microemulsion gel was prepared and evaluated,including particle size,polydispersity index,transmission electron microscopy,conductivity,refractive index,pH,viscosity,stability,and the nasal mucous membrane irritation.4 LC-MS/MS method was used to determinate the concentration of ginsenoside Rd in SD rat plasma and brain tissue.The distribution of ginsenoside Rd in plasma and brain were evaluated after intranasal administration.SD rats were selected as experimental animals and were fasted overnight with access to water before dosing.Blood and brain tissue samples were collected at 8,20 min and 1,2,6,8,10,24,48,96 h after intranasal administration of ginsenoside Rd solution,microemulsion and microemulsion gel(six rats for one time point).The drug concentration-time curves were ploted by Microso Office Excel and the pharmacokinetic parameters were calculated by Kinetica analysis software.The bioavailability,drug targeting efficiency and brain drug directl transport percentage were carried out by equations.Results1 The RSD of ginsenoside Rd in pH 3.00、5.85、6.80、7.13 and 9.80 phosphate buffer solution(PBS)after 24h were all less than 3%,illustrating that the contents were not changed and the drug was stable.However,the RSD in pH 1.00 PBS was greater than 3%.The oil-water partition coefficient of ginsenoside Rd was 1.08-1.34 in n-octanol/buffer system.2 An optimized proportion of ginsenoside Rd microemulsion could be oleic acid:ethyl oleate:Tween 80:Transcutol P:water of 1:4:27.5:27.5:40.The drug contents were found to be 100 mg/mL.It showed globule size of 14.70 ± 0.29 nm,with PDI value of 0.192±0.010,zeta potential of-0.367±0.821 mV,conductivity of 18.63±0.06 μs/cm,refractive index of 1.4199± 0.0003,pH of 5.06 ± 0.01,viscosity of 34.67±0.21 mpa·s,transmittance of 99.02 ± 0.11%and style of oil-in-water.Morphological examination on ginsenoside Rd microemulsion using TEM has indicated that the globules were almost spherical in shape.There are negligible changes in the physicochemical parameters such as ginsenoside Rd content,globule size and PDI after 1,3 months of storage at room temperature and refrigerated condition.The microemulsion had slow-release characteristics.No obvious nasal mucosa irritation was found.3 An optimized proportion of ginsenoside Rd microemulsion could be oleic acid:ethyl oleate:Tween 80:Transcutol P:water:carbomer 940 of 1:4:27.5:27.5:40:0.5.The drug contents were found to be 100 mg/mL.It showed globule size of 13.11±0.27 nm,with PDI value of 0.123±0.068,zeta potential of-0.233 ± 0.754 mV,conductivity of 18.30±0.02 μs/cm,refractive index of 1.4226±0.0001,pH of 4.92±0.01 and viscosity of 59.93±0.35 mpa·s.Morphological examination on ginsenoside Rd microemulsion gel using TEM has indicated that the globules were almost spherical in shape.There are negligible changes in the physicochemical parameters such as ginsenoside Rd content,globule size and PDI after 1 months of storage at room temperature and refrigerated condition.The microemulsion gel had remarkable slow-release characteristics.No obvious nasal mucosa irritation was found.4 The LC-MS/MS method of ginsenoside Rd in plasma was linear over the concentration range of 1～1000 ng/mL,and the lower limit of quantification was 1.0 ng/mL for ginsenoside Rd.The method was validated and showed good specificity and linearity,intra-day precision of 4.39%、2.98%and 3.08%,inter-day precision of 6.08%、6.97%and 8.46%,accuracy of 87.77%～112.67%,recovery of 93.82%～101.50%.The matrix effects,dilution integrity and stability also met the requirements.The method linear over the concentration range of 1000～10000 ng/mL was partial validated and also showed good linearity,intra-day precision of 2.19%、1.15%and 1.94%,inter-day precision of 2.56%、2.67%and 2.66%,accuracy of 100.02%～113.13%,recovery of 88.03%～89.58%.5 The LC-MS/MS method of ginsenoside Rd in brain was linear over the concentration range of 1～1000 ng/mL,and the lower limit of quantification was 1 ng/mL for ginsenoside Rd.The method was validated and showed good specificity and linearity,intra-day precision of 5.08%、3.63%and 4.16%,inter-day precision of 9.65%、4.26%and 3.48%,accuracy of 88.35%～107.80%,recovery of 68.30%～75.69%.The matrix effects and stability also met the requirements.6 After intranasal administration of ginsenoside Rd microemulsion,microemulsion gel,solution and intravenous administration of ginsenoside Rd solution,the pharmacokinetic parameters of plasma were analyzed with Tmax of 2.00 h,2.00 h,2.00 h,0.13 h,Cmax of 26833 ng/mL,46940 ng/mL,4740 ng/mL,173033 ng/mL,AUC0-96h of 333122 ng h/mL,493531 ng h/mL,73736 ng h/mL,889342 ng h/mL,AUC0-∞ of 343758 ng h/mL,506015 ng h/mL,74917 ng h/mL,900394 ng h/mL,T1/2 of 15.79 h,20.95 h,16.05 h,15.93 h,MRT of 19.25 h,20.40 h,19.54 h,11.11h,respectively.7 After intranasal administration of ginsenoside Rd solution,microemulsion and microemulsion gel and intravenous administration of ginsenoside Rd solution,the pharmacokinetic parameters of brain tissue were analyzed with Tmax of 2.00 h,2.00 h,2.00 h,0.13 h,Cmax of 451.73 ng/mL,1008.25 ng/mL,69.75 ng/mL,988.25 ng/mL,AUC0-96h of 3477.86 ngh/mL,6934.21 ngh/mL,626.19 ngh/mL,7088.34 ngh/mL,AUC0-∞ of 3983.36 ng h/mL,8912.10 ng h/mL,868.56 ng h/mL,8073.38 ng h/mL,T1/2 of 7.05 h,12.09 h,13.85 h,8.28 h,MRT of 10.48 h,14.96 h,18.70 h,11.07 h,respectively.8 After intranasal administration of ginsenoside Rd microemulsion,microemulsion gel and solution,the absolute bioavailability were 37.46%,55.49%and 8.29%,respectively.The relative absolute bioavailability of ginsenoside Rd microemulsion and microemulsion gel were 451.78%and 669.32%.The AUCbrain/AUCpiasma of ginsenoside Rd microemulsion gel was greater than that of microemulsion,and the AUCbrain/AUCplasma of solution was least.The DTP%of ginsenoside Rd microemulsion and microemulsion gel were 130.99%and 176.28%,and the DTE were 23.66%and 43.27%,respectively.Conclusion1 Ginsenoside Rd was stable in alkaline solution,but easy to be damaged under the condition of strong acid.As a result,it was better to choose solid form during the process,transport and storage of the pure drug or related preparation.If it was in liquid form,we should avoid as far as possible from low pH condition.According to the oil-water partition coefficient of ginsenoside Rd in different PBS,it could be speculated that low bioavailability of ginsenoside Rd was caused not by the permeability,and likely by low solubility and slow dissolution rate.2 The Optimized ginsenoside Rd microemulsion and microemulsion gel conformed to the requirements of the preparation.3 The LC-MS/MS method of ginsenoside Rd in plasma was established and the peak time was 8.06 min.The linear range were 1～1000 ng/mL and 1000～10000 ng/mL.The specificity was good and extraction recovery of low,medium and high quality sample was accordant.The accuracy and precision met the requirements.4 The LC-MS/MS method of ginsenoside Rd in brain tissue was established and the peak time was 8.10 min.The linear range were 1～1000 ng/mL.The specificity was good and extraction recovery of low,medium and high quality sample was accordant.The accuracy and precision met the requirements.5 After intranasal administration of ginsenoside Rd microemulsion and microemulsion gel in rats,the drug bioavailability were both improved to some extent.The DTP%and DTE%of microemulsion gel were biggest,showing that it could transport drugs into the brain tissue through nasal brain pathways in large extent.Ginsenoside Rd microemulsion and microemulsion gel has been successfully prepared.Both of the two preparations could improve the bioavailability and concentration of drug in brain tissue,and microemulsion gel had a higher brain-targeting ability,which might be the foundation for future treatment of ischemic stroke by nasal administration route. |
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