| Ellagic acid(EA),one such polyphenolic phytonutrient,possesses a wide array of pharmacological activities like antioxidant activity,radical scavenging,anticarcinogenic and anti-inflammatory effects.However,the pharmacokinetics study reveals EA for oral use is poorly absorbed,rapidly eliminated from the body due to short plasma half life and finally fails in attaining required tissue concentrations.The results can be attributed to poor aqueous solubility of EA.Hence,it becomes one of the key problems on how to improve EA water solubility,enhance oral absorbability and improve bioavailability.Nano-pharmaceutics,a recently springing-up formulation,not only can enhance solubility of poorly soluble drugs to improve bioavailability,but also has the advantages including targeting,sustain release,low side effects and strengthening efficacy.In this study,micronized EA(m-EA)was prepared by antisolvent precipitation.The m-EA significantly improved water solubility,dissolution rate,which brought about the enhancement of oral absorption,along with the improvement of bioavailability and drug efficacy.Antisolvent precipitation and single factor design that was used to optimize this technique were utilized to prepare m-EA freeze-dried powder which was evaluated in vivo and vitro.The major contents and results are as follows:1.Single factor experiments were used to optimize the technique and the particle size,zeta potential,specific surface area,drug loading efficiency and short-time stability were evaluated The optimum conditions for preparation of m-EA through single factor experiments were 2min of precipitation time,30C of precipitation temperature,2500rpm of stirring intensity,5 of the volume ratio of antisolvent to solvent,30mL/h of the dripping speed of solvent adding into antisolvent and 30mg/mL concentration of EA.Under these conditions,m-EA freeze-dried powder with a mean particle size(MPS)of 429.2±7.6nm and zeta potential of-22.56±1.76mV were obtained and EA content in the freeze-dried powder was 15.07±0.46%.The results of specific surface area indicated 1.2520m2/g was for raw EA and 14.3129m2/g was for m-EA freeze-dried powder.The m-EA freeze-dried powder prepared under optimum conditions was in thin strip form and m-EA dispersed in water with uniform particle size revealed regular cubes or spheres through scanning electron microscopy(SEM);The residual amount of NMP in m-EA freeze-dried powder was determined by gas chromatograph(GC)and the residual content in m-EA freeze-dried powder was 405ppm;According to liquid chromatography mass spectrum(LC-MS)and Fourier transform infrared spectroscopy(FITR)the chemical structure of EA had no change via antisolvent precipitation and freeze-drying process.The results of differential scanning calormetry(DSC),and X-ray diffraction(XRD)indicated that EA in m-EA freeze-dried powder had low crystallinity via antisolvent precipitation.2.Antioxidant activity of raw EA and m-EA freeze-dried powder in vitro was compared by examining DPPH radical scavenging ability and reducing power of potassium ferricyanide.The experiments showed that the scavenging effects of m-EA freeze-dried powder reached 69.64±3.33%when its concentration was 2mg/mL.However,the scavenging effects of raw EA were only 46.72±1.36%at the same concentration.Their half-inhibition concentrations(IC50)were 2.04mg/mL and 1.11mg/mL,respectively.So it proved that micronization process improved the defects of poor water solubility of EA to greatly increase antioxidant activity.3.The results of dissolution study in vitro on raw EA and m-EA freeze-dried powder showed the raw EA powder due to poor water solubility,cannot completely dissolved very slowly.The cumulative dissolution percentage reached only about 16%at 24h.However,m-EA freeze-dried powder rendered a good dissolution effect.It reached about 32.53%of the maximum cumulative dissolution percentage at 24h and the dissolution amount was twice that of raw EA.The water solubility of raw EA and m-EA freeze-dried powder at 37℃ was 1.82±0.09μg/mL and 11.67±0.46p,g/mL,respectively.4.Penetration experiment in vitro was performed on raw EA and m-EA freeze-dried powder.The results showed that penetration rate of m-EA freeze-dried powder was faster than raw EA;at the concentration of 0.5mg/mL,the penetration amount per unit area of m-EA freeze-dried powder was 1.27 times that of raw EA;at the concentration of 1mg/mL,the penetration amount per unit area of m-EA freeze-dried powder was 1.47 times that of raw EA.5.The content of EA in rat plasma was examined by high performance liquid chromatography(HPLC)in bioavailability study of EA.The results showed that raw EA was accorded with two compartment model and m-EA freeze-dried powder was accorded with one compartment model.Raw EA and m-EA freeze-dried powder groups attained their maximum of EA concentration in rat plasma,namely,248.3±24.1ng/mL and 726.7±36.4ng/mL after 1h and 1.5h of taking drugs,respectively.The results indicated the oral bioavailability of m-EA freeze-dried powder increased about 1.78 times compared with raw EA through comparing area under the curve.Nonetheless raw EA persisted longer in the body than m-EA freeze-dried powder seen from half life.6.Binding ability of three bile salts of raw EA was examined by ultraviolet spectrophotometry,which was as a basis to predict the activities of reducing lipid of EA.The results indicated that binding ability of bile salts of raw EA was very prominent,which was dozens of times that of common fruits and vegetables,namely 63.59±2.14μmol/100mg of sodium cholate(SC),18.64±1.56μmol/100mg of sodium taurocholate(STC)and 18.31±1.26μmol/100mg of sodium glycocholate(SGC).Binding ability of three bile salts of raw EA will increase the amount of cholesterol into bile salts in the liver to keep balance with bile salts.The results cause the decrease of cholesterol in liver,which make lower cholesterol from liver to blood and reduce lipid at last.7.Raw EA and m-EA freeze-dried powder with different doses all could significantly inhibit the weight growth of the hyperlipidemia rats via 21 days treatment(P<0.05 or P<0.01);EA can be used to treat the higher levels of total cholesterol(TC),total triglyceride(TG)and low density lipoprotein cholesterol(LDL-C)and lower level of high density lipoprotein cholesterol(HDL-C)of hyperlipidemia rats caused by high fat emulsion.The m-EA freeze-dried powder with the same dosage 30mg/kg with raw EA had a better anti-hyperlipidemia effect.Through the above experimental results,we proved the prepared m-EA freeze-dried powder had a uniform particle size distribution,low residual solvent for safety in use and stable physical and chemical properties.The results of penetration test in vitro indicated m-EA freeze-dried powder showed merits of transdermal compared with raw EA and could be applied to cosmetics industry as transdermal preparation.Moreover,it exhibited higher bioavailability,antioxidant ability and anti-hyperlipidemia activity than raw EA.So the m-EA freeze-dried powder shows potential advantages as a new oral preparation and provides technical support and theoretical basis for development of anti-oxidative drugs and hypolipidemic agents. |
PDF Full Text Request