Study On The Preparation Of Zedoary Turmeric Oil Microspheres With Self-emulsification Ability By Emulsion-solvent-diffusion Method And In Vivo Evaluation

Solidification of liquid systems has been a challenge that has attracted wide attention at present. Various attempts have been reported in literature to transform liquids into solids. The main goals in this study field are to obtain the solid dose form with high content of liquid drug and to improve the stability and bioavailability of the drug. In this study, the microspheres with self-emulsifying ability containing zedoary turmeric oil (ZTO), which is as a poorly water-soluble model drug with pharmacological action including suppression of tumors and anti-bacterial, were prepared by the quasi-emulsion solvent diffusion method. To evaluate the drug content in microspheres, two pure components were separated from ZTO as the standards. Then, the absorption of three indexical components (curdione, germacrone, curcumol) found in ZTO in rat intestine was investigated to direct the formulation design. And, pharmacokinetic of ZTO was studied in rats and rabbits after intravenous administration of ZTO emulsion. In pre-formulation studies, the oily excipients were selected to improve the stability of ZTO in microspheres. Then, two kinds of ZTO microspheres (conventional ZTO microspheres and sustained-release ZTO microspheres) were prepared and evaluated with the appearance, drug stability and release. The efficiency of self-emulsification of microspheres was also investigated. Finally, the bioavailabilities of two types of microspheres were evaluated in rabbits after oral administration.Two components were separated from ZTO by the column chromatography. The both contents were evaluated to be > 95% by the methods of TLC and HPLC. The chemical structures of two components were confirmed to be germacrone and curdione, respectively, by the methods of IR, MS, ¹⁴C-NMR and ¹H-NMR.To study the absorption of ZTO in rat intestine, In situ single pass perfusion model was used and the concentrations of three components in perfusate were determined by HPLC in combination with diode array detection. It was shown that the P_apps of curcumol, curdione and germacrone were all low and had no significant difference (P>0.05) at zedoary oil concentration of 0.4, 0.8 and 1.2 mg-mL^-1 in transmucosal fluid or in four different regions of intestine of rat [duodenum, jejunum, ileum, colon ]. The absorption rates of germacrone and curdione were faster than curcumol's in this study. It was concluded that ZTO concentration in transmucosal fluid had no significant effect on the P_apps within the scope of 0.4-1.2 mg·mL^-1. The absorption of curcumol, curdione and germacrone showed the passive diffusion process, and didn't contain a special absorption window. An economical HPLC method to determine the amount of germacrone in plasma was presented to investigate the pharmacokinetic of ZTO in rats. To obtain sufficient sensitivity, the method had been improved for the ZTO pharmacokinetics study in rabbits. It was found that the plasma germacrone concentration-time datas fit to a two-compartment intravenous model in rats and rabbits after intravenous administration of ZTO microcmulsion. The phannacokinetic parameters of T_1/2α and T_1/2β were 9.890 and 41.53 min, respectively, after administration of 8.9 mg·kg^-1 of germacrone in rats. T_1/2α were 0.159, 0.153 and 0.148h, respectively, and T_1/2β were 6.66, 9.502 and 8.88h, respectively, after administration of three germacrone dose levels of 3.2, 6.4 and 9.6mg.kg(-1) in rabbits. Germacrone exhibited linear phannacokinetics after intravenous administration to rabbits over the dose range of 3.2-9.6 mg·kg^-1. Rapid in vivo distribution of germacrone was shown from the concentration-time profiles in rats and rabbits, while slow in vivo elimination of germacrone were shown in rabbits, which can be the reason that germacrone has undergone the circulation between liver and intestine.To improve the stability of ZTO in microsphcres, some oily excipients as ZTO stabilizer were added into the formulations. The stability studies were carded out at the opening environment of 60℃, airtight environment of 60℃, opening environment of RH 75ï¼…and opening environment of illumination (4500LX), in which the ZTO stability was evaluated by determining the contents of ZTO and three indexical components in microspheres, such as germacrone, curdione and curcumol. It was shown that castor oil is the most helpful to the ZTO stability in selected oily excipients.Two kinds of ZTO microsphcrcs with oil (containing oil drug and excipicnt) content of～50ï¼…, such as conventional and sustained-release microspheres, had been prepared by the emulsion-solvent-diffusion method. The enterosoluble polymer, such as hydroxypropyl methylcellulose acetate succinate (HPMCAS-LG), was used in the formulation of conventional microsphcrcs, while HPMCAS-HG as a retarding agent was employed in the formulation of sustained-release microspheres. Aerosi1200 and Talc were used as adsorbent and dispersing agent, respectively, in both formulations. Satisfying incorporation efficiency over 90ï¼…was obtained for ZTO and three indexical components in conventional and sustained-release microspheres. The appearance, drug release and stability of both kinds of resultant microspheres were investigated. It was shown that microspheres were spherical and exhibited uniform and compact internal structure by the observation of SEM. The ZTO release rate was restrained in the pH1.2 HCL solution (containing 1.2ï¼…, W/V, tween-80 to meet the sink condition) and, subsequently, rapid release was shown in the pH5.8 phosphate buffer (containing 1.2ï¼…, W/V, tween-80) for the conventional microspheres. The amounts of HPMCAS-LG, Aerosi1200 and Talc in the formulation had significant effect on ZTO release rate in the pH1.2 HCL solution (containing 1.2ï¼…, W/V, tween-80), while did not show the obvious different to release rate in pH 5.8 phosphate buffer (containing 1.2ï¼…, W/V, tween-80). The ZTO release rate from the sustained-release microspheres was mainly controlled by the ratio of HPMCAS to Aerosi1200 when microspheres were put into the medium of distilled water containing 1.2ï¼…(W/V) tween-80. Stability studies of two kinds of microspheres, which were packed under simulating them at counter, were carded out. It was shown that above microspheres were stable under the normal temperature (25℃, RH 60ï¼…) and cool environment (4-8℃), while had not sufficient stability under accelerating environment (40℃, RH 75ï¼…).To investigate the character of microsphere self-emulsification, we dealt with a system consisting of Aerosil, HPMCAS, ZTO and pH6.8 phosphate buffer. A series of emulsions was prepared by ultrasonic-emulsifying method and evaluated with the stability and droplet size in order to investigate the effect of different types of Aerosil and polymer as well as the ratio of them to stabilize ZTO emulsion. In addition, the ZTO microspheres containing different ratios of Aerosil to polymer were prepared. The stability and droplet size of resultant emulsion produced from self-emulsification of microspheres were compared with them of emulsions prepared by ultrasonic-emulsifying method. It was shown that emulsion stability was defined strongly by the type of Aerosil and polymer as well as the ratio of these excipients. Droplet size and stability of emulsions, which were produced by ultrasonic-emulsifying method and microsphere self-emulsification, increased on the increase of Aerosil/polymer ratio, while the smaller size of droplets and stabler emulsion were obtained by microsphere self-emulsification comparing with them of emulsions prepared by ultrasonic-emulsifying method when at the same Aerosil/polymer ratio. An ideal range of Aerosil/HPMCAS ratio seems to be given in the case of almost complete coverage of the Aerosil particles without having a substantial amount of unbonded polymer in the bulk solution. The best ratio of Aerosil to HPMCAS-LG or -HG was predictable from the Langumuir-fit of the adsorption isotherms, which is very useful for the design of our microsphere formulation.The bioavailability studies were carded out in rabbits after oral administration of conventional ZTO microspheres, sustained-release ZTO microspheres and conventional self-emulsifying ZTO formulation (conventional SES). The relative bioavailabilities of conventional and sustained-release ZTO microspheres were greatly improved over the conventional SES, which were 157.7ï¼…and 135.6ï¼…, respectively. It was evident that the sustained-release ZTO microspheres achieved a more prolonged absorption profile compared with other two preparations. More rapid rate of self-emulsification resulted in shorter time of getting maximum germacrone concentration after oral administrations of above three ZTO preparations. Decrease of droplet size of resultant emulsions produced from self-emulsifying preparations significantly enhanced the in vivo absorption of ZTO and thus improved the bioavailability of oral administration.Some investigations, which have not reported at precent, have been carried out in this work, such as the study of absorption of ZTO in intestine, pharmacokinetics of ZTO in rats and rabbits and the study of stabilizing ZTO emulsions by a united use of Aerosil and polymer. A novel formulation of oily drug, ZTO microspheres with self-emulifying ability, has been designed here. The microspheres, as a solid dose form of liquid drug, have three advantages, such as high content of oily drug in microspheres, greatly dispersed degree of drug and self-emulsifying ability of microspheres, which were significantly different from the conventional self-emulsifying formulation.