| A temperature-sensitive diosgenin imprinted silica gel microspheres（DG-TMIP）was prepared by surface-imprinting technique based on silica gel as carrier using diosgenin as template,methacrylic acid（MAA）as functional monomer and N-isopropylacrylamide（NIPAM）as temperature sensitive monomer.The crystal structure,chemical functional groups and morphological structure of DG-TMIP were characterized by scanning electron microscopy（SEM）,infrared spectroscopy（FTIR）,X-ray diffraction（XRD）and N2 adsorption-desorption surface analysis method（BET）.The adsorption behavior,solid phase extraction capacity,drug loading and drug release control capacity for the diosgenin-imprinted microspheres were studied.（1）For the preparation of DG-TMIP,synthesis conditions were optimized by using factors design.Firstly,solvent was needed to be considered in order to make the template dissolve completely.The solvent ratio for ethanol-acetonitrile mixed solvent was optimized and 20%ethanol acetonitrile solution was adopted for the preparation of DG-TMIP.A L9（34）orthogonal experiment design for four factor from three levels was utilized and the optimized synthesis conditions were as follows:0.2 mmol functional monomer,0.2 mmol temperature sensitive monomer,2 mmol crosslinking agent and10mL solvent.The influence of interaction between factors on the adsorption capacity of the imprinted polymer obtained was also studied.Results indicated that there is a negative correlation between the dosage of functional monomer and solvent,between the dosage of functional monomer and crosslinking agent,between the dosage of crosslinking agent and temperature sensitive monomer,and between the dosage of solvent and temperature sensitive monomer.Additionally,a positive correlation was observed between the amount of functional monomer and the amount of temperature sensitive monomer when using a higher amount of functional monomer and between the solvent volume and the amount of crosslinking agent when using a higher solvent volume during preparation.Through using factor design method to optimize the preparation conditions,the number of experiments was reduced greatly,improving the experimental efficiency during simultaneous optimization of multi-factors.（2）DG-TMIP was prepared under the optimized and its morphological structure,functional groups and crystal structure were characterized by SEM,FTIR,XRD and BET.This imprinted polymer was shown with abundant micropore on the surface.When using acetonitrile as solvent,the imprinted polymer has high adsorption capacity for diosgenin,with a value of 116.4 mg/g.When the used DG-TMIP as adsorbent for solid phase extraction,the optimized washing solvent was 2×1 mL acetonitrile,and the optimized elution solvent is 1mL acetic acid ethanol mixed solvent containing 20%,40%,60%and 80%acetic acid.When the simulated sample solution is extracted under this condition,the recovery of diosgenin is high,especially in the elution step,most of diosgenin can be recovered.When the optimized extraction conditions were used to extract the crude extract of Dioscorea zingiberensis,85.32%of diosgenin can be recovered only through elution-1 and elution-2 steps,and the contents of diosgenin in the collected effluents were high,with values of 68.55%and 84.19%,respectively.This indicated that the effective enrichment and separation of diosgenin could be carried out by two-step elution.In addition,this molecularly imprinted polymer can be utilized for more times with high reproducibility.（3）The extraction,adsorption and drug release-controlled ability for the DG TMIP were measured.Firstly,the adsorption capacity and selectivity for this DG-TMIP toward template molecules were was determined under various temperatures.When temperature was at 30℃,the equilibrium adsorption capacity of the imprinted microspheres toward diosgenin was the highest,with a value of 25.58 mg g-1,higher than that obtained at 35℃and 25℃.This result indicated that this temperature-sensing imprinted polymers possessed the maximum adsorption capacity near the critical temperature of the MIP.The character for this temperature-sensitive imprinted materials can be utilized to adjust the adsorption and release of target compounds by changing the temperature.The Scatchard analysis for the adsorption isotherms showed that there mainly were two kinds of adsorption sites in DG-TMIP.The adsorption process for the MIP under 30℃and 35℃is more suitable to be described by the second-order adsorption kinetic model,while that under 25℃is more in line with the first-order adsorption kinetic model.The temperature-sensitive imprinted polymer had stronger adsorption capacity not only toward diosgenin,but also toward its structural analogues.However,the adsorption capacity for the template was significantly higher than that for its structural analogues.Diosgenin adsorbed on the surface of this molecularly imprinted microspheres can be slowly released by optimizing desorption conditions,with a desorption percentage of 103.8%within 12 hours under the optimized conditions.However,the non-imprinted polymers microspheres has no this capability.Environmental conditions such as temperature,solvent type and ionic strength were shown with an important influence on the release rate of diosgenin from this temperature-sensitive imprinted polymer.This capability for this impritned polymer is conducive to the development of a new drug carrier for diosgenin,controlling its release rate and improving its disease treatment effect and bioavailability.（4）A chromatographic method was selected to study the retention and release effect of the MIP toward diosgenin by using the MIP as stationary phase in liquid chromatography.The effects of such conditions as mobile phase composition,column temperatures and ionic strength of eluent on the retention behavior of the MIP column.The results showed that when 0.1 mol/L pH7.4 Na H2PO4-Na2HPO4 buffer was used as the mobile phase,the imprinted column had the highest selective retention ability to the template,with a retention factor value of 8.239 and selection factors values of 8.214 and3.191,relative to ursolic acid and cholesterol,respectively.When the pH value of mobile phase decreases,the retention ability for the MIP toward template molecule decreases.The effect of column temperature on the retention capacity is related to the critical temperature of imprinted polymer and the mobile phase with low ion strength benefits the retention of the template on the MIP.Diosgenin loaded on the surface of the TMIP can be released by eluting using solvent.However,the eluent composition,temperature and eluent ion strength can influence release rate of diosgenin from molecularly imprinted column.Results indicated the release rate of drug molecules became slow and the release time was long when the release was performed near critical temperature using eluent with higher pH value and with lower ion strength.This study possessed potential value in the field of controlled release of drug from the surface of molecularly imprinted polymers as the drug carriers. |
