| As a by-product of the olive tree,olive leaves have a relatively high yield,but due to their low utilization,they were often treated as waste materials for burning,which wasted resources and damaged the environment.There are many biologically active substances in olive leaves.Among them,hydroxytyrosol has anti-oxidant,anti-inflammatory,anti-bacterial and anti-cancer effects.Hydroxytyrosol was currently mainly extracted from olive residue,but it was difficult to separate and purify.Molecular imprinting is a technology with specific recognition function developed in recent years.It imitates the interaction between enzyme-substrate and antibody-antigen,and can specifically recognize template molecules.In this paper,surface molecular imprinting technology was used to synthesize a magnetic molecularly imprinted polymer with high selectivity to hydroxytyrosol,which can quickly separate,purify and enrich hydroxytyrosol,and applied to olive leaves.The main research contents are as follows:(1)Single-factor experiments were performed on ultrasonic-assisted extraction and shaking-assisted extraction,and the extraction rate of hydroxytyrosol was used as the standard to optimize the extraction temperature,extraction time,material-to-liquid ratio and hydrochloric acid concentration.Under the optimal conditions of single factor,comparing the extraction rate of hydroxytyrosol with the two methods,the results showed that ultrasonic assisted extraction was more efficient.The response surface experiment was used to further optimize the ultrasonic-assisted extraction,and it was concluded that the optimal conditions for extracting hydroxytyrosol from olive leaves were that the concentration of hydrochloric acid was 0.496mol/L,the ratio of material to liquid was 1:21.23g/m L,and the ultrasonic time was 78.6.min,the extraction temperature was 83.8 ?.(2)On the basis of the optimal extraction conditions,a high-performance liquid chromatograph was used to establish a method for determining the content of hydroxytyrosol,and the method was investigated,and the content of hydroxytyrosol in olive leaves was found to be 0.37 mg/g.(3)The functional monomer and porogen were screened by quantum chemistry software,and the best functional monomer and porogen were 1-vinylimidazole and ethanol,respectively.Fe3O4 nanoparticles coated with silica were synthesized,and used as a carrier.1-vinylimidazole as a functional monomer,EDGMA as a crosslinking agent,ethanol as a porogen,and azo diisobutyronitrile was used as the initiator to synthesize magnetic molecularly imprinted polymer on its surface.It was characterized by Fourier transform infrared spectroscopy(FTIR),X-ray diffractometer(XRD),scanning electron microscope(SEM)and sample vibration magnetometer(VSM).The results showed that the magnetic molecularly imprinted polymer was successfully synthesized and Fe3O4 and Fe3O4@MIPs saturation magnetization is 53.90 and 15.70 emu/g.It has strong magnetic responsiveness under the action of a magnetic field,which facilitates the movement and aggregation of particles,and can meet the requirements of magnetic separation.(4)The dynamic adsorption and static adsorption of the synthesized magnetic molecularly imprinted polymer were studied.The results showed that the Fe3O4@MIPs had a high affinity for hydroxytyrosol and could reach adsorption equilibrium within 45 min.The maximum adsorption capacity was 36.27 mg/g,which was about twice that of Fe3O4@NIPs.The dynamic adsorption and static adsorption of Fe3O4@MIPs was fitted.It was found to accord with the pseudo-second-order kinetic equation.As the concentration of hydroxytyrosol increased,Fe3O4@MIPs absorption capacity of hydroxytyrosols was also increasing.It conformed to the Langmuir model,and the calculated maximum adsorption capacity was 38.31 mg/g.The adsorption results of Fe3O4@MIPs on hydroxytyrosol and its structural analogs indicated that it had high selectivity to hydroxytyrosol.The elution conditions were optimized.The best conditions for elution of hydroxytyrosol were at 25 ? or room temperature,with methanol/acetic acid(8:2,v/v)as the elution solvent,and shaking in a constant temperature oscillator with 45 min and the elution rate is relatively high,basically maintaining above 96%.The regeneration performance was analyzed.After10 times of repeated adsorption and elution,the adsorption and selectivity of the magnetic molecularly imprinted polymer Fe3O4@MIPs did not decrease significantly,indicating that the structure of Fe3O4@MIPs was very stable.The MISPE column was prepared using Fe3O4@MIPs,and the separation conditions of the MISPE column were optimized.It was found that the best activation solvent was methanol,the loading solvent was water,the washing solvent was water,and the elution solvent was methanol/acetic acid(8:2,v/v).The MISPE column was applied to the separation and purification of hydroxytyrosol in olive leaves,and the purity of the purified hydroxytyrosol was determined by HPLC.The results showed that the MISPE column had a good separation and purification effect on hydroxytyrosol in olive leaves.After analyzing and calculating the purity of the purified hydroxytyrosol for many times,it was found that the purity of the purified hydroxytyrosol after the MISPE column was basically maintained at more than 95%.(5)The the activity of purified hydroxytyrosol was studied through inhibitory effect on tyrosinase.The results showed that hydroxytyrosol had a lower inhibitory effect on tyrosinase than that of kojic acid.When the concentration of hydroxytyrosol was 100 ?g / m L,its inhibition rate reached 62.79 %.According to the concentration and inhibition rate,the approximate value of the half inhibitory concentration(IC50)was calculated.The half inhibitory concentration of hydroxytyrosol was 82.01±0.365?g/m L.The antioxidant experiment results showed that the ability to clear DPPH free radicals was not as good as Vc.The scavenging rate of ABTS+ free radicals was higher than that of Vc.In the determination of copper ion reducing ability,the reducing ability of hydroxytyrosol is also higher than that of Vc. |
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