| Objective:(1)A series of hydrophobic deep-eutectic solvents(HDESs)were developed and they were used for the first effevtively extraction step of quercetagetin,an active ingredient of flavonoids in the traditional Chinese medicine of Tagetes erecta Linn(T.erecta)flower.A new type of molecularly imprinted polymers(MIPs),which had been prepared and used as the adsorbents for dispersive solid-phase extraction(d-SPE),had been appled for the second high-efficiency enrichment step of quercetagetin from HDESs extract.Based on MIPs,a new restricted access MIPs(RAMIPs)was prepared,and thus the d-SPE based on RAMIPs coupled with HPLC/UPLC-MS/MS were applied for the analysis of quercetagetin in the plasma of mouse.(2)Prepare Covalent organic framework materials with high crystallinity by taking green and environmentally friendly Deep-eutectic solvent(DES)as the reaction solvent.A new extraction method of Solid phase microextraction(SPME)based on COF-DES-1 was developed by using SPME as fiber coating.The method was combined with UPLC-MS/MS to analyze the metabolism of luteolin in vivo.Covalent organic framework(Named as COF-DES-1)with high crystallinity was prepared by using green and environmentally friendly DES as the reaction solvent.Based on COF-DES-1,a new extraction method of solid phase microextraction(SPME)was developed by using COF-DES-1 as the SPME fiber coating.The SPME method coupled withh UPLC-MS/MS was used to analyze the metabolism of luteolin in vivo.Methods:(1)Firstly,tetrabutylammonium bromide(TBAB),DLmenthol and L menthol were separately used as hydrogen bond receptors(HBA).PPG-400,hexafluoroisopropanol,pyruvate,levulinic acid and acetic acid were separately used as HBD donors.The DESs were prepared by using HBA and HBD through a solvothermal method.Secondly,HDESs was used as extraction solvent to extract quercetagetin from Tagetes erecta Linn(T.erecta)flower by using ultrasonic-assisted extraction method.By using the extraction efficiency of quercetagetin as the index,the optimal extraction conditions were obtained by optimizing the type of HDESs,solid-liquid ratio,extraction time and extraction temperature.Further,the content of quercetagetin in HDES extract under the optimal extraction conditions was analyzed by using HPLC-DAD method.Then,by separately using quercetagetin,2-vinylpyridine(2-VP),acetonitrile,ethylene glycol dimethacrylate(EGDMA)and azodiisobutyronitrile(AIBN)as template,functional monomers,solvent,cross-linker and initiator,MIPs which showed selective adsorption ability to quercetagetin were synthesized through the precipitation polymerization method.By optimizing the factors affecting the selective adsorption ability of MIPs including the types of functional monomers,the ratio of functional monomers and template,the ratio of template and cross-linker,standing or stirring,MIPs with the best adsorption ability for quercetagetin were obtained.Further,RAMIPs which had restriced access ability and selective adsorption ability,were prepared by modifying the bovine serum albumin(BSA)onto the surface of the optimal MIPs.The physicochemical properties of MIPs and RAMIPs were characterized through scanning electron microscopy(SEM),fourier transform infrared spectroscopy(FTIR)and thermogravimetric analysis(TGA).The selective adsorption properties of MIPs and RAMIPs were investigated by static,dynamic and selective adsorption experiments.Furthermore,in order to explore the potential applications of MIPs and RAMIPs in complex biological samples,the protein exclusion abilities of MIPs and RAMIPs were explored.Subsequently,the optimal desorption conditions of quercetagetin for the desorption from MIPs were optimized and the reusability of MIPs was also investigated.Finally,MIPs and RAMIPs were separately used for the highefficiency enrichment of quercetagetin from HDES extraction and the plasma of mouse,coupling with HPLC/UPLC-MS/MS analysis.(2)Firstly,by using DES which was consisted of choline chloride(Ch Cl)and hexafluoroisopropyl alcohol(HFIP)(molar ratio 2:3),as reaction solvent,1,3,5-tris(4-aminophenyl)benzene and 2,5-dihydroxyterephthalaldehyde as building blocks,COF-DES-1 with high crystallinity was prepared through a freeze-thaw cycle-solvothermal method.Secondly,SEM,FT-IR,powder X-ray diffraction(PXRD),X-ray photoelectron spectroscopy(XPS),TGA and contact angle experiments were used to characterize the physicochemical properties of the asprepared COF-DES-1.Then,by using physical adhesion method,COFDES-1 was attached onto stainless steel fiber to prepare SPME fiber,and the morphology of fiber and coating thickness were characterized by SEM.Further,static,dynamic and selective adsorption experiments were used to investigate the selective adsorption properties of SPME fiber for luteolin.Moreover,macromolecular exclusion experiment was conducted to investigate the exclusion effects of SPME extraction on a variety of proteins.Subsequently,the optimal conditions for the desorption of luteolin from SPME fiber were optimized and the reusability of was also investigated.Finally,SPME extraction combined with UPLC-MS/MS analysis was used to analyze luteolin and its metabolites in the plasma of mouse.Results:(1)A series of HDESs(DES-1 to DES-10)were successfully prepared and used for the first extraction step of quercetagetin from Tagetes erecta Linn(T.erecta)flower.The optimum extraction conditions of quercetagetion were DES-9 as extraction solvent,solid-to-liquid ratio of 5:1,extraction temperature of 30 ℃,extraction time 50 of min.Under the optimal extraction conditions,the extraction yield of quercetagetion was calculated as 18.40 mg·g-1,which was significantly higher than those of the extraction yields by using conventional organic solvents such as methanol,ethanol,70% ethanol,etc.The optimal extraction conditions of MIPs were as follows: 2-VP was used as functional monomer,the molar ratio of template,functional monomer and cross-linker was 1:6:30,and the MIPs was synthesized under static condition.Moreover,by introducing BSA,RAMIPs was successfully prepared.SEM results showed that MIPs was highly crosslinked,while RAMIPs had relative good dispersion.The size of RAMIPs was larger than that of MIP,indicating that the introduction of BSA changed some properties of MIPs.FT-IR characterization results showed that the as-prepared MIPs and RAMIPs contained-OH,C-H,C=O,C-O-C and other functional groups,which proved the successful synthesis of MIPs and RAMIPs.The results of TGA showed that the weight loss from 30 ℃ to 80 ℃ was mainly due to the volatilization of solvent.When the temperature increased from 280 ℃ to 430 ℃,some structures of MIPs and RAMIPs were destroyed,and the weight loss increases rapidly.When the temperature exceeded 450 ℃,the structures of two kinds of polymers were completely destroyed.In general,the structures of MIPs and RAMIPs were stable and had good thermal stability before 280 ℃.The results of static adsorption experiments showed that MIPs and RAMIPs had good adsorption properties for quercetagetion,and the maximum adsorption capacities were calculated as 27.2 mg·g-1 and 23.1 mg·g-1,respectively.The dynamic adsorption experiment results showed that the adsorption equilibrium time of MIPs and RAMIPs for quercetagetion was 60 min.The results of selective adsorption experiments showed that MIPs and RAMIPs had good selective adsorption properties for quercetagetion.The MIPs and RAMIPs also had good exclusion effects on macromolecules.Due to the introduction of BSA,the exclusion effects of RAMIPs was better than that of MIPs.In addition,the optimal desorption solvent of MIPs was methanolacetic acid(8:2,v/v),the optimal desorption time was 15 min,and the optimal desorption times was three times.The reusability experiment results showed that MIPs maintained good adsorption capacity after 8 adsorption-desorption cycles,revealing that MIPs had good reuse performance.Finally,MIPs was used as d-SPE adsorbent for the second enrichment step of quercetagetion from DES extract.The enrichment efficiency of quercetagetion was 65.27%-78.77%.These results revealed that the MIPs could be effectively used to enrich quercetagetion in actual TCMs samples.The results of RAMIPs which used as d-SPE adsorbent for the enrichment of quercetagetion in mouse plasma showed that this method can be effectively used for the enrichment of quercetagetion in complex biological samples,and the enrichment effect was significant.Besides,the enrichment effect was much better than that of the traditional protein precipitation-liquid-liquid extraction method,revealing that RAMIPs has great application potential in complex biological samples.(2)By using HDES which based on Ch Cl-HFIP(molar ratio of 2:3)as reaction solvent,COF-DES-1 with high crystallinity was successfully prepared by freeze-thaw cycle method-solvothermal method at the reaction temperature of 80 ℃ for 2 days.SEM results showed that COF-DES-1 had a spherical morphology.FT-IR results showed that a peak of C=N bond formed by the Schiff base reaction between TPB and DHA appeared in the FT-IR spectrum of COF-DES-1 at 1600 cm-1.The XPS analysis of COFDES-1 showed that a peak at 399.2 e V was attributed to the C=N bond.Both the results of FT-IR and XPS proved the successful synthesis of COFDES-1.PXRD results showed that there was a significant peak at 2.83°,and two small peaks at 4.82° and 5.65°.These phenomena indicated that COF-DES-1 had good crystallinity.TGA results showed that COF-DES-1 had good thermal stability under 320 ℃.The results of contact Angle showed that the water contact Angle of COF-DES-1 was 89.38°,indicating that COF-DES-1 had a certain hydrophilicity.Moreover,SEM results of COF-DES-1 SPME fiber showed that COF-DES-1 could uniformly adhere to the surface of SPME fibes,and the adhesion layer thickness was about 128 μm.Further,the static adsorption results showed that SPME fibers had good adsorption capacity for luteolin,and the maximum adsorption capacity was 145.31 μg.The dynamic adsorption results showed that SPME fiber could reach adsorption equilibrium at 120 min.The selective adsorption experiments showed that SPME fibers had good specific adsorption capacity for luteolin.Besides,SPME fiber also showed the good exclusion effects on a variety of proteins,and the exclusion effciencies were high than 93%.In addition,the optimal desorption solvent of luteolin was acetone,the optimal desorption time was 90 min and the optimal desorption times was three times,respectively.After five adsorption-desorption cycles,the SPME fiber still had a good adsorption effect on luteolin,indicating that the COF-DES-1 based SPME fiber had good reusability.Finally,the results of the analysis of luteolin and its metabolites in mouse plasma showed that the COF-DES-1 based SPME extraction method was effective in the enrichment of luteolin and its metabolites.The enrichment efficiencies of the SPME method were higher than that of the traditional protein precipitation-liquid-liquid extraction method,revealing that COF-DES-1 based SPME extraction method had good application value in complex biological samples.Conclusions:(1)HDESs had excellent performance and can replace traditional organic solvents for the extraction of flavonoids from TCMs.MIPs and RAMIPs had good selective adsorption effect on quercetagetion.The method of primary extraction based on HDES and secondary enrichment based on MIPs can be effectively used for the efficient separation and enrichment of quercetagetion from complex TCMs.Moreover,RAMIPs can be effectively used to enrich quercetagetion from complex biological samples.(2)The COF-DES-1 coated SPME fiber had good selective adsorption ability and macromolecular exclusion ability for luteolin,which can be effectively used for the enrichment of luteolin and its metabolites in complex biological samples.COF-DES-1 based SPME fiber had great application potential in the enrichment of flavonoid aglycones or their metabolites in complex biological samples. |
