单分散的芯壳型分子印记硅胶对葛根中异黄酮苷高选择性富集

1、单分散的芯壳型分子印记硅胶对葛根中异黄酮苷高选择性富集 Monodisperse core-shell microstructured molecularimprinting silica for highly selective enrichment of510152025isoflavonoid glycosides from Radix PuerariaeJing Luo Qing Zhang Liying Zhang Jiawei Zhao Shuhu Du School of Pharmacy Nanjing Medical University Nanjing 210029 Abs

2、tract This paper reports the preparation of puerarin PR imprinted layer-coated silicamicroparticles toward selective recognition of PR and fast affinity-enrichment of the main isoflavonoidglycosides from the crude extract of Radix puerariae Before the preparation quantum mechanics QM method was appl

3、ied to identify three kinds of common functional monomers capable of interactionwith PR and then predict optimal functional monomer acrylamide AA and the relative molar ratio oftemplate to functional monomer PRAA 1 4 The obtained PR-imprinted silica microparticles wereevaluated by transmission elect

4、ron microscope TEM and rebinding experiments exhibiting goodmorphology and high binding affinity to PR Meanwhile the rebinding amount of the imprintedmicroparticles to PR was nearly 21-folds that of non-imprinted microparticles When the PR-imprintedmicrospheres were used as packing materials for sol

5、id-phase extraction the recovery yields of PRdaidzin DD and genistin GS were simultaneously up to 90 by one-step extraction from the crudeextract of Radix puerariae Additionally the PR-imprinted microparticles could be re-used for at least 5times without losing any extraction efficiency These result

6、s indicate that the PR-imprintedmicroparticles have highly selective adsorption capabilities to PR DD and GS from the crude extract ofRadix puerariae The method of molecularly imprinted polymers MIPs coupled with solid-phaseextraction SPE provides a good solution of the enrichment and separation of

7、active extracts fromcomplicated traditional Chinese medicine TCM with certain structuresKey words Molecular imprinting Quantum mechanics Solid-phase extraction Puerarin IsoflavonoidglycosidesFoundations the Specialized Research Fund for the Doctoral Program of Higher Education of China No201XXXXXXXX

8、XX1 the National Natural Science Foundation of China No21075066 Brief author introductionJing Luo Nov17th1985 famalecomee of Pharmaceutical chemistryCorrespondance author Shuhu Du Oct1962 MaleProfessorImprinting technique E-mailshuhuducom-1-0 IntroductionRadix Puerariae RP including Pueraria lobata

9、Wild Ohwi and Pueraria thomsonii benth30354045whose Chinese names are Yege and Fenge respectively1 has been used in clinical Chinesemedicine for the treatment of fever pain diabetes measles acute dysentery or diarrhea2 and alsoserves as food in oriental countries Modern studies have shown that RP is

10、 rich in activeisoflavonoids such as puerarin PR daidzein 8-C-glucoside daidzin DD daidzein7-O-glucoside genistin GS genistein 7-O-glucoside daidzein DE and genistein GE Fig1 3 and exhibit multiple biological and pharmacological activities for instance antioxidanteffect45 estrogenic activity67 anti-

11、cancer property810 neuro-protective action1112 andliver-protective effect13 Due to these activities considerable researches on separation andpurification of isoflavonoids from the herb have been widely explored through the traditionalseparation techniques Up to now a number of extraction or separati

12、on methods including lowtemperature soaking14 ultrasonic extraction15 reflux extraction16 pressurized liquidextraction17 microwave-assisted extraction18 macroporous adsorption resin chromatography19and the high speed counter-current chromatography20 have been developed However all thesemethods are u

13、sually very arduous and time-consuming and the used absorbents lack molecularselectivity based on surface physical adsorption Therefore the development of stableantibody-like materials with specific binding properties for target molecules will provide novelsorbents to effectively extract active cons

14、tituents from herbFig 1The chemical structures of main isoflavonoids from Radix puerariae glc -D-glucoseMolecular imprinting involves the preparation of polymers with specific molecular recognition50properties and is attracting widespread interest especially in solid-phase extraction SPE chromatogra

15、phic separation biomimetic sensor bioassay drug delivery2124 and so on Up to datethe PR-imprinted polymers are mainly prepared by bulk polymerization25 Although the methodpermitted the easy preparation of molecularly imprinted polymers MIPs the bulky MIPsexhibited some disadvantages including incomp

16、lete template removal slow mass transfer and-2-5560657075irregular materials shape The measures to solve above problems generally require that imprintedpolymers are prepared in the optimizing forms that control templates to be situated at the surfaceor in the proximity of materials surface providing

17、 the complete removal of templates goodaccessibility to the target molecules and low mass-transfer resistance Many surface-imprintingstrategies have recently been explored ie imprinted layer-coated at silica nanoparticles26magnetic particles27 polystyrene beads28 and quantum dots29 As an alternative

18、 approachpolymerization occurred at the nanoparticles surface has emerged for producing high-qualityimprinted nanoparticles But the application of the imprinted nanoparticles in SPE orhigh-performance liquid chromatography HPLC is limited due to high column pressureAdditionally these technologies we

19、re primarily used for analyzing or enriching a targetconstituent and not for analyzing or enriching multiple target constituents from complicatedtraditional Chinese medicine TCM or complicated matricesThe main objective of the present work was to prepare imprinted polymer at the surface ofmicrometer

20、-scale silica particles and extend significantly the scope of application ofsurface-imprinted polymers To realize this goal large-sized and monodispersed spherical silicaparticles were first synthesized by sol precipitation Then the silica microparticles were modifiedthrough using 3- methacryloxy pr

21、opyltrimethoxysilane MPTS followed by the graftcopolymerization of acrylamide AA and ethylene glycol dimethacrylate EGDMA in thepresence of PR template Finally uniform PR-imprinted polymer layer was successfully coated atthe surface of silica microparticles After the templates were removed from the

22、polymer networkby solvent extraction the recognition sites of PR were created in the polymer coating layer Theobtained PR-imprinted microparticles displayed higher recognition selectivity and binding affinityto PR and its analogues compared to non-imprinted microparticles When the imprinted material

23、swere used as the sorbents of SPE for the simultaneous enrichment and detection of isoflavonoidglycosides from RP a better satisfactory result was obtained80111ExperimentalChemicals and materialsPuerarin PR daidzin DD genistin GS daidzein DE and genistein GE were supplied byNational Institute for th

24、e Control of Pharmaceutical and Biological Products Beijing China Acrylamide AA methacrylic acid MAA and azo bis -isobutyronitrile AIBN purified85through recrystallization in ethanol before use were purchased from Sinopharm ChemicalReagentCoLtd ShanghaiChina Tetraethoxysilane TEOS 3- methacryloxy pr

25、opyltrimethoxysilane MPTS 4-vinylpyridine 4-VP divinylbenzene DVB and ethyleneglycol dimethacrylate EGDMA were purchased from Sigma-Aldrich Steinheim Germany Theacetonitrile of HPLC grade was from Merck Darmstadt Germany All other reagents were of90analytical grade Deionized water was prepared using

26、 a Milli-Q water purification system Bedford USA -3-AB-8 macroporous adsorption resin polystyrene resin 03125 mm particle size waspurchased from Nankai University Chemical Plant Tianjin China Fengge FG and Yege YG were obtained from Jiangsu Pharmaceutical Co Ltd Nanjing China 08 g of YG powder was95

27、100accurately weighed into a conical flask and is extracted by heating under reflux with 30 mL of65 ethanol for 30 min on a water bath The extracted solution were filtered evaporated todryness under vacuum And then the residue was re-dissolved in 10 mL of methanol-water 1 9vv before loaded onto the

28、SPE cartridge FG extract was simultaneously prepared by theprocedures as mentioned above except that the total volume 10 mL of methanol-water 1 9 vv was replaced by 20 mL of methanol-water 1 9 vv in the re-dissolved process of residue12InstrumentationAn IKA Vortex Genius 3 homogenizer Staufer German

29、y was applied during the bindingexperiment The morphology and size of PR-imprinted silica microparticles were examined at 80kV by a JEM-1010 transmission electron microscope TEM from JEOL Tokyo Japan Thermal105110115analysis of PR-imprinted microparticles was performed using a thermogravimetric anal

30、yzer TGAModel TGA-2050 TA Instruments to determine the weight loss wt Measurements generallyon samples of 1015 mg contained in a platinum crucible were carried out in an airflow rate of 90mL min-1 by a controlled 10 C min-1 heating rate from 25 to 750 C The PR amount wasanalyzed by means of Shimadzu

31、 HPLC system Tokyo Japan equipped with LC-10ATvp pumpSPD-10Avp detector CTO-10ASvp column oven and Shimadzu shim-pack C18 column 150 mm 46 mm id 5 m The mobile phase was constituted of 01 formic acid A and acetonitrile B at a flow rate of 10 mL min1 with a linear gradient as follows 0 min 00 B 15 mi

32、n 315B 18 min 365 B 23 min 360 B 25 min 500 B 28 min 100 B returning to the initialconditions within 3 min and maintaining these conditions for 5 min A 20 L injection volumewas used for all analyses And then the analytes were monitored with ultraviolet detector at awavelength of 250 nm All solutions

33、 used for HPLC were filtered through a 045 m filter beforeuse13Preparation of silica microparticlesTEOS was applied to prepare monodispersed spherical silica particles by the hydrolysis in120125aqueous ammonia according to the reported Stber method30 Typically 90 mL of ammonia wasfirstly dissolved i

34、n 50 mL of ethanol Then the mixture of 50 mL of TEOS in 30 mL of ethanolwas added via a syringe pump with a constant flow rate about 100 L min-1 into above solutionunder vigorous magnetic stirring 750 rpm at room temperature The whole mixture was allowedto react for 24 h after adding The resultant s

35、ilica microparticles were separated from the mixedsolution by centrifugation and then washed with ethanol to remove residual ammonia14 Chemical modification of silica microparticlesPrior to the coating procedure the silica microparticles were chemically modified using MPTSto obtain polymerizable dou

36、ble bonds Typically 01 g of silica microparticles and 08 mL of-4-MPTS were added into anhydrous toluene to make 20 mL of mixture solution and stirred under130135dry nitrogen at 50 C for 24 h The resulting MPTS-modified silica MPTS-silica microparticleswere separated by centrifugation rinsed with met

37、hanol and dried under vacuum at roomtemperature15 Molecular modeling studiesQuantum mechanics QM method was employed to select the optimal functional monomer andmolar ratio of template to functional monomer The full geometry optimization of templatemonomer and template-monomer complex with various r

38、atios was carried out with densityfunctional theory DFT at B3LYP6-31G d p level Then the corresponding energeticcalculations were performed at the B3LYP6-31G d p level The binding energy Ebind of PRwith the monomer in vacuum was calculated according to the following formula140Ebind Ecomplex Etemplat

39、e Emonomer 1 where Etemplate and Emonomer were the energies of the template and functional monomerrespectively and Ecomplex was the total energy of the template-monomer complex All thecalculations were performed with the Gaussian 09 program3116Preparation of PR-imprinted microparticles145150155Befor

40、e polymerization the prearranged solution was prepared by 2082 mg of PR 05 mmol and 1422 mg of AA 20 mmol dissolved in 25 mL of methanol-acetonitrile 1 4 vv andstored in dark for 12 h 100 mg of MPTS-silica microparticles were dispersed in 25 mL ofmethanol-acetonitrile 1 4 vv by ultrasonic vibration

41、The prearranged solution 075 mL ofEGDMA 40 mmol and 164 mg of AIBN 01 mmol were then dissolved into the abovesolution The mixing solution was purged with nitrogen for 10 min while cooled in ice bath Thethree-step-temperature polymerization reaction was carried out in an incubating shaker with a rate

42、of 300 rpm First the prepolymerization was carried out at 50 C for 6 h and the finalpolymerization was completed at 60 C for 24 h Subsequently the products were further aged at85 C for 6 h separated by centrifugation and rinsed with methanol The obtained microparticleswere ultrasonically cleaned by

43、methanol-acetic acid 9 1 vv to remove template then washedwith methanol for several times and dried in vacuum oven at 60 C for 48 hThe non-imprinted microparticles SiO2NIP were also prepared under the same chemicalconditions but without the addition of PR template17Binding properties of PR-imprinted

44、 microparticles160In order to evaluate the recognition property of the PR-imprinted microparticles to targetmolecules three kinds of adsorption experiments were performedSteady-state binding capacity of SiO2PR-MIP microparticles to PR was measured bysuspending 20 mg of SiO2PR-MIPSiO2NIP microparticl

45、es in 50 mL of acetonitrile withvarious PR concentrations 0130 mmol L1 respectively After incubation under a-5-165170reciprocating shaking-table at room temperature the microparticles in the solution were filteredthrough 045 m nylon membranes when the PR concentration reached adsorption equilibriumA

46、nd then the filtrate was dealt with a nitrogen drying step and the residue was re-dissolved in 50mL of acetonitrile 20 L of the obtained solutions were injected to HPLC for analysis Thebinding amount of PR was determined by measuring the difference between the total amount andthe residual amount in

47、solutionThe binding kinetics was tested by monitoring the temporal evolution of PR concentration inthe solution 20 mg of SiO2PR-MIPSiO2NIP microparticles was added to 50 mL ofPR1room temperature respectively and then filtered by 045 m nylon membranes The binding175amount of PR was determined by the

48、method as mentioned aboveThe selectivity of SiO2PR-MIPSiO2NIP microparticles was investigated using DD GSDE and GE as the structural analogues of PR template18Extraction performance of MISPETwo hundred milligrams of SiO2PR-MIP microparticles SiO2NIP microparticles and the180185commercial resins whic

49、h were crushed and sieved to produce micrometer-sized particles for theirapplications were packed into a 20-mL polypropylene SPE cartridge respectively The eachcartridge was attached with a reservoir and a stop cock at the bottom end The microparticles wererinsed with methanol and then with water by

50、 water-driven vacuum pump respectivelySubsequently the extracted solution of FG or YG 05 mL was loaded into the MISPE cartridgeat a flow rate of 05 mL min-1 respectively After the loading the sample solution was soaked inthe cartridge for 4 h And then the cartridge was washed with 10 mL of methanol-

51、water 1 10vv followed by the elution with 10 mL of methanol-acetic acid 9 1 vv The solution of eachstep was evaporated to dryness and the residue was reconstituted in 50 mL of methanol-water 1 9 vv and 20 L of the reconstituted sample was injected for HPLC analysis respectively190221Results and disc

52、ussionPreparation of silica microparticlesSilica is an important support material because it has heat resistance corrosion resistance anddo not swell in organic solvents Uniform silica particles with the different sizes were usuallyprepared by the hydrolysis of TEOS with aqueous ammonia Generally th

53、e smaller the diameter195200of silica particles packed the SPE column is the higher the column pressure is In order to achievethe rapid separation the large-sized monodispersed spherical silica microparticles were needed inthis work To obtain the silica microparticles there were two methods includin

54、g single stepprocess32 and seed particles growth process33 However the formation of secondary particlescauses a multimodal distribution of particle size during seed particles growth process So thesingle step process is finally carried out in which one reactant TEOS dissolved in ethanol isadded into

55、the other one ammonia dissolved in ethanol at a flow rate It was found that the-6-particle size was a decreasing function of the addition rate of TEOS When the flow rate was keptat 100 L min1 the monodispersed and uniform silica microparticles with a size of 1 m wereobtained Fig 2 205Fig 2Transmissi

56、on electron micrographs of silica microparticles22Chemical modification and characterization of silica microparticlesIn order to coat silica microparticles surface with functional polymers silica microparticleswere first modified with MPTS according to the method in our previous report34 Coating the210surface of silica microparticles with functional polymers is commonly done by the chemicalimmobilization of azo-initiatorschain-transfer agents at the surface of silica microparticlesfollowed by initiating a p