Preparation Of Molecularly Imprinted Polymers For Biochanin A And Its Application In Dalbergia Odorifera Leaves

Dalbergia odorifera（alias Huanghuali）is the source plant of Dalbergia odorifera.At the same time,its heartwood has been regarded as a superior material for making high-end furniture,crafts and musical instruments since ancient times.Driven by excessive profits,Dalbergia odorifera has been cut down indiscriminately,and its timbering cycle is relatively long,resulting in the plant being listed as a national second-class key protected wild plant and an endangered tree species.In view of this,the comprehensive development of forestry waste-Dalbergia odorifera leaves has very important practical significance for promoting the protection of Dalbergia odorifera resources.Modern research shows that biochanin A（BCA）is one of the main components in the leaves of Dalbergia odorifera.It has many biological activities such as anti-cancer,anti-inflammatory,anti-oxidation,neuroprotective,anti-diabetes and liver protection.At present,the application of macroporous adsorption resin,silica gel chromatography and ultrasonic extraction technology in the enrichment process of BCA often has the problems of poor selectivity,low efficiency and high comprehensive cost.Molecular imprinting technology has the advantages of strong selectivity to target components,easy preparation of polymers,simple operation of separation experiments,less organic solvent consumption,and recyclability of polymers.In view of this,based on the previous research experience in the preparation of molecularly imprinted polymers for flavonoids,this paper carried out the study of biochanin A molecularly imprinted polymers（BCA-MIPs）and applied it to the leaves of Dalbergia odorifera,in order to promote the efficient utilization of Dalbergia odorifera leaves resources.Objective:Preparation of biochanin A molecularly imprinted polymer and construction of“macroporous adsorption resin+molecularly imprinted SPE”technology to improve the purification efficiency of biochanin A in Dalbergia odorifera leaves.Methods:Using the precipitation polymerization method,Firstly,the structure of the complex and polymer formed by BCA and 1-vinylimidazole was studied by UV and FT-IR,respectively.Secondly,the imprinting factor（IF）and imprinting adsorption capacity（W）were used as the investigation indexes.The single factor experiment combined with the multi-index comprehensive scoring method was used to investigate the ratio of template molecule to crosslinking agent,the selection of porogen,the amount of porogen,the polymerization temperature,the time of passing nitrogen and the amount of initiator on the adsorption capacity of BCA-MIPs.Secondly,the morphology and thermal stability of BCA-MIPs were characterized by scanning electron microscopy and thermogravimetric analysis.Finally,the adsorption performance evaluation of isothermal adsorption,adsorption kinetics and recognition was carried out.Based on the preparation of BCA-MIPs as SPE filler,the effects of sample column volume,washing solution,solvent and amount of eluent and reuse times on the capture of BCA in Dalbergia odorifera leaves were investigated.Through the pilot scale-up test,the difference between the small test and the pilot scale-up of 50times was compared.On this basis,the“macroporous adsorption resin+molecularly imprinted SPE”hyphenated technology was constructed.Referring to the previous relevant literature,the BCA content was used as the evaluation index to compare the effects of macroporous adsorption resin,molecularly imprinted SPE,“molecularly imprinted SPE+macroporous adsorption resin”hyphenated and“macroporous adsorption resin+molecularly imprinted SPE”hyphenated on the BCA content,impurity removal effect and chemical HPLC chemical fingerprint of Dalbergia odorifera leaves.Results:The optimal ratio of template molecule BCA to functional monomer1-vinylimidazole was determined to be 1:2 by UV analysis.By multi-index comprehensive scoring method combined with single factor experiment preparation process optimization,It was determined that when the functional monomer:crosslinking agent was 1:5,acetonitrile was the porogen,the volume of the porogen was 30 m L,the polymerization temperature was 60°C,nitrogen was introduced for 10 min,and 30 mg initiator was added as the best preparation condition.At the same time,BCA interacts with 1-vinylimidazole by hydrogen bonding.The static adsorption capacity Q is 6.52 mg·g^-1,IF is 1.64,W is 1.94 mg·g^-1,and it has good thermal stability within 340°C.The isothermal adsorption process of BCA-MIPs conforms to the Langmuir adsorption model.Scatchard equation analysis showed that there was one binding site in BCA-MIPs,and the maximum binding amount of affinity site was 7.72 mg·g^-1.The adsorption kinetics study showed that the adsorption process of BCA-MIPs conformed to the pseudo-second-order kinetic equation,and the adsorption of BCA by BCA-MIPs was mainly chemical adsorption.In addition,BCA-MIPs have good recognition ability for BCA.The BCA-MIPs were used as SPE fillers to determine the optimum conditions of molecularly imprinted SPE:the sample concentration of Dalbergia odorifera leaves extract was 990.07μg·m L^-1,the sample column volume was 2.57 m L,followed by4.19 m L of methanol washing,34.90 m L of methanol and acetic acid（9:1,v/v）elution,the best capture effect of BCA can be obtained.After elution and regeneration,BCA-MIPs can be reused for 9 times.In addition,there was no significant difference in the BCA-MIPs prepared by the pilot-scale amplification of 50 times compared with the small-scale sample directional separation of BCA,which could increase the purity of BCA in Dalbergia odorifera leaves to 9.00%.Finally,it was determined that the purity of BCA in the original extract of Dalbergia odorifera leaves could be increased from 1.51%to 13.48%by the combination of“D101 macroporous adsorption resin+BCA-MIPs SPE”.Conclusion:Compared with the previous literature,this study innovated the combination technology of“macroporous adsorption resin+molecularly imprinted SPE”,which has the advantages of simple preparation process,low production cost and good purification effect.It is helpful to improve the separation and purification efficiency of BCA in Dalbergia odorifera leaves,which is expected to realize industrial production and promote the efficient utilization of Dalbergia odorifera leaves resources.