Synthesis Of Molecularly Imprinted Polymer And Application In The Study On Anti-inflammatory Effect Of Re Du Ning Injection

Objects:Molecularly imprinted polymer(MIP)is a new tailor-made adsorption material to separate template and analogs from complex matrix without affecting other components of the system.The binding sites and the three dimensional cavity of MIP have high affinity for tenplate by interacting with its complementary functional groups or structural elements.The polymer also has a good stability of physical and chemical properties.It can be grafting to the silica carrier by sol-gel process and retain the specific adsorption performance at the same time to realize semi-preparative scale,even preparative scale separation and preparation.Re Du Ning Injection(RDNI),a traditional Chinese medicine(TCM)injection which prepared by Artemisia annua L.,Lonicera japonica Thunb.,and Gardenia jasminoides E.,is widely used to relieve fever caused by viral infection diseases in clinical,such as upper respiratory tract infection and hand-foot-mouth disease.The main constituents and contents in RDNI are caffeoylquinic acid compounds(CACs),coumarins,iridoids and flavonoids.In this work,we adopted a sol-gel coating process based on surfece imprinting technology to synthesize the MIP.The synthesized core-shell MIP was used to separate caffeic acid(CA)and CACs from RDNI.The experiments of lipopolysaccharide(LPS)-induced prostaglandin E2(PGE2)release in RAW264.7 cells were performed to study the anti-inflammatory effects of separated samples,compound combinations,compounds and sample combinations.Finally,the combination index(Cl)was calculated to evaluate the synergistic and antagonistic effect of compound combinations,compound and sample combinations of RDNI at the systems level.Methods:We exploratory studied the preparation condition of sol-gel molecular imprinting polymer,where emodin,3-Aminopropyltriethoxysilane(APTS),tetraethoxysilane(TEOS),silica beads and tetrahydrofuran(THF)were as template,functional monomer,cross-linker supporting matrix and solvent,respectively.Two kinds of CA-MIPs were synthesized by using the above sol-gel method.The consumption of APTS,TEOS and the particle size of silica beads were optimized.MIP1,non-imprinted polymer 1(NEP1),MIP2 and NIP2 were used as stationary phase of liquid chromatography,respectively.MIP1 and MIP2 column were used to separate caffeic acid and caffeoylquinic acid compounds from RDNI without affecting other components,respectively,and NIP columns were used to examine the non-specific adsorption of separation materials and preparation methods.Finally,samples of RDNI for cell experiment were prepared.The synergistic and antagonistic anti-inflammatory effects of active ingredients in RDNI were studied by the experiments of LPS-induced PGE2 release in RAW264.7 cells at system-level.The combination of CA and SCO,IsoB and SCO both showed synergistic effect inside or outside the RDN system through interact with different target proteins in the pathway of LPS-induced PGE2 release process.Results and Discussion:The synthetic MIP showed the specific adsorption for emodin and was used as a stationary phase of high performance liquid chromatography(HPLC)column to evaluate the separating ability for emodin.The results indicated a good separation for emodin and:its anakigues.Finally,95%purity of emodin(380 mg)was separated from alcohol extract of Rheum palmatum L.in five days(sample 4 times every day,each separating time about 2 hours)by using MIP as stationary phase of glass column of liquid chromato graphy at semi-preparative scale and the column showed high affinity for its template.After several separation of sample,the column can be refreshing by washing with MeOH-CH3COOH(9:1,v/v).The elution of 10 mL RDNI through NIP1 and NIP2 column were freeze dried.Compared with freeze dried powder of 10 mL RDNI(744.1 mg),there were no significant change of NEP1 sample(730.7 mg)and NIP2 sample(732.8 mg).It indicated that the non-specific adsorptions of separation materials and preparation methods of MIP1 and MIP2 had no significant impact on the composition of RDNI.731.4 mg of sample S1(samples of RDNI which lack of CA)and 2.1 mg CA were prepared from 10 mL RDNI,and the purity of CA was 90.5%.552.0 mg sample S2(samples of RDNI which lack of CACs)and 181.3 mg sample S3(sample of CACs in RDNI)were prepared from 10 mL RDNI.The determination results of HPLC fingerprints also exhibited good separation of CA and CACs by MIPs.It indicated that MIP was an ideal separation material for preparative scale separation of compound and its’analog without affecting other compounds,and provides a new method for the study of effect-site of TCM.We also enriched and separated the trace CA from extract of Flos Lonicerae.Finally,146 μg CA was separated from 13 mL solution of extract of Flos Lonicerae and the purity was 92%.It indicated that rapid enrichment and separation of trace components in TCM can be realized by using the core-shell MIP.It provides an effective way to study the high-activity but low-content compounds in TCM.The strength of synergistic effect and sample concentration showed a certain dose-effect relationship.The combination of CA and IsoB,SCO and S2 both showed antagonistic effect inside or outside the RDNI system,but the antagonism was weakened in the RDNI system.Conclusions:We have studied the synergistic and antagonistic anti-inflammatory effects of active compounds in a traditional Chinese medicine(RDNI)at system-evel by using molecular imprinting technology for the first time.The core-shell MIPs were used as stationary phase of liquid chromatography to separate CA and CACs from RDNI without affecting other compounds.The in vitro experiment results showed that both synergism and antagonism among compounds and separated samples existed inside and outside the RDNI(TCM)system.Compounds with different skeletons can produce synergistic effect through binding with different target proteins in the pathway.Compounds with similar skeletons can produce antagonistic effect due to competitive binding with the same target protein in the pathway.It also indicated that TCM as a system could regulate the synergism and antagonism by interactions among compounds.And molecular imprinting technology eould be an effective approach to study the interaction mechanism of TCM.This work would provide a novel approach for studying the interaction mechanism of TCM at system-level and provide new insights for the further study of TCM to enhance effectiveness by improving synergism and reducing antagonism.