Based On Dextransucrase Glycosylation Of Phenethyl Acetoacetate And Its Glycoside Products Activity Study

Bio-glycosylation is an efficient strategy to improve the biological activity and physicochemical properties of natural compounds for therapeutic drug development.In this study,two caffeic acid phenethyl ester(CAPE)glucosides(G-CAPE and 2G-CAPE)were synthesized by transglycosylation with dextransucrase from Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor.The products were purified and the structures were characterized.The physicochemical properties,anti-inflammatory activity and cytotoxicity of the two CAPE glucosides were measured.The water solubility of G-CAPE and 2G-CAPE is 35 and 90 times higher,respectively,than that of CAPE.Compared to CAPE,the mono-glycoside product showed superior anti-inflammatory effects,and its inhibition rate of NO,IF-6,and TNF-a is 93.4%,76.81%,and 56.58%in RAW 264.7 macrophages,respectively,at 20 μM.Also the cytotoxicity of both products was significantly improved.These glycosylation-modified CAPEs circumvent some of the flaws in CAPE application in anti-inflammatory drugs.Investigate the anti-inflammatory mechanism of G-CAPE.By acting on TLR4 on the cell membrane,G-CAPE blocks the activation of NF-κB in the downstream signaling pathway of the receptor,which blocks the transmission of inflammatory signals and inhibits the expression of COX-2 and iNOS inflammatory mediators,thereby inhibits the secretion of various inflammatory factors to show nanti-inflammatory effect.Molecular simulations showed that the glycosyl moiety of G-CAPE forms four hydrogen bond with the residues of Arg90,Glu92,and Cys133 of MD2,which enhances the binding affinity of G-CAPE to MD2,thereby enhancing anti-inflammatory activity.The CAPE enzymatic glycosylation reaction is optimized.To obtain more G-CAPE,the optimal reaction temperature for the enzymatic reaction is 30 ℃,the most suitable pH is 5.0,and the optimal sucrose concentration is 750 mM.In this study,the glycosylation of CAPE by dextran invertase improved some defects in the application of anti-inflammatory drug CAPE,and the anti-inflammatory activity of the product G-CAPE was significantly improved.Further research on the anti-inflammatory mechanism of G-CAPE has important implications for the development of anti-inflammatory drugs.