| Ferulic acid,as a phenolic acid compound,has a broad physiological activity,such as antioxidation,bacteriostatic,antithrombotic,anti-tumor etc.and has been applied to many fields.However,due to its poor lipophilic,the application of ferulic acid is particularly limited in fatty food system.Therefore,the combination of ferulic acid and liposoluble molecules are expected to not only improve its physiological activity,but also enhance its lipophilicity,in order to expand its application.In this work,a series of ferulic acid alkyl esters derivatives(FAEs)were prepared by attaching ferulic acid with fatty alcohols of different carbon chain lengths via both chemical and enzymatic esterification methods,and then were purified by silica gel column chromatography.The structures of FAEs were characterized by NMR,IR,and MS.The relationship between physiological activity(antioxidation and antibacterial activity)and molecular structure of FAEs was studied systematically.The work accomplished is summarized as follows:(1)FAEs were prepared by chemical and enzymatic synthesis,including ethyl ferulate(FAC2),butyl ferulate(FAC4),hexyl ferulate(FAC6),octyl ferulate(FAC8),decyl ferulate(FAC 10),dodecyl ferulic acid(FAC12),tetradecyl ferulate(FAC14),hexadecyl ferulate(FAC16),and octadecyl ferulate(FAC 18).(2)The reaction process was monitored by thin layer chromatography(TLC).The raw products of FAEs were isolated and purified by silica gel column chromatography.The mobile phase used was PE/EA in the ratio of 6:1 and 4:1.(3)The structure of the pure FAEs was identified by 1H-NMR?13C-NMR and MS.(4)Studies on the relationship between antioxidant activity and structure-of FAEs.The testing systems include in vitro(DPPH-radical scavenging,FRAP iron ion reduction,ORAC oxygen free radical absorption),peanut oil emulsion system and liposome.In the DPPH-scavenging experiment,the abilities of FA and FAEs to scavenge DPPH-were both significantly increased with the concentration ranging from 50,100,200,400 ?M.The ability of FA to scavenge DPPH· was shown to be stronger than that of FAEs and BHT.Nevertheless,the ability of FAEs to scavenge DPPH-chains is similar.In terms of the FRAP experiment,with FAEs concentration increasing from 20,100,200 to 400 ?M,the antioxidation ability of FAEs was increased.Meanwhile,the antioxidatant ability of FAEs decreased as the carbon chain length increased.In the ORAC experiment,the higher the concentration of FAEs(50,100,200,400 ?M),the longer the quenching time of fluorescein sodium.However,at the same concentration,the antioxidant activity of FAEs did not show a linear trend.With the increase of alkyl chain length,the ORAC value of medium-length carbon chain FAC6 was slightly higher.In the emulsion oxidation test,antioxidant activities of FAC 12 and FAC 14 for both 10M and 25?M FAEs were better than the other FAEs within 6 days and 12 days.After storage for 12 and 15 days,100 ?M FAEs,the best antioxidant effect is produced by FAC 12,FAC 14,FAC 16.In the liposome oxidation experiment,it was clearly found that with the increase of carbon chain length of FAEs,the antioxidant effect showed the phenomenon of CUT-OFF effect.At the concentration of 25 ?M,the antioxidant effects of FAC6,FAC8,FAC10,FAC12 and FAC14 were the best;At the concentration of 50 ?M,the antioxidant effect of FAC6 and FAC8 were the best;At the concentration of 100 ?M,the antioxidant effect of FAC6,FAC8 and BHT were the best,and the antioxidation effect of BHT was better than that of FAC6,FAC8.According to the trend of antioxidant activity of FAEs,the antioxidation activities of FAEs with different carbon chain length were shown to be obviously different in different systems accordingly.In vitro antioxidant test,DPPH·and FRAP were of homogeneous non-competitive mode,in which the main determining factor is the solubility of FAEs in homogeneous phase.On the other hand,in the ORAC test of homogeneous competition mode,the main factors to determine the antioxidant activity includes the solubility of FAEs in homogeneous phase as well as their ability to react with free radical.Therefore,for emulsion systems,the main reason for the phenomenon of CUT-OFF effect is that the oxidation of oil occurs at the water-oil interface.If FAEs have a carbon chain of suitable length,they are able to distribute themselves on the water-oil interface,thus can effectively prevent or delay the oxidation of oil droplets.For liposome system,the phenomenon of CUT-OFF effect is mainly due to the peroxy free radical produced by AAPH in water phase which attacks lecithin on the surface of liposome membrane.This means that it is required for the FAEs to have a carbon chain of appropriate length to be located near the surface of the membrane so as for the antioxidant effect to be optimal.(4)The relationship between antibacterial activity and structure of FAEs was studied.Two common food-borne pathogenic bacteria,Listeria monocytogenes,Escherichia coli were tested for minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC).The best antibacterial effect for L.monocytogenes,is FAC4 and FAC6,with MIC and MBC both being 0.1 mM and 0.4 mM,respectively.The best antibacterial effect for E.coli.,is FAC6,with MIC 1.6 mM and MBC 25.6 mM.E.coli was more sensitive than L.monocytogenes to FA,but after simple esterification(FAC2),the bacterial sensitivity was reversed.The inhibitory effect of FAC2 against L.monocytogenes was more significant.With the increase of FAEs carbon chain length,the lipophilic degree of FAEs increased,and the MIC decreased initially and then increased.Moreover,scanning electron microscope(SEM)was conducted to further investigate the antibacterial activity of FAEs.1ŚMIC FAC6 treatment for 4 h destroyed severely the outer structure of L.monocytogenes cells,with the partial disappearance of cell walls and the formation of holes on the surface.In contrast,ubnder the same conditions,FA only induces surface deformation and contraction of L.monocytogenes cells. |
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