Study On Antioxidant Andα-Amylase/Glucosidase Inhibition Properties Of Ca Acylated Derivatives Synthetized By Enzyme

Chlorogenic acid（CA）,a member of polyphenols,has a variety of biological activities,such as antioxidant,anti-inflammation,regulation of glucose metabolism and so on.Due to the poor stability and lipophilicity of CA,its utilization is limited in organism.In this study,CA was taken as the research object.CA acylated derivatives with different lipophilicities were prepared by enzymatic modification to the molecular structure of CA.Then,the structure of CA derivatives was identified to confirm the acylation sites of CA derivatives.Meanwhile,the in vitro digestive stabilities,in vitro antioxidant activities,and the inhibitory activities ofα-amylase andα-glucosidase of CA derivatives were determined to provide a theoretical basis for the application of CA derivatives as novel lipophilic antioxidants andα-amylase andα-glucosidase inhibitors.The main conclusions are as follows:The acylation of CA and vinyl butyrate was catalyzed by lipase.Eleven kinds of solvent systems and nine kinds of lipases were investigated using the conversion of CA as an indicator.The optimal solvent system and lipase were methyl tert butyl ether（MTBE）and Lipozyme RM,respectively.Three different components of butyryl-CA were obtained using a preparative high performance liquid chromatography.The structures of the products were identified as 3-O-butyryl-CA,4-O-butyryl-CA and di-butyryl-CA using liquid chromatography-mass spectrometry（LC-MS）,fourier transform infrared spectroscopy（FTIR）,and nuclear magnetic resonance spectroscopy（NMR）,and the main acylation product was4-O-butyryl-CA.Then,the reaction conditions were discussed,and the optimal reaction conditions were listed as follows:reaction temperature,55°C;the lipase dosage,0.28 U/（mg CA）;molar ratio of CA and vinyl butyrate,1:10;reaction speed,400 r/min;reaction time,4 d.It was found that the acyl donor had the greatest influence on the regioselectivity of CA acylation.When the acyl donor was vinyl laurate,the component was only 4-O-lauroyl-CA.The lipophilicity of 4-O-monosubstituted CA derivatives with different chain lengths was investigated.The effects of the lipophilicity of CA derivatives on in vitro simulated digestive stabilities,DPPH·and ABTS⁺·scavenging abilities,ferric ion reducing abilities and cellular antioxidant activities（CAA）were studied.The results showed that compared with CA,the stabilities of CA derivatives decreased slightly in the simulated gastric digestion stage.The stabilities of CA derivatives increased in some degree in the intestinal digestion stage,and the stability of C4-CA was better than that of CA and other derivatives.The radical scavenging capacities results shown that the DPPH·scavenging capacities of CA derivatives were significantly lower than CA except C4-CA,while only the ABTS⁺·scavenging capacities of C2-CA was significantly decreased.In assays of ferric ion reducing capacities,the antioxidant capacities of CA derivatives decreased significantly.In CAA tests,with the increasing of lipophilicity,the abilities of CA derivatives to cross cell membrane enhanced and the antioxidant capacities of CA derivatives increased.The celluar antioxidant capacities of C6-CA,C8-CA,and C12-CA were higher than that of CA.The inhibition ofα-amylase andα-glucosidase by 4-O-monosubstituted CA derivatives with different chain lengths were studied.The inhibition mechanism was discussed by inhibition kinetics and fluorescence quenching.The results showed that C12-CA possessed the strongest inhibitory activity onα-amylase andα-glucosidase.The inhibitory activities of CA derivatives onα-amylase were higher than CA and increased with the extension of chain length.However,the inhibitory activity of CA onα-glucosidase was only lower than that of C8-CA and C12-CA.The results of inhibition kinetics and fluorescence quenching showed that the inhibitory type and quenching mechanism forα-amylase andα-glucosidase of CA and its derivatives had no difference.But the competitive inhibition constant(K_ic),uncompetitive inhibition constant(K_iu),and fluorescence quenching constant(K_FQ)of CA and its derivatives changed.The K_iu and K_ic values of C12-CA were the smallest,and the K_FQ value of it was the highest,which indicated that C12-CA had the greatest effect on the conformation ofα-amylase andα-glucosidase.