Enzymatic Acylation Of Anthocyanin With Fatty Acid And The Evaluation Of The Properties And Cytotoxicity Of The Product

Cyanidin-3-O-glucoside(C3G)is a water-soluble anthocyanin widely distributed in the nature.It has a variety of bioactivities such as antioxidation,anti-cancer benefits,and the ability to prevent many chronic diseases.However,its instability and poor lipophilicity limit its application.In order to expand the utilization of agricultural waste and the application of anthocyanins in various industries,the crude extract of low-purity black bean peel was used as raw material in this study to prepare the lipophilic C3G-lauric acid(C3G-La).The stability and physiological activity,as well as the cytotoxicity and digestive characteristics of the products were evaluated.The main results are as follows:1.The preparation of C3G-LaCrude extract of black bean peels(C3G purity 35.8%)and lauric acid were vigorously stirred with Novozymes 435 in tert-butanol(containing 10%DMF in volume)to yield product with an acylation efficiency of 66.59%.The purity of C3G-La could reach 82.4%after a simple extraction by ethyl acetate.When the product was subsequently purified by silica gel column chromatography,the purity of C3G-La reached as high as 97.5%.The molecular weight of C3G-La was identified to be 631by HPLC-MS/MS.By NMR characterization,C3G-La was found to be formed by the esterification reaction between the hydroxyl group on the glucosyl moiety of C3G and the carboxyl group on lauric acid.The oil-water partition coefficient test showed that the log P value of C3G-La was 3.28,which was much higher than that of C3G(-1.06),indicating that the lipophilicity of C3G was significantly improved after the acylation reaction.2.The stability and physiological activity of C3G-LaThe storage stability experiment showed that C3G-La was more stable than C3G in acidic environment(p H?7),while C3G was more stable than C3G-La in alkaline environment(7<p H?11).Nevertheless,both of them were extremely unstable in strong alkali environment(p H?13).In the physiological activity experiment,H₂O₂ was used to establish the oxidative damaged model of LO2 cells,and the protective ability and mechanism of C3G-La against oxidative damage of LO2 cells were investigated.Pretreatment of C3G-La(300?M)resulted in an increase in cell viability from 51.03%to 81.70%(p<0.05)in H₂O₂-injured cells,and the dead cell area portion was smaller than that of C3G-treated group in live/dead staining assay.The cell viability of H₂O₂-injured cells pretreated with C3G(300?M)was 80.59%.Thus,the antioxidative protection effect of C3G-La on LO2 cells was as better as C3G(p>0.05).The intracellular ROS level of C3G-La pretreated cells decreased to 1.09,which was significantly lower than untreated cells(1.27),while the intracellular ROS level of C3G-La pretreated cells decreased to 1.13.Moreover,C3G-La pretreatment significantly reduced both the MDA level and the extracellular LDH level in H₂O₂-injured LO2 cells,and enhanced the activity of SOD.The measurement of Mitochondrial Membrane Potential(MMP)showed that the MMP value of H₂O₂-injured cells pretreated with C3G-La was significantly increased,which suggested that the C3G-La could inhibit cell apoptosis by maintaining the stability of mitochondrial metabolism.From cell apoptosis analysis,the pretreatment of C3G-La and C3G reduced the apoptosis rate of H₂O₂-injured cells.The expression levels of apoptosis related proteins were determined,and it was found that C3G-La could down-regulate the expression level of Bax and up-regulate the expression level of Bcl2 in H₂O₂-injured cells,thus reducing the apoptosis rate of H₂O₂-injured cells.Further analyses showed that C3G-La could reduce the oxidative stress level and cell apoptosis by regulating the expression of proteins related in AKT and Nrf2 pathways,i.e.P-AKT?NQO1?HO-1.3.The digestive tract cytotoxicity and in vitro digestive characteristics of C3G-LaC3G-La had significant impact on the growth of GES-1 and Caco-2 cells in a dose-dependent manner.Its impact on the growth of HEEC cells was much less than on GES-1 and Caco-2 cells.C3G-La showed obvious cytotoxicity at high administration levels.Flow cytometry was applied to investigate the effects of C3G-La and C3G on cell apoptosis,cell cycle and intracellular ROS level of GES-1 and Caco-2 cells.Treatment of C3G-La at high administrational level induced apoptosis of the cells significantly,while the variation of the treatment levels of C3G hardly influence the cell apoptosis.The distribution of cell cycle of GES-1 and Caco-2 was also influenced by the administration level of C3G-La.As the concentration of C3G-La increased gradually,the proportion of G0/G1 phase was increased and the proportion of S phase and G2/M phase was decreased.However,the cell cycle distribution of these two cells had no distinct change in response to the variation of treatment levels of C3G.Similarly,the intracellular ROS level was affected by the administration level of C3G-La but hardly by that of C3G.Although both C3G-La and C3G led to cytotoxicity at high concentrations,C3G-La showed a more toxic effect on cell proliferation.However,in this work,the monolayer model of Caco-2 was established and found that the cytotoxicity of C3G-La might be attenuated by the efflux mechanism.In addition,in the in vitro simulated digestion system,C3G-La showed high stability under gastric phase with a residual rate of 94.83%after gastric digestion,which was higher than that of C3G(72.86%).After intestinal digestion,a large amount of C3G-La was decomposed,and the residual rate was only 25.88%,while the residual rate of C3G was58.13%.This study developed a simple and efficient method for the preparation of lipophilic anthocyanin?fatty acid esters directly from low-purity anthocyanin crude extracts.The acylation efficiency could reach 67%,and the product displayed well sustained stability and antioxidant activity.The results provide important data and theoretical basis for the industrial preparation and application of acylated anthocyanins.