Enzymatic Acylation Of Cyanidin-3-O-Glucoside And Its Activity Research

Anthocyanins are the main coloring substances in plants with the characteristics of multiple variety and wide distribution.They have a variety of physiological activities,and are recognized as the most abundant natural pigments to replace aniline synthetic pigments.However,the structural stability of anthocyanins is poor,and susceptible to external environmentits in the processing process.In terms of physiological activity,due to the low lipophilicity,it is not easy for anthocyanins to reach the target point through the cell membrane of the phospholipid bilayer,which greatly reduces the physiological value.Through acylation modification,the structure of anthocyanins can be optimized,and their stability and lipid solubility can be improved.It has important practical significance for expanding the application of anthocyanins in the food processing,nutrition,and health care industries.In this study,lipase-catalyzed acylation modification of cyanidin-3-glucoside(C3G)with fatty acid was performed,and the Uv-visible absorption spectrum,lipophilicity,stability,antioxidant and anti-tumor capabilities of C3 G before and after acylation were compared.The main conclusions of this paper are as follows:(1)C3G was acylated with fatty acids,fatty acid methyl esters,and vinyl acetate,and the transesterification was performed with different lipases and in different reaction media to study the best route of acylation.The conversion yield of C3G's direct esterification reaction is from 35.9% to 50.3%,which is higher than that of the transesterification reaction with fatty acid methyl esters(3.4%-12.7%).while the preservation rate of the two was exactly opposite.The preservation rate of the direct esterification is only about 6%,and transesterification reached more than 85%.The acylation product of vinyl acetate with C3 G was not detected.Considering the conversion yield and preservation rate,fatty acid methyl esters were selected as the most suitable acyl donor.A better conversion yield and preservation rate were obtained in t-butanol and t-amyl alcohol,but the operation in t-amyl alcohol was more convenient.Used Lipozyme 435 as biocatalyst obtained the highest conversion yield.(2)The C3 G acylated derivatives were separated and purified with semi-preparativeliquid chromatography,and obtained acylated products with purity close to 100%.The structure of the acylated product was further identified by LC-MS/MS and NMR technology.The products produced by transesterification of C3 G with methyl n-octanoate,methyl laurate and methyl myristate was determined as cyanidin-3-(6?-n-octanoyl)-glucoside,cyanidin-3-(6?-lauroyl)-glucoside,cyanidin-3-(6?-myristoyl)-glucoside respectively.(3)Hydrophobic solvent(pyridine,petroleum ether,n-hexane,isooctane)was respectively added to tert-amyl alcohol(v:v,2:8)to study the acylation reaction of C3 G and methyl myristate in the mixed solvent.The conversion yield of the transesterification reaction was significantly improved in the mixed solvent system compare to single solvent.Changing the proportion of hydrophobic solvent,20% pyridine-tert-amyl alcohol solvent as the best volume ratio,and increased the acylation conversion yield from 10.21 ± 0.18% to 15.72 ±0.32%.On this basis,temperature,substrate molar ratio,enzyme addition,reaction time and other conditions of acylation were optimized.The optimal conditions shown by the orthogonal results are: temperature 60 ?,reaction time 22 h,substrate molar ratio is 1: 250.Under the optimal conditions,33.39% conversion yield was obtained with methyl myristate as acyl donor,and the conversion yields with methyl n-octanoate and methyl laurate as acyl donors reached 17.03% and 28.32%,respectively.(4)The UV-visible absorption spectrum,lipophilicity,stability and antioxidant activities of C3 G and the three C3 G acylated derivatives.was researched.The maximum absorption wavelength of the acylated derivatives was red-shifted compared with C3 G.For every two additional carbon atoms in the fatty acyl chain length,the maximum absorption wavelength of the acylated product increased by 7 nm.The acylation of C3 G with medium-chain fatty acids increased the maximum absorbance of C3 G.The log P value of C3 G is-1.36,showing extremely strong hydrophilicity,while the Log P value of acylated C3 G all increased significantly,ranging from 0.8 to 1.1.Cyanidin-3-(6?-n-octanoyl)-glucoside and cyanidin-3-(6?-lauroyl)-glucoside showed stronger thermal stability and ultraviolet light stability than C3 G,but under incandescent light,only cyanidin-3-(6?-n-octanoyl)-glucoside with higher stability than C3 G.The thermal and light stability of the derivatives wasweakened with the increase of the fatty acyl carbon chain length.Compared with C3 G,the in vitro antioxidant activity of the acylated derivatives have a certain degree of reduction,but they still remained good in vitro antioxidant properties.In addition,the intracellular antioxidant activity of cyanidin-3-(6?-n-octanoyl)-glucoside and cyanidin-3-(6?-lauroyl)-glucoside is 1.14 and 1.96 times higher than that of C3 G,respectively.(5)Comparing the anti-proliferative and toxic effects of C3 G and its acylated derivatives on Hep G2 cells and A549 cells.It was found that the toxicity and anti-proliferative effects of C3 G acylated derivatives on Hep G2 cells and A549 cells were both stronger than C3 G and proportional to their lipophilicity.To study the effect of cyanidin-3-(6?-myristoyl)-glucoside on the cell cycle and apoptosis of Hep G2 cells.The results showed that cyanidin-3-(6?-myristoyl)-glucoside blocked the cell cycle of Hep G2 in G1 phase.Low concentration of cyanidin-3-(6?-myristoyl)-glucoside(? 80 mg/m L)promoted early apoptosis of Hep G2 cells,while high concentration of cyanidin-3-(6?-myristoyl)-glucoside(? 80 mg/m L)mainly promoted late apoptosis.RT-q PCR was used to detect the effect of cyanidin-3-(6?-myristoyl)-glucoside on the expression of anti-proliferation-related genes in Hep G2 cells.The results showed that cyanidin-3-(6?-myristoyl)-glucoside inhibited the proliferation of Hep G2 cells through three ways.One is to down-regulate the expression of pi3 k and m-TOR to regulate the pi3k/Akt/m TOR signal transduction pathway,thereby inhibiting the proliferation of Hep G2 cells.The second is to up-regulate Bax and down-regulate the expression level of Bcl-2 to induce the apoptosis.The third is to block the cell cycle of Hep G2 cells in G1 phase through down-regulate the expression of CDK4 in Hep G2 cells,and inhibit their further growth and proliferation.