Development and Optimization of Lipase-Catalyzed Synthesis of Phospholipids Containing 3,4-Dimethoxycinnamic Acid by Response Surface Methodology

Abstract

The interesterification reaction of egg-yolk phosphatidylcholine (PC) with ethyl ester of 3,4-dimethoxycinnamic acid (E3,4DMCA) catalyzed by Novozym 435 in hexane as a reaction medium was shown to be an effective method for the synthesis of corresponding structured O-methylated phenophospholipids. The effects of substrate molar ratios, time of the reaction and enzyme load on the process of incorporation of 3,4DMCA into PC were evaluated by using the experimental factorial design of three factors and three levels. The results showed that a substrate molar ratio is a crucial variable for the maximization of the synthesis of 3,4-dimethoxycinnamoylated phospholipids. Under optimized parameters of 1/10 substrate molar ratio PC/E3,4DMCA, enzyme load 30% (w/w), hexane as a medium and incubation time of 3 days, the incorporation of aromatic acid into phospholipid fraction reached 21 mol%. The modified phosphatidylcholine (3,4DMCA-PC) and modified lysophosphatidylcholine (3,4DMCA-LPC) were obtained in isolated yields of 3.5% and 27.5% (w/w), respectively. The developed method of phosphatidylcholine interesterification is the first described in the literature dealing with 3,4DMCA and allows us to obtain new O-methylated phenophospholipids with potential applications as food additives or nutraceuticals with pro-health activity.

Biological Properties, Health Benefits and Enzymatic Modifications of Dietary Methoxylated Derivatives of Cinnamic Acid

“…Ferulated lysophospholipids were also further successfully produced during one-step lipase-catalyzed transesterification of egg yolk PC with higher reaction yield [ 89 ]. Moreover, in the following years, methoxylated phenolic acids such as p -methoxy [ 90 ], 3,4-dimethoxy [ 91 ] and anisic acid [ 92 ] were successfully introduced into phospholipid structure via Novozym 435-catalyzed acidolysis or transesterification reaction ( Figure 3 ).…”

“…The regioselectivity of this reaction, towards the sn -1 position, was confirmed by the results of GC analyses. The acid profile in the obtained phospholipid fractions (containing natural (PC, LPC) and structured phospholipids (modified PC, modified LPC)) revealed a decrease in the concentration of saturated fatty acids, which present mainly in the sn -1 of natural PC [ 91 ]. The hydroxy group of phosphatidylcholines released in this way undergoes the next transesterification with an acyl residue donor molecule, yielding the first reaction product which is modified phosphatidylcholine.…”

Recent Developments in Therapeutic and Nutraceutical Applications of p-Methoxycinnamic Acid from Plant Origin

“…Based on the analysis of formed products via thin-layer chromatography (TLC) and high-performance liquid chromatography Extensive studies on the chemical and enzymatic production of phenolipids and their biological activities were performed using phospholipids, which are characterized by unique physicochemical properties [77][78][79][80][81][82][83]. In our research group, we developed the biotechnological method of synthesis of phospholipid derivatives of p-MCA [84]. In the enzymatic interesterification reaction of egg yolk phosphatidylcholine (PC) with ethyl p-methoxycinnamate (Ep-MCA), catalyzed by Novozym 435 under optimized parameters, after three days p-methoxycinnamoylated lysophosphatidylcholine (p-MCA-LPC) and p-methoxycinnamoylated phosphatidylcholine (p-MCA-PC) were obtained in isolated yields of 32% and 3% (w/w), respectively (Figure 5).…”

“…In the enzymatic interesterification reaction of egg yolk phosphatidylcholine (PC) with ethyl p-methoxycinnamate (Ep-MCA), catalyzed by Novozym 435 under optimized parameters, after three days p-methoxycinnamoylated lysophosphatidylcholine (p-MCA-LPC) and p-methoxycinnamoylated phosphatidylcholine (p-MCA-PC) were obtained in isolated yields of 32% and 3% (w/w), respectively (Figure 5). Based on the analysis of formed products via thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), the pathway of possible changes occurring during the reaction of enzymatic interesterification was also proposed [84].…”

Biocatalytic Process Optimization

“…Conversion yields of 88%-94% were obtained for 100-400 mM DHA + EPA concentrate at a constant enzyme activity of 200 U, substrate ratio of 1:1 (DHA + EPA: EA), and reaction time of 300 min. Rychlicka et al [24] developed a biotechnological method of synthesizing 3,4-dimethoxycinnamoylated phospholipids via the interesterification of egg-yolk phosphatidylcholine with the ethyl ester of 3,4-dimethoxycinnamic acid. RSM and a Box-Behnken design were used to evaluate reaction conditions.…”