Enzymatic synthesis of steryl ferulates

Abstract

Steryl ferulates are plant sterols esterified to ferulic acid, a common phenolic acid. This esterification leads to sterol esters with improved biological properties, such as antioxidant activity. Commercially available and extracted steryl ferulates from rice bran are often limited in their sterol profiles. For further research and later food applications, a simple enzymatic esterification could address the lack of availability of single steryl ferulates. Whereas several enzymatic procedures for the esterification of steryl fatty acid esters have been published, no fully enzymatic procedure for steryl ferulates has been reported so far. We optimized both direct esterification of b-sitosterol with ferulic acid as well as transesterification with ethyl ferulate yielding steryl ferulates. The reaction was catalyzed by a lipase from Candida rugosa, which lead to yields of 35 and 55% for the direct esterification and transesterification, respectively. Moreover, both reactions followed a similar time course over incubation. The enzyme activity was rather low, which is probably due to the specificity of the different isoenzymes of C. rugosa lipase. However, successful conditions for a fully enzymatic synthesis of steryl ferulates are reported for the first time.Practical applications: This enzymatic procedure leads to steryl ferulates, which do not need thorough purification, as no toxic catalysts were applied. This is especially an advantage when animal or human studies are conducted, which are needed for further evaluation of the potential health benefits of steryl ferulates. Further, it is less labor intensive than earlier published procedures using vinyl esters as substrates, which have to be synthesized and chromatographically purified.

Enzymatic synthesis of steryl hydroxycinnamates and their antioxidant activity

“…Transesterification of ferulic acid was optimized systematically in an earlier study , and the process was slightly adjusted by the addition of some butanone or slight changes of the substrate concentrations for other hydroxycinnamic acids to improve the yield. Overall, the yield for the steryl ferulate was the highest with almost 55% (Table ).…”

“…Interestingly, of the three coumaric acids m ‐coumaric acid and o ‐coumaric acid were transesterified to the according steryl ester in similar efficiency (18.8 and 18.7%, respectively), but p ‐coumaric acid was transesterified not to a quantifiable extent. For the m ‐ and o ‐coumaric acid, addition of some butanone increased the yield from below 10% to almost 19%, compared to the ferulic acid where it only decreased the yield . Commercial methyl caffeate had to be used as starting material for the transesterification of caffeic acid.…”

“…Transesterification of hydroxycinnamic acids was achieved using the product from the ethylation reaction. The residue after evaporation was used directly for the transesterification reaction as published earlier for the ferulic acid . β‐Sitosterol, C. rugosa lipase and solvents were added to the ethyl hydroxycinnamates followed by incubation in an oil bath.…”

“…Phloretic acid was transesterified in a similar manner as published earlier . The steryl esters applied in the antioxidant assay, namely steryl ferulate and steryl sinapate, were directly esterified as previously reported . The purification was achieved with a base–acid work‐up as described for the C18 esters.…”

“…However, besides linear alcohols, also the esterification of ferulic acid with sterols is of interest. Three approaches have been studied so far: a chemical synthesis, which was optimized only recently , a chemoenzymatic approach including the transesterification of vinyl phenolic acid esters , or a fully enzymatic approach . In this latest study, two enzymatic methods were compared, the direct esterification of ferulic acid (yield of 35%) and the transesterification of ferulic acid from ethyl ferulate to steryl ferulates (yield of 55%).…”