Enzymatic synthesis and flash chromatography separation of 1,3-diferuloyl-sn-glycerol and 1-feruloyl-sn-glycerol

Authors: Compton, David - Dave; Appell, Michael; Kenar, James - Jim; Evans, Kervin

Submitted to: Methods and Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2020
Publication Date: 1/16/2020
Citation: Compton, D.L., Appell, M., Kenar, J.A., Evans, K.O. 2020. Enzymatic synthesis and flash chromatography separation of 1,3-diferuloyl-sn-glycerol and 1-feruloyl-sn-glycerol. Methods and Protocols. 3(1):8. https://doi.org/10.3390/mps3010008.
DOI: https://doi.org/10.3390/mps3010008

Interpretive Summary: Ferulic acid is a natural antioxidant found throughout the plant kingdom. This compound is usually chemically bound to other plant components, such as sugars and oils, and can be present in many different structural forms. They are of special interest due to their health benefits and ability to be used as cosmetic ingredients. One of the problems with studying ferulic acid-containing compounds is that methods to purify them in greater than milligram quantities is lacking. This research demonstrates a rapid method using a technique called flash chromatography to purify two different ferulic acid-containing compounds that are produced as part of an industrial process to synthesize these important natural products using vegetable oils. The successful development and transfer to industry of these method contributes to the development of the national bioeconomy and supports greater economic returns to US agricultural producers and rural economies through expanded, value-added markets for agricultural materials and byproducts.

Technical Abstract: Ethyl ferulate (4-hydroxy-3-methoxycinnamic acid ethyl ester) was transesterified with Enova Oil (a soy-based vegetable oil containing 80 – 85 % diacylglycerol) using Novozym 435 at 60 'C. The resultant feruloylated vegetable oil reaction product produced a precipitate (96.4 g, 4.02 wt%) after 7 d of standing at room temperature. Preliminary characterization of the precipitate identified the natural phenylpropenoids 1,3-Diferuloyl-sn-glycerol (F2G) and 1-Feruloyl-sn-glycerol (FG) as the major components. A flash chromatography method was developed and optimized (e.g., mass of sample load, flow rate, binary solvent gradient slope, separation run length) using a binary gradient of hexane and acetone mobile phase and silica gel stationary phase to separate and isolate F2G and FG. The optimized parameters afforded F2G (1.188 ± 0.052 g, 39.6 ± 1.7 %) and FG (0.313 ± 0.038 g, 10.4 ± 1.3%) from 3.0 g of the transesterification precipitate, n = 10 trials. Overall, all flash chromatography separations combined, F2G (39.1 g, 40.6 %) and FG (9.4 g, 9.8 %) were isolated in a combined yield of 48.5 g (51.4 %), relative to the 96.4 g of transesterification precipitate collected. The optimized flash chromatography method was a necessary improvement over previously reported preparative HPLC and column chromatography methods used to purify milligram to low gram quantities of F2G and FG to be able to processes ~ 100 g of material to a timely, efficient manner.