Enzymatic Synthesis and Bioactivity Evaluation of Alkyl Dihydrocaffeates

Abstract Dihydrocaffeic acid (DHCA) is a phenolic compound known for its antioxidant properties but suffers from limitations due to its hydrophilicity. This study focused on enzymatically synthesizing DHCA esters with alkyl chains ranging from C4 to C12 to enhance their lipid compatibility, while also assessing their safety and effectiveness. The esters were produced using Candida antarctica lipase B, yielding between 37.40 ± 4.41% and 59.34 ± 0.31%. Antioxidant assays, including DPPH, ABTS, CUPRAC, and lipid systems, indicated that the antioxidant activity was dependent on chain length: shorter esters excelled in polar assays (e.g., C4-DHC IC 50 = 0.14 ± 0.01 mM in DPPH), whereas longer chains (C8-DHC and C12-DHC) were more effective in lipid-rich environments. In vegetable oils, both DHCA and its esters significantly reduced oxidation, similarly to butylated hydroxytoluene (BHT), under both storage and accelerated conditions. Cytotoxicity tests performed on HaCaT cells showed that short-chain esters (C4-DHC and C8-DHC) were toxic at concentrations of 0.1 mM and above, while C12-DHC maintained 61.6 ± 15.0% viability at 0.1 mM. Phytotoxicity effects varied by species, with C12-DHC exhibiting minimal inhibition of germination. The results highlighted the critical role of alkyl chain length in influencing antioxidant effectiveness and safety. Longer esters, specifically those with chain lengths of C8–C12, provided a favorable balance of lipid solubility, oxidative stability, and biocompatibility, making them sustainable options for food additives. Further in vivo studies are also needed to verify their safety in cosmetics and agriculture. Graphical abstract