A novel “cut and sew” procedure for the natural reconstitution of essential oils prior to biological assays

Background: Given the increasing interest in natural compounds for therapeutic applications, understanding the contribution of individual components within complex essential oils (EOs) is crucial. The isolation is often achieved through bio-guided fractionation, typically using flash chromatography to target chemical classes. To refine this, some methods rely on customized reconstitution using reference standards, which is challenging due to issues with preserving natural abundances and enantiomeric ratios. Results: This study compares conventional flash chromatography with an advanced preparative multidimensional gas chromatography (prep-GC) system, equipped with a novel collection setup. This prep-GC approach enabled the isolation of sub-fractions at the milligram scale for biological assays. Critically, it preserved the natural distribution of chiral components, eliminating the need for standard-based reconstitution and its associated challenges. Mentha × piperita L. EO, known for its inhibitory activity against butyrylcholinesterase (BChE), an enzyme relevant to Alzheimer's disease, was chosen as a case study. The novel approach allowed for a comprehensive investigation by isolating the oxygenated fraction devoid of menthol. This facilitated a direct comparison of the biological activity of the whole EO against the total oxygenated fraction (from flash chromatography) and the menthol-devoid fraction (from prep-GC), as well as pure (−)-menthol, pure (−)-menthone, and their mixture. Significance: The results clearly indicate that (−)-menthol, (−)-menthone, and other terpenes within the oxygenated fraction jointly contribute to the overall BChE inhibitory activity. These findings highlight the complex interplay among Mentha × piperita L. EO constituents. Furthermore, this study underscores the potential of this new prep-GC technology for uncovering the biological roles of minor constituents in complex natural mixtures, by removing the contribution of the main representative components.