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

Objective: Menthol is the most representative molecule in Mentha × piperita L. essential oils (EOs), accounting for between 30 and 55% of the entire chemical composition. A literature review by Kamatou et al. extensively described its bioactivity, reporting more than ten different biological properties. Of course, the presence of menthol plays a prominent role in the resulting biological activity of the entire EO. In this study, aiming to expand the knowledge on the minor peppermint EO constituents, by minimizing menthol’s effect, a novel analytical approach was developed, aiming to remove menthol from the oxygenated fraction. In a second step, the EO, menthol, the entire oxygenated fraction, and the oxygenated collection devoid of menthol were evaluated in terms of biological activity. Methods: GC-MS and GC-FID analyses were carried out to evaluate Mentha piperita × L. essential oil volatile composition. Multidimensional preparative gas chromatography was exploited to guarantee the isolation of target fractions from the essential oil, while enantioselective multidimensional gas chromatography was used to evaluate the preservation of the enantiomeric ratios from the essential oil to the fraction collected. Results: Multidimensional preparative gas chromatography, operated by means of Dean's switch transfer devices, efficiently guaranteed the collection of the oxygenated terpenes, drastically reducing menthol and hydrocarbons’ content. Moreover, the chiral GC analyses confirmed the preservation of the enantiomeric excesses of the terpenols of interest from the EO until the collections, representing a consistent step ahead with respect to common practices of standards’ addition. Finally, biological tests revealed a synergistic/complementary effect between menthol and the oxygenated fraction devoid of the latter. Conclusions: The isolation of distinct sub-fractions of the sample facilitated the successful elucidation of the inhibitory effect attributed to each fraction, circumventing the challenges associated with the conventional use of standard compounds to reconstitute the fraction to be biologically tested. Specifically, while comparable inhibitory activity was observed for both the total essential oil and the entire oxygenated fraction, approximately half of this activity was attributed to pure menthol and the oxygenated fraction devoid of menthol. These findings significantly advance the current understanding of the contribution of individual essential oil components to the overall biological effect.