*3.3. The Cholinergic Activity*

Finally, the inhibition activity of this EO against AChE can be discussed. Observing our results, shown in Table 2, the inhibition capacity of *J. rugosa* EO was compared to the ones of galanthamine and *L. nobilis* EO. However, whereas the biological activity of galanthamine is clearly extremely high, mainly because it is a pure compound, the biological activity of *L. nobilis* EO is decidedly lower. Nevertheless, *L. nobilis* EO is considered as an

active mixture in this kind of assay, and it can be subsequently used as a better positive control while working with EOs [51]. In our case, the inhibition power of *J. rugosa* EO is about 68% compared to that of *L. nobilis* EO, clearly resulting in less activity but not inactive. This fact could be explained by the presence of at least one active minor sesquiterpene in the mixture. If that is the case, the EO may be considered useless as it is, but suitable to be studied, through a bio-guided fractionation, in search of new sesquiterpene inhibitors. The interest in this aspect resides in that, to the best of the authors' knowledge, the most active EOs are characterised by an important monoterpene fraction (except for the case where the EO is dominated by (*E*)-β-caryophyllene) [18,22]. However, due to their toxicity, hydrocarbon monoterpenes can hardly be used as pharmaceutical active principles, which cannot be assumed for sesquiterpenes. Therefore, the discovery of new sesquiterpene inhibitors of AChE is a matter of some pharmaceutical interest. Consequently, this volatile fraction is suitable for further investigation, according to two main lines: a) the purification and structure elucidation of the major undetermined compounds, by mean of preparative chromatography and NMR spectroscopy; b) a bio-guided preparative fractionation, intended to investigate the presence of new sesquiterpene AChE inhibitors among the minor components. Due to the low distillation yield of this EO, a non-classical approach should be applied. On the one hand, a tentative method for purification and structure elucidation could be the use of preparative thin-layer chromatography (TLC) and microprobe NMR spectroscopy. In this way, about 1 mg of a pure compound would be enough to be submitted to a complete series of NMR experiments. On the other hand, the bioguided investigation could be faced through a bioautographic method. Based on a TLC analysis, a bioautographic assay can be carried out with few micrograms of EO. Since the active compounds possibly are known sesquiterpenes, the combined use of bioautography, preparative TLC and GC-MS should afford the desired information.

In regards to the traditional use of *J. rugosa*, some previous studies have described the antioxidant and anti-inflammatory activities of the non-volatile fraction, mainly attributed to flavonoids [13,14]. Since these properties are fully consistent with the ethnobotanical use, the EO could probably be exempted to be considered the active fraction.

#### **4. Materials and Methods**

#### *4.1. General Information*

The chemical and enantioselective analyses of the *J. rugosa* EO were performed with a gas chromatography-mass spectrometry (GC-MS) system, consisting of a 6890 N Agilent Technologies gas chromatograph with an autoinjector model 7683. The instrument was coupled to an Agilent Technologies simple quadrupole mass spectrometry detector (MSD) model 5973 INERT (Santa Clara, CA, USA), and a common flame ionization detector (FID). The MSD operated in SCAN mode (scan range 35–350 *m*/*z*), with an electron ionization (EI) source at 70 eV. The qualitative and quantitative analyses were carried out with both non-polar and polar stationary phase capillary columns from Agilent Technologies. The non-polar column was based on 5% phenyl-methylpolysiloxane phase (DB-5ms, 30 m long, 0.25 mm internal diameter, and 0.25 μm film thickness), while the polar column was provided with a polyethylene glycol stationary phase (HP-INNOWax, 30 m × 0.25 mm × 0.25 μm). The enantioselective analysis was run with an enantioselective capillary column, based on 30% diethyl-*tert*-butyldimethylsilyl-*β*-cyclodextrin in PS-086 as chiral stationary phases as a chiral selector (25 m × 250 μm internal diameter × 0.25 μm phase thickness, purchased from Mega, MI, Italy). For all the analyses, GC purity grade helium (Indura, Guayaquil, Ecuador) was used as the carrier gas, set at the constant flow, with a rate of 1 mL/min. For the biological assays, a Spectronic Genesys 6 spectrophotometer was used, purchased from Thermo-Fisher Scientific (Waltham, MA, USA).

All solvents for GC analysis, the mixture of *n*-alkanes C9–C25 for linear retention indices (LRI), internal standard (*n*-nonane), and reagents for the inhibition activity assays were purchased from Sigma-Aldrich. The calibration standard was isopropyl caproate, obtained by synthesis in the authors' laboratory and purified to 98.8% (GC-FID).
