*4.4. GC-MS Qualitative Analyses*

The EO was analysed by injecting 1 μL of analytical sample into the GC instrument that operated in split mode (40:1). The injector temperature was set at 220 ◦C. The elution with the DB-5ms column was conducted according to the following oven temperature program: 60 ◦C was kept for 5 min, followed by a first thermal gradient to 100 ◦C at a rate of 2 ◦C/min, a second gradient to 150 ◦C at a rate of 3 ◦C/min, then a third gradient to 200 ◦C at a rate of 5 ◦C/min; in the end, the oven temperature was maintained at 250 ◦C for 15 min at a rate of 15 ◦C/min. With the HP-INNOWax column, the same conditions and oven program were applied, except for the final temperature, which was set at 230 ◦C.

In order to identify the components of the EO, a homologous series of *n*-alkanes, from *n*-nonane to *n*-pentacosane, was injected in each column. The linear retention index (LRI) of each constituent was calculated according to Van Den Dool and Kratz [48]. This way each volatile metabolite was identified by comparing the corresponding LRI value and EI-MS spectrum with tabulated data for DB-5ms [25] and HP-INNOWax [26–47].

#### *4.5. GC-FID Quantitative Analyses*

The quantitative analyses were performed, with both columns, under the same conditions and configurations described for the qualitative ones. All the samples were injected in quadrupled and the percentage of each analyte in the EO was calculated as the average value over the four injections. The quantification was achieved by external calibration and use a process internal standard, calculating the relative response factor (RRF) of each EO constituent, based on its combustion enthalpy [52]. The original method was modified, isopropyl caproate instead of the methyl octanoate reported in the literature, was chosen as the calibration standard for this analysis. This approach is based on the principle that the RRF of an organic compound only depends on the molecular formula and number of aromatic rings, being the same for isomers.

Two external calibration curves were build-up, according to what is described in previous articles [18–22]. All calibration curves achieved an R2 > 0.995.

#### *4.6. Enantioselective Analysis of the EO*

The enantioselective analysis of the EO was carried out by GC-MS on the same samples of the qualitative and quantitative analyses. The injector temperature was the same as for the EO qualitative analysis, whereas the injector operated in split mode, with a ratio of 50:1. The following oven thermal program was applied: The initial temperature was 60 ◦C for 2 min, followed by a thermal gradient of 2 ◦C/min until 220 ◦C, maintained for 2 min. In addition, a mixture of *n*-alkanes (C9–C25) was injected under the same conditions as for conventional analysis to determine LRIs. The enantiomeric pairs of chiral sesquiterpenes

were identified based on the EI-MS spectra and elution order, determined according to literature data for the same chiral selector [53,54].

#### *4.7. AChE Inhibition Spectrophotometric Assay*

The protocol followed in this study was that of Rhee et al., with slight modifications [55]. Enzyme solution 0.22 U/mL was prepared in 50 mM Tris-HCl, pH 8, containing 0.1% bovine serum albumin (BSA). Acetyltiocholine (ATCI) solution 1.5 mM was prepared in Millipore water. Ellman's reagent solution 3mM was prepared in 50 mM Tris–HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl2 hexahydrate. Essential oils stock solutions 15 mg/mL and 30 mg/mL were prepared in dimethyl sulfoxide (DMSO). *Laurus nobilis* essential oil solution 15 mg/mL and galanthamine solution 0.4 mg/mL in DMSO were used as a positive control.

The reagents were placed in the cuvette in the following order: 150 μL of ATCI solution, 900 μL of buffer B, 5 μL of the essential oil/galanthamine solution and finally 150 μL of enzyme solution. BSA 1%, Tween-20 0.1 and 0.5% and Tween-80 0.1 and 0.5% were tested as buffer B detergents to increase the essential oil solubility in the aqueous reaction mixture. The reaction mixture was incubated for 6 minutes at room temperature (25 ◦C). Absorbance values were collected after 6 minutes of incubation at 412 nm. The absorbance corresponding to 100% of AChE activity was measured by replacing the EOs/galanthamine solution with 5 μL of pure DMSO. Control and sample blank solutions were prepared by replacing the 150 μL of enzyme solution with 150 μL of buffer B. The percentage of AChE inhibition was measured according to the equation below:

% Inhibition = ΔA (Control) − ΔA (Sample)/ΔA (Control) × 100

ΔA (Control) or (Sample) = A412 (Control) or (Sample) − A412 (Control Blank) or (Sample Blank)

#### **5. Conclusions**

The fresh leaves of *Jungia rugosa* Less afforded, by steam distillation, an essential oil in quite a low yield (0.09% by weight). This volatile fraction was composed exclusively of sesquiterpenes, whose major constituents were γ-curcumene (more than 45%) and βsesquiphellandrene (about 17%). The other two unknown oxygenated sesquiterpenoids were detected among the main constituents (about 7% and 5% of the whole mixture). The EO also manifested a weak inhibition activity against AChE.

**Author Contributions:** Conceptualization, O.M. and G.G.; investigation, K.C., F.C. and V.V.; data curation, O.M., B.S. and G.G.; writing—original draft preparation, K.C.; writing—review and editing, O.M. and G.G.; and supervision, O.M., B.S. and G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw data are available from the authors (K.C.).

**Acknowledgments:** We are grateful to the Universidad Técnica Particular de Loja (UTPL) for supporting this investigation (2nd call funding TFT, Apr–Aug 2020) and open access publication.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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