Linalool

Animals were injected intraperitoneally with 100 mg/kg of linalool (Sigma Aldrich, St. Louis, MO, USA) or 200 mg/kg of L. angustifolia essential oil (linalool 33.1%) (obtained and analyzed in our previous work [22 (link)]). linalool and essential oil were solubilized in physiological saline solution with 2% Tween-80 and 1% DMSO. The control groups were injected with physiological saline (NaCl 0.9%), which was also used to dissolve the drug. The doses of linalool and L. angustifolia essential oil used to test the effects on behavior of mice in different situations were selected on the basis of previous studies [12 (link),21 (link),58 (link)].

One gram (1 g) of geraniol (98%, Sigma-Aldrich) was subjected to hydrodistillation in a Clevenger-type apparatus for 3 h using a 500 mL flask and approximately 250 mL of distilled water. After this time, hydrolate was subjected to extraction with ethyl acetate (3 × 50 mL) and the organic phase was dried with anhydrous Na₂SO₄, concentrated under reduced pressure in a rotary evaporator (Buchi, Flawil, Switzerland), transferred to an amber glass bottle, carefully concentrated in nitrogen and stored in freezer (Electrolux, Curitiba, Brazil) until analysis by GC/MS. To prove the conversion of geraniol to linalool, 7 mg of linalool (97%, Sigma-Aldrich, São Paulo, Brazil) was added to 3.33 mg/mL hydrolate solution (geraniol hydrodistillation solution, after conversion) and after GC/MS analysis was observed if peak of linalool increased.

For chemical analyses, EOs were diluted in cyclohexane MS SupraSolv^® for gas chromatography (Sigma-Aldrich, Steinheim, Germany). For enantiomers identification, the analytical standards of (−)-menthol, (+)-menthol, (−)-menthone, (+)-menthone, (−)-α-terpineol, (+)-α-terpineol, (−)-limonene, (+)-limonene, (−)-terpinen-4-ol, (+)-terpinen-4-ol, (−)-trans-caryophyllene, (−)-carvone, (+)-carvone, (−)-linalool, (±)-linalool, (−)-menthyl acetate, (+)-menthyl acetate, (−)-dihydrocarvone, (+)-dihydrocarvone, (−)-borneol, and undecane-2-one (all Sigma-Aldrich, Steinheim, Germany) were used.

Dimethyl sulfoxide (DMSO), acetylcholinesterase (AChE) from Electrophorus electricus and butyrylcholinesterase (BChE) from equine serum, acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), galanthamine hydrobromide, and all the buffer components (TWEEN20, bovine serum albumin, Trizma base, Trizma HCl, MgCl₂·6H₂O), cyclohexane, nonane, petroleum ether, ethyl acetate, 1,8-cineole, linalool, linalyl acetate, 4-terpineol, and α-terpineol were purchased from Merck Life Science S.r.l. (Milan, Italy).
Six batches of Elettaria cardamomum (L.) Maton EO were obtained via steam distillation from botanically identified plant material (dried fruits, geographical origin Guatemala) and supplied by Erboristeria Magentina (Poirino, Torino, Italy). The hydrodistilled EO was obtained by submitting 100 g of dried fruits (same geographical origin) purchased from a local herbal shop to hydrodistillation for 4 h in Clevenger apparatus (a pic of the plant used to obtain the hydrodistilled EO is reported in the Supplementary Material Figure S2). The EO was collected and stored in the dark at +4 °C until use.

Pure reference standards of (E)-5-methyl-2-hepten-4-one (filbertone), ethyl-2-methylbutyrate, α-pinene, linalool, and limonene used for identity confirmation were supplied by Merck (Milan, Italy). Working solutions were prepared in cyclohexane at a final concentration of 100 mg/L.
n-Alkanes (n-C9 to n-C25), adopted for linear retention indices (I^T) calibration, were from Merck (Milan, Italy): the test mixture was prepared in cyclohexane at a final concentration of 100 mg/L.

Thymol methyl ether, Carvacrol methyl ether, (+)-2-carene, 1-octen-3-ol, myrcene, trans-anethole, α-terpineol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carvacrol, γ-terpinene, 3-carene, p-cymene, camphene, α-terpinene, terpinolene, (+/−)-α-pinene, (−/−)-β-pinene, R-(+)-limonene, S-(−)-limonene, were purchased from Extrasynthese (Genay, France). Thymol, linalool, α-phellandrene were purchased from Merck Millipore (Boston, MA, USA). All reagents and solvents have European Pharmacopoeia quality.