Menthol

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.

To determine intracellular Ca²⁺-concentrations, DAOY cells were seeded on cover slips, mounted in a cell chamber and perfused as described in the section “Whole-cell patch clamp”. Fluorescence was measured every 2 s on an inverted microscope (IX71, Olympus, Chromaphor) using a Fluar 20 × /0.75 objective (Olympus) and Till Vision real-time imaging software (Till Photonics). Cells were loaded for 30 min at 37 °C with 2 μM Fura-2-AM (Molecular Probes) in the bath solution. Fura-2 was excited at 340/380 nm and the emission was recorded between 470 and 550 nm using a sensicam CCD camera (PCO imaging). Acquisition and data analysis were done using the Till Vision software and Excel.
The bath solutions used for the capsaicin, menthol, ATP and ionomycin stimulations consisted of the conditioning bath solution described under “Whole-cell patch clamp” containing the following chemicals: 100 μM capsaicin (Sigma Aldrich), 200 μM menthol (Sigma Aldrich), 100 μM ATP (Sigma Aldrich), or 1 μM ionomycin (Santa Cruz Biotechnology, Dallas, USA), respectively. The 30 mM K⁺ solution contained the following compounds (in mM): NaCl 85, KCl 30, D-glucose 5, HEPES 5, glucose 5.5, MgCl₂ 1, sodium gluconate 25, calcium gluconate 3. pH was adjusted to 7.4 with NAOH and HCl. The pH 6.0 solution consisted of the acidic bath solution described in “Whole-cell patch clamp”.

For in vitro assays, purified pulegone and menthol (Sigma-Aldrich (Steinheim, Germany) were prepared with a final 1% ethanol concentration for cell incubation.
For in vivo assays, purified pulegone and menthol (Sigma-Aldrich (Steinheim, Germany) were emulsified in warmed (37°C) carboxymethyl cellulose solution (CMC, 1%; in NaCl 0.9%) for subsequent intraperitoneal (i.p.) injections.

Standard 2‐hexanone, hexanal, 1H‐1‐ethyl‐pyrrole, 2‐hexenal, cis‐3‐hexen‐1‐ol, heptanal, 6‐methyl‐5‐hepten‐2‐one, 2‐pentyl‐furan, 2‐ethyl‐1‐hexanol, benzeneacetaldehyde, cis‐linalool oxide, trans‐linalool oxide, linalool, 3‐octen‐2‐ol, phenylethyl alcohol, menthol, α‐terpineol, safranal, decanal, camphene, geraniol and indole were purchased from Sigma Co. Ltd. Benzyl alcohol, trans‐β‐damascenone, trans‐α‐ionone, cis‐geranylacetone, trans‐β‐ionone, 2,4‐ditert‐butylphenol, cedrol, and caryophyllene oxide were purchased from Alfa Aesar Co. Ltd. Standard chemical series of C₈–C₂₀ alkanes that were used to determine the liner retention index (RI) and the internal standard cyclohexanone were obtained from Sigma Co. Ltd. Cis‐pyranoid‐linalool oxide and trans‐pyranoid‐linalool oxide were purchased from Sinopharm Chemical Reagent Co. Ltd.