Menthol

The authentic compounds used were 1,8-cineole (Wako Pure Chemical Industrials, Ltd., Osaka, Japan), (S)-(−)-limonene (Wako), menthone (Wako), menthofuran (Extrasynthese, Genay, France), menthol (Wako), pulegone (Wako), and (E)-β-caryophyllene (Wako). An authentic chemical solution (0.19 mg mL⁻¹ 1,8-cineole; 0.02 mg mL⁻¹ (S)-(−)-limonene; 0.27 mg mL⁻¹ menthone; 0.13 mg mL⁻¹ menthofuran; 0.4 mg mL⁻¹ menthol; 0.49 mg mL⁻¹ pulegone; 0.15 mg mL⁻¹ (E)-β-caryophyllene) in 3 mL of hexane in a glass vial (4 mL) was prepared to emit VOCs that approximately corresponded to the headspace levels of major VOCs released from candy mint (4 gFW)²⁰ . Similarly, an authentic chemical solution of (S)-(−)-limonene (see above), 1,8-cineole (see above), and (R)-(−)-carvone (1.6 mg mL⁻¹, Wako) was prepared to emit VOCs that mimicked the headspace level of the VOCs from spearmint (4 gFW). A blend of authentic VOCs consisting of candy mint VOCs (1,8-cineole, (S)-(−)-limonene, menthone, menthofuran, menthol, pulegone, and (E)-β-caryophyllene) and spearmint VOCs ((S)-(−)-limonene [0.02 mg mL⁻¹], 1,8-cineole [0.1 mg mL⁻¹] and (R)-(−)-carvone) at the respective ratios corresponding to the headspace levels was also prepared in 3 mL of hexane in a glass vial. Note that germacrene D was excluded from our assays because it was not available as a pure authentic chemical.

Capsaicin (Wako), menthol (Wako), bradykinin (Wako), ProTx-II (Wako), histamine (Wako), pyrilamine (Sigma), chloroquine (Wako), Nav1.7 inhibitor (GLPBIO), AMG9810 (Wako), PEAQX (GLPBIO) and NBQX (MedChemExpress) were dissolved in DMSO, ethanol or sterile, distilled water at the maximum concentration guaranteed by the manufacturer. The working concentration was prepared as 100 × dissolved in sterile, distilled water.

Standard solutions of pure chemicals of menthol and menthone (FUJIFILM Wako Chemicals, Osaka, Japan), pulegone, and menthofuran (Sigma-Aldrich, Tokyo, Japan) were dissolved in pure methanol. The dilution series of each solution was prepared with methanol. A volume of 2 µL of the standard solution was added to the glass vial with 2 mL CaCl₂, and the compound was measured using GC–MS with the same method described above. Three to four concentrations of each solution were used to make standard curves. Peak areas of each compound obtained both from samples and standards were used for calculations.