α β thujone

Dimethyl sulfoxide (DMSO), fMLF, Histopaque 1077, (−)-α-thujone, α/β-thujone, and dillapiole were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Menthone, isomenthone, and pulegone were from Toronto Research Chemicals (North York, ON, Canada). n-Hexane was purchased from Merck (Darmstadt, Germany). Fluo-4AM was purchased from Invitrogen (Carlsbad, CA, USA). Hanks’ balanced salt solution (HBSS; 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 5.56 mM glucose, and 10 mM HEPES, pH 7.4) was purchased from Life Technologies (Grand Island, NY, USA). HBSS without Ca²⁺ and Mg²⁺ is designated as HBSS^–; HBSS containing 1.3 mM CaCl₂ and 1.0 mM MgSO₄ is designated as HBSS⁺.

The purified standard of α, β-thujone (80% purity; 70% α-thujone and 10% β-thujone) was from Sigma-Aldrich (Steinheim, Germany). The reference standard solution contained 0.724 g/mL of α- and β-thujone (taking into consideration a purity of 80.0% and density of 0.925 g/mL). An aliquot of 25 μL of the standard solution was diluted in 25 mL of methanol to a final concentration of 0.724 mg/mL. The extracts were dissolved in methanol with an approximate of concentration 2 to 6 mg/mL, filtered (Nylon filter, 0.45 μm pore size), and then used for reversed phase liquid chromatography (DIONEX UltiMate 3000 UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) with fluorescence detection experiments. The peak areas were recorded, and the amount of thujone was calculated using the calibration plot on LC.
The LC analysis was carried out using a Zorbax Eclipse XDB-C18 (Agilent, 5 µm, 15 cm × 0.46 cm i.d.) column heated at 35 °C and an acetonitrile to water ratio of 55 to 45 as the mobile phase; the flow rate was 1 mL/min, and the injection volume was 20 µL. The fluorescence detector was set at 220/290 nm (λex/λem). The thujones were detected as only one peak as the sum of the two isomers. The quantitative analysis was performed both by using linear regression analysis, obtained by injecting various concentrations of thujone solution in methanol, and by the standard addition method.