Thymol

Aspergillus fumigatus strain Af293 was used in this study. The strain was cultured in PDB at 37 °C on a rotary shaker at 120 rpm throughout the study. In some experiments, glucose minimal medium (GMM) (1%(w/v) glucose, 0.052% KCl, 0.052% MgSO₄·7H₂O, 0.152% KH₂PO₄, 0.0022% ZnSO₄·7H₂O, 0.0011% H₃BO₃, 0.0005% MnCl₂·4H₂O, 0.0005% FeSO₄·7H₂O, 0.00016% CoCl₂·5H₂O, 0.00016% CuSO₄·5H₂O, 0.00011% (NH₄)6Mo₇O₂₄·4H₂O, 0.005% Na₄EDTA, pH 6.5) containing variable nitrogen sources, such as sodium nitrate, ammonium sulphate, and proline (70 mM) were used, which were designated GMM(NO₃), GMM(NH₄), and GMM(Pro), respectively. The antifungal chemicals used were commercially obtained, as follows: thymol (NACALAI TESQUE, Inc., Kyoto, Japan), farnesol (Sigma-Aldrich Co., St. Louis, MO, USA), citral (NACALAI TESQUE, Inc.), nerol (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan), salicylic acid (NACALAI TESQUE, Inc.), PCA (Wako Pure Chemicals Industries, Osaka, Japan), pyocyanin (Cayman Chemical Company, Ann Arbor, MI, USA), itraconazole (Wako Pure Chemicals Industries), voriconazole (Tokyo Chemical Industry Co., Ltd), and amphotericin B (Sigma-Aldrich Co.). Micafungin was a generous gift from Dr. Keietsu Abe, Tohoku University.

The blood levels of propofol and the protein-unbound free propofol level were measured as described in our previous study^{8 (link)}. Serum was collected from the blood samples by centrifuging the blood samples at 1,400 G for 10 min. To measure the protein-unbound propofol level, protein-free serum was extracted using centrifugal filter units (Centrifree®; Merck Millipore, Bedford, USA) by centrifuging the samples at 2,000 G for 30 min. For preparation, thymol (Nacalai Tesque, Inc., Kyoto, Japan), as an internal standard (IS); 0.1 M potassium dihydrogen phosphate solution (Merck KGaA, Darmstadt, Germany); and heptane solution (Sigma-Aldrich, Co., MO, USA) were added to each sample. After the resultant mixture was centrifuged at 13,000 G for 3 min, the propofol concentration of the supernatant was measured with a high-performance liquid chromatography (HPLC) system (Shimadzu Co., Tokyo, Japan).
The peak area ratio of propofol to the IS was obtained from the HPLC chromatogram at each control concentration, and calibration curves for the blood propofol level (0.156, 0.312, 0.625, 1.25, and 2.5 μg/mL) and the protein-unbound free propofol level (0.005, 0.01, 0.02, and 0.04 μg/mL) were constructed.