Assessment of olfactory identification threshold

The olfactory ability was assessed by identifying the threshold for a rose odor using 2-phenylethyl alcohol (CAS no. 60-12-8; Tokyo Chemical Industry, Tokyo, Japan), a dominant odor compound in natural rose petals. The odorant concentration, dilution solvent, and methods for the assessment were all the same as described previously [9]. Briefly, a serial tenfold odorant dilution of eight steps, from 1 to 8 (low-to-high) concentrations, was prepared with a starting concentration of 631 mg/ml [16]. In this study, we analyzed the lowest concentration step at which the subjects correctly identified the odor. When the subjects had naming the odor difficultly, they were asked to identify the odor using a card with the correct odorant names written on it (rose flower, faint sweet, flower, or plants). The olfactory test took place in a quiet, well-ventilated room at temperature of 22–25 °C. A portable local ventilation equipment (SMST-DD-W-HD, Shonan Maruhachi S-Tech Co., Ltd., Kanagawa, Japan) was set close to the odorant bottles. Subjects were asked to avoid using perfume on the day of testing, and eating or drinking anything except for water 30-min before testing.