As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.


Figure S1. HPLC profiles of sequential extracts from saffron, and authentic safranal. A, n-hexane extract; B, chloroform extract; C, safranal. For HPLC, we used Pump L-2310 (Hitachi, Tokyo, Japan), Rheodyne (Cotati, CA, USA) Model 7725i equipped with a 5 μL sample loop, and an L-4000 UV detector (Hitachi). Two mobile phase solvents were employed. The mobile phase constituted of 15% HPLC grade acetonitrile in ultrapure water (Solvent A) and HPLC grade methanol (Solvent B) at a constant solvent flow rate of 300 μL/min. For analysis, we used a reversed phase column (Cosmosil 5C18-MS-II, 2.0 × 150 mm, Nacalai Tesque) and the following mobile gradient: 0–60 min, linear gradient from 95% solvent A to 0% solvent A.

Figure S2. Cellular PTP activity in safranal-treated C2C12 myotubes. After C2C12 myotubes were treated with the indicated concentrations of safranal in serum-free DMEM for 3 h, cellular PTP activity was measured according to the published procedure [1]. Briefly, cell lysate was incubated with 4 mM pNPP, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mg/mL BSA. The reaction was allowed to proceed for 20 min at room temperature and p-nitrophenol produced was continuously measured by UV absorption at a wavelength of 405 nm with a microplate reader. The results shown are means ± SE (n = 3).

Figure S3. GC/MS analysis of (A) control plasma at 30 min after single oral administration of vehicle, (B) plasma sample at 30 min after single oral administration of safranal, and (C) EI mass spectrum of the peak eluting at 13.3 min. KK-Ay mice were fasted for 18 h and orally administered 20 mg/kg body weight safranal or vehicle. Blood samples were collected from anesthetized mice by cardiac puncture into heparinized tubes at 30 min after administration. After centrifugation at 2000 × g for 5 min, plasma (200 μL) was diluted with 150 μl of saline and 50 μL of 1.0 M HCl followed by the addition of 600 μL of chloroform/methanol (1 : 2, v/v). Then, the mixture was ultrasonicated with a sonicator for 30 sec, and further mixed with 200 μL of chloroform. After vortexing for 2 min and centrifuging the mixture for 5 min at 130,00 × g, the lower phase (organic phase) was collected, and 400 μL of chloroform was added to the other phase. After mixing and centrifuging the solution, the lower phase was collected and combined with the first extract. The collected sample was mixed with 5 ml of n-hexane and 5 ml of H2O. After mixing and centrifuging the solution, n-hexane layer was applied to a Sep-Pak silica cartridge (Waters, Milford, MA, USA) pre-equibrated with 10 mL of n-hexane. The column was then washed sequentially with 10 mL of n-hexane and 5 mL of n-hexane/ethyl acetate (98 : 2, v/v), and safranal was eluted with 4 mL of n-hexane/ethyl acetate (3 : 4, v/v). The eluate was transferred to a 5-mL volumetric flask and diluted with n-hexane to a final volume of 5 mL. GC/MS analysis was performed as described in the Materials and Methods section. The detection was operated in the selected ion monitoring (SIM) mode, and the concentration of safranal was calculated from the area of the peak at m/z 107. The recovery of the safranal spiked into plasma was ca. 95%.

Figure S4. Effects of safranal administration on body weight (A) and food intake (B). KK-Ay mice were orally administered vehicle alone or 20 mg/kg/day safranal for 2 weeks. Data are shown as the mean ± SE. *p < 0.05 versus vehicle-treated control group.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.