Vitamin d3 cholecalciferol

Mouse asthma model was constructed as previously described and also illustrated in Figure 1A (31 (link)). Briefly, mice were sensitized by intraperitoneal injection with 50 µg of house dust mite extract (HDM, Greer Laboratories, Lenoir, NC) supplemented with 2 mg of adjuvant aluminum hydroxide (Al(OH)₃) (Thermo Fisher Scientific, Cleveland, OH, USA) in 200 µl PBS at day 1, 3 and 7. During the sensitization, mice were also administered intranasally every day with or without 10 µg of vanadium pentoxide (V₂O₅, the most common form of vanadium; Sigma-Aldrich, Inc., St. Louis, Mo, USA) per mouse in 20 µl of PBS. The mice were challenged intranasally with 50 µg of HDM per mouse in 20 µl of PBS from day 15 to 21 with or without 10 µg of V₂O₅ per mouse. All mice were sacrificed 24 h after the last challenge. In some cases, the mice were pre-treated with 100 µg/kg cholecalciferol (vitamin D₃, Sigma-Aldrich, St. Louis, MO) dissolved in coconut oil (Sigma-Aldrich, St. Louis, MO) via oral administration 1 h before every single challenge. PBS-treated age- and gender-matched mice were taken as control.

Solid pharmaceutical
secondary standard of cholecalciferol (vitamin D₃) was
obtained from Sigma-Aldrich (St. Louis, MO), and a 500 mg L^–1 stock solution of it was prepared by dissolving in toluene and stored
at −20 °C. All other reagents were purchased from Merck
KGaA, Darmstadt, Germany. Standard solutions were prepared daily and
stored at 4 °C until the start of experiments. For the pill sample,
one soft gelatine caplet (50,000 IU, containing 1250 μg of vitamin
D3) was weighed (0.175 g) and dissolved in 100 mL of toluene to contain
roughly 12.5 mg L^–1 of the analyte. For DES–SBSE
analysis, it was diluted to 50 μg L^–1 with
toluene. For the analysis of vitamin D₃ in Ddrops, 125
μL of the oily sample [containing 400 IU (10 μg of vitamin
D3)] was placed into a 5 mL measuring flask and diluted with hexane
to mark to achieve 50 μg L^–1 of the vitamin.
Then, the procedure was performed with and without spiking. No special
pretreatment was performed for the milk sample.

Ergosterol determination of freeze-dried material was based on Niemenmaa et al. [26 (link)]. In summary, 200 mg of the material was saponified in 10 % KOH in methanol for 1 h at 80 °C. After cooling, hexane and distilled water were added for extraction. The samples were shaken for 10 min and centrifuged for 15 min at 4,000 rpm. The hexane phase was collected and the hexane-water extraction was repeated once. The hexane phases of the 2 extractions were pooled and evaporated under vacuum. The extracted ergosterol was dissolved in 1 mL methanol before ergosterol content was determined by a high performance liquid chromatography (HPLC) fitted with a reversed phase C18 column (250 × 4.6 mm, Phenomex aqua 5 μm). The liquid phase was 90 % methanol and 10 % (1:1) 2-propanol/hexane. Areas under the peak were corrected for the extraction efficiency based on the internal standard cholecalciferol (vitamin D₃) (9.6 μg added) (Sigma Aldrich, St. Louis, Missouri, USA), using Empower 2 software (Waters Corporation, Milford, Massachusetts, USA). Mycelium of C. subvermispora and L. edodes grown on malt extract plates covered with cellophane was freeze dried and subjected to ergosterol extraction. The ergosterol content of the pure mycelium was used to calculate the amount of fungal biomass formed.