Thymol

Plant samples were collected in June 2018 at the flowering stage. At each region, 6 to 11 individual shrubs were collected depending on the population size with a minimum distance of 100 m. Voucher specimens (no. MPH-1799) were authenticated and deposited in the Herbarium of Medicinal Plants and Drugs Research Institute (MPH), Shahid Beheshti University, Tehran, Iran. Geographical data and altitude for each sampling area were recorded using GPS (Table 1). Besides, climate data for five years were taken from metrological stations closest to the habitats (Table 2). Carvacrol, linalool, p-cymene, and γ-terpinene were purchased from Sigma-Aldrich-Fluka (Germany), and thymol and α-pinene from Roth (Germany).

Oleogel was produced from essential oil of thyme, obtained from Sigma-Aldrich Chemie GmbH (Germany), olive oil, obtained from Henry Lamotte GmbH (Germany), and colloidal anhydrous silica Aerosil 200, obtained from Degussa AG (Germany). For identification of active compounds in oleogels thymol and carvacrol were used, which were obtained from Carl Roth GmbH & Co. Kg (Germany). For HPLC analysis, acetonitrile was used, which was obtained from Chromasolv (USA). Antifungal activity of oleogels was performed on Mueller-Hinton broth II agar, obtained from BBL (USA), and culture Candida albicans ATCC 60193, obtained from Becton Dickinson and Company (USA).

For all experiments, freshly prepared ultrapure water was taken from the Sartorius arium® pro UV water-treatment system (Sartorius AG, Göttingen, Germany). All chemicals were of analytical grade, unless noted otherwise. Acetonitrile, OPD, 2,3-dimethylquinoxaline, MGO, GO, 2-KDG, 3-DG, 5-HMF, D-(+)-glucose, methanol, and ammonium acetate (for mass spectrometry) were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Acetic acid (glacial) 100%, toluene, sulfuric acid 95–98%, and ethanol 96% were obtained from Merck (Merck KGaA, Darmstadt, Germany). Furthermore, 1,4-dioxane and thymol were purchased from Roth (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). The 3-DGal was obtained from Cayman (Cayman Chemical Company, Ann Arbor, Michigan, USA), and 3,4-DGE was purchased from Carbosynth (Carbosynth Ltd, Compton, Berkshire, UK).
thymol-sulfuric acid reagent was freshly prepared according to the monograph "Glucose" from the Ph. Eur., section C "Thin Layer Chromatography", subitem “Detection” [21 ]:
0.5 g of thymol were solved in a mixture of 5 mL of sulfuric acid and 95 mL of ethanol 96% (v/v). No cooling was required for this step.
The aqueous phase during LC-MS/MS measurement was a 5 mM ammonium acetate buffer solution adjusted to pH = 3.5 using 0.1% (v/v) acetic acid. It was freshly prepared according to Thomas et al (2013) [22 (link)].