Uvaol

Commercially available standards of ten studied pentacyclic triterpenoids—friedelin (tech. grade), betulinic acid (≥97.0%), oleanolic acid (≥97.0%), ursolic acid (≥90.0%), lupeol (≥90.0%), β-amyrin (≥98.5%), α-amyrin (≥98.5%), erythrodiol (≥97.0%), betulin (≥98.0%), and uvaol (≥95.0%)—were purchased from Sigma-Aldrich (Steinheim, Germany).
HPLC gradient grade methanol and isopropanol (Chimmed, Moscow, Russia), and carbon dioxide (≥99.99%, Kriogen, Moscow, Russia) were used for the preparation of mobile phase. Methanol was also used for analyte solution preparation and PLE extraction of plant materials.
The stock solutions of triterpenoids in methanol (250 mg·L⁻¹) were prepared from an accurate sample. Calibration solutions of analytes were prepared by mixing and successive dilutions of the stock solutions with methanol. The solutions were stored in the dark at 4 °C for no more than one week. Stability was checked once a day.

Ten commercially available pentacyclic triterpenoids—betulinic acid (≥97.0%), oleanolic acid (≥97.0%), ursolic acid (≥90.0%), lupeol (≥90.0%), α-amyrin (≥98.5%), β-amyrin (≥98.5%), 3β-taraxerol (≥95%), betulin (≥98.0%), erythrodiol (≥97.0%), and uvaol (≥95.0%) were purchased from Sigma-Aldrich (Steinheim, Germany).
HPLC gradient grade methanol, acetonitrile, ethyl acetate, isopropanol (Chimmed, Moscow, Russia), formic acid (≥96%, Sigma-Aldrich, St. Louis, MO, USA), and ammonium formate (10 M aqueous solution, Sigma-Aldrich, St. Louis, MO, USA) were used for the preparation of the mobile phase and in the plant material pressurized liquid extraction (PLE) procedure. Deionized water with a resistivity of 18.2 MΩ cm was obtained using a Simplicity UV system (Millipore, Molsheim, France).
The stock solutions of PCTs in methanol (250 mg L⁻¹) were prepared from an accurately weighed sample. Calibration solutions of analytes were obtained by mixing successive dilutions of the stock solutions with methanol. All prepared solutions were stored in the dark at 4 °C for no more than one week. Their stability was checked once a day.

Asiatic acid (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), β-boswellic acid (Cayman Chemical Company, Ann Arbor, MI, USA), corosolic acid (Cayman Chemical Company), madecassic acid (Cayman Chemical Company), AKBA (Alexis Corporation, Lausen, Switzerland), ursolic acid (Sigma-Aldrich), and uvaol (Sigma-Aldrich) were used in this study. Recombinant human IL-1α was provided by Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan).

The compound tested in the experiments performed was uvaol, provided by Sigma^® (St. Louis, MO, USA), with a purity greater than 95%. A stock solution of uvaol with a concentration of 1 mg/mL (in 40 µL of DMSO + 960 µL culture medium) was prepared and subsequently diluted in culture medium until reaching the concentrations required for each test.

Phorbol 12-myristate 13-acetate (PMA), water soluble unesterified cholesterol, apolipoprotein (apo) A1, resazurin sodium salt, cycloheximide, and digitonin were purchased from Sigma-Aldrich (Vienna, Austria). Pioglitazone was obtained from Molekula (Munich, Germany). [³H]-cholesterol (1 mCi, 37 MBq) was provided by Perkin Elmer Life Sciences (Vienna, Austria). The olive oil-derived compounds (uvaol, erythrodiol, maslinic acid, oleanolic acid, betulinic acid, oleuropein, 3-hydroxytyrosol, and chlorogenic acid) were supplied by Sigma-Aldrich (Vienna, Austria). Oleocanthal (>80% purity) was isolated from olive oil as recently described (Adhami et al., 2015 (link)) and was and kindly provided by Prof. Liselotte Krenn (University of Vienna). Bovine serum albumin (BSA-fatty acid free) was from Roth (Karlsruhe, Germany). All tested compounds were dissolved in DMSO, aliquoted and stored at −20°C until use. An equal amount of DMSO was always tested in each condition in all experiments to assure that the solvent vehicle does not influence the results.

KCl, NaCl, HEPES, EDTA, nonactin, pentane, ethanol, methanol, calcein, triton X-100, sephadex G-50, digitonin, tribulosin, dioscin, diosgenin, solasodine, escin, uvaol, lupeol, betulin, nystatin (NyS), and gramicidin A (GrA) were purchased from Sigma-Aldrich (St. Louis, USA). Syringomycin E (SyrE) was isolated and purified as described previously [37 (link)], and kindly offered by Dr. J.Y. Takemoto (Utah State University, USA). The chemical structures of tested agents are presented in Figure 6. The purity of saponins and related compounds was ≥95% (except for betulin (≥98%), lupeol (≥94%), diosgenin (≥93%), and digitonin (~50%)). All experiments were performed at room temperature (25 °C).
Lipids, 1,2-diphytanoil-sn-glycero-3-phosphocholine (DPhPC), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (HOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG), and cholesterol (CHOL) were obtained from Avanti Polar Lipids (Pelham, NY, USA).

All solvents used for extraction and analysis, i.e., chloroform, diethyl ether, and methanol (purchased from Chempur, Piekary Śląskie, Poland), were of analytical grade. Authentic standards of α-amyrin and ursolic acid methyl ester were purchased from Roth (Karlsruhe, Germany), and β-amyrin, lupeol, uvaol, betulinic acid, oleanolic acid, campesterol, sitosterol, and stigmasterol were purchased from Sigma-Aldrich (Steinheim, Germany).