Ethyl acetate

All reagents were commercially available materials and were used without further purification. (+)-(S)-2-(6-Methoxynaphthalen-2-yl)propanoic acid (≥98%) was provided from AmBeed (Arlington Heights, IL, USA). L-proline (≥98.5%) was obtained from Carl Roth (Karlsruhe, Germany). Trimethylsilyl chloride (≥99%) (TMSCl) was purchased from Sigma-Aldrich (Steinheim am Albuch, Germany). Ammonium hydroxide solution 25% (NH₃∙H₂O) was of analytical grade obtained from StanLab (Lublin, Poland). Methanol (MeOH), ethanol (EtOH), propan-2-ol (i-PrOH), propan-1-ol (PrOH), butan-1-ol (BuOH), dimethyl sulfoxide, chloroform, ethyl acetate, diethyl ether, toluene, and n-hexane of high purity were purchased from Chempur (Gliwice, Poland). Deuterated chloroform (CDCl₃) (99.8%) (+0.03% TMSCl) was provided by Eurisotop (Cheshire, England). Acetonitrile was provided by J.T. Baker (Radnor, Pennsylvania, USA). PBS (pH 7.4), potassium dihydrogen phosphate (p.a.) was purchased from Merck (Darmstadt, Germany). For cell culture studies, murine fibroblast cell line, L929, and all cell culture reagents were purchased from Sigma Aldrich (Poznań, Poland), while all sterile, single-use cell culture plasticware was purchased from VWR (Gdańsk, Poland).

Intestinal Acetone powder from a rat source of α-glucosidase (EC 3.2.1.20), α-amylase from porcine pancreas type VI-B (EC 3.2.1.1), pancreatin from porcine pancreas, pepsin from the gastric mucosa of pigs, bile from bovine and ovine, TRIS-HCl, acarbose, caffeic acid, chlorogenic acid, (+)-catechin, cinchonine, (+)-catechin, (−)-epicatechin, kaempferol 3-glucoside, naringin, sodium chloride, maltose, formic acid, methanol, and acetonitrile were obtained from Sigma Aldrich (Steinheim, Germany). Acetone, ethanol, ethyl acetate, hydrochloric acid, sodium hydroxide, sodium bicarbonate, iodine, potassium iodide, disodium phosphate, and monosodium phosphate were purchased from Chempur (Piekary Śląskie, Poland). Potato starch and calcium chloride were purchased from POCH (Gliwice, Poland) and glucose test from Biomaxima SA (Lublin, Poland). Quercetin 3-glucoside, quercetin 3-rutinoside, quercetin 3-rhamnoside, and isorhamnetin 3-glucoside were obtained from Extrasynthese (Lyon, France). Procyanidin C1, procyanidin B1, procyanidin B2, neochlorogenic acid, cryptochlorogenic acid, and 3,5-dicaffeoylquinic acid were purchased from PhytoLab (Vestenbergsgreuth, Germany). Kaempferol was purchased from ICN Biomedicals (Costa Mesa, CA, USA). Ultrapurity water was prepared in the laboratory using a Simplicity Water Purification System (Millipore, Marlborough, MA, USA).

Phenolic acids: SA, CA, and FA, β-sitosterol ≥ 70% (mainly impurities campesterol and β-sitostanol), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH, 95%), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), acetic anhydride, 4-(dimethylamino)pyridine (DMAP), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), dicyclohexylcarbodiimide (DCC), potassium persulfate and potassium carbonate were obtained from Merck, Warszawa, Poland. Methanol, ethanol, ethyl acetate, dichloromethane, chloroform, hexane, and hydrochloric acid were purchased from Chempur, Piekary Śląskie, Poland. Thin layer chromatography (TLC) plates with fluorescent indicator UV₂₅₄, trade name ALUGRAM R SIL G/UV₂₅₄ (Macherey-Nagel, Dueren, Germany) and silica gel (pore size 60Å, 230–400 mesh, Kieselgel, Macherey-Nagel, Dueren, Germany) were purchased from Alchem, Toruń, Poland.

Analytical standards of ciprofloxacin, enrofloxacin, nalidixic acid, metronidazole and cefuroxime (purity of all >98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) (Table 1). Analytical grade acetonitrile, methanol, acetone, ethyl acetate and hydrochloric acid were purchased from Chempur (Piekary Śląskie, Poland). Analytical grade acetic acid was purchased from POCH S.A (Gliwice, Poland). Analytical grade ammonium acetate and citrate buffer (pH = 2, pH = 3, pH = 4, pH = 5, pH = 6) were purchased from Merck (Darmstadt, Germany). Hypergrade water, methanol, acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany).
Oasis HLB cartridges (500 mg, 6 mL) from Waters (Eschborn, Germany) were used for solid-phase extraction (SPE). Nylon syringe filters (0.45 µm, 25 mm, PURELAND) were used to filter the sample. Plant material samples (parsley root) were purchased from stores in the Silesian Voivodship (administrative unit) in Poland. Soil samples were collected in Gliwice, Poland.

LC-MS-grade reagents water (≥95%), methanol (≥99%), formic acid (≥98%), acetylcholine chloride (≥99%) and acetylcholinesterase (EC 3.1.1.7 from electric eel, lyophilized powder 200–1000 U/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA), n-hexane (≥99%) and ethyl acetate (≥99%) from Chempur (Piekary Śląskie, Poland), and lactucin (≥95%) and lactucopicrin (≥95%) from Extrasynthese (Genay CEDEX, France). Nylon syringe filters were purchased from Chromacol (Herts, UK). Ultrapure distilled water (18.2 MΩ cm) was obtained on a Millipore Milli-Q Plus system (Bedford, MA, USA). Fresh chicory roots (Cichorium intybus L.) were obtained from a local chicory breeder Bakor cultivating the vegetable using the hydroponic method (Skierniewice, Poland).

Pseudomonas putida (DSM 291), Pseudomonas moorei (DSM 12647), Sphingomonas mali (DSM 10565) and Bacillus subtilis (DSM 3657) were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Germany). Methylisothiazolinone, chloroxylenol, 3-indoleacetic acid, iron(III) chloride, phospholipid standards and ammonium format were purchased from Merck (Poland). SYTOX™ Green Nucleic Acid Stain, General Oxidative Stress Indicator (CM-H₂DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester) and AlamarBlue Cell Viability Reagent were obtained from Thermo Fisher Scientific (Poland). Difco™ nutrient broth and Mueller Hinton broth were purchased from Becton Dickinson (Poland). DMSO and phosphate-buffered saline (PBS) were obtained from BioShop (Canada). Methanol, ethyl acetate, hydrochloric acid, acetic acid, crystal violet and sodium sulfate anhydrous were obtained from Chempur (Poland). All of the other reagents with a high analytical purity grade were purchased from Avantor (Poland).

The following compounds were used for the preparation of betulin, and the synthesis of its derivatives: 4-dimethylaminopyridine (DMAP), acetic anhydride (Ac₂O), carbon tetrachloride (CCl₄), tetrahydrofuran (THF), dichloromethane (DCM), palladium on carbon (10% Pd/C), pyridinium chlorochromate (PCC), 3-chloroperbenzoic acid (mCPBA), lithium aluminum hydride (LiAlH₄), tert-butyl(chloro)diphenylsilane (TBDPSCl), purchased from Sigma Aldrich (Steinheim am Albuch, Germany), KOH, hexane, methanol, ethyl acetate, acetone, MgSO₄, ethanol, purchased from Chempur (Piekary Śląskie, Poland). All reagents were of analytical grade.
For the determination of the antioxidant activity and to assess the skin permeation of the compounds obtained: acetonitrile for HPLC was from J.T. Baker (Berlin, Germany). 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TE), were purchased from Sigma Aldrich (St. Louis, MO, USA); Folin–Ciocalteu reagent and gallic acid (GA), were from Merck (Darmstadt, Germany), potassium persulfate and chloride, sodium chloride, methanol, ethanol, acetone and phosphate buffered saline (PBS, pH = 7.4) were purchased from Chempur (Piekary Śląskie, Poland). All reagents were of analytical grade.