Methanol

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.

Graft copolymers (GP1–GP3) based on PILs with choline cations and chloride counterions (Table 1) were synthesized through controlled radical polymerization and characterized according to previously described procedures [31 (link),43 (link)]. Sodium piperacillin (NaPIP; 99%) and tazobactam (TAZ; 94%) were purchased from Alfa Aesar (Warsaw, Poland) and used without prior purification. Methanol was obtained from Chempur (Piekary Śląskie, Poland). Phosphate-buffered saline (PBS) was obtained from Sigma-Aldrch (Poznań, Polska). DMEM-F12 medium and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were received from Aldrich (Poznań, Poland). Human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Cat# ATCC^® CRL-9609; Manassas, VA, USA).

The substances used in the preparation of the systems, including fisetin (FIS) (>95%) and Poly(1-vinylpyrrolidone-co-vinyl acetate (CPV)), were obtained from Sigma Aldrich Chemie (Berlin, Germany).
Trifluoric acid and methanol HPLC grades were provided by Merck (Darmstadt, Germany). High-quality pure water was prepared using a Direct-Q 3 UV purification system (Millipore, Molsheim, France).
Activity testing reagents: 2,2-Diphenyl-1-picrylhydrazyl, iron (III) chloride hexahydrate, 2,2′-casino-bis (3-ethylbenzothiazoline-6-sulfonic acid), neocuproine, 2,4,6-Tri(2-pyridyl)-s-triazine, and Trolox were supplied by Sigma-Aldrich (Schnelldorf, Germany). Sodium chloride and sodium hydrogen phosphate were purchased from Avantor Performance Materials (Gliwice, Poland). Ammonium acetate (NH₄Ac) and methanol were supplied by Chempur (Piekary Śląskie, Poland). Cupric chloride dihydrate, ethanol (96%), isopropanol (99%), acetic acid (99.5%), and sodium acetate trihydrate were obtained from POCH (Gliwice, Poland).