Methanol

Lyophilized papain (~30,000 USP units/mg) was purchased from Himedia, India. Sodium acetate (anhydrous, ≥99%), trichloroacetic acid (≥99%), and 2,4,6-Tris(2-pyridyl)-s-triazine (≥98%) were obtained from the Sigma-Aldrich group (Schnelldorf, Germany). Citric acid (99%), potassium sulfate (≥99%), copper sulfate (≥99%) sulfuric acid (≥99%), phenolphthalein (≥98%), methanol (≥98%), glucose (≥99%), phenol (≥99%), and sodium hydroxide (≥99%) were purchased from Reanal (Budapest, Hungary). Ultrasil P3-11 was purchased from Ecolab-Hygiene Kft (Budapest, Hungary). Ferric chloride (≥99%), sulfuric acid (≥99.9%), amyl alcohol (≥99.9%), ascorbic acid (99.7%), bacteriological agar powder, and soybean casein digestive medium were procured from Merck (Darmstadt, Germany). Acrylamide (≥99%), sodium-dodecyl sulfate (≥99%), ammonium persulfate (≥99%), Tetramethylethylenediamine (≥99%), tris(hydroxymethyl)aminomethane (≥99%), glycine (≥99%), ethyl alcohol (≥99%), coomassie blue stain (≥99%), acetic acid (≥99%), isopropanol (≥99%), glycerol (≥99%), 2-βmercaptoethanol (≥99%), and bromophenol blue (≥99%) were purchased from BIO-RAD (BIO-RAD, USA). Milli-Q ultrapure deionized water (18.2 MΩ·cm; Merck-Millipore, Molsheim, France) was used throughout the experiment.

High purity synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt (DOPG) was purchased from NOF (Tokyo, Japan) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS) was purchased from Avanti Polar Lipids Inc (Sigma-Aldrich, Budapest, Hungary). Liposomes were prepared by using the lipid thin film hydration technique. Lipids were dissolved in chloroform (LabScan, Budapest, Hungary) containing 50% vol methanol (Reanal, Budapest, Hungary), which was then evaporated using a rotary evaporator. The resulting lipid film was kept in a vacuum for at least 8 h to remove residual traces of solvent. The dried lipid film was hydrated with the assay buffer. After repeated heating (37 °C) and cooling (−196 °C) steps (at least 10 times), the solutions were extruded through polycarbonate filters with 100 nm pore size (at least 11 times) using a LIPEX extruder (Northern Lipids Inc., Burnaby, BC, Canada). Final lipid concentration was 13 mM. For mimicking mammalian, bacterial, and cancer cell membranes, pure DOPC, DOPC/DOPG (80/20 n/n%), and DOPC/DOPS (80/20 n/n%) referred to as PC, PC/PG, and PC/PS, respectively, were used thoroughly in the study.

High-purity
synthetic DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine),
DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],
sodium salt), and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane,
chloride salt) were purchased from NOF Corporation (Tokyo, Japan).
The lipid thin film hydration technique was employed to prepare liposomes.
Briefly, lipids were dissolved in chloroform (LabScan, Budapest, Hungary)
containing 50 vol % of methanol (Reanal, Budapest, Hungary), which
were then evaporated by a rotary evaporator. To remove any remaining
solvent traces, the lipid film was kept in vacuum for at least 8 h.
PBS buffer was used to hydrate the dried lipid film. The solutions
after repeated heating (37 °C) and cooling (−196 °C)
cycles (at least 10 times) were extruded through polycarbonate filters
with a 100 nm pore size (at least 11 times) using a LIPEX extruder
(Northern Lipids Inc., Burnaby, Canada). The corresponding lipid stock
solution concentration was 13 mM. It was further diluted to 0.635
mM for all of the measurements, which involved liposomes. Pure PC,
PG, and 80:20% mixtures of PC/PG and PC/DOTAP were used in the study.

Throughout the experiments, deionized Milli-Q (Millipore, Molsheim, France) water with a resistivity of 18.2 MΩ·cm was used. All of the chemicals were of analytical grade, if not stated otherwise. Copper(II) sulfate, neocuproine (2,9-Dimethyl-1,10-phenanthroline) hydrochloric acid salt, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES sodium salt), ascorbic acid, bovine serum albumin (BSA), and hydrochloric acid were purchased from Sigma Aldrich Ltd. (Budapest, Hungary); NaCl 0.9% was obtained from B. Braun (Budapest, Hungary); chloroform and methanol were purchased from Reanal (Budapest, Hungary). Synthetic high-purity 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG 2000) were obtained from Avanti Polar Lipids (Alabaster, AL, USA) or Sigma Aldrich. The chemicals were used without further purification. Disposable PD-10 and PD MidiTrap G-25 desalting columns were obtained from GE Healthcare Life Sciences (Budapest, Hungary). Concentrated nitric acid (65% w/w) and H₂O₂ (30% w/w) of Suprapur quality needed for the sample preparation of cell lines for Total-Reflection X-ray Fluorescence (TXRF) measurement were supplied by Merck (Darmstadt, Germany).

Caffeine and rutin standards, HPLC grade acetonitrile, methanol, acetic acid, formic acid, dimethyl sulfoxide, Phosphate Buffer Saline (PBS), porcine polar brain lipid extract, cholesterol, phosphatidylcholine, and deuterated methanol (methanol-d₄, 99.8 atom% D, contains 0.03% (v/v) TMS) were purchased from Merck (Darmstadt, Germany). Ethyl acetate, methanol, and n-dodecane of reagent grade were purchased from Reanal-Ker (Budapest, Hungary). HPLC-grade water was prepared with a Millipore Direct Q5 water purification system (Merck, Darmstadt, Germany). All aqueous eluents for HPLC were filtered through MF-Millipore membrane filters (0.45 μm, mixed cellulose esters) (Merck, Darmstadt, Germany) and degassed in an ultrasonic bath before use.

The underground organs of three Plantago species (P. lanceolata L., P. major L., and P. media L.) were collected from different Hungarian locations in June of 2018. Leaf and fruit samples of the Forsythia species (F. europaea Degen & Bald, F. suspensa Vahl, and F. × intermedia cultivars ‘Lynwood’, ‘Melisa’, ‘Minigold’, ‘Primulina’, ‘Spectabilis’, and ‘Week End’) were collected from the Botanical Garden of Szent István University (Budapest, Hungary, Villányi street 29–43): the leaves and unripe, green fruits in July of 2018 and the ripe, yellow-brown, closed fruits as well as the ripe, yellow-brown, opened fruits in October of 2018. The collected samples were immediately lyophilized on the day of collection. The voucher specimens of all dried samples are deposited in the Department of Plant Anatomy, Eötvös Loránd University, Budapest, Hungary. The materials and reagents applied in the analysis and isolation of plant metabolites, such as acetonitrile (ACN), distilled water (DW), formic acid, dimethyl sulfoxide (DMSO), methanol (Reanal, Budapest, Hungary), DMSO-d₆, and methanol-d₄ (VWR chemicals, Leuven, Belgium) were all of analytical reagent grade of the highest purity available.

Petroleum ether (Carlo Erba, Spain) boiling at 40–70 °C was used for Soxhlet extraction. Chemicals for the determination of polyphenol and antioxidant content were 97% ethanol (Reanal, Hungary), 99% methanol (Reanal, Hungary), anhydrous sodium carbonate (Riedel–de Haen, Germany), Folin–Ciocalteu reagent (Merck, Germany), 2-4-6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich, USA), acetic acid (Reanal, Hungary), anhydrous iron chloride (Merck, Germany), 98% Trolox (Sigma-Aldrich, USA), gallic acid (Sigma-Aldrich, USA).