Ethanol

The following drugs were used: acetylcholine chloride (ACh), sodium nitroprusside (SNP), noradrenaline hydrochloride (NA) (Sigma-Aldrich, St. Louise, MO, USA); potassium chloride (KCl) (Chempur, Piekary Slaskie, Poland); pinacidil and U-46619 (Cayman Chemical, Ann Arbor, MI, USA). The stock solutions (10 mM) of drugs were prepared in distilled water, except for Noradrenaline (NA) which was dissolved in a NaCl (0.9%) + ascorbic acid (0.01% w/v) solution; pinacidil was dissolved in DMSO, and U-46619 in ethanol. These solutions were kept at −20 °C, and appropriate dilutions were made in KHS (in mM: NaCl 115; CaCl₂ 2.5; KCl 4.6; KH₂PO₄ 1.2; MgSO₄ 1.2; NaHCO₃ 25; glucose 11.1) on the day of the experiment. At a concentration of 0.01%, the solvents did not alter the reactivity of isolated aortic rings.
Myo-inositol, trichloroacetic acid (TCA), thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 1-(trimethylsilyl) imidazole, pyridine and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich (St. Louis, MO, USA); while ethanol was purchased from Stanlab (Lublin, Poland). Other reagents were of analytical grade and were provided by commercial suppliers, including POCh (Gliwice, Poland), and Chempur (Piekary Slaskie, Poland).

The preparation of hierarchical material based on commercial FAU-type zeolite was based on dispersing the commercial FAU zeolite (Alfa Aesar) in the amount of 0.50 g in a mixture containing 100.00 g distilled water, 1.25 g ammonia (StanLab), 60.00 g ethanol (StanLab, 96%) and 0.35 g CTABr (cetyltrimethylammonium bromide) (Fluka Analytical). The whole mixture was placed in a polyethylene bottle and ultrasonicated for a period of 30 min at 65 °C. After this time, 0.56 g of tetraethyl orthosilicate (TEOS) (Aldrich Chemistry) was added to the solution as a silicon source, followed by 0.0122 g of ruthenium source, which was chloropentaaminoruthenium(III) chloride (Sigma Aldrich, >98%). In a further step of the synthesis, the whole mixture was stirred on a magnetic stirrer for 24 h at 65 °C, after which the resulting precipitate was drained on a glass funnel using a filter strainer, washed with a mixture of distilled water-ethyl alcohol at a ratio of 1:1, and allowed to air-dry at room temperature. After drying, the material was calcined for 5 h at 550 °C (Figure S16 in Supplementary Materials).

Melatonin, indole-3-propionic acid (IPA), 17β-estradiol, sodium chlorate (NaClO₃), ammonium thiocyanate (NH₄SCN), potassium selenocyanate (KSeCN), potassium nitrate (KNO₃), sodium fluoride (NaF), potassium perchlorate (KClO₄) and bisphenol A (BPA) were purchased from Sigma (St. Louis, MO, USA). Ethanol (96%) was purchased from Stanlab (Lublin, Poland). The LPO-586 kit for LPO was obtained from Enzo Life Science (Farmingdale, NY, USA). All the used chemicals were of analytical grade and came from commercial sources.

For SEM examination, the scaffolds with imposed cells of the studied lines were fixed in 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany) diluted in cacodylate buffer (0.2 M, pH 7.4, Serva Electrophoresis). After 1 h, the samples were washed three times in cacodylate buffer for 5 min at room temperature and postfixed for 1 h at 4 °C in 1% osmium tetroxide (Serva Electrophoresis) diluted in cacodylate buffer. The samples were next bathed three times with cacodylate buffer for 5 min. Dehydration of the samples was performed by increasing concentrations of ethanol (Stanlab, Lublin, Poland) for 15 min in each solution (50%, 70%, 80%, 96%) at 4 °C. Subsequently, the studied cell lines were incubated in absolute alcohol three times for 15 min at room temperature. Finally, the specimens were transferred to pure acetone, air-dried, and covered with 30 nm of gold in a high-vacuum sputter coater (Edwards, Burgess Hill, United Kingdom). Observations were taken with a JSM-6610A scanning electron microscope (JEOL, Tokyo, Japan) using 20 kV accelerating voltage and a secondary electron detector, revealing topography contrast.

The dry plant material was grounded in a laboratory mill (A11 IKA, Germany) to obtain a homogenous drug powder. Drug material (2 g) was extracted for 30 min with 20 mL of 50% (v/v) ethanol (Stanlab, Lublin, Poland) solution using an ultrasound-assisted method (U-504 Ultron, Moorpark, CA, USA). Subsequently, the extract was filtered through a paper filter. ethanol was removed under vacuum using a rotary evaporator (Heidolph G3, Heidolph, Schwabach, Germany) at 40 °C. The extracts were prepared in triplicates. Obtained ethanol-free water extract was frozen at −80 °C and lyophilised for 24 h using FreeZone 2.5 lyophilizer (Labconco, Kansas City, MO, USA). Lyophilised extracts were diluted in sterile water, shortly before the experiment, to the final concentrations of 125, 250, 500 and 1000 µg/mL.

The preparation of unmodified hierarchical zeolite MFI (ZSM-5), BEA (β) or FAU (Y) was based on dispersing weighed amount (0.75 g) of commercial zeolite of ZSM-5 (Acros Organics B.V.B.A., Geel, Belgium), β (Alfa Aesar, Ward Hill, MA, USA) or Y (Alfa Aesar, Ward Hill, MA, USA) type in a mixture containing 150.00 g of distilled water, 1.875 g of ammonia (StanLab, Lublin, Poland), 90.00 g of ethanol (StanLab, Lublin, Poland), and 0.525 g of cetyltrimethylammonium bromide (CTABr) (Fluka Analytical, Buchs, Switzerland). This process was carried out in a polyethylene bottle, for a period of 30 min, at 65 °C, using an ultrasonic bath. After 30 min, 0.84 g of tetraethyl orthosilicate (TEOS) (Aldrich Chemistry, Saint Louis, MO, USA) was added to the solution as a source of silicon. The entire solution was then stirred on a magnetic stirrer for 4 h at 65 °C.
After this time, the precipitate obtained was filtered on a glass funnel using a filter paper, washed with a mixture of distilled water and ethyl alcohol in a volumetric ratio of 1:1, and left to dry in air at room temperature. After drying, the precipitate was calcined in order to remove the templating agent (CTABr). The calcination was carried out for 5 h at 550 °C.

The preparation of a hierarchical material based on commercial FAU-type zeolite was based on dispersing commercial FAU zeolite (Alfa Aesar, Haverhill, MA, USA) in an amount of 0.50 g in a mixture containing 100.00 g distilled water, 1.25 g ammonia (StanLab, Lublin, Poland), 60.00 g of ethanol (StanLab, 96%), and 0.35 g of CTABr (cetyltrimethylammonium bromide) (Fluka Analytical). The whole mixture was placed in a polyethylene bottle and ultrasonicated for a period of 30 min at 65 °C. After this time, 0.56 g of tetraethyl orthosilicate (TEOS, silicon source) (Aldrich Chemistry, Saint Louis, MI, USA) was added to the solution as a source of silicon. In a further step of the synthesis, the whole mixture was stirred on a magnetic stirrer for 4 h at 65 °C, after which the resulting precipitate was drained off on a glass funnel using a filter strainer, washed with a mixture of distilled water—ethyl alcohol—in a ratio of 1:1, and left to dry in the air at room temperature. After drying, the material was calcined for 5 h at 550 °C.
TEOS molecules are incorporated into the interblock space and undergo simultaneous hydrolysis to form stable SiO₂ filaments between the individual sheets and the surfactants (CTABr), used for intercalation or swelling [45 (link)].