Methanol

The ability to neutralise the DPPH (2.2-diphenyl-1-picrylhydrazyl) radical was determined on the basis of colorimetric changes in the concentration of the stable DPPH radical in relation to the control sample [34 (link),35 (link)]. The phenolic extract solution (0.3 mL) was mixed with a methanol (Stanlab, Lublin, Poland) solution containing DPPH (Aldrich, St. Louis, MO, USA) radicals (0.4 mM, 4 mL). Measurement of the absorbance was made at a wavelength λ = 517 nm, after 20 min incubation at room temperature, without light (Thermo Scientific, Helios Zeta UV-VIS, Madison, WI, USA). The ability of the tested extracts to counteract the oxidation reaction was calculated from the formula: $\%\mathrm{inhibition}=100-\left\{\frac{\left[\left({\mathrm{A}}_{\mathrm{w}}-{\mathrm{A}}_0\right)\times 100\right]}{{\mathrm{A}}_{\mathrm{k}}}\right\}$
where: A_w—absorbance of a specific sample (the tested extract); A₀—absorbance of the zero sample; A_k—absorbance of the control sample (with a synthetic DPPH radical).

The support 3-(glycidoxy)propyl-functionalized silica-gel (SiO₂-epoxy) was obtained from SiliCycle Inc. (Quebec, QC, Canada). According to the specification card, the particle sizes ranged between 40 and 63 μm, the molecular loading (grafting level) was 1.21 mmol g⁻¹, the specific surface area equaled 494 m² g⁻¹, the pore diameter was 60 Å and pore volume was 0.74 mL g⁻¹. All the reagents were commercially available products used with no further purification. Ethylenediamine, triethylenetetramine, tris(2-aminoethyl)amine, 4,7,10-trioxa-1,13-tridecanediamine, methyl acrylate, folic acid, salicylic acid, nicotinic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the solvents and salts used for the buffers preparation were of the purity grade p.a. Methanol and hydrochloric acid (HCl) were purchased from STANLAB (Lublin, Poland). N,N-dimethylformamide (DMF), dichloromethane (DCM), acetic acid (AcOH), sodium acetate (AcONa) and potassium chloride (KCl) were obtained from Eurochem (Tarnow, Poland). Di- and monosodium phosphates (Na₂HPO₄ and NaH₂PO₄) were purchased from POCH (Gliwice, Poland).

Isopropanol (p.a., POCh. S.A., Gliwice, Poland), acetone, methanol (p.a., P.P.H. STANLAB, Lublin, Poland) and deionized water (DI, with conductivity of 0.05 µS) were used during the sonication process. WO₃ NFs were synthesized using anodic oxidation of tungsten foil (0.127 mm, 99.9% purity, Sigma-Aldrich, Darmstadt, Germany) in the aqueous electrolyte composed of sulfuric acid solution (96%, p. a., P.P.H. STANLAB, Lublin, Poland) and sodium fluoride (p. a., P.P.H. STANLAB, Lublin, Poland).

The biomass harvested from the tested N. officinale microshoot cultures was immediately frozen in liquid N₂ and lyophilized (Labconco Corporation, Kansas City, MO, USA). The biomass was pulverized in a mixing ball mill (MM400, Retch, Haan, Germany). Samples (0.2 g) were weighed out and extracted twice in 4 mL of methanol (STANLAB, Lublin, Poland) under sonication for 20 min in an ultrasonic bath (POLSONIC 2, Warsaw, Poland). Then, the samples were centrifuged (8 min, 2000× g; MPW-223E; MPW, Warsaw, Poland) and filtered (0.22 μm syringe filters; Millex^®GP; Merck Millipore, Burlington, MA, USA). If not otherwise stated, the extract was used for further analyses.

As incubation was completed, the BC discs and the medium over the biofilm were removed. A total of 1 mL of 0.1% (w/v) solution of tetrazolium chloride (TTC, 2,3,5-triphenyl-tetrazolium chloride, AppliChem Gmbh, Damstadt, Germany) in TSB was added for 2 h (37 °C) (Plate 3). Next, the solution was gently removed, and 1 mL of methanol (Stanlab, Lublin, Poland): acetic acid (Chempur, Piekary Slaskie, Poland) mixture (9:1) was added. The plate was shaken for 30 min/400 rpm at RT. From each well, three samples for 100 µL were transferred to 96-well plates (Jet Bio-Filtration Co. Ltd., Guanzhou, China), and absorbance was measured at 490 nm with a spectrophotometer (Multiskan Go, Thermo Fisher Scientific, Vantaa, Finland). Each EO and control were tested in six replicates. Compared to the biofilms treated with NaCl, reduction in biofilm metabolic activity was defined as a percentage.

2-(2-Butoxyethoxy)ethanol (Acros Organics, Geel, Belgium, 99+%), 2,6-di-tert-butyl-4-methylphenol (BHT, Acros Organics, 99%), o-dichlorobenzene (Acros Organics, 99%), C₆D₄Cl₂ (99 atom % D, Deutero GmbH, Kastellaun, Germany), acetone (analytical grade, POCH, Gliwice, Poland) and methanol (Stanlab, Lublin, Poland, analytical grade) were used as received.

Naringenin and alkyl iodides (1-iodoheptane, 1-iodooctane, 1-iodononane, 1-iodoundecane) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA); hydroxylamine hydrochloride from LOBA Feinchemie GmbH (Fischamed, Austria); anhydrous sodium acetate, potassium carbonate and N,N-dimethylformamide (DMF) from Chempur (Piekary Śląskie, Poland).
Anhydrous ethanol for reactions was prepared according to standard procedures. All organic solvents used for extraction and purification (diethyl ether, hexane, ethyl acetate, methylene chloride, chloroform, methanol) were of analytical grade and were purchased from Stanlab (Lublin, Poland).