Iron 2 chloride

The following reagents were used as received for the synthesis of the COPP sample: iron (III) chloride (FeCl₃·6H₂O, 99%, Merck), iron (II) chloride (FeCl₂·4H₂O, ≥ 99%, Merck) and sodium hydroxide (NaOH, ≥ 99%, Merck). The NaOH solution (0.17275 mol, 1.15 M) was dropwise added to the aqueous solution containing FeCl₃ (0.043 mol, 0.2 M) and FeCl₂ (0.0215 mol, 0.1 M). The co-precipitation reaction was carried out in an Argon atmosphere under continuous stirring at 60 °C. The precipitate was separated by normal filtration, thoroughly washed with deionized water and absolute ethanol (no chlorine ions were detected in the filtrate, AgNO₃ solution was used), and finally air-dried for 24 h at 80 °C.

All the chemicals, including Folin-Ciocalteu’s phenol reagent, polyvinylpyrrolidone (PVP), sodium carbonate, tannic acid (98% purity), aluminum chloride hexahydrate (AlCl₃·6H₂O; Ph Eur purity), quercetin (98% purity), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH; 95% purity), 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH; 97% purity), 2,2-azino-bis (3-thylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; 98% purity), trolox (97% purity), ferrozine (97% purity), iron (II) sulfate heptahydrate (FeSO₄·7H₂O; 99% purity), iron (III) chloride (FeCl₃·6H₂O; 97% purity), hydroxylamine hydrochloride (98% purity), rutin (99% purity), bovine serum albumin, glucose, fructose, sodium azide, iron (II) chloride (FeCl₂·4H₂O; 99% purity), tert-butyl hydroperoxide (tBOOH; 80% purity), and methanol were purchased from Merck (Darmstadt, Germany). RPMI 1640 medium and fetal bovine serum were provided by Aurogene (Rome, Italy).

Cysteine (98.8%), alanine (97.6%), NaH₂PO₄ (AR), and Na₂HPO₄ (AR) were procured from Sigma-Aldrich. Sodium hypochlorite (NaOCl, AR), iron(ii) chloride (AR), 1,10-ortho-phenanthroline (AR), hydrochloric acid (HCl, AR), acetic acid (CH₃COOH, AR), and sodium hydroxide (NaOH, AR) were procured from Merck.

n-Hexane, ethyl acetate, and methanol (all HPLC grade) were obtained from Fisher Chemical (Argenta, Poznań, Poland). A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2′-azinobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), ferrozine, Trolox, Folin-Ciocalteu reagent, gallic acid (≥98%), luteolin (>99%), EDTA, iron (II) chloride, potassium persulfate, sodium carbonate (all ACS grade), and acetonitrile and formic acid (both MS grade) were obtained from Merck (Merck, Warsaw, Poland). Melatonin and Melatonin-d₄ standards were purchased from Cayman Chemical (Biokom, Janki, Poland). Sulfuric, perchloric, and nitric acids, and hydrogen peroxide (all ACS grade) were purchased from Avantor (Gliwice, Poland). Ultrapure water was prepared with a Milli-Q purification system (Millipore, Burlington, MA, USA).

Sodium dihydrogen phosphate (NaH₂PO₄), potassium ferrocyanide (K₃Fe(CN)₆), trichloroacetic acid (TCA), iron-III-chloride (FeCl₃), potassium peroxydisulfate (K₂O₈S₂) copper-II-chloride (CuCl₂), sodium acetate (NaCH₃COO), hydrochloric acid (HCl), tris, iron-II-sulfate (FeSO₄), iron-II-chloride (FeCl₂), disodium hydrogen phosphate (Na₂HPO₄), methionine, ethanol, ethylenediaminetetraacetic acid (EDTA), ammonium thiocyanate (NH₄SCN), sodium bicarbonate (NaHCO₃), sodium hydroxide (NaOH), disodium sulfate (Na₂SO₄), sodium perchlorate (NaClO₄), and disodium carbonate (Na₂CO₃) were obtained from Merck (Merck, made in Germany). Cis-9, cis-12-octadecanoic acid (linoleic acid), 2,2-diphenyl-1-picrylhydrazyl (DPPH), polyoxyethylene sorbitan monolaurate (Tween 20), nitroblue tetrazolium (NBT), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt (ferrozine), riboflavin (vitamin B2), N-N-Dimethyl-p-phenyl-enediamine dihydrochloride (DMPD), neocuproine hydrate (C₄H₁₂N₂·XH₂O), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium sulfate (ABTS), ID-8 (C₆H₁₄N₂O₄), 3,4-dihydroxy-5-methoxybenzoic acid (C₈H₈O₅), silychristin (C₂₅H₂₂O₁₀), malvin (C₂₉H₂₈O₁₇), pelargonin (C₂₇H₃₁O₁₅), oenin (C₂₃H₂₅O₂), arachidonoyl dopamine (C₂₈H₄₁NO₃), callistephin (C₂₁H₂₁O₁₀), and 2,4,6-trihydroxy-benzaldehyde (C₆H₂CHO) were purchased from Sigma Aldrich Company (St Louis, MO, USA).

Acetone and methanol were obtained from Valente and Ribeiro (Lisbon, Portugal). Ethanol (96%) was purchased from “AGA-Álcool e Géneros Alimentares”, S.A. (Lisbon, Portugal). Monobasic potassium phosphate was from Merck (Darmstadt, Germany), while dibasic potassium phosphate, iron (II) chloride (FeCl₂), ferrozine, ethylenediaminetetraacetic acid (EDTA), pyrocatechol violet (PV), copper sulfate (CuSO₄), sodium acetate, choline chloride, glucose, fructose, sucrose, and xylose were provided by VWR International (Leuven, Belgium). Panreack AppliChem ITW Reagents (Barcelona, Spain) supplied fluorescein and Acros Organics (Geel, Belgium) provided 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and Trolox. Sigma-Aldrich (Lisbon, Portugal) delivered DPPH and butylated hydroxytoluene (BHT).