Folin ciocalteau s phenol reagent

Sodium persulfate, ferrous chloride, Folin-Ciocalteau’s phenol reagent, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), and (+)-catechin were purchased from Sigma (Poznań, Poland). Acetonitrile high-performance liquid chromatography (HPLC) grade and methanol were obtained from P.O.Ch. Company (Gliwice, Poland).

Cocoa shells, a by-product of the Kandit chocolate factory (Osijek, Croatia) were obtained in 2017. Before the extraction, the plant material was ground using a standard laboratory mill. DPPH and ethyl acetate were purchased from Sigma-Aldrich Chemie (Steiheim, Germany). All other solvents were of analytical grade and purchased from J.T. Baker (J.T. Baker, Phillipsburg, NJ, USA). Folin-Ciocalteau’s phenol reagent was purchased from Sigma-Aldrich Chemie (Steiheim, Germany). Caffeine (purity 99%), theobromine (purity 98.5%), catechin (purity 99%), epicatechin (purity 97%) gallic acid (purity 99%) and chlorogenic acid (purity 98%) were purchased from Sigma-Aldrich. Theophylline (purity 99%), furfural (purity 99%) and 5-HMF (purity 98%) were purchased from Acros Organics (Geel, Belgium). Sugars (glucose, rhamnose, arabinose, mannose, xylose and fucose) and carboxylic acids (levulinic acid, lactic acid and formic acid) with high-purity (>99%) for HPLC analysis were obtained from Sigma-Aldrich.

All chemicals were obtained as pure commercial products and used without further purification. Standards of fatty acids and fatty acid methyl esters, Desferal (deferoxamine mesylate salt), Trolox, Folin-Ciocalteau’s phenol reagent, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), kojic acid, allopurinol, XO from cow’s milk, xanthine, and all solvents used, of the highest available purity, were from Sigma-Aldrich (Milan, Italy). The methanolic HCl (3 N) was purchased from Supelco (Bellefonte, PA).

Methyl alcohol and acetonitrile (Romil Chemical Ltd., Heidelberg, Germany), trifluoroacetic acid TFA (Merck; Darmstadt, Germany) and acetic acid AcOH (Merck) were HPLC grade. Concentrated aqueous hydrochloric acid (HCl) solution (32%) was from Merck. Deionised water was purified on a Milli-Q system (Millipore, Bedford, MA, USA). L(+)-Tartaric acid and ethyl alcohol of analytical grade were provided by Merck. NaH₂PO₄/Na₂HPO₄ 0.1 M solution buffer, which was prepared using monosodium phosphate (Sigma-Aldrich, Steinheim, Germany) and sodium phosphate dibasic (Merck), and sodium hydroxide NaOH (Panreac, Barcelona, Spain). Solution for phloroglucinolysis was prepared using phloroglucinol and l-ascorbic acid 99%+ reagent (Sigma-Aldrich, St. Louis, MO, USA), hydrochloric acid 37% and MeOH with HPLC grade (VWR, Fontenay-sous-Bois, France). Stop solution was prepared using sodium acetate (VWR) and Milli-Q water. Folin-Ciocalteau’s phenol reagent, sodium carbonate 99.5%+ granular and gallic acid monohydrate 98%+ were provided by Sigma-Aldrich. Standards of malvidin-3-O-glucoside (Mv-3-glc) and (+)-catechin were supplied by Extrasynthèse (Genay, France). All solvents used were previously filtered through 0.45 μm nylon membranes (Lida, Kenosha, WI, USA).

3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt (Ferrozine^®), α,α-diphenyl-β-picrylhydrazyl (DPPH), Folin–Ciocalteau’s phenol reagent, gallic acid, ascorbic acid, d-glucose, gelatin, Coomassie Brilliant Blue R-250, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA) were all purchased from Sigma Aldrich-Merck (St. Louis, MO, USA). 30% Acrylamide/Bis 37.5:1 solution, ammonium persulfate (APS), 1,2-Bis (dimethylamino) ethane (TEMED), Tris/Glycine buffers, nitrocellulose membranes (0.45 μM) blotting membranes and Clarity Western ECL solution were purchased from Bio-Rad. Disposable plastics were from Corning. Photometric measurements from multi-well plates were recorded on an iMARK microplate reader (Bio-Rad, Hercules, CA, USA). When not otherwise specified, all chemicals and solvents, such as ethanol, methanol and n-hexane, were of the highest analytical grade and were purchased from Sigma Aldrich-Merck.

Medium molar mass chitosan (CS) was purchased from Sigma Aldrich Brazil Ltd. (São Paulo, SP, Brazil). Its degree of acetylation (DA = 15%) was determined by ¹H NMR spectrometry in D₂O/DCl at 65 °C. Its viscosity average molar mass ( ${\overline{M}}_v)$ of ~2.97 × 10⁵ was determined previously [69 (link)]. Glycerol (Gly) and choline chloride (ChCl) were purchased from Vetec Química Fina Ltda. (Rio de Janeiro, RJ, Brazil) and Sigma Aldrich Brazil Ltd. (São Paulo, SP, Brazil), respectively. Açaí pulp oil (AO) was purchased from Beraca Ingredientes Naturais S.A. (Ananindeua, PA, Brazil). Folin-Ciocalteau’s phenol reagent, 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid and (±)-6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) as well as 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma–Aldrich (Milan, Italy). All other chemicals used in this study were of analytical grade. Ultrapure water from a Millipore Direct-Q 3 UV system was used.