Gallic acid

Potentiometric measurements were carried out using an Expert-pH pH-meter (OOO Ekoniks-expert, Moscow, Russia) with the EMF measurement function and the RS-232 interface. A two-electrode electrochemical cell was used. The working electrode was the EPV-1 redox platinum electrode, and the reference electrode was the EVL-1M silver/silver chloride electrode (Ag/AgCl/3 mol/dm³ KCl) (Gomel ZIP, Gomel, Belarus). The studies were carried out in a thermostated cell at 37 °C using the LOIP LT-205a circulation thermostat (ZAO LOIP, Saint Petersburg, Russia).
The following reagents were used in the work: 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), α-tocopherol, 2,6-ditretbutylphenol, ionol, uric acid, glutathione (Sigma-Aldrich, St. Louis, MO, USA); K₄[Fe(CN)₆], K₃[Fe(CN)₆], KH₂PO₄, Na₂HPO₄·12H₂O (Reakhim, Moscow, Russia); ascorbic acid, L-cysteine, catechol, gallic acid, chlorogenic acid, caffeic acid (Panreac, Barcelona, Spain). All reagents were analytical grade. The studies were carried out in phosphate buffer solution (PS) pH = 7.4 KH₂PO₄/Na₂HPO₄·12H₂O.

Ground F. vesiculosus from July 2017 was purchased from Algaplus Lda (Aveiro, Portugal). Acetone, ethanol, acetonitrile HPLC grade, hydrochloric acid, and glacial acetic acid were acquired from Fisher Chemical (Pittsburgh, PA, USA). The enzymes α-glucosidase from Saccharomyces cerevisiae (EC No.—3.2.1.20) and xanthine oxidase from bovine milk (EC No.—1.17.3.2), together with 2,4-dimethoxybenzaldehyde (DMBA), phloroglucinol, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS-NH₄), nitrotetrazolium blue chloride (NBT), phenazine methosulfate (PMS), 4-nitrophenyl α-D-glucopyranoside (pNPG), allopurinol, ascorbic acid, and formic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium persulfate, potassium di-hydrogen phosphate, potassium hydroxide, sodium di-hydrogen phosphate 1-hydrate, and gallic acid were acquired from Panreac (Barcelona, Spain). β-nicotinamide adenine dinucleotide (β-NADH), trolox, 3,5-dinitrosalicylic acid (DNS), acarbose, and 4-nitrophenol were purchased from Acros Organics (Hampton, NH, USA) and dimethylsulfoxide (DMSO) were acquired from Honeywell Riedel-de Haën (Charlotte, NC, USA), and xanthine were purchased from AlfaAesar (Ward Hill, MA, USA). All reagents were of analytical grade or of the highest available purity.

Ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH^●), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS^●+), neocuproine, trichloroacetic acid (TCA), and potassium ferricyanide were used for the determination of the antioxidant activities and were purchased from Sigma Chemical Co. (Sigma-Aldrich GmbH, Stern-heim, Germany). The following chemicals were used for the enzyme inhibition assays: α-glucosidase from Saccharomyces cerevisiae, 4-nitrophenyl α-D-glucopyranoside (pNPG), α-amylase from porcine pancreas, β-nicotinamide adenine dinucleotide (β-NADH), phenazinemethosulphate (PMS), nitrotetrazolium blue chloride (NBT) and Ascorbic acid were obtained from Sigma (St. Louis, MO, USA), while acarbose and potato starch were purchased from Fluka (Bucharest, Romania) and Fisher (Pittsburgh, PA, USA), respectively. Sodium nitroprusside, sulfanilamide, and 3,5-dinitrosalicylic acid (DNS) were obtained from Acros Organics (Hampton, NH, USA). Folin–Ciocalteu reagent, Na₂CO₃, and gallic acid were purchased from Panreac (Barcelona, Spain). Standards used for the elucidation of the phenolic compounds and for elaboration of the calibration curves were obtained from EXTRA SYNTHESE (Genay Cedex, France). Acetonitrile HPLC-grade and formic acid were purchased from Panreac (Barcelona, Spain). All other chemicals were of analytical grade.

HCl (hydrochloric acid), 2,6-DCFI (2,6-dichloroindophenol), FCR (Folin–Ciocalteu reagent), K₂S₂O₈ (potassium persulfate), ethanol, and gallic acid were purchased from Panreac (Barcelona, Spain). Na₂HPO₄ (sodium hydrogen phosphate), KH₂PO₄ (potassium dihydrogen phosphate), and NaHCO₃ (sodium hydrogen carbonate) were obtained from Scharlab (Barcelona, Spain). Na₂CO₃ (sodium carbonate), acetone, acetonitrile, and ascorbic acid were provided by VWR International (Lovaina, Belgium). Formic acid was purchased from Merck (Darmstadt, Germany). Acetic acid, hexane, and methanol were supplied by J.T. Baker (Deventer, The Netherlands). HPO₃ (metaphosphoric acid), Trolox ((+/−)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), AAPH (2,2′-azobis (2-methyl)propionamidine), fluoresceine, protocatechuic acid, (+)-catechin, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, naringin, hesperetin, hesperidin, and apigenin were obtained from Sigma-Aldrich (St. Louis, USA). Rutin trihydrate and quercetin were obtained from HWI Analytik GmbH (Ruelzheim, Germany). Analytical grade chemicals and distilled water were used.

The total polyphenol content (TPC) of the CE was determined spectrophotometrically according to Arnous et al. (2002), with some modifications [19 (link)]. Briefly, in a hemolysis tube, 2300 μL of ultrapure water, 100 μL of previously prepared extract at different concentrations, 150 μL of Folin–Ciocalteu’s reagent (Carlo Erba, Milan, Italy), and 450 μL of 20% Na₂CO₃ (Carlo Erba, Milan, Italy) were mixed. After 2 h in the dark at room temperature, the absorbance of the blue color was measured at 750 nm against a blank sample with a UV–VIS 50 Bio Varian spectrophotometer. A calibration curve with gallic acid (Panreac, Barcelona, Spain), as standard was produced. The TPC was expressed as the milligram equivalent of gallic acid per gram of dry matter (mg GAE/g DM). All TPC measurements were performed in triplicate.

All reagents used in this work were analytical grade and used without changes. The analytical procedures were performed using water purified by a Milli-Q system from Millipore (Bedford, MA, USA). LC-MS grade methanol and acetic acid were purchased from Fisher Chemicals (Waltham, MA, USA) and Sigma-Aldrich (Steinheim, Germany), respectively.
For TPC and TFC and antioxidant capacity, the following reagents were provided from the indicated suppliers: gallic acid, sodium acetate, ferric chloride and hydrochloric acid were purchased from Panreac (Barcelona, Spain) and ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate)], ferric sulphate, Folin–Ciocalteu reagent, potassium persulfate, TPTZ (2,4,6-tripyridyl-S-triazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), vanillin and (+)-catechin from Sigma-Aldrich (St. Louis, MO, USA).

All chemical standards were HPLC-grade pure. Folin-Ciocalteu reagent, gallic acid, aluminum chloride, sodium carbonate, potassium iodide, and bisulfite were purchased from Panreac (Barcelona, Spain). Quercetin and 2,2-diphenyl-1-picrilhidrazil (DPPH) were purchased from Alfa Aesar (Massachusetts, USA) and methanol was obtained from Merck (Darmstandt, Germany). Hydrolyzed starch for diastase determination was purchased from Carlo Erba (Barcelona, Spain). The calibration of the HANNA Honey Color C221 colorimeter was with glycerin was provided by Glycerol HANNA instruments (Woonsocket, Rhode Island, USA). Standards of glucose, fructose, sucrose, maltose, trehalose, turanose and melezitose were obtained from Sigma–Aldrich (Madrid, Spain).