Ellagic

Trifluoracetic acid, acetonitrile, methanol, and ethanol were provided from Penta (Prague, Czech Republic). α-Amylase and neutral detergent solution package (containing sodium lauryl sulphate, EDTA disodium, sodium borate, sodium phosphate dibasic together with triethylene glycol) were purchased from Ankom Technology (Macedon, NY, USA). Folin-Ciocalteu reagent, AlCl₃·6H₂O were purchased from Penta (Prague, Czech Republic). The substance 2,2’-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS), radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), and standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were provided from Sigma-Aldrich (St. Louis, MI, USA). Phenolic and sugar standards were as follows: Epigallocatechin, catechin, epicatechin, rutin, quercetin, kaempferol, neochlorogenic, chlorogenic, gallic, protocatechuic, p‑hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, ellagic, o-coumaric, cinnamic acids and protocatechuic acid ethyl ester; D(+)-maltose, D(+)-glucose, D(−)-fructose, D(+)-rhamnose, D(+)-xylose, D(+)-saccharose, all were purchased from Sigma-Aldrich (St. Louis, MI, USA). All standards and solvents used in this study were of HPLC-grade (purity ≥ 98.5–99.0%). Total dietary fiber and resistant starch assay kits were provided from Megazyme International Ireland Ltd. (Wicklow, Ireland).

HPLC-grade methanol (≥99.9% purity) was supplied by Honeywell Riedel-de Haën AG, Seelze, Germany. The commercial enzymes, peroxidase from horseradish and tyrosinase from mushroom, ABTS (2,20- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), 1,2-dihydroxybenzene (Catechol), guaiacol, sodium carbonate, arbutin, rutin, gallic, caffeic, ellagic, syringic and trifluoroacetic (TFA) acids were purchased from Sigma-Aldrich (Sintra, Portugal). (+)-catechin, phloridzin, procyanidin B1, quercetin-3-O-glucoside, quercetin-3-O-galactoside, luteolin-7-O-glucoside, kaempferol-3-O-glucoside, epicatechin gallate and protocatechiuc acid were supplied at Extrasynthese (Genay Cedex, France). Potassium persulfate and Folin-Ciocalteu from Merk (Algés, Portugal) and acetonitrile was obtained from Fisher Scientific (Oeiras, Portugal).

About 100 g of air-dried powder aerial parts of the two samples of H. curassavicum (Coastal and Inland) were extracted in 70% hydro-methanol at room temperature (27 ± 2°C), filtered, and dried under vacuum to give dark black gum (1.6 and 1.85 g, respectively).
The standard phenolic, gallic, cinnamic, chlorogenic, ferulic, coffeic, syringic, ellagic, coumaric acids, vanillin, caffeine, propyl gallate, and flavonoids, quercetin, rutin, catechin, pyrocatechol, naringenin, 4‘.7-dihydroxy isoflavone, were purchased from Sigma-Aldrich (Germany). Trifloroacetic acid and acetonitrile (HPLC gradient grades) were purchased from Sigma-Aldrich (Germany). The used di-distilled water in HPLC was obtained by Hamilton water distillation apparatus (Hamilton Laboratory Glass Ltd., Kent, England).
HPLC analysis was performed using an Agilent 1260 series. The separation was carried out using a C18 column (4.6 × 250 mm i.d., 5 μm). The mobile phase contained water (A) and 0.02% trifloroacetic acid in acetonitrile (B) with a flow rate of 1 mL min⁻¹. The mobile phase was automated successively in a linear gradient as follows: 0 min (80% A), 0–5 min (80% A), 5–8 min (40% A), 8–12 min (50% A), 12–14 min (80% A), and 14–16 min (80% A). The multi-wavelength detector was monitored at 280 nm. The injection volume was 10 μL for each of the sample solutions. The column temperature was maintained at 35 °C.