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5 protocols using potassium persulfate

1

Antioxidant Assays and Zebrafish Analyses

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The fatty acids methyl esters (FAMEs) were purchased from Supelco Co. (Bellefonte, PA, USA). The standards of L-ascorbic acid (≥99%), gallic acid (>97.5%), DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-tripyridyl-S-triazine, ≥98%), ABTS (diammonium salt of 2,2′-azino-bis(3-ethylbenzthiazolin-6-yl)sulfonic acid, >99.0%), and dichloro-dihydro-fluorescein diacetate (≥97%, DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Organic solvents (dimethyl sulfoxide (DMSO, p.a.), ethanol (p.a.), methanol (p.a.)), hydrochloric acid (HCl, p.a.), iron (III) chloride (FeCl3, p.a.), Folin–Ciocalteu reagent and NaHCO3 (p.a.) were obtained from Kemika (Zagreb, Croatia), while potassium persulfate (>98%) was purchased from Scharlab (Barcelona, Spain). Hydrogen peroxide (H2O2, 30%) was obtained from Alkaloid Skopje (Skopje, North Macedonia).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
Zebrafish D. rerio adults of wild-type WIK strain were obtained from the European Zebrafish Resource Center of the Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Used solvents were of HPLC grade and were obtained from J.T. Baker (Bridgewater, NJ, USA).
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2

Antioxidant Compounds Extraction and Analysis

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The reagents sodium carbonate, di-sodium hydrogen phosphate, sodium carbonate, potassium ferricyanide, potassium persulfate, ethylenediamine tetra acetic acid (EDTA) and gallic acid were purchased from Scharlau (Barcelona, Spain). Folin–Ciocalteu’s phenol reagent, absolute ethanol and trichloroacetic acid (TCA) were purchased from Panreac (Castellar del Vallès, Barcelona, Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical, Iron (II) Chloride 4-hydrate, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylacid (Trolox), (L)-Dehydroascorbic acid and malondialdehyde (MDA) were purchased from Sigma Chemical Co. (Steinheim, Germany, and St. Louis, MO, USA). Propylgallate (PG) was purchased from Acrōs Organics (Fair Lawn, NJ, USA). 2-Thiobarbituric acid (TBA) was purchased from Merck KGaA (Darmstadt, Germany). The following standards were used: phenolic acids—hydroxybenzoic acid, vanillic acid, protocatechuic acid; cinnamic acids—chlorogenic acid, 4-coumaric acid; flavanols—catechin; flavonols—kaempferol, quercetin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-rutinoside, rutin or quercetin-3-O-rutinoside; flavanones—naringenin, eriodictyol, eriodictyol-7-O-glucoside (Merck KGaA, Darmstadt, Germany).
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3

Quantitative Antioxidant Assay Protocols

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The standards of gallic acid (>97.5%), L-ascorbic acid (≥99%), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (diammonium salt of 2,2′-azino-bis(3-ethylbenzthiazolin-6-yl)sulfonic acid, >99.0%), TPTZ (2,4,6-tripyridyl-S-triazine, ≥98%), dichloro-dihydro-fluorescein diacetate (≥97%, DCF-DA), AAPH (2,2-azobis (2-methylpropionamidine) dihydrochloride, 97%) and 2′,7′-dichlorofluorescin diacetate were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Dimethyl sulfoxide (DMSO, p.a.), methanol (p.a.), ethanol (p.a.), iron (III) chloride (FeCl3, p.a.), hydrochloric acid (HCl, p.a.), NaHCO3 (p.a.) and Folin–Ciocalteu reagent were obtained from Kemika (Zagreb, Croatia). Hydrogen peroxide (H2O2, 30%) was purchased from Alkaloid (Skopje, Macedonia) and potassium persulfate (>98%) from Scharlau, (Regensburg, Germany).
The solvents used for SPE were of HPLC grade and were obtained from J.T. Baker (New Jersey, PA, USA).
Acetonitrile with 0.1% (v/v) formic acid and water with 0.1% (v/v) formic acid, both hypergrade for HPLC-MS LiChrosolv®, were purchased from Supelco Co. (Bellefonte, PA, USA).
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4

ABTS Radical Scavenging Activity Assay

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The anti-free radical activity was also evaluated by ABTS, 2,2-azinobis (3 ethylbenzothiazoline-6-sul-fonic acid) diammonium salt (Sigma-Aldrich Chemie, Steinheim, Germany) radical cation decolourisation test using spectrophotometric method [15] (link). ABTS was dissolved in distilled water to a concentration of 7 nM.
The solution of the ABTS cation radical was obtained by incubating for 12 to 16 h in the dark and at room temperature a mixture of equal volumes of the stock ABTS solution with a solution of potassium persulfate (99%, Scharlab SL, Spain) at 2.45 nM. ABTS cation solution was diluted with ethanol to an absorbance of 0.700 ± 0.02 at 734 nm before use. Then 1500 µl of ABTS cation solution were mixed with 50 µl of extract solution or reference (ascorbic acid) at different concentrations (same of the DPPH). The samples were kept in the dark for 15 minutes at room temperature and absorption was measured at 734 nm on a spectrophotometer. All determinations were carried out in triplicate. Results were expressed as percentage inhibition (PI) as previously described for DPPH test.
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5

Antioxidant Assays Protocol

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The standards of gallic acid (>97.5%), L-ascorbic acid (≥99%), DPPH (2,2-diphenyl-1picrylhydrazyl), ABTS (diammonium salt of 2,2´-azino-bis(3-ethylbenzthiazolin-6-yl) sulfonic acid, >99.0%), dichloro-dihydro-fluorescein diacetate (≥97%, DCF-DA), AAPH (2,2-azobis (2-methylpropionamidine) dihydrochloride, 97%) and 2´,7´-dichloro fluorescin diacetate were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Dimethyl sulfoxide (DMSO, p.a.), methanol (p.a.), ethanol (p.a.), hydrochloric acid (HCl, p.a.), sodium carbonate (Na2CO3, p.a.), dibasic sodium phosphate (Na2HPO4*2H2O, p.a.), monobasic sodium phosphate (NaH2PO4*H2O, p.a.) and Folin-Ciocalteu reagent were obtained from Kemika (Zagreb, Croatia) while potassium persulfate (>98%) was purchased from Scharlau, (Regensburg, Germany).
The solvents used for solid-phase extraction were of HPLC grade and were obtained from Honeywell (Charlotte, NC, USA).
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