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4 protocols using sulphuric acid

1

Comprehensive Chemical Reagent Procurement

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Copper(II) sulphate pentahydrate, citric acid monohydrate, anhydrous sodium carbonate, potassium hexacyanoferrate(II) trihydrate, zinc acetate, calcium carbonate, potassium iodide, sulphuric acid, potassium thiocyanate, starch, phenolphthalein, sodium hydroxide, sodium thiosulfate and sodium chloride were purchased from Kemika (Zagreb, Croatia). Myrtenol standard was obtained from Sigma Aldrich (St. Lois, MO, USA) and C7-C30 saturated alkanes standard from Merck (Darmstadt, Germany). Helium for gas chromatography was obtained from Messer, Austria.
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2

Analytical Protocols for Polyphenol Identification

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Sodium potassium l(+)-tartrate tetrahydrate was obtained from VWR Chemicals (Vienna, Austria). Potassium iodide was obtained from BDH Prolabo Chemicals (Leuven, Belgium). Iodine, sodium hydroxide (1 and 0.1 M), sodium thiosulfate (0.1 M) and sodium hydroxide pellets were obtained from Gram-mol (Zagreb, Croatia). Sulphuric acid (96%), starch (p.a., soluble) and phenolphthalein were obtained from Kemika (Zagreb, Croatia). Bromothymol blue was obtained from Merck (Darmstadt, Germany). (+)-Catechin, (-)-epicatechin, 2,5-dihydroxybenzoic acid (2,5-DHBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3-hydroxytyrosol, caffeic acid, ellagic acid, quercetin, naringenin, luteolin-7-O-glucoside, pinobanksin, p-coumaric acid and syringic acid were obtained from Sigma-Aldrich, Merck (St. Louis, MO, USA). Gallic acid was obtained from Alfa Aesar (Thermo Fischer Scientific, Tewksbury, MA, USA). Ferulic acid and resveratrol were obtained from Extrasynthese (Genay, France). Honeywell research chemicals (Charlotte, NC, USA) supplied ethanol (HPLC grade) and acetonitrile (LC-MS grade). Sigma-Aldrich supplied formic acid (LC-MS grade), ultrapure water (LC-MS grade) and ethanol (96%).
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3

Antioxidant Compound Characterization

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Acetic anhydride, acetic acid, sulphuric acid (96%), iron(III) chloride, potassium iodide and mercury(II) chloride were purchased from Kemika (Zagreb, Croatia). Methanol, dichloromethane, ethyl acetate, cyclohexane and potassium peroxodisulphate were obtained from VWR Chemicals (Radnor, PA, USA) while chloroform was obtained from Honeywell (Charlotte, NC, USA). ABTS and linoleic acid were purchased from Alfa Aesar (Kandel, Germany). Trolox® was obtained from Acros Organics (Geel, Belgium). DPPH, β-carotene and lipoxidase type I-B from soybean were purchased from Sigma-Aldrich (Darmstadt, Germany). Finally, Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were obtained from Capricorn Scientific (Ebsdorfergrund, Germany).
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4

Triterpene Identification in Plant Extracts

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In order to monitor the presence of triterpene substances in different fractions obtained by Soxhlet extraction, a TLC method was performed using pre-coated silica gel GF254 plates (20x20 cm, thickness 0.25 mm, Merck, Dürmsttadt). The solvent system used as eluent was benzene (Merck, Germany): ethyl acetate (Sigma-Aldrich, US): formic acid (36:12:5) (Kemika, Zagreb). Detection of triterpene substances was achieved by observation under UV354/254, spraying with 4anisaldehyde (Merck, Germany) -sulphuric acid (Kemika, Zagreb) and heating with a heat gun until full color development. Since the triterpene compounds are the subject of the study, the triterpene standards were used for their identification in different extracts.
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