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5 protocols using nahco3

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

Analytical Reagents for Research Protocols

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All reagents and solvents used for the analyses were of analytical grade. Pepsin was obtained from Sigma Aldrich GmbH (Taufkir-chen, Germany), salts (NaCl, KCl, NaH2PO4, Na2SO4, NaHCO3, CO(NH2)2, CaCl2), ethanol, and hydrochloride acid were obtained from Kemika (Zagreb, Croatia). DPPH was obtained from Merck KgaA (Darmstadt, Germany).
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3

Cyclodextrin-based Formulation Development

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PR was purchased from Henan Lihua Pharmaceutical Company (Henan, China). hydroxypropyl-γ-cyclodextrin (HPGCD) was donated by Cyclolab Ltd. (Budapest, Hungary), ZnHA and ZnGlu from Gedeon Richter Plc (Budapest, Hungary). Hydroxypropyl-β-cyclodextrin (HPBCD) was obtained from Wacker-Chemie GmbH (Munich, Germany). Dimethyl sulfoxide (DMSO), BK, NaCl, boric acid, and borax (for borate buffer) were obtained from Molar Chemical Ltd. (Halásztelek, Hungary), KCl, NaHCO3, D-glucose monohydrate from Kemika (Zagreb, Croatia), CaCl2 × 2H2O and MgCl2 × 6H2O from Sigma-Aldrich, Chemie GmbH (Steinheim, Germany). Ringer-buffer was used as medium during the experiments. The pH of Ringer buffer was set to 7.4.
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4

Bronze Alloy Patination and Coating

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Experiments were performed on 0.5 cm thick discs, which were cut from a bronze rod with a 1.2 cm diameter, received from Strojopromet Ltd., Zagreb, Croatia. The composition of the bronze alloy is given in Table 1.
K2Sn (Sigma-Aldrich Corp.,Saint Louis, MO, USA) was used for bronze patination. 16-phosphonohexadecanoic acid (PA, 97%, Sigma-Aldrich Corp. Saint Louis, MO, USA) dissolved in ethanol (96% p.a., Lach-ner d.o.o., Zagreb, Croatia) was used for patinated bronze pretreatment. Paraloid B-72 (C.T.S., Altavilla Vicentina, Italia) dissolved in ethyl acetate (p.a., T.T.T., Sveta Nedjelja, Croatia) was used for bronze coating. Artificial rain was prepared by dissolving 0.2 g/L Na2NO3 (p.a., T.T.T., Sveta Nedjelja, Croatia), 0.2 g/L NaHCO3 (p.a., Kemika, Zagreb, Croatia), and 0.2 g/L Na2SO4 (p.a., Kemika, Zagreb, Croatia) in deionized water. The pH of the obtained artificial rain was adjusted to 5 with 0.5 M H2SO4. The conductivity of the artificial rain solution was 0.56 mS cm−1.
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5

Establishing Methods for Serum Analysis

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Reagents. -Ultrapure water (electrolytic conductivity 0.055 μS cm -1 ) was used throughout. Hydrochloric acid 35 % (Lach-Ner, Czech Republic), glycerol (Gram-mol, Croatia), NaHCO 3 (Kemika, Croatia), Na 2 HPO 4 × 2H 2 O (Merck, Germany), KCl (Lach-Ner), MgCl 2 × 6H 2 O (Kemika), CaCl 2 × 2H 2 O (Merck Alkaloid, Macedonia), NaCl (Zorka, Serbia) and HNO 3 65 % (Kemika) were all of pro analysi grade.
Human albumin 20 % and human intravenous immunoglobulin 5 % were both from the Institute of Immunology (Zagreb, Croatia).
Serum samples. -All experiments were approved by the Ethical Committ ees of the Srebrnjak Children's Hospital, Zagreb, Croatia, and Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia. To establish new methods, the pool of remnant children's sera was used. All sera samples were obtained from the Children's Hospital Srebrnjak, Zagreb, Croatia, in 2006 and 2007, and were stored at -18 °C. Before analysis, the serum sample was thawed overnight and mixed for 10 min.
HCl-acidifi ed serum pool was prepared by adding 3 mol dm -3 HCl into the serum pool to a fi nal dilution ratio 1:5 (AS/5) or 1:10 (AS/10).
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