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Differentiation between stoichiometric and anticatalytic antioxidant properties of benzoic acid analogues: a structure/redox potential relationship study.

Chemico-biological interactions (2013-09-26)
Thierry Franck, Ange Mouithys-Mickalad, Thierry Robert, Gianangelo Ghitti, Ginette Deby-Dupont, Philippe Neven, Didier Serteyn
ABSTRACT

We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities of phenolic acid derivatives were studied by measuring their capacity to scavenge the radical cation 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS(+)) and reactive oxygen species (ROS) produced by stimulated neutrophils. The anticatalytic antioxidant capacity of the molecules was evaluated on the activity of myeloperoxidase (MPO), an oxidant enzyme present in and released by the primary granules of neutrophils. The ROS produced by PMA-stimulated neutrophils were measured by lucigenin-enhanced chemiluminescence (CL) and the potential interaction of the molecules with MPO was investigated without interferences due to medium by Specific Immuno-Extraction Followed by Enzyme Detection (SIEFED). The antioxidant activities of the phenolic compounds were correlated to their redox potentials measured by differential pulse voltammetry (DPV), and discussed in relation to their molecular structure. The ability of the phenolic molecules to scavenge ABTS radicals and ROS derived from neutrophils was inversely correlated to their increased redox potential. The number of hydroxyl groups (three) and their position (catechol) were essential for their efficacy as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential. Likewise, for the inhibition of MPO activity the number of OH groups and mainly the elongation of the carboxylic group were essential, probably by facilitating the interaction with the active site or the structure of the enzyme. The redox potential measurement, combined with ABTS and CL techniques, seems to be a good technique to select stoichiometric antioxidants but not anticatalytic ones, as seen for MPO, what rather involves a direct interaction with the enzyme.

MATERIALS
Product Number
Brand
Product Description

Supelco
Mettler-Toledo Calibration substance ME 18555, Benzoic acid, analytical standard, for the calibration of the thermosystem 900, traceable to primary standards (LGC)
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Pyrocatechol, purified by sublimation, ≥99.5%
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Pyrocatechol, suitable for plant cell culture, BioReagent, ≥99%, powder
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Benzoic acid, meets analytical specification of Ph. Eur., BP, USP, FCC, E210, 99.5-100.5% (alkalimetric)
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Benzoic acid, SAJ first grade, ≥99.5%
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Benzoic acid, SAJ special grade, ≥99.5%
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Benzoic acid, Pharmaceutical Secondary Standard; Certified Reference Material
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Melting point standard 121-123°C, analytical standard
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Benzoic acid, purified by sublimation, ≥99%
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Benzoic acid, natural, ≥99.5%, FCC, FG
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Benzoic acid, ≥99.5%, FCC, FG
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Benzoic acid, ReagentPlus®, 99%
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1,2-Dihydroxybenzene, ReagentPlus®, ≥99%
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Benzoic acid, certified reference material for titrimetry, certified by BAM, ≥99.5%
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SS Benzoic Acid, 2000 μg/mL in dichloromethane, analytical standard
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Gallic acid, certified reference material, TraceCERT®
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Benzoic acid, Standard for quantitative NMR, TraceCERT®