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Cited by 149Free Radicals and AntioxidantsBioactive Compounds and Antitumor AgentsRedox biology and oxidative stress

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Apocynin: Chemical and Biophysical Properties of a NADPH Oxidase Inhibitor

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Petrônio, Maicon, Zeraik, Maria, Fonseca, Luiz, Ximenes, Valdecir·2013·Molecules
DOI 10.3390/molecules18032821PMC PMC6269682
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Abstract

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M−1 cm−1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M−1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

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Figures

Figure 1 UV-Vis spectrum of apocynin and molar absorption coefficients. (a) UV-Vis spectra of apocynin dissolved in water (6.25–100 μM); (b) Linear regression at 275 nm, r2 = 0.9994; (c) Linear regression at 303 nm, r2 = 0.9989.
Figure 1 UV-Vis spectrum of apocynin and molar absorption coefficients. (a) UV-Vis spectra of apocynin dissolved in water (6.25–100 μM); (b) Linear regression at 275 nm, r2 = 0.9994; (c) Linear regression at 303 nm, r2 = 0.9989.CC BY · PubMed Central
Figure 2 Molecular structures of apocynin and protocatechuic acid and their hydrophobicity indexes (log P) and acidity constants (pKa).
Figure 2 Molecular structures of apocynin and protocatechuic acid and their hydrophobicity indexes (log P) and acidity constants (pKa).CC BY · PubMed Central
Figure 3 Cyclic voltammograms for apocynin (a) and protocatechuic acid (b) (0.1 mM) obtained in 0.2 M sodium phosphate buffer at pH 7.0. The scan rate was 5 mV s.
Figure 3 Cyclic voltammograms for apocynin (a) and protocatechuic acid (b) (0.1 mM) obtained in 0.2 M sodium phosphate buffer at pH 7.0. The scan rate was 5 mV s.CC BY · PubMed Central
Figure 4 Reactivity of apocynin (APO) and protocatechuic acid (PA) with peroxyl radical (ROO•). (a,c) Bleaching of the triene (eleostearic acid) by ROO• and the lag phase provoked by the addition of the tested substances. (b,d) AUC versus concentration.
Figure 4 Reactivity of apocynin (APO) and protocatechuic acid (PA) with peroxyl radical (ROO•). (a,c) Bleaching of the triene (eleostearic acid) by ROO• and the lag phase provoked by the addition of the tested substances. (b,d) AUC versus concentration.CC BY · PubMed Central
Figure 5 Reactivity of apocynin (APO) and protocatechuic acid (PA) with Nitric oxide. The reaction mixtures were constituted of 10 mM sodium nitroprusside and various concentration of the tested substances in 10 mM PBS, pH 7.4. The production of NO was measured by the Griess method.
Figure 5 Reactivity of apocynin (APO) and protocatechuic acid (PA) with Nitric oxide. The reaction mixtures were constituted of 10 mM sodium nitroprusside and various concentration of the tested substances in 10 mM PBS, pH 7.4. The production of NO was measured by the Griess method.CC BY · PubMed Central
Figure 6 Reactivity of apocynin and protocatechuic acid with hydrogen peroxide. The reaction mixtures were constituted of 1 mM tested substances, 1 mM H2O2 and 100 nM HRP in 0.1 M PBS, pH 7.4, at 25 °C.
Figure 6 Reactivity of apocynin and protocatechuic acid with hydrogen peroxide. The reaction mixtures were constituted of 1 mM tested substances, 1 mM H2O2 and 100 nM HRP in 0.1 M PBS, pH 7.4, at 25 °C.CC BY · PubMed Central
Figure 7 Reactivity of apocynin and protocatechuic acid with hypochlorous acid. The reaction mixtures were constituted of 100 μM HOCl and various concentrations of the tested substances in 0.01 M PBS, pH 7.4, at 25 °C. The consumed HOCl was measured using the taurine-chloramine/TMB assay.
Figure 7 Reactivity of apocynin and protocatechuic acid with hypochlorous acid. The reaction mixtures were constituted of 100 μM HOCl and various concentrations of the tested substances in 0.01 M PBS, pH 7.4, at 25 °C. The consumed HOCl was measured using the taurine-chloramine/TMB assay.CC BY · PubMed Central
Figure 8 Fluorescence quenching of HSA by apocynin. (a) Emission spectra of HSA (1 μM) (λex = 295 nm) in the presence of apocynin (0–40 μM). The arrow indicates the addition of apocynin; (b) Stern-Volmer plot; (c) Plot for apparent binding constant determination.
Figure 8 Fluorescence quenching of HSA by apocynin. (a) Emission spectra of HSA (1 μM) (λex = 295 nm) in the presence of apocynin (0–40 μM). The arrow indicates the addition of apocynin; (b) Stern-Volmer plot; (c) Plot for apparent binding constant determination.CC BY · PubMed Central
Figure 9 Effect of apocynin on HSA synchronous fluorescence. (a) Quencher of synchronous fluorescence (scanning at fixed Δλ = 15 nm). (b) Quencher of synchronous fluorescence (scanning at fixed Δλ = 60 nm). The mixtures consisted of 1.0 μM HSA and apocynin (0–30 μM) in 10 mM PBS, pH 7.2 at 25 °C. The arrow indicates the addition of apocynin.
Figure 9 Effect of apocynin on HSA synchronous fluorescence. (a) Quencher of synchronous fluorescence (scanning at fixed Δλ = 15 nm). (b) Quencher of synchronous fluorescence (scanning at fixed Δλ = 60 nm). The mixtures consisted of 1.0 μM HSA and apocynin (0–30 μM) in 10 mM PBS, pH 7.2 at 25 °C. The arrow indicates the addition of apocynin.CC BY · PubMed Central
Figure 10 Displacement of fluorophores from HSA by apocynin. (a) Displacement of dansylamide (DNSA); (b) Displacement of dansylglycine (DG). The mixtures consisted of 10.0 μM HSA, 5.0 μM fluorophores and apocynin (0, 5, 10, 20, 30, 40 and 50 μM) in 10 mM PBS, pH 7.2 at 25 °C.
Figure 10 Displacement of fluorophores from HSA by apocynin. (a) Displacement of dansylamide (DNSA); (b) Displacement of dansylglycine (DG). The mixtures consisted of 10.0 μM HSA, 5.0 μM fluorophores and apocynin (0, 5, 10, 20, 30, 40 and 50 μM) in 10 mM PBS, pH 7.2 at 25 °C.CC BY · PubMed Central
Figure 11 Effect of apocynin on circular dichroism spectra of HSA. (a) Far-UV-CD: The mixtures consisted of 1.0 μM HSA and 30 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C. The inset is a zoom of graph “a”; (b) Near-UV-CD: The mixtures consisted of 30 μM HSA and 60 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C.
Figure 11 Effect of apocynin on circular dichroism spectra of HSA. (a) Far-UV-CD: The mixtures consisted of 1.0 μM HSA and 30 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C. The inset is a zoom of graph “a”; (b) Near-UV-CD: The mixtures consisted of 30 μM HSA and 60 μM apocynin in 10 mM PBS, pH 7.2 at 25 °C.CC BY · PubMed Central

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Metadata

Authors
Petrônio, M., Zeraik, M., Fonseca, L., & Ximenes, V.
Affiliations
Universidade Estadual Paulista (Unesp) (BR)
Corresponding author
Ximenes, Valdecir
ORCID
Venue
Molecules
Publisher
MDPI AG
Published
2013-03-01
Volume
18
Issue
3
Pages
2821-2839
ISSN
1420-3049 (ISSN-L)
DOI
10.3390/molecules18032821
PMC
PMC6269682
OpenAlex
W2036509107
Cited by
149
References
49
Citations by year
2026: 8 · 2025: 12 · 2024: 8 · 2023: 11 · 2022: 14 · 2021: 14 · 2020: 9 · 2019: 12 · 2018: 11 · 2017: 11 · 2016: 6 · 2015: 12 · 2014: 14 · 2013: 7
FWCI
5.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecules (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Acervo Digital da Universidade Estadual Paulista (Universidade Estadual Paulista) (submittedVersion, other-oa)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
Funders
  • Fundação de Amparo à Pesquisa do Estado de São Paulo
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Free Radicals and Antioxidants
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Bioactive Compounds and Antitumor Agents
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
apocynin, NADPH oxidase inhibitors, NOX4, superoxide, hydrogen peroxide scavenging, free radical scavenging, hypochlorous acid, protocatechuic acid, human serum albumin binding, fluorescence quenching, antioxidant activity, physicochemical properties
MeSH terms
Acetophenones, Enzyme Inhibitors, Hydrogen Peroxide, Hydrogen-Ion Concentration, Hypochlorous Acid, Kinetics, Oxidation-Reduction, Free Radical Scavengers, NADPH Oxidases, Hydrophobic and Hydrophilic Interactions

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