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Updated: May 28, 2026
Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases
Published on: February 24, 2018
Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by
Ricardo Lagoa1, Ilaria Graziani, Carmen Lopez-Sanchez
1Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, Badajoz, Spain.
Abstract:
Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H₂O₂ production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H₂O₂ production by mitochondria from both tissues (IC₅₀ approximately 1-2 μM), even when H₂O₂ production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.
Insights
Flavonoids like quercetin and kaempferol inhibit mitochondrial hydrogen peroxide production, suggesting mitochondria are a key target for their protective effects against oxidative stress and related diseases.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Flavonoids show protective effects against cell damage and diseases linked to oxidative stress and mitochondrial dysfunction.
- Epidemiological studies suggest a role for flavonoids in mitigating diseases associated with oxidative stress.
Purpose of the Study:
- To investigate the effects of five major flavonoid subclasses on mitochondrial hydrogen peroxide (H₂O₂) production and mitochondrial complex activities.
- To determine if mitochondria are a primary target for flavonoid-mediated cellular protection.
Main Methods:
- Screening of five flavonoids for their impact on H₂O₂ production in isolated rat brain and heart mitochondria.
- Assessing the effects of flavonoids on mitochondrial oxygen consumption and the activity of respiratory chain complexes (I, II, III, and IV).
- Investigating the interaction of flavonoids with ubiquinone (coenzyme Q) binding sites in Complex I.
Main Results:
- Quercetin, kaempferol, and epicatechin potently inhibited mitochondrial H₂O₂ production (IC₅₀ ≈ 1-2 μM).
- Quercetin, kaempferol, and apigenin inhibited Complex I activity, with inhibition dependent on coenzyme Q concentration, suggesting competitive binding.
- Epicatechin, quercetin, and kaempferol could stoichiometrically reduce purified cytochrome c, while other complexes and oxygen consumption remained largely unaffected.
Conclusions:
- Mitochondria are a plausible primary target for flavonoid action.
- Flavonoids may exert their protective effects against oxidative stress and mitochondrial dysfunction-related pathologies, at least partly, through direct interaction with mitochondrial components.
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