Eupafolin: Effect on mitochondrial energetic metabolism

doi:10.1016/j.bmc.2007.10.029

Bioorganic & Medicinal Chemistry

Volume 16, Issue 2, 15 January 2008, Pages 854-861

https://doi.org/10.1016/j.bmc.2007.10.029 Get rights and content

Abstract

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3′,4′-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25–200 μM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated ∼30% by eupafolin (100 μM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08 V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA–Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.

Graphical abstract

In this study the effects of eupafolin (6-methoxy 5,7,3′,4′-tetrahydroxyflavone), a flavone extracted from dry leaves of Eupatorium litoralle, on mitochondrial metabolism and redox properties were evaluated.

Introduction

Flavonoids are a class of secondary plant phenolics of low molecular weight,¹ being derivatives of benzo-γ-pyrone,² distributed in all vascular plants³ and present in most edible fruits and vegetables of the human diet.⁴

Many flavonoids are able to act as antioxidants through a free radical scavenging mechanism, transferring electrons to free radicals with the formation of less reactive flavonoid phenoxyl radicals. Their antioxidant effect can also be caused by their ability to chelate transition metals.5, 6, 7, 8 Antioxidants may protect cells against free radicals that promote cellular damage involved in different disorders, including ischemic conditions and tumor development.⁹ They possess a remarkable spectrum of other biological activities, affecting cell functions such as growth, differentiation, and apoptosis.10, 11, 12, 13

Some flavonoids contain a catechol ring, a characteristic that has been observed in compounds that can undergo autooxidation, generating reactive oxygen species (e.g., catecholamines)¹⁴ making them capable of acting as prooxidants.¹⁵

Structure–activity studies on flavonoids demonstrated that the inhibition of succinate oxidase appears to be linked to their abilities to participate in oxidation–reduction reactions and that the most potent inhibitors possess hydroxyl substituents configurations, namely, hydroquinone and catechol.¹⁶ Inhibition of mitochondrial enzymes by flavonoids may contribute to their cytotoxic and antineoplastic activities.17, 18

Standard reduction potentials of individual electron carriers of the mitochondrial respiratory chain have been determined and vary from −0.320 to +0.816 V. Hence it is important to verify the reduction potential of flavonoids and if some of the biological activities may be dependent on their direct action on electron flow in the respiratory chain.

Eupafolin (6-methoxy 5,7,3′,4′-tetrahydroxyflavone) (Fig. 1), the flavone used in this study, was extracted from Eupatorium litoralle. Some medicinal plant extracts used in Brazil also contain this flavone.19, 20, 21, 22, 23, 24 Eupafolin possesses several known biological properties, among them: (i) it promotes iron release from ferritin, and donates electrons to the stable free radical DPPH²⁵; (ii) it protects cultured neurons against glutamate-induced oxidative stress¹²; and (iii) it inhibits xanthine oxidase activity.²⁶ Eupafolin also has antiproliferative activity against MK-1 (human gastric adenocarcinoma), B16-F10 (murine melanoma), and HeLa (human uterine carcinoma)⁵ cells. Although some effects of eupafolin have been demonstrated, its mechanism of action is not well known. The effects of eupafolin on mitochondrial metabolism and redox properties were evaluated, in order to contribute to the understanding of its cytotoxicity mechanism, especially on mitochondrial metabolism.

Section snippets

Chemicals

Glutamic acid, succinic acid, NADH, ATP, ADP, EGTA, EDTA, FCCP, DPPH, rotenone, d-mannitol, sucrose, Hepes, BSA, phosphoenolpyruvate (PEP), pyruvate kinase, valinomycin, oligomycin, cytochrome c, and Tris were purchased from Sigma (St. Louis, MO, USA). Potassium hydroxide, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, ammonium heptamolybdate, and ferrous sulfate were purchased from Merck (Brazil). Other reagents were of analytical grade. Solutions were

Effects of eupafolin on mitochondrial oxygen uptake

To investigate the mechanism of action of eupafolin on mitochondrial functions, we assessed its effect on isolated rat liver mitochondria. The rates of oxygen consumption on addition of ADP (state 3) and after its exhaustion (state 4) in the presence of eupafolin (25–200 μM), the respiratory control coefficient (RCC), and the ADP/O ratio were measured. Table 1, Table 2 show the effects of eupafolin on these parameters when succinate or glutamate was the oxidizable substrate, respectively. With

Discussion

Flavonoids have won recent interest because of their broad pharmacological activity. Putative therapeutic effects of many traditional medicines may be ascribed to the presence of flavonoids. The pharmacological effect of flavonoids is especially due to their inhibition of certain enzymes and their antioxidant activity.36, 37, 38 Flavonoids possess well-recognized antioxidant and prooxidant properties.³⁹ Eupafolin has some biological effects, such as cytotoxicity to tumoral cells. Many

Conclusion

Eupafolin, a flavone present in plants, promotes an intense inhibitory effect on the oxygen consumption rate in mitochondria isolated from rat liver. We believe that this effect occurs due to modifications in the mitochondrial respiratory chain, mainly between complexes I and III.

This flavonoid also suffers oxidation at a potential range of the respiratory chain, and promoted the reduction of cytochrome c in a non-enzymatic way, depending on the presence of NADH and iron to generate ROS which

Acknowledgments

This investigation was supported by the Brazilian Research Council (CNPq, Fundação Araucária and CAPES). The authors thank Dr. Aguinaldo José Nascimento for suggestions for the statistical analysis and Dr. Philip Albert James Gorin for the language corrections.

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^☆: Contract/grant sponsor: Brazilian Research Council (CNPq, Fundação Araucária and CAPES).

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Eupafolin: Effect on mitochondrial energetic metabolism☆

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

Effects of eupafolin on mitochondrial oxygen uptake

Discussion

Conclusion

Acknowledgments

J. Nutr. Biochem.

Nutr. Res.

Free Radic. Biol. Med.

FEBS Lett.

Toxicology

Eur. J. Pharmacol.

Free Radic. Biol. Med.

Toxicol. Lett.

Archiv. Biochem. Biophys.

Phytochemistry

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Phytochem.

Free Radic. Biol. Med.

Method Enzymol.

J. Biol. Chem.

BBA—General Subjects

J. Biol. Chem.

J. Biol. Chem.