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Modulation of oxidative stress and TNFα secretion by peritoneal macrophages of arthritic rats fed with a flavonoid-enriched diet


The inflammatory response increases the oxidative stress and cytokine production, such as TNFα, in macrophages. Flavonoids, present in vegetables, have potential antioxidant and anti-inflammatory properties. Previous studies have shown that a flavonoid-enriched cocoa extract added to macrophages in vitro decreases the secretion of NO and TNFα [1].


The objective of the present study was to ascertain the production of oxidant products and a pro-inflammatory cytokine (TNFα) by peritoneal macrophages obtained from arthritic rats after a long-term cocoa diet.


Female LOU rats were fed with a 10% cocoa diet or standard chow. After 2 weeks of diet, collagen-induced arthritis (CIA) was induced in part of animals from each group. One month later, peritoneal macrophages were obtained by injecting ice-cold sterile PBS into the peritoneal cavity. Cells were cultured and stimulated by addition of 1 µg/mL LPS. TNFα secretion was quantified by ELISA in 24 h supernatants. In the same samples, NO2- concentration (stable end product of NO) was measured by a modification of Griess reaction. To determine intracellular ROS production, macrophages were incubated with reduced 2’,7’-dichlorofluorescein diacetate (H2DCF-DA) for 30 min at 37 ºC. Fluorescence of DCF oxidized by ROS was measured by fluorometry every 30 min up to 2 h.


Macrophages from CIA animals fed cocoa decreased TNFα and NO production by ~57% and ~36% with respect to those from CIA reference group under LPS-stimulation conditions (P<0.05). ROS production by macrophages from CIA animals fed standard diet was higher with respect to healthy reference animals (P<0.05). Cocoa diet avoided ROS secretion increase in CIA animals (P<0.05), raising similar values to healthy animals.


A long-term cocoa diet reduced the production of oxidants and TNFα in peritoneal macrophages obtained from collagen-induced arthritic rats.


  1. Ramiro E, Franch A, Castellote C, Pérez-Cano FJ, Permanyer J, Izquierdo-Pulido M, Castell M: Flavonoids from Theobroma cacao down-regulate inflammatory mediators. J Agric Food Chem. 2005, 53: 8506-8511. 10.1021/jf0511042.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ramos-Romero, S., Pérez-Cano, F.J., Ramírez-Santana, C. et al. Modulation of oxidative stress and TNFα secretion by peritoneal macrophages of arthritic rats fed with a flavonoid-enriched diet. J Transl Med 8 (Suppl 1), P24 (2010).

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