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Poster presentation | Open | Published:

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. 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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Correspondence to M Castell.

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  • Flavonoid
  • Peritoneal Macrophage
  • Dichlorofluorescein
  • Griess Reaction
  • Dichlorofluorescein Diacetate