Elsevier

Nitric Oxide

Volume 15, Issue 1, August 2006, Pages 30-39
Nitric Oxide

Inhibition of nitric oxide biosynthesis by anthocyanin fraction of blackberry extract

https://doi.org/10.1016/j.niox.2005.10.003Get rights and content

Abstract

Anthocyanins are natural colorant belonging to the flavonoid family, widely distributed among flowers, fruits, and vegetables. Some flavonoids have been found to possess anticarcinogenic, cytotoxic, cytostatic, antioxidant, and anti-inflammatory properties. Since increased nitric oxide (NO) plays a role in inflammation, we have investigated whether the pharmacological activity of the anthocyanin fraction of a blackberry extract (cyanidin-3-O-glucoside representing about 88% of the total anthocyanin content) was due to the suppression of NO synthesis. The markedly increased production of nitrites by stimulation of J774 cells with lipopolysaccharide (LPS) for 24 h was concentration-dependently inhibited by the anthocyanin fraction (11, 22, 45, and 90 μg/ml) of the extract. Moreover, this inhibition was dependent on a dual mechanism, since the extract attenuated iNOS protein expression and decreased the iNOS activity in lungs from LPS-stimulated rats. Inhibition of iNOS protein expression appeared to be at the transcriptional level, since the extract and similarly cyanidin-3-O-glucoside (10, 20, 40, and 80 μg/ml, amounts corresponding to the concentrations present in the extract) decreased LPS-induced NF-κB activation, through inhibition of IκBα degradation, and reduced ERK-1/2 phosphorylation in a concentration-dependent manner. In conclusion, our study demonstrates that at least some part of the anti-inflammatory activity of blackberry extract is due to the suppression of NO production by cyanidin-3-O-glucoside, which is the main anthocyanin present in the extract. The mechanism of this inhibition seems to be due to an action on the expression/activity of the enzyme. In particular, the protein expression was inhibited through the attenuation of NF-κB and/or MAPK activation.

Section snippets

Anthocyanins contained in blackberry extract

Blackberry fruits were obtained as commercial products. Fruit extract (43.6 g) was obtained by pounding in a mortar about 1 kg of fruits. The extract was centrifuged at 6000 rpm for 20 min, the juice filtered on glass wool, and the pulps extracted overnight with about 1 L of ethyl alcohol (EtOH)/HCl (99:1, v/v) at room temperature. The filtered extract was concentrated under reduced pressure and diluted with water (1:1, v/v). Both the filtered juice and the pulp extract were applied to a C18 silica

Effect of anthocyanin fraction of blackberry extract on LPS-induced nitrite production

To asses whether the anthocyanin fraction of the blackberry extract was able to reduce NO production we utilized the Griess assay to measure nitrite, the stable end-product of NO, in supernatants of the murine macrophage cell line J774 stimulated with LPS (10 μg/ml). A marked increase of nitrite production in the cell medium was observed at 24 h (23.10 ± 0.55 nmol/106 cells; p < 0.001, n = 3) in respect to unstimulated macrophages (3.12 ± 0.40 nmol/106 cells). When the anthocyanin fraction of the

Discussion

Recently, much attention has focused on the protective function of naturally occurring antioxidants in biological systems, and on the mechanisms of their action. Phenolic compounds, which are widely distributed in plants, are considered to play an important role as dietary antioxidants for the prevention of oxidative damage caused, in living systems, by active oxygen radicals.

Anthocyanin pigments are widely distributed in the human diet through crops, beans, fruits, and vegetables, suggesting

Acknowledgments

The authors are grateful to Dr. Antonio Leonardi (Department of Biology and Cellular and Molecular Pathology “L. Califano,” University of Naples Federico II, Naples, Italy) for his advice on EMSA. Thanks are due to the Italian MIUR for financial support (COFIN 2003).

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