Food and Chemical Toxicology 49 (2011) 1224–1228
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An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice Rafik Bachoual a,⇑, Wifak Talmoudi a, Tarek Boussetta b,c, Françoise Braut b,c, Jamel El-Benna b,c a
Faculté des Sciences de Gabès, Université de Gabès, Tunisia INSERM, U773, F-75018, France c Université Paris 7 Denis Diderot, Faculté de Médecine, site Bichat, Paris F-75018, France b
a r t i c l e
i n f o
Article history: Received 2 November 2010 Accepted 25 February 2011 Available online 3 March 2011 Keywords: Punica granatum Pomegranate Inflammation Neutrophils ROS MPO
a b s t r a c t Punica granatum peel aqueous extract (PGE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. In this study we investigated the effect of PGE on human neutrophil reactive oxygen species (ROS) production in vitro and on LPS-induced lung inflammation in vivo in mice. Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H2O2 was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethyl benzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. PGE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionylleucyl-phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. PGE had no effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. PGE did not scavenge H2O2 but directly inhibited myeloperoxidase activity in vitro. In vivo studies showed that PGE also attenuated LPS-induced lung inflammation in mice. So this study reveals that PGE inhibits neutrophil MPO activity and attenuates LPS-induced lung inflammation in mice. Inhibition of MPO activity by PGE could explain its anti-inflammatory action. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Punica granatum, a member of the Punicacea family, is a small tree originating from Asia and now widely cultivated in the Mediterranean basin. It produces orange-red flowers between May and April. The ripe pomegranate fruit is composed of juicy seeds, surrounded by a leathery yellow peel. In Tunisian (Boukef et al., 1982) and Indian (Nagaraju and Rao, 1990) folk medicine, dried pomegranate peel is used to treat disorders such as colitis, headache, aphthae, diarrhea, dysentery and ulcers (Kirthikar and Basu, 2000). Pomegranate extracts have been shown to have anti-oxidant activity (Aviram et al., 2000), and to protect against cancer (Mehta and Lansky, 2004). Pomegranate peel extract delays the proliferation of human breast and prostate cancer cell lines (Toi et al., 2003). Schubert et al. (1999) showed that flavonoids contained in pomegranate seed oil and fermented juice exhibit antioxAbbreviations: fMLF, FN-formyl-methionyl-Leucyl-phenylalanine; MP, myeloperoxydase; PMA, phorbol myristate acetate; ROS, reactive oxygen species; DCFHDA, 20 ,70 -dichlorofluoroscein-diacetate. ⇑ Corresponding author. Address: Faculté des Sciences de Gabès, Campus Universitaire Cité Erriadh, 6072 Gabès, Tunisia. Tel./fax: +216 75 392 421. E-mail address: Rafi
[email protected] (R. Bachoual). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.02.024
idant and anti-inflammatory properties, inhibiting ecosanoid enzymes. Inflammatory disorders are due to excessive production of pro-inflammatory mediators such as TNFa, GM-CSF, IL-1, IL-6, IL8, leukotriene B4 and PAF, the activity of inflammatory cells such as neutrophils, monocytes and macrophages, and excessive production of reactive oxygen species (ROS) (Babior, 2000; Nathan, 2006; Ley, 2002). Polymorphonuclear neutrophils play a key role in host defenses against invading microorganisms (Hampton et al., 1998), but excessive neutrophil activation participates in tissue damage associated with inflammatory disorders (Babior, 1984, 2000). In response to a variety of agents, neutrophils migrate to inflammatory sites, where they release proteases, bactericidal peptides, and large quantities of ROS in a process known as the respiratory burst (Babior, 1984). Oxygen reduction by neutrophil NADPH oxidase, a multicomponent enzyme system, yields superoxide anion ðO 2 Þ (El-Benna et al., 2005), while myeloperoxidase (MPO) produces hypochloric acid from hydrogen peroxide (Klebanoff, 2005). The aim of this work was to examine the effect of P. granatum peel aqueous extract (PGE) on ROS production by human neutrophils, and its effect on LPS-induced lung inflammation in mice.
R. Bachoual et al. / Food and Chemical Toxicology 49 (2011) 1224–1228
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2. Materials and methods
2.9. Bronchoalveolar lavage (BAL) and lung sampling
2.1. Chemicals and reagents
The mice were anesthetized by an intraperitoneal injection of 50 mg of urethane (sigma) and killed by exsanguination. The lungs were lavaged twice with 1 ml of physiological saline, removed from the chest cavity, and immediately placed at 80 °C until use. The lavage fluid (1.8 ml) was immediately placed on ice. Free alveolar cells were recovered from the lavage fluid by centrifugation at 400g for 15 min at 4 °C. The total protein concentration in the supernatant was measured with the Quick-Start Bradford assay (Bio-Rad, Marnes-la-Coquette, France). The cell pellet was suspended in 150 lL of physiological saline and an aliquot was used to determine the total white cell count with a hemocytometer. For differential counts, the cell suspension was cytospun (Cytospin-2, Shandon Products Ltd.), fixed in methanol, and stained with Diff Quick solution (Medion Diagnostics, Plaisir, France). One hundred cells were counted with an oil immersion lens (1000).
Luminol, cytochrome c, fMLF, PMA, zymosan, superoxide dismutase (SOD), catalase and HRPO Escherichia coli (O55:B5) lipopolysaccharide (LPS) were from Sigma–Aldrich (Saint-Quentin Fallavier, France). Ficoll and Dextran T500 were from GE Healthcare. HBSS, HEPES and glucose were from Gibco. 20 ,70 -dichlorofluoroscein-diacetate (DCFH-DA) was from Acros Fine Chemicals. Stock solutions of DCFHDA (50 mmol/l) and fMLF (102 mol/l) were prepared in dimethyl sulfoxide (DMSO) and stored at 20 °C. The different solutions were diluted in phosphate-buffered saline (PBS) immediately before use.
2.2. Preparation of PGE P. granatum peel (Hammouri variety) dried at 37 °C was collected from Institut des regions Arides (IRA, Medenine, Tunisia). The peel was blended and suspended in sterile 0.9% NaCl, then centrifuged at 2000 rpm for 3 min. The supernatant (PGE) was used for all experiments.
2.10. Statistical analysis Data are reported as mean ± standard error. The Newman-Keuls multiple comparisons test was used, and P values
