Polyphenolic Extract from Tarocco (Citrus sinensis L. Osbeck) Clone "Lempso" Exerts Anti-Inflammatory and Antioxidant Effects via NF-kB and Nrf-2 Activation in Murine Macrophages

Abstract

Citrus fruits are often employed as ingredients for functional drinks. Among Citrus, the variety, "Lempso", a typical hybrid of the Calabria region (Southern Italy), has been reported to possess superior antioxidant activity when compared to other common Citrus varieties. For these reasons, the aim of this study is to investigate in vitro the nutraceutical value of the Tarocco clone, "Lempso", highlighting its anti-inflammatory and antioxidant potential. A post-column 2,2'-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging assay for the screening of antioxidant compounds in these complex matrices was developed. Subsequently, polyphenolic extract was tested on a murine macrophage cell line under inflammatory conditions. The extract resulted was able to significantly inhibit nitric oxide (NO) and cytokine release and inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) expression. The inhibition of these pro-inflammatory factors was associated to Nuclear factor-kB (NF-kB) inhibition. Our results also indicate an anti-oxidant potential of the extract as evidenced by the inhibition of reactive oxygen species (ROS) release and by the activation of the nuclear factor E2-related factor-2 (Nrf-2) pathway in macrophages. The obtained results highlight the anti-inflammatory and antioxidant potential of Lempso extract and its potential use, as a new ingredient for the formulation of functional beverages with high nutraceutical value, providing health benefits to consumers.

Keywords: Anthocyanins; LPS; macrophages J774A.1; on-line HPLC-DPPH; oxidative stress.

Figure 1

On-line High Performance Liquid Chromatography-Photodiode Array-2,2′-Diphenyl-1-picrylhydrazyl (HPLC-PDA-DPPH) profiles of anthocyanins in Lempso extract. Black and red lines denote the control and post column reaction, respectively.

Figure 2

Effect of Lempso flavonoidic extract (LFE) (10–50 μg mL⁻¹) on nitric oxide (NO) release, evaluated as NO₂⁻ (µM), by macrophages J774A.1 stimulated with lipopolysaccharide from E. coli (LPS) (Panel A). Effect of LFE (10–50 μg mL⁻¹) on LPS-induced inducible nitric oxide synthase (iNOS) (Panel B) and cycloxygenase-2 (COX-2) (Panel C) in macrophages J774A.1. Values are expressed as mean ± s.e.m of at least three experiments, each in triplicate. Comparisons were performed using one-way analysis of variance and multiple comparisons were made by Bonferroni’s test. *** and ** denote p < 0.001 and p < 0.01 vs. LPS alone.

Figure 3

Effect of LFE (10–50 μg mL⁻¹) on LPS –induced tumor necrosis factor (TNF)-α (Panel A) and interleukin (IL)-6 (Panel B) production in J774A.1 macrophages. TNF-α and IL-6 production was measured in the supernatants of J774A.1 cells treated with LFE (10–50 μg mL⁻¹) and LPS (1 μg mL⁻¹) for 18 h by means of Enzyme-Linked Immuno Sorbent Assay (ELISA). Values are expressed as mean ± s.e.m of at least three experiments, each in triplicate. Comparisons were performed using one-way analysis of variance and multiple comparisons were made by Bonferroni’s test. *** denotes p < 0.001 vs. LPS alone. Effect of LFE (25 μg mL⁻¹) on LPS-induced p65 nuclear translocation (Panel C) in J774A.1 macrophages. Nuclear translocation of Nuclear factor-kB (NF-kB) p65 subunit was detected using immunofluorescence assay at confocal microscopy. Scale bar, 10 µm. Blue and green fluorescence indicate localization of nucleus (DAPI) and p65, respectively. Analysis was performed by confocal laser scanning microscopy.

Figure 4

Effect of LFE (10–50 μg mL⁻¹) on LPS-induced reactive oxygen species (ROS) in LPS-stimulated J774A.1 macrophages (Panel A). Effect of LFE (25 μg mL⁻¹) on NF-E2-related factor-2 (Nrf-2) (Nrf2) nuclear translocation in J774A.1 macrophages (Panel B). Nuclear translocation of Nrf2 was detected using immunofluorescence assay at confocal microscopy. Scale bar, 10 µm. Blue and green fluorescence indicates localization of nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of LFE (25 μg mL⁻¹) on LPS-induced heme oxygenase-1 (HO-1) (Panel C) and on NAD(P)H quinone dehydrogenase 1 (NQO1) (Panel D) in macrophages J774A.1. Values are expressed as mean ± s.e.m of at least three experiments, each in triplicate. Comparisons were performed using one-way analysis of variance and multiple comparisons were made by Bonferroni’s test. °°° denotes p < 0.001 vs. control. *** and * denote p < 0.001 and p < 0.05 vs. LPS alone.