Bioavailable Citrus sinensis Extract: Polyphenolic Composition and Biological Activity

Abstract

Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrussinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value.

Keywords: Citrus sinensis polyphenols; anti-inflammatory; antioxidant; bioavailability; gastro-intestinal digestion.

Figure 1

Total polyphenols content in Citrus sinensis extract after the in vitro gastro-intestinal digestion process (Panel A); Comparison of the polyphenol profile of Cs (Citrus sinensis, Panel B) and CsB (bioavailable Citrus sinensis, Panel C).

Figure 2

Effect of Cs and CsB (250–10 μg/mL) on NO release, evaluated as NO₂⁻ (µM), by macrophages J774A.1 stimulated with LPS (Panel A and Panel B); Effect of Cs and CsB (250–10 μg/mL) on LPS-induced iNOS (Panel C) and COX-2 (Panel D) in macrophages J774A.1. Values, mean ± s.e.m., are expressed as % of inhibition vs. J774A.1 treated with LPS alone. *** and ** denote p < 0.001 and p < 0.01 vs. LPS alone. °°° denotes p < 0.001 vs. Cs.

Figure 3

Effect of Cs and CsB (250–10 μg/mL) on LPS –induced TNF-α (Panel A and Panel C) and IL-6 (Panel B and Panel D) production in J774A.1 macrophages. TNF-α and IL-6 production was measured in the supernatants of J774A.1 cells treated with Cs and CsB (250–10 μg/mL) and LPS (1 µg/mL) for 18 h by means of ELISA. Values, mean ± s.e.m., are expressed as % of inhibition vs. J774A.1 treated with LPS alone. ***, ** and * denote p < 0.001, p < 0.01 p < 0.05 vs. LPS alone. °°°, °° and ° denote p < 0.001, p < 0.01 and p < 0.05 vs. Cs.

Figure 4

Effect of Cs and CsB (150–50 μg/mL) on p65 nuclear translocation in normal (A) and in inflammatory conditions (B) in J774A.1 macrophages. Nuclear translocation of NF-κB 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 5

Effect of Cs and CsB (250–10 μg/mL) on LPS-induced ROS in LPS-stimulated J774A.1 macrophages (Panel A and Panel B); Effect of Cs and CsB (250–10 μg/mL) on LPS-induced HO-1 (Panel C) in macrophages J774A.1. Values, mean ± s.e.m., are expressed as % of inhibition vs. J774A.1 treated with LPS alone and as mean fluorescence intensity. ^### denotes p < 0.001 vs. control. *** and ** denote p < 0.001 and p < 0.01 vs. LPS alone. °°°, °° and ° denote p < 0.001, p < 0.01 and p < 0.05 vs. Cs.

Figure 6

Effect of Cs and CsB (250–10 μg/mL) on NO release, evaluated as NO₂⁻ (µM), by peritoneal macrophages stimulated with LPS (Panel A); Effect of Cs and CsB (250–10 μg/mL) on LPS-induced iNOS (Panel B); COX-2 (Panel C) and nitrotyrosine (Panel D) in peritoneal macrophages. Values, mean ± s.e.m., are expressed as % of inhibition vs. peritoneal macrophages treated with LPS alone. *** and * denotes p < 0.001 and p < 0.05 vs. LPS alone. °°°, °° and ° denote p < 0.001, p < 0.01 and p < 0.05 vs. Cs.

Figure 7

Effect of Cs and CsB (250–10 μg/mL) on LPS-induced ROS in LPS-stimulated peritoneal macrophages (Panel A); Effect of Cs and CsB (250–10 μg/mL) on LPS-induced HO-1 (Panel B) in peritoneal macrophages. Values, mean ± s.e.m., are expressed as % of inhibition vs. peritoneal macrophages treated with LPS alone. ^### denotes p < 0.001 vs. control. *** denotes p < 0.001 vs. LPS alone. °°° and °° denote p < 0.001 and p < 0.01 vs. Cs.