Citrus Pomace as a Source of Plant Complexes to Be Used in the Nutraceutical Field of Intestinal Inflammation

Abstract

This study aims to recover the main by-product of Citrus fruits processing, the raw pomace, known also as pastazzo, to produce plant complexes to be used in the treatment of inflammatory bowel disease (IBD). Food-grade extracts from orange (OE) and lemon (LE) pomace were obtained by ultrasound-assisted maceration. After a preliminary phytochemical and biological screening by in vitro assays, primary and secondary metabolites were characterized by proton nuclear magnetic resonance (¹H-NMR) and liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) analyses. The intestinal bioaccessibility and antioxidant and anti-inflammatory properties were investigated by in vitro simulated gastro-intestinal digestion followed by treatments on a lipopolysaccharide (LPS)-stimulated human colorectal adenocarcinoma cell line (Caco-2). The tight junctions-associated structural proteins (ZO-1, Claudin-1, and Occludin), transepithelial electrical resistance (TEER), reactive oxygen species (ROS)-levels, expression of some key antioxidant (CAT, NRF2 and SOD2) and inflammatory (IL-1β, IL-6, TNF-α, IL-8) genes, and pNFkB p65 nuclear translocation, were evaluated. The OE and LE digesta, which did not show any significant difference in terms of phytochemical profile, showed significant effects in protecting against the LPS-induced intestinal barrier damage, oxidative stress and inflammatory response. In conclusion, both OE and LE emerged as potential candidates for further preclinical studies on in vivo IBD models.

Keywords: Citrus by-products; anti-inflammatory activity; antioxidant activity; food-grade extracts; in vitro simulated gastro-duodenal digestion; intestinal bioaccessibility; nutraceutics; phytochemistry; primary metabolites; secondary metabolites.

Figure 1

¹H NMR profiling of LE (top) and OE (bottom). Full spectra (A) and extended spectral regions from δ 5.95 to 7.30 (B), from δ 3.2 to 5.5 (C), and from δ 3.1 to 0.7 (D). 1 = sucrose, 2 = α-glucose, 3 = β-glucose, 4 = citric acid, 5 = tyrosine, 6 = asparagine, 7 = fructose, 8 = malic acid, 9 = GABA, 10 = aspartic acid, 11 = succinic acid, 12 = proline, 13 = alanine.

Figure 2

Distribution percentage of phytochemical classes identified in orange and lemon raw pomace extracts (OE and LE, respectively).

Figure 3

Representative LC-DAD chromatograms of orange raw pomace extract (OE, panel A) and lemon raw pomace extract (LE, panel B) pre- (black) and post-gastro-duodenal digestion (orange and green chromatogram, respectively) acquired at 292 nm.

Figure 4

OE and LE effects on Caco-2 cell viability. Cell viability evaluated by XTT assay and expressed as percentage of cell viability in Caco-2 cells untreated or treated with different concentrations of OE for 24 h (A) and 48 h (B); and in Caco-2 cells untreated or treated with different concentrations of LE for 24 h (C) and 48 h (D). Values are the mean ± SD of three independent experiments repeated at least in quintuplicate. Data were analyzed by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01; ns: non-significant.

Figure 5

Cell viability and proliferation in Caco-2 cells under different treatments. Cell viability evaluated by XTT assay and expressed as percentage of cell viability in Caco-2 cells untreated or treated with different concentrations of LPS for 24 h (A) and 48 h (B); and in Caco-2 cells untreated (Ctrl) or treated with LPS, LPS + 200 µg/mL OE and LPS + 200 µg/mL LE for 24 h (C) and 48 h (D). Cell proliferation monitored by using the Incucyte live cell imaging system was expressed as fold change of mean cell confluence in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE for 24 h (E) and 48 h (F). Values are the mean ± SD of three independent experiments repeated at least in quintuplicate. Data were analyzed by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significant.

Figure 6

Intestinal permeability of Caco-2 cells under different treatments. (A) TEER values expressed as percentage of initial values of unit area resistance calculated by dividing resistance values by the effective membrane area in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. Values are the mean ± SD of three independent experiments. Data were analyzed by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001. (B) Representative immunofluorescence by confocal imaging of ZO-1, Claudin-1, and Occludin in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. 40× magnification.

Figure 7

Oxidative stress of Caco-2 cells under different treatments. Fold change of the relative mean fluorescence (A) and representative images (B) of CM-H2DCFDA (green) staining in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. Hoechst nuclear staining (blue). 40× magnification. Relative mRNA expression of CAT (C), SOD2 (D), and NRE2L2 (E) genes measured by qPCR in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. Values are the mean ± SD of three independent experiments. Data were analyzed by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significant.

Figure 8

Inflammatory response of Caco-2 cells under different treatments. Relative mRNA expression of IL-1β (A), IL-6 (B), IL-8 (C), and TNF-α (D) genes measured by qPCR in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. Representative immunofluorescence by confocal imaging (E) and QFIA (F) of pNFκB p65 (red) in Caco-2 cells Ctrl, LPS, LPS + 200 µg/mL OE, and LPS + 200 µg/mL LE. Hoechst nuclear staining (blue). 40× magnification. Values are the mean ± SD of three independent experiments. Data were analyzed by 2-tailed Student’s t test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significant.