Citrus limon Peel Powder Reduces Intestinal Barrier Defects and Inflammation in a Colitic Murine Experimental Model

Abstract

This study examines the ameliorative effects of lemon (Citrus limon) peel (LP) powder on intestinal inflammation and barrier defects in dextran sulfate sodium (DSS)-induced colitic mice. The whole LP powder was fractionated into methanol (MetOH) extract and its extraction residue (MetOH residue), which were rich in polyphenolic compounds and dietary fibers, respectively. Mice were fed diets containing whole LP powder, MetOH extract, and MetOH residue for 16 d. DSS administration for 9 d induced bodyweight loss, reduced colon length, reduced the colonic expression of tight junction proteins including zonula occludens-1 and -2, and claudin-3 and -7, and upregulated colonic mRNA expression of interleukin 6, chemokine (C-X-C motif) ligand 2, and C-C motif chemokine ligand 2. Feeding LP powder restored these abnormalities, and the MetOH residue, but not MetOH extract, also showed similar restorations. Feeding LP powder and MetOH residue increased fecal concentrations of acetate and n-butyrate. Taken together, LP powder reduced intestinal damage through the protection of tight junction barriers and suppressed an inflammatory reaction in colitic mice. These results suggest that acetate and n-butyrate produced from the microbial metabolism of dietary fibers in LP powder contributed to reducing colitis.

Keywords: colitis; dietary fiber; inflammatory cytokine; lemon peel; tight junction.

Figure 1

Representative chromatograms of polyphenols in standard solution, whole peel powder, and its methanol extract from UPLC/MS/MS analysis. The standard solution (50 μM, a), whole peel powder (b), and its methanol extract (c) were analyzed. Arrows indicate the peaks of each polyphenol.

Figure 2

Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on bodyweight change and clinical score in dextran sodium sulfate (DSS)-induced colitic mice. Bodyweight change (a) and clinical score (b) of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration. Values are the mean ± SEM (n = 7). Means without a common letter differ, p < 0.05.

Figure 3

Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on colon length and mucosal structure in dextran sodium sulfate (DSS)-induced colitic mice. Colon length (a,b) of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration. Representative images of the cecum and colon of seven mice in each group are shown in (a). Scale bar represents 10 mm. Colonic sections of mice were stained with hematoxylin and eosin. Representative images of seven mice in each group are shown in (c). Scale bar represents 100 μm. Values are the mean ± SEM (n = 7). Means without a common letter differ, p < 0.05.

Figure 4

Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on tight junction protein expression in the colon of dextran sodium sulfate (DSS)-induced colitic mice. Protein expression of zonula occludens (ZO)-1, ZO-2, occludin, claudin-3, and claudin-7 in the colon of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as determined by immunoblot analysis. Values are the mean ± SEM (n = 7). Means without a common letter differ, p < 0.05.

Figure 5

Effects of whole LP powder, methanol (MetOH extract), and MetOH extraction residue (MetOH residue) on claudin-3 expression in the colon of dextran sodium sulfate (DSS)-induced colitic mice. Immunolocalization of claudin-3 in the colon of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as analyzed by immunofluorescence microscopy. Representative images of seven mice in each group are shown.

Figure 6

Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on inflammatory cytokine expression in the colon of dextran sodium sulfate (DSS)-induced colitic mice. Gene expression of Il6, Il17A, Cxcl2, and Ccl2 in the colon of mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Values are the mean ± SEM (n = 7). Means without a common letter differ, p < 0.05.

Figure 7

Effects of whole LP powder, methanol (MetOH) extract, and MetOH extraction residue (MetOH residue) on organic acid concentrations in feces of dextran sodium sulfate (DSS)-induced colitic mice. Fecal concentrations of acetate, propionate, n-butyrate, iso-butyrate, n-valerate, iso-valerate, lactate, and succinate in mice fed diets with and without whole LP powder, MetOH extract, and MetOH residue, with or without DSS administration, as determined by liquid chromatograph/tandem mass spectrometry (LC/MS/MS) analysis. Values are the mean ± SEM (n = 7). Means without a common letter differ, p < 0.05.