Toxicity and Anti-Inflammatory Activity of Phenolic-Rich Extract from Nopalea cochenillifera (Cactaceae): A Preclinical Study on the Prevention of Inflammatory Bowel Diseases

Abstract

Phenolic compounds have been scientifically recognized as beneficial to intestinal health. The cactus Nopalea cochenillifera, used as anti-inflammatory in traditional medicine, is a rich source of these bioactive compounds. The present study aimed to investigate the phytochemical profile of N. cochenillifera extract and evaluate its acute toxicity and anti-inflammatory effect on 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis in rats. The total phenolic content per gram of dry extract was 67.85 mg. Through HPLC-IES-MSⁿ, a total of 25 compounds such as saccharides, organic acids, phenolic acids and flavonoids were characterized. The dose of 2000 mg/kg of extract by an oral route showed no signs of toxicity, mortality or significant changes in biochemical and hematological parameters. Regarding intestinal anti-inflammatory effects, animals were treated with three different doses of extract or sulfasalazine. Macroscopic analysis of the colon indicated that the extract decreased the disease activity index. Levels of IL-1β and TNF-α decreased, IL-10 increased and MDA and MPO enzyme levels decreased when compared with the control group. In addition, a down-regulation of MAPK1/ERK2 and NF-κB p65 pathway markers in colon tissue was observed. The epithelial integrity was improved according to histopathological and immunohistological analysis. Thus, the extract provided strong preclinical evidence of being effective in maintaining the remission of colitis.

Keywords: Cactaceae; colitis; flavonoids; functional foods; herbal medicines; inflammatory chronic diseases.

Figure 1

Relative body weight (A) and food consumption (B) of female rats treated with a single dose (2000 mg/kg) of Nopalea cochenillifera hydroethanolic extract (NCHE) observed for 14 days. Data are expressed as Mean ± SEM (n = 6/group). No statistical differences were detected between the treated group and the control group.

Figure 2

Photomicrographs of histopathological analyses of the liver, kidneys and spleen (eosin-hematoxylin stain; 10× and 40× magnification) of tissues from rats in the control group and treated with N. cochenillifera hydroethanolic extract (NCHE) in a single oral dose (2000 mg/kg) after 14 days.

Figure 3

Effect of treatments with N. cochenillifera hydroethanolic extract (NCHE) (100, 200 and 300 mg/kg) and sulfasalazine (SSZ) (250 mg/kg) on colitis induced in rats by 2.4-Dinitrobenzene sulfonic acid (DNBS). (A) Experimental design; (B) colon damage; (C) weight loss; (D) disease activity index; (E) weight/length ratio; (F) macroscopic damage score. H: Healthy group. Data are expressed as means ± SEM. Groups with a different letter differ statistically (p < 0.05).

Figure 4

Effect of N. cochenillifera hydroethanolic extract (NCHE) (100, 200 and 300 mg/kg) and sulfasalazine (SSZ) (250 mg/kg) on colonic (A) malondialdehyde (MDA) levels and (B) myeloperoxidase activity. H: Healthy group. Data are expressed as means ± SEM. Groups with a different letter differ statistically (p < 0.05).

Figure 5

Effect of N. cochenillifera hydroethanolic extract (NCHE) (100, 200 and 300 mg/kg) and sulfasalazine (SSZ) (250 mg/kg) on colonic levels of the cytokines: Tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin 10 (IL-10). H: Healthy group. Data are expressed as means  ±  SEM. Groups with a different letter differ statistically (p  <  0.05).

Figure 6

Effect of N. cochenillifera hydroethanolic extract (NCHE) (100, 200 and 300 mg/kg) and sulfasalazine (SSZ) (250 mg/kg) on the gene expression of mitogen-activated protein kinase 1 (MAPK1/ERK2), nuclear factor kappa B p65 (NF-κB p65), zonula occludens type I (ZO-1) and mucin type II (MUC-2) in colonic tissue of the experimental trial of acute colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS) in rats. Healthy group. Data are expressed as means ± SEM. Groups with a different letter differ statistically (p < 0.05).

Figure 7

Histopathological analyses of representative colonic tissue (longitudinal section) using Hematoxylin/Eosin staining. Groups represented: H—healthy group (A), DNBS—DNBS control (B), Sulfasalazine (SSZ) (250 mg/kg) (C) and NCHE—N. cochenillifera hydroethanolic extract (100, 200 and 300 mg/kg) (D–F) and histopathological score (G). The figure represents microscopic damage assessment (10× and 40× magnification). Normal intestinal layers (star), inflammatory cell infiltrate intense (***) and moderate (**), total mucosal ulceration (circle), Initial mucosal regeneration (arrow). Data are expressed as means  ±  SEM. Groups with a different letter differ statistically (p  <  0.05).

Figure 8

Immunohistochemical analyses of representative colonic tissue (longitudinal section). NF-κB p65 and COX-2 and their respective immunohistochemical scores. Groups represented: H—healthy group (A), DNBS—DNBS control (B), Sulfasalazine (SSZ) (250 mg/kg) (C) and NCHE—N. cochenillifera hydroethanolic extract (100, 200 and 300 mg/kg) (D–F). The figure represents a microscopic damage assessment (10× and 40× magnification), the lines indicate diffuse active colitis with intense antibody reactivity, the asterisks represent dense infiltrate of inflammatory cells and arrows indicate antibody reactivity. Data are expressed as means  ±  SEM. Groups with a different letter differ statistically (p  <  0.05).

Figure 9

Schematic representation of the proposed mechanisms of action of dinitrobenzene sulfonic acid (DNBS) and Nopalea cochenillifera hydroethanolic extract (NCHE) in DNBS-induced colitis. NCHE inhibits the activation of nuclear factor kappa B (NF-κB) and mitogen activated protein kinase 1 (MAPK1) signaling pathways induced by DNBS. The suppression of the translocation of NF-κB to the nucleus can also be a possible reason for reduced levels of cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Through recognition of Pathogen Associated Molecular Patterns (PAMPs) by toll-like receptors (TLRs), gastric mucosal epithelial cells, through signal transduction pathways, can attract neutrophils and dendritic cells, favoring the production of inflammatory cytokines (IL-1β and TNF-α). It is suggested that NCHE may act by reducing neutrophil chemotaxis and neutralizing reactive oxygen species (ROS) produced by DNBS inflammation, decreasing myeloperoxidase (MPO) and malondialdehyde (MDA) levels in the tissue, respectively. NCHE can also improve DNBS-induced intestinal barrier dysfunction through positive regulation of mucin type 2 (MUC-2) and tight junction protein zonula occludens-1 (ZO-1) expression.