Prophylactic effects of Clausena excavata Burum. f. leaf extract in ethanol-induced gastric ulcers

Abstract

Clausena excavata is a natural herb with both antioxidant and anti-inflammatory properties. It has been used for decades in folkloric practice for the amelioration of various ailments. In this study, the gastroprotective activity of methanolic extract of C. excavata leaves (MECE) was determined in the Sprague Dawley rat ethanol-induced gastric ulcer model. Rats were pretreated with a single dose of vehicle (5% Tween 20), 20 mg/mL omeprazole, 400 and 200 mg/mL of MECE dissolved in 5% Tween 20. Ulcer was induced with 5 mL/kg of ethanol and stomach tissue was obtained after 1 hour. Histological examination was done on hematoxylin and eosin, periodic acid-Schiff, and immunochemically stained gastric mucosal tissues. Prostaglandin E2, superoxide dismutase, catalase, glutathione peroxidase, and lipid peroxidation levels of the gastric tissue homogenates were also determined. Significantly (P<0.05) smaller ulcer areas, less intense edema, and fewer leukocytes' infiltration were observed in MECE- and omeprazole-treated than in untreated gastric mucosa with ulcer. The gastric pH, mucus production, superoxide dismutase, catalase, and glutathione peroxidase contents increased, while the lipid peroxidation content decreased as a result of MECE treatment. Bcl-2-associated X protein was underexpressed, while heat shock protein 70 and transforming growth factor-beta protein were overexpressed in the ulcerated gastric mucosa tissues treated with omeprazole and MECE. Similarly, there was a reduction in the levels of tumor necrotic factor-alpha and interleukin-6, while the level of interleukin-10 was increased. This study showed that the gastroprotective effect of MECE is achieved through inhibition of gastric juice secretion and ulcer lesion development, stimulation of mucus secretion, elevation of gastric pH, reduction of reactive oxygen species production, inhibition of apoptosis in the gastric mucosa, and modulation of inflammatory cytokines.

Keywords: Rutaceae; antioxidant enzymes; antiulcer; cytokines; histopathology; immunohistochemical proteins.

Figure 1

Cytotoxic effect of MECE on J774A.1 cells. Abbreviation: MECE, methanolic extract of Clausena excavata leaves.

Figure 2

Protective effect of methanolic extract of Clausena excavata leaf on ethanol-induced gastric mucosal lesions in rats. Notes: Pretreatment with (A) 5% Tween 20, 5 mL/kg body weight (normal control); (B) 5 mL/kg body weight, absolute ethanol (ulcer control) showing severe ulceration of the gastric mucosa (arrows); (C) 20 mg/kg body weight omeprazole; (D) 200 and (E) 400 mg/kg body weight extract showing very mild ulceration of the gastric mucosa (arrow).

Figure 3

Rat gastric tissue stained showing mucus production. Notes: (A) Normal gastric tissue (negative control); (B) untreated ethanol-induced gastric ulcer (positive control); and pretreatment with (C) 20 mg/kg body weight omeprazole, (D) 200 and (E) 400 mg/kg body weight methanolic Clausena excavata leaf extract. Magenta coloration in the epithelial cells indicates increased gastric gland secretion. The most intense coloration was observed in gastric tissue treated with 400 mg/kg body weight extract (periodic acid-Schiff stain, ×200).

Figure 4

Untreated gastric ulcer with extensive hemorrhages (black arrows), and sub-mucosal oedema (red arrows). Notes: (A) Normal gastric tissue; (B) untreated gastric ulcer with extensive hemorrhages (black arrows); and pretreated with (C) 20 mg/kg body weight omeprazole, (D) 200 (E) and 400 mg/kg body weight methanolic Clausena excavata leaf extract (H&E stain, ×200). Abbreviation: H&E, hematoxylin and eosin.

Figure 5

Rat gastric tissues showing Bax protein expression. Notes: (A) Negative control; (B) untreated ethanol-induced ulcer showing overexpression of Bax proteins in the epithelial cells; pretreatment with (C) 20 mg/kg body weight omeprazole, (D) 200 and (E) 400 mg/kg body methanolic Clausena excavata leaf extract showing reduced Bax protein expression in the apical epithelial cells (immunoperoxidase stain, ×200).

Figure 6

Rat gastric tissue showing HSP70 expression. Notes: (A) Negative control; (B) untreated ethanol-induced ulcer showing underexpression of HSP70 proteins in the epithelial cells; pretreatment with (C) 20 mg/kg body weight omeprazole, (D) 200 and (E) 400 mg/kg body methanolic Clausena excavata leaf extract. Increased HSP70 expression was seen after treatment with 400 mg/kg body weight extract (immunoperoxidase stain, ×100). Abbreviation: HSP70, heat shock protein 70.

Figure 7

Rat gastric tissues showing TGF-β protein expression. Notes: (A) Negative control; (B) untreated ethanol-induced ulcer showing underexpression of TGF-β proteins in the epithelial cells; pretreatment with (C) 20 mg/kg body weight omeprazole, (D) 200 and (E) 400 mg/kg body weight methanolic Clausena excavata leaf extract showing increased TGF-β protein expression in the apical epithelial cells (immunoperoxidase stain, ×200). Abbreviation: TGF-β, transforming growth factor-beta.

Figure 8

Expression of prostaglandin and antioxidant enzyme parameters in rat gastric tissues with ulcer after Clausena excavata leaf extract treatment. Notes: (A) Prostaglandin E2, (B) superoxide dismutase, (C) catalase, (D) glutathione peroxidase, (E) LPO activities in ulcerated rat gastric mucosa tissues after pretreatment with MECE. All values are expressed as mean ± standard error of the mean. ^*Significant difference at P<0.05 upon comparison with the ulcer control. Abbreviations: Bwt, body weight; LPO, lipid peroxidation; MECE, methanolic extract of C. excavata leaves.

Figure 9

Effect of MECE on TNF-α, IL-6, and IL-10 levels in gastric tissue of ethanol-induced ulcerated rats. Notes: Results are expressed as mean ± SEM. ^*P<0.05, statistically significant as compared with other groups. Abbreviations: Bwt, body weight; IL, interleukin; MECE, methanolic extract of Clausena excavata leaves; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.