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

Figures & data

Figure 1 Cytotoxic effect of MECE on J774A.1 cells.

Abbreviation: MECE, methanolic extract of Clausena excavata leaves.

Table 1 Effect of methanolic Clausena excavata leaf extract treatment on gastric secretory effects of rats

Pretreatment	Volume of gastric juice (mL)	pH of gastric juice	Total acidity (mEq/L)
5% Tween 20 v/v (normal controla)	5.2±0.5	1.5±0.1	3,717.9±146.7
Omeprazole (20 mg/kg Bwt)	2.2*±0.3	3.8*±0.1	1,976.7*±389.0
MECE (200 mg/kg Bwt)	2.6*±0.2	2.6*±0.1	1,579.0*±236.0
MECE (400 mg/kg Bwt)	1.7*±0.3	2.7*±0.3	1,102.4*±82.2

Notes: All the values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with normal control.

Abbreviations: Bwt, body weight; MECE, methanolic extract of Clausena excavata leaves.

Table 2 Effect of methanolic Clausena excavata leaf extract treatment on pH of gastric content, gastric ulcer area, and ulcer inhibition in rats

Pretreatment	pH of gastric content	Mucus content	Ulcer area (mm^2)	Inhibition (%)
5% Tween 20 v/v (normal controla)	6.8±0.2	0.9±0.5	0	NA
Ethanol (5 mL/kg Bwt) (untreated control)	3.1±0.1	0.5±0.1	460.8±65.1	NA
Omeprazole (20 mg/kg Bwt)	7.1*±0.3	0.8*±0.1	59.4*±23.8	86.3
MECE (200 mg/kg Bwt)	6.2*±0.1	0.7*±0.2	34.2*±14.5	92.6
MECE (400 mg/kg Bwt)	6.0*±0.3	1.1*±0.03	56.2*±23.9	87.8

Notes: All the values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with untreated control.

Abbreviations: Bwt, body weight; MECE, methanolic extract of Clausena excavata leaves; NA, not applicable.

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).

Table 3 Gastric tissue lesion score in rat gastric tissue with ulcer treated with methanolic Clausena excavata leaf extract

Treatment	Hemorrhage (no of squares)	Submucosal edema (no of squares)	Epithelial cell loss (no of squares)	Inflammatory cell infiltration (no of squares)	Total lesion area (mm^2)
5% Tween 20 v/v (normal controla)	0	0	0	0	0
Ethanol (5 mL/kg Bwt) (untreated control)	2.8±0.2	2.6±0.3	2.7±0.4	2.3±0.1	10.4
Omeprazole (20 mg/kg Bwt)	1.5*±0.4	1.5*±0.2	1.4*±0.1	1.6*±0.3	6.1*
MECE (200 mg/kg Bwt)	1.6*±0.3	1.4*±0.1	1.5*±0.3	1.3*±0.4	5.8*
MECE (400 mg/kg Bwt)	0.7*±0.1	0.9*±0.1	0.6*±0.2	0.7*±0.1	2.9*

Notes: One unit square is the 2×2 mm² area of tissue with lesion. All values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with untreated control.

Abbreviations: Bwt, body weight; MECE, methanolic extract of Clausena excavata leaves.

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.

Table 4 Area of rat gastric mucosal tissue positive for Bax protein staining after treatment with methanolic Clausena excavata leaf extract

Treatment	Stained area (×10^3) (µm^2)	% Area
5% Tween 20 v/v (normal controla)	0.00	0
Ethanol (5 mL/kg Bwt) (untreated control)	48.0*±0.4	40
Omeprazole (20 mg/kg Bwt)	21.2*±2.6	17
MECE (200 mg/kg Bwt)	15.1*±2.1	12
MECE (400 mg/kg Bwt)	4.2*±1.3	4

Notes: All values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with normal control.

Abbreviations: Bwt, body weight; MECE, methanolic extract of C. excavata leaves.

Table 5 Area of rat gastric mucosal tissue positive for HSP70 protein immunoperoxidase staining after treatment with methanolic Clausena excavata leaf extract

Treatment	Stained area (×10^3) (μm^2)	% area
5% Tween 20 v/v (normal controla)	5.4±2.2	4
Ethanol (5 mL/kg Bwt) (untreated control)	6.3±3.1	5
Omeprazole (20 mg/kg Bwt)	32.4*±2.9	22
MECE (200 mg/kg Bwt)	36.4*±3.3	25
MECE (400 mg/kg Bwt)	46.3*±3.7	33

Notes: All the values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with normal control.

Abbreviations: Bwt, body weight; HSP70, heat shock protein 70; MECE, methanolic extract of C. excavata leaves.

Table 6 Area of rat gastric mucosal tissue positive for TGF-β protein immunoperoxidase staining after treatment with methanolic Clausena excavata leaf extract

Treatment	Stained area (×10^3) (µm^2)	% Area
5% Tween 20 v/v (normal controla)	3.6±0.8	3
Ethanol (5 mL/kg Bwt) (untreated control)	4.5±0.1	4
Omeprazole (20 mg/kg Bwt)	44.2*±0.9	34
MECE (200 mg/kg Bwt)	31.6*±2.3	20
MECE (400 mg/kg Bwt)	35.1*±3.4	24

Notes: All the values are expressed as mean ± standard error of the mean.

a Treated with 5% Tween 20 only.

* Significant differences at P<0.05 compared with normal control.

Abbreviations: Bwt, body weight; MECE, methanolic extract of C. excavata leaves; TGF-β, transforming growth factor-beta.

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.