Anti-ulcerogenic effect of methanolic extracts from Enicosanthellum pulchrum (King) Heusden against ethanol-induced acute gastric lesion in animal models

Abstract

A natural source of medicine, Enicosanthellum pulchrum is a tropical plant which belongs to the family Annonaceae. In this study, methanol extract from the leaves and stems of this species was evaluated for its gastroprotective potential against mucosal lesions induced by ethanol in rats. Seven groups of rats were assigned, groups 1 and 2 were given Tween 20 (10% v/v) orally. Group 3 was administered omeprazole 20 mg/kg (10% Tween 20) whilst the remaining groups received the leaf and stem extracts at doses of 150 and 300 mg/kg, respectively. After an additional hour, the rats in groups 2-7 received ethanol (95% v/v; 8 mL/kg) orally while group 1 received Tween 20 (10% v/v) instead. Rats were sacrificed after 1 h and their stomachs subjected to further studies. Macroscopically and histologically, group 2 rats showed extremely severe disruption of the gastric mucosa compared to rats pre-treated with the E. pulchrum extracts based on the ulcer index, where remarkable protection was noticed. Meanwhile, a significant percentage of inhibition was shown with the stem extract at 62% (150 mg/kg) and 65% (300 mg/kg), whilst the percentage with the leaf extract at doses of 150 and 300 mg/kg was 63% and 75%, respectively. An increase in mucus content, nitric oxide, glutathione, prostaglandin E2, superoxide dismutase, protein and catalase, and a decrease in malondialdehyde level compared to group 2 were also obtained. Furthermore, immunohistochemical staining of groups 4-7 exhibited down-regulation of Bax and up-regulation of Hsp70 proteins. The methanol extract from the leaves and the stems showed notable gastroprotective potential against ethanol.

Figure 1. Chromatographic profiles of E. pulchrum extracts

(A–B) leaf extract constituents profile. (C–D) stem extract constituent profile.

Figure 2. Histological sections in acute toxicity test (H&E staining, 20x).

Histological sections of renal (first row) and liver (second row) cells in acute toxicity test. Rats treated with 5 mL/kg vehicle (10% Tween 20) (A and D). Rats treated with 1500 mg/kg (5 mL/kg) of leaf extract (B and E). Rats treated with 1500 g/kg (5 mL/kg) of stem extract (C and F). There is no significant difference in structures of renal and liver cells between treated and control groups.

Figure 3. Effect of E. pulchrum extracts on macroscopic appearance of the gastric mucosa in ethanol-induced gastric mucosal lesions in the groups (n = 6).

(A) Normal control group shows no lesions to the gastric mucosa; (B) Ulcer control group displays severe lesions to the gastric mucosa (black arrow); (C) Omeprazole (20 mg/kg), a positive control group shows mild lesions to the gastric mucosa; (D–E) Rats receiving 150 and 300 mg/kg of stem extract show moderate lesions to the gastric mucosa, (F–G) Rats pretreated with 150 and 300 mg/kg of leaf extract show mild lesions in the gastric mucosa.

Figure 4. Effects of E. pulchrum extracts on glutathione (GSH), superoxide dismutase (SOD) and nitric oxide (NO).

Groups 1–3 represent negative control of Tween80, ethanol 95% and positive control (omeprazole, 20 mg/mL), respectively. The experimental groups from 4–7 received 150 and 300 mg/kg of stem extract and the same dosage of leaf extract. All values are expressed as standard error of mean where p<0.05 is considered significant (one between groups ANOVA with post-hoc analysis). *P<0.05 compared to group 2 and ^# P<0.05 compared to group 3. All values represent mean ± S.E.M (n = 6) and the biological assays were done in triplicate.

Figure 5. Histological effects of E. pulchrum extracts on gastric tissue (H&E staining 20×) in the groups (n = 6).

Normal group shows healthy gastric mucosa (A). The ulcer control group has severe disruption to the gastric mucosa (black arrow); necrotic lesions penetrating deeply into the mucosa, extensive edema of the submucosal layer (blue arrow) and leucocyte infiltration (yellow arrow) (B). The omeprazole group shows mild damage to the surface epithelium mucosa (C). In the experimental groups, rats pre-treated with 150 and 300 mg/kg of the stem extract show mild mucosal disruption with edema and leucocyte infiltration of the submucosal layer (D–E). In the groups pretreated with 150 and 300 mg/kg of the leaf extract, rats showed a moderate mucosal disruption with edema and leucocyte infiltration in the submucosal layer (F–G).

Figure 6. Effects of E. pulchrum extracts on gastric tissue glycoprotein (PAS staining 20×) in the groups (n = 6).

The negative control group (A); the ulcer control group (B); the omeprazole-treated group (20 mg/kg) control group (C); the experimental groups pre-treated with 150 and 300 mg/kg of stem extract (D–E) and groups pre-treated with leaf extract (F–G). Magenta color (green arrow) in the apical epithelial cells of the groups pre-treated with omeprazole (C), and extracts (D–G) shows increased intensity of mucus in gastric glands.

Figure 7. Effects of E. pulchrum extracts on the immunohistochemistry analysis of expression of Hsp70 proteins (20×) in the groups (n = 6).

Normal control group (A), ulcer control group (B), omeprazole-treated group 20 mg/kg (C), rats receiving 150 and 300 mg/kg of the stem extract (D–E), rats receiving 150 and 300 mg/kg of the leaf extract (F–G). Immunohistochemistry staining of Hsp70 shows over-expression of Hsp70 protein in the experimental groups (D–G). The orange arrow points to Hsp70 protein accumulation.

Figure 8. Effects of E. pulchrum extracts on the immunohistochemistry analysis of the expression of the Bax protein in the gastric mucosa of rats in the groups (n = 6).

(A) Normal control group; (B) ulcer control group; (C) positive control group, omeprazole (20 mg/kg); (D–E) Rats pre-treated with 150 and 300 mg/kg stem extract; (F–G) Rats pre-treated with 150 and 300 mg/kg leaf extract of E. pulchrum. Bax protein expression (red arrow) was downregulated in rats pre-treated with postive control and E. pulchrum extracts (magnification 20×).

Figure 9. Effect of leaf and stem extracts of E. pulchrum on Hsp70, Bax and β-actin.

Groups 1 represents negative control, group 2 represents ulcer control group and group 3 represents reference standard drug. The experimental groups 4–5 received 300 mg/kg of stem and leaf extract, respectively. The statistical significance is expressed as p<0.05.