Synergistic gastroprotective activity of methanolic extract of a mixture of Melastoma malabathricum and Muntingia calabura leaves in rats

Abstract

Background: Melastoma malabathricum L. (family Melastomaceae; MM) and Muntingia calabura L. (family Elaeocarpaceae; MC) have been separately reported to possess gastroprotective activity. In an attempt to develop a pharmaceutical product with antiulcer potential, the synergistic gastroprotective activity of methanolic extract of a mixture of MM and MC (MMMC) at various ratios was evaluated in rat models.

Methods: Rats were pre-treated orally with 2% Tween 80 (vehicle), 100 mg/kg ranitidine (reference drug) or MMMC (ratios of 1:1, 1:3 and 3:1 (v/v); doses of 15, 150 or 300 mg/kg) and then subjected to the ethanol-induced gastric ulcer or pyloric ligation assays. Stomach of rats from the former assay was collected and subjected to the macroscopic and microscopic observations, and enzymatic and non-enzymatic antioxidant studies while the gastric juice content and tissue from the latter assay were subjected to the antisecretory activity study. The UHPLC analysis of MMMC was also performed.

Result: MMMC, in the ratio 1:1, demonstrated the most effective (P < 0.001) gastroprotective activity indicated by the highest reduction in ethanol-induced ulcer area formation. These macroscopic findings were supported by the microscopic observations. Except for pH and total acidity, MMMC also significantly (P < 0.001) reduced the volume of gastric content but increased the gastric wall mucus content in the pyloric-ligation test. MMMC also demonstrated remarkable antioxidant activity indicated by the highest total phenolic content (TPC) value and oxygen radical absorbance capacity (ORAC) activity with the recorded IC₅₀ value of approximately 53 μg/mL for the 2,2- diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. MMMC also improved the catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), prostaglandin E2 (PGE2) and malondialdehyde (MDA) activities of the gastric tissue intoxicated by ethanol. UHPLC analysis of MMMC confirmed the presence several flavonoid-based bioactive compounds.

Conclusion: MMMC, at the ratio of 1:1 (v/v), exerts gastroprotective activity partly by activating its antisecretory and antioxidant activities, and via modulation of the gastric tissue endogenous antioxidant system.

Keywords: Antioxidant; Antisecretory; Gastric ulcer; Melastoma malabathricum; Muntingia calabura; Synergistic effect.

Fig. 1

The IC₅₀ value of MMMC at different ratio assessed using the DPPH scavenging assay. a 1:1 (v/v) MMMC. b 1:3 (v/v) MMMC. c 3:1 (v/v) MMMC

Fig. 2

Gross examination of the ethanol intoxicated gastric mucosa tissue of rats orally pretreated with various test solutions. (A1) Rats pretreated with 2% Tween 80 (ulcer control). Severe lesions observed with extensive visible hemorrhagic necrosis of gastric mucosa. (B1) Rats pretreated with ranitidine (100 mg/kg, positive control). Moderate lesions of gastric mucosa were seen compared to the lesions in ulcer control group. (C1, D1, E1) Rats pretreated with low dose of 1:1, 1:3 and 3:1 MMMC, respectively (15 mg/kg). Very moderate lesions were seen in group the D1 and E1. Meanwhile, group C1 shows mild lesions. (F1, G1, H1) Rats pretreated with 150 mg/kg of 1:1, 1:3 and 3:1 MMMC respectively. Group F1 and G1 exhibit mild lesions when compared to the group treated with 2% Tween 80. Very moderate lesion was formed in group H1. (I1, J1, K1) Rats pretreated with high dose of 1:1, 1:3 and 3:1 MMMC (300 mg/kg). Very moderate lesion was seen in group K1. Meanwhile, group I1 and J1 shows a very mild lesion, which indicates highly protection of the extract against gastric ulcers

Fig. 3

Effect of orally administered vehicle (Tween 80, 2%), ranitidine (100 mg/kg) or different ratio of 15, 150 and 300 mg/kg MMMC on ulcer area formation in the ethanol-induced gastric ulcer model. The ulcerated area (mm²) was expressed as Mean ± SEM for six animals. One way ANOVA was followed by Dunnett’s post-hoc test, ***p < 0.001 vs vehicle

Fig. 4

Histological evaluation of the ethanol intoxicated gastric mucosa tissue of rats orally pretreated with various test solutions. (A2) Rats pretreated with 2% Tween 80 (ulcer control) showing severe destruction to surface epithelium and necrotic lesions. (B2) Rats pretreated with ranitidine (100 mg/kg, positive control). Moderate disruption to gastric mucosa layer with moderate edema and hemorrhage, in addition of leukocyte infiltration was observed. (C2, D2, E2) Stomach receiving low dose of 1:1, 1:3 and 3:1 MMMC, respectively (15 mg/kg). Mild lesion on mucosa was seen in group C2 with mild hemorrhage and edema. Meanwhile, in group D2 and E2 show a very moderate effect on mucosa with mild hemorrhage and edema. (F2, G2, H2) Stomach pretreated with 150 mg/kg of 1:1, 1:3 and 3:1 MMMC respectively. Mild effect on mucosa and edema were formed in group F2 and G2. Group H2 exhibit very moderate lesion and mild edema and hemorrhage. (I2, J2, K2) Stomach pretreated with 300 mg/kg of 1:1, 1:3 and 3:1 MMMC with group K2 showing a moderate effect on mucosa with moderate hemorrhage and mild edema. Group I2 exhibit very mild effect mucosa with edema indicating that the 1:1 MMMC at its highest dose was highly able to protect the gastric mucosa. The yellow arrow indicates disruption to the surface epithelium. H-hemorrhage; E-edema; L-leukocyte infiltration (H&E staining, 10 x)

Fig. 5

Effect of orally administered vehicle (Tween 80, 2%), ranitidine (100 mg/kg) or, different ratio of 15, 150 and 300 mg/kg MMMC on gastric wall mucus production in the ethanol-induced gastric ulcer model. The gastric wall mucus content (μg Alcian Blue/g wet tissue) was expressed as Mean ± SEM for six animals. One way ANOVA was followed by Dunnett’s post-hoc test, ***p < 0.001, **p < 0.01 vs vehicle

Fig. 6

UHPLC analysis of MMMC in negative ion mode. Total ion chromatography (TIC) profile of the 1:1 (v/v) MMMC