Mechanism(s) of action underlying the gastroprotective effect of ethyl acetate fraction obtained from the crude methanolic leaves extract of Muntingia calabura

Abstract

Background: Muntingia calabura L. (family Muntingiaceae), commonly known as Jamaican cherry or kerukup siam in Malaysia, is used traditionally to treat various ailments. The aim of this study is to elucidate the possible underlying gastroprotective mechanisms of ethyl acetate fraction (EAF) of Muntingia calabura methanolic leaves extract (MEMC).

Methods: MEMC and its fractions were subjected to HPLC analysis to identify and quantify the presence of its phyto-constituents. The mechanism of gastroptotection of EAF was further investigated using pylorus ligation-induced gastric lesion rat model (100, 250, and 500 mg/kg). Macroscopic analysis of the stomach, evaluation of gastric content parameters such as volume, pH, free and total acidity, protein estimation, and quantification of mucus were carried out. The participation of nitric oxide (NO) and sulfhydryl (SH) compounds was evaluated and the superoxide dismutase (SOD), gluthathione (GSH), catalase (CAT), malondialdehyde (MDA), prostaglandin E2 (PGE2) and NO level in the ethanol induced stomach tissue homogenate was determined.

Results: HPLC analysis confirmed the presence of quercetin and gallic acid in EAF. In pylorus-ligation model, EAF significantly (p <0.001) prevent gastric lesion formation. Volume of gastric content and total protein content reduced significantly (p < 0.01 and p < 0.05, respectively), while free and total acidity reduced in the doses of 250 and 500 mg/kg (p <0.001 and p <0.05, respectively). EAF also augmented the mucus content significantly (p < 0.001). Pre-treatment with N-nitro-L-arginine methyl ester (L-NAME) or N-ethylmaleimide (NEM) reversed the gastroprotective activity of EAF. EAF treatment markedly ameliorated the SOD, GSH and CAT activity and PGE2 and NO level while attenuating MDA level, relative to the vehicle group.

Conclusions: In conclusion, the underlying gastroprotective mechanisms of EAF could be associated with the antisecretory, participation of mucus, antiperoxidative, improvement of antioxidant status, modulation of NO and SH compounds, stimulation of PGE2 as well as presence of quercetin and gallic acid.

Fig. 1

a and b: HPLC analysis of MEMC, PEF and EAF carried out at the wavelength 330 nm revealed the presence of the quercetin at λ_max 255.5-370.6 nm at RT 3.696 min. Spiking of quercetin with the extracts increased the peak area, corresponding to the same λ_max value of the compounds. c and d:HPLC fingerprinting of MEMC, PEF and EAF at the 280 nm wavelength revealed the presence of the gallicacid at λ_max 216.6-272.0 nm at RT 4.204 min. Spiking of gallic acid with the extracts increased the peak area, corresponding to the same λ_max value of the compounds

Fig. 2

Total ion chromatography (TIC) of the indicatedEAF sample, obtained from the UHPLC instrument in negative ion mode

Fig. 3

MS spectra and structure of ermanin, kaempferide,pinobaksin andpinostrobin detected in the Muntingia calabura ethyl acetate fraction

Fig. 4

Effect of oral administration of vehicle (Tween 80, 8 %), ranitidine (100 mg/kg) or EAF (100, 250, and 500 mg/kg) on pylorus ligation-induced ulcer. The ulcerated area (mm²) was expressed as Mean ± SEM for 6 animals. One way ANOVA was followed by Dunnett's post hoc test, ***p < 0.001 vs. vehicle

Fig. 5

Effect of oral administration of vehicle (Tween 80, 8 %), ranitidine (100 mg/kg) or EAF (100, 250 and 500 mg/kg) on gastric wall mucus produced in the stomach. The gastric wall mucus content (mg Alcian blue/g wet tissue) was expressed as Mean ± SEM for 6 animals. One way ANOVA was followed by Dunnett's post hoc test, ***p < 0.001vs. vehicle

Fig. 6

Effect of vehicle (Tween 80, 8 %, p.o.), carbenoxolone (CBX, 100 mg/kg, p.o.) and EAF (500 mg/kg, p.o) on gastric lesions induced by absolute ethanol in rats pre-treated with saline i.p., L-NAME (70 mg/kg, i.p.) or NEM (10 mg/kg, i.p.). Each column represents the Mean ± SEM of 6 animals. One-way ANOVA followed by Newman–Keuls test, **p < 0.01,***p < 0.001 vs. saline vehicle; †††p <0.001 groups pre-treated with L-NAME vs. vehicle L-NAME; §§ p < 0.01, §§§ p <0.001 groups pre-treated with NEM vs. vehicle NEM; ###p <0.001 saline pre-treatment vs. its corresponding L-NAME pre-treatment; +p < 0.05, +++p < 0.001 saline pre-treatment vs. its corresponding NEM pre-treatment