Ethnopharmacological relevance: Madhuca indica J. R Gmel. (Sapotaceae) has shown antioxidant, anti-inflammatory, analgesic, anti-diabetic and hepatoprotective potential. It has been traditionally used as laxative, tonic, anti-burn, anti-earthworm, wound healing and headache. Aim of the study: To investigate the efficacy and possible mechanism of Madhuca indica J. F. Gmel. leaves methanolic extract (MI-ALC) and its isolated chloroform fraction (D3) against experimental induced gastric ulcers. Materials and methods: D3 was isolated from MI-ALC, well characterized (HPTLC, FT-IR, H-1-NMR and LC-MS) and evaluated for its gastroprotective activity by using acetic acid induced ulcer in male Wistar rats (150-200 g). D3 (2.5, 5 and 10 mg/kg, p.o.) were administered for the period of 14 days. At the end of treatment, rats were sacrificed to collect the stomach sample for evaluation of antioxidant (SOD, GSH, and MDA) enzyme, oxido-inflammatory (TNF-alpha, IL-1, iNOs) and prostaglandins (COX-II) markers by using RT-PCR. Results: The structure and molecular weight (MW) of the isolated compound (D3) were confirmed by 1D and 2D spectral data and characterized as 3,5,7,3',4'-Pentahydroxy flavone with MW C15H10O7. Administration of 3,5,7,3',4'-Pentahydroxy flavone (5 and 10 mg/kg) significantly and dose-dependently inhibited (P < 0.01 and P < 0.001) acetic acid induced an alteration in the antioxidant enzyme. It also significantly and dose-dependently down-regulated gastric oxido-inflammatory and prostaglandins markers. Histopathological aberration induced in the stomach also attenuated by 3,5,7,3',4'-Pentahydroxy flavone treatment. Conclusion: Finding of present investigation suggests that MI-ALC possessed potent antiulcer activity due to the presence of 3,5,7,3',4'-Pentahydroxy flavone via its oxido-inflammatory and prostaglandins modulatory potential. (C) 2016 Elsevier Ireland Ltd. All rights reserved.