First Report of (E)-1-Phenyl-3,7-dimethyl-2-octen-1-ol and N-propyl-4-methoxycinnamamide from Aegle marmelos unripe fruit: Isolation, characterization and mechanistic evaluation of proton pump inhibitory and antioxidant activities

Aegle marmelos (L.) Corr. (Rutaceae) is a medicinally important tree widely used across South Asia for the treatment of gastrointestinal disorders including peptic ulcers, diarrhea, and dysentery. Beyond its antiulcer applications, the plant has been reported to possess antioxidant, anti-inflammatory, antimicrobial, and cytoprotective activities. However, the specific bioactive compounds and mechanisms underlying proton pump inhibition remain insufficiently characterized at the molecular level.To isolate and characterize bioactive constituents from the unripe fruit of A. marmelos responsible for proton pump inhibitory and antioxidant activities, and to provide a mechanistic scientific basis for its traditional use in gastric acid-related disorders. Bioassay-guided fractionation of the dichloromethane fraction (DCMF; yield: 4.61 g from 500 g dry powder, 0.92% w/w) from unripe Aegle marmelos fruit was performed by silica gel column chromatography. Isolated compounds were characterized by FTIR, 1 H and 13 C NMR (600/150 MHz, CDCl3), and LC-MS analyses. Proton pump inhibitory activity was assessed by an in vitro H+/K+-ATPase assay using sheep gastric microsomes, with omeprazole as the positive reference. Antioxidant activity was determined by the DPPH radical scavenging assay. In silico molecular docking against the gastric H+/K+-ATPase crystal structure (PDB: 7 ×22), molecular dynamics (MD) simulations (100 ns, GROMACS), PASS biological activity prediction, and Lipinski drug-likeness evaluation were performed. Two pure compounds were isolated: AM-DCMF-32, identified as ( E )-1-phenyl-3,7-dimethyl-2-octen-1-ol (C 16 H 24 O; MW 232.36 g/mol; yield 28.4 mg), and AM-DCMF-65, identified as N-propyl-4-methoxycinnamamide (C 13 H 17 NO 2 ; MW 219.28 g/mol; yield 19.6 mg). AM-DCMF-32 demonstrated potent H + /K + -ATPase inhibitory activity (63.48 ± 2.36% at 250 µg/mL) approaching that of omeprazole (69.21 ± 4.14%), and significant DPPH radical scavenging activity (IC 50 = 17.7 µg/mL). AM-DCMF-65 showed moderate inhibition (57.37 ± 1.01% at 250 µg/mL; IC 50 = 25.51 µg/mL). Molecular docking revealed binding affinities of −5.9 and −6.2 kcal/mol for AM-DCMF-32 and AM-DCMF-65, respectively (omeprazole: −6.4 kcal/mol). Both compounds satisfied Lipinski's Rule of Five (zero violations). This study reports the first isolation of ( E )-1-phenyl-3,7-dimethyl-2-octen-1-ol and N-propyl-4-methoxycinnamamide from A. marmelos. Both compounds exhibited in vitro proton pump inhibitory and DPPH radical scavenging activities. These findings provide scientific support for the traditional use of A. marmelos in gastric disorders and identify two compounds reported for the first time from this plant that warrant further pharmacological, toxicological, and in vivo investigations to establish their therapeutic potential as antiulcer agents.