Phytochemical analysis and biological activities of Aegle marmelos unripe fruit: An integrated in vitro, in vivo and...

In India, Bangladesh and other South Asian countries, unripe fruits of Aegle marmelos are traditionally consumed as decoctions, powders, or dried preparations for the management of diarrhea, dysentery, abdominal inflammation, gastric irritation, and inflammatory bowel conditions. Despite its long-standing ethnomedicinal use, systematic studies correlating phytochemical composition, fraction-specific bioactivity and computational pharmacological prediction of unripe fruit extracts remain limited.

The present study aimed to comparatively evaluate the antioxidant and anti-inflammatory activities of solvent fractions obtained from unripe Aegle marmelos fruit, identify the most bioactive fraction through a bioactivity-guided approach, and characterize its phytochemical constituents. In addition, computational analyses were performed to explore the potential interactions of selected phytochemicals with the cyclooxygenase-2 (COX-2) enzyme.

Sequential extraction of unripe A. marmelos fruit was performed using n-hexane, dichloromethane (DCMF), chloroform, ethyl acetate, and methanol. Total phenolic and flavonoid contents were quantified, and antioxidant activity was assessed using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, total antioxidant capacity, and iron-reducing power assays. Anti-inflammatory activity was evaluated via heat- and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte membrane stabilization assays and the carrageenan-induced paw edema model in vivo. The most active fraction (DCMF) underwent GC-MS analysis for chemical profiling. In silico studies, including PASS prediction, ADMET profiling, molecular docking, and molecular dynamics simulations, were performed to investigate interactions of identified compounds with COX-2. Elemental composition and heavy metal content were also determined.

DCMF exhibited the highest total phenolic (20.85 ± 0.30 mg GAE/g) and flavonoid (25.84 ± 1.62 mg CE/g) contents, as well as the greatest total antioxidant capacity and reducing power among the tested fractions. In contrast, EAF showed the strongest DPPH radical scavenging activity (IC₅₀ = 16.22 μg/mL). DCMF demonstrated substantial membrane stabilization (75.47 ± 0.47% inhibition at 400 μg/mL) and significantly inhibited carrageenan-induced paw edema (62% inhibition at 5 h; 100 mg/kg), approaching the effect of diclofenac sodium (67.66% at 5 h; 10 mg/kg). GC-MS/MS analysis of DCMF identified 29 compounds, with methyl palmitate (34.61%), methyl oleate (22.04%), caryophyllene oxide (17.90%), and β-sitosterol (7.62%) as the predominant constituents. Computational analyses suggested favorable predicted interactions of selected compounds with the COX-2 binding site, which remained relatively stable during molecular dynamics simulations. Elemental analysis detected essential minerals, including Fe (1.32 ppm) and K (0.4788 ppm), while toxic heavy metals were not detected.

The integrated phytochemical, pharmacological and computational findings highlight unripe A. marmelos fruit as a valuable source of bioactive compounds with significant antioxidant and anti-inflammatory properties. The results provide scientific support for the traditional use of A. marmelos in inflammatory conditions and highlight the unripe fruit as a promising source of bioactive phytochemicals for future investigation. Further studies focusing on the isolation of active constituents and clinical validation are warranted.