PHYTOCHEMICAL PROFILING, GC–MS ANALYSIS, AND ANTIBACTERIAL ACTIVITY OF THE ACETONE EXTRACT OF AEGLE MARMELOS (L.) LEAVES

Krishnagowdu Saravanan*, Selvaraj Thiraviam, Murugan Sanjay, Sundarasamy Dhanapal, Pratima Gorai, Shubham Gorai, Lakshmanan Archana Devi. Subbiah Selvarani

ABSTRACT

Background and objectives: Aegle marmelos (L.) Corrêa (Rutaceae), the bael tree, occupies a central position in Indian traditional medicine, yet the mid-polarity metabolite fraction of its foliage remains comparatively under-characterised because most published surveys have relied on aqueous, methanolic or ethanolic extraction. Acetone spans a polarity window that recovers both lipophilic terpenoids and moderately polar phenolics, and is therefore mechanistically attractive for antibacterial screening. The present study was undertaken to (i) compare the qualitative metabolite profile of acetone, n-hexane and petroleum ether extracts of A. marmelos leaves, (ii) resolve the chemical composition of the acetone extract by gas chromatography–mass spectrometry (GC–MS), and (iii) evaluate the concentration-dependent activity of the extract against Escherichia coli. Methods: Mature leaves were collected, shade-dried, pulverised and extracted sequentially with acetone, n-hexane and petroleum ether. Extracts were screened for terpenoids, coumarins, tannins, quinones, phenolics and flavonoids using standard colourimetric assays. The acetone extract was resolved on a 5%-phenyl polysiloxane capillary column with electron-impact ionisation, and constituents were assigned by comparison of mass spectra with the NIST reference library. Antibacterial activity against E. coli was determined by the agar well diffusion method at 2.5, 5.0, 7.5 and 10.0 µg/mL with amikacin as the reference control (n = 3), and data were analysed by one-way ANOVA followed by Tukey’s HSD test. Results: The acetone extract displayed the broadest qualitative profile, being the only solvent to recover tannins and phenolics in addition to terpenoids, coumarins and quinones. GC–MS resolved 35 chromatographic entries corresponding to 34 distinct constituents distributed across nine structural classes, dominated in equal measure by fatty acids with their esters and by monoterpenoids (six constituents each, 17.6%), followed by sesquiterpenoids (11.8%); on a relative peak-area basis fatty acids and esters (26.6%), monoterpenoids (23.0%) and phytosterols (18.6%) together accounted for more than two-thirds of the detected signal. Stigmasterol (relative peak area 100.00), n-hexadecanoic acid (91.83), (+)-4-carene (88.91), α-terpineol (48.63) and caryophyllene (45.86) were the principal constituents. The extract inhibited E. coli at all four concentrations, with a maximum zone of inhibition of 9.67 ± 0.50 mm at 7.5 µg/mL — significantly greater than the amikacin control (6.33 ± 0.90 mm; p < 0.05) — followed by an unexpected decline to 4.40 ± 1.00 mm at 10.0 µg/mL. Treatment effects were highly significant overall (F4,10 = 15.73, p = 0.00026). Conclusion: The acetone extract of A. marmelos leaves is a chemically rich, terpenoid- and fatty-acid-dominated matrix with demonstrable activity against a Gram-negative enteric pathogen. The non-monotonic concentration–response relationship is most plausibly attributed to solubility- and diffusion-limited behaviour of lipophilic constituents in agar rather than to a true biphasic pharmacological effect, and this interpretation is explicitly framed as a hypothesis requiring dilution-based confirmation. The findings justify bioassay-guided fractionation and broth microdilution determination of MIC/MBC values as the immediate next steps.

Keywords: Aegle marmelos; bael; acetone extract; phytochemical screening; GC–MS; stigmasterol; n-hexadecanoic acid; caryophyllene; agar well diffusion; Escherichia coli; antimicrobial resistance.