Influence of Sequential Solvent Extraction on the Antibacterial Activity of Origanum vulgare Leaf Extracts

Natural products remain an invaluable source of structurally diverse molecules with promising pharmaceutical applications. Origanum vulgare L. is an aromatic and medicinal species rich in phenolic monoterpenes, flavonoids, and other specialized metabolites associated with antimicrobial activity. In this study, the influence of sequential solvent extraction on the recovery of antibacterial metabolites from O. vulgare leaf extracts was investigated using solvents of increasing polarity (n-hexane, ethyl acetate, and methanol). The resulting crude extracts were evaluated against Escherichia coli by the agar disk diffusion method at concentrations of 200, 100, 50, 25, and 12.5 mg/mL. None of the extracts produced inhibition zones against E. coli, whereas the positive controls (doxycycline and ceftriaxone) exhibited inhibition halos ranging from 2.0 to 3.3 cm, confirming the reliability of the assay. The lack of antibacterial activity against this Gram-negative bacterium is likely associated with the permeability barrier of the outer membrane, which limits the penetration of hydrophobic phytochemicals. Previous studies have demonstrated that ethanolic extracts and essential oils of O. vulgare, rich in carvacrol and thymol, exhibit significant antibacterial activity against Staphylococcus aureus, producing inhibition zones of up to 2.16 cm. These findings emphasize the influence of solvent polarity on the extraction of bioactive metabolites and provide a foundation for future phytochemical characterization and bioactivity-guided fractionation to identify the compounds responsible for the antibacterial properties of O. vulgare.