Phytochemical characterization and in vitro evaluation of antibacterial and cytotoxic activities of indigenous Iranian Nigella sativa L. seeds

Nigella sativa L. is a medicinal plant with well-documented pharmacological properties; however, comparative information regarding the biological activities of its essential oil and native protein fraction remains limited. This study aimed to isolate and characterize the essential oil and protein fraction of N. sativa seeds and to comparatively evaluate their antibacterial activity against Staphylococcus aureus and Escherichia coli as well as their cytotoxic effects on MCF-7 human breast cancer cells and normal L-929 fibroblasts. Essential oil was extracted by Soxhlet extraction followed by hydrodistillation using a Clevenger apparatus, whereas the protein fraction was isolated using lysis buffer extraction and ammonium sulfate precipitation. Protein content and integrity were evaluated by the Bradford assay and SDS-PAGE, respectively, and thymoquinone was quantified by high-performance liquid chromatography. Antibacterial activity was assessed using disk diffusion and growth inhibition assays, while cytotoxicity was determined by MTT and Neutral Red uptake assays. The essential oil exhibited pronounced antibacterial activity, producing complete growth inhibition of S. aureus at 40 mg/mL and approximately 44% inhibition of E. coli . In contrast, the native protein fraction displayed only moderate antibacterial activity against S. aureus and no detectable activity against E. coli . The essential oil also demonstrated selective cytotoxicity toward MCF-7 cells, with an estimated IC 50 of approximately 0.26 mg/mL, while causing minimal toxicity in normal L-929 fibroblasts at biologically active concentrations. Conversely, the protein fraction showed no significant cytotoxic effects in either cell line. These findings identify the essential oil as the principal bioactive fraction responsible for the antibacterial and selective anticancer activities of N. sativa seeds, whereas the native protein fraction exhibited limited biological activity in its crude form. Further purification of protein-derived bioactive components and mechanistic in vivo investigations are warranted to fully explore the therapeutic potential of N. sativa.