Synergistic antimicrobial activity of essential oils mixture of Moringa oleifera, Cinnamomum verum and Nigella sativa against Staphylococcus aureus using L-optimal mixture design

Abstract

The urgent need to address the growing problem of antimicrobial resistance in multidrug-resistant bacteria requires the development of pioneering approaches to treatment. The present study aims to evaluate the antimicrobial potential of the essential oils (EOs) of Moringa oleifera (moringa), Cinnamomum verum (cinnamon), and Nigella sativa (black seed) and the synergistic effect of the mixture of these oils against Staphylococcus aureus MCC 1351. Statistical modeling revealed cinnamon oil had the highest individual antimicrobial potency, followed by black seed oil. The combination of the three EOs exhibited significant synergistic effects compared to the individual oils, with a Fractional Inhibitory Concentration (∑FIC) index of 0.27. L-Optimal mixture design of response surface methodology (RSM) identified the optimal mixture as moringa: cinnamon: black seed oils by the ratio of (1:1:1) in run 15 (0.338:0.307:0.355 mL) (v/v). This mixture exhibited significant antibacterial efficacy, outperforming individual oils and conventional antibiotics like tetracycline. Specifically, the combination reduced the MIC values from 3.12, 0.78, and 6.25 to 0.25, 0.06, and 0.78 μg/mL for moringa, cinnamon, and black seed oil, respectively. Synergistic interactions between oils further boosted efficacy, with moringa-cinnamon and cinnamon-black seed pairings exhibiting the strongest synergies. The developed predictive models for IZD and MIC showed excellent fit, with R² values of 0.9843 and 0.9958, respectively. Pareto chart analysis highlighted the predominant individual and synergistic effects, with the Moringa-Cinnamon interaction exhibiting the highest positive synergy. Notably, the oil mixture of run 15 demonstrated excellent biocompatibility, maintaining 97.6% viability of normal human skin fibroblasts (HSF) after 24 h exposure to 200 μL EOs of the mixture per mL. Gas chromatography mass spectrometry (GC/MS) identified abundant bioactive phytochemicals like cinnamaldehyde, linoleic acid, and palmitic acid methyl esters underlying the observed antimicrobial effects. This rationally designed, synergistic phytochemical combination presents a promising natural therapeutic against antibiotic-resistant S. aureus while exhibiting minimal cytotoxicity. The results underscore how combining essential oils could help address the issue of antibiotic resistance in S. aureus.