Unlocking the therapeutic potential of Nigella sativa extract: phytochemical analysis and revealing antimicrobial and antioxidant marvels

Abstract

The growing global threat of antimicrobial resistance endangers both human and animal life, necessitating the urgent discovery of novel antimicrobial solutions. Medicinal plants hold promise as sources of potential antimicrobial compounds. In this study, we investigated the phytochemical constituents and microbicidal capabilities of the ethanolic extract from Nigella sativa (black seed). Gas chromatography analysis (GC) identified 11 compounds, among them thymoquinone, and thymol, contributing to antimicrobial and antioxidant properties. Antimicrobial assays demonstrated notable inhibition zones against broad spectra of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Enterobacter, and Bacillus subtilis, along with potent antifungal activity against Aspergillus niger, Penicillium, and Candida albicans. Notably, when combined with antibiotics, the extract displayed exceptional synergistic antimicrobial efficacy. The black seed extract demonstrated membrane-damaging activity and disrupted virulence factors that protect microbes from antimicrobial agents, including the formation of bacterial biofilm and protease secretion. Thymoquinone, the primary active constituent of the extract, exhibited similar antimicrobial and ant virulence properties. In silico analysis targeting key regulators of quorum sensing and biofilm formation in P. aeruginosa, such as RhlG, LasR, and PqsR, showed a remarkable affinity of thymol and thymoquinone for these targets. Moreover, the N. sativa extract exhibited dose-dependent cytotoxicity against both the promastigote and amastigote forms of Leishmania tropica parasites, hinting at potential antiparasitic activity. In addition to its antimicrobial properties, the extract displayed potential antioxidant activity at a concentration of 400 μg/mL.

Keywords: Antimicrobial; Antioxidant; Antiparasitic; Black seed; Synergism; Traditional medicine.

Fig. 1

Preparation of N. sativa seed extract. The seeds were ground and mixed with ethanol for extraction of biocomponents

Fig. 2

Fourier transform infrared analysis of Nigella sativa ethanolic extract (a). GC analysis and identified compounds in ethanolic extract of N. sativa (b) and chemical structure of the identified compounds (c)

Fig. 3

Broad spectrum antimicrobial activity of N. sativa extract. (a) antibacterial activity, (b) antifungal activity, and (c) pictures from the antifungal assay of N. sativa extract (NSE). Data were represented as (Mean ± SEM) of 3 independent measurements, and significance was analyzed by two-tailed unpaired T test. No significance (ns); * p ≤ 0.05; ** p ≤ 0.01

Fig. 4

N. sativa and thymoquinone disrupt the bacterial biofilm and compromise the protease activity of P. aeruginosa. a) Effect of N. sativa and thymoquinone on P. aeruginosa biofilm formation and (b & c) protease inhibition using the skim milk agar method. Two hundred microliters of supernatant from different cultures with N. sativa or thymoquinone was tested for protease activity on skim milk agar by a well diffusion assay. Data are presented as the mean ± standard error of the mean (SEM). ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001. A two-tailed unpaired T test was employed to analyze significance

Fig. 5

2D and 3D interactions of thymol (a) and thymoquinone (b) with the binding site of the crystal structure of the P. aeruginosa RhlG/NADP active-site complex (PDB: 2B4Q)2D and 3D interactions of thymol (c) and thymoquinone (d) with the binding site of the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer (PDB: 2UV0)

Fig. 6

2D and 3D interactions of thymol (a) and thymoquinone (b) with the binding site of the crystal structure of the PqsR coinducer binding domain of P. aeruginosa with the ligand NHQ (PDB: 4JVD). The structures of thymol and thymoquinone (c)

Fig. 7

(a) antioxidant activity and (b) anti-leishmanial activity of N. sativa extract (NSE)