In vitro and in silico β-lactamase inhibitory properties and phytochemical profile of Ocimum basilicum cultivated in central delta of Egypt

Abstract

Context: Some studies reported the chemical content and antimicrobial properties of Ocimum basilicum L. (Lamiaceae), relevant to the ecological variations in some areas of Egypt and other countries, yet no research was conducted on the plant cultivated in the central delta region of Egypt. Also, no previous data reported on inhibition of β-lactamases by O. basilicum.

Objective: To assess β-lactamases inhibition by O. basilicum extracts and the individual constituents.

Materials and methods: Dried aerial parts of O. basilicum were extracted by hydrodistillation for preparation of essential oil and by methanol for non-volatile constituents. Essential oil content and the methanol extract were analysed by GC-MS and UPLC-PDA-MS/MS, respectively. Methyl cinnamate was isolated and analysed by NMR. Broth microdilution method was used to investigate the antimicrobial against resistant clinical isolates of Escherichia coli identified by double disc synergy, combination disc tests and PCR. The most active oil content was further tested with a nitrocefin kit for β-lactamase inhibition and investigated by docking.

Results: O. basilicum was found to contain methyl cinnamate as the major content of the essential oil. More interestingly, methyl cinnamate inhibited ESBL β-lactamases of the type CTX-M. The in vitro IC₅₀ using nitrocefin kit was 11.6 µg/mL vs. 8.1 µg/mL for clavulanic acid as a standard β-lactamase inhibitor.

Discussion and conclusions: This is the first study to report the inhibitory activity of O. basilicum oil and methyl cinnamate against β-lactamase-producing bacteria. The results indicate that methyl cinnamate could be a potential alternative for β-lactamase inhibition.

Keywords: Methyl cinnamate; antimicrobial.

Figure 1.

Analysis of essential oil and methanol extract of O. basilicum. (A) Gas chromatogram of O. basilicum essential oil with the structure of methyl cinnamate and 1,8-cineol linked to their respective peaks by arrows. UPLC-PDA-MS total ion chromatogram fingerprint of O. basilicum in (B) ESI + and (C) ESI– modes.

Figure 2.

Phenotypic and genotypic screening for ESBL producing E. coli isolates using (a) double disc synergy test showing typical keyhole pattern denoting a positive ESBL producing isolate and (b) combination disc test showing increased zone diameter around the discs after addition of clavulanic acid confirming positive ESBL as referred by arrows. (c) Electrophoregram showing amplified amplicon bands at 500 bp denoting positive bla_CTX-M gene.

Figure 3.

Screening for β-lactamase inhibitors among test botanical agents by colorimetric method showing (a) changes in colours due to different effects against test enzyme and (b) quantitative determination of percent reduction in enzymatic activity showing methyl cinnamate and EO were able to strongly inhibit β-lactamase activity.

Figure 4.

Representation of half maximal inhibitory concentration (IC₅₀) values for both enzyme inhibitors: (a) methyl cinnamate as a test compound and (b) clavulanic acid as a standard β-lactamase inhibitor. Data presented as percent inhibition (%INH) of CTX-M against log the compound concentrations and nitrocefin as a substrate.

Figure 5.

The amino acid residues involved in ligand interaction for (a) clavulanic acid and (b) methyl cinnamate.