Antibacterial, Antibiofilm, and Antioxidant Activity of 15 Different Plant-Based Natural Compounds in Comparison with Ciprofloxacin and Gentamicin

Abstract

Plant-based natural compounds (PBCs) are comparatively explored in this study to identify the most effective and safe antibacterial agent/s against six World Health Organization concern pathogens. Based on a contained systematic review, 11 of the most potent PBCs as antibacterial agents are included in this study. The antibacterial and antibiofilm efficacy of the included PBCs are compared with each other as well as common antibiotics (ciprofloxacin and gentamicin). The whole plants of two different strains of Cannabis sativa are extracted to compare the results with sourced ultrapure components. Out of 15 PBCs, tetrahydrocannabinol, cannabidiol, cinnamaldehyde, and carvacrol show promising antibacterial and antibiofilm efficacy. The most common antibacterial mechanisms are explored, and all of our selected PBCs utilize the same pathway for their antibacterial effects. They mostly target the bacterial cell membrane in the initial step rather than the other mechanisms. Reactive oxygen species production and targeting [Fe-S] centres in the respiratory enzymes are not found to be significant, which could be part of the explanation as to why they are not toxic to eukaryotic cells. Toxicity and antioxidant tests show that they are not only nontoxic but also have antioxidant properties in Caenorhabditis elegans as an animal model.

Keywords: Caenorhabditis elegans; antibacterial agents; antibiotics; antioxidant; biofilms; cannabinoids; essential oils; herbal; mechanism; natural compounds; plant-based compounds; toxicity.

Figure 1

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC) of 15 different plant-based natural compounds (PBCs) against various pathogens of concern. Genta = gentamicin; Cipro = ciprofloxacin; Cin = cinnamaldehyde; TT = tea tree oil; Coum = coumaric acid; CB = Canada balsam; RSV = resveratrol; Cur = curcumin; n = number of trials.

Figure 2

Hydrogen peroxide and free iron [(ferrous) Fe²⁺] concentration. (A) H₂O₂ concentrations in the samples with the naked eye (down) and plate reader (upper), with ciprofloxacin and gentamycin used as the internal controls. (B) Standard 1 mM solution of hydrogen peroxide serially diluted with double-distilled (DD) water 1:2 for a total of 11 samples. DD water was used as the blank working reagent (WR) as a negative control, and bacteria treated with 250 μM H₂O₂ as a positive control. (C) Standard curve after calculating standard curve based on standard concentrations and OD means; H₂O₂ concentrations of all samples were calculated based on R² value. (D) Free iron [(ferrous) Fe²⁺] concentration in a P. aeruginosa lysate after 1 h treatment with selected PBCs. Bacterial incubated in 95 °C for 10 min to break the Fe-S complex as a positive control. The naked eye (upper) heatmap (down) is illustrated in the panels. Con+: control-positive (Bacteria + WR + H₂O₂); Con−: control-negative (Bacteria + WR); Cin: cinnamaldehyde; THC: tetrahydrocannabinol; CBD: cannabidiol; Cip: ciprofloxacin; Gen: gentamycin.

Figure 3

The fluorescence microscopy of PI-staining of P. aeruginosa exposed with selected PBCs, antibiotics, and controls for 1 h. Higher red fluorescence has higher membrane disruption and permeability. The densitometry and intensity measurement heat map is shown in the bottom panel (n = 3). Con+: control-positive (bacteria boiled for 10 min to disrupt the membrane); Con−: control-negative (untreated); Cin: cinnamaldehyde; THC: tetrahydrocannabinol; CBD: cannabidiol; Cip: ciprofloxacin; Gen: gentamycin.

Figure 4

Heatmap on survival ratio of C. elegans and toxicity of the 15 different plant-based natural compounds (PBCs) as an antibacterial agent in the C. elegans model. All treated groups with PBCs were compared with the untreated group and the results are reported in the ratio (%). The red colour shows the lower advantage and the groups with the blue colour had the higher advantage in comparison with the control group. n = number of trials; N = number of animals; Regen = regeneration.

Figure 5

DCFH-DA staining for reactive oxygen species (ROS) level. Fluorescence intensity of all C. elegans ROS levels was measured after exposure to control (PBS) (A), ciprofloxacin (B), THC (C), CBD (D), cinnamaldehyde (E), and carvacrol (F) by DCFH-DA probe. Heatmap on ROS level of C. elegans in the treated and untreated groups showed in panel (G). n = trial number.

Figure 6

DHE staining for O₂⁻ level. Fluorescence intensity of all C. elegans O₂⁻ levels was measured after exposure to control (PBS) (A), ciprofloxacin (B), THC (C), CBD (D), cinnamaldehyde (E), and carvacrol (F) by DHE probe. Heatmap on O₂⁻ level of C. elegans in the treated and untreated groups shown in panel (G). n = trial number.

Figure 7

NDA staining for reduced glutathione level. Fluorescence intensity of all C. elegans GSH levels was measured after exposure to control (PBS) (A), ciprofloxacin (B), THC (C), CBD (D), cinnamaldehyde (E), and carvacrol (F) by NDA probe. Heatmap on glutathione level of C. elegans in the treated and untreated groups shown in panel (G). n = trial number.

Figure 8

Mechanism illustration of plant-based components (PBCs) in bacterial and eukaryotic cells. Overall, PBCs target the bacterial cell membrane (but not eukaryotic cells) because of their different membrane structure. GSH = glutathione; GSSG = oxidized glutathione.