Antimycobacterial and Anticancer Properties of Myrtus communis Leaf Extract

Abstract

Background: Plant-derived products or extracts are widely used in folk/traditional medicine to treat several infections, ailments, or disorders. A well-known medicinal herb, Myrtus communis is an evergreen fragrant plant native to the Mediterranean region that has been used for ages in traditional medicine around the world.

Materials and methods: The microplate alamarBlue assay and the well diffusion method were used to evaluate the zone of inhibition and MIC, respectively. The double-disc diffusion method was used to investigate the synergy between antibiotics and the extract. The crystal violet method was used to investigate biofilm development. The SulphoRhodamine-B assay and DNA flow cytometry were used to investigate the proliferation and subsequent distribution of cells among different phases of the cell cycle. The apoptotic and necrotic phases of the cancer cells were examined using flow cytometry in conjunction with Annexin V-FITC/PI labeling. Using the IBM SPSS statistical program, a one-way ANOVA with Tukey's post hoc test was employed for statistical analysis.

Results: The ethanolic leaf extract of M. communis showed a strong growth inhibition effect (zone of inhibition: 20.3 ± 1.1-26.3 ± 2.5 mm, MIC: 4.88-312.5 µg/mL, and MBC: 39.07-1250 μg/mL) against several rapidly growing and slow-growing mycobacterial strains in a dose-dependent manner. Damage to the cell wall of bacterial cells was determined to be the cause of the antimycobacterial action. The extract inhibited biofilm formation (MBIC of 9.7 µg/mL) and eradicated already-formed mature and ultra-mature biofilms of M. smegmatis, with MBEC values of 78 µg/mL and 156 µg/mL, respectively. Additionally, the extract exhibited potent anticancer effects against diverse cancer cell lines of the breast (MCF-7), liver (HepG2), cervix (HeLa), and colon (HCT116) (IC₅₀ for HCT116: 83 ± 2.5, HepG2: 53.3 ± 0.6, MCF-7: 41.5 ± 0.6, and HeLa: 33.3 ± 3.6) by apoptosis after arresting the cells in the G₁ phase of the cell cycle.

Conclusions: These results suggest that M. communis leaf extract is a potential source of secondary metabolites that could be further developed as potential anticancer and antimycobacterial agents to treat diverse types of cancers and mycobacterial infections.

Keywords: Myrtus communis; antibacterial; anticancer; apoptosis; biofilm.

Figure 1

Zone of inhibition and MIC of M. communis leaf extract. (A) Zone of inhibition of Mycobacterium kansasii. (B) Zone of inhibition of Mycobacterium fortuitum ATCC6841. (C) Two-fold dilutions of the extract (µg/mL) used in the culture wells showed an inhibitory effect on the growth of M. kansasii ATCC35775 at 4.884 µg/mL. The pink color of the well indicates growth, while the blue color indicates inhibition of the growth of bacteria. ★: well contains ethanol; ▽: well contains extract.

Figure 2

Growth of S. aureus and M. smegmatis in the presence of the extract: The growth of (A) S. aureus and (B) M. smegmatis was monitored in the presence of the MIC, 2× MIC, and 4× MIC of the extract by recording fluorescence readings at 30 min (S. aureus) and 1 h (M. smegmatis) intervals, respectively, using alarmBlue as a growth indicator.

Figure 3

SEM images of M. smegmatis cells: Tomographic images of untreated (A) and treated (B) M. smegmatis cells. The arrows show that the cell wall is intact in untreated cells but damaged in treated cells.

Figure 4

Synergy between antibiotics and extract. Bacitracin and vancomycin exhibited bridging of the zone of inhibition in combination with the extract against M. smegmatis.

Figure 5

Inhibition of S. aureus and M. smegmatis biofilm formation by M. communis leaf extract. The growth of (A) M. smegmatis and (B) S. aureus in the presence of different concentrations of the extract was measured by optical density at 600 nm. The biofilms formed at these concentrations were estimated by crystal violet absorbance at 570 nm. The inlets show the crystal violet-stained biofilms of one of the three replicate wells. *: p < 0.002, ꟸ: p < 0.006.

Figure 6

Eradication of mature biofilms by M. communis leaf extract. Mature and extra-mature biofilms of M. smegmatis (A, B) and S. aureus (C, D) were subjected to different concentrations of extract treatment. The biofilms that remained after treatment were developed by the crystal violet method and estimated by absorbance at 570 nm. The percentage of biofilms eradicated was calculated and plotted against the extract concentrations. The inlets show the crystal violet-stained biofilms in one of the three replicate wells. *: p < 0.002; ꟸ: p < 0.006.

Figure 7

IC₅₀ values of the M. communis leaf extract against cancer cell lines. The percentage of viable cells after 72 h of treatment with various extract concentrations.

Figure 8

Cell cycle analysis of cancer cell lines after M. communis leaf extract treatment. (A–D): The cell cycle distribution of cells of MCF-7 (A), HepG2 (B), HCT116 (C), and HeLa (D) analyzed by DNA flow cytometry after their individual treatment with a plant extract (treated). Similarly, the cell cycle distribution of all cancer cell lines was analyzed after their treatment with DMSO only (control). (E) A bar graph showing the percentage of cells for each cell line in different phases of the cell cycle after their treatment with the plant extract (T) or DMSO (C).

Figure 9

M. communis leaf extract induces apoptosis. (A–D): Scatter plots of flow cytometry analysis showing the distribution of cells of MCF-7 (A), HepG2 (B), HCT116 (C), and HeLa (D) into the early and late phases of apoptosis and necrosis after treatment with the plant extract (treated) or DMSO (control) and subsequent staining with Annexin V-FITC/PI. A representation of the four quadrants is given in the scatter plot in the top left corner.