Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells

Abstract

R. officinalis L. is an aromatic plant commonly used as condiment and for medicinal purposes. Biological activities of its extract were evaluated in this study, as antimicrobial effect on mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory capacity, and genotoxicity. Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed of C. albicans with each bacterium were formed in microplates during 48 h and exposed for 5 min to R. officinalis L. extract (200 mg/mL). Its cytotoxic effect was examined on murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7), and cervical carcinoma cells (HeLa) after exposure to different concentrations of the extract, analyzed by MTT, neutral red (NR), and crystal violet (CV) assays. The anti-inflammatory activity was evaluated on RAW 264.7 non-stimulated or stimulated by lipopolysaccharide (LPS) from Escherichia coli and treated with different concentrations of the extract for 24 h. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) were quantified by ELISA. Genotoxicity was verified by the frequency of micronuclei (MN) at 1000 cells after exposure to concentrations of the extract for 24 h. Data were analyzed by T-Test or ANOVA and Tukey Test ( P ≤ 0.05). Thus, significant reductions in colony forming units per milliliter (CFU/mL) were observed in all biofilms. Regarding the cells, it was observed that concentrations ≤ 50 mg/mL provided cell viability of above 50%. Production of proinflammatory cytokines in the treated groups was similar or lower compared to the control group. The MN frequency in the groups exposed to extract was similar or less than the untreated group. It was shown that R. officinalis L. extract was effective on mono- and polymicrobial biofilms; it also provided cell viability of above 50% (at ≤ 50 mg/mL), showed anti-inflammatory effect, and was not genotoxic. Impact statement Rosmarinus officinalis L. extract effectively contributed to in vitro control of important species of microorganisms such as Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa in mono- and polymicrobial biofilms that are responsible for several infections in oral cavity as in other regions of the body. Furthermore, this extract promoted also cell viability above 50% at concentrations ≤ 50 mg/mL, excellent anti-inflammatory effect, showing inhibition or reduction of the synthesis of proinflammatory cytokines, being also non-genotoxic to cell lines studied. Thus, this extract may be a promising therapeutic agent that can be added in some medical and dental formulations such as toothpastes, mouthwashes, irrigating root canals, ointments, soaps, in order to control pathogenic microorganisms and biofilms, with anti-inflammatory effect and absence of cytotoxic and genotoxic.

Keywords: Rosmarinus officinalis L.; anti-inflammatory activity; antibiofilm activity; antimutagenic activity; antiproliferative activity.

Figure 1

Action of R. officinalis L. extract on monomicrobial biofilms. Mean values (± standard deviation) of CFU/mL of C. albicans, S. aureus, E. faecalis, S. mutans and P. aeruginosa biofilms presented in untreated group (0.9% NaCl) and treated group with R. officinalis L. extract (200 mg/mL) for 5 min. P values follow on the columns (n = 10. T-Test, P ≤ 0.05). (A color version of this figure is available in the online journal.)

Figure 2

Reduction percentage of monomicrobial biofilms. After exposure to R. officinalis L. extract (200 mg/mL) for 5 min, significant reductions were observed in the biofilms of C. albicans (Ca), S. aureus (Sa), E. faecalis (Ef), S. mutans (Sm) and P. aeruginosa (Pa). The groups were reunited according to their homogeneity. Statistically significant differences can be observed among groups with different superscript letters. (n = 10. ANOVA, Tukey test, P ≤ 0.05)

Figure 3

Action of R. officinalis L. extract on polymicrobial biofilms. Mean (± standard deviation) of CFU/mL of polymicrobial associations of C. albicans with S. aureus, E. faecalis, S. mutans and P. aeruginosa presented in the untreated group (0.9% NaCl) and treated groups with R. officinalis L. extract (200 mg/mL) for 5 min. P values follow on the columns (n = 10. T-Test, P ≤ 0.05). (A color version of this figure is available in the online journal.)

Figure 4

Reduction percentage of polymicrobial biofilms. Data obtained in polymicrobial associations of C. albicans with S. aureus (Ca + Sa), E. faecalis (Ca + Ef), S. mutans (Ca + Sm) and P. aeruginosa (Ca + Pa) after exposure to R. officinalis L. extract (200 mg/mL) for 5 min. Statistically significant difference between the reductions in yeast and bacteria in each association can be observed among groups with superscript asterisks. (n = 10. T-Test, P ≤ 0.05). (A color version of this figure is available in the online journal.)

Figure 5

Cell viability verified by MTT, NR and CV assays. After exposure of RAW 264.7, FMM-1, MCF-7 and HeLa at concentrations of 25, 50 and 100 mg/mL of R. officinalis L. extract, cell viability of the cultures, compared to the control group (0 mg/mL), were analyzed by: (a) reduction of MTT salt to formazan; (b) Incorporation of neutral red (NR) in the lysosomes; and (c) DNA staining with crystal violet (CV). Statistically significant differences among experimental groups can be observed with different superscript letters (n = 10. ANOVA, Tukey Test, P ≤ 0.05). Optical microscopy (200×). (A color version of this figure is available in the online journal.)

Figure 6

Cell viability percentage obtained in each concentration of the R. officinalis L. Data were obtained in RAW 264.7, FMM-1, MCF-7 and HeLa cultures, after exposure for 5 min to concentrations of R. officinalis L. extract (25, 50 e 100 mg/mL), and analyzed by MTT (a), NR (b) and CV (c) assays. Different superscript letters indicate statistically significant differences among experimental groups. (n = 10. ANOVA, Tukey Test, P ≤ 0.05)

Figure 7

Micronuclei (MN). MN are DNA fragments located around and close to the cell nucleus (indicated by white arrows), and may have variable size but always smaller than the cell nucleus and varied amount, as shown in the figure bottom right, which shows two MN. Its presence characterizes DNA damage, provided by intrinsic or extrinsic causes. After fixing the cells, previously treated or not with different concentrations of R. officinalis L. extract, DAPI dye was added and the nuclei and MN were observed through fluorescence microscopy (200×) and then the frequency of MN was determined after counting 1000 nuclei. (A color version of this figure is available in the online journal.)

Figure 8

Micronuclei (MN) frequency presented by cells. RAW 264.7, FMM-1, MCF-7 and HeLa were exposed to concentrations of 25, 50 and 100 mg/mL of R. officinalis L. extract. After 24 h, MN frequency was counted. (a) MN frequency presented by the four cell lineages per 1000 cells counted. (b) MN frequency obtained in each experimental group (0, 25, 50 and 100 mg/mL). Statistically significant difference among treated groups and control groups can be observed with different superscript letters (n = 2. ANOVA, Tukey Test, P ≤ 0.05). (A color version of this figure is available in the online journal.)