Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract

Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Figure 1. POH effect on macrophage viability and proliferation. The RAW 264.7 cells were treated with POH (10-250 µM) and cultured in vitro for 24, 48, or 72 h. The viability was determined by the trypan blue (A) and MTS (B) assays at 24 h. The bars represent the means, and the vertical lines represent the standard deviations of three independent experiments. C): POH effect on proliferation of RAW 264.7 cells at 24, 48, and 72 h. Each horizontal line represents an experimental condition, and the vertical lines represent the standard deviations of three independent experiments. The asterisks (****) indicates a significant difference compared to the negative control (2-way ANOVA, p<0.0001)

Figure 2. POH effect on ROS production induced by PMA (A) and LPS (B) in RAW 264.7 macrophages. The cells were treated with 10 µM and 100 µM of POH for 30 minutes, followed by a 20-minute stimulation with 50 ng/mL PMA or 10 µg/mL LPS. The ROS production was assessed by flow cytometry by measuring the oxidation of the substrate 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, which emits fluorescence in the green channel (FITC) upon excitation at 488 nm. Representative dot-plots of flow cytometry analysis depicting the percentage of ROS-positive (FITC-positive) cells. Significant differences are based on the ANOVA with a post-hoc Tukey test. * p<0.05, ** p<0.01, *** p<0.001 versus control or as indicated in the figure

Figure 3. Expression of TNF and Arginase-1 assessed by the RT-qPCR in RAW 264.7 cells. M1 stimuli were E. coli LPS (1 μg/mL) and IFNγ (100 ng/mL) in pre-treated cells (30 min) with 10 µM or 100 µM of POH (control cells were treated with the same vehicle of the vehicle used to dilute POH). Columns represent the means for two independent experiments