Chemical composition, immunostimulatory, cytotoxic and antiparasitic activities of the essential oil from Brazilian red propolis.

Most studies of Brazilian red propolis have explored the composition and biological properties of its ethanolic extracts. In this work, we chemically extracted and characterized the essential oil of Brazilian red propolis (EOP) and assessed its adjuvant, antiparasitic and cytotoxic activities. The chemical composition of EOP was analyzed using gas chromatography with mass spectrometry (GC-MS). EOP was tested for in vitro activity against Trichomonas vaginalis (ATCC 30236 isolate); trophozoites were treated with different concentrations of EOP (ranging from 25 to 500 μg/mL) in order to establish the MIC and IC50 values. A cytotoxicity assay was performed in CHO-K1 cells submitted to different EOP concentrations. BALB/c mice were used to test the adjuvant effect of EOP. The animals were divided in 3 groups and inoculated as follows: 0.4 ng/kg BW EOP (G1); 50 μg of rCP40 protein (G2); or a combination of 0.4 ng/kg BW EOP and 50 μg of rCP40 (G3). Total IgG, IgG1 and IgG2a levels were assessed by ELISA. The major constituent compounds of EOP were methyl eugenol (13.1%), (E)-β-farnesene (2.50%), and δ-amorphene (2.3%). Exposure to EOP inhibited the growth of T. vaginalis, with an IC50 value of 100 μg/mL of EOP. An EOP concentration of 500 μg/mL was able to kill 100% of the T. vaginalis trophozoites. The EOP kinetic growth curve showed a 36% decrease in trophozoite growth after a 12 h exposure to 500 μg/mL of EOP, while complete parasite death was induced at 24 h. With regard to CHO-K1 cells, the CC50 was 266 μg/mL, and 92% cytotoxicity was observed after exposure to 500 μg/mL of EOP. Otherwise, a concentration of 200 μg/mL of EOP was able to reduce parasite proliferation by 70% and was not cytotoxic to CHO-K1 cells. As an adjuvant, a synergistic effect was observed when EOP was combined with the rCP40 protein (G3) in comparison to the administration of each component alone (G1 and G2), resulting in higher concentrations of IgG, IgG1 and IgG2a. EOP is constituted by biologically active components with promising antiparasitic and immunostimulatory activities and can be investigated for the formulation of new vaccines or trichomonacidal drugs

Kinetic growth curve of the <i>Trichomonas vaginalis</i> 30236 isolate after exposure to EOP at 500 μg/mL for 1 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h.

<p>Data represent mean ± standard deviation of at least three experiments, all in triplicate. Different letters indicate a significant difference (p < 0.05).</p

Anti-rCP40 total IgG (4A), IgG1 (4B) and IgG2a (4C) levels in immunized mice treated with EOP and recombinant endoglycosidase CP40 from <i>C</i>. <i>pseudotuberculosis</i> (rCP40), either separately or together.

<p>Measurements were taken on day 0 and then 21 and 42 days post-immunization. Data represent means ± standard deviations of IgG levels in the sera collected from 6 animals/group. Different letters in the same experimental day indicate a significant difference (p < 0.05).</p

CHO-K1 cell viability after treatment with different concentrations of EOP.

<p>Cell proliferation in CHO-K1 was investigated by the MTT assay, and metronidazole was used as a positive control. Data are expressed as the mean ± SEM of the viabilities of CHO-K1 cells in three independent experiments. (*) indicates a difference between the treatments in CHO-K1 cells. The differences were considered significant at p < 0.05. VC = vehicle control; MTZ = metronidazole.</p

Volatile compounds identified by gas chromatography followed by mass spectrometry (GC-MS) of the Brazilian red propolis essential oil.

<p>Volatile compounds identified by gas chromatography followed by mass spectrometry (GC-MS) of the Brazilian red propolis essential oil.</p