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Braz J Med Biol Res
2017 Jul 10;508:e5163. doi: 10.1590/1414-431X20175163.
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Almeida VG
,
Avelar-Freitas BA
,
Santos MG
,
Costa LA
,
Silva TJ
,
Pereira WF
,
Amorim MLL
,
Grael CFF
,
Gregório LE
,
Rocha-Vieira E
,
Brito-Melo GEA
.
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Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.
Figure 2. Apoptotic or necrotic effect of P. brasiliensis extracts. Percentage of viable, apoptotic, or necrotic cells in cultures of peripheral blood mononuclear cells (PBMCs) treated with or without P. brasiliensis extracts after 4-h incubation. Evaluation was done by flow cytometry (n=8). Culture treated with dimethyl sulfoxide (DMSO) constituted the solvent control. ACE: P. brasiliensis ethyl acetate extract; AQU: P. brasiliensis aqueous extract; ETA: P. brasiliensis ethanolic extract.
Figure 3. Effect of P. brasiliensis aqueous extract on cytokine production by lymphocytes. Peripheral blood mononuclear cells (PBMCs, 5×105 cells; n=8) were stimulated with phorbol myristate acetate (PMA) in the presence of 25, 50, or 100 µg/mL of P. brasiliensis aqueous extract (AQU). Non-stimulated cultures constituted the negative control (PBMC), and cultures stimulated with PMA + ionomycin in the absence of plant extract constituted the positive control. Production of IFN-γ (A), TNF-α (B), and IL-10 (C) was determined by flow cytometry. Results are reported as means±SD. Different letters indicate significant differences (P≤0.05, ANOVA with Tukey post hoc).
Figure 4. Antiproliferative effect of P. brasiliensis aqueous extract. Peripheral blood mononuclear cells (PBMCs, 5×05 cells; n=6) labeled with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with phytohemagglutinin (PHA) in the absence or presence (25, 50, or 100 µg/mL) of P. brasiliensis aqueous extract (AQU). Non-stimulated cultures constituted the negative control (PBMC), and cultures stimulated with PHA in the absence of plant extract constituted the positive control. After 5 days, the proliferation index of lymphocytes (A), CD4+ T cells (B), and CD8+ T cells (C) was calculated by cytometry analysis. Data are reported as means±SD. Different letters indicate significant differences (P≤0.05, ANOVA with Tukey post hoc).
Figure 5. Representative ESI-MS and LC-PDA-MS analysis of the aqueous extract of Pseudobrickellia brasiliensis. A, ESI-MS full scan; B, total ion chromatogram (TIC), and C, chemical, retention time (in min), and m/z.
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