Inhibitory effect of the Pseudobrickellia brasiliensis (Spreng) R.M. King & H. Rob. aqueous extract on human lymphocyte proliferation and IFN-γ and TNF-α production in vitro

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.


Introduction
The use of medicinal plants as an alternative therapy is still a common practice in some regions of Brazil. However, many of these plants, though well-known in popular culture, have not been submitted to investigations for identifying their chemical compounds or assayed to determine their biological activity, which may justify their use.
Pseudobrickellia brasiliensis (Spreng) R.M. King & H. Rob. (Asteraceae) is a native species of the Brazilian Cerrado (1) known for its medicinal properties. The alcoholic extract obtained from the leaves of this species is used as a topical anti-inflammatory, healing, and analgesic agent and is currently an effective treatment of chronic inflammatory processes (2)(3)(4)(5).
The inflammatory response is a complex network of biological events involving vascular changes, high expression/activity of enzymes involved in the production of lipid mediators, activation of leukocytes, and production of cytokines. These events have several functions that aim to eliminate harmful agents and restore the homeostasis of the tissue (6,7). T lymphocytes have a pivotal role in cytokine production during chronic inflammation (8,9). These cells, once activated in secondary lymphoid organs, undergo clonal proliferation, and then migrate to the injured tissue in the late stage of the inflammatory process. These cells are responsible for the mononuclear histological aspect, characteristic of the chronic inflammatory process, and contribute to the high expression of cytokines and chemokines in the inflammatory focus (10). Among the proinflammatory cytokines produced, interleukin (IL)-2 acts as an autocrine growth factor and induces clonal proliferation of T and B lymphocytes, and tumor necrosis factor (TNF)-a and interferon (IFN)-g contribute to the activation of phagocytes, promote high expression levels of adhesion molecules in endothelial cells, and accelerate the events that culminate in tissue injury. In contrast, the production of anti-inflammatory cytokines such as IL-4, IL-5, and IL-10 promote regulatory or immunomodulatory functions and contribute to the control of the aggressive events observed in the long-term inflammatory process (11,12). The balance between pro-and anti-inflammatory cytokines influences the course of inflammation.
P. brasiliensis is used as an anti-inflammatory agent in traditional medicine. This study aimed to verify whether non-toxic P. brasiliensis extracts inhibit pro-inflammatory cytokine production and proliferation of human peripheral blood lymphocytes in vitro. The main chemical compounds of these extracts were also identified. Determination of the chromatographic profile of P. brasiliensis extract by high-performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS)

Material and Methods
Sample solution. A sample solution was prepared using 10 mg of previously dried P. brasiliensis aqueous extract and dissolved in HPLC-grade methanol:water (1:9; 1 mL) on the sonication bath for 5 min. The solution was filtered through a 0.2 mm pore size syringe filter with a diameter of 13 mm.
Direct infusion ESI-MS and ESI-MS/MS analyses. Direct infusion ESI-MS and ESI-MS/MS analyses were performed using a Bruker Daltonics micrOTOF-Qt II, ESI-Qq-TOF mass spectrometer system operating in negative ion mode.

Biological samples and peripheral blood mononuclear cell (PBMC) separation
PBMCs were obtained from 31 healthy adult blood donors (10 men and 21 women; 25 ± 5 years old). Subjects with any infectious or autoimmune disease, or those using antibiotics, anti-inflammatory drugs, corticosteroids, or other immunosuppressive drugs were not considered for blood donation. Informed written consent was obtained from all participants. The study was approved by the Ethical Committee of the UFVJM, Diamantina, MG, Brazil (register code 02/009). Blood samples were collected in vacuum tubes containing heparin (Vacutainer; Becton Dickinson, USA).

Cell viability analyses
PBMCs (5 Â 10 5 ; n=4) were cultured in RPMI-1640 medium (Sigma-Aldrich Corporation) supplemented with 2 mM L-glutamine, 10% FCS (Gibco, Invitrogen Corporation), and antibiotic-antimycotic cocktail (penicillin G 100 IU/ mL, streptomycin 100 mg/mL and amphotericin B 250 ng/mL, Sigma-Aldrich Corporation). Cells were treated with ACE, ETA, or AQU (12.5, 25, 50, or 100 mg/mL) extracts or an equal volume of DMSO (Sigma-Aldrich Corporation) (solvent control) in a humidified incubator at 37°C with 5% CO 2 air atmosphere for 24 h or 5 days. Untreated PBMCs constituted the cell culture control (CON). PBMCs treated with cadmium chloride (CdCl 2 ; 20mM) constituted the positive control of cell death. PBMCs were washed with PBS (200 g, 7 min, 4°C) and re-suspended in 0.5 mL PBS. Then, 10 mL cell suspension was mixed with 190 mL of 0.002% trypan blue (Sigma-Aldrich Corporation) and analyzed by flow cytometry (FACScan, Becton Dickinson, USA) (14). Ten thousand events were acquired in the region corresponding to lymphocytes. CellQuestt software (Becton Dickinson) was used for data collection and analyses. Cell viability was calculated by dividing the number of viable cells by the total cell number.
The evaluation of apoptosis or necrosis induced by P. brasiliensis extract was performed by flow cytometry using a commercial kit for apoptotic cell detection (Annexin V FITC Apoptosis Detection Kit II, BD Pharmingen, USA). PBMCs (5 Â 10 5 ; n=8) were incubated in RPMI-1640 containing ACE, ETA, or AQU (50 mg/mL) extracts or an equal volume of DMSO (solvent control) at 37°C in a humidified incubator with a 5% CO 2 air atmosphere for 4 h. Untreated PBMCs constituted the negative cell culture control (CON). Cells were washed (200 g, 10 min, 4°C) and re-suspended in 100 mL binding buffer. Cells were incubated with 2 mL of annexin V-FITC and 2 mL of propidium iodide, then analyzed by flow cytometry (FACScan, Becton Dickinson). At least 20,000 events were acquired in the region corresponding to lymphocytes. CellQuestt software (Becton Dickinson) was used for data collection and analyses.

Statistical analyses
GraphPad Prism, version 5.0 for Windows (GraphPad Software, USA) was used for statistical analysis. Oneway ANOVA and Tukey's post hoc test were used for analyses. P values lower than 0.05 were considered to be statistically significant. Data are reported as means ± SD.

Results and Discussion
Since plant extracts and solvents are considered external factors that can directly influence cell death or activate mechanisms linked to programmed cell death (17,18), we initially investigated the cytotoxicity of the P. brasiliensis extracts through cell viability and apoptosis/ necrosis analyses in cell cultures treated with different extracts of the plant. At different concentrations, the AQU extract did not affect cell viability after 24 h ( Figure 1A) or 5 days ( Figure 1B), compared to the viability of CON cultures. ETA and ACE reduced cell viability to about 0.3% after 24 h, relative to control cell cultures (data not shown). Although the trypan blue exclusion test is a technique widely used to evaluate cell viability, this assay does not detect cell modifications observed in the early stages of cell death. Therefore, in an attempt to determine whether the presence of the plant extracts could trigger early apoptotic/necrotic mechanisms, we performed an additional cell analysis using propidium iodide and annexin V. The cell viability of the cultures treated with 50 mg/mL AQU did not significantly differ from that of CON culture ( Figure 2). In contrast, cell cultures treated with 50 mg/mL ACE and ETA showed a low percentage of viable cells and a high percentage of necrotic and apoptotic lymphocytes, in comparison to the CON and DMSO cultures. These data are in agreement with the cell viability data obtained with the trypan blue assay, and allow us to conclude that toxicity demonstrated by ACE and ETA is not attributable to the solvent used for dilution. Moreover, the treatment with AQU extract showed no toxicity to lymphocytes in vitro. Assays to evaluate the cytotoxic activity of medicinal plants in vitro are essential to assess the continuity of subsequent experimental steps. Therefore, based on cell toxicity, the sequential analyses of the chemical compounds and immune parameters were performed using only the AQU extract.
Regarding the balance between pro-and anti-inflammatory cytokines and its influence on the course of inflammation mediated by T lymphocytes, we decided to evaluate TNF-a, IFN-g, and IL-10 production by PMAstimulated lymphocytes using a flow cytometry platform. The results were presented as mean fluorescence intensity (MFI), which allows the correlation of the signal emitted by fluorescent-conjugated specific monoclonal antibodies and the cytokine expression by specific cells. Three different concentrations of AQU (25,50, and 100 mg/mL) were used to verify the inhibitory effect of the plant extract on the proinflammatory cytokine production. According to the results, the expression of IFN-g in lymphocytes was 4.8-fold lower in cultures treated with PMA plus AQU (100 mg/mL) than in cultures treated with PMA alone ( Figure 3A). Similarly, in the cell cultures treated with 100 mg/mL AQU, the TNF-a expression was 3.8-fold lower than in the PMA-stimulated cell cultures ( Figure 3B). There were no differences in the  IL-10 production by lymphocytes between cell cultures submitted to different experimental conditions. In summary, the P. brasiliensis aqueous extract exerted a strong inhibitory action on pro-inflammatory cytokine production in stimulated T lymphocytes in vitro.
Because lymphocyte proliferation is a pivotal and essential event in immune-mediated inflammatory response (19), we also evaluated the proliferative response of cells treated with AQU extract. All tested concentrations of the AQU extract reduced the proliferative response of T lymphocytes, including CD4+ and CD8+ cells (Figure 4). These data support the finding that AQU extract exhibits an anti-inflammatory effect in vitro, and strengthens the validity of the data collected in the cytokine analyses. The inhibitory effects observed suggest that AQU extract components may interfere with cellular activation mechanisms directly involved in cytokine production and lymphocyte capacity expansion.
The results obtained in our biological assays of the AQU extract of the plant revealed an anti-proliferative action on human lymphocytes and an inhibitory effect on pro-inflammatory cytokine production in vitro. Chemical analysis of AQU extract was carried out to verify the presence of chemical compounds with anti-inflammatory activities that have already been described in the literature. Previous studies with P. brasiliensis apolar extracts revealed the presence of triterpenes and sesquiterpenes, such as hidroxigermacreno, lupeol, b-amyrin acetate, spathulenol, cadinene, cadinol, oplopanona acetate, and a-acetoxy-b-amyrin (20). Moreover, the essential oil from the leaves contains the major compounds terpinen-4-ol, g-terpinene, a-terpinene, and a-terpineol (21). However, no study has been conducted to identify the chemical compounds present in the polar extract of P. brasiliensis, a similar formulation used in folk medicine.
To identify the chemical components in the AQU extract, we performed LC-PDA-MS, ESI-MS, and ESI-MS/ MS (direct infusion) analyses ( Figure 5). The results indicated the presence of quinic acid and its derivatives, 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid. These  Hepatoprotective, antioxidant, anti-inflammatory, and analgesic actions of quinic acid and its derivatives have been previously described (22)(23)(24)(25)(26). The quinic acid derivative obtained from Pimpinella brachycarpa (Umbeliferae) showed an inhibitory effect against nitric oxide (NO) production in microglia (26). Chlorogenic acids, including 5-caffeoylquinic and dihidrocafeilquinic acids, are among the main constituents of the aqueous extract of Brazilian green propolis collected from Apis mellifera hives. This extract showed an anti-inflammatory effect in rats that received both subcutaneous implantation of polyester-polyurethane sponges cut into disks and oral treatment with 500 mg/kg of green propolis (27).
The in vitro anti-inflammatory effects of 5-o-caffeoylquinic acid were demonstrated by its inhibitory action on NO production, expression of COX-2 and iNOS, and production of pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) and adhesion molecules in LPS-stimulated murine microglia/macrophages. It was also demonstrated that 5-o-caffeoylquinic acid inhibited the nuclear translocation of NF-kB (28).
Luteolin is found in various plants of the Asteraceae family that exhibit anti-inflammatory activity. It is a flavonoid with potent anti-inflammatory activity both in vitro and in vivo (29)(30)(31), and anti-carcinogenic (32) and antioxidant (33,34) proprieties. The anti-inflammatory action of luteolin was demonstrated through its inhibitory effect on the expression of pro-inflammatory cytokine TNF-a via the inhibition of the NF-kB signaling pathway (35). Furthermore, luteolin inhibits the expression of IL-6 by inhibiting the JNK and AP-1 pathways (36,37). The anti-inflammatory effect of luteolin may also be related to its inhibitory effect on COX-2, LOX, and iNOS enzymes (29,38,39).
Although the inhibitory effect of AQU on T lymphocyte proliferation and pro-inflammatory cytokine production was not associated specifically with the compounds identified in our study, the presence of active molecules linked to anti-inflammatory action already described in literature provides strong evidence for the therapeutic potential of the plant. Many phytomedicines are composed of a mixture of several components, exerting their therapeutic effects through the synergistic action of compounds. Further studies with fractionated AQU extract will be performed to elucidate the structure of the substances responsible for the effect demonstrated here, as well as the performance of the active fractions in inflammatory models in vivo.
In summary, our results showed that the aqueous extract of P. brasiliensis demonstrated no cytotoxic activity at the tested concentrations. It exerted an anti-inflammatory effect in vitro by decreasing the expression of the proinflammatory cytokines TNF-a and IFN-g and by inhibiting lymphocyte proliferation. These data suggest that some of the therapeutic effects of P. brasiliensis may be because of its actions on essential mechanisms for inflammation maintenance.