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

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 (2-5).

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 pro-inflammatory cytokines produced, interleukin (IL)-2 acts as an autocrine growth factor and induces clonal proliferation of Tand B lymphocytes, and tumor necrosis factor (TNF)-[alpha] and interferon (IFN)-[gamma] 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.

Material and Methods

Plant material, identification, and preparation of P. brasiliensis extract

P. brasiliensis leaves were collected in Diamantina, MG, Brazil (18[degrees]12, 1640 South, 43[degrees]34, 398' East). The botanical identification was performed by Dr. P.A. Oliveira from the Felfili Dendrologic Herbarium (HDJF) of the Federal University of Jequitinhonha and Mucuri Valleys (UFVJM), where the specimen was deposited under voucher No. 833. P. brasiliensis leaves were dried and macerated sequentially in n-hexane, ethyl acetate, ethanol, and water for 72 h at room temperature, then Altered. Ethanol (ETA) and ethyl acetate (ACE) extracts were concentrated under vacuum on a rotary evaporator (Fisatom, model 801, Brazil) at 40-42[degrees]C, under reduced pressure. Aqueous extract (AQU) was Altered, frozen, and lyophilized. ACE and ETA extracts were diluted to 2.0 mg/mL with dimethyl sulfoxide (DMSO; Sigma-Aldrich Corporation, USA). AQU extract was diluted with phosphate buffered saline (PBS; 1.50 M NaCl; 0.08 M [Na.sub.2]HP[O.sub.4]; 0.02 M Na[H.sub.2]P[O.sub.4], pH 7.4). Small stock aliquots were kept at -20[degrees]C until their use. New dilutions of the extract in DMSO or PBS were performed to obtain the required concentration in cell culture medium.

Authorization for plant collection was provided by the Brazilian Ministry of the Environment (registration No. 35605-1) and the National Council of Technological and Scientific Development (CNPq/CGEN 010832/2014-9).

Determination of the chromatographic profile of P. brasiliensis extract by high-performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS)

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 [micro]m pore size syringe filter with a diameter of 13 mm.

Chromatographic system. The ultra-fast liquid chromatography (UFLC) system used consisted of a Shimadzu Prominence UFLC system (Japan) equipped with two pumps (LC-20AD), an on-line degasser (DGU-20A), a column oven (CTO-20A), an autosampler (SIL-20AHT), and a PDA detector (SPD-20A) coupled with a TOF mass spectrometry detector (micrOTOF-Q[TM]; Bruker Daltonics, USA). It used a VP-ODS (Shimadzu) analytical column (150 x 2 mm, particle size 4.6 mm) with a GPV-ODS guard column (5 x 2 mm; Shimadzu). Ultrapure water (Type 1; Direct-Q[R] 5 System, Merck Millipore, USA) and acetonitrile (HPLC grade) were used as the mobile phase. Analysis was performed at a flow rate of 0.8 mL/min and monitored at 190-600 nm.

The gradient mobile phases used were as follows: 0-5 min: water (90%), acetonitrile (10%); 45-50 min: acetonitrile (100%), and 52-60 min: water (90%) and acetonitrile (10%).

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-Q[TM] 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 [+ or -] 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).

PBMCs were isolated from heparinized human peripheral blood samples by the Ficoll-Histopaque (specific gravity, 1.077) gradient density method (13). The peripheral blood (15 mL) was diluted with PBS and centrifuged in a Ficoll-Histopaque 1.077 (Sigma-Aldrich Corporation, USA) gradient at 400 g at room temperature for 30 min. PBMCs were collected, washed with PBS, and centrifuged (240 g, 7 min). Cells were re-suspended at a concentration of 1 x [10.sup.7] cells/mL in

PBS or RPMI-1640 medium (Sigma-Aldrich Corporation) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Invitrogen Corporation, USA), 2 mM L-glutamine (Sigma-Aldrich Corporation), and antibiotic/antimycotic cocktail (penicillin G 100 IU/mL, streptomycin 100 mg/mL and amphotericin B 250 ng/mL; Sigma-Aldrich Corporation).

Cell viability analyses

PBMCs (5 x [10.sup.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 [micro]g/mL and amphotericin B 250 ng/mL, Sigma-Aldrich Corporation). Cells were treated with ACE, ETA, or AQU (12.5, 25, 50, or 100 [micro]g/mL) extracts or an equal volume of DMSO (Sigma-Aldrich Corporation) (solvent control) in a humidified incubator at 37[degrees]C with 5% C[O.sub.2] air atmosphere for 24 h or 5 days. Untreated PBMCs constituted the cell culture control (CON). PBMCs treated with cadmium chloride (Cd[Cl.sub.2]; 20mM) constituted the positive control of cell death. PBMCs were washed with PBS (200 g, 7 min, 4[degrees]C) and re-suspended in 0.5 mL PBS. Then, 10 [micro]L cell suspension was mixed with 190 [micro]L 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. CellQuest[TM] 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 x [10.sup.5]; n=8) were incubated in RPMI-1640 containing ACE, ETA, or AQU (50 [micro]g/mL) extracts or an equal volume of DMSO (solvent control) at 37[degrees]C in a humidified incubator with a 5% C[O.sub.2] air atmosphere for 4 h. Untreated PBMCs constituted the negative cell culture control (CON). Cells were washed (200 g, 10 min, 4[degrees]C) and re-suspended in 100 [micro]L binding buffer. Cells were incubated with 2 [micro]L of annexin V-FITC and 2 [micro]L of pro-pidium iodide, then analyzed by flow cytometry (FACScan, Becton Dickinson). At least 20, 000 events were acquired in the region corresponding to lymphocytes. CellQuest[TM] software (Becton Dickinson) was used for data collection and analyses.

Effect of P. brasiliensis aqueous extract on cytokine production

PBMCs (5 x [10.sup.5]) (n=8) were cultured in RPMI-1640 containing 10% FCS (Gibco, Invitrogen Corporation), 2 mM L-glutamine, antibiotic-antimycotic cocktail (Sigma-Aldrich Corporation), 25 ng/mL phorbol-12-myristate-13-ace-tate-PMA (Sigma-Aldrich Corporation), 1 ng/mL ionomycin (Sigma-Aldrich Corporation), 1 [micro]g/mL Brefeldin-A (Sigma-Aldrich Corporation), and either PBS (negative control) or AQU (25, 50, or 100 [micro]g/mL). Untreated PBMCs were used as the non-stimulated cell culture control (CON). The cells were maintained at 37[degrees]C in a humidified incubator with a 5% C[O.sub.2] air atmosphere for 4 h. Cells were then fixed, permeabilized, and incubated with the monoclonal antibody (mAb) phycoerythrin (PE) conjugated specifically for IFN-[gamma] (phycoerythrin-conjugated IFN-[gamma] mAb-IFN-[gamma]-PE, clone 45.B3), TNF-[alpha] (phycoerythrin-conjugated TNF-[alpha] mAb-TNF-[alpha]-PE, clone MAb11), and IL-10 (phycoerythrin-conjugated IL-10 mAb-IL10-[alpha]-PE, clone JES3-9D7) cytokines (all from Biolegend, USA). The expression of cytokines by lymphocytes was evaluated by flow cytometry (FACScan, Becton Dickinson). At least 30, 000 events were acquired in a region corresponding to the PBMCs. The collection and analyses of data were performed with CellQuest[TM] software (Becton Dickinson).

Effect of P. brasiliensis aqueous extract on the lymphocyte proliferative response

The effect of AQU on the proliferative lymphocyte response to PHA stimulation was evaluated by 5-(and-6)-carboxyfluorescein succinimidyl ester (CFSE) fluorescence decay assay (15). PBMCs (1 x [10.sup.7]) were re-suspended in PBS/BSA 0.1% and labeled with 10 [micro]M CFSE (Sigma-Aldrich Corporation) for 10 min at 37[degrees]C. CFSE-stained PBMCs (5 x [10.sup.5]; n=6) were cultured in RPMI-1640 containing 10% FCS (Gibco, Invitrogen Corporation), 2 mM L-glutamine, antibiotic-antimycotic cocktail (Sigma-Aldrich Corporation), either with or without phytohemagglutinin (PHA) (1 [micro]g/mL). Cells also were stimulated with PHA in combination with different concentrations of AQU (25, 50, or 100 [micro]g/mL). Untreated PBMCs were used as the non-stimulated cell culture control (CON). The cells were kept in a humidified incubator with a 5% C[O.sub.2] air atmosphere for 5 days at 37[degrees]C. After incubation, the cells were harvested, washed in PBS, and stained with specific mAb for human CD3 (anti-CD3 biotin, clone HIT3a), CD4 (CD4 mAb phycoerythrin-cyanine conjugated - CD4/ PeCy5, clone RPA-T4), and CD8 (CD8 mAb phycoerythrin-cyanine conjugated - CD8/PeCy5, clone RPA-T8) (both from BD Biosciences, USA). After washing with PBS (0.015 M PBS, pH 7.4, 0.5% BSA and 0.1% sodium azide), the suspensions were incubated for 15 min in the dark at 4[degrees]C with streptavidin-phycoerythrin conjugated (BD Biosciences). Samples were analyzed on a BD FACScan with CellQuest[TM] software, and a total of 50, 000 events were acquired for each tube. Proliferation was measured on the basis of the dilution of CFSE (diminished staining intensity). The proliferative index was then calculated from CFSE fluorescence histograms using the following formula (16): Proliferative index = (100-Y)/Y, where Y (%) = X0 + X1/2 + X2/4 + X3/8 + X4/16 + X5/32 + X6/64; X0 represents the percentage of T cells that did not divide and X1-6 represent the maximum gradual division.

Statistical analyses

GraphPad Prism, version 5.0 for Windows (GraphPad Software, USA) was used for statistical analysis. One-way 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 [+ or -] 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 [micro]g/mL AQU did not significantly differ from that of CON culture (Figure 2). In contrast, cell cultures treated with 50 [micro]g/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-[alpha], IFN-[gamma], and IL-10 production by PMA-stimulated 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 [micro]g/mL) were used to verify the inhibitory effect of the plant extract on the pro-inflammatory cytokine production. According to the results, the expression of IFN-[gamma] in lymphocytes was 4.8-fold lower in cultures treated with PMA plus AQU (100 [micro]g/mL) than in cultures treated with PMA alone (Figure 3A). Similarly, in the cell cultures treated with 100 [micro]g/mL AQU, the TNF-[alpha] 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, [beta]-amyrin acetate, spathulenol, cadinene, cadinol, oplopanona acetate, and [alpha]-acetoxy-[beta]-amyrin (20). Moreover, the essential oil from the leaves contains the major compounds terpinen-4-ol, [gamma]-terpinene, [alpha]-terpinene, and [alpha]-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 derivatives are known as chlorogenic acids, which are a variety of polyphenolic acids present in food and medicinal plants. Furthermore, the presence of the ions m/z 609 and m/z 285 suggests the presence of the flavonoid luteolin (m/z 285) and its derivative luteolin dihexoside (m/z 609).

Hepatoprotective, antioxidant, anti-inflammatory, and analgesic actions of quinic acid and its derivatives have been previously described (22-26). 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-[alpha], IL-1[beta], 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-[kappa]B (28).

Quinic acid derivatives, including 3, 5-dicaffeoylquinic acid, are also present in Lychnophora ericoides (Aster-aceae), popularly known as arnica-da-serra. The 3, 5-dicaffeoylquinic acid purified from the polar extract of L. ericoides demonstrated analgesic activity in animal models of acetic acid-induced abdominal contortion (22).

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 (29-31), 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-[alpha] 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 pro-inflammatory cytokines TNF-[alpha] and IFN-[gamma] 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.

http://dx.doi.org/10.1590/1414-431X20175163

Acknowledgments

We are grateful to the Brazilian funding agencies FAPEMIG, CNPq and CAPES for financial support, to the Research Support Center in Natural and Synthetic Products (NPPNS), School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, for LC-PDA-MS, ESI-MS and ESI-MS/MS analysis, as well as to the Multicentric Post-Graduate Program in Physiology Science of the Universidade Federal dos Vales do Jequitin-honha e Mucuri. The authors would like to acknowledge the Integrated Center of the Post-Graduate Program and Health Research of the Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, for providing equipment and technical support for experiments.

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V.G. Almeida1 [2], B.A. Avelar-Freitas [2], M.G. Santos1 [2], L.A. Costa1 [2], T.J. Silva [2], W.F. Pereira [2], M.L.L. Amorim [3], C.F.F. Grael [3], L.E. Gregorio [4], E. Rocha-Vieira1, [2] and G.E.A. Brito-Melo [1, 2]

[1] Programa Multicentrico de Pos-Graduacao em Ciencias Fisiologicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brasil

[2] Laboratorio de Imunologia, Centro Integrado de Pos-Graduacao e Pesquisa em Saude do Vale do Jequitinhonha, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brasil

[3] Laboratorio de Farmacognosia, Departamento de Farmacia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brasil

[4] Laboratorio de Insumos Naturais e Sinteticos, Departamento de Ciencias Farmaceuticas, Universidade Federal de Sao Paulo, Diadema, SP, Brasil
Figure 1. Analysis of the cytotoxic effect of P. brasiliensis aqueous
extract with the trypan blue exclusion assay. Percentage of viable
cells in cultures of peripheral blood mononuclear cells (PBMCs)
treated with or without P. brasiliensis aqueous extract (AQU) after 24
h (A) or 5 days (B) of incubation. Evaluation was done with trypan
blue exclusion assay (n=8). Culture treated with cadmium chloride
(Cd[Cl.sub.2]) constituted the positive control of cell death.
Results are reported as means [+ or -] SD. Different letters
indicate significant differences (P [less than or equal to] 0.05,
ANOVA with Tukey post hoc).

A. 24 Hours

PBMC (5X[10.sup.5] cells)   +     +     +     +     +
Cd[Cl.sub.2] (mM)           -    20     -     -     AQU
([micro]g/Ml)               -     -    25    50    100

B. 5 days

PBMC (5X[10.sup.5] cells)   +     +     +     +     +
Cd[Cl.sub.2] (mM)           -    20     -     -     AQU
([micro]g/Ml)               -     -    25    50    100

Note: Table made from bar graph.

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.

PBMC (5X[10.sup.5])   +     +     +     +     +
DMSO                  -     +     -     -     ACE
50 (50 [micro]g/ML    -     -     +     -     AQU
50 (50 [micro]g/ML    -     -     -     +     ETA
50 (50 [micro]g/ML    -     -     -     -     +

Note: Table made from bar graph.

Figure 3. Effect of P. brasiliensis aqueous extract on cytokine
production by lymphocytes. Peripheral blood mononuclear cells (PBMCs,
5 x [10.sup.5] cells; n=8) were stimulated with phorbol myristate
acetate (PMA) in the presence of 25, 50, or 100 [micro]g-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-[gamma] (A), TNF-[alpha] (B), and
IL-10 (C) was determined by flow cytometry. Results are reported as
means [+ or -] SD. Different letters indicate significant differences
(P [less than or equal to] 0.05, ANOVA with Tukey post hoc).

A. IFN-[gamma]

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PMA + ionomicyn              -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

B. T TNF-[alpha]

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PMA + ionomicyn              -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

C. IL-10

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PMA + ionomicyn              -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

Note: Table made from bar graph.

Figure 4. Antiproliferative effect of P. brasiliensis aqueous extract.
Peripheral blood mononuclear cells (PBMCs, 5 x [0.sup.5] cells; n=6)
labeled with carboxyfluorescein succinimidyl ester (CFSE) were
stimulated with phytohemagglutinin (PHA) in the absence or presence
(25, 50, or 100 [micro]g-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
[+ or -] SD. Different letters indicate significant differences
(P [less than or equal to] 0.05, ANOVA with Tukey post hoc).

A. T cell

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PHA                          -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

B. T CF4

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PHA                          -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

C. T CD8

PBMC (5X[10.sup.5] cells)    +     +     +     +     +
PHA                          -     +     +     +     +
AQU ([micro]g/ML)            -     -    25    50    100

Note: Table made from bar graph.

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.

C

Chemical                     Retention    m/z
                                time

Quinic acid                     2.4       191
Luteolin                        3.8       285
Monocaffeoylquinic acid         16.7      353
(5-caffeoylquinic acid)
Dicaffeoylquinic acid           37.5      515
Luteolin hexoside-hexoside      --        609

Note: Table made from line graph.
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Article Details
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Title Annotation:Research Article
Author:Almeida, V.G.; Avelar-Freitas, B.A.; Santos, M.G.; Costa, L.A.; Silva, T.J.; Pereira, W.F.; Amorim,
Publication:Brazilian Journal of Medical and Biological Research
Date:Aug 1, 2017
Words:5486
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