4 ARTIGO I SUBMETIDO À FITOTERAPIA
4.1 Pro-inflammatory activity of Astronium fraxinifolium Schott on
Milena Aguiar Bragaa, Raphael de Oliveira Rodriguesaa, Juliana Navarro Ueda Yaochitea,
Greyce Luri Sasaharaa, Ana Flávia Seraine Custódio Vianab, Paulo Iury Gomes Nunesb, Karina
Moura de Melob, Flávia Almeida Santosb, Maria Jânia Teixeirac, João Tavares Calixto Juniord,
Ana Livya Moreira Rodriguese, Selene Maia de Moraise, Aparecida Tiemi Nagao-Diasa
a Department of Clinical Analysis and Toxicology, Faculty of Pharmacy, Universidade Federal
do Ceara (UFC), Rua Capitao Francisco Pedro 1210, 60430-370 Fortaleza, CE, Brazil
b Department of Physiology and Pharmacology, Faculty of Medicine, Universidade Federal do
Ceara (UFC), Rua Cel Nunes de Melo 1127, 60430-270 Fortaleza, CE, Brazil
c Department of Pathology and Legal Medicine, Faculty of Medicine, Universidade Federal do
Ceara (UFC,) Rua Monsenhor Furtado S/N, 60430-350 Fortaleza, CE, Brazil
d Department of Biological Sciences, Universidade Regional do Cariri (URCA), Rua Cel.
Antônio Luis 1161, 63105-000 Crato/CE Brazil
eLaboratory of Natural Products, Universidade Estadual do Ceará, Science and Technology
Center, Itaperi 60714903 - Fortaleza, CE – Brazil
ABSTRACT
The study aimed to perform the phytochemical characterization of ethanolic extract from sapwood of Astronium fraxinifolium (EEAF) and to investigate its immunomodulating capacity on LPS-stimulated RAW 264.7 cells. High performanc liquid chromatography was used to identify the constituents of the extract. The antioxidant activity of EEAF was evaluated by its capacity of inhibiting the production of free radical DPPH and ABTS. For the analysis of its immunomodulatory properties, production of nitric oxide (NO), TNF-α and TGF-β was determined in supernatants from LPS-stimulated RAW 264.7 cells after treatment with EEAF at different concentrations. Expression of messenger RNA for COX-2 and iNOS, and also COX-2 protein identification were analyzed. Between constituents identified in the extract
caffeic acid, quercetin followed by orientin and ρ-coumaric acid were the major compounds. EEAF demonstrate significant antioxidant activity in comparison to the reference standard. High production of NO and also high expression of COX-2 mRNA and protein were observed when LPS-stimulated cells were treated with EEAF. Moreover, increased levels of TNF-α and low secretion of TGF-β were demonstrated in supernatants from LPS-stimulated cells treated with EEAF at different concentrations. In opposition to many different types of medicinal plants that show anti-inflammatory properties, the ethanolic extract from sapwood of A. fraxinifolium presents a powerful pro-inflammatory capacity. This property may be extremely relevant in regards to the treatment of chronic infectious diseases based on medicinal plants. For this reason, studies are being conducted in order to evaluate proinflammatory properties of EEAF in cells infected with Leishmania.
Keywords: Astronium fraxinifolium. Inflammatory mediators. RAW 264.7 cells. LPS.
Abbreviations: LPS, lipopolysaccharide; HPLC, High Performance Liquid Chromatography;
EEAF, Ethanolic extract from sapwood of Astronium fraxinifolium; tR, retention time; LOD, limit of detection; LOQ, limit of quantification; DPPH, 2,2'- diphenyl-1-picrylhydrazyl; ABTS, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; IC50, inhibitory concentration; OD,
optical density; MTT, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide; DMSO, dimethylsulfoxide; ELISA, enzyme linked immunonosorbent assay; NO, nitric oxide; DEXA, dexametasone; TNF-α, tumor necrosis factor alpha; TGF-β, transforming growth factor beta; RNA, ribonucleic acid; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; RT- PCR, reverse transcriptase-polymerase chain reaction technique; cDNA, complementary deoxyribonucleic acid; PMSF, phenylmethylsulfonyl fluoride
Introduction
Astronium fraxinifolium Schott belongs to the Anacardiaceae family and can be
found on rocky and dry lands, with geographical distribution in Central and South American countries (SALOMÃO; SILVA, 2006). It is popularly known as Gonçalves-alves, gonçaleiro and aroeira-do-campo (LORENZI, 1992). Flowering occurs during the months of August and September. A. fraxinifolium is of great importance for economy due to high quality of timber production (LUNA, 2012). There is a great advantage in its use because it is not at risk of
extinction (BRASIL, 2014). For medicinal purposes, the plant has been generally employed for the treatment of bronchitis, pulmonary tuberculosis, diarrhea and hemorrhoids (LORENZI, 1992). Antimicrobial properties, such as antibacterial (MONTANARI et al., 2012), antifungal Bonifácio et al. (2015) and leishmanicidal activities Lima et al. (2014) have been described. According to phytochemical studies, the plant leaves present (Z)-β-ocimene, (E)-β-ocimene, bicyclogermacrene, limonene, α-terpinolene and viridiflorene (MONTANARI et al., 2012). Limonene shows anti-inflammatory and antioxidant properties (ZLOTEK; MICHALAK- MAJEWSKA; SZYMANOWSKA, 2016). Alpha-terpinolene is antifungal and leishmanicidal (RAMOS et al., 2014). In respect to the anti-inflammatory properties of A. fraxinifolium, scientific knowledge is still poor and needs to be better elucidated. Inflammation is a response of the immune system against invasive stimuli, infection or tissue damage. It is a complex process regulated by various chemical mediators and immune cells (GUO et al., 2016). Macrophages play a key role in the homeostasis of the organism, through the phagocytosis of pathogens and production of chemokines, cytokines and lipid mediators (ARULSELVAN et
al., 2016). Lipopolysaccharide (LPS) is one of the major components of the cell outer
membrane of Gram-negative bacteria, which induces immune cell activation and triggers an inflammatory response against bacterial invasion (YANG et al., 2016). LPS-stimulated macrophages have been widely used to provide an in vitro inflammatory environment, which contributes, for instance to study the role of anti-inflammatory compounds (DONG et al., 2017). Taking into account the limited scientific knowledge regarding the immunomodulatory properties of Astronium fraxinifolium, the present study aimed to evaluate its capacity of immunomodulating LPS-stimulated RAW 264.7cells.
Material and methods
Plant material
Sapwood from Astronium fraxinifolium Schott (register at SISGEN A166486, 04/24/2018) was collected in the city of Lavras da Mangabeira (06o45’12”S 38o57’52W5
239m), Ceara, Brazil. A voucher specimen (54265) was deposited at the Herbarium Prisco Bezerra, Department of Biology, Universidade Federal do Ceará, Brazil. The plant material (1000 g) was dried in an oven with forced air circulation for a period of 48 h at 65°C. Ehanolic extract was obtained by macerating the plant material with absolute ethanol for 7 days at room
temperature. After concentration of the material on a rotary evaporator under reduced pressure at 40°C, the final extract (yield 11 of 1.8%) was submitted to phytochemical analysis.
Phenol quantification
Phenolic compounds in the extract were determined according to the method of Folin-Ciocalteau (SINGLETON; JOSEPH; ROSSI, 1965). The assay was performed in triplicate, using gallic acid (Sigma, USA) as standard reference.
Flavonoids quantification
Flavonoid content was determined according to the method described by Vennat et
al. 1992 (VENNAT; GROSS; POURRAT, 1992). Quercetin (Sigma, USA) was used as
reference standard. Tannin quantification
Tannin content was determined by the Folin-Denis colorimetric method, according to Association of Official Analytical Chemists (AOAC, 1995). Tannic acid (Sigma, USA) was used as reference standard.
High Performance Liquid Chromatography (HPLC)
A chromatographic analysis of the plant material was performed by high performance liquid chromatography (HPLC-DAD) on a Prominence HPLC Auto Sampler (SIL-20A) (Shimadzu, Kyoto, Japan), equipped with a Shimadzu LC-20AT pumps connected to a DGU degasser 20A5 with a CBM 20A integrator, SPD-M20A diode array detector and LC solutions in software 1.2 SP1. Ethanolic extract from sapwood of A. fraxinifolium (EEAF) and reference standards were injected into a Phenomenex C18-reverse phase column (4.6 mm x 250 mm). The elution was performed using mobile phases A and B, that is, respectively, ultrapure water, previously acidified to pH 3.0 with 2% acetic acid, and acetonitrile, as described by Reis
et al. (2014), with some modifications. The volume of 50 μL of the extract (15 mg/mL) was
injected at a solvent flow rate of 0.6 mL/min. Reference standards were tested at 0.025-0.300 mg/mL. Quantification of the compounds was performed analyzing the peaks and their
retention time (tR), considering that the optical reading for gallic acid (Merck, Germany) was 254 nm; 280 nm for catechin (Sigma, USA); 325 nm for ρ-cumaric acid (Merck, Germany) and caffeic acid (Merck, Germany); 366 nm for quercetin (Sigma, USA), rutin (Sigma, USA), luteolin (Sigma, USA) and orientin (Sigma, USA). The chromatographic procedures were performed at room temperature and in triplicate. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope by means of three independent analysis curves, as defined by Boligon et al. 2014. Evaluation of the antioxidant activity
a) Inhibition of DPPH activity
Antioxidant activity of the EEAF extract was evaluated by the 2,2'-diphenyl-1- picrylhydrazyl (DPPH) radical scavenging method [19], with some modifications.
Quercetin was used as the reference standard. The antioxidant activity was expressed according to the equation: Inhibition percentage (%) = [Abs (DPPH) − Abs (sample) / Abs (DPPH)] × 100. The assay was carried out in sextuplicate. The IC50 indicated the
concentration of the sample required to inhibit 50% of the DPPH activity. Abs (DPPH) was the absorbance of the DPPH solution and Abs (sample) was the absorbance of the solution containing the EEAF extract at a particular concentration. IC50 values (concentration of sample
required to scavenge 50% of free radicals) were calculated by the regression equation of the concentration of the EEAF extract, and percentage inhibition of free radical formation/percentage inhibition DPPH.
b) Inhibition of ABTS activity
To confirm the antioxidant activity of the EEAF extract, 2,2-azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) method was performed according to the procedure described by Miller & Rice-Evans, 1997 [20], with some modifications. The percentage of inhibition was assessed according to the Equation: Inhibition percentage (%) = [(ODABTS - ODextract) / ODABTS] x 100. ODABTS was the absorbance of the ABTS solution and
ODextract was the absorbance of the solution containing the EEAF extract at a particular
concentration. The assays were carried out in triplicate. Evaluation of EEAF Cytotoxicity
The MTT tetrazolium assay (MOSMANN, 1983) was used to evaluate cell viability after treatment with the EEAF. Murine RAW 264.7 macrophage cell line obtained from the Cell Bank of Rio de Janeiro (BCRJ, Brazil) was used.
At total of 5 x 105 cells/mL RAW 264.7 cells were seeded in 96-well culture
microplates in DMEM (LGC, Brazil) medium supplemented with 10% heat inactivated fetal bovine serum (LGC, Brazil) and gentamicin (50 μg/mL,Sigma,USA), and incubated overnight at 37° C and 5% CO2. After this period, the cells were treated with different concentrations of
EEAF (15.63 to 500.00μg/mL) and incubated for 24 h. Thereafter, the supernatant was discarded and the adherent cells were washed with phosphate-buffered saline (PBS). Subsequently, a new supplemented DMEM medium was added to the cells followed by 3-[4,5- dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) dissolved in PBS at 500 μg/mL. After incubation for 4 h at 37°C and 5% CO2, the supernatant was discarded and
100 μL of 100% dimethylsulfoxide (DMSO, Sigma, USA) were added to the plates. The plates were vigorously shaken for 15 min and the optical density was read at 570 nm wavelength using an ELISA plate reader. The assays were performed in triplicate.
Plant activity on LPS-activated cells
RAW 264.7 cells were incubated at 5 x 105cells/mL with 1 μg/mL of LPS from E.
coli O128:B12 (Sigma, USA) in a 24-well culture plate for 24 h at 37° C and 5% CO2. Nontoxic
concentrations of EEAF (31.25; 62.5 and 125 μg/mL) and controls (0.1% DMSO and 4.0 μg/mL dexamethasone Sigma, USA) were added to the wells. After a new 24 h period of incubation, the cells or their supernatants were collected and analyzed.
a) NO measurement
The presence of nitric oxide (NO) was indirectly assessed by the amount of nitrite in the cell culture supernatant using Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl) ethylenediaminedihydrochloride), according to the procedure described by Green et al. 1982. The standard curve was obtained using different concentrations of sodium nitrite (Sigma, USA).
b) TNF-α and TGF-β levels measurement
Quantification of tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) levels in cell supernatants were performed by using sandwich-enzyme immuno assays, according to the manufacturer's recommendations (Novex®, Life Technologies, USA).
c) Expression of messenger RNA for COX-2, iNOS and TGF-β
Expression of messenger RNAs for COX-2, iNOS and TGF-β was evaluated by a reverse transcriptase-polymerase chain reaction technique (RT-PCR). RNA was extracted from cells previously stimulated and treated with various concentrations of EEAF (31.25; 62.5 and 125 μg/mL) RAW 264.7 cells using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. After extraction, 0.5 μg of total RNA was reverse transcribed into complementary DNA (cDNA) with the aid of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), according to the manufacturer's recommendations. The sequences of nucleotides for designing target-specific primers were obtained from NCBI and analyzed by the OligoPerfect™ Designer software (Thermo Fisher Scientific, USA). The following primers were used: COX-2, F: 5'- AGAAGGAAATGGCTGCAGAA-3 'and R: 5'- GCTCGGCTTCCAGTATTGAG-3'; iNOS, F: 5'-CACCTTGGAGTTCACCCAGT-3' and R: 5'-ACCACTCGTACTTGGGATGC-3'; TGF-β, F: 5’- TTGCTTCAGCTCCACAGAGA-3 'and R: 5'-TGGTTGTAGAGGGCAAGGAC-3'; β-actin (housekeeping gene), F: 5'- AGCCATGTACGTAGCCATCC-3 'and R: 5'- CTCTCAGCTGTGGTGGTGAA-3'. The number of amplification cycles was adjusted according to the target, in order to obtain an exponential increase of the amplicons for each biomarker. The annealing temperature and the number of cycles used for the amplification of each target are described in Table 1. Reactions were performed using an Applied Biosystems 2720 Thermal Cycler (Biosystems, EUA). The amplicons were electrophoresed on a 2.5% agarose gel followed by staining with SYBR Safe stain (Invitrogen, USA). The bands were visualized by a ChemiDoc ™ MP Imaging System (BIO-RAD, EUA) and their intensity was analyzed the Image Lab™ software (version 5.1, BIO-RAD, USA).
d) COX-2 detection by the Western blot technique
After treatment with the plant material or with the reference drug (4.0 μg/mL dexamethasone Sigma, USA), LPS-stimulated RAW 264.7 cells, were lysed with RIPA buffer solution (Sigma, USA), which was supplemented with protease inhibitor cocktail P8340 (Sigma, USA) at 1:100 (v/v), phenylmethylsulfonyl fluoride (PMSF) (Sigma, USA) at 2mM and sodium orthovanadate (Sigma,USA) at 1 mM. Quantification of total proteins was done by the Lowry Method [23], using the DC Protein Assay kit (Bio-Rad Laboratories, USA). Cell proteins (20 μg per well) were electrophoresed on an 8% sodium dodecyl sulphate- polyacrylamide gel, under a voltage of 125 V for 1 h and 20 min at room temperature. The separated polypeptides were blotted onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, USA), under a constant amperage of 400 mA, at 4° C for 2 h. The membranes were blocked with 5% skim milk solution containing 5% bovine serum albumin (BSA, Sigma, USA) overnight. After, the membranes were incubated for 2 h with primary rabbit antibody to cyclooxygenase-2 (COX-2, Santa Cruz Biotechnology®, USA) at a dilution of 1:500 in 5% BSA. Anti-β-actin antibody (Cell Signaling Technology, USA) was used as an internal reference standard. The membranes were washed (5 times) with Tris-buffered solution containing 1% Tween 20 and incubated with the secondary peroxidase-conjugated anti- rabbit IgG (Cell Signaling Technology, USA), at 1:3000 dilution in 5% BSA for 2 h. For the chemiluminescence detection, luminol solution containing H2O2 (Amersham ECL ™ Prime reagent) was used. The membranes were photographed by a ChemiDoc™ MP Imaging System (BIO-RAD, USA) and the Image Lab™ software (version 5.1, BIO- RAD, USA) was used for the image acquisition and edition.
Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM) and analyzed by Student's t-test or analysis of variance (ANOVA), followed by Tukey's post-test. All analyzes were performed using GraphPad Prism version 7.0 for Windows (GraphPad Software, San Diego, CA, USA). The values considered statistically significant presented a value of p ≤ 0.05.
Results
Quantification of phenols, tannins and flavonoids in the EEAF
Spectrophotometric analysis of total phenols, flavonoids and tannins was performed in the ethanolic extract from A. fraxinifolium (EEAF). Amounts equivalent to 600 ± 1.53 mg of total phenols, 23,12± 0.0079 mg of flavonoids and 130,6±0,0045 mg of tannins were found in 1000 mg of EEAF.
Quantification of the EEAF compounds by HPLC
High performance liquid chromatography (HPLC) was used to analyze the EEAF compounds (Figure 1). Molecules from eight peaks were identified and quantified according to known concentrations of standard solutions and their retention time (tR), that is, caffeic acid (tR = 23.11 min, peak 3, 5.12 ± 0.01 mg/mL) and quercetin(tR = 39.91 min, peak 7, 2.19 ± 0.01 mg/mL), followed by orientin (tR = 32.15 min, peak 5, 1.16 ± 0.03 mg/mL) and ρ-coumaric acid (tR = 29.86 min, peak 4, 1.15 ± 0.01 mg/mL) (Table 2).
Evaluation of antioxidant activity
The 50% inhibitory capacity of free radicals of the EEAF was equivalent to 5.607± 0.012µg/mL for DPPH and 3.544 ± 0.16 μg/mL for ABTS. The reference standard, that is, quercetin, was almost the same activity than the A. fraxinifolium extract, 5.0 ± 0.18 µg/mL for DPPH and 3 1.79 ± 0.02 μg/mL for ABTS.
Evaluation of EEAF citotoxicity
Concentration equivalent to 500 μg/mL of EEAF on RAW 264.7 cells was considered to be toxic (Figure 2).
Quantification of nitric oxide after EEAF treatment
LPS-stimulated RAW 264.7 cells were treated with 4μM dexametasone or with EEAF at 31.25 μg/mL, 62.5 μg/mL or 125 μg/mL. Nitrite concentration was measured after 24
h of incubation. The mean concentrations of nitric oxide in supernatants from LPS-stimulated cells treated with EEAF at 62.5 μg/mL, 125 μg/mL and untreated LPS-stimulated cells were 14.3 μM, 13.15 μM and 12.23 μM, respectively (Figure 3). No statistically significant difference was found among the groups. The EEAF did not promote any nitrite production in unstimulated cells (Figure 4).
Quantification of cytokine levels after EEAF treatment
As demonstrated in the Figure 5, all the concentrations of the EEAF induced a significant secretion of TNF-α in comparison to LPS-stimulated cells without treatment (p <0.001; Tukey’s test). It was also observed statistical difference among the treated groups (p <0.001). On the contrary, untreated LPS-stimulated cells showed much higher concentrations of TGF-β than those treated with EEAF at different concentrations in comparison to EEAF at 31.25 μg/mL (p<0.01), at 62.5 μg/mL and 125 μg/mL (p<0.001) (Tukey’s test, Figure 6). COX-2 and iNOS mRNA expression after EEAF treatment.
The treatment of LPS-stimulated RAW 264.7 cells with EEAF at 62.5 μg/mL promoted an increase in COX-2 mRNA expression when compared to LPS-stimulated cells without treatment (p <0.05, T test, Figure 7). No significant differences among the groups were observed when iNOS mRNA expression was evaluated (Figure 8).
COX-2 detection by Western blot technique
An increase of COX-2 expression was observed in LPS-stimulated cells treated with EEAF at 31.25μg/mL when compared to LPS-stimulated group without treatment (p<0.05, T test, Figure 9).
Discussion
Few studies are found in literature regarding Astronium fraxinifolium [5] reported that essential oil from leaves of this plant presents antibacterial activity against Escherichia
coli, Bacillus cereus and Staphylococcus aureus. The methanolic extract from the leaves
(PINTO; SOUZA; OLIVEIRA, 2010). Our research group demonstrated that ethanolic extract from the plant showed leishmanicidal activity by in vitro and in vivo studies (LIMA et al., 2014).
Studies related to the isolation and identification of secondary metabolites of species of Anacardiaceae are incipient. It is estimated that less than 7% of the species were phytochemically characterized (CORREIA; DAVID, J. P.; DAVID, J. M., 20016). Montanari
et al. (2012) identified the constituents in the essential oil from leaves of A. fraxinifolium.
According to Silva et al. (2010), the main components of the genus Astronium sp. included flavonoids, proanthocyanidins, such as profisetinidine and prorobinetinidine, and a high content of polyphenols, such as tannins and lignins. The authors suggested that the condensed and hydrolyzable flavonoids and tannins found in the genus Astronium are responsible for certain characteristics such as astringency, and complexing with macromolecules, such as proteins, polysaccharides, and metal ions.
In the present work, we observed that ethanolic extract from sapwood of
A.fraxinifolium (EEAF) presented 600 mg total phenols per 1000 mg extract, 23.12 mg
flavonoids per 1000 mg extract and 130.6 mg tannins per 1000 mg extract. Phenols may comprise simple phenols, phenolic acids, coumarins, flavonoids, stilbenes, condensed and hydrolyzable tannins, lignans and lignins (NACZK; SHAHIDI, 2004). The profile of the EEAF obtained after elution by HPLC comprised caffeic acid and quercetin in greater amounts followed by, orientin, ρ-coumaric acid, gallic acid, luteolin, rutin and catechin. Caffeic acid may present several biological properties, such as antiviral Touaibia, Jean-François and Doiron (2011) and antioxidant activities (SUN-WATERHOUSE; THAKORLAL; ZHOU, 2011). It also presents anti-inflammatory activity by reducing prostaglandins and leukotriene synthesis (ERDEMLI, et al., 2015), by inhibiting COX-2 expression, and by inhibiting nuclear factor kappa B (NF-κB), and consequently interleukin-1β and TNF-α production, on LPS-stimulated RAW 264.7 macrophages (JUMAN et al., 2012).
Quercetin is a flavonoid that has antioxidant, anti-inflammatory, antibacterial, antiviral, gastroprotective, immunomodulatory, neuroprotection functions and is used in the treatment of obesity and cardiovascular diseases (WANG et al., 2016). Its antiinflammatory action can be attributed to the inhibition of the enzymes phospholipase A2, lipoxygenase, cyclooxygenase and inhibition of nitric oxide production, through the modulation of the enzyme iNOS (YOON; BAEK, 2005). Luteolin also decreases NO production by modulating the expression of the enzyme iNOS and by inhibiting the enzyme cyclooxygenase. Quercetin,
luteolin and rutin are able to inhibit the secretion of proinflammatory cytokines, such as TNF- α and IL-1 (CAZAROLLI et al., 2008).
In the present study, the EEAF exhibited antioxidant activity. The same amount of EEAF would be necessary to neutralize the free radicals DPPH and ABTS in comparison to quercetin, the reference standard used in the experiments. The quercetin was one of the constituents identified in the extract.
Most of the phenols produced by plants have therapeutic functions. However, there are some highly toxic compounds present in resin channels of leaves and other parenchymal tissues in certain species of Anacardiaceae, which are responsible for protection against insects and vertebrates (AGUILAR-ORTIGOZA; SOSA, 2004).
About 25% of the genera of the family Anacardiaceae are classified as toxic and may cause severe contact dermatitis due to the production of allergenic substances, such as phenols of the types alkylcatecols, alkylresorcinols and biflavonoids. In the genus Astronium, alkylcatecols and biflavonoids are usually found in leaves and bark. Alkyl-catechols, for