Microsoft Word - 28-Bio_41999 1575 Bioscience Journal Original Article Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 Schinus terebinthifolius ESSENTIAL OIL AND FRACTIONS IN THE CONTROL OF Aedes aegypti Schinus terebinthifolius ÓLEO ESSENCIAL E FRAÇÕES NO CONTROLE DO Aedes aegypti Wanessa de Campos BORTOLUCCI1; Hérika Line Marko de OLIVEIRA1; Eloísa Schineider SILVA1; Caio Franco de Araújo Almeida CAMPO2; José Eduardo GONÇALVES2,3; Ranulfo PIAU JUNIOR4; Nelson Barros COLAUTO1; Giani Andrea LINDE1; Zilda Cristiani GAZIM1,* 1. Postgraduate Program in Biotechnology Applied to the Agriculture, Paranaense University- Unipar, Umuarama, PR, Brasil; 2. Postgraduate Program in Clean Technologies, Cesumar University, Maringá, Paraná, Brazil; 3. Cesumar Institute of Science, Technology and Innovation – ICETI, Paraná, Brazil; 4. Postgraduate Program of Animal Science, Paranaense University- Unipar, Umuarama, PR, Brasil. cristianigazim@prof.unipar.br ABSTRACT: Several technologies have been developed to control Aedes aegypti, mainly studies on isolated plant molecules. The Schinus terebinthifolius (Raddi) (Anacardiaceae), popularly known as pink pepper is a plant widely used in reforestation of degraded areas and its fruits are used as condiments. The objective of this work was to investigate the potential of essential oils (EOs) and fractions (FRs) obtained from fresh fruits and leaves of S. terebinthifolius. The EOs were obtained by hydrodistillation (2 hours), fractionated on a chromatographic column using as the stationary phase silica gel 60 (0.063-0.2mm), mobile phases: n- hexane, dichloromethane, ethyl acetate and methanol and chemically evaluated by gas chromatography coupled to mass spectrometer (GC/MS). EOs and FRs were tested against larvae of the third stage and pupae of Ae. aegypti by Immersion Test at concentrations ranging from 500.00 to 0.003 mg mL-1 (v/v). The hexane FRs obtained from fruits and leaves were the ones that showed the greatest activity on the larvae (LC99.9= 0.60 mg mL-1 and LC99.9 0.64 mg mL-1, respectively) and pupae (LC99,9 = 2.51 mg mL-1 and 2.61 mg mL-1, respectively). These results were confirmed by the anticholinesterase activity where the hexane (fruit and leaf) FRs presented the highest inhibitory potential on the acetylcholinesterase enzyme (0.156 mg mL-1 and 0.312 mg mL-1, respectively), suggesting the likely mechanism of action. The larvicidal potential can be explained by the presence of the major compounds bicyclogermacrene and germacrene D in the hexane FRs, indicating in this way that they may replace or even act in synergisms with conventional chemical larvicides. In this way the present study opens the field for new researches, aiming the development of products with the compounds bicyclogermacrene and germacrene D, as an alternative in the control of this culicide. KEYWORDS: Larval Immersion Test. Acetylcholinesterase. Bicyclogermacrene. Rose pepper. Germacrene D. ABBREVIATIONS: GC/MS gas chromatography coupled to mass spectrometer; LC lethal concentration; LC50 lethal concentration to eliminate 50% of larvae and pupae; LC99.9 lethal concentration to eliminate 99.9% of larvae and pupae; EO essential oil; FR fraction. INTRODUCTION Aedes aegypti L. has been responsible for epidemics of dengue, chikungunya and microcephaly related to the infection by Zika virus (DINIZ et al., 2014) in South America and tropical countries, and it has become a worldwide public health problem with high rate of morbidity and mortality (BRASIL, 2016; TEIXEIRA et al., 2016). In Brazil, 251,711 million cases of dengue were recorded in 2017, of which 44,915 cases were registered by March 2018 (21.6 cases/100 thousand inhabitants), 12,102 cases of chikungunya (5.8 cases/100 thousand inhabitants) and 705 cases of people infected with zika virus (0.3 cases/100 thousand inhabitants) (BRASIL, 2018). Although the authorities have shown efforts to combat this vector through awareness programs to eradicate reproduction sites, the incidence of these pathologies is still alarming regarding the number of cases and seriousness (NASCIMENTO et al., 2008). The control of this vector is done by utilizing chemical insecticides from organophosphates and pyrethroids. Organophosphates are characterized by the short- term effect, and are chemically more unstable, which demands frequent applications, but it has toxic effect to vertebrate even in small doses Received: 28/04/18 Accepted: 05/12/18 1576 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 (BRAGA; VALLE, 2007). These organophosphates usually act directly on the acetylcholinesterase enzyme, causing its accumulation on the synaptic cleft, resulting in hyperexcitability and consequently the insect death (ČOLOVIĆ; KRSTIĆ, 2013; KING; AARON, 2015). Pyrethoids have also been very utilized as an alternate substitute of organophosphates; they are non-cumulative and chemically stable but are toxic to fish, bees and aquatic arthropods (COSMOSKI et al., 2015; MANRIQUE-SAIDE et al., 2015; PORTO et al., 2017). Besides the environmental impact that they cause, another disadvantage of the utilization of chemical insecticides is their high cost as well as the occurrence of resistance in samples of Ae. aegypti populations (ZARA et al., 2016; RODRIGUES et al., 2017). Therefore, to minimize the problems caused by organophosphates and pyrethroids, studies have advanced on the search of bioactive molecules that can substitute as well as act in synergism with conventional insecticides. Thus, Schinus terebinthifolius Raddi, from the Anacardiaceae family, popularly known as rose pepper, “aroeira”, is a native plant of the Atlantic Forest, and is easily found in the northeastern region of Paraná where it has been utilized in reforestation areas (CESÁRIO; GAGLIANONE, 2013; OLIVEIRA et al., 2014; FERREIRA-FILHO et al., 2015). Due to its medicinal properties, this species has been studied and reported in the literature regarding its acaricidal, larvicidal and fungicidal activities (MWANGI et al., 2009; SILVA et al., 2012; HUSSEINS et al., 2015). Its fruits and leaves are rich in essential oil, presenting great yield, mainly in the fruits (EL-MASSRY et al., 2009). Thus, this study aimed to evaluate the chemical composition of the essential oil and fractions of rose pepper fruits and leaves against Ae. aegypti third-stage larvae and pupae. MATERIAL AND METHODS Implantation of culture S. terebinthifolius leaves and fruits were collected in Juranda, northearten region of Paraná State, Brazil (S 24º 21’ 28.2456” and WO 52º 36’6276” and altitude of 419m). The botanical identification was done and the specimen voucher was deposited on the Herbarium of the West Paraná State University – UNIOESTE, under the registration number 1717. This species is registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the registration number A22FA69. Obtainment of vegetal matter, extraction and fractioning of rose pepper fruit and leaf essential oil The ripe fruits were harvested in December (fruitification), and the leaves were monthly collected from April to December, 2015. The essential oil was obtained by hydrodistillation of fresh fruits and leaves for 2 h (SANTOS et al., 2013). The EO was withdrawn with n-hexane and filtered with anhydrous Na2SO4, stored in an amber flask, weighed and kept under refrigeration (- 4ºC) until total evaporation of n-hexane (BRASIL, 2010). Next, the yield (%) of the fruit and leaf essential oil was calculated in triplicate. For the fractioning, EO from rose pepper leaves and fruits (6.0 g) was utilized and submitted to column chromatography (CC) using silica gel 60 as the stationary phase (0.063-0.2 mm) and n- hexane, dichloromethane, ethyl acetate and methanol as the mobile phases. Chemical characterization of essential oil and fractions from rose pepper fruits and leaves The chemical identification of EO and fractions (FRs) was done by Gas Chromatographer (Agilent 7890 B) coupled to Mass Spectrometer (Agilent 5977A) (GC-MS), equipped with a silica funded capillary column HP-5MS UI Agilent (30 m x 0.250 mm x 0.25 μm). The analysis conditions were: injector temperature 220 oC, injection volume of and the specimen voucher 2 µ L and injection ration in split mode (1:30). The column initial temperature was 60 oC (2 min), with heating ramp of 2oC/min until 180 oC (4 min). From 180°C to 260°C a heating ramp of 10°C/min was stablished. From 260°C to 300°C a heating ramp of 40°C/min was utilized (CAVALCANTE et al., 2015). The transfer line was kept at 285 ºC, and the ionization source and quadrupole at 230 oC and 150oC, respectively. The utilized carrier gas was He with 1 mL/min flow. The detection system was EM in “Scan” mode, in the mass/charge (m/z) range from 40 – 550, with 3-min Solvent Delay. The samples of oils and fractions were diluted in a 1:10 rate with dichloromethane. The chemical components were identified by comparing their mass spectra with mass spectra from WILEY 275 libraries and also by comparing their retention indexes (RI) which were obtained by utilizing a homologous series of n- alkane standard (C7 - C26) (ADAMS, 2012). 1577 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 Biological activity of essential oils and fractions from rose pepper leaves and fruits on Aedes aegypti larvae and pupae Ae. aegypti third-stage larvae and pupae from the Núcleo de Controle de Endemias Transmissíveis por Vetores - Secretaria de Vigilância Sanitária from Juranda PR were utilized. The essential oil and fraction from S. terebinthifolius leaves and fruits were tested using an initial concentration of 500.00, 400.00, 300.00, 200.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.125, 1.562, 0.781, 0.390, 0.195, 0.097, 0.048, 0.024, 0.012, 0.006, 0.003 mg mL-1 (v/v), diluted in an aqueous solution of polysorbate (80) at 2.00%. Ten third-stage larvae and ten pupae of Ae. aegypti were separated using a pasteur pipette and placed in assay tubes containing 1.00 mL of different concentrations of EO and fractions in triplicate (CAVALCA et al., 2010). For the negative control, an aqueous solution of polysorbate (80) at 2.00% was utilized, and for the positive control, a commercial organophosphate at 400.00 mg/L (CAMARGO et al., 1998) was used. The larvae and pupae were exposed to fruit and leaf EO and fractions at different concentrations for 24 hours, and those that showed absence of movements and did not respond to stimuli were considered dead (CAVALCA et al., 2010). The larval mortality rate (%) and the average mortality (%) were calculated according to Equation Anticholinesterase activity of essential oil and fractions from rose pepper fruits and leaves The anticholinesterase activity was determined by the bioautographic method described by Marston et al. (2002) with modifications (YANG et al., 2009). The EO and fractions from rose pepper leaves and fruits were evaluated from an initial concentration of 50.00, 40.00, 30.00, 20.00, 10.00, 5.00, 2.50, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.019, and 0.009 mg mL-1, diluted in methanol. The samples were plotted in aluminum chromoplates (10 x 10 cm, silica gel 60 F254, 0.2 mm thick); after plotting, the plates were dried and sprayed with a solution of acetylcholinesterase enzyme diluted in a TRIS buffer solution; next, it was sprayed with α- naftyl acetate solution. The plates were kept at 37 °C for 20 minutes. After this period, the chromoplates were sprayed with a colorimetric reagent, Fast Blue B salt, resulting in the emergence of the purple color. The anticholinesterase activity was determined by the emergence of white stains after 5 minutes, demonstrating the inhibitory action of the evaluated concentrations on the enzyme activity, contrasting with the purple color of the colorimetric reagent (COLLINS et al., 1997). A commercial organophosphate at 400.00 mg/L (CAMARGO et al., 1998) was utilized as positive control. STATISTICAL ANALYSIS The experiments were done in triplicate, and the mortality percentage (%) of Ae. aegypti larvae and pupae was obtained by calculating the mean ± standard deviation (SD) and the coefficient of variation (CV) utilizing Microsoft Excel® (Excel® Version 2010). The values of Lethal Concentration (LC50 and LC99.9) and their respective confidence intervals (CI) were calculated by analysis of Probitos (ED 50 Plus version 1.0). The obtained data were submitted to analysis of variance (ANOVA) and the differences between the averages were determined by Duncan’s test (P ≤ 0.05). RESULTS AND DISCUSSION The results found for the physical and chemical characteristics of EO from fruits and leaves indicate that the EO from S. terebinthifolius fruits has transparent color and characteristic odor of the species whereas the EO from fruits had light yellow color and strong terebintine odor. The fruit EO yield (%) was (7.25a ± 0.61 %) (v/p) and for leaf EO was (0.57b ± 0.10 %) (v/p), making the superior yield of fruit oil evident when compared to leaf oil. The results are in agreement with the ones by Jeribi et al. (2012) who found a yield of 5.03% (v/p) for fruit oil, and Barbosa et al. (2007) and El-Massry et al. (2009) who found 0.44% and 0.50% (v/p) for rose pepper leaves, respectively. The data regarding the chemical composition of rose pepper fruit and leaf EOs are shown in Table 1. 54 compounds were identified in the leaf EO and the predominant class was sesquiterpene hydrocarbons (72.63%), and the major ones were: bicyclogermacrene (27.57%), β- phellandrene (7.30%), germacrene D (7.16%) and isolongifolene (7.11%). 80 compounds were found in the fruit EO and the main class was sesquiterpene hydrocarbons (43.53%), and the major ones were: β- pinene (30.32%), germacrene D (14.23%), bicyclogermacrene (5.97%) and α-pinene (3.58%). 1578 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 Table 1. Chemical composition of Schinus terebinthifolius leaves and fruit essential oil Relative Area (%) Identification methods Peak aCompounds bRI calc. EO Leaves EO Fruits Monoterpene hydrocarbons 1 n.i. 838 0.31 - a,b,c 2 α-Thujene 916 - 0.04 a,b,c 3 α-Pinene 926 6.30 3.58 a,b,c 4 n.i 938 - t a,b,c 5 Sabinene 962 0.22 1.76 a,b,c 6 β-Pinene 970 1.91 30.32 a,b,c 7 Myrcene 980 0.12 t a,b,c 8 α-Phellandrene 993 0.07 0.09 a,b,c 9 δ-3-Carene 1008 t 0.48 a,b,c 10 Limonene 1015 0.16 t a,b,c 11 p-Cimene 1020 t 0.28 a,b,c 12 β-Phellandrene 1024 7.30 1.85 a,b,c 13 1,8-Cineole 1027 0.36 0.13 a,b,c 14 β-cis-Ocimene 1045 t 0.05 a,b,c Oxygenated monoterpenes 15 p-Mentha-2,8-dien-1-ol 1086 t 0.13 a,b,c 16 trans-Pinocarveol 1137 - 0.08 a,b,c 17 Terpinen-4-ol 1169 - 0.21 a,b,c 18 α-Terpineol 1189 0.12 - a,b,c Sesquiterpene hydrocarbons 19 δ-Elemene 1335 1.68 0.60 a,b,c 20 n.i. 1338 - 0.08 a,b,c 21 α-Cubebene 1344 0.32 0.14 a,b,c 22 Cyclosativene 1369 - 0.08 a,b,c 23 α-Ylangene 1372 0.35 - a,b,c 24 α-Copaene 1375 5.08 1.44 a,b,c 25 β-Cubebene 1386 - 0.08 a,b,c 26 Isolongifolene 1389 7.11 - a,b,c 27 β-Elemene 1390 - 1.70 a,b,c 28 Cyperene 1393 1.65 - a,b,c 29 β-Longipinene 1399 4.35 - a,b,c 30 Longifolene 1407 t 2.57 a,b,c 31 α-Gurjunene 1409 0.11 0.55 a,b,c 32 α-Cedrene 1410 0.17 t a,b,c 33 trans-Caryophyllene 1417 0.94 2.57 a,b,c 34 β-Copaene 1430 t 0.24 a,b,c 35 γ-Elemene 1433 0.11 0.27 a,b,c 36 α-Guaiene 1437 t 0.43 a,b,c 37 Aromadendrene 1439 0.46 0.13 a,b,c 38 β-cis-Farnesene 1442 6.38 - a,b,c 1579 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 39 α-Humulene 1451 - 0.51 a,b,c 40 allo-Aromadendrene 1457 0.40 1.70 a,b,c 41 γ-Gurjunene 1475 - 1.06 a,b,c 42 γ-Muurolene 1477 - 0.13 a,b,c 43 γ-Himachalene 1482 - 0.18 a,b,c 44 Germacrene D 1485 7.16 14.23 a,b,c 45 β-Selinene 1489 0.16 - a,b,c 46 β-cis-Guaiene 1492 - t a,b,c 47 Valencene 1496 - 0.13 a,b,c 48 Bicyclogermacrene 1500 27.57 5.97 a,b,c 49 α-Muurolene 1501 0.37 1.49 a,b,c 50 Cuparene 1504 - 1.24 a,b,c 51 Germacrene A 1509 1.66 0.18 a,b,c 52 γ-Cadinene 1512 - 0.87 a,b,c 53 δ-Cadinene 1523 0.51 3.48 a,b,c 54 α-Cadinene 1539 1.98 0.14 a,b,c 55 Selina-3,7(11)-diene 1540 1.57 0.15 a,b,c 56 α-Calacorene 1545 0.53 0.13 a,b,c 57 Germacrene B 1560 2.01 0.35 a,b,c 58 n.i. 1563 0.11 0.15 a,b,c Oxygenated sesquiterpenes 59 Palustrol 1567 - 0.21 a,b,c 60 Caryophyllenylalcohol 1569 2.41 1,14 a,b,c 61 Spathulenol 1577 1.02 2.82 a,b,c 62 ar-Turmerol 1576 0.39 0.17 a,b,c 63 Caryophyllene oxide 1584 0.75 0.12 a,b,c 64 Globulol 1590 - 1.11 a,b,c 65 Viridiflorol 1592 0.09 0.22 a,b,c 66 Guaiol 1600 - 0.11 a,b,c 67 Ledol 1603 0.30 1.02 a,b,c 68 cis-Isolongifolanone 1610 0.11 0.41 a,b,c 69 1,10-di-epi-Cubenol 1619 0.30 0.24 a,b,c 70 γ-Eudesmol 1629 1.49 0.35 a,b,c 71 epi-α-Cadinol 1638 0.21 0.40 a,b,c 72 α-Muurolol 1644 - 0.59 a,b,c 73 epi-α-Muurolol 1646 0.26 0.17 a,b,c 74 β-Eudesmol 1649 - 2.07 a,b,c 75 α-Eudesmol 1651 - 0.73 a,b,c 76 α-Cadinol 1653 1.58 2.35 a,b,c 77 trans-Bisabolol-11-ol 1668 - 0.20 a,b,c 78 α-Santalol 1674 t 1.06 a,b,c 79 α-Bisabolol 1684 - 0.25 a,b,c 80 2,3-dihydro-Farnesol 1688 0.43 0.42 a,b,c 81 Germacrone 1693 - 0.08 a,b,c 82 2,6-trans-Farnesal 1703 - 0.07 a,b,c 83 14-hydroxy-4,5-dihydro-β- 1706 - 0.12 a,b,c 1580 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 Caryophyllene 84 2-trans, 6-cis, Farnesal 1714 - 0.10 a,b,c 85 β-trans-Santalol 1716 - 0.43 a,b,c 86 Aristolone 1762 - 0.10 a,b,c 87 β-Costol 1766 - 0.09 a,b,c 88 y-Eudesmol acetate 1783 - 0.36 a,b,c 89 Isolongifolol acetate 1819 - 0.20 a,b,c 90 5-cis, 9-trans-Farnesyl acetone 1886 - 0.19 a,b,c 91 n.i. 2292 - 0.12 a,b,c 92 n.i. 2306 - 0.25 a,b,c Total identified 98.53 99.59 Monoterpene Hydrocarbons 16.44 38.58 Oxygenated Monoterpenes 0.12 0.42 Sesquiterpene Hydrocarbons 72.63 43.53 Oxygenated Sesquiterpenes 9.34 17.06 aChemical compounds (%) are listed in order of elution from an DB-5 column. bRI - Retention index calculated by using n- alkanes (C7 a C26) in the column (HP-5MS); c Identification based on comparison with the mass spectra of the Wiley 275 Libraries; Relative area (%): percentage of the area occupied by the compounds in the chromatogram; n.i: not identified; (-): absent. The chemical composition of essential oils can be directly affected by biotic and abiotic factors; and according to Morais (2009) it can be altered in function of the location of the culture implantation, cultivation method, harvest time, vegetal cycle, climate, season of the year, whether or not the plant is dried or fresh, among others. Therefore, there was a difference in the chemical composition as well as in the amount of the major compounds of the fruit EO when compared to other authors who worked on the same species. In this experiment, which was carried out in the northeastern region of Paraná state, Brazil, we obtained β-pinene (30%) as a major compound. Cavalcante et al. (2015) obtained α- pinene (27.70%) in a culture implemented in the state of Rio de Janeiro, Brazil. Oliveira et al. (2014) obtained p-mentha-2,4 (8)-diene (35.34%) while Santos et al. (2014) had limonene (67.15%) in the state of Sergipe, Brazil. Colle et al. (2014) and Pratti et al. (2015) obtained δ-carene (30.37%) and (55.36%), respectively in the state of Espirito Santo, Brazil, and Bendaoud et al. (2010) obtained α- phellandrene (34.38%) in a culture implemented in Tunisia. Two distinct situations were verified in the essential oil from leaves. The major compound bicyclogermacrene (37.57%) found in this experiment was also found in a culture implemented in Tunisia by Jeribi et al. (2014): (bicyclogermacrene 23.56%) and by Ennigrou et al. (2011): (bicyclogermacrene 27.11%), respectively. The second situation was the alteration of the major compound, and it was observed regarding the cultures implemented by Santos et al. (2014) in the state of Sergipe, Brazil, who obtained δ-carene (81.79%); Cavalcante et al. (2015) had β- caryophyllene (35.2%) in the state of Rio de Janeiro, Brazil; Santana et al. (2012) obtained germacrene D (23.80%) in the state of São Paulo, Brazil, while Santos et al. (2009) found limonene (14.21%) in the state of Rio Grande do Sul, Brazil. After obtaining essential oil from fruits and leaves, the oils were submitted to fractioning in chromatographic column (CC), and only compounds that presented relative area (%) greater than 4.0% were selected, whose results are Table 2. Table 2. Chemical composition and relative area (%) of the fractions obtained from the chromatographic column fractioning of the essential oils (EO) from Schinus terebinthifolius leaves and fruits. EO LEAVES FR1 FR2 FR3 FR4 Bicyclogermacrene (30.94%) Germacrene D Spathulenol (28.93%) epi-α-cadinol Viridiflorol (10.25%) Phthalic acid (26.13%) Xilenol 1581 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 (10.94%) (11.43%) (17.26%) EO FRUIT FR1 FR2 FR3 FR4 Germacrene D (22.82%) Bicyclogermacrene (14.28%) α-muurolol (16.29%) α-cadinol (4.48%) Spathulenol (4.24%) β-pinene (32.06%) α-cadinol (20.88%) EO: essential oil; FR1 – Hexane fraction; FR2 – Dichloromethane fraction; FR3 – Ethyl acetate fraction; FR4 – methanol fraction. Compounds that presented relative area (%) greater than 4.0% were selected. The hexane fraction (FR1) indicated the presence of sesquiterpene hydrocarbons in fruits and leaves and their major compounds were bicyclogermacrene and germacrene D, which present antimicrobial, larvicidal and acaricidal activity already reported in the literature (COSTA et al. 2005; SANTANA et al. 2012). The results indicated that fractions (leaves and fruits) presented greater potential against larvae with (LC99.9 0.64 mg mL-1) and (LC99.9 0.60 mg mL-1), respectively (Table 3). Kiran and Devi (2007) found LC95 of 0.897 mg mL-1 against Ae. aegypti larvae for isolated germacrene D. In another experiment, Santana et al. (2015), evaluated Piper arboretum EO and obtained as major compounds germacrene D (31.83%) and bicyclogermacrene (21.40%) against Ae. aegypti larvae, and found LC90 of 0.204 mg mL-1, justifying the great potential of hexane FR (FR1) when compared to other isolated FRs. Dichloromethane fraction (FR2) and ethyl acetate fraction (FR3) obtained from fruits and leaves consist of oxygenated sesquiterpenes, and their compounds are spathulenol, α-cadinol, epi-α- cadinol, α-muurolol and viridiflorol, and according to Vidal et al. (2016), these compounds have great antimicrobial potential. Phthalic acid found in (FR4) from leaves is also reported to have antimicrobial potential (AJOKE et al., 2014), and the compound β-pinene (32.06%) (FR4) from fruits acts is reported to attract pollinizers (TAIZ; ZEIGER, 2013). The chemical compounds identified in FRs (2, 3 and 4) as well as their biological potential reported in the literature justify their low action against larvae and pupae in this experiment. EOs and fractions were tested against Ae. aegypti larvae and pupae and the results of the Lethal Doses (LCs) are shown in Table 3. Table 3. Mean mortality ± standard deviation and confidence interval of lethal concentrations (LC50 and LC99.9) of essential oil and fractions of Schinus terebinthifolius leaves and fruits against Ae. aegypti larvae and pupae by analysis of Probitos Ae. aegypti larvae Ae. aegypti pupae Mortality LC50 (mg mL-1) (CI) LC99.9 (mg mL-1) (CI) LC50 (mg mL-1) (CI) LC99.9 (mg mL- 1) (CI) Positive control 0.398 ± 0.050ab (0.348 – 0.448) 1.14 ± 0.060a (1.080 – 1.200) 234.37 ± 22.090D (212,28 – 265,46) 443.64 ± 14.870D (428.77 – 458.51) EO leaves 0.370 ± 0.008ab (0.334 – 0.301) 2.304 ± 0.045b (2.224 – 2.384) 0.733 ± 0.024A (0.689 – 0.778) 2.518 ± 0.095A (2.224 – 2.384) FR1- leaves 0.075 ± 0.006a (0.060 – 0.083) 0.642 ± 0.010a (0.617 – 0.656) 0.552 ± 0.019A (0.505 – 0.578) 2.617 ± 0.083A (2.413 – 2.735) FR2- leaves 1.603 ± 0.053ab (1.427 – 1.676) 10.110 ± 0.233d (9.819 – 10.278) 6.591 ± 0.014A (6.250 – 6.788) 22.580 ± 0.250B (21.968 – 22.933) FR3- leaves 2.011 ± 0.006b (1.859 – 2.098) 11.030 ± 0.023e (10.602 – 11.276) 2.015 ± 0.113A (1.959 – 2.048) 20.068 ± 0.113B (19.791 – 20.227) 1582 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 FR4- leaves 8.659 ± 0.695c (6.959 – 9.641) 22.483 ± 0.606f (20.998 – 23.340) 27.927 ± 1.273B (26.102 – 28.980) 93.502 ± 0.746C (90.387 – 95.300) EO fruits 0.374 ± 0.023ab (0.367 – 0.384) 0.895 ± 0.073a (0.691 – 0.730) 0.389 ± 0.0438A (0.362 – 0.405) 1.099 ± 0.043A (1.018 – 1.147) FR1- fruits 0.030 ± 0.001a (0.028 – 0.031) 0.602 ± 0.004a (0.591 – 0.608) 0.058 ± 0.001A (0.057 – 0.059) 2.338 ± 0.029A (2.267 – 2.379) FR2- fruits 2.056 ± 0.031b (1.979 – 2.100) 6.161 ± 0.054c (6.028 – 6.237) 2.061 ± 0.048A (1.943 – 2.128) 20.189 ± 0.233B (19.784 – 20.422) FR3- fruits 1.720 ± 0.053ab (1.589 – 1.795) 10.640 ± 0.104e (10.602 – 11.276) 1.960 ± 0.031A (1.890 – 2.009) 20.025 ± 0.249B (19.414 – 20.377) FR4- fruits 37.232 ± 1.372d (33.609 – 39.324) 92.727 ± 0.023g (91.899 – 93.204) 46.190 ± 0.655C (44.587 – 47.116) 91.440 ± 1.395C (88.026 – 93.411) FR1 – Hexane fraction; FR2 – Dichloromethane fraction; FR3 – Ethyl acetate fraction; FR4 – methanol fraction; EO: essential oil; LC50: lethal concentration that kills 50% of the exposed Ae. aegypti larvae and pupae; LC99.9: lethal concentration that kills 99.9% of the exposed Ae. aegypti larvae and pupae; CI: confidence interval; Equal letters in the same column indicate that there was no significant difference between treatments by Duncan’s test (p≤0.05) When comparing the action of EOs and FR1 from fruits and leaves against larvae and pupae, the obtained results indicated greater potential on larvae because biocompounds act on the cell wall of the larvae as well as on the ingestion and absorption by the gastrointestinal tract (PROCÓPIO et al., 2015). However, in the pupal stage, the pupae do not feed themselves, and there is a greater difficulty for EOs as well as FRs to penetrate (CHAUBEY, 2012; PROCÓPIO et al., 2015; PIETA et al., 2017). The acetylcholinesterase enzyme activity was done by the bioautographic methods to verify the possible action mechanism of EOs and FRs from fruits and leaves against Ae. aegypti larvae and pupae (Table 4). Table 4. Inhibitory activity of the Acetylcholinesterase enzymeat different concentrations (mg/mL) of the essential oil and fractions from Schinus terebinthifolius leaves and fruits by bioautographic method. Concentration mg mL-1 EO FR 1 FR 2 FR 3 FR 4 leaves fruits leaves fruits leaves fruits leaves fruits leaves fruits PC 10.00 + + + + + + + + + + + 5.00 + + + + + + + + + + + 2.50 + + + + + + + + - - + 1.25 + + + + + + - - - - + 0.625 + + + + + - - - - - + 0.312 + - + + - - - - - - + 0.156 - - - + - - - - - - - 0.078 - - - - - - - - - - - EO: essential oil; FR1 – Hexane fraction; FR2 – Dichloromethane fraction; FR3 – Ethyl acetate fraction; FR4 – methanol fraction. PC: positive control [commercial solution based on organophosphate]; (+): inhibition of acetylcholinesterase enzyme; (-) Absence of inhibition of acetylcholinesterase enzyme. The results shown in Table 4 made evident that the probable action mechanism by which EO and FRs acted on the larvae was by the inhibition of Acetylcholinesterase enzyme the same way organophosphates do (ČOLOVIĆ; KRSTIĆ, 2013; KING; AARON, 2015). The (FR1) from fruit EO was the one that presented greater anticholinesterase potential (0. 156 mg mL-1), and was more active than the positive control, followed by (FR1) from leaf EO (0. 312 mg mL-1). Comparing the concentrations found in LC (Table 3) and the lower concentration that inhibited 1583 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 acetylcholinesterase enzyme (Table 4), it was evident that the in vitro test (bioautographic) was more effective than the in vivo test (larval immersion) for EO and FRs. This difference can be explained by the absence of physiological conditions that cause interference in the in vivo biochemical reactions because the bioautographic method is done in a controlled ambient with pre- stablished conditions, without the interference of permeability of the cell wall, molecular absorption characteristics as well as the ones regarding the solubility in hydrophilic and lipophilic media inherent to a living being (BENSON, 2005; BRAIN et al., 2007). Thus, this study opens perspectives to new research studies aiming the development of products with bicyclogermacrene and germacrene D as an alternative to control this culicidae. CONCLUSION S. terebinthifolius has a great concentration of EO in its leaves and fruits, and its major compounds are bicyclogermacrene in the leaves and germacrene D in the fruits. These compounds presented high potential against Ae. aegypti larvae, indicating that essential oil and isolated molecules can be an alternative source to control this culicidae. ACKNOWLEDGEMENTS The authors thank Universidade Paranaense, Centro Universitário de Maringá, Instituto Federal do Paraná - campus Umuarama, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES), finance code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the fellowship and financial support. RESUMO: Diversas tecnologias têm sido desenvolvidas para o controle do Aedes aegypti, destacando pesquisas com moléculas isoladas de plantas. A Schinus terebinthifolius (Raddi) (Anacardiaceae), conhecida popularmente como pimenta rosa é uma planta muito utilizada no reflorestamento de áreas degradadas e seus frutos são utilizados como condimentos. O objetivo deste trabalho foi investigar o potencial dos óleos essenciais (OEs) e frações (FRs) obtidos dos frutos e folhas frescos de S. terebinthifolius. Os OEs foram obtidos por hidrodestilação (2 horas), fracionados em coluna cromatográfica utilizando como fase estacionária sílica gel 60 (0,063-0,2mm), fases móveis: n-hexano, diclorometano, acetato de etila e metanol e avaliados quimicamente por cromatografia gasosa acoplada à espectrometria de massas (CG/EM). Os OEs e FRs foram testados frente a larvas do terceiro estádio e pupas do Ae. aegypti pelo Teste de Imersão em concentrações que variaram de 500,00 à 0,003 mg/mL (v/v). As FRs hexano obtidas dos frutos e folhas, foram as que apresentaram maior atividade sobre as larvas (CL99,9= 0,60 mg mL-1 e CL99,9 0,64 mg mL-1, respectivamente) e pupas (CL99,9= 2,51mg mL-1 e 2,61 mg mL-1, respectivamente). Estes resultados foram confirmados pela atividade anticolinesterase onde as FRs hexano (fruto e folha), foram as que apresentaram maior potencial inibitório sobre a enzima acetilcolinesterase (0,156 mg mL-1 e 0,312 mg mL-1, respectivamente), sugerindo desta forma o provável mecanismo de ação. O potencial larvicida encontrado pode ser explicado pela presença dos compostos majoritários biciclogermacreno e germacreno D nas FRs hexano, indicando desta forma, que estes possam vir a substituir, ou até mesmo agir em sinergismos com os larvicidas químicos convencionais. Desta forma o presente estudo abre campo para novas pesquisas, visando o desenvolvimento de produtos com os compostos bicyclogermacrene e germacrene D, como alternativa no controle deste culicídeo. PALAVRAS-CHAVE: Teste de Imersão Larval. Acetilcolinesterase. Bicyclogermacrene. Pimenta rosa. Germacrene D. REFERENCES ADAMS, R. P. Identification of essential oil components by Gas Chromatography / Mass Spectrometry. 4th ed., Allued: Publishing Corporation, 2012. 804p. AJOKE, F. L.; KAITA, H.; ILYAS, M. Antibacterial Activity of 1,2-Benzenediccarboxylic Acid, Dioctyl Ester Isolated from the Ethyl Acetate Soluble Sub-portion of the unripe Fruits of Nauclealatifolia. International Journal of Pure & Applied Bioscience, v. 2, p. 223-230, 2014. 1584 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 BARBOSA, L. C. A.; DEMUNER, A. J.; CLEMENTE, A. D.; PAULA, V. F.; ISMAIL, F. M. D. Seasonal variation in the compositions of volatile oils from Schinus terebinthifolius RADDI. Química Nova, v. 30, n. 8, p. 1959-1965, 2007. http://dx.doi.org/10.1590/S0100-40422007000800030 BENDAOUD, H.; ROMDHANE, M.; SOUCHARD, J. P.; CAZAUX, S.; BOUAJILA, J. Chemical composition and anticancer and antioxidant activities of Schinus molle L. and Schinus terebinthifolius Raddi berries essential oils. Journal of Food Science, v. 75, n. 6, p. 466-472, 2010. https://doi.org/10.1111/j.1750- 3841.2010.01711.x BENSON, H. A. E. Transdermal Drug Delivery: Penetration Enhancement Techniques. Current Drug Delivery, v. 2, p. 23-33, 2005. https://doi.org/10.2174/1567201052772915 BRAGA, I. A.; VALLE, D. Aedes aegypti: Surveillance, Resistance Monitoring, and Control Alternatives in Brazil. Epidemiologia e Serviços de Saúde, v. 16, n. 4, p. 295-302, 2007. https://doi.org/10.5123/S1679- 49742007000400006 BRAIN, K. R.; GREEN, D. M.; APIA, M. In-vitro human skin penetration of the fragrance material geranyl nitrile. Toxicology In Vitro, v. 21, p. 133-138, 2007. https://doi.org/10.1016/j.tiv.2006.08.005 BRASIL. Farmacopéia Brasileira. Agência Nacional de Vigilância Sanitária. 5a ed., Brasília: Anvisa, 2010. 546 p. BRASIL. Ministério da Saúde. Secretaria de Vigilância em Saúde (SVS/MS). Boletim Epidemiológico. Monitoramento dos casos de dengue, febre de chikungunya e febre pelo vírus Zika até a Semana Epidemiológica 49, 2016. Ministério da Saúde: Brasília, v. 49, n. 38, p. 1-10. Available in: http://combateaedes.saude.gov.br/images/pdf/2016-Dengue_Zika_Chikungunya-SE49.pdf. Access in:16 dez. 2017. BRASIL. Secretaria de Vigilância em Saúde. Boletim Epidemiológico. Monitoramento dos casos de dengue, febre de chikungunya e febre pelo vírus Zika até a semana epidemiológica 9 de 2018. Ministério da Saúde: Brasília, v. 49, n. 13, p. 1-13, 2018. Available in: http://portalarquivos2.saude.gov.br/images/pdf/2018/abril/06/2018-012.pdf. Access in:15 Abr. 2018. CAMARGO, M. F.; SANTOS, A. H.; OLIVEIRA, A. W. S.; ABRÃO, N.; ALVES, R. B. N.; ISAC, E. Avaliação da ação residual do larvicida temephó sobre o Aedes aegypti (Diptera, Culicidae) em diferentes tipos de recipientes. Revista de Patologia Tropical, v. 27, p. 270-272, 1998. https://doi.org/10.5216/rpt.v27i1.17197 CAVALCA, P. A. M.; LOLIS, M. I. G. A,; REIS, B.; BONATO, C. M. Homeopathic and Larvicide Effect of Eucalyptus cinerea Essential Oil against Aedes aegypti. Brazilian Archives of Biology and Technology, v. 53, n. 4, p. 835-843, 2010. http://dx.doi.org/10.1590/S1516-89132010000400012 CAVALCANTE, A. S.; ALVES, M. S.; SILVA, L. C. P.; PATROCINIO, D. S.; SANCHES, M. N.; CHAVES, D. A. S.; SOUZA, M. A. A. Volatiles Composition and extraction kinetics from Schinus terebinthifolius a Schinus molle leaves and fruit. Revista Brasileira de Farmacognosia, v. 25, n. 4, p. 356-362, 2015. http://dx.doi.org/10.1016/j.bjp.2015.07.003 CESÁRIO, L. F.; GAGLIANONE, M. C. Pollinators of Schinus terebinthifolius Raddi (Anacardiaceae) in vegetational formations of restinga in northern Rio de Janeiro state. Bioscience Journal, v. 29, n. 2, p. 458- 467, 2013. CHAUBEY, M. K. Acute, lethal and synergistic effects of some terpenes against Tribolium castaneum Herbst (Coleptera: Tenebrionidae). Ecologia Balkanica, v. 4, p. 53-62, 2012. COLLE, E. R.; SANTOS, R. B.; LACERDA JUNIOR, V.; MARTINS, J. D. L.; GRECO, S. J.; CUNHA NETO, A. Chemical composition of essential oil from ripe fruit of Schinus terebinthifolius Raddi and 1585 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 evaluation of its activity against wild strains of hospital origin. Brazilian Journal of Microbiology, v. 45, n. 3, p. 821-828, 2014. http://dx.doi.org/10.1590/S1517-83822014000300009 COLLINS, C. H.; BRAGA, G. L.; BONATO, P. S. Introdução a Metodologia Cromatográfica. 7th ed., Campinas: Unicamp, 1997, 279 p. ČOLOVIĆ, M.; KRSTIĆ, D. Acetylcholinesterase inhibitors: Pharmacology and toxicology. Current Neuropharmacololy, v. 11, n. 3, p. 315–335, 2013. http://dx.doi.org/10.2174/1570159X11311030006 COSMOSKI, A. C. O. F.; ROEL, A. R.; PORTO, K. R. A.; MATIAS, R.; HONER, M. R.; MOTTI, P. R. Phytochemistry and larvicidal activity OF Spermacoce latifolia Aubl. (Rubiaceae) in the control of Aedes aegypti L. (Culicidae). Bioscience Journal, v. 31, n. 5, p. 1512-1518, 2015. DOI: http://dx.doi.org/10.14393/BJ-v31n5a2015-26333 COSTA, J. G. M.; RODRIGUES, F. F. G.; ANGÉLICO, E. C.; SILVA, M. R.; MOTA, M. L.; SANTOS, N. K. A.; CARDOSO, A. L. H.; LEMOS, T. L. G. Chemical-biological study of the essential oils of Hyptis martiusii, Lippia sidóides and Syzigium aromaticum against larvae of Aedes aegypti and Culex. Revista Brasileira de Farmacognosia, v. 15, n. 4, p. 304-309, 2005. http://dx.doi.org/10.1590/S0102-695X2005000400008 DINIZ, M. M. C. S.; HENRIQUES, A. D. S.; LEANDRO, R. S.; AGUIAR, D. L.; BESERRA, E. B. Resistência de Aedes aegypti ao temefós e desvantagens adaptativas. Revista de Saúde Pública, v. 48, n. 5, p. 775-782, 2014. http://dx.doi.org/10.1590/S0034-8910.2014048004649 EL-MASSRY, K. F.; EL-GHORAB, A.; SHAABAN, H. A.; SHIBAMOTO, T. Chemical Compositions and Antioxidant/Antimicrobial Activities of Various Samples Prepared from Schinus terebinthifolius Leaves Cultivated in Egypt. Journal of Agricultural and Food Chemistry, v. 57, n. 12, p. 5265-5270, 2009. http://dx.doi.org/10.1021/jf900638c ENNIGROU, E. A.; HOSNI, K.; CASABIANCI, H.; VULLIET, E.; SAMIRA, S. Leaf volatile oil constituents of Schinus terebinthifolius and Schinus molle from Tunisia. FOODBALT, v. 1, p. 90-92, 2011. FERREIRA-FILHO, P. J.; PINÃ-RODRIGUES, F. C. M.; SILVA, J. M. S.; GUERREIRO, J. C.; GHIOTTO, T. C.; PIOTROWSKI, I.; DIAS, L. P.; WILCKEN, C. F.; ZANUNCIO, J. The exotic wasp Megastigmus transvaalensis (Hymenoptera: Torymidae): first record and damage on the Brazilian peppertree, Schinus terebinthifolius drupes, in São Paulo, Brazil. Anais da Academia Brasileira de Ciências, v. 87, n. 4, p. 2091- 2095, 2015. http://dx.doi.org/10.1590/0001-3765201520140478 HUSSEINS, A. H.; AHL, S. A.; ABBAS, Z. K.; SABRA, A. S.; TKACHENKO, K. G. Essential Oil Composition of Hyssopus officinalis L. Cultivated in Egypt. International Journal of Plant Sciences, v. 1, n. 2, p. 49-53, 2015. JERIBI, C.; KAROUI, I. J.; HASSINE, D. B.; ABDERRABBA, M. Comparative Study of Bioactive Compounds and Antioxidant Activity of Schinus terebinthifolius RADDI Fruits and Leaves Essential Oils. International Journal of Science and Research, v. 12, n. 3, p. 453-458, 2014. KING, A. M.; AARON, C. K. Organophosphate and Carbonate Poisoning. Emergency Medicine Clinics of North America, v. 33, p. 133–151, 2015. http://dx.doi.org/10.1016/j.emc.2014.09.010 KIRAN, S. R.; DEVI, P.S. Evaluation of mosquitocidal activity of essential oil and sesquiterpenes from leaves of Chloroxylon swietenia DC. Journal of Parasitology Research, v. 101, n. 2, p. 413-418, 2007. http://dx.doi.org/10.1007/s00436-007-0485-z MANRIQUE-SAIDE, P.; MANRIQUE-SAIDE, P.; CHE-MENDOZA, A.; BARRERA-PEREZ, M.; GUILLERMO-MAY, G.; HERRERA-BOJORQUEZ, J.; DZUL-MANZANILLA, F.; GUTIERREZ-CASTRO, C.; LENHART, A.; VAZQUEZ-PROKOPEC, G.; SOMMERFELD, J.; MCCALL, P. J.; KROEGER, A.; 1586 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 ARREDONDO-JIMENEZ, J. I. Use of insecticide-treated house screens to reduce infestations of dengue virus vectors, Mexico. Emerging Infectious Disseases, v. 21, n. 2, p. 308-311, 2015. http://dx.doi.org/10.3201/eid2102.140533 MARSTON, A.; KISSLING, J.; HOSTETTMANN, K. A. A rapid TLC bioautography method for the detection of acetylcholinesterase and butyrylcholinesterase inhibitors in plants. Phytochemistry, v. 13, p. 51-54, 2002. http://dx.doi.org/10.1002/pca.623 MORAIS, L. A. S. Influência dos fatores abióticos na composição química dos óleos essenciais. Horticultura Brasileira, v. 27, p. 4050-4063, 2009. MWANGI, J. W.; THOITHI, G. N.; KIBWAGE, I. O. Essential Oil Bearing Plants from Kenya: Chemistry, Biological Activity and Applications. African Natural Plant Products, v. 1021, p. 495- 525. 2009. http://dx.doi.org/10.1021/bk-2009-1021.ch027 NASCIMENTO AM, ALMEIDA DL, LIMA EM, RONCHI SN, CRUZ ZMA AND SILVA AG. Mopho Mutagenesis risk and larvicide activity of the essential oil of Christmas berry, Schinus terebinthifolius Raddi against Aedes aegypti (L.). Natureza on line, v. 6, n. 2, p. 86-90, 2008. OLIVEIRA, L. F. M.; OLIVEIRA JUNIOR, L. F. G.; SANTOS, M. C.; NARAIN, N.; LEITE NETO, M. T. S. Distillation time and volatile profile of the essential oil of Brazilian pepper (Schinus terebinthifolius) in Sergipe, Brazil. Revista Brasileira de Plantas Medicinais v. 16, n. 2, p. 243-249, 2014. http://dx.doi.org/10.1590/S1516-05722014000200012 PIETA, L.; ESCUDERO, F. L. G.; JACOBUS, A. P.; CHEIRAN, K. P.; GROSS, J.; MOYA, M. L. E.; SOARES, G. L. G.; MARGIS, R.; FRAZZON, A. P. G.; FRAZZON, J. Comparative transcriptomic analysis of Listeria monocytogenes reveals upregulation of stress genes and downregulation of virulence genes in response to essential oil extracted from Baccharis psidioides. Annals of Microbiology, v. 67, p. 479-490, 2017. http://dx.doi.org/10.1007/s13213-017-1277-z PORTO, K. R. A.; MOTTI, P. R.; YANO, M.; CARDOSO, C. A. L.; MATIAS, R. Screening of plant extracts and fractions on Aedes aegypti larvae found in the state of Mato Grosso do Sul (Linnaeus, 1762) (Culicidae). Anais da Academia Brasileira de Ciências, v. 89, n. 2, p. 895-906, 2017. http://dx.doi.org/10.1590/0001- 3765201720150017. PRATTI, D. L. A.; RAMOS, A. C.; SCHERER, R.; CRUZ, Z. M. A.; SILVA, A. G. Mechanistic basis for morphological damage induced by essential oil from Brazilian pepper tree, Schinus terebinthifolius, on larvae of Stegomyia aegypti, the dengue vector. Parasites & Vectors, v. 8, p. 1-10, 2015. http://dx.doi.org/10.1186/s13071-015-0746-0 PROCÓPIO, T. F.; FERNANDES, K. M.; PONTUAL, E. V.; XIMENES, R. M.; DE OLIVEIRA, A. R.; SOUZA, C. S.; MELO, A. M.; NAVARRO, D. M.; PAIVA, P. M.; MARTINS, G. F.; NAPOLEÃO, T. H.; Schinus terebinthifolius Leaf Extract Causes Midgut Damage, Interfering with Survival and Development of Aedes aegypti Larvae. PLOS ONE, v. 10, n. 5, p. 1-19, 2015. http://dx.doi.org/10.1371/journal.pone.0126612 RODRIGUES, T. A. D.; ARRUDA, E. J.; FERNANDES, A. F.; CARVALHO, C. T.; LIMA, A. R.; CABRINI, I. Copper II – polar amino acid complexes: toxicity to bacteria and larvae of Aedes aegypti. Anais da Academia Brasileira de Ciências, v. 89, n. 3, p. 2273-2280, 2017. http://dx.doi.org/10.1590/0001- 3765201720160775 SANTANA, H. T.; TRINDADE, F. T. T.; STABELI, R. G.; SILVA, A. A. E.; MILITÃO, J. S. T. L.; FACUNDO, V. A. Essential oils of leaves of Piper species display larvicidal activity against the dengue vector, Aedes aegypti (Diptera: Culicidae). Revista Brasileira de Plantas Medicinais, v. 17, p. 105-111, 2015. http://dx.doi.org/10.1590/1983-084X/13_052 1587 Schinus terebinthifolius essential oil… BORTOLUCCI, W. C. et al. Biosci. J., Uberlândia, v. 35, n. 5, p. 1575-1587, Sep./Oct. 2019 http://dx.doi.org/10.14393/BJ-v35n5a2019-41999 SANTANA, J. S.; SARTORELLI, P.; GUADAGNIN, R. C.; MATSUO, A. L.; FIGUEIREDO, C. R.; SOARES, M. G.; SILVA, A. M.; ALLAGO, J. H. Essential oils from Schinus terebinthifolius leaves – chemical composition and in vitro cytotoxicity evaluation. Pharmaceutical Biology, v. 50, n. 10, p. 1248- 1253, 2012. http://dx.doi.org/10.3109/13880209.2012.666880 SANTOS, A. C. A.; ROSSATO, M.; AGOSTINI, F.; SERAFINI, L. A.; SANTOS, P. L.; MOLON, R.; DELLACASSA, E.; MOYNA, P. Chemical Composition of the Essential Oils from Leaves and Fruits of Schinus molle L. and Schinus terebinthifolius Raddi from Southern Brazil. Journal of Essential Oil Bearing Plants, v. 12, p. 16-25, 2009. https://doi.org/10.1080/0972060X.2009.10643686 SANTOS, I. T. B. F.; SANTOS, T. S.; SILVA, F. L. S.; GAGLIARDI, P. R.; OLIVEIRA JÚNIOR, L. F. G.; BLANK, A. F. Óleo essencial de Schinus terebinthifolius Raddi como controle alternativo de Colletotrichum gloeosporioides e Lasiodiplodia theobromae, fungos fitopatogênicos de pós-colheita. Revista GEINTEC, v. 4, n. 4, p. 1409-1417, 2014. http://dx.doi.org/10.7198/S2318-3403201400020019 SANTOS, M. R. A.; LIMA, R. A.; SILVA, A. G.; LIMA, D. K. S.; SALLET, L. A. P.; TEIXEIRA, C. A. D.; FACUNDO, V. A. Chemical composition and insecticidal activity of the essential oil of Schinus terebinthifolius Raddi (Anacardiaceae) on coffee berry borer (Hypothenemus hampei) Ferrari. Revista Brasileira de Plantas Medicinais, v. 15, n. 4, p. 757-762, 2013. https://doi.org/10.1590/S1516- 05722013000500017 SILVA, A. C. R.; LOPES, P. M.; BARROS, A. M. M.; COSTA, D. C.; ALVIANO, C. S.; ALVIANO, D. S. Biological Activities of α-Pinene and β-Pinene Enantiomers. Molecules, v. 17, p. 6305-6316, 2012. http://dx.doi.org/10.3390/molecules17066305 TAIZ, L.; ZEIGER, E. Fisiologia vegetal. 5th ed., Porto Alegre: Artmed, 2013, 917 p. TEIXEIRA, M. G.; COSTA, M. C.; OLIVEIRA, W. K.; NUNES, M. L.; RODRIGUES, L. C. The Epidemic of Zika Virus – Related Microcephaly in Brazil: Detection, Control, Etiology, and Future Scenarios. American Journal of Public Health, v. 106, n. 4, p. 601-605, 2016. http://dx.doi.org/10.2105/AJPH.2016.303113 VIDAL, C. S.; TINTINO, C. D. M. O.; TINTINO, S. R.; GALVÃO, H. B.; COSTA, J. G. M.; COUTINHO, H. D. M.; MENEZES, I. R. A. Chemical composition, antibacterial and modulatory action of the essential oil of Croton rhamnifolioides leaves Pox and Hoffman. Bioscience Journal, v. 32, n. 6, p. 1632-1643, 2016. http://dx.doi.org/10.14393/BJ-v32n1a2016-33918 YANG, Z.; ZHANG, X.; DUAN, D.; SONG, Z.; YANG, M. L. S. Modified TLC bioautographic method for screening acetylcholinesterase inhibitors from plant extracts. Journal of Separation Science, v. 32, p. 3257- 3259, 2009. http://dx.doi.org/10.1002/jssc.200900266 ZARA, A. L. S. A.; SANTOS, S. M.; FERNANDES-OLIVEIRA, E. S.; CARVALHO, R. G.; COELHO, G. E. Aedes aegypti control strategies: a review. Epidemiologia e Serviços de Saúde, v. 25, n. 2, p. 391-404, 2016. http://dx.doi.org/10.5123/s1679-49742016000200017