Abstract

INTRODUCTION

Plants are important sources of biologically active substances and have become industrial models for the development of new drugs, whether of natural origin or chemical synthesis planned from natural products (Barreiro, Bolzani, 2009Barreiro EJ, Bolzani VDS. Biodiversidade: fonte potencial para a descoberta de farmacos. Quím Nova. 2009;32(3):679-688.). Interest in aromatic species has grown steadily in recent years, particularly with regard to essential oils, which are of great economic interest and are widely described in the literature (Stefanello et al., 2011Stefanello MEA, Riva D, Simionatto EL, Carvalho JE, Ruiz ALTG, Salvador MJ. Chemical composition and cytotoxic activity of essential oil from Myrcia laruotteana fruits. J Essent Oil Res. 2011;23(5):7-10.). Essential oils may be alternatives for synthetic compounds used in pharmaceutical formulations, chemistry applications, foods, cosmetics, agricultural implements and cleaning materials.

Myrtaceae is one of the largest families of angiosperms and comprises approximately 140 genus and 5500 species, located on continents in the southern hemisphere (Retamales, Scharaschkin, 2015Retamales HA, Scharaschkin T. Comparative leaf anatomy and micromorphology of the Chilean Myrtaceae: Taxonomic and ecological implications. Flora. 2015;217:138-154.). The species of the family are abundant in Brazil and present an economic importance, as a source of fruits, spices, timber and paper, and utilization as a therapeutic agent, folk remedy, and as an ornamental plant (Moresco et al., 2014Moresco HH, Pereira M, Bretanha LC, Micke GA, Pizzolatti MG, Brighente IMC. Myricitrin as the main constituent of two species of Myrcia. J Appl Pharm Sci. 2014;4(2):1-7.).

The Myrcia genus comprises a large portion of economically useful species of the family. Myrcia species are important sources of essential oils and are widely used in folk medicine, primarily for treatment of diabetes, an illness of worldwide importance. Essential oils are produced in leaves, stems, flowers and fruits, and are comprised mainly of sesquiterpenes (Cascaes et al., 2015Cascaes MM, Guilhon GM, Andrade EH, Zoghbi MD, Santos L da S. Constituents and Pharmacological Activities of Myrcia (Myrtaceae): A Review of an Aromatic and Medicinal Group of Plants. Int J Mol Sci. 2015;16(10):23881-904.).

Several studies have highlighted the main properties of essential oils derived from the Myrcia species. Myrcia ovata leaf essential oil (EO) extracts exert anti-inflammatory (Santos et al., 2014Santos GCM, Gomes GA, Gonçalves GM, Sousa LM, Santiago GMP, Carvalho MG, et al. Essential oil from Myrcia ovata: Chemical composition, antinociceptive and anti-inflammatory properties in mice. Planta Med. 2014;80(17):1588-1596.) and larvicidal (Lima et al., 2011Lima MAA, Oliveira FFM, Gomes GA, Lavor PL, Santiago GMP, Nagao-Dias AT, et al. Evaluation of larvicidal activity of the essential oils of plants from Brazil against Aedes aegypti (Diptera: Culicidae). Afr J Biotechnol. 2011;10(55):11716-20.) activities. EO extracts of Myrcia fallax flowers (Alarcón et al., 2009Alarcón LD, Peña AE, Gonzales CN, Quintero A, Meza M, Usubillaga A, et al. Composition and antibacterial activity of the essential oil of Myrcia fallax (Rich.) DC. from Venezuela. Rev Soc Quím Perú. 2009;75(2):221-227.), Myrcia myrtillifolia leaves (Cerqueira et al., 2007Cerqueira MD, Souza-Neta LC, Passos MGVM, Lima EO, Roque NF, Martins D, et al. Seasonal Variation and Antimicrobial Activity of Myrcia myrtifolia essential oils. J Braz Chem Soc. 2007;18(5):998-1003.), and Myrcia splendens stems (Jiménez et al., 2012Jiménez D, Araque M, Rojas L, Cordero A, Briceño B. Componentes volátiles y actividad antibacteriana del vástago de Myrcia splendens (Sw.) DC. Rev Fac Farm. 2012;54:7-11.) exhibit antimicrobial activities. EO of Myrcia tomentosa, Myrcia bella, and Myrcia lingua leaves have antioxidant properties (Takao, Imatomi, Gualtieri, 2015Takao LK, Imatomi L, Gualtieri SCJ. Antioxidant activity and phenolic content of leaf infusions of Myrtaceae species from Cerrado (Brazilian Savanna). Braz J Biol. 2015;75(4):948-952.). EO of Myrcia laruotteana fruits have antiproliferative properties (Stefanello et al., 2011Stefanello MEA, Riva D, Simionatto EL, Carvalho JE, Ruiz ALTG, Salvador MJ. Chemical composition and cytotoxic activity of essential oil from Myrcia laruotteana fruits. J Essent Oil Res. 2011;23(5):7-10.).

The species Myrcia hatschbachii D. Legrand is native and endemic to Brazil, with confirmed occurrences in the Southern Region (Sobral et al., 2015Sobral M, Proença C, Souza M, Mazine F, Lucas E. Myrtaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro, 2015. [citad 2018 Feb 02]. Available from: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB1070.
http://floradobrasil.jbrj.gov.br/jabot/f... ). The aim of this study was to investigate the composition of essential oil, and evaluate, for the first time, its phytotoxic potential, toxicity aspects using in vitro models, antioxidant properties and antibacterial activity. This species can be cultivated for extraction of essential oil and may have unidentified beneficial properties.

MATERIAL AND METHODS

Plant material

Leaves of Myrcia hatschbachii D. Legrand were collected at the Federal University of Parana, Curitiba, Brazil (25° 26’ S, 49° 14’ W) in autumn (April). Species identification was performed by comparison with the specimen held at the herbarium of the Municipal Botanical Museum of Curitiba, under registry number 72379, by the curator José Tadeu Weidlich Motta.

Essential oil extraction

Plant material was dried at room temperature for 30 days, and 600 g of leaves were extracted using hydrodistillation by water vapor drag for 6 hours using Clevenger apparatus. EO yield was 0.17%.

Analysis of chemical composition of essential oil

Identification of EO components was performed using a gas chromatograph coupled to a mass spectrometer (Shidmazu® CG-EM QP2010 Plus), equipped with a capillary column (RTX-5MS; 30 m × 0.25 mm × 0.25 µm). The instrument was operated in split mode with the injector set at 250 °C. The interphase and ion source were maintained at 250 °C. Scan data were collected across the range of 40-350 m/z. Helium was used as the carrier gas with a constant flow of 1.02 mL/minute. Initial column pressure was 59 KPa, and the programmed temperature rose from 60 °C to 250 °C at a rate of 3 °C/minute. Following data collection, chemical constituents were characterized using the Kovats retention index and comparison to existing literature (Adams, 2007Adams, RP. Identification of essential oil components by gas chromatography / mass spectrometry. 4 ed. Carol Stream IL: Allured Publishing; 2007. 804 p.).

Antioxidant activity

Antioxidant activity was evaluated by the DPPH• radical (2,2-diphenyl-1-picrylhydrazyl) scavenging method (adapted from Mensor et al., 2001Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TCD, Coube CS, Leitão SG. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res. 2001;15(2):127-130.) and the phosphomolybdenum complex formation assay (adapted from Prieto, Pineda, Aguilar, 1999Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999;269(2):337-341.). For both methods, EO solutions and standards were prepared (ascorbic acid, rutin and butylated hydroxytoluene [BHT]) at 200 µg/mL in methanol, with the addition of polysorbate 80. Data were evaluated by ANOVA and statistical differences between results were determined using Tukey’s test (p<0.05).

The following procedure was performed for the DPPH• radical scavenging method. The reaction was performed in a microplate by mixing 142 µL of standard or EO solution with 58 µL of methanolic solution of DPPH•. A blank was prepared to account for background color of the sample and methanol was used as a negative control. The reaction was incubated for 30 minutes in the dark at room temperature. Absorbance (Abs) was measured at 540 nm using a spectrophotometer. Antioxidant activity percentage (AA%) of the EO was calculated as follows: $\mathrm{AA}\%=100-\left[\left(\left(\mathit{Abs}\mathit{sample}-\mathit{Abs}\mathit{blank}\right)\times 100\right)-\mathit{Abs}\mathit{control}\right]$.

Phosphomolybdenum complex formation assay was performed as follows. An aqueous reactive solution of sulfuric acid (0.6 mol/L), sodium phosphate (28 mmol/L), and ammonium molybdate (4 mmol/L) was prepared. In test tubes, 0.3 mL of standard or EO solution was pipetted, followed by addition of 3 mL of the aqueous reactive solution. A blank was prepared using the solvents used to prepare samples. The tubes were closed and placed in a water bath at 95 °C for 90 minutes. Following incubation, samples were allowed to equilibrate to room temperature. Absorbance (Abs) was measured at 690 nm using a spectrophotometer. Results were expressed as antioxidant activity (AA%) of the sample compared to ascorbic acid, BHT, and rutin (standards with 100% antioxidant activity) as follows: $\mathrm{AA}\%=\left[\left(\mathit{Abs}\mathit{sample}-\mathit{Abs}\mathit{blank}\right)/\left(\mathit{Abs}\mathrm{standard}-\mathit{Abs}\mathit{blank}\right)\right]\times 100$.

Lethality against brine shrimp

Evaluation of toxicity against brine shrimp (Artemia salina) was performed as previously described by Meyer et al. (1982)Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DJ, McLaughlin JL. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 1982;45(5):31-34.. Artificial saline was prepared by dissolving 14.31 g of sea salt in 400 mL of distilled water, with pH between 8.0 and 10.0. Two hundred milligrams of Artemia salina eggs were added to the saline solution, and the mixture was maintained at 27 °C to 30 °C, and constantly agitated and illuminated (20 W) for 48 h to facilitate hatching. EO samples were solubilized in polysorbate 80 and prepared at concentrations of 1000, 750, 500, 250, 100, 50 and 10 µL/mL in methanol. The positive control, quinidine sulfate, was prepared at the same concentrations. Samples were placed in a laboratory oven at 40 °C to evaporate solvent. Following evaporation, the samples were resuspended in saline solution and 10 nauplii were incubated. Live and dead nauplii were counted after 24 hours. Data were analyzed using the Probit method, using SPSS version 23.0. The lethal concentration, LC₅₀, and the confidence interval at 95% were defined.

Hemolytic activity

In vitro hemolytic activity was performed as previously described by Banerjee et al. (2008)Banerjee A, Kunwar A, Mishra B, Priyadarsini KI. Concentration dependent antioxidant/pro-oxidant activity of curcumin: Studies from AAPH induced hemolysis of RBCs. Chem Biol Interact. 2008;174(2):134-139.. Phosphate buffered (PBS) solution, sample solutions and controls were prepared. Sheep red blood cells solution (2% v/v) was prepared in cold PBS. EO samples were taken from a stock solution, in which the oil was solubilized in 50 µL of DMSO (Dimethyl sulfoxide) and 10% methanol in PBS. Dilutions were made in PBS to obtain 250, 500, 750, and 1000 µg/mL solutions. Triton (1%) in PBS and clean water were used as positive controls. Phytochemical standards were prepared by diluting saponin to the same concentrations as the samples. The reactions for the sample, phytochemical standard, positive controls, and negative controls (solvent dilution) consisted of 200 µL of each respective solution with addition of 200 µL of 2% red blood cells. A blank was prepared to account for the color of the sample by combining 200 µL of each sample solution with 200 µL of PBS. Samples were incubated at 37 °C for 3 hours and centrifuged at 3000 rpm for 5 minutes. The supernatant was transferred to a microplate and absorbance was measured at 540 nm using a spectrophotometer. The percentage of the hemolytic activity was calculated using triton (1%) and clean water as 100%. Data were analyzed by ANOVA and the statistical difference between results was determined using Tukey’s test (p<0.05).

Antibacterial activity

Antibacterial activity of EO was evaluated by determining the Minimal Inhibitory Concentration (MIC), using the microdilution method, according to CLSI - Clinical and Laboratory Standards Institute (2008)Clinical and Laboratory Standard Institute. CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M07-A8; 2008.. EO were tested against strains of Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25913, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhimurium ATCC 14028. Bacterial suspensions were prepared at 1.0 × 10⁸ UFC/mL in saline solution, corresponding to 0.5 on the McFarland scale, and inoculated in microplates (5 µL) with a final concentration of 10⁴ UFC/mL. EO samples were prepared in 5% polysorbate and tested in the range of 1000-3.9 µg/mL. Negative controls were prepared by adding 100 µL of 5% polysorbate solution to 100 µL of Mueller-Hinton Broth (MHB) and 5 µL of the bacterial preparation. The positive control was composed of 100 µL of MHB and 5 µL of bacterial preparation. The sterility control was prepared by combining 100 µL of MHB and 100 µL of EO. Microplates were placed in a bacteriological incubator at 35 °C for 16 to 20 hours. After this period, 20 µL of 0.5% triphenyltetrazolium chloride solution (TTC) was added and the microplates were incubated for 3 hours. MIC results were characterized as follows: good antibacterial activity (up to 100 µg/mL), moderate activity (between 100-500 µg/mL), weak activity (between 500-1000 µg/mL), and inactive (greater than 1000 µg/mL) (Ayres et al., 2008Ayres MCC, Brandão MS, Vieira-Júnior GM, Menor JCAS, Silva HB, Soares MJS, etal. Atividade antibacteriana de plantas úteis e constituintes químicos da raiz de Copernicia prunifera. Rev Bras Farmacogn. 2008;18(1):90-97.; Santos et al., 2008Santos AO dos, Nakamura TU, Dias Filho BP, Veiga Junior VF, Pinto AC, Nakamura CV. Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus. Mem Inst Oswaldo Cruz. 2008;103(3):277-281.).

Phytotoxic activity

Phytotoxic activity was evaluated as described by Silva, Overbeck and Soares (2014)Silva ER, Overbeck GE, Soares GLG. Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: evaluation of seasonal effects. South Afr J Bot. 2014;93:14-18., in which the influence of EO on germination and growth of Lactuca sativa was evaluated. In gerbox boxes, two paper filters were added at the base and one paper filter was placed on the box cover. A stock solution was prepared by addition of polysorbate 80 to EO. From this stock solution, dilutions were made in distilled water to obtain concentrations of 1%, 0.1%, 0.01%, 0.001%. Controls consisted of 1% polysorbate 80 in water. In a laminar flow cabinet, 5 mL of distilled water was added to the paper filter at the base of the gerbox boxes. The boxes were divided into 4 quadrants, each containing 5 lettuce seeds (Lactuca sativa, variety Grand Rapids). After addition of seeds, 3 mL of each sample solution was added to the paper filter glued to the box cover. The gerbox boxes were closed and maintained in BOD incubators at 20 ± 5 °C, for 7 days. During germination, daily readings were collected for 7 days with withdrawal of germinated seeds. Growth verifications were made on the last day through measurement of hypocotyl and radicle growth. Mean differences were determined using the Scott-Knott (p<0.05) method.

RESULTS AND DISCUSSION

In the identification of the chemical constituents of EO (Table I) 40 compounds were detected, and among these, 34 terpenes were identified (corresponding to 91.06% of identified components present in the EO). Among the 34 identified compounds, 47.81% were sesquiterpenes, 30.05% oxygenated sesquiterpenes, 7.20% monoterpenes and 6.00% oxygenated monoterpenes. Sesquiterpenes are prominent in the majority of species from the Myrcia genus (Cerqueira et al., 2009Cerqueira MD, Marques EJ, Martins D, Roque NF, Cruz FG, Guedes MLS. Variação sazonal da composição do essential oil de Myrcia salzmannii Berg. (Myrtaceae). Quím Nova. 2009;32(6):1544-1548.; Rosa et al., 2016Rosa CS, Veras KS, Silva PR, Lopes Neto JJ, Cardoso HLM, Alves LPL, et al. Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med. 2016;18(1):19-26., Silva, Uetanabro, Lucchese, 2013Silva AN, Uetanabaro APT, Lucchese AM. Chemical Composition and Antibacterial Activity of Essential Oils from Myrcia alagoensis (Myrtaceae). Nat Prod Commun. 2013;8(2):269-271.).

The major compounds in Myrcia hatschbachii EO were the sesquiterpenes trans-calamenene (19.10%), (E)-caryophyllene (10.96%) and spathulenol (5.03%).

Trans-calamenene was also the main constituent of the species Myrcia obtecta (Stefanello et al., 2010Stefanello MEA, Cervi AC, Wisniewski Junior A, Simionatto EL. Composição e variação sazonal do óleo essencial de Myrcia obtecta (O.Berg) Kiaersk. var. obtecta, Myrtaceae. Rev Bras Farmacogn. 2010;20(1):82-86.). (E)-caryophyllene was previously found in leaves of Myrcia sylvatica (Rosa et al., 2016Rosa CS, Veras KS, Silva PR, Lopes Neto JJ, Cardoso HLM, Alves LPL, et al. Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med. 2016;18(1):19-26.), Myrcia cuprea (Zoghbi et al., 2003Zoghbi MGB, Andrade EHA, Silva MHL, Carreira LMM, Maia JGS. Essential oils from three Myrcia species. Flavour Fragr J. 2003;18(5fig):421-424.) and Myrcia salzmannii (Cerqueira et al., 2009Cerqueira MD, Marques EJ, Martins D, Roque NF, Cruz FG, Guedes MLS. Variação sazonal da composição do essential oil de Myrcia salzmannii Berg. (Myrtaceae). Quím Nova. 2009;32(6):1544-1548.), and is associated with antibacterial (Huang et al., 2012Huang M, Sanchez-Moreiras AM, Abel C, Sohrabi R, Lee S, Gershenzon J, et al. The major volatile organic compound emitted from Arabidopsis thaliana, the sesquiterpene β-caryophyllene, is a defense against a bacterial pathogen. New Phytol. 2012;193(4):997-1008.; Lang, Buchbauer, 2012Lang G, Buchbauer G. A review on recent research results (2008-2010) on essential oils as antimicrobials and antifungals. A review. Flavour Fragr J. 2012;27(1):13-39.) and anticarcinogenic activities (Alcântara et al., 2010Alcântara JM, Yamaguchi KK de L, Veiga Junior VF da, Lima ES. Composição química dos óleos essenciais de espécies de Aniba e Licaria e suas atividades antioxidantes e antiagregante plaquetária. Quím Nova. 2010;33(1):141-145.). The oxygenated sesquiterpene, spathulenol, also has antibacterial (Lang and Buchbauer, 2012Lang G, Buchbauer G. A review on recent research results (2008-2010) on essential oils as antimicrobials and antifungals. A review. Flavour Fragr J. 2012;27(1):13-39.) and anticarcinogenic activities (Alcântara et al., 2010Alcântara JM, Yamaguchi KK de L, Veiga Junior VF da, Lima ES. Composição química dos óleos essenciais de espécies de Aniba e Licaria e suas atividades antioxidantes e antiagregante plaquetária. Quím Nova. 2010;33(1):141-145.), and has been found in the species Myrcia bracteata and Myrcia sylvatica (Zoghbi et al., 2003Zoghbi MGB, Andrade EHA, Silva MHL, Carreira LMM, Maia JGS. Essential oils from three Myrcia species. Flavour Fragr J. 2003;18(5fig):421-424.).

A previous study of composition of EO of fresh leaves from the species Myrcia hatschbachii, collected between the months of November and January, was made for comparison to other species within the same genus. The predominant compounds identified were (E)-caryophyllene (23.3%), δ-cadinene (8.1%), and bicyclogermacrene (6.9%) (Limberger et al., 2004Limberger RP, Sobral M, Henriques AT, Menut C, Bessiere JM. Óleos voláteis de espécies de Myrcia nativas do Rio Grande do Sul. Quím Nova. 2004;27(6):916-919.). The two latter compounds were not identified in our study. On the other hand, trans-calamenene (major constituent) has not been reported by Limberger (2004)Limberger RP, Sobral M, Henriques AT, Menut C, Bessiere JM. Óleos voláteis de espécies de Myrcia nativas do Rio Grande do Sul. Quím Nova. 2004;27(6):916-919.. The variation between the compositions of EO might be related to season and collection site, growth conditions, age of the plant, weather conditions, intensity of the solar radiation, soil composition or genetic variability (Rosa et al., 2016Rosa CS, Veras KS, Silva PR, Lopes Neto JJ, Cardoso HLM, Alves LPL, et al. Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med. 2016;18(1):19-26.; Sá et al., 2012Sá FAS, Borges LL, Paula JAM, Sampaio BL, Ferri PH, Paula JR. Essential oil in aerial parts of Myrcia tomentosa composition and variability. Rev Bras Farmacogn. 2012;22(6):1233-1240.).

Antioxidant activity as evaluated by the DPPH• radical scavenging method and phosphomolybdenum complex formation assay is summarized in Table II.

Results from the DPPH• radical scavenging method demonstrated that EO was less active than standards. However, due to the fact that this method is more sensitive to polar substances, and that EO has a lipophilic characteristic, composed majoritarily by sesquiterpenes, it was necessary to evaluate antioxidant activity by phosphomolybdenum complex formation assay. EO presented results statistically superior to rutin, which is a flavonoid with known antioxidant properties (Zhang et al., 2016Zhang R, Zhang BL, He T, Yi T, Yang JP, He B. Increase of rutin antioxidant activity by generating Maillard reaction products with lysine. Bioorganic Med Chem Lett. 2016;26(11):2680-2684.).

The LC₅₀ of EO of Myrcia hatschbachii is summarized in Table III.

According to Meyer et al. (1982)Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DJ, McLaughlin JL. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 1982;45(5):31-34., results are considered significant when LC₅₀ is less than 1000 µg/mL. According to Amarante et al. (2011)Amarante CB, Müller AH, Póvoa MM, Dolabela MF. Estudo fitoquímico biomonitorado pelos ensaios de toxicidade frente à Artemia salina e de atividade antiplasmódica do caule de aninga (Montrichardia linifera). Acta Amaz. 2011;41(3):431-434., samples where LC₅₀ is between 100 and 500 µg/mL, such as the analyzed oil, are considered moderately toxic. In a study performed with the species Myrcia myrtifolia, EO treatment resulted in an LC₅₀ of 479.16 µg/mL, suggesting potential usage as antimicrobial (Cerqueira et al., 2007Cerqueira MD, Souza-Neta LC, Passos MGVM, Lima EO, Roque NF, Martins D, et al. Seasonal Variation and Antimicrobial Activity of Myrcia myrtifolia essential oils. J Braz Chem Soc. 2007;18(5):998-1003.).

To evaluate hemolytic activity, the percentage of hemolysis was calculated against 1% triton and clean water, which were used as 100% positive controls. Linear regression was used to determine the concentration necessary to achieve 50% hemolytic activity. The regression equations were as follows: $y=0.0334x+19.149\left(R^2=0.9902\right)\mathrm{for}1\%\mathrm{triton}\mathrm{and}y=0.0321x+18.402\left(R^2=0.9902\right)$ for clean water. The abscissa represents the sample concentration and the ordinate is the percentage of hemolysis. The results of this determination were 923.68 µg/mL for 1% triton and 984.36 µg/mL for clean water. Hemolysis was dose-dependent.

The biological activities shown using in vitro toxicity tests against Artemia salina and hemolytic activity presented a toxic potential of Myrcia hatschbachii EO and indicate an opportunity of research and evaluation of antitumoral capacity.

EO toxicity might be related to properties of its major constituents. Previous studies have shown that (E)-caryophyllene and spathulenol have anticarcinogenic properties through apoptotic signaling and inhibition of tumor cell proliferation (Alcântara et al., 2010Alcântara JM, Yamaguchi KK de L, Veiga Junior VF da, Lima ES. Composição química dos óleos essenciais de espécies de Aniba e Licaria e suas atividades antioxidantes e antiagregante plaquetária. Quím Nova. 2010;33(1):141-145.). Moreover, the remaining constituents might act synergistically with the major components, influencing biological activity of the oil.

The toxicity assay against Artemia salina might be used as a model to evaluate acute oral toxicity of plant extracts. In a study using 20 species, toxicity against Artemia salina was compared to toxicity tests made in mice, demonstrating good correlation between the methods in vitro and in vivo (Parra et al., 2001Parra AL, Yhebra RS, Sardiñas IG, Buela LI. Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicicty of plant extracts. Phytomedicine. 2001;8(5):395-400.). This experiment is also an indication of potential anticancer activity, and can be used as a pre-screen to investigate activity against solid tumors. The concentration capable of causing lethality of 50% of the brine shrimp is usually ten times the effective dose of inhibition of the cellular growth in antitumoral tests (McLaughlin, Rogers, Anderson, 1998Mclaughlin JL, Rogers LL, Anderson JE. The use of biological assays to evaluate botanicals. Drug Inf J. 1998;32:513-524.; Rosa et al., 2016Rosa CS, Veras KS, Silva PR, Lopes Neto JJ, Cardoso HLM, Alves LPL, et al. Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med. 2016;18(1):19-26.).

In vitro evaluation of hemolytic activity of natural products helps to determine possible damage caused to the membranes of erythrocytes by the constituents of EO. When red blood cells undergo lysis, hemoglobin is released (Sobrinho et al., 2016Sobrinho ACN, Souza EB, Rocha MFG, Albuquerque MRJR, Bandeira PN, Santos HS, et al. Chemical composition, antioxidant, antifungal and hemolytic activities of essential oil from Baccharis trinervis (Lam.) Pers. (Asteraceae). Ind Crops Prod. 2016;84:108-115.). Thus, hemolytic activity is considered a preliminary test of toxicity of vegetal samples, especially when evaluating potential therapeutic activities.

Antioxidant activity also showed relevant results for Myrcia hatschbachii EO. Compounds with antioxidant capacity are of interest with regard to treatment and prevention of cancers. An example of these compounds is ascorbic acid. At higher concentrations, ascorbic acid might enhance production of ATP and induce apoptosis in tumor cells through a pro-oxidant mechanism (Mata et al., 2016Mata AMOF da, Carvalho RM, Alencar MVOB, Cavalcante AACM, Silva BB. Ascorbic acid in the prevention and treatment of cancer. Rev Assoc Med Bras. 2016;62(7):680-686.).

Regarding antibacterial activity, Myrcia hatschbachii EO treatment resulted in a MIC of 500 µg/mL (moderate activity) for Enterococcus faecalis and 1000 µg/mL (weak activity) for Staphylococcus aureus. These bacteria are important pathogens in foodborne illnesses (Jesus et al., 2016Jesus IC de, Frazão GGS, Blank AF, Santana LC de A. Myrcia ovata Cambessedes essential oils: A proposal for a novel natural antimicrobial against foodborne bacteria. Microb Pathog. 2016;99:142-147.). For the remaining microorganisms, EO showed MIC results greater than 1000 µg/mL. EO of Myrcia alogenensis (Silva et al., 2013Silva AN, Uetanabaro APT, Lucchese AM. Chemical Composition and Antibacterial Activity of Essential Oils from Myrcia alagoensis (Myrtaceae). Nat Prod Commun. 2013;8(2):269-271.) and Myrcia myrtifolia (Cerqueira et al., 2007Cerqueira MD, Souza-Neta LC, Passos MGVM, Lima EO, Roque NF, Martins D, et al. Seasonal Variation and Antimicrobial Activity of Myrcia myrtifolia essential oils. J Braz Chem Soc. 2007;18(5):998-1003.) exhibited activity against Staphylococcus aureus. In contrast, EO from Myrcia fallax (Alarcón et al., 2009Alarcón LD, Peña AE, Gonzales CN, Quintero A, Meza M, Usubillaga A, et al. Composition and antibacterial activity of the essential oil of Myrcia fallax (Rich.) DC. from Venezuela. Rev Soc Quím Perú. 2009;75(2):221-227.) and Myrcia splendens (Jiménez et al., 2012Jiménez D, Araque M, Rojas L, Cordero A, Briceño B. Componentes volátiles y actividad antibacteriana del vástago de Myrcia splendens (Sw.) DC. Rev Fac Farm. 2012;54:7-11.) were active against Staphylococcus aureus and Enterococcus faecalis.

Phytotoxic activity was evaluated by examining the influence of EO of Myrcia hatschbachii on germination and growth of Lactuca sativa seeds. Regarding the germination results, no statistical differences were observed compared to water and 1% polysorbate controls. However, at the concentration of 1%, EO negatively affected germination speed index (GSI). Results are summarized in Table IV.

Growth results of both hypocotyl and radicle, showed the most expressive activity in 1% preparations. All concentrations of EO tested inhibited growth of the hypocotyl of Lactuca sativa. Regarding the radicle, the oil presented more significant results growth inhibition results at the concentration of 1%, but the concentrations 0.001% and 0.01% also presented negative influence according to the statistical analysis, compared to the controls water and polysorbate at 1% in water.

A previous study examining Myrcia guianensis EO showed greater potential as an inhibitor of seed germination than radicle or hypocotyl growth in seeds of Mimosa pudica and Senna obtusifolia (Souza Filho et al., 2006Souza Filho APS, Santos RA, Santos LS, Guilhon GMP, Santos AS, Arruda MSP, et al. Allelophatic Potential of Myrcia guianensis. Planta Daninha. 2006;24(4):649-656.).

Our results showing inhibition of growth of the hypocotyl and radicle of Lactuca sativa demonstrate the phytotoxic potential of Myrcia hatschbachii and support further investigation of this species for development and production of natural herbicides.

Study of metabolites as allelochemicals has increased recently, focused mainly on production of bioherbicides. These agents might be used as alternatives for synthetic agrochemicals, which are associated with health risks and negative environmental impact. Thus, the search for bioherbicides becomes an alternative for sustainable agriculture with the utilization of medicinal plants at the farming of foods free of agrochemicals. It also contributes to the reduction of costs at the production chain, as they tend to be cheaper than the traditional herbicides (Santiago et al., 2017Santiago JA, Cardoso MG, Cruz FA, Palmieri MJ, Souza RV, Soares LI, et al. Cytogenotoxic effect of essential oil from Backhousia citriodora L. (Myrtaceae) on meristematic cells of Lactuca sativa L. South Afr J Bot. 2017;112:515-520.).

A general assay to predict possible therapeutic or biological activity of plant species would aid in research and development of new products and new drugs (Rosa et al., 2016Rosa CS, Veras KS, Silva PR, Lopes Neto JJ, Cardoso HLM, Alves LPL, et al. Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med. 2016;18(1):19-26.).

This study demonstrates that EO of Myrcia hatschbachii could be used in agriculture for development of bioherbicides based on phytotoxic potential. In vitro toxicity tests point to the execution of antitumoral activity tests, searching for prospection of antineoplastic drugs. Finally, these EO also exhibit antibacterial activity against two important foodborne bacteria: Staphylococcus aureus and Enterococcus faecalis.

ACKNOWLEDGMENTS

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors are grateful to the Department of Chemistry of the Federal University of Parana, Brazil, for the help in GC / MS analysis.

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