Chemical composition and biological activities of essential oil from flowers of Psidium guajava (Myrtaceae)

Composição química e atividades biológicas do óleo essencial das flores de Psidium guajava (Myrtaceae)

C. C. Fernandes J. L. Rezende E. A. J. Silva F. G. Silva L. Stenico A. E. M. Crotti V. R. Esperandim M. B. Santiago C. H. G. Martins M. L. D. Miranda About the authors

Abstract

Xylella fastidiosa is a plant-pathogenic bacterium that lives inside host xylem vessels, where it forms biofilm which is believed to be responsible for disrupting the passage of water and nutrients. Pectobacterium carotovorum is a Gram-negative plant-specific bacterium that causes not only soft rot in various plant hosts, but also blackleg in potato by plant cell wall degradation. Chagas disease, which is caused by Trypanosoma cruzi, has been commonly treated with nifurtimox and benzonidazole, two drugs that cause several side effects. As a result, the use of natural products for treating bacterial and neglected diseases has increased in recent years and plants have become a promising alternative to developing new medicines. Therefore, this study aimed to determine, for the first time, the chemical composition of essential oil from Psidium guajava flowers (PG-EO) and to evaluate its in vitro anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi and cytotoxic activities. PG-EO was obtained by hydrodistillation in a Clevenger apparatus while its chemical composition was determined by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Major compounds identified in PG-EO were α-cadinol (37.8%), β-caryophyllene (12.2%), nerolidol (9.1%), α-selinene (8.8%), β-selinene (7.4%) and caryophyllene oxide (7.2%). Results showed that the PG-EO had strong trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi (IC50 = 14.6 μg/mL), promising antibacterial activity against X. fastidiosa (MIC = 12.5 μg/mL) and P. carotovorum (MIC = 62.5 μg/mL), and moderate cytotoxicity against LLCMK2 adherent epithelial cells in the concentration range (CC50 = 250.5 μg/mL). In short, the PG-EO can be considered a new source of bioactive compounds for the development of pesticides and trypanocide drugs.

Keywords:
Neglected tropical diseases; bactericide efficacy; guava; α-cadinol; LLCMK2

Resumo

Xylella fastidiosa é uma bactéria patogênica que vive dentro dos vasos do xilema hospedeiro, onde forma um biofilme responsável por interromper a passagem de água e nutrientes. Pectobacterium carotovorum é uma bactéria Gram-negativa que causa não só podridão macia em várias plantas hospedeiras, mas também canela-preta na batata por degradação da parede celular da planta. A doença de Chagas, causada pelo Trypanosoma cruzi, é comumente tratada com nifurtimox e benzonidazol, duas drogas que causam vários efeitos colaterais. Como resultado, o uso de produtos naturais para o tratamento de doenças bacterianas e negligenciadas aumentou nos últimos anos e as plantas continuam sendo uma alternativa promissora para o desenvolvimento de novos medicamentos. Portanto, este estudo teve como objetivo determinar, pela primeira vez, a composição química do óleo essencial de flores de Psidium guajava (PG-EO) e avaliar suas propriedades anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi e citotóxica in vitro. PG-EO foi obtido por hidrodestilação em um aparelho Clevenger, enquanto sua composição química foi determinada por cromatografia em fase gasosa com detecção por ionização por chama (CG-DIC) e por cromatografia em fase gasosa acoplada à espectrometria de massa (CG-EM). Os principais compostos identificados no PG-EO foram α-cadinol (37,8%), β-cariofileno (12,2%), nerolidol (9,1%), α-selineno (8,8%), β-selineno (7,4%) e óxido de cariofileno (7,2%). Os resultados mostraram que o PG-EO apresentou forte atividade tripanocida contra as formas tripomastigotas de T. cruzi (CI50 = 14,6 μg/mL), promissora atividade antibacteriana contra X. fastidiosa (MIC = 12,5 μg/mL) e P. carotovorum (MIC = 62,5 μg/mL) e citotoxicidade moderada contra células epiteliais aderentes (LLCMK2) na faixa de concentração (CC50 = 250,5 μg/mL). Em suma, o PG-EO pode ser considerado uma nova fonte de compostos bioativos para o desenvolvimento de pesticidas e drogas tripanocidas.

Palavras-chave:
Doenças tropicais negligenciadas; eficácia bactericida; goiaba; α-cadinol; LLCMK2

1. Introduction

Xylella fastidiosa, a bacterium found in xylem, is a pathogen that lives in several plant species, such as weeds and fruit trees of economic interest (Coletta-Filho et al., 2016COLETTA-FILHO, H.D., FRANCISCO, C.S., LOPES, J.R.S., OLIVEIRA, A.F. and SILVA, L.F.O., 2016. First report of olive leaf scorch in Brazil, associated with Xylella fastidiosa subsp. pauca. Phytopathologia Mediterranea, vol. 55, no. 1, pp. 130-135. http://dx.doi.org/10.14601/Phytopathol_Mediterr-17259.
http://dx.doi.org/10.14601/Phytopathol_M...
). It is responsible for one of the most important plant diseases that has been reported over the last decades, i. e., a disease whose early symptoms are mostly silent, rather than apparent. An exception is citrus variegated chlorosis (CVC), a disease which is characterized by little yellow spots on leaves and severe fruit deterioration (Coletta-Filho et al., 2016COLETTA-FILHO, H.D., FRANCISCO, C.S., LOPES, J.R.S., OLIVEIRA, A.F. and SILVA, L.F.O., 2016. First report of olive leaf scorch in Brazil, associated with Xylella fastidiosa subsp. pauca. Phytopathologia Mediterranea, vol. 55, no. 1, pp. 130-135. http://dx.doi.org/10.14601/Phytopathol_Mediterr-17259.
http://dx.doi.org/10.14601/Phytopathol_M...
). Fruits get more acid and smaller, since their maturation time is shorter; as a result, their commercial value decreases. In Brazil, the disease was first detected in 1987. In 2002, about a third of the country’s orange groves had been infected by X. fastidiosa, while in 2016, about 43% of all produce had been overrun by the bacterium. It led to losses and posed negative impact on the economy. CVC may have arrived in Brazil from Argentina in the 1980’s when the transportation of infected trees enabled the phytopathogen to quickly disseminate all over South America, where it is now endemic (Coletta-Filho et al., 2016COLETTA-FILHO, H.D., FRANCISCO, C.S., LOPES, J.R.S., OLIVEIRA, A.F. and SILVA, L.F.O., 2016. First report of olive leaf scorch in Brazil, associated with Xylella fastidiosa subsp. pauca. Phytopathologia Mediterranea, vol. 55, no. 1, pp. 130-135. http://dx.doi.org/10.14601/Phytopathol_Mediterr-17259.
http://dx.doi.org/10.14601/Phytopathol_M...
).

Xylella fastidiosa is transmitted by vector insects, such as cicadas that belong to the subfamily Cicadellinae (Hemiptera: Cicadellidae). Synthetic insecticides have been used for controlling these vectors so as to decrease bacterial transmission and the number of infected plants (Bleve et al., 2018BLEVE, G., GALLO, A., ALTOMARE, C., VURRO, M., MAIORANO, G., CARDINALI, A., D’ANTUONO, I.D., MARCHI, G. and MITA, G., 2018. In vitro activity of antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS Microbiology Letters, vol. 365, no. 5, pp. fnx281. http://dx.doi.org/10.1093/femsle/fnx281. PMid:29390137.
http://dx.doi.org/10.1093/femsle/fnx281...
). However, resistance to conventional insecticides is the main cause of increase in the number of sick trees. Besides, bacterial infection has been observed even when good practices are applied to culture management. The literature has shown some strategies used against X. fastidiosa, such as the production of genetically-modified plants with either proteins or peptides that are capable of killing pathogens and the search for little molecules that may reach the xylem sap flow and inhibit its growth and movement (Bleve et al., 2018BLEVE, G., GALLO, A., ALTOMARE, C., VURRO, M., MAIORANO, G., CARDINALI, A., D’ANTUONO, I.D., MARCHI, G. and MITA, G., 2018. In vitro activity of antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS Microbiology Letters, vol. 365, no. 5, pp. fnx281. http://dx.doi.org/10.1093/femsle/fnx281. PMid:29390137.
http://dx.doi.org/10.1093/femsle/fnx281...
).

Soft rot caused by Pectobacterium carotovorum is considered a bacterial disease that affects economically important plants, such as lettuce and potato, not only in Brazil, but all over the world (Felix et al., 2014FELIX, K.C.S., OLIVEIRA, W.J., MARIANO, R.L.R. and SOUZA, E.B., 2014. Lettuce genotype resistance to “soft rot” caused by Pectobacterium carotovorum subsp. carotovorum. Scientia Agrícola, vol. 71, no. 4, pp. 287-291. http://dx.doi.org/10.1590/0103-9016-2013-0301.
http://dx.doi.org/10.1590/0103-9016-2013...
). In Pernambuco, a state in northeastern Brazil, lettuce has been successfully cultivated all along the year but soft rot has caused severe damage, mainly when temperature and humidity are high. Pectobacterium carotovorum has also been considered a highly aggressive bacterium which infects potato cultures in tropical and subtropical regions worldwide since 2004. Control of this type of disease has been hindered mainly by the wide range of host plants and the capacity that these bacteria have to survive in residues of cultures. The use of resistant cultivars is considered the most cost-effective and technically viable strategy, since pathogenic populations have resisted to pesticides (Felix et al., 2014FELIX, K.C.S., OLIVEIRA, W.J., MARIANO, R.L.R. and SOUZA, E.B., 2014. Lettuce genotype resistance to “soft rot” caused by Pectobacterium carotovorum subsp. carotovorum. Scientia Agrícola, vol. 71, no. 4, pp. 287-291. http://dx.doi.org/10.1590/0103-9016-2013-0301.
http://dx.doi.org/10.1590/0103-9016-2013...
; Czajkowski et al., 2015CZAJKOWSKI, R., PÉROMBELON, M.C.M., JAFRA, S., LOJKOWSKA, E., POTRYKUS, M., VAN DER WOLF, J.M. and SLEDZ, W., 2015. Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Annals of Applied Biology, vol. 166, no. 1, pp. 18-38. http://dx.doi.org/10.1111/aab.12166. PMid:25684775.
http://dx.doi.org/10.1111/aab.12166...
).

Pesticides have been widely used for controlling plant diseases around the world. Although the use of such products has positive short-term effect on producers, their long-term employment has many negative effects on society and the environment, such as soil and water pollution, deposition of agrochemical residues on food and the emergence of resistant pathogens, as mentioned before (Guimarães et al., 2015GUIMARÃES, P.G., MOREIRA, I.S., CAMPOS FILHO, P.C., FERRAZ, J.L.A.A., NOVAES, Q.S. and BATISTA, R., 2015. Antibacterial activity of Schinopsis brasiliensis against phytopathogens of agricultural interest. Fitos, vol. 9, no. 3, pp. 161-252. http://dx.doi.org/10.5935/2446-4775.20150013.
http://dx.doi.org/10.5935/2446-4775.2015...
). In order to reduce negative effects of pesticides, natural products have been investigated so as to control phytopathogens. Therefore, there has been intense search for new antimicrobial agents from plants as the result of increasing resistance of pathogenic microorganisms to synthetic products (Guimarães et al., 2015GUIMARÃES, P.G., MOREIRA, I.S., CAMPOS FILHO, P.C., FERRAZ, J.L.A.A., NOVAES, Q.S. and BATISTA, R., 2015. Antibacterial activity of Schinopsis brasiliensis against phytopathogens of agricultural interest. Fitos, vol. 9, no. 3, pp. 161-252. http://dx.doi.org/10.5935/2446-4775.20150013.
http://dx.doi.org/10.5935/2446-4775.2015...
). Essential oils are some of the natural products that exhibit a wide array of biological properties, such as insecticidal, antimicrobial, antioxidant and bioregulatory properties (Pandey et al., 2013PANDEY, A.K., SINGH, P., PALNI, U.T. and TRIPATHI, N.N., 2013. Application of Chenopodium ambrosioides Linn. essential oil as botanical fungicide for the management of fungal deterioration in pulses. Biological Agriculture and Horticulture, vol. 29, no. 3, pp. 197-208. http://dx.doi.org/10.1080/01448765.2013.822828.
http://dx.doi.org/10.1080/01448765.2013....
). Regarding their application to agriculture, their antifungal activity against phytopathogens, such as Sclerotinia sclerotiorum and Colletotrichum gloeosporioides, should be highlighted (Valadares et al., 2018VALADARES, A.C.F., ALVES, C.C.F., ALVES, J.M., DE DEUS, I.P.B., OLIVEIRA FILHO, J.G., SANTOS, T.C.L., DIAS, H.J., CROTTI, A.E.M. and MIRANDA, M.L.D., 2018. Essential oils from Piper aduncum inflorescences and leaves: chemical composition and antifungal activity against Sclerotinia sclerotiorum. Anais da Academia Brasileira de Ciências, vol. 90, no. 3, pp. 2691-2699. http://dx.doi.org/10.1590/0001-3765201820180033. PMid:30304214.
http://dx.doi.org/10.1590/0001-376520182...
; Sarkhosh et al., 2018SARKHOSH, A., SCHAFFER, B., VARGAS, A.I., PALMATEER, A.J., LOPEZ, P., SOLEYMANI, A. and FARZANEH, M., 2018. Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit. Biological Agriculture and Horticulture, vol. 34, no. 1, pp. 18-26. http://dx.doi.org/10.1080/01448765.2017.1358667.
http://dx.doi.org/10.1080/01448765.2017....
). On the other hand, some essential oils have also exhibited satisfactory activity against several phytopathogenic bacteria, such as Xanthomonas vesicatoria and Agrobacterium tumefaciens (Vasinauskiene et al., 2006VASINAUSKIENE, M., RADUSIENE, J., ZITIKAITE, I. and SURVILIENE, E., 2006. Antibacterial activities of essential oils from aromatic and medicinal plants against growth of phytopathogenic bacteria. Agronomy Research (Tartu), vol. 4, pp. 437-440.; Gormez et al., 2015GORMEZ, A., BOZARI, S., YANMIS, D., GULLUCE, M., SAHIN, F. and AGAR, G., 2015. Chemical composition and antibacterial activity of essential oils of two species of Lamiaceae against phytopathogenic bacteria. Polish Journal of Microbiology, vol. 64, no. 2, pp. 121-127. http://dx.doi.org/10.33073/pjm-2015-018. PMid:26373171.
http://dx.doi.org/10.33073/pjm-2015-018...
).

Neglected tropical diseases are a group of 17 diseases considered to be chronic common infections among the poorest people from less developed countries. The World Health Organization recognizes the urgency of developing new tools and technologies to combat these diseases which are considered to be the world’s greatest health problems (Hotez et al., 2016HOTEZ, P.J., PECOUL, B., RIJAL, S., BOEHME, C., AKSOY, S., MALECELA, M., TAPIA-CONYER, R. and REEDER, J.C., 2016. Eliminating the neglected tropical diseases: translational science and new technologies. PLoS Neglected Tropical Diseases, vol. 10, no. 3, pp. e0003895. http://dx.doi.org/10.1371/journal.pntd.0003895. PMid:26934395.
http://dx.doi.org/10.1371/journal.pntd.0...
).

Chagas disease or American trypanosomiasis is caused by the flagellate protozoan Trypanosoma cruzi, which is transmitted to the human host, mainly by infected triatomines, commonly known as barbeiro (Delmondes and Stefani, 2018DELMONDES, P.H. and STEFANI, R., 2018. In silico study of the antichagasic activity of aromatic compounds. Orbital: the Electronic. Journal of Chemistry, vol. 10, no. 5, pp. 395-401. http://dx.doi.org/10.17807/orbital.v10i5.1018.
http://dx.doi.org/10.17807/orbital.v10i5...
). This disease affects about 7-8 million Latin American people and the main mode of transmission to people is through feces of the infected vectors (Lavorato et al., 2015LAVORATO, S.N., SALES JÚNIOR, P.A., MURTA, S.M.F., ROMANHA, A.J. and ALVES, R.J., 2015. In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures. Memorias do Instituto Oswaldo Cruz, vol. 110, no. 4, pp. 566-568. http://dx.doi.org/10.1590/0074-02760150007. PMid:26061148.
http://dx.doi.org/10.1590/0074-027601500...
). There are other means of contamination such as contaminated blood transfusion, congenital transmission, organ transplantation, and even by ingestion of infected fruits, such as açaí (Lavorato et al., 2015LAVORATO, S.N., SALES JÚNIOR, P.A., MURTA, S.M.F., ROMANHA, A.J. and ALVES, R.J., 2015. In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures. Memorias do Instituto Oswaldo Cruz, vol. 110, no. 4, pp. 566-568. http://dx.doi.org/10.1590/0074-02760150007. PMid:26061148.
http://dx.doi.org/10.1590/0074-027601500...
; Passos et al., 2012PASSOS, L.A.C., GUARALDO, A.M.A., BARBOSA, R.L., DIAS, V.L., PEREIRA, K.S., SCHMIDT, F.L., FRANCO, R.M.B. and ALVES, D.P., 2012. Sobrevivência e infectividade do Trypanosoma cruzi na polpa de açaí: estudo in vitro e in vivo. Epidemiologia e Serviços de Saúde : Revista do Sistema Unico de Saúde do Brasil, vol. 21, no. 2, pp. 223-232. http://dx.doi.org/10.5123/S1679-49742012000200005.
http://dx.doi.org/10.5123/S1679-49742012...
).

In the treatment of Chagas disease, two nitroheterocyclic drugs are widely used: nifurtimox and benznidazole (Andrade et al., 2015ANDRADE, M.A., CARDOSO, M., GOMES, M.S., DE AZEREDO, C.M., BATISTA, L.R., SOARES, M.J., RODRIGUES, L.M. and FIGUEIREDO, A.C., 2015. Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis. Brazilian Journal of Microbiology, vol. 46, no. 1, pp. 189-194. http://dx.doi.org/10.1590/S1517-838246120130683. PMid:26221107.
http://dx.doi.org/10.1590/S1517-83824612...
). These drugs have several side effects, such as: anorexia, nausea, gastrointestinal disorders, allergic dermatopathy, polyneuritis, bone marrow depression, peripheral neuropathy among others (Oliveira et al., 2008OLIVEIRA, M.F., NAGAO-DIAS, A.T., PONTES, V.M.O., SOUSA JÚNIOR, A.S., COELHO, H.L.L. and COELHO, I.C.B., 2008. Tratamento etiológico da Doença de Chagas no Brasil. Revista de Patologia Tropical, vol. 37, no. 3, pp. 209-228. http://dx.doi.org/10.5216/rpt.v37i3.5063.
http://dx.doi.org/10.5216/rpt.v37i3.5063...
).

Due to these complications caused by available drugs, the search for new chemotherapeutic agents that are effective and have low toxicity becomes increasingly relevant. In this sense, the wide biodiversity of bioactive compounds found in essential oils extracted from plants has increasingly aroused the interest of researchers worldwide (Ngahang Kamte et al., 2018NGAHANG KAMTE, S.L., RANJBARIAN, F., CIANFAGLIONE, K., SUT, S., DALL’ACQUA, S., BRUNO, M., AFSHAR, F.H., IANNARELLI, R., BENELLI, G., CAPPELLACCI, L., HOFER, A., MAGGI, F. and PETRELLI, R., 2018. Identification of highly effective antitrypanosomal compounds in essential oils from the Apiaceae family. Ecotoxicology and Environmental Safety, vol. 156, no. 30, pp. 154-165. http://dx.doi.org/10.1016/j.ecoenv.2018.03.032. PMid:29549739.
http://dx.doi.org/10.1016/j.ecoenv.2018....
). These essential oils can be obtained from different plant species and have various biological activities, such as: antibacterial, anticancer, anti-inflammatory, antimutagenic, antifungal, antioxidant and antiprotozoal (Raut and Karuppayil, 2014RAUT, J.S. and KARUPPAYIL, S.M., 2014. A status review on the medicinal properties of essential oils. Industrial Crops and Products, vol. 62, no. 1, pp. 250-264. http://dx.doi.org/10.1016/j.indcrop.2014.05.055.
http://dx.doi.org/10.1016/j.indcrop.2014...
).

Psidium guajava L. belongs to the family Myrtaceae, which comprises about 80 genera and 3,000 species distributed in the tropics and subtropics, mainly in the Americas, Asia and Australia. The genus Psidium has around 150 species of bushes; P. guajava is the most well-known and most widely distributed one all over the world (Pereira et al., 2017PEREIRA, F.M., USMAN, M., MAYER, N.A., NACHTIGAL, J.C., MAPHANGA, O.R.M. and WILLEMSE, S., 2017. Advances in guava propagation. Revista Brasileira de Fruticultura, vol. 39, no. 4, pp. e-358. http://dx.doi.org/10.1590/0100-29452017358.
http://dx.doi.org/10.1590/0100-294520173...
). Guava is considered one of the most important cultures in horticulture in the world’s tropical and subtropical regions due to its commercial and nutritional characteristics which result from its aggregated value and high content of vitamin C (Panneerselvam et al., 2012PANNEERSELVAM, P., MOHANDAS, S., SARITHA, B., UPRETI, K.K., POOVARASAN., MONNAPPA, A. and SULLADMATH, V.V., 2012. Glomus mosseae associated bacteria and their influence on stimulation of mycorrhizal colonization, sporulation, and growth promotion in guava (Psidium guajava L.) seedlings. Biological Agriculture and Horticulture, vol. 28, no. 4, pp. 267-279. http://dx.doi.org/10.1080/01448765.2012.741108.
http://dx.doi.org/10.1080/01448765.2012....
). This species was chosen to be used in this study because of its high yield of essential oils, a fact that has drawn the attention of researchers who aim at improving their use in different cases (Mendes et al., 2018MENDES, L.A., SOUZA, T.S., MENINI, L., GUILHEN, J.H.S., BERNARDES, C.O., FERREIRA, A. and FERREIRA, M.F.S., 2018. Spring alterations in the chromatographic profile of leaf essential oils of improved guava genotypes in Brazil. Scientia Horticulturae, vol. 238, no. 19, pp. 295-302. http://dx.doi.org/10.1016/j.scienta.2018.04.065.
http://dx.doi.org/10.1016/j.scienta.2018...
).

Based on previously described facts and on several benefits shown by essential oils, this study aimed at determining, for the first time, the chemical composition and the in vitro antibacterial, trypanocidal and cytotoxic effects of essential oil extracted from P. guajava flowers (Figure 1) against X. fastidiosa, P. carotovorum, T. cruzi and LLCMK2 adherent epithelial cells.

Figure 1
Leaves and Flowers of P. guajava (Myrtaceae).

2. Material and Methods

2.1. Plant material

Flowers were collected at the Instituto Federal Goiano, Rio Verde, Goiás, Brazil (17°48′12.006″S and 50°54′19.083″W), at 5 pm on 21st March 2017. The plant material was identified and samples were deposited as voucher specimens in the herbarium at the State University of Montes Claros in Minas Gerais, Brazil (identification number 4481).

2.2. Essential oil extraction

Essential oil from Psidium guajava flowers (PG-EO) was extracted from fresh flowers by hydrodistillation for 3 h in a Clevenger-type apparatus. Hydrodistillation was performed in triplicate. To this end, the plant material was divided into three 100-g samples and 500 mL distilled water was added to each sample. After manual collection of the essential oil, traces of water which remained in the oil were removed with anhydrous sodium sulfate, followed by filtration. PG-EO was stored in an amber bottle and kept in a refrigerator at 4°C until analysis. Calculation of PG-EO yield was based on the weight of fresh flowers and expressed as the average of triplicate analyses.

2.3. Chemical analyses of essential oil (PG-EO)

Gas chromatography-flame ionization detection (GC-FID) and gas chromatography–mass spectrometry (GC–MS) analyses were performed by Shimadzu QP2010 Plus and GCMS2010 Plus (Shimadzu Corporation, Kyoto, Japan) systems. GC-MS and GC-FID conditions and the identification of PG-EO have been previously reported (Lemes et al., 2018LEMES, R.S., ALVES, C.C.F., ESTEVAM, E.B.B., SANTIAGO, M.B., MARTINS, C.H.G., SANTOS, T.C.L., CROTTI, A.E.M. and MIRANDA, M.L.D., 2018. Chemical composition and antibacterial activity of essential oils from Citrus aurantifolia leaves and fruit peel against oral pathogenic bacteria. Anais da Academia Brasileira de Ciências, vol. 90, no. 2, pp. 1285-1292. http://dx.doi.org/10.1590/0001-3765201820170847. PMid:29898096.
http://dx.doi.org/10.1590/0001-376520182...
). Identification of volatile components of PG-EO (Table 1) was based on their retention indices on an Rtx-5MS capillary column under the same operating conditions used for GC relative to a homologous series of n-alkanes (C8-C20). Structures were computer-matched with Wiley 7, NIST 08 and FFNSC 1.2 spectral libraries and their fragmentation patterns were compared with data found in the literature (Adams, 2007ADAMS, R.P., 2007. In identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. 4th ed. Carol Stream: Allured Publishing Corporation, 804 p.).

Table 1
Chemical composition of essential oil from Psidium guajava flowers (PG-EO).

2.4. Pathogen preparation and identification

Both strains X. fastidiosa 9a5c and P. carotovorum Pca (424) used by this study were collected from CVC-affected Valencia sweet orange twigs in Macaubal (São Paulo, Brazil) and infected potatoes bought in Ipuíuna (Minas Gerais, Brazil), respectively. Strains were kept in the culture collection at the Laboratory of Research in Applied Microbiology (LaPeMA), University of Franca, São Paulo, Brazil, under cryopreservation at −80°C in periwinkle wilt (PW) broth with glycerol at 20% (v/v).

2.5. Minimum inhibitory concentrations and in vitro determination

Minimum inhibitory concentrations (MICs), i. e., the lowest compound concentrations that are able to inhibit microorganism growth, was determined in triplicate by using the microdilution broth method on a 96‐well polystyrene tissue culture plate (TPP, Trasadingen, Switzerland). The methodology recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012CLINICAL AND LABORATORY STANDARDS INSTITUTE – CLSI, 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard: M07-A9. 9th ed. Wayne: CLSI.) was followed. PG-EO samples (1 mg) were dissolved in 125 μL dimethylsulfoxide (DMSO; Merck, Darmstadt, HE, Germany) and diluted in PW broth. Then, samples were tested at concentrations ranging from 0.48 to 1,000 μg/mL. Inoculums were adjusted to produce cell concentrations of 1 × 106 CFU/mL, as advocated by the CLSI (2012)CLINICAL AND LABORATORY STANDARDS INSTITUTE – CLSI, 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard: M07-A9. 9th ed. Wayne: CLSI.. A growth control with no antibiotic and a sterility control with no inoculum were also included. DMSO at 5% was the maximum DMSO concentration (v/v) in the samples that allowed X. fastidiosa 9a5c and P. carotovorum Pca (424) to grow normally. Streptomycin (Sigma, St. Louis, MO, USA) was used as the reference antibiotic drug. The 96‐well microplate was kept in biological oxygen demand (BOD, Cientec, Brazil) at 28°C for seven days. After incubation, 30 μL of a 0.02% aqueous resazurin (Sigma‐Aldrich) solution was added to each well on the microplate. Resazurin is a dye that allows microbial growth to be observed. Blue and red represent absence and presence of microbial growth, respectively (Sarker et al., 2007SARKER, S.D., NAHAR, L. and KUMARASAMY, Y., 2007. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods (San Diego, Calif.), vol. 42, no. 4, pp. 321-324. http://dx.doi.org/10.1016/j.ymeth.2007.01.006. PMid:17560319.
http://dx.doi.org/10.1016/j.ymeth.2007.0...
). The microplate was incubated for additional 24 h so that observation and a descriptive analysis could be carried out.

2.6. In vitro trypanocidal and cytotoxic activities

To obtain the trypomastigotes of T. cruzi, LLCMK2 adherent epithelial cells were cultured in RPMI medium supplemented with 2 x 10-6 mol/L L-glutamine, 10-5 mol/L NaHCO3, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% inactivated fetal bovine serum. The procedure was accomplished in culture bottles at 37 °C, under 5% ambient CO2 and relative humidity of 95%. The trypomastigote forms were maintained in RPMI medium and the parasites were transferred to fresh medium every 48 h to furnish free parasite forms. The assay conducted after 24 h was based on the methodology of Rashed et al. (2016)RASHED, K., FERREIRA, D.S., ESPERANDIM, V.R., MARÇAL, M.G., SEQUEIRA, B.M., FLAUZINO, L.G.B., CUNHA, W.R., 2016. In vitro trypanocidal activity of the Egyptian plant Schinopsis lorentizii against trypomastigote and amastigote forms of Trypanosoma cruzi. Journal of Applied Pharmaceutical Science, vol. 6, no. 6, pp. 55-60.. Approximately 1 x 106 trypomastigotes was added to each well in a 96-well microtiter plate. Then, the essential oil was added at concentrations ranging from 12.5 to 200 µg/mL. After 24 h incubation, the biological activity of the samples was evaluated by the colorimetric MTT tetrazolium salt assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/mL). Readings were conducted by a microplate reader at 517 nm wavelength. Positive and negative controls were benznidazole (from 12.5 to 200 µg/mL) and 0.5% dimethyl sulfoxide (DMSO), respectively. Assays were performed in triplicate.

LLCMK2 adherent epithelial cells were grown in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 5% inactivated fetal calf serum. They were kept at 37°C in 5% CO2. A cell suspension was seeded at a concentration of 1 x 106 cells/mL in a 96-well microplate with RPMI 1640 medium. Thereafter, cells were treated with essential oil at different concentrations (6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL). Plates were incubated at 37°C for 24 h and the biological activity was evaluated by the MTT colorimetric method [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] in a microplate reader at 540 nm. RPMI 1640 medium was the positive control whereas DMSO and RPMI 1640 media were the negative ones. All experiments were performed in triplicate. The percentage of cell viability was determined by the following formula: % cell viability = 1 - [(Y-N)/(N-P)] x 100, where Y = absorbance of wells containing cells and essential oil at different concentrations; N = negative control; and P = positive control (Rashed et al., 2016RASHED, K., FERREIRA, D.S., ESPERANDIM, V.R., MARÇAL, M.G., SEQUEIRA, B.M., FLAUZINO, L.G.B., CUNHA, W.R., 2016. In vitro trypanocidal activity of the Egyptian plant Schinopsis lorentizii against trypomastigote and amastigote forms of Trypanosoma cruzi. Journal of Applied Pharmaceutical Science, vol. 6, no. 6, pp. 55-60.).

3. Results and Discussion

Thirteen volatile compounds were identified in PG-EO, while its yield was 1.0%. Major compounds of PG-EO were α-cadinol (37.8%), β-caryophyllene (12.2%), nerolidol (9.1%), α-selinene (8.8%), β-selinene (7.4%) and caryophyllene oxide (7.2%) (Table 1). They were determined by GC-FID and GC–MS.

It is important to mention that chemical constituents which predominate in PG-EO had already been previously identified at different concentrations in the essential oil from leaves of this species found in Brazil (Mendes et al., 2018MENDES, L.A., SOUZA, T.S., MENINI, L., GUILHEN, J.H.S., BERNARDES, C.O., FERREIRA, A. and FERREIRA, M.F.S., 2018. Spring alterations in the chromatographic profile of leaf essential oils of improved guava genotypes in Brazil. Scientia Horticulturae, vol. 238, no. 19, pp. 295-302. http://dx.doi.org/10.1016/j.scienta.2018.04.065.
http://dx.doi.org/10.1016/j.scienta.2018...
; Souza et al., 2017SOUZA, T.S., FERREIRA, M.F.S., MENINI, L., SOUZA, J.R.C.L., PARREIRA, L.A., CECON, P.R. and FERREIRA, A., 2017. Essential oil of Psidium guajava: influence of genotypes and environment. Scientia Horticulturae, vol. 216, no. 14, pp. 38-44. http://dx.doi.org/10.1016/j.scienta.2016.12.026.
http://dx.doi.org/10.1016/j.scienta.2016...
). When the chemical composition of PG-EO is compared with the one of oils extracted from the same species in other regions around the world, some similarities can be found. In China, major constituents were α-cubebene, caryophyllene, aromadendrene, α-cadinene and calamenene in essential oil from P. guajava leaves (Xu et al., 2017XU, C., LIANG, Z., TANG, D., XIAO, T., TSUNODA, M., ZHANG, Y., ZHAO, L., DENG, S. and SONG, Y., 2017. Gas chromatography-mass spectrometry (GC-MS) analysis of volatile components from guava leaves. Journal of Essential Oil Bearing Plants, vol. 20, no. 6, pp. 1536-1546. http://dx.doi.org/10.1080/0972060X.2017.1417746.
http://dx.doi.org/10.1080/0972060X.2017....
). Oils extracted from Tunisian P. guajava stems and leaves exhibited the following major constituents: α-humulene, germacrene D, valerenol, viridiflorol and β-caryophyllene (Khadhri et al., 2014KHADHRI, A., EL MOKNI, R., ALMEIDA, C., NOGUEIRA, J.M.F. and ARAÚJO, M.E.M., 2014. Chemical composition of essential oil of Psidium guajava L. growing in Tunisia. Industrial Crops and Products, vol. 52, no. 1, pp. 29-31. http://dx.doi.org/10.1016/j.indcrop.2013.10.018.
http://dx.doi.org/10.1016/j.indcrop.2013...
). In India, major components of oil from waste leaves of P. guajava were cineole, caryophyllene, copaene, azulene and eucalyptol (Kamran et al., 2012KAMRAN, A., MISHRA, R.K., GUPTA, R., KUMAR, A., BAJAJ, A.K. and DIKSHIT, A., 2012. Therapeutic effects of essential oil from waste leaves of Psidium guajava L. against cosmetic embarrassment using phylogenetic approach. American Journal of Plant Sciences, vol. 3, no. 6, pp. 745-752. http://dx.doi.org/10.4236/ajps.2012.36090.
http://dx.doi.org/10.4236/ajps.2012.3609...
). Major components of this essential oil in Nepal were (E)-nerolidol and (E)-caryophyllene (P. guajava leaves) while limonene and β-caryophyllene (P. guajava leaves) were identified in Nigeria (Satyal et al., 2015SATYAL, P., PAUDEL, P., LAMICHHANE, B. and SETZER, W.N., 2015. Leaf essential oil composition and bioactivity of Psidium guajava from Kathmandu, Nepal. American Journal of Essential Oils and Natural Products, vol. 3, no. 2, pp. 11-14.; Ogunwande et al., 2003OGUNWANDE, I.A., OLAWORE, N.O., ADELEKE, K.A., EKUNDAYO, O. and KOENIG, W.A., 2003. Chemical composition of the leaf volatile oil of Psidium guajava L. growing in Nigeria. Flavour and Fragrance Journal, vol. 18, no. 2, pp. 136-138. http://dx.doi.org/10.1002/ffj.1175.
http://dx.doi.org/10.1002/ffj.1175...
). In Cuba, major constituents identified in essential oil from P. guajava leaves were the following terpenes: β-caryophyllene, (E)-nerolidol and selin-11-en-4α-ol (Pino et al., 2001PINO, J.A., AGUERO, J., MARBOT, R. and FUENTES, V., 2001. Leaf oil of Psidium guajava L. from Cuba. The Journal of Essential Oil Research, vol. 13, no. 1, pp. 61-62. http://dx.doi.org/10.1080/10412905.2001.9699607.
http://dx.doi.org/10.1080/10412905.2001....
). Even though this study is the first report of the chemical composition of PG-EO (flowers), it may be noticed that the chemical composition found by this study is similar to the chemical composition which has already been described in the literature for other species belonging to the same genus and Myrtaceae family (Stefanello et al., 2011STEFANELLO, M.É.A., PASCOAL, A.C.R.F. and SALVADOR, M.J., 2011. Essential oils from neotropical Myrtaceae: chemical diversity and biological properties. Chemistry & Biodiversity, vol. 8, no. 1, pp. 73-94. http://dx.doi.org/10.1002/cbdv.201000098. PMid:21259421.
http://dx.doi.org/10.1002/cbdv.201000098...
).

In the literature, differences in chemical compositions of essential oils from this botanical species may be due to the method of extraction, region of origin of the plant, climate, soil composition, plant organ, age, seasonality and circadian cycle. These factors affect the quality and quantity of the composition of essential oils, besides biotic and abiotic agents. It is known that chemotypical variation guides the use of plants, since chemical characterization and chemotype identification enable more refined chemical and agricultural analyses to be carried out (Souza et al., 2017SOUZA, T.S., FERREIRA, M.F.S., MENINI, L., SOUZA, J.R.C.L., PARREIRA, L.A., CECON, P.R. and FERREIRA, A., 2017. Essential oil of Psidium guajava: influence of genotypes and environment. Scientia Horticulturae, vol. 216, no. 14, pp. 38-44. http://dx.doi.org/10.1016/j.scienta.2016.12.026.
http://dx.doi.org/10.1016/j.scienta.2016...
; Bouyahya et al., 2019BOUYAHYA, A., BELMEHDI, O., EL JEMLI, M., MARMOUZI, I., BOURAIS, I., ABRINI, J., FAOUZI, M.E.A., DAKKA, N. and BAKRI, Y., 2019. Chemical variability of Centaurium erythraea essential oils at the three developmental stages and investigation of their in vitro antioxidant, antidiabetic, dermatoprotective and antibacterial activities. Industrial Crops and Products, vol. 132, pp. 111-117. http://dx.doi.org/10.1016/j.indcrop.2019.01.042.
http://dx.doi.org/10.1016/j.indcrop.2019...
).

Minimum inhibitory concentrations values determined for PG-EO against X. fastidiosa and P. carotovorum were 12.5 μg/mL and 62.5 μg/mL, respectively. Streptomycin was the positive control and its MIC value was 0.0368 μg/mL. Regarding the antibacterial activity of natural products, Holetz et al. (2002)HOLETZ, F.B., PESSINI, G.L., SANCHES, N.R., CORTEZ, D.A.G., NAKAMURA, C.V. and DIAS FILHO, B.P., 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infections diseases. Memorias do Instituto Oswaldo Cruz, vol. 97, no. 7, pp. 1027-1031. http://dx.doi.org/10.1590/S0074-02762002000700017. PMid:12471432.
http://dx.doi.org/10.1590/S0074-02762002...
showed that samples with good, moderate and weak antibacterial activity and inactivity have MIC values below 100 µg/mL, from 100 to 500 µg/mL, from 500 to 1000 µg/mL and above 1000 µg/mL, respectively. Results showed that PG-EO exhibited high in vitro antibacterial activity against X. fastidiosa (MIC = 12.5 µg/mL) and P. carotovorum (MIC = 62.5 µg/mL) and may have promising activity against phytopathogenic bacteria. It should be highlighted that PG-EO exhibited more promising in vitro anti-Xyllela fastidiosa activity than the ones of seventeen essential oils whose MIC values had already been reported by the literature (Santiago et al., 2018SANTIAGO, M.B., MORAES, T.S., MASSUCO, J.E., SILVA, L.O., LUCARINI, R., SILVA, D.F., VIEIRA, T.M., CROTTI, A.E.M. and MARTINS, C.H.G., 2018. In vitro evaluation of essential oils for potential antibacterial effects against Xylella fastidiosa. Journal of Phytopathology, vol. 116, no. 11-12, pp. 790-798. http://dx.doi.org/10.1111/jph.12762.
http://dx.doi.org/10.1111/jph.12762...
). In addition, essential oil from P. guajava leaves has already been described as a natural product which has potential activity against Sclerotinia sclerotiorum, a phytopathogen that also causes damage to agriculture (Silva et al., 2018SILVA, E.A.J., SILVA, V.P., ALVES, C.C.F., ALVES, J.M., SOUCHIE, E.L. and BARBOSA, L.C.A., 2018. Chemical composition of the essential oil of Psidium guajava leaves and its toxicity against Sclerotinia sclerotiorum. Semina: Ciências Agrárias, vol. 39, no. 2, pp. 865-874. http://dx.doi.org/10.5433/1679-0359.2018v39n2p865.
http://dx.doi.org/10.5433/1679-0359.2018...
).

The excellent antibacterial activity exhibited by PG-EO against both phytopathogenic bacteria under investigation may be justified by the high concentrations of its major constituents, since they have already had their antibacterial potential described by the literature. For instance, α-cadinol, β-caryophyllene, α-selinene, β-selinene and caryophyllene oxide have already been identified as major constituents of essential oil from Teucrium yemense, whose satisfactory antibacterial activity has been shown by the disc diffusion test and the broth microdilution test (Ali et al., 2017ALI, N.A.A., CHHETRI, B.K., DOSOKY, N.S., SHARI, K., AL-FAHAD, A.J.A., WESSJOHANN, L. and SETZER, W.N., 2017. Antimicrobial, antioxidante, and cytotoxic activities of Ocimum forskolei and Teucrium yemense (Lamiaceae) essential oils. Medicines (Basel, Switzerland), vol. 4, no. 2, pp. 1-17. http://dx.doi.org/10.3390/medicines4020017. PMid:28930232.
http://dx.doi.org/10.3390/medicines40200...
). The third major constituent found in PG-EO – nerolidol – may have significantly contributed to good results of anti-Xylella fastidiosa and anti-Pectobacterium carotovorum activities, since this sesquiterpene has relevant antibacterial activity and has been considered a promising chemical or drug candidate in the field of agriculture by the specialized literature (Chan et al., 2016CHAN, W.K., TAN, L.T.H., CHAN, K.G., LEE, L.H. and GOH, B.H., 2016. Nerolidol: a sesquiterpene alcohol with multi-faceted pharmacological and biological activities. Molecules (Basel, Switzerland), vol. 21, no. 5, pp. 529. http://dx.doi.org/10.3390/molecules21050529. PMid:27136520.
http://dx.doi.org/10.3390/molecules21050...
). According to Bajpai et al. (2011)BAJPAI, V.K., KANG, S.-R., XU, H., LEE, S.-G., BAEK, K.-H. and KANG, S.-C., 2011. Potential roles of essential oils on controlling plant pathogenic bacteria Xanthomonas species: a review. The Plant Pathology Journal, vol. 27, no. 3, pp. 207-224. http://dx.doi.org/10.5423/PPJ.2011.27.3.207.
http://dx.doi.org/10.5423/PPJ.2011.27.3....
, certain essential oils act in many ways on various types of disease complex and may be applied onto the important crop plants in the same way as other agricultural chemicals. These oils can be used as a leading factor in a wide range of activities against many plant pathogenic bacteria, where these pathogens have developed resistance against the specific bactericide (Bajpai et al., 2011BAJPAI, V.K., KANG, S.-R., XU, H., LEE, S.-G., BAEK, K.-H. and KANG, S.-C., 2011. Potential roles of essential oils on controlling plant pathogenic bacteria Xanthomonas species: a review. The Plant Pathology Journal, vol. 27, no. 3, pp. 207-224. http://dx.doi.org/10.5423/PPJ.2011.27.3.207.
http://dx.doi.org/10.5423/PPJ.2011.27.3....
).

It should be emphasized that, in addition to the classes (sesquiterpene hydrocarbons and oxygenated sesquiterpenes) to which the constituents belong, other factors, such as isomerism and synergism among components, must be taken into account when antibacterial activity is evaluated (Costa et al., 2017COSTA, E.C.C., CHRISTOFOLI, M., COSTA, G.C.S., PEIXOTO, M.F., FERNANDES, J.B., FORIM, M.R., PEREIRA, K.C., SILVA, F.G. and CAZAL, C.M., 2017. Essential oil repellent action of plants of the genus Zanthoxylum against Bemisia tabaci biotype B (Homoptera: aleyrodidae). Scientia Horticulturae, vol. 226, no. 19, pp. 327-332. http://dx.doi.org/10.1016/j.scienta.2017.08.041.
http://dx.doi.org/10.1016/j.scienta.2017...
). In short, the results in this study showed that PG-EO had a strong antibacterial activity against X. fastidiosa and P. carotovorum. It could be attributed also to its constituents of sabinene, α-pinene, β-pinene, limonene and β-caryophyllene (Table 1), which appear to make the cell membrane permeably and disintegrate the outer membrane of Gram-negative bacteria (Zhang et al., 2017ZHANG, J., YE, K.P., ZHANG, X., PAN, D.D., SUN, Y.Y. and CAO, J.X., 2017. Antibacterial activity and mechanism of action of black pepper essential oil on meat-borne Escherichia coli. Frontiers in Microbiology, vol. 7, no. 4, pp. 2094. http://dx.doi.org/10.3389/fmicb.2016.02094. PMid:28101081.
http://dx.doi.org/10.3389/fmicb.2016.020...
).

In relation to the trypanocidal activity investigated, the essential oil from flowers of P. guajava has been shown to be active against trypomastigote forms of Trypanosoma cruzi. There was reduction in the viability of trypomastigote cells with increased concentration of essential oil. Thus, the essential oil exhibited satisfactory trypanocidal activity with IC50 = 14.6 μg/mL compared to positive control using benznidazole (positive control) with IC50 = 9.8 μg/mL (Table 2).

Table 2
Trypanocidal activity in vitro of the essential oil from flowers of P. guajava (Myrtaceae).

A current study with the essential oil of Eugenia dysenterica dried leaves reports that essential oil samples with IC50 <10 μg/mL had trypanocidal activity considered to be highly active, active (IC50 > 10 < 50 µg/mL), moderately active (IC50 > 50 < 100 µg/mL) and inactive (IC50 > 100 µg/mL) against trypomastigote forms of T. cruzi (Santos et al., 2019SANTOS, L.S., ALVES, C.C.F., ESTEVAM, E.B.B., MARTINS, C.H.G., SILVA, T.S., ESPERANDIM, V.R. and MIRANDA, M.L.D., 2019. Chemical composition, in vitro trypanocidal and antibacterial activities of the essential oil from the dried leaves of Eugenia dysenterica DC from Brazil. Journal of Essential Oil Bearing Plants, vol. 22, no. 2, pp. 347-355. http://dx.doi.org/10.1080/0972060X.2019.1626293.
http://dx.doi.org/10.1080/0972060X.2019....
).

The antiparasitic activity presented by the essential oil of P. guajava flowers can be attributed to the synergism between the constituents present in the essential oil analyzed (Bakkali et al., 2008BAKKALI, F., AVERBECK, S., AVERBECK, D. and IDAOMAR, M., 2008. Biological effects of essential oils – a review. Food and Chemical Toxicology, vol. 46, no. 2, pp. 446-475. http://dx.doi.org/10.1016/j.fct.2007.09.106. PMid:17996351.
http://dx.doi.org/10.1016/j.fct.2007.09....
). In addition, among the constituents present in the oil, there are those that already have recognized trypanocidal activity reported in the literature, such as terpenes: α-cadinol (37.8%), β-caryophyllene (12.2%), nerolidol (9.1%), α-selinene (8.8%), β-selinene (7.4%), and caryophyllene oxide (7.2%) (Table 1) previously identified in the essential oils of the species Annona vepretorum, A. squamosa, Cymbopognon giganteus, C. nardus, C. citratus, C. schoenantus, Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, oils that also had significant trypanocidal effect (Meira et al., 2014MEIRA, C.S., GUIMARÃES, E.T., MACEDO, T.S., DA SILVA, T.B., MENEZES, L.R.A., COSTA, E.V. and SOARES, M.B.P., 2014. Chemical composition of essential oils from Annona vepretorum Mart. and Annona squamosa L. (Annonaceae) leaves and their antimalarial and trypanocidal activities. The Journal of Essential Oil Research, vol. 27, no. 2, pp. 160-168. http://dx.doi.org/10.1080/10412905.2014.982876.
http://dx.doi.org/10.1080/10412905.2014....
; Kpoviessi et al., 2014KPOVIESSI, S., BERO, J., AGBANI, P., GBAGUIDI, F., KPADONOU-KPOVIESSI, B., SINSIN, B., ACCROMBESSI, G., FRÉDÉRICH, M., MOUDACHIROU, M. and QUETIN-LECLERCQ, J., 2014. Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin. Journal of Ethnopharmacology, vol. 151, no. 1, pp. 652-659. http://dx.doi.org/10.1016/j.jep.2013.11.027. PMid:24269775.
http://dx.doi.org/10.1016/j.jep.2013.11....
; Nibret and Wink, 2010NIBRET, E. and WINK, M., 2010. Trypanocidal and antileukaemic effects of the essential oils of Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, and their main individual constituents. Phytomedicine, vol. 17, no. 12, pp. 911-920. http://dx.doi.org/10.1016/j.phymed.2010.02.009. PMid:20359874.
http://dx.doi.org/10.1016/j.phymed.2010....
).

The various biological activities of essential oils, including against trypanosomatids, are mainly due to their terpenic composition and to the aforementioned synergism among their constituents (Borges et al., 2012BORGES, A.R., AIRES, J.R.A., HIGINO, T.M.M., MEDEIROS, M.G.F., CITÓ, A.M.G.L., LOPES, J.A.D. and FIGUEIREDO, R.C.B.Q., 2012. Trypanocidal and cytotoxic activities of essential oils from medicinal plants of northeast of Brazil. Experimental Parasitology, vol. 132, no. 2, pp. 123-128. http://dx.doi.org/10.1016/j.exppara.2012.06.003. PMid:22771867.
http://dx.doi.org/10.1016/j.exppara.2012...
). The terpenes are responsible for the hydrophobic nature of the essential oils, allowing their diffusion through the cell membrane of the parasite thus affecting the metabolic pathways and intracellular organelles (Raut and Karuppayil, 2014RAUT, J.S. and KARUPPAYIL, S.M., 2014. A status review on the medicinal properties of essential oils. Industrial Crops and Products, vol. 62, no. 1, pp. 250-264. http://dx.doi.org/10.1016/j.indcrop.2014.05.055.
http://dx.doi.org/10.1016/j.indcrop.2014...
).

Cytotoxicity was assessed against LLCMK2 cells because the cell medium is the same as the parasites are cultured. The execution of this assay is justified since the efficacy of the sample against the parasites is proved without lysing the healthy cells at the same time as the parasites are lysed. Cultures of LLCMK2 adherent epithelial cells were treated with the essential oil at the concentrations of 6.25, 12.5, 25.0, 50.0, 100, 200 and 400 μg/mL for 24 h. The results showed that the essential oil from flowers of P. guajava presented moderate toxicity at the concentration evaluated, presenting CC50 = 250.5 μg/mL (Table 3) compared to the benznidazole positive control (CC50 = 147.3 μg/mL) and with data already reported in the literature (Carneiro et al., 2017CARNEIRO, N.S., ALVES, J.M., ALVES, C.C.F., ESPERANDIM, V.R. and MIRANDA, M.L.D., 2017. Óleo essencial das flores de Eugenia klotzchiana (Myrtaceae): composição química e atividades tripanocida e citotóxica in vitro. Revista Virtual de Química, vol. 9, no. 3, pp. 1381-1392. http://dx.doi.org/10.21577/1984-6835.20170080.
http://dx.doi.org/10.21577/1984-6835.201...
).

Table 3
Cytotoxic activity of the essential oil from flowers of P. guajava (Myrtaceae)

It is important to evaluate the cytotoxicity of a given sample because it makes it possible to elucidate the biological mechanism that generates the cytotoxic effect and the mechanism of action of different compounds during their interaction with tissues (51). Toxicity levels are reported in the literature as highly toxic CC50 < 10 μg/mL, toxic (10 < CC50 < 100 μg/mL), moderately toxic (100 < CC50 < 1000 μg/mL), and nontoxic (CC50 > 1000 μg/mL) (Andrade et al., 2018ANDRADE, P.M., MELO, D.C., ALCOBA, A.E.T., FERREIRA JÚNIOR, W.G., PAGOTTI, M.C., MAGALHÃES, L.G., SANTOS, T.C.L.D., CROTTI, A.E.M., ALVES, C.C.F. and MIRANDA, M.L.D., 2018. Chemical composition and evaluation of antileishmanial and cytotoxic activities of the essential oil from leaves of Cryptocarya aschersoniana Mez. (Lauraceae Juss.). Anais da Academia Brasileira de Ciências, vol. 90, no. 3, pp. 2671-2678. http://dx.doi.org/10.1590/0001-3765201820170332. PMid:30304213.
http://dx.doi.org/10.1590/0001-376520182...
; Lima et al., 2012LIMA, J.P.S., PINHEIRO, M.L.B., SANTOS, A.M.G., PEREIRA, J.L.S., SANTOS, D.M.F., BARISON, A., SILVA-JARDIM, I. and COSTA, E.V., 2012. In vitro antileishmanial and cytotoxic activities of Annona mucosa (Annonaceae). Revista Virtual de Química, vol. 4, no. 6, pp. 692-702. http://dx.doi.org/10.5935/1984-6835.20120052.
http://dx.doi.org/10.5935/1984-6835.2012...
; Camacho et al., 2003CAMACHO, M.R., PHILLIPSON, J.D., CROFT, S.L., SOLIS, P.N., MARSHALL, S.J. and GHAZANFAR, S.A., 2003. Screening of plant extracts for antiprotozoal and cytotoxic activities. Journal of Ethnopharmacology, vol. 89, no. 2-3, pp. 185-191. http://dx.doi.org/10.1016/S0378-8741(03)00269-1. PMid:14611881.
http://dx.doi.org/10.1016/S0378-8741(03)...
). The moderate cytotoxicity exhibited by the essential oil of P. guajava flowers against the LLCMK2 adherent epithelial cells is an indicator that this essential oil can be well tolerated by the biological system, however, further studies are still necessary to evaluate its toxicity in vivo.

4. Conclusion

In summary, results demonstrated that PG-EO exhibited a mixture of sesquiterpenes in its chemical composition. Its major constituents were α-cadinol, β-caryophyllene, nerolidol, α-selinene, β-selinene and caryophyllene oxide, since they exhibited the highest concentrations. The high concentration of α-cadinol (37.8%) in PG-EO investigated by this study is the prospect of a new source of the secondary metabolite as a raw material in the synthesis of a new bactericide. In addition, results of this study show that there is good prospect of using these essential oil experimentally to control phytopathogens in both greenhouse and field conditions. On the other hand, PG-EO can also be considered the source of an important secondary metabolite which may be applied to agriculture, i. e., nerolidol. From an environmentally sustainable perspective, PG-EO may become an alternative to the use of synthetic pesticides in agriculture and may act as a natural insecticide which is capable of protecting cultures of economic interest. The PG-EO also showed satisfactory trypanocidal activity against Trypanosoma cruzi trypomastigote forms and exhibited moderate cytotoxicity against LLCMK2 adherent epithelial cells. In sum, results provide support for further studies of PG-EO which aim at isolating bioactive compounds and investigating its in vivo antibacterial, cytotoxic and trypanocidal properties.

Acknowledgments

The authors are grateful to FAPEG, CNPq, CAPES, UNIFRAN and IF GOIANO – Campus Rio Verde for their financial support.

  • (With 1 figure)

References

  • ADAMS, R.P., 2007. In identification of essential oil components by gas chromatography/quadrupole mass spectroscopy 4th ed. Carol Stream: Allured Publishing Corporation, 804 p.
  • ALI, N.A.A., CHHETRI, B.K., DOSOKY, N.S., SHARI, K., AL-FAHAD, A.J.A., WESSJOHANN, L. and SETZER, W.N., 2017. Antimicrobial, antioxidante, and cytotoxic activities of Ocimum forskolei and Teucrium yemense (Lamiaceae) essential oils. Medicines (Basel, Switzerland), vol. 4, no. 2, pp. 1-17. http://dx.doi.org/10.3390/medicines4020017 PMid:28930232.
    » http://dx.doi.org/10.3390/medicines4020017
  • ANDRADE, M.A., CARDOSO, M., GOMES, M.S., DE AZEREDO, C.M., BATISTA, L.R., SOARES, M.J., RODRIGUES, L.M. and FIGUEIREDO, A.C., 2015. Biological activity of the essential oils from Cinnamodendron dinisii and Siparuna guianensis. Brazilian Journal of Microbiology, vol. 46, no. 1, pp. 189-194. http://dx.doi.org/10.1590/S1517-838246120130683 PMid:26221107.
    » http://dx.doi.org/10.1590/S1517-838246120130683
  • ANDRADE, P.M., MELO, D.C., ALCOBA, A.E.T., FERREIRA JÚNIOR, W.G., PAGOTTI, M.C., MAGALHÃES, L.G., SANTOS, T.C.L.D., CROTTI, A.E.M., ALVES, C.C.F. and MIRANDA, M.L.D., 2018. Chemical composition and evaluation of antileishmanial and cytotoxic activities of the essential oil from leaves of Cryptocarya aschersoniana Mez. (Lauraceae Juss.). Anais da Academia Brasileira de Ciências, vol. 90, no. 3, pp. 2671-2678. http://dx.doi.org/10.1590/0001-3765201820170332 PMid:30304213.
    » http://dx.doi.org/10.1590/0001-3765201820170332
  • BAJPAI, V.K., KANG, S.-R., XU, H., LEE, S.-G., BAEK, K.-H. and KANG, S.-C., 2011. Potential roles of essential oils on controlling plant pathogenic bacteria Xanthomonas species: a review. The Plant Pathology Journal, vol. 27, no. 3, pp. 207-224. http://dx.doi.org/10.5423/PPJ.2011.27.3.207
    » http://dx.doi.org/10.5423/PPJ.2011.27.3.207
  • BAKKALI, F., AVERBECK, S., AVERBECK, D. and IDAOMAR, M., 2008. Biological effects of essential oils – a review. Food and Chemical Toxicology, vol. 46, no. 2, pp. 446-475. http://dx.doi.org/10.1016/j.fct.2007.09.106 PMid:17996351.
    » http://dx.doi.org/10.1016/j.fct.2007.09.106
  • BLEVE, G., GALLO, A., ALTOMARE, C., VURRO, M., MAIORANO, G., CARDINALI, A., D’ANTUONO, I.D., MARCHI, G. and MITA, G., 2018. In vitro activity of antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS Microbiology Letters, vol. 365, no. 5, pp. fnx281. http://dx.doi.org/10.1093/femsle/fnx281 PMid:29390137.
    » http://dx.doi.org/10.1093/femsle/fnx281
  • BORGES, A.R., AIRES, J.R.A., HIGINO, T.M.M., MEDEIROS, M.G.F., CITÓ, A.M.G.L., LOPES, J.A.D. and FIGUEIREDO, R.C.B.Q., 2012. Trypanocidal and cytotoxic activities of essential oils from medicinal plants of northeast of Brazil. Experimental Parasitology, vol. 132, no. 2, pp. 123-128. http://dx.doi.org/10.1016/j.exppara.2012.06.003 PMid:22771867.
    » http://dx.doi.org/10.1016/j.exppara.2012.06.003
  • BOUYAHYA, A., BELMEHDI, O., EL JEMLI, M., MARMOUZI, I., BOURAIS, I., ABRINI, J., FAOUZI, M.E.A., DAKKA, N. and BAKRI, Y., 2019. Chemical variability of Centaurium erythraea essential oils at the three developmental stages and investigation of their in vitro antioxidant, antidiabetic, dermatoprotective and antibacterial activities. Industrial Crops and Products, vol. 132, pp. 111-117. http://dx.doi.org/10.1016/j.indcrop.2019.01.042
    » http://dx.doi.org/10.1016/j.indcrop.2019.01.042
  • CAMACHO, M.R., PHILLIPSON, J.D., CROFT, S.L., SOLIS, P.N., MARSHALL, S.J. and GHAZANFAR, S.A., 2003. Screening of plant extracts for antiprotozoal and cytotoxic activities. Journal of Ethnopharmacology, vol. 89, no. 2-3, pp. 185-191. http://dx.doi.org/10.1016/S0378-8741(03)00269-1 PMid:14611881.
    » http://dx.doi.org/10.1016/S0378-8741(03)00269-1
  • CARNEIRO, N.S., ALVES, J.M., ALVES, C.C.F., ESPERANDIM, V.R. and MIRANDA, M.L.D., 2017. Óleo essencial das flores de Eugenia klotzchiana (Myrtaceae): composição química e atividades tripanocida e citotóxica in vitro. Revista Virtual de Química, vol. 9, no. 3, pp. 1381-1392. http://dx.doi.org/10.21577/1984-6835.20170080
    » http://dx.doi.org/10.21577/1984-6835.20170080
  • CHAN, W.K., TAN, L.T.H., CHAN, K.G., LEE, L.H. and GOH, B.H., 2016. Nerolidol: a sesquiterpene alcohol with multi-faceted pharmacological and biological activities. Molecules (Basel, Switzerland), vol. 21, no. 5, pp. 529. http://dx.doi.org/10.3390/molecules21050529 PMid:27136520.
    » http://dx.doi.org/10.3390/molecules21050529
  • CLINICAL AND LABORATORY STANDARDS INSTITUTE – CLSI, 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard: M07-A9 9th ed. Wayne: CLSI.
  • COLETTA-FILHO, H.D., FRANCISCO, C.S., LOPES, J.R.S., OLIVEIRA, A.F. and SILVA, L.F.O., 2016. First report of olive leaf scorch in Brazil, associated with Xylella fastidiosa subsp. pauca. Phytopathologia Mediterranea, vol. 55, no. 1, pp. 130-135. http://dx.doi.org/10.14601/Phytopathol_Mediterr-17259
    » http://dx.doi.org/10.14601/Phytopathol_Mediterr-17259
  • COSTA, E.C.C., CHRISTOFOLI, M., COSTA, G.C.S., PEIXOTO, M.F., FERNANDES, J.B., FORIM, M.R., PEREIRA, K.C., SILVA, F.G. and CAZAL, C.M., 2017. Essential oil repellent action of plants of the genus Zanthoxylum against Bemisia tabaci biotype B (Homoptera: aleyrodidae). Scientia Horticulturae, vol. 226, no. 19, pp. 327-332. http://dx.doi.org/10.1016/j.scienta.2017.08.041
    » http://dx.doi.org/10.1016/j.scienta.2017.08.041
  • CZAJKOWSKI, R., PÉROMBELON, M.C.M., JAFRA, S., LOJKOWSKA, E., POTRYKUS, M., VAN DER WOLF, J.M. and SLEDZ, W., 2015. Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Annals of Applied Biology, vol. 166, no. 1, pp. 18-38. http://dx.doi.org/10.1111/aab.12166 PMid:25684775.
    » http://dx.doi.org/10.1111/aab.12166
  • DELMONDES, P.H. and STEFANI, R., 2018. In silico study of the antichagasic activity of aromatic compounds. Orbital: the Electronic Journal of Chemistry, vol. 10, no. 5, pp. 395-401. http://dx.doi.org/10.17807/orbital.v10i5.1018
    » http://dx.doi.org/10.17807/orbital.v10i5.1018
  • FELIX, K.C.S., OLIVEIRA, W.J., MARIANO, R.L.R. and SOUZA, E.B., 2014. Lettuce genotype resistance to “soft rot” caused by Pectobacterium carotovorum subsp. carotovorum. Scientia Agrícola, vol. 71, no. 4, pp. 287-291. http://dx.doi.org/10.1590/0103-9016-2013-0301
    » http://dx.doi.org/10.1590/0103-9016-2013-0301
  • GORMEZ, A., BOZARI, S., YANMIS, D., GULLUCE, M., SAHIN, F. and AGAR, G., 2015. Chemical composition and antibacterial activity of essential oils of two species of Lamiaceae against phytopathogenic bacteria. Polish Journal of Microbiology, vol. 64, no. 2, pp. 121-127. http://dx.doi.org/10.33073/pjm-2015-018 PMid:26373171.
    » http://dx.doi.org/10.33073/pjm-2015-018
  • GUIMARÃES, P.G., MOREIRA, I.S., CAMPOS FILHO, P.C., FERRAZ, J.L.A.A., NOVAES, Q.S. and BATISTA, R., 2015. Antibacterial activity of Schinopsis brasiliensis against phytopathogens of agricultural interest. Fitos, vol. 9, no. 3, pp. 161-252. http://dx.doi.org/10.5935/2446-4775.20150013
    » http://dx.doi.org/10.5935/2446-4775.20150013
  • HOLETZ, F.B., PESSINI, G.L., SANCHES, N.R., CORTEZ, D.A.G., NAKAMURA, C.V. and DIAS FILHO, B.P., 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infections diseases. Memorias do Instituto Oswaldo Cruz, vol. 97, no. 7, pp. 1027-1031. http://dx.doi.org/10.1590/S0074-02762002000700017 PMid:12471432.
    » http://dx.doi.org/10.1590/S0074-02762002000700017
  • HOTEZ, P.J., PECOUL, B., RIJAL, S., BOEHME, C., AKSOY, S., MALECELA, M., TAPIA-CONYER, R. and REEDER, J.C., 2016. Eliminating the neglected tropical diseases: translational science and new technologies. PLoS Neglected Tropical Diseases, vol. 10, no. 3, pp. e0003895. http://dx.doi.org/10.1371/journal.pntd.0003895 PMid:26934395.
    » http://dx.doi.org/10.1371/journal.pntd.0003895
  • KAMRAN, A., MISHRA, R.K., GUPTA, R., KUMAR, A., BAJAJ, A.K. and DIKSHIT, A., 2012. Therapeutic effects of essential oil from waste leaves of Psidium guajava L. against cosmetic embarrassment using phylogenetic approach. American Journal of Plant Sciences, vol. 3, no. 6, pp. 745-752. http://dx.doi.org/10.4236/ajps.2012.36090
    » http://dx.doi.org/10.4236/ajps.2012.36090
  • KHADHRI, A., EL MOKNI, R., ALMEIDA, C., NOGUEIRA, J.M.F. and ARAÚJO, M.E.M., 2014. Chemical composition of essential oil of Psidium guajava L. growing in Tunisia. Industrial Crops and Products, vol. 52, no. 1, pp. 29-31. http://dx.doi.org/10.1016/j.indcrop.2013.10.018
    » http://dx.doi.org/10.1016/j.indcrop.2013.10.018
  • KPOVIESSI, S., BERO, J., AGBANI, P., GBAGUIDI, F., KPADONOU-KPOVIESSI, B., SINSIN, B., ACCROMBESSI, G., FRÉDÉRICH, M., MOUDACHIROU, M. and QUETIN-LECLERCQ, J., 2014. Chemical composition, cytotoxicity and in vitro antitrypanosomal and antiplasmodial activity of the essential oils of four Cymbopogon species from Benin. Journal of Ethnopharmacology, vol. 151, no. 1, pp. 652-659. http://dx.doi.org/10.1016/j.jep.2013.11.027 PMid:24269775.
    » http://dx.doi.org/10.1016/j.jep.2013.11.027
  • LAVORATO, S.N., SALES JÚNIOR, P.A., MURTA, S.M.F., ROMANHA, A.J. and ALVES, R.J., 2015. In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures. Memorias do Instituto Oswaldo Cruz, vol. 110, no. 4, pp. 566-568. http://dx.doi.org/10.1590/0074-02760150007 PMid:26061148.
    » http://dx.doi.org/10.1590/0074-02760150007
  • LEMES, R.S., ALVES, C.C.F., ESTEVAM, E.B.B., SANTIAGO, M.B., MARTINS, C.H.G., SANTOS, T.C.L., CROTTI, A.E.M. and MIRANDA, M.L.D., 2018. Chemical composition and antibacterial activity of essential oils from Citrus aurantifolia leaves and fruit peel against oral pathogenic bacteria. Anais da Academia Brasileira de Ciências, vol. 90, no. 2, pp. 1285-1292. http://dx.doi.org/10.1590/0001-3765201820170847 PMid:29898096.
    » http://dx.doi.org/10.1590/0001-3765201820170847
  • LIMA, J.P.S., PINHEIRO, M.L.B., SANTOS, A.M.G., PEREIRA, J.L.S., SANTOS, D.M.F., BARISON, A., SILVA-JARDIM, I. and COSTA, E.V., 2012. In vitro antileishmanial and cytotoxic activities of Annona mucosa (Annonaceae). Revista Virtual de Química, vol. 4, no. 6, pp. 692-702. http://dx.doi.org/10.5935/1984-6835.20120052
    » http://dx.doi.org/10.5935/1984-6835.20120052
  • MEIRA, C.S., GUIMARÃES, E.T., MACEDO, T.S., DA SILVA, T.B., MENEZES, L.R.A., COSTA, E.V. and SOARES, M.B.P., 2014. Chemical composition of essential oils from Annona vepretorum Mart. and Annona squamosa L. (Annonaceae) leaves and their antimalarial and trypanocidal activities. The Journal of Essential Oil Research, vol. 27, no. 2, pp. 160-168. http://dx.doi.org/10.1080/10412905.2014.982876
    » http://dx.doi.org/10.1080/10412905.2014.982876
  • MENDES, L.A., SOUZA, T.S., MENINI, L., GUILHEN, J.H.S., BERNARDES, C.O., FERREIRA, A. and FERREIRA, M.F.S., 2018. Spring alterations in the chromatographic profile of leaf essential oils of improved guava genotypes in Brazil. Scientia Horticulturae, vol. 238, no. 19, pp. 295-302. http://dx.doi.org/10.1016/j.scienta.2018.04.065
    » http://dx.doi.org/10.1016/j.scienta.2018.04.065
  • NGAHANG KAMTE, S.L., RANJBARIAN, F., CIANFAGLIONE, K., SUT, S., DALL’ACQUA, S., BRUNO, M., AFSHAR, F.H., IANNARELLI, R., BENELLI, G., CAPPELLACCI, L., HOFER, A., MAGGI, F. and PETRELLI, R., 2018. Identification of highly effective antitrypanosomal compounds in essential oils from the Apiaceae family. Ecotoxicology and Environmental Safety, vol. 156, no. 30, pp. 154-165. http://dx.doi.org/10.1016/j.ecoenv.2018.03.032 PMid:29549739.
    » http://dx.doi.org/10.1016/j.ecoenv.2018.03.032
  • NIBRET, E. and WINK, M., 2010. Trypanocidal and antileukaemic effects of the essential oils of Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, and their main individual constituents. Phytomedicine, vol. 17, no. 12, pp. 911-920. http://dx.doi.org/10.1016/j.phymed.2010.02.009 PMid:20359874.
    » http://dx.doi.org/10.1016/j.phymed.2010.02.009
  • OGUNWANDE, I.A., OLAWORE, N.O., ADELEKE, K.A., EKUNDAYO, O. and KOENIG, W.A., 2003. Chemical composition of the leaf volatile oil of Psidium guajava L. growing in Nigeria. Flavour and Fragrance Journal, vol. 18, no. 2, pp. 136-138. http://dx.doi.org/10.1002/ffj.1175
    » http://dx.doi.org/10.1002/ffj.1175
  • OLIVEIRA, M.F., NAGAO-DIAS, A.T., PONTES, V.M.O., SOUSA JÚNIOR, A.S., COELHO, H.L.L. and COELHO, I.C.B., 2008. Tratamento etiológico da Doença de Chagas no Brasil. Revista de Patologia Tropical, vol. 37, no. 3, pp. 209-228. http://dx.doi.org/10.5216/rpt.v37i3.5063
    » http://dx.doi.org/10.5216/rpt.v37i3.5063
  • PANDEY, A.K., SINGH, P., PALNI, U.T. and TRIPATHI, N.N., 2013. Application of Chenopodium ambrosioides Linn. essential oil as botanical fungicide for the management of fungal deterioration in pulses. Biological Agriculture and Horticulture, vol. 29, no. 3, pp. 197-208. http://dx.doi.org/10.1080/01448765.2013.822828
    » http://dx.doi.org/10.1080/01448765.2013.822828
  • PANNEERSELVAM, P., MOHANDAS, S., SARITHA, B., UPRETI, K.K., POOVARASAN., MONNAPPA, A. and SULLADMATH, V.V., 2012. Glomus mosseae associated bacteria and their influence on stimulation of mycorrhizal colonization, sporulation, and growth promotion in guava (Psidium guajava L.) seedlings. Biological Agriculture and Horticulture, vol. 28, no. 4, pp. 267-279. http://dx.doi.org/10.1080/01448765.2012.741108
    » http://dx.doi.org/10.1080/01448765.2012.741108
  • PASSOS, L.A.C., GUARALDO, A.M.A., BARBOSA, R.L., DIAS, V.L., PEREIRA, K.S., SCHMIDT, F.L., FRANCO, R.M.B. and ALVES, D.P., 2012. Sobrevivência e infectividade do Trypanosoma cruzi na polpa de açaí: estudo in vitro e in vivo. Epidemiologia e Serviços de Saúde : Revista do Sistema Unico de Saúde do Brasil, vol. 21, no. 2, pp. 223-232. http://dx.doi.org/10.5123/S1679-49742012000200005
    » http://dx.doi.org/10.5123/S1679-49742012000200005
  • PEREIRA, F.M., USMAN, M., MAYER, N.A., NACHTIGAL, J.C., MAPHANGA, O.R.M. and WILLEMSE, S., 2017. Advances in guava propagation. Revista Brasileira de Fruticultura, vol. 39, no. 4, pp. e-358. http://dx.doi.org/10.1590/0100-29452017358
    » http://dx.doi.org/10.1590/0100-29452017358
  • PINO, J.A., AGUERO, J., MARBOT, R. and FUENTES, V., 2001. Leaf oil of Psidium guajava L. from Cuba. The Journal of Essential Oil Research, vol. 13, no. 1, pp. 61-62. http://dx.doi.org/10.1080/10412905.2001.9699607
    » http://dx.doi.org/10.1080/10412905.2001.9699607
  • RASHED, K., FERREIRA, D.S., ESPERANDIM, V.R., MARÇAL, M.G., SEQUEIRA, B.M., FLAUZINO, L.G.B., CUNHA, W.R., 2016. In vitro trypanocidal activity of the Egyptian plant Schinopsis lorentizii against trypomastigote and amastigote forms of Trypanosoma cruzi Journal of Applied Pharmaceutical Science, vol. 6, no. 6, pp. 55-60.
  • RAUT, J.S. and KARUPPAYIL, S.M., 2014. A status review on the medicinal properties of essential oils. Industrial Crops and Products, vol. 62, no. 1, pp. 250-264. http://dx.doi.org/10.1016/j.indcrop.2014.05.055
    » http://dx.doi.org/10.1016/j.indcrop.2014.05.055
  • SANTIAGO, M.B., MORAES, T.S., MASSUCO, J.E., SILVA, L.O., LUCARINI, R., SILVA, D.F., VIEIRA, T.M., CROTTI, A.E.M. and MARTINS, C.H.G., 2018. In vitro evaluation of essential oils for potential antibacterial effects against Xylella fastidiosa. Journal of Phytopathology, vol. 116, no. 11-12, pp. 790-798. http://dx.doi.org/10.1111/jph.12762
    » http://dx.doi.org/10.1111/jph.12762
  • SANTOS, L.S., ALVES, C.C.F., ESTEVAM, E.B.B., MARTINS, C.H.G., SILVA, T.S., ESPERANDIM, V.R. and MIRANDA, M.L.D., 2019. Chemical composition, in vitro trypanocidal and antibacterial activities of the essential oil from the dried leaves of Eugenia dysenterica DC from Brazil. Journal of Essential Oil Bearing Plants, vol. 22, no. 2, pp. 347-355. http://dx.doi.org/10.1080/0972060X.2019.1626293
    » http://dx.doi.org/10.1080/0972060X.2019.1626293
  • SARKER, S.D., NAHAR, L. and KUMARASAMY, Y., 2007. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods (San Diego, Calif.), vol. 42, no. 4, pp. 321-324. http://dx.doi.org/10.1016/j.ymeth.2007.01.006 PMid:17560319.
    » http://dx.doi.org/10.1016/j.ymeth.2007.01.006
  • SARKHOSH, A., SCHAFFER, B., VARGAS, A.I., PALMATEER, A.J., LOPEZ, P., SOLEYMANI, A. and FARZANEH, M., 2018. Antifungal activity of five plant-extracted essential oils against anthracnose in papaya fruit. Biological Agriculture and Horticulture, vol. 34, no. 1, pp. 18-26. http://dx.doi.org/10.1080/01448765.2017.1358667
    » http://dx.doi.org/10.1080/01448765.2017.1358667
  • SATYAL, P., PAUDEL, P., LAMICHHANE, B. and SETZER, W.N., 2015. Leaf essential oil composition and bioactivity of Psidium guajava from Kathmandu, Nepal. American Journal of Essential Oils and Natural Products, vol. 3, no. 2, pp. 11-14.
  • SILVA, E.A.J., SILVA, V.P., ALVES, C.C.F., ALVES, J.M., SOUCHIE, E.L. and BARBOSA, L.C.A., 2018. Chemical composition of the essential oil of Psidium guajava leaves and its toxicity against Sclerotinia sclerotiorum. Semina: Ciências Agrárias, vol. 39, no. 2, pp. 865-874. http://dx.doi.org/10.5433/1679-0359.2018v39n2p865
    » http://dx.doi.org/10.5433/1679-0359.2018v39n2p865
  • SOUZA, T.S., FERREIRA, M.F.S., MENINI, L., SOUZA, J.R.C.L., PARREIRA, L.A., CECON, P.R. and FERREIRA, A., 2017. Essential oil of Psidium guajava: influence of genotypes and environment. Scientia Horticulturae, vol. 216, no. 14, pp. 38-44. http://dx.doi.org/10.1016/j.scienta.2016.12.026
    » http://dx.doi.org/10.1016/j.scienta.2016.12.026
  • STEFANELLO, M.É.A., PASCOAL, A.C.R.F. and SALVADOR, M.J., 2011. Essential oils from neotropical Myrtaceae: chemical diversity and biological properties. Chemistry & Biodiversity, vol. 8, no. 1, pp. 73-94. http://dx.doi.org/10.1002/cbdv.201000098 PMid:21259421.
    » http://dx.doi.org/10.1002/cbdv.201000098
  • VALADARES, A.C.F., ALVES, C.C.F., ALVES, J.M., DE DEUS, I.P.B., OLIVEIRA FILHO, J.G., SANTOS, T.C.L., DIAS, H.J., CROTTI, A.E.M. and MIRANDA, M.L.D., 2018. Essential oils from Piper aduncum inflorescences and leaves: chemical composition and antifungal activity against Sclerotinia sclerotiorum. Anais da Academia Brasileira de Ciências, vol. 90, no. 3, pp. 2691-2699. http://dx.doi.org/10.1590/0001-3765201820180033 PMid:30304214.
    » http://dx.doi.org/10.1590/0001-3765201820180033
  • VASINAUSKIENE, M., RADUSIENE, J., ZITIKAITE, I. and SURVILIENE, E., 2006. Antibacterial activities of essential oils from aromatic and medicinal plants against growth of phytopathogenic bacteria. Agronomy Research (Tartu), vol. 4, pp. 437-440.
  • XU, C., LIANG, Z., TANG, D., XIAO, T., TSUNODA, M., ZHANG, Y., ZHAO, L., DENG, S. and SONG, Y., 2017. Gas chromatography-mass spectrometry (GC-MS) analysis of volatile components from guava leaves. Journal of Essential Oil Bearing Plants, vol. 20, no. 6, pp. 1536-1546. http://dx.doi.org/10.1080/0972060X.2017.1417746
    » http://dx.doi.org/10.1080/0972060X.2017.1417746
  • ZHANG, J., YE, K.P., ZHANG, X., PAN, D.D., SUN, Y.Y. and CAO, J.X., 2017. Antibacterial activity and mechanism of action of black pepper essential oil on meat-borne Escherichia coli. Frontiers in Microbiology, vol. 7, no. 4, pp. 2094. http://dx.doi.org/10.3389/fmicb.2016.02094 PMid:28101081.
    » http://dx.doi.org/10.3389/fmicb.2016.02094

Publication Dates

  • Publication in this collection
    14 Aug 2020
  • Date of issue
    Jul-Sep 2021

History

  • Received
    29 Oct 2019
  • Accepted
    30 Jan 2020
Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
E-mail: bjb@bjb.com.br