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Vernonanthura polyanthes leaves aqueous extract enhances doxorubicin genotoxicity in somatic cells of Drosophila melanogaster and presents no antifungal activity against Candida spp.

O extrato aquoso das folhas de Vernonanthura polyanthes potencializa a genotoxicidade da doxorrubicina em células somáticas de Drosophila melanogaster e não apresenta atividade antifúngica contra Candida spp.

Abstract

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (Asteraceae), known as “assa-peixe”, has been used in ethnomedicine for the treatment of various diseases such as bronchitis, pneumonia, hemoptysis, persistent cough, internal abscesses, gastric and kidney stone pain. Moreover, some studies demonstrated that species of Genus Vernonia present antifungal activity. Due to the biological relevance of this species, the aim of this study was to investigate the toxic, genotoxic, antigenotoxic and antifungal potential of V. polyanthes leaves aqueous extract in somatic cells of Drosophila melanogaster or against Candida spp. The aqueous extract of the plant showed no toxic, genotoxic and antigenotoxic activity in the experimental conditions tested using the wing somatic mutation and recombination test (SMART/wing). However, when the extract was associated with doxorubicin, used in this work as a positive control, the mutagenic potential of doxorubicin was enhanced, increasing the number of mutations in D. melanogaster somatic cells. In the other hand, no inhibitory activity against Candida spp. was observed for V. polyanthes leaves aqueous extract using agar-well diffusion assay. More studies are necessary to reveal the components present in the V. polyanthes leaves aqueous extract that could contribute to potentiate the doxorubicin genotoxicity.

Keywords:
assa-peixe; Asteraceae; Cerrado; SMART/wing test; Vernonia polyanthes

Resumo

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (Asteraceae), conhecida como “assa-peixe”, tem sido utilizada na medicina popular para o tratamento de várias doenças, como bronquite, pneumonia, hemoptise, tosse persistente, abcessos internos, afecções gástricas e cálculo renal. Além disso, alguns estudos já demonstraram que espécies do Gênero Vernonia apresentam atividade antifúngica. Devido à relevância biológica dessa espécie, o objetivo deste estudo foi investigar os efeitos citotóxico, genotóxico, antigenotóxico e antifúngico do extrato aquoso das folhas de V. polyanthes em células somáticas de Drosophila melanogaster ou contra Candida spp. O extrato aquoso da planta não apresentou atividade citotóxica, genotóxica e antigenotóxica nas condições experimentais testadas usando o teste de recombinação e mutação somática em asa (SMART-asa). No entanto, quando o extrato foi associado com a doxorrubicina, utilizada neste trabalho como controle positivo, o potencial mutagênico da doxorrubicina foi potencializado, aumentando o número de mutações em células somáticas de D. melanogaster. Por outro lado, nenhuma atividade inibitória contra Candida spp. foi observada utilizando o extrato aquoso das folhas de V. polyanthes por meio do método de difusão em ágar. Mais estudos são necessários para desvendar os componentes presentes no extrato aquoso das folhas de V. polyanthes que possam contribuir para potencializar a genotoxicidade da doxorrubicina.

Palavras-chave:
assa-peixe; Asteraceae; Cerrado; teste SMART/asa; Vernonia polyanthes

1 Introduction

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. [synonymous of Vernonanthura phosphorica (Vell.) H. Rob. according to Vega and Dematteis (2010)Vega, A.J. and Dematteis, M., 2010. The transfer of Vernonia perangusta to the genus (Vernonieae, Asteraceae) and the correct name for VernonanthuraVernonanthura phosphorica.Phytotaxa, vol. 8, no. 1, pp. 46-50. http://dx.doi.org/10.11646/phytotaxa.8.1.5.
http://dx.doi.org/10.11646/phytotaxa.8.1...
] belongs to the plant family Asteraceae and is popularly known as “assa-peixe”. Due to the complexity of the Vernonia Genus, including around 1000 species (Keeley and Jones, 1979Keeley, S.C. and Jones, S., 1979. Distribution of pollen types in . Vernonia (Vernonieae - Compositae)Systematic Botany, vol. 4, no. 3, pp. 195-202. http://dx.doi.org/10.2307/2418418.
http://dx.doi.org/10.2307/2418418...
), Robinson (1999)Robinson, H., 1999. Generic and subtribal classification of American Vernonieae. Smithsonian Contributions to Botany, no. 89, pp. 1-116. http://dx.doi.org/10.5479/si.0081024X.89.
http://dx.doi.org/10.5479/si.0081024X.89...
segregated most of its South America species in 22 new genera, including Vernonathura, and after Vernonia polyanthes was combined as Vernonanthura polyanthes (Vega and Dematteis, 2010Vega, A.J. and Dematteis, M., 2010. The transfer of Vernonia perangusta to the genus (Vernonieae, Asteraceae) and the correct name for VernonanthuraVernonanthura phosphorica.Phytotaxa, vol. 8, no. 1, pp. 46-50. http://dx.doi.org/10.11646/phytotaxa.8.1.5.
http://dx.doi.org/10.11646/phytotaxa.8.1...
), the focus of this work. Metabolomic investigation corroborates the taxonomical classification, suggesting that Vernonia polyanthes should be considered as belonging to the Vernonanthura genus (Martucci et al., 2014Martucci, M.E.P., Vos, R.C.H., Carollo, C.A. and Gobbo-Neto, L., 2014. Metabolomics as a Potential Chemotaxonomical Tool: Application in the Genus . Vernonia SchrebPLoS One, vol. 9, no. 4, pp. 1-8. http://dx.doi.org/10.1371/journal.pone.0093149. PMid:24736747.
http://dx.doi.org/10.1371/journal.pone.0...
).

The species belonging to Vernonia Genus can be found in a wide range of habitats of broad ecological diversity and climatic conditions, but especially in tropical regions. Many of them are used as food and medicine (Toyang and Verpoorte, 2013Toyang, N.J. and Verpoorte, R., 2013. Review of the medicinal potentials of plants of the genus (Asteraceae). VernoniaJournal of Ethnopharmacology, vol.146, no. 3, pp. 681-723. PMid:23395623.). It has been reported that plants belonging to Vernonia Genus presents a plenty of bioactivities, including antiplasmodial, antileishmanial, antischistosomial, cytotoxicity, antimicrobial and anti-inflammatory (Toyang and Verpoorte, 2013Toyang, N.J. and Verpoorte, R., 2013. Review of the medicinal potentials of plants of the genus (Asteraceae). VernoniaJournal of Ethnopharmacology, vol.146, no. 3, pp. 681-723. PMid:23395623.). The Vernonanthura species represent the “assa-peixes” used in Brazil in the composition of syrups for the treatment of flus and colds (Leitão et al., 2014Leitão, F., Leitão, S.G., Fonseca-Kruel, V.S., Silva, I.M. and Martins, K., 2014. Medicinal plants traded in the open-air markets in the State of Rio de Janeiro, Brazil: an overview on their botanical diversity and toxicological potential. Revista Brasileira de Farmacognosia, vol. 24, no. 2, pp. 225-247. http://dx.doi.org/10.1016/j.bjp.2014.04.005.
http://dx.doi.org/10.1016/j.bjp.2014.04....
). V. polyanthes is a plant common in Minas Gerais, São Paulo, Mato Grosso and Goiás, occurring primarily in Cerrado biome. This species is a shrub with oval leaves, rough and hairy spear-shaped. The white or pink inflorescences are arranged at the apices of the branches in small capitula (Alves and Neves, 2003ALVES, V F.G. and NEVES, L.J., 2003. Anatomia Foliar de Vernonia Polyanthes Less (Asteraceae). Universidade Rural do Rio de Janeiro. Revista da Universidade Rural: Série Ciências da Vida, vol. 22, pp. 1-8.).

V. polyanthes leaves and roots in decoction or infusion are used in ethnomedicine to treat bronchitis, persistent coughs, pneumonia, kidney stones, gastric disorders, malaria, fever, wounds, fractures, sprains, bruises and dislocations. In addition, the plant is indicated by common sense as diuretic and anti-rheumatic (Jorgetto et al., 2011Jorgetto, G.V., Boriolo, M.F.G., Silva, L.M., Nogueira, D.A., José, T.D.S., Ribeiro, G.E., OLIVEIRA, N.M.S. and FIORINI, J.E., 2011. Ensaios de atividade antimicrobiana in vitro e mutagênica . in vivo com extrato de Vernonia polyanthes Less (Assa-peixe)Revista Instituto Adolfo Lutz, vol. 70, pp. 53-61.; Oliveira et al., 2011Oliveira, A.K.M., Oliveira, N.A., Resende, U.M. and Martins, P.F.R.B., 2011. Ethnobotany and traditional medicine of the inhabitants of the Pantanal Negro sub-region and the raizeiros of Miranda and Aquidauna, Mato Grosso do Sul, Brazil. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 71, no. 1, suppl., pp. 283-289. http://dx.doi.org/10.1590/S1519-69842011000200007. PMid:21537601.
http://dx.doi.org/10.1590/S1519-69842011...
; Slongo and Hoscheid, 2012Slongo, J.M. and Hoscheid, J., 2012. Avaliação fitoquímica preliminar de extratos de folhas e raízes de Vernonanthura polyanthes obtidos por maceração, infusão e decocção. Revista de Fitoterapia, vol. 12, pp. 77.). It has been demonstrated that V. polyanthes is a potential vasodilatation agent, able to manage blood pressure (Romanezi da Silveira et al., 2003Romanezi Da Silveira, R., Foglio, M.A. and Gontijo, J.A.R., 2003. Effect of the crude extract of Less. on blood pressure and renal sodium excretion in unanesthetized rats. Vernonia polyanthesPhytomedicine, vol. 10, no. 2-3, pp. 127-131. http://dx.doi.org/10.1078/094471103321659825. PMid:12725565.
http://dx.doi.org/10.1078/09447110332165...
). Moreover, the plant presents antiulcer effect (Barbastefano et al., 2007Barbastefano, V., Cola, M., Luiz-Ferreira, A., Farias-Silva, E., Hiruma-Lima, C.A., Rinaldo, D., Vilegas, W. and Souza-Brito, A.R.M., 2007. . Vernonia polyanthes as a new source of antiulcer drugsFitoterapia, vol. 78, no. 7-8, pp. 545-551. http://dx.doi.org/10.1016/j.fitote.2007.07.003. PMid:17904766.
http://dx.doi.org/10.1016/j.fitote.2007....
), and antinociceptive and anti-inflammatory activities (Temponi et al., 2012Temponi, L.G., Poli, L.P., Sakuragui, C.M. and Coelho, M.A.N., 2012. Araceae do Parque Estadual de Ibitipoca, Minas Gerais, Brasil. Rodriguésia, vol. 63, no. 4, pp. 957-969. http://dx.doi.org/10.1590/S2175-78602012000400013.
http://dx.doi.org/10.1590/S2175-78602012...
). Phytochemicals studies showed that V. polyanthes presents flavonoids (flavonols and flavones), sesquiterpene lactones, chlorogenic acids and phenolic acid (protocatechuic acid) (Igual et al., 2013Igual, M.O., Martucci, M.E.P., Costa, F.B. and Gobbo-Neto, L., 2013. Sesquiterpene lactones, chlorogenic acids and flavonoids fromleaves of Less (Asteraceae). Vernonia polyanthesBiochemical Systematics and Ecology, vol. 51, pp. 94-97. http://dx.doi.org/10.1016/j.bse.2013.08.018.
http://dx.doi.org/10.1016/j.bse.2013.08....
; Martucci et al., 2014Martucci, M.E.P., Vos, R.C.H., Carollo, C.A. and Gobbo-Neto, L., 2014. Metabolomics as a Potential Chemotaxonomical Tool: Application in the Genus . Vernonia SchrebPLoS One, vol. 9, no. 4, pp. 1-8. http://dx.doi.org/10.1371/journal.pone.0093149. PMid:24736747.
http://dx.doi.org/10.1371/journal.pone.0...
).

Due to V. polyanthes biological activities and extensive popular use, it is necessary to study the toxic, genotoxic and antigenotoxic activity of the leaves aqueous extract. Moreover some studies suggest that Cerrado plants could provide the basis for the production of new drugs (Silva et al., 2003Silva, I.R., Bittencourt, A.C.S.P., DOMINGUEZ, J.M.L. and SILVA, S.B.M., 2003. Uma contribuição à gestão ambiental da costa do descobrimento (litoral sul do Estado da Bahia): avaliação da qualidade recreacional das praias. Geografia, vol. 28, pp. 397-414.). In that way, results obtained from genotoxicity tests are crucial to ensure the safe use of the plant (Maciel et al., 2002Maciel, M.A.M., Pinto, A.C., Veiga, V.E. Jr., Grynberg, N.F. and Echevarria, A., 2002. Plantas medicinais: a necessidade de estudos multidisciplinares. Química Nova, vol. 23, no. 3, pp. 429-438. http://dx.doi.org/10.1590/S0100-40422002000300016.
http://dx.doi.org/10.1590/S0100-40422002...
). One of the most widely used genotoxicity test is the wing Somatic Mutation And Recombination Test (SMART/wing) for genetic and chromosomal mutations and also somatic recombination detection in somatic cells of Drosophila melanogaster (Rocha et al., 2013Rocha, L.D.L.S., Faria, J.C.N.M., Cruz, A.H.S., REIS, A.A.S. and SANTOS, R.S., 2013. Drosophila: um importante modelo biológico para a pesquisa e o ensino de genética. Scire Salutes, vol. 3, no. 1, pp. 37-48. http://dx.doi.org/10.6008/ESS2236-9600.2013.001.0004.
http://dx.doi.org/10.6008/ESS2236-9600.2...
).

The changes observed in SMART/wing test are induced in cells of the imaginal discs that after several mitotic divisions give rise to the wings of adults. The test is based on the identification of mutant phenotypes that are originated due to the occurrence of DNA damage, caused by the loss of larval heterozygous cells (Fonseca and Pereira, 2004Fonseca, C.A. and Pereira, D.G., 2004. Aplicação da genética toxicológica em planta com atividade medicinal. Infarma, vol. 16, pp. 7-8.; Ribeiro et al., 2003Ribeiro, L.R., SALVADORI, D.M.F. and MARQUES, E.K., 2003. Mutagênese ambiental. Canoas: ULBRA. 356 p., 2009Ribeiro, V., Viera, I.L.B.F., Passos, D.C.S., Silva, E.M., Vale, C.R., Felício, L.P., Ferreira, H.D., Vieira, P.M. and Carvalho, S., 2009. Ausência da mutagenicidade de . Solanum paniculatum L. em células somáticas de Drosophila melanogaster: SMART/ASARevista de Biologia Neotropical, vol. 6, pp. 27-33.; Mendanha et al., 2010Mendanha, D.M., Ferreira, H.D., Felício, L.P., Silva, E.M., Pereira, D.G., Nunes, W.B. and Carvalho, S., 2010. Modulatory effect of (Malpighiaceae) against damage induced by doxorubicin in somatic cells of Byrsonima verbascifoliaDrosophila melanogaster.Genetics and Molecular Research, vol. 9, no. 1, pp. 69-77. http://dx.doi.org/10.4238/vol9-1gmr678. PMid:20092036.
http://dx.doi.org/10.4238/vol9-1gmr678...
; Abreu et al., 2011Abreu, B.R.R., Thomé, S., Lehmann, M. and Dihl, R.R., 2011. Investigação da atividade mutagênica de dois agentes antibacterianos em células somáticas de Drosophila melanogaster.Revista de Iniciação Científica da ULBRA, vol. 1, pp. 69-76.; Alte et al., 2012Alte, M.A., Thomé, S., Abreu, B.R.R., Lehmann, M. and Dihl, R.R., 2012. Avaliação da atividade tóxico-genética da enrofloxacina. Revista de Iniciação Científica da ULBRA, vol. 10, pp. 21-26.). The test makes use of three D. melanogaster different strains: (i) multiple wing hairs (mwh); (ii) flare-3 (flr3); and (iii) ORR/ORR. SMART/wing test is considered to be quick and inexpensive and gives reliable results, which are unambiguous and highly reproducible (Felício et al., 2014Felício, D.C., Pereira, D.S., Assumpção, A.M., Jesus-Moraleida, F.R., Queiroz, B.Z., Silva, J.P., Rosa, N.M.B., DIAS, J.M.D. and PEREIRA, L.S.M., 2014. Systemic inflammation and physical function in community elderly women. Inflammation & Cell Signaling, vol. 1, pp. 1-3.).

Many species of Asteraceae, including that ones belonging to Vernonia Genus, exhibit significant antifungal activity (Toyang and Verpoorte, 2013Toyang, N.J. and Verpoorte, R., 2013. Review of the medicinal potentials of plants of the genus (Asteraceae). VernoniaJournal of Ethnopharmacology, vol.146, no. 3, pp. 681-723. PMid:23395623.; Leitão et al., 2014Leitão, F., Leitão, S.G., Fonseca-Kruel, V.S., Silva, I.M. and Martins, K., 2014. Medicinal plants traded in the open-air markets in the State of Rio de Janeiro, Brazil: an overview on their botanical diversity and toxicological potential. Revista Brasileira de Farmacognosia, vol. 24, no. 2, pp. 225-247. http://dx.doi.org/10.1016/j.bjp.2014.04.005.
http://dx.doi.org/10.1016/j.bjp.2014.04....
). Many studies are being carried out to find plant species with potential antifungal activity (Silva et al., 2012Silva, S.M.F.Q., Pinheiro, S.M.B., Queiroz, M.V.F., Pranchevicius, M.C., Castro, J.G.D., Perim, M.C. and Carreiro, S.C., 2012. Atividade de extratos brutos de duas espécies vegetais do Cerrado sobre leveduras do gênero in vitroCandida.Ciência & Saúde Coletiva, vol. 17, no. 6, pp. 1649-1656. http://dx.doi.org/10.1590/S1413-81232012000600028. PMid:22699655.
http://dx.doi.org/10.1590/S1413-81232012...
; Höfling et al., 2010Höfling, J.F., Anibal, P.C., Obando-Pereda, G.A., Peixoto, I.A., Furletti, V.F., Foglio, M.A. and Gonçalves, R.B., 2010. Antimicrobial potential of some plant extracts against . Candida speciesBrazilian Journal of Biology = Revista Brasileira de Biologia, vol. 70, no. 4, pp. 1065-1068. http://dx.doi.org/10.1590/S1519-69842010000500022. PMid:21180915.
http://dx.doi.org/10.1590/S1519-69842010...
), because synthetic drugs may exhibit high toxicity to patients (Araújo et al., 2004Araújo, J.C.L.V., Lima, E.O., Ceballos, B.S.O., Freire, K.R.L., Souza, E.L. and Santos-Filho, L., 2004. Ação antimicrobiana de óleos essenciais sobre microrganismos potencialmente causadores de infecções oportunistas. Revista de Patologia Tropical, vol. 33, pp. 55-64.). Substances more efficient against pathogenic microorganisms and less toxic to the patient could be isolated from vegetal species (Ostrosky, 2009Ostrosky, E.A., 2009. Avaliação da eficácia e segurança do extrato de folhas de R. rosaefolius Sm. visando a aplicação como conservante em produtos cosméticos. São Paulo: Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 175 p. PhD Thesis.). Studies performed by Ogundare et al. (2006)Ogundare, A.O., Adetuyi, F.C. and Akinyosoye, F.A., 2006. Antimicrobial activities of Vernonia tenoreana.African Journal of Biotechnology, vol. 5, pp. 1663-1668. demonstrated that bark extracts from Vernonia tenoreana inhibited the growth of Candida albicans. Furthermore, it was demonstrated that other Cerrado plants, such as Lafoensia pacari (pacari) and Brossimum gaudichaudii (mama-cadela) extracts, also inhibited the growth of Candida spp. yeasts (Silva et al., 2012Silva, S.M.F.Q., Pinheiro, S.M.B., Queiroz, M.V.F., Pranchevicius, M.C., Castro, J.G.D., Perim, M.C. and Carreiro, S.C., 2012. Atividade de extratos brutos de duas espécies vegetais do Cerrado sobre leveduras do gênero in vitroCandida.Ciência & Saúde Coletiva, vol. 17, no. 6, pp. 1649-1656. http://dx.doi.org/10.1590/S1413-81232012000600028. PMid:22699655.
http://dx.doi.org/10.1590/S1413-81232012...
). These results point to the need for more studies on the utilization of Cerrado species for antifungal purposes. Candida spp. are of great importance for public health, since candidiasis is the third systemic mycosis in number of deaths (Prado et al., 2009Prado, M., Silva, M.B., Laurenti, R., Travassos, L.R. and Taborda, C.P., 2009. Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006. Memórias do Instituto Oswaldo Cruz, vol. 104, no. 3, pp. 513-521. http://dx.doi.org/10.1590/S0074-02762009000300019. PMid:19547881.
http://dx.doi.org/10.1590/S0074-02762009...
) and vulvovaginal candidiasis is the first most common cause of fungal vaginal infections (Sobel, 2007Sobel, J.D., 2007. Vulvovaginal candidosis. Lancet, vol. 369, no. 9577, pp. 1961-1971. http://dx.doi.org/10.1016/S0140-6736(07)60917-9. PMid:17560449.
http://dx.doi.org/10.1016/S0140-6736(07)...
).

Due to V. polyanthes therapeutic potential, the present study aimed to evaluate the toxic, genotoxic and antigenotoxic potential of V. polyanthes leaves aqueous extract in somatic cells of Drosophila melanogaster. The V. polyanthes leaves aqueous extract inhibitory activity against Candida spp. was also tested.

2 Material and Methods

2.1 Plant sample obtaining

The plant sample was collected near Pôr do Sol lake in the city of Iporá, Goiás (16° 25' S, 51° 06' W), in June 2012. After the botanical identification, a voucher specimen (no 6519) was deposited in the herbarium of Universidade Estadual de Goiás, Câmpus Henrique Santillo. The collected leaves were dehydrated and pulverized.

2.2 D. melanogaster strains and crosses

The test makes use of three different strains of D. melanogaster: (i) multiple wing hairs: y;mwh j (mwh, 3-0.3); (ii) flare-3: (flr3, 3-38.8) (flr3/In(3LR)TM3, ri pp sep I(3)89Aa bx34e and BdS); and (iii) ORR/ ORR: flr3/In(3LR)TM3, ri pp sep I(3)89Aa bx34e and BdS.

Two crosses were performed. In standard cross (ST), 80 female from flr3 lineage were crossed with 40 males from mwh lineage. In high bioactivation (HB) crossing, 80 female ORR lineage were crossed with 40 mwh males. In both crosses the following offspring was obtained: marked trans-heterozygous (MH) with genetic constitution mwh +/+ flr3, which can be easily identified by the smooth wings (Ribeiro et al., 2003Ribeiro, L.R., SALVADORI, D.M.F. and MARQUES, E.K., 2003. Mutagênese ambiental. Canoas: ULBRA. 356 p., 2009Ribeiro, V., Viera, I.L.B.F., Passos, D.C.S., Silva, E.M., Vale, C.R., Felício, L.P., Ferreira, H.D., Vieira, P.M. and Carvalho, S., 2009. Ausência da mutagenicidade de . Solanum paniculatum L. em células somáticas de Drosophila melanogaster: SMART/ASARevista de Biologia Neotropical, vol. 6, pp. 27-33.; Valadares, 2007Valadares, B.L.B., 2007. Avaliação de derivados sintéticos da testosterona pelo teste de mutação e recombinação somática (SMART) em Drosophila melanogaster. Uberlândia: Programa de Pós-graduação em Genética e Bioquímica, Universidade Federal de Uberlândia, 113 p. PhD Thesis.; Mendanha et al., 2010Mendanha, D.M., Ferreira, H.D., Felício, L.P., Silva, E.M., Pereira, D.G., Nunes, W.B. and Carvalho, S., 2010. Modulatory effect of (Malpighiaceae) against damage induced by doxorubicin in somatic cells of Byrsonima verbascifoliaDrosophila melanogaster.Genetics and Molecular Research, vol. 9, no. 1, pp. 69-77. http://dx.doi.org/10.4238/vol9-1gmr678. PMid:20092036.
http://dx.doi.org/10.4238/vol9-1gmr678...
).

2.3 Survival curve

To evaluate the toxic potential of the V. polyanthes leaves aqueous extract, D. melanogaster third-stage larvae originated from the two crosses (HB and ST) were collected 72 h after the beginning of oviposition, and washed in water with help of fine mesh stainless steel strainer. After, larvae were subjected to chronic treatment with different concentrations of V. polyanthes leaves aqueous extract. For this, 50 D. melanogaster larvae were placed in glass tubes (2.5 cm diameter and 8.0 cm height) containing 0.9 g of mashed potatoes medium culture (Yoki SA, mashed potato flakes) hydrated with 3 mL of 7 different concentrations of V. polyanthes leaves aqueous extract for 48 h. The aqueous extract of the plant was prepared similar to how people prepare the V. polyanthes tea: 3 g of dried and pulverized leaves boiled for 5 min in 150 mL of distilled water (ANVISA, 2011AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA – ANVISA, 2011. Formulário de fitoterápicos da farmacopeia brasileira. Brasília. 126 p.). From this stock solution at 0.02 g/mL, dilutions were made to obtain the following concentrations: 0.016 g/mL, 0.012 g/mL, 0.008 g/mL, 0.004 g/mL, 0.002 g/mL and 0.001 g/mL. Sterile distilled water was used as negative control. After seven days, the larvae that suffered metamorphosis into adult flies were counted as survivors. The test was performed in triplicate.

2.4 Evaluation of genotoxicity and antigenotoxicity of V. polyanthes leaves aqueous extract

ST and HB crosses were performed. The larvae from both crosses were subjected to chronic treatment, as described for the survival curve. For the assessment of genotoxicity and antigenotoxicity of the V. polyanthes leaves aqueous extract, three concentrations were tested: 0.012 g/mL, 0.004 g/mL and 0.001 g/mL. For the antigenotoxicity test, these extract concentrations were associated with 0.000125 g/mL doxorubicin (DXR, Doxolen lyophilized, Eurofarma Laboratórios Ltda., São Paulo, Brazil, CAS No. 23214-92-8). Sterile distilled water was used as negative control and doxorubicin, a potent clastogenic agent of direct action (Dhawan et al., 2003Dhawan, A., Kayani, M.A., Parry, J.M., Parry, E. and Anderson, D., 2003. Aneugenic and clastogenic effects of doxorubicin in human lymphocytes. Mutagenesis, vol. 18, no. 6, pp. 487-490. http://dx.doi.org/10.1093/mutage/geg024. PMid:14614182.
http://dx.doi.org/10.1093/mutage/geg024...
), was used as positive control. When the adult flies were obtained, its wings were used for the preparation of the slides.

2.5 Preparation of the slides

The wings of flies were removed using an entomological forceps and a magnifying glass. The slides were kept on a hot plate between 40-45 °C over a 24 h period, after this period, the slides were covered with coverslips containing Faure solution (30 g arabic gum, 16 mL glycerol, 200 g chloral hydrate (Cl3CCH(OH)2) and 50 mL distilled water) for 24 h at 40-45 °C. After this period, the slides were examined for spots using a light microscope at 400x magnification.

2.6 Statistical test

The χ2 test was used to evaluate the mutagenic potential (Frei and Würgler, 1988Frei, H. and Würgler, F.E., 1988. Statistical methods to decide whether mutagenicity test data from . Drosophila assays indicate a positive, negative, or inconclusive resultMutation Research, vol. 203, no. 4, pp. 297-308. http://dx.doi.org/10.1016/0165-1161(88)90019-2. PMid:3136327.
http://dx.doi.org/10.1016/0165-1161(88)9...
). For the antimutagenic analysis, the frequencies of each type of spot for each treatment group were compared pairwise (DXR x V. polyanthes extract + DXR), using the nonparametric Mann-Whitney U-test and Wilcoxon rank sum tests (Frei and Würgler, 1995Frei, H. and Würgler, F.E., 1995. Optimal experimental design and sample size for the statistical evaluation of data from somatic mutation and recombination tests (SMART) in Drosophila.Mutation Research, vol. 334, no. 2, pp. 247-258. http://dx.doi.org/10.1016/0165-1161(95)90018-7. PMid:7885379.
http://dx.doi.org/10.1016/0165-1161(95)9...
).

2.7 Fungal strains and cultivation conditions

For the antifungal potential analysis of V. polyanthes leaves aqueous extract, two species were selected: Candida albicans (ATCC 28367) and Candida parapsilosis (ATCC 22019). The fungus was maintained on Agar Sabouraud Dextrose (Himedia Laboratories Pvt. Ltd., India) at room temperature.

2.8 Agar-well diffusion assay

The fungal inoculum was prepared by suspension of colonies from a 48 h culture in 2 mL of sterile saline solution (0.9% NaCl). The suspension was set at 0.5 of the McFarland scale (Silva et al., 2012Silva, S.M.F.Q., Pinheiro, S.M.B., Queiroz, M.V.F., Pranchevicius, M.C., Castro, J.G.D., Perim, M.C. and Carreiro, S.C., 2012. Atividade de extratos brutos de duas espécies vegetais do Cerrado sobre leveduras do gênero in vitroCandida.Ciência & Saúde Coletiva, vol. 17, no. 6, pp. 1649-1656. http://dx.doi.org/10.1590/S1413-81232012000600028. PMid:22699655.
http://dx.doi.org/10.1590/S1413-81232012...
). 30 μL of this suspension was spread on Agar Sabouraud Dextrose supplemented with 10 μg/mL gentamicin. Then, six 6 mm diameter wells were made using a sterile tip (Alves et al., 2006Alves, P.M., Leite, P.H.A.S., Pereira, J.V., Pereira, L.F., Pereira, M.S.V., Higino, J.S. and Lima, E.O., 2006. Atividade antifúngica do extrato de da cavidade oral: uma avaliação Psidium guajava Linn. (goiabeira) sobre leveduras do gênero Candidain vitro.Revista Brasileira de Farmacognosia, vol. 16, no. 2, pp. 192-196. http://dx.doi.org/10.1590/S0102-695X2006000200010.
http://dx.doi.org/10.1590/S0102-695X2006...
). Each well was filled with 50 µL of V. polyanthes leaves aqueous extract in seven different concentrations: 0.16 g/mL, 0.08 g/mL, 0.04 g/mL, 0.02 g/mL, 0.012 g/mL, 0.004 g/mL and 0.001 g/mL. Nystatin oral suspension 100,000 IU/mL (Germed Pharmaceutical Ltda. Brazil) in a final dosage of 0.02 g/mL (CLSI, 2008CLINICAL AND LABORATORY STANDARDS INSTITUTE – CLSI, 2008. Method for broth dilution antifungal susceptibility testing of yeasts: approved standard-third edition M27-A3. Wayne: CLSI.) and distilled water were used as positive and negative control, respectively. The plates were incubated at room temperature for 48 h and, then, the zones of inhibition were measured using a ruler. The zone sizes obtained in the presence of the V. polyanthes leaves aqueous extracts were compared to the values obtained for the positive control. This assay was performed in quadruplicate.

3 Results

To assess the V. polyanthes toxicity, a survival curve of D. melanogaster larvae treated with different concentrations of V. polyanthes leaves aqueous extract was performed. After compiling the results of three independent experiments, the mean percentage of survivors was calculated. The survival curve showed that, under the conditions used in this work, V. polyanthes leaves aqueous extract presented no toxic activity in D. melanogaster larvae of both crosses, ST and HB, when compared to the negative control (see Figure 1).

Figure 1
Survival curve of D. melanogaster larvae derived from the standard (ST) and high bioactivation (HB) crosses. The larvae obtained from ST and HB crosses were subjected to chronic treatment with different concentrations of V. polyanthes leaves aqueous extract. After seven days, the larvae that reached the adult stage were counted as survivors. Data are presented as means of triplicates percentage of surviving larvae + the standard deviation of triplicates. D1: 0.02 g/mL; D2: 0.016 g/mL; D3: 0.012 g/mL; D4: 0.008 g/mL; D5: 0.004 g/mL; D6: 0.002 g/mL; D7: 0.001 g/mL; Negative control: sterile distilled water.

The V. polyanthes leaves aqueous extract genotoxicity was evaluated by ST (mwh/flr3) and HB (mwh/ORR) crosses, obtaining marker-heterozygous (MH) flies (mwh +/+ flr3), presenting wild-type wings. According to the results, there was a significant increase in simple small spots (SSS), simple big spots (SBS) and twin spots (TWS), comparing to the negative control (distilled water). However, when compared the number of SSS, SBS and TWS of the treatments with the higher numbers obtained by the positive control (doxorubicin), one can suggest that V. polyanthes aqueous extract presented neither direct genotoxic effect, based on the analysis of ST crosses, nor indirect genotoxic effect, according to HB crosses, in somatic cells of D. melanogaster (as shown in Table 1).

Table 1
Frequency of mutant spots observed in marker-heterozygous descendants of D. melanogaster from standard (ST) and high bioactivation (HB) crosses treated with different concentrations of V. polyanthes leaves aqueous extract.

V. polyanthes leaves aqueous extracts co-treatment with DXR (positive control) was used to evaluate the plant extract antigenotoxicity. After promoting the same crosses described for genotoxicity, a significant increase in SSS, SBS and TWS numbers was observed for the co-treatments, when compared to the positive control used alone (as shown in Table 2). In this way, the V. polyanthes leaves aqueous extracts may present no antigenotoxic activity, contrariwise, in the concentrations used in this work, the plant extract enhanced the doxorubicin genotoxicity in D. melanogaster somatic cells.

Table 2
Frequency of mutant spots observed in marker-heterozygous descendants of D. melanogaster from standard (ST) and high bioactivation (HB) crosses treated with different concentrations of V. polyanthes leaves aqueous extract associated with doxorubicin.

V. polyanthes leaves aqueous extract antifungal activity was tested in seven different concentrations (0.16 g/mL, 0.08 g/mL, 0.04 g/mL, 0.02 g/mL, 0.012 g/mL, 0.004 g/mL and 0.001 g/mL). The conditions used in this work showed no inhibitory activity against C. albicans and C. parapsilosis, when compared with the positive control nystatin. The nystatin inhibition zone diameter was 13 mm ± 1 and 14 mm ± 1 against C. albicans and C. parapsilosis, respectively (see Figure 2).

Figure 2
V. polyanthes leaves aqueous extract presents no antifungal activity against Candida spp. C. albicans and C. parapsilosis were cultivated on Sabouraud Dextrose agar for 48 h. Then, the yeasts were spreaded on this media in presence of seven different concentrations of V. polyanthes leaves aqueous extract. After 48 h, the zones of inhibition were measured using a ruler. Nystatin and distilled water were used as positive (+) and negative (-) control, respectively. D1: 0.02 g/mL; D2: 0.012 g/mL; D3: 0.004 g/mL; D4: 0.001 g/mL; D5: 0.04 g/mL; D6: 0.08 g/mL; D7: 0.16 g/mL.

4 Discussion

V. polyanthes leaves aqueous extract presented no toxic, genotoxic or antigenotoxic activity in D. melanogaster somatic cells under the conditions used in this work. Similarly, Vernonia condensata leaves aqueous extract did not present mutagenic potential using Salmonella/microsome assay (Monteiro et al., 2001Monteiro, M.H.D., Gomes-Carneiro, M.R., Felzenszwalb, I., Chahoud, I. and Paumgartten, F.J.R., 2001. Toxicological evaluation of a tea from leaves of Vernonia condensata.Journal of Ethnopharmacology, vol. 74, no. 2, pp. 149-157. http://dx.doi.org/10.1016/S0378-8741(00)00363-9. PMid:11167033.
http://dx.doi.org/10.1016/S0378-8741(00)...
). Moreover, the aqueous extract of Vernonia amygdalina and the methanol extract of Vernonia cineria present no toxicity in murine model (Amole et al., 2006AMOLE, O.O., IZEGBU, M.C., ONAKOYA, J.A.A. and DADA, M.O., 2006. Toxicity studies of the aqueous extract of Vernonia amygdalina. Biomedical Research, vol. 17, pp. 39-40.; Rajamurugan et al., 2011Rajamurugan, R., Selvaganabathy, N., Kumaravel, S., Ramamurthy, C., Sujatha, V., Suresh Kumar, M. and Thirunavukkarasu, C., 2011. Identification, quantification of bioactive constituents, evaluation of antioxidant and acute toxicity property from the methanol extract of leaf extract. in vivoVernonia cinereaPharmaceutical Biology, vol. 49, no. 12, pp. 1311-1320. http://dx.doi.org/10.3109/13880209.2011.604334. PMid:22077167.
http://dx.doi.org/10.3109/13880209.2011....
). However, previous study with Swiss albinus mice demonstrated that V. polyanthes leaves hydroalcoholic extract presented a moderated genotoxic effect only in concentrations higher than 1500 mg/kg (Jorgetto et al., 2011Jorgetto, G.V., Boriolo, M.F.G., Silva, L.M., Nogueira, D.A., José, T.D.S., Ribeiro, G.E., OLIVEIRA, N.M.S. and FIORINI, J.E., 2011. Ensaios de atividade antimicrobiana in vitro e mutagênica . in vivo com extrato de Vernonia polyanthes Less (Assa-peixe)Revista Instituto Adolfo Lutz, vol. 70, pp. 53-61.). And when a sesquiterpene lactone, glaucolide B, was isolated from Vernonia eremophila, the molecule presented genotoxic and cytotoxic activities in vitro using human cultured lymphocytes, but did not present genotoxicity in vivo when bone marrow cells from BALB/c mice were analyzed (Burim et al., 1999Burim, R.V., Canalle, R., Lopes, J.L.C. and Takahashi, C.S., 1999. Genotoxic action of the sesquiterpene lactone glaucolide B on mammalian cells in vitro and in vivo.Genetics and Molecular Biology, vol. 22, no. 3, pp. 401-406. http://dx.doi.org/10.1590/S1415-47571999000300020.
http://dx.doi.org/10.1590/S1415-47571999...
).

These data suggest that the plant specie, the preparation method of the leaves extract and the model organism could influence the toxicity/genotoxicity responses. Some compounds may be present in one species and absent in another. It has been observed, for example, that V. polyanthes was the only species with presence of flavones chrysoeriol-7-O-glycuronyl, acacetin-7-O-glycuronyl and sesquiterpenes lactones piptocarphin A and piptocarphin B among ten Vernonia species studied by Martucci et al. (2014)Martucci, M.E.P., Vos, R.C.H., Carollo, C.A. and Gobbo-Neto, L., 2014. Metabolomics as a Potential Chemotaxonomical Tool: Application in the Genus . Vernonia SchrebPLoS One, vol. 9, no. 4, pp. 1-8. http://dx.doi.org/10.1371/journal.pone.0093149. PMid:24736747.
http://dx.doi.org/10.1371/journal.pone.0...
. Glaucolide A was detected only in V. brasiliana and V. polyanthes in this same study (Martucci et al., 2014Martucci, M.E.P., Vos, R.C.H., Carollo, C.A. and Gobbo-Neto, L., 2014. Metabolomics as a Potential Chemotaxonomical Tool: Application in the Genus . Vernonia SchrebPLoS One, vol. 9, no. 4, pp. 1-8. http://dx.doi.org/10.1371/journal.pone.0093149. PMid:24736747.
http://dx.doi.org/10.1371/journal.pone.0...
). Furthermore, the extraction method using methanol described by Igual et al. (2013)Igual, M.O., Martucci, M.E.P., Costa, F.B. and Gobbo-Neto, L., 2013. Sesquiterpene lactones, chlorogenic acids and flavonoids fromleaves of Less (Asteraceae). Vernonia polyanthesBiochemical Systematics and Ecology, vol. 51, pp. 94-97. http://dx.doi.org/10.1016/j.bse.2013.08.018.
http://dx.doi.org/10.1016/j.bse.2013.08....
, for example, favors extraction of compounds stored in glandular trichomes, such as sesquiterpene lactones. In this way, the aqueous extract used in this work may have not favored the extraction of toxic/genotoxic compound(s).

Although V. polyanthes leaves aqueous extract presented no toxic, genotoxic or antigenotoxic activity, it enhanced doxorubicin genotoxicity in D. melanogaster somatic cells. Similar results have been demonstrated to Piper cubeba, Lycopersicon esculentum and Tabebuia impetiginosa using the same organism model. When (-)-cubebin, a compound isolated from Piper cubeba seeds, was combined with DXR, the number of mutant spots increased (Rezende et al., 2011Rezende, A.A., e Silva, M.L., Tavares, D.C., Cunha, W.R., Rezende, K.C., Bastos, J.K., Lehmann, M., de Andrade, H.H., Guterres, Z.R., Silva, L.P. and Spanó, M.A., 2011. The effect of the dibenzylbutyrolactolic lignan (_)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila malanogaster.Food and Chemical Toxicology, vol. 49, no. 6, pp. 1235-1241. http://dx.doi.org/10.1016/j.fct.2011.03.001. PMid:21385598.
http://dx.doi.org/10.1016/j.fct.2011.03....
). The extract of organic tomato Lycopersicon esculentum when combined with DXR also potentiates the genotoxic effect of this drug (Dutra et al., 2009Dutra, E.S., Dias, C.D., Araújo, B.C., Castro, A.J.S. and Nepomuceno, J.C., 2009. Effect of organic tomato (Lycopersicon esculentumDrosophila) extract on the genotoxicity of doxorubicin in the . wing spot testGenetics and Molecular Biology, vol. 32, no. 1, pp. 133-137. http://dx.doi.org/10.1590/S1415-47572009005000008. PMid:21637658.
http://dx.doi.org/10.1590/S1415-47572009...
). T. impetiginosa alone did not act as a genotoxin or an antigenotoxin, but it induced the DXR genotoxicity when associated with this mutagen (Sousa et al., 2009Sousa, N.C., Rezende, A.A.A., Silva, R.M.G., Guterres, Z.R., Graf, U., Kerr, W.E. and Spanó, M.A., 2009. Modulatory effects of (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Tabebuia impetiginosaDrosophila melanogaster.Genetics and Molecular Biology, vol. 32, no. 2, pp. 382-388. http://dx.doi.org/10.1590/S1415-47572009005000042. PMid:21637695.
http://dx.doi.org/10.1590/S1415-47572009...
). Moreover, it was demonstrated, using micronucleus assay, that hydroalcoholic extract of Mikania glomerata (Asteraceae) aerial parts significantly increased DXR genotoxicity (Barbosa et al., 2012Barbosa, L.C., Morais, M.D., Paula, C.A., Ferreira, M.C.S., Jordão, A.A., Silva, M.L.A., Bastos, J.K., Silva-Filho, A.A. and Cecchi, A.O., 2012. . Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levelsJournal of Toxicology and Environmental Health. Part A., vol. 75, no. 16-17, pp. 1102-1109. http://dx.doi.org/10.1080/15287394.2012.697842. PMid:22852859.
http://dx.doi.org/10.1080/15287394.2012....
).

Plant extracts contain multiple components that can exert toxicity separately or synergistically (Cai et al., 2004Cai, Z., Tsung, E.F., Marinescu, V.D., Ramoni, M.F., Riva, A. and Kohane, I.S., 2004. Bayesian approach to discovering pathogenic SNPs in conserved protein domains. Human Mutation, vol. 24, no. 2, pp. 178-184. http://dx.doi.org/10.1002/humu.20063. PMid:15241800.
http://dx.doi.org/10.1002/humu.20063...
; Romero-Jiménez et al., 2005Romero-Jiménez, M., Campos-Sánchez, J., Analla, M., Muñoz-Serrano, A. and Alonso-Moraga, A., 2005. Genotoxicity and anti-genotoxicity of some traditional medicinal herbs. Mutation Research, vol. 585, no. 1-2, pp. 147-155. http://dx.doi.org/10.1016/j.mrgentox.2005.05.004. PMid:16005256.
http://dx.doi.org/10.1016/j.mrgentox.200...
). In this way, co-administration of herb and therapeutic drugs may cause clinical risk to patients and so need to be investigated (Barbosa et al., 2012Barbosa, L.C., Morais, M.D., Paula, C.A., Ferreira, M.C.S., Jordão, A.A., Silva, M.L.A., Bastos, J.K., Silva-Filho, A.A. and Cecchi, A.O., 2012. . Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levelsJournal of Toxicology and Environmental Health. Part A., vol. 75, no. 16-17, pp. 1102-1109. http://dx.doi.org/10.1080/15287394.2012.697842. PMid:22852859.
http://dx.doi.org/10.1080/15287394.2012....
). But the components and mechanisms used by some plants to enhance DXR genotoxic effect were not revealed yet. DXR is a chemotherapeutic agent that induces single- and double-stranded DNA breaks (Rezende et al., 2011Rezende, A.A., e Silva, M.L., Tavares, D.C., Cunha, W.R., Rezende, K.C., Bastos, J.K., Lehmann, M., de Andrade, H.H., Guterres, Z.R., Silva, L.P. and Spanó, M.A., 2011. The effect of the dibenzylbutyrolactolic lignan (_)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila malanogaster.Food and Chemical Toxicology, vol. 49, no. 6, pp. 1235-1241. http://dx.doi.org/10.1016/j.fct.2011.03.001. PMid:21385598.
http://dx.doi.org/10.1016/j.fct.2011.03....
), after being metabolized by cytochrome P450 complex (Dutra et al., 2009Dutra, E.S., Dias, C.D., Araújo, B.C., Castro, A.J.S. and Nepomuceno, J.C., 2009. Effect of organic tomato (Lycopersicon esculentumDrosophila) extract on the genotoxicity of doxorubicin in the . wing spot testGenetics and Molecular Biology, vol. 32, no. 1, pp. 133-137. http://dx.doi.org/10.1590/S1415-47572009005000008. PMid:21637658.
http://dx.doi.org/10.1590/S1415-47572009...
). Interestingly, this enzyme complex is also responsible for DXR detoxification (Niitsu et al., 2000Niitsu, N., Okabe-Kado, J., Nakayama, M., Wakimoto, N., Sakashita, A., Maseki, N., Motoyoshi, K., Umeda, M. and Honma, Y., 2000. Plasma levels of the differentiation inhibitory factor nm23-H1 protein and their clinical implications in acute myelogenous leukemia. Blood, vol. 96, no. 3, pp. 1080-1086. PMid:10910925.). It is proposed that plant components could (i) directly interact with cytochrome P450 enzymes involved in DXR metabolism or (ii) generate oxygen free radicals that could increase this enzymatic activity, both contributing to DXR mutagenicity enhancing (Sousa et al., 2009Sousa, N.C., Rezende, A.A.A., Silva, R.M.G., Guterres, Z.R., Graf, U., Kerr, W.E. and Spanó, M.A., 2009. Modulatory effects of (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Tabebuia impetiginosaDrosophila melanogaster.Genetics and Molecular Biology, vol. 32, no. 2, pp. 382-388. http://dx.doi.org/10.1590/S1415-47572009005000042. PMid:21637695.
http://dx.doi.org/10.1590/S1415-47572009...
). In that way, the V. polyanthes extract used in this work may contain compound(s) that influence the DXR genotoxicity.

V. polyanthes leaves aqueous extract presented no antifungal activity against C. albicans and C. parapsilosis under the conditions used in this work. Similar results were observed by Sartori et al. (2003)Sartori, M.R.K., Pretto, J.B., Cruz, A.B., Bresciani, L.F.V., Yunes, R.A., Sortino, M., Zacchino, S.A. and Cechinel FILHO, V., 2003. Antifungal activity of fractions and two pure compounds of flowers from Wedelia paludosa (Acmela brasiliensis) (Asteraceae). Die Pharmazie, vol. 58, no. 8, pp. 567-569. PMid:12967035. using a methanolic extract of Acmela brasiliensis (Asteraceae) flowers. None of the fractions tested possessed activity against the yeasts tested, including C. albicans and Candida tropicalis. However, all fractions were active against dermatophytes (Sartori et al., 2003Sartori, M.R.K., Pretto, J.B., Cruz, A.B., Bresciani, L.F.V., Yunes, R.A., Sortino, M., Zacchino, S.A. and Cechinel FILHO, V., 2003. Antifungal activity of fractions and two pure compounds of flowers from Wedelia paludosa (Acmela brasiliensis) (Asteraceae). Die Pharmazie, vol. 58, no. 8, pp. 567-569. PMid:12967035.). Similarly, Vernonia cinerea leaves acetonic extract presented antifungal activity only against a strain of Candida glabrata (H05 flu R), but not against another strain of this fungus (H05 flu S) and against C. albicans, Candida krusei and A. fumigatus (Mini et al., 2010Mini, N.V., Ida, B., Seema, D., Shital, D., Riva, D.S. and Astrida, R., 2010. Antimicrobial activity of ten common herbs, commonly known as ‘Dashapushpam’ from Kerala, India. African Journal of Microbiological Research, vol. 4, pp. 2357-2362.). Vernonia cinerea leaves methanolic extract was also tested and exhibited antifungal activity against C. albicans (Latha et al., 2011Latha, L.Y., Darah, I., Jain, K. and Sasidharan, S., 2011. Effects of Less methanol extract on growth and morphogenesis of Vernonia cinereaCandida albicans.European Review for Medical and Pharmacological Sciences, vol. 15, no. 5, pp. 543-549. PMid:21744750.). The crude leaf and tuber dichloromethane, methanolic and aqueous extracts from Vernonia guineensis exhibited weak antifungal activity against Candida albicans, A. fumigatus and Trichophyton mentagrophytes; except for the dichloromethane and methanolic tuber extract, which showed moderate activity against A. fumigatus (Toyang et al., 2012Toyang, N.J., Ateh, E.N., Keiser, J., Vargas, M., Bach, H., Tane, P., Sondengam, L.B., Davis, H., Bryant, J. and Verpoorte, R., 2012. Toxicity, antimicrobial and anthelmintic activities of Benth. (Asteraceae) crude extracts. Vernonia guineensisJournal of Ethnopharmacology, vol. 144, no. 3, pp. 700-704. http://dx.doi.org/10.1016/j.jep.2012.10.016. PMid:23107821.
http://dx.doi.org/10.1016/j.jep.2012.10....
).

These results suggest that different extracts of Vernonia species could present antifungal activity against different fungi species. So, more studies have to be developed using different V. polyanthes parts, or different forms of extraction, against different fungi groups, such as dermatophytes, to elucidate if this plant could inhibit any fungus growth.

In this study, we showed that V. polyanthes leaves aqueous extract could enhance DXR genotoxicity by components and mechanisms not revealed yet. Future work will focus on determine the substance(s) involved in this potentiating effect, since an understanding of components and mechanisms involved in herb–drug interactions is essential for clinical risk assessment (Barbosa et al., 2012Barbosa, L.C., Morais, M.D., Paula, C.A., Ferreira, M.C.S., Jordão, A.A., Silva, M.L.A., Bastos, J.K., Silva-Filho, A.A. and Cecchi, A.O., 2012. . Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levelsJournal of Toxicology and Environmental Health. Part A., vol. 75, no. 16-17, pp. 1102-1109. http://dx.doi.org/10.1080/15287394.2012.697842. PMid:22852859.
http://dx.doi.org/10.1080/15287394.2012....
). Moreover, further studies are needed to confirm the efficacy and/or risks of the use of this plant tea by the population, as well as to find alternatives to the rational use of this natural resource.

Acknowledgements

We would like to thank Dr. Leonardo Luiz Borges for pulverizing the V. polyanthes dehydrated leaves, and Dr. Maria do Rosário Rodrigues Silva for providing Candida spp. strains. We are also grateful to Universidade Estadual de Goiás (UEG) for the scholarships (PBIT/UEG) granted to Isis de Jesus Guerra and Jamira Dias Rocha during the execution of this work.

  • (With 2 figures)

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Publication Dates

  • Publication in this collection
    03 May 2016
  • Date of issue
    Oct-Dec 2016

History

  • Received
    23 Mar 2015
  • Accepted
    31 July 2015
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