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Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone
Experimental Parasitology 133 (2013) 334–338
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Research Brief
Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone Nadja Soares Vila-Nova a, Selene Maia de Morais a,b,⇑, Maria José Cajazeiras Falcão b, Terezinha Thaize Negreiros Alcantara b, Pablito Augusto Travassos Ferreira b, Eveline Solon Barreira Cavalcanti b, Icaro Gusmão Pinto Vieira c, Cláudio Cabral Campello a, Mary Wilson d,e,f a
Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Avenida Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil Curso de Química, Centro de Ciências e Tecnologia, Universidade Estadual do Ceara, Avenida Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil PADETEC – Parque de Desenvolvimento Tecnologico, Universidade Federal do Ceará, Av. Humberto Monte, 2977, Bairro Parquelandia Bloco 310, CEP 60.440-593 Fortaleza, Ceará, Brazil d Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA e Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA f VA Medical Center, Iowa City, IA 52242, USA b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Acetogenins were isolated from A.
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muricata and scoparone from P. floribundum. All compounds inhibited the promastigote growth in the three species. Annonacinone presented high leishmanicidal activity. Corossolone, and scoparone demonstrated moderate leishmanicidal actions. Toxicity was tested using A. salina assay, corossolone was the most toxic compound.
a r t i c l e
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Article history: Received 3 September 2012 Received in revised form 12 November 2012 Accepted 26 November 2012 Available online 8 December 2012 Keywords: Leishmanicidal Acetogenin Coumarin Leishmania Artemia salina
a b s t r a c t Leishmaniasis is a zoonotic disease that can manifest itself in visceral and cutaneous form. The aim of this study was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied to compounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmania mexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form were used to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundum and two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities in relation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. major and L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetogenin annonacinone (EC50 = 6.72 8.00 lg/mL) indicated high leishmanicidal activity; corossolone
⇑ Corresponding author at: Curso de Química, Universidade Estadual do Ceará, Av. Paranjana, 1700, Campus do Itaperi, CEP 60740-903, Serrinha, Fortaleza, Ceará, Brazil. Tel.: +55 85 31019933. E-mail address: [email protected] (S.M. de Morais). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.11.025
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(EC50 = 16.14 18.73 lg/mL) and scoparone (EC50 = 9.11 27.51 lg/mL) moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate these compounds for further studies aiming the development of new leishmanicidal agents. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Leishmaniasis is a tropical zoonotic disease caused by more than 20 protozoa species of Leishmania genus (Singh et al., 2012) manifesting itself in visceral and cutaneous form. The geographical distribution of leishmaniasis relate to the growth of sandfly vector, which are dominant in tropical and temperate regions (Roy et al., 2012). The chemotherapy used in the treatment of leishmaniasis is based on the use of drugs that are toxic heavy metals, known as antimoniates, among which the most used are meglumine antimoniate (GlucantimeÒ) and sodium stibogluconate (PentostanÒ). When this type of treatment is not effective, other medications such as pentamidine and amphotericin B and its lipossomal formulation, are also used. All these medications are administrated intravenously and/or intramuscular require clinical supervision or hospitalization due to the severity of side effects (Chan-Bacab and Pena-Rodríguez, 2001; Rath et al., 2003). In dogs, one of the main reservoirs, treatment is not a recommended measure in Brazil, since it does not diminish the importance of these animals as reservoir of the parasite. It only provides clinical remission and not a parasitological cure (Sessa et al., 1994; Alves and Bevilacqua, 2004; Ait-Oudhia et al., 2012). In the early 1990s, the World Health Organization (WHO) reported that 65–80% of the population of developing countries depended on medicinal plants as the only form of access to basic health care (Veiga et al., 2005). A vast number of Brazilian plants are able to produce secondary metabolites with antiparasitic activity; many of these metabolites have not been properly isolated and chemically or biologically evaluated. The Annonaceae family is a great producer of acetogenins that has shown promising results in the search for new drugs against various protozoa, revealing good potential as a source of agents for the treatment of leishmaniasis (Rocha et al., 2005). Annona muricata is a well-known plant in Brazil, the compounds there are most found in this plant are acetogenins and alkaloids (Vila-Nova et al., 2011; Tempone et al., 2005), and has antiprotozoal, antioxidant and anticancer properties (Rondon et al., 2011; Boyom et al., 2009; Lima et al., 2011; Chen et al., 2012a). Platymiscium floribundum is a regional plant in Ceará state used by the population as anti-inflammatory and the isoflavonoids isolated from this plant have demonstrated activity against cancer cells (Militão et al., 2006; Falcao et al., 2005), and the crude extract demonstrated antifungal and acetylcholinesterase activity (Cardoso-Lopes et al., 2008), pterocarpans isolated from the heartwood demonstrated antimitotic effect on sea urchin eggs (Militão et al., 2005), but a few of studies have been developed in the search of others chemotherapeutic values as well as isolating others compounds from this plant. Due to the shortcomings described above, there is a pressing need for new leishmanicidal treatments, and compounds from Northeastern Brazilian plants can be promising sources for future drugs. 2. Material and methods 2.1. Plants The heartwood and sapwood of the stem of P. floribundum and leaves of A. muricata were collected in the state of Ceará, Brazil.
Aerial parts of the two plants were deposited in the Prisco Bezerra Herbarium under the reference number 31052 and 43951, respectively. 2.2. Chromatographic procedures The active chemical constituents were isolated from plant extracts by silica gel (d 0.063–0.200 mm; 70–230 mesh) and Sephadex (LH-20) column chromatography and solvents for elution were petroleum ether, hexane, chloroform, ethyl acetate, acetone and methanol from VETEC (Brazil). The fractions collected in the columns were compared by thin layer chromatography (TLC), using silica gel (60 G F 254) on glass plates (3 cm 8 cm). The TLC plates were analyzed in a iodine chamber, under UV Light (at 312 and 365 nm), Vilbert Loumart, CN-15 LM model, and by spraying the reagent 2.5% vaniline in sulfuric acid diluted in ethanol (1:1), followed by heating in an oven at 100 °C. 2.3. Isolation of compounds and spectroscopic identification A. muricata seeds (2 kg) were triturated and left in contact with methanol for 1 week, then the solvent was filtered, evaporated to dryness and a light yellow solid was obtained (402 g). This material was chromatographed on a filtering silica gel column yielding two main compounds. The trunk heartwood of P. floribundum (800 g) was cold extracted using hexane, chloroform and ethanol successively. Respective extracts were obtained by elimination of solvents in a rotatory evaporator. The chloroform extract was introduced on the top of a glass chromatographic column, filled with silica gel, and the solvents hexane and ethyl acetate, in mixtures of increasing polarity, were used to elute the column. Fiftysix 10 mL fractions were collected and compared by TLC. This procedure conducted to the isolation of a unique compound, revealed by a simple spot on TLC plate. The structures of compounds were determined by spectroscopic analysis of infrared spectra, recorded on a FT-IR PerkinElmer 1000 spectrophotometer, values expressed in cm 1, and nuclear magnetic resonance spectra, recorded on a Bruker Avance DRX-500 spectrometer in CDCl3. 2.4. Leishmanicidal assay L. major, L. mexicana and L. donovani mCherry-Luciferaseexpressing (mCherry-Luc) species were generated with the integrating vector pIR1SAT, provided by Dr. Steve Beverley, as described (Thalhofer et al., 2010). The promastigote forms were cultured in hemoflagellate-modified MEM (HOMEM) medium, supplemented with 10% fetal bovine serum at 26 °C. For the leishmanicidal assay, an adapted methodology from Tempone et al. (2005) was used. The compounds were dissolved in DMSO at a concentration of 0.2% and diluted with HOMEM medium in 96-well microplates. The assay was performed at concentrations of 100, 50, 25, 12.5 and 6.25 lg/mL. Control wells contained DMSO or no additives. Each concentration was tested in triplicate in replicate experiments. Promastigotes at a logarithmic phase were counted in a Neubauer hemocytometer and seeded at 1 106/well. The plates were incubated at 26 °C for 24 h, and the viability of the promastigotes, based on their morphology, was observed under a
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light microscope. Pentamidine (Sigma–Aldrich, St. Louis, MO, USA) was used as the positive control drug. The optical density (OD) was determined in a Fluostar Omega (BMG Labtech) at 620 nm. The number of living promastigotes was determined indirectly by the optical density (OD – 620 nm) representing the percentage of survival. The EC50 values (±SD) were calculated using a nonlinear regression curve using the statistical software GhaphPad Prism 4.0. 2.5. Toxicity tests using Artemia salina A. salina (Artemiidae), also known as brine shrimp larva, is an invertebrate normally used as a safe, practical and economic substitute assay to determine toxicity of compounds from natural products (Barbosa et al., 2009). The toxicity tests were developed using A. salina assay by Vanhaecke et al. (1981) with modifications. Dried cysts of A. salina (15–20 g in 50 mL of seawater) were incubated in a hatcher (a mini-aquarium with two compartments, one at darkness and another illuminated) at 28–30 °C with strong aeration, under a continuous light regime during 24 h. A. salina cysts were placed for hatching in the dark compartment of the aquarium. The phototropic nauplii that migrated to the illuminated compartment were collected with a Pasteur pipette and placed in another flask, when then they were used in the toxicity bioassay. A solution is prepared containing the chemical compound and DMSO (1% v/v) in seawater. Each test consisted of exposing groups of 30 larvae aged 24 h to various concentrations of the toxic compound (10,000, 1000, 100, 10 and 1 ppm). The toxicity was determined after 24 h (nauplii in instar II/III) of exposure. Each concentration was tested in triplicate in replicate experiments. The numbers of survivors were counted, larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation. 3. Statistical analysis The data were tested for compliance with the requirements for carrying out the analysis of variance and normal distribution and homogeneity of variances among treatments. The ANOVA was then performed using the GLM procedure of SAS (2002) and Tukey’s test was used to compare means. For the toxicity assay using A. salina, the EC50 and its confidence intervals were calculated by Probit analysis using the software StatPlus 2009 Professional 5.8.4. 4. Results and discussion The assay of acute toxicity against A. salina has been used in a great diversity of medicinal plants research and its chemical compounds in order to determine their pharmacological assessment, including pursuing anticancer agents, which are those with high toxicity. Barbosa et al. (2009) demonstrated that the A. salina assay was efficient in the search for antileishmanial substances. The evaluation of plant extract toxicity by the brine shrimp bioassay, an LC50 value lower than 1000 lg/mL is considered bioactive (Meyer
et al., 1982). This test was used to find active compounds from plants in our laboratory, as well as to a toxicity screening of the isolated compounds. The assay demonstrated that A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic compound of all three (Table 1). The spectral data of the compounds isolated from A. muricata were the same of previous reported data (Vila-Nova et al., 2011) for the acetogenins, corossolone, and annonacinone. The compound isolated from P. floribundum was characterized as scoparone (6,7dimethoxycoumarin) by comparison of 1H and 13C-nuclear magnetic ressonance data with earlier described by Ma et al. (2006). Corossolone: 13C NMR (CDCl3, 125 MHz): 174.84 (C1), 131.31 (C2), 33.64 (C3), 70.01 (C4), 37.30 (C5), 29.20–29.92 (C6–7), 23.99 (C8), 42.79 (C9), 211.60 (C10), 42.88 (C11), 23.84 (C12), 25.42 (C13), 33.64 (C14), 74.32 (C15), 82.75 (C16), 28.96 (C17– 18), 82.89 (C19), 74.02 (C20), 33.59 (C21), 25.78 (C22), 29.20– 29.92 (C23–29), 32.11 (C30), 22.88 (C31), 14.32 (C32), 152.12 (C33), 78.21 (C34), 19.29 (C35). 1H NMR (CDCl3, 500 MHz): 2.39 (H3a), 2.50 H3b), 3.83 (H4), 1.25–1.29 (H5), 1.25–1.29 (H6–7), 1.55 (H8), 2.38 (H9), 2.38 (H11), 1.55 (H12), 1.25–1.29 (H13), 1.38 (H14), 3.49 (H15), 3.80 (H16), 1.64; 1.98 2 H18), 3.80 (H19), 3.40 (H20), 1.39 (H-21), 1.25–1.29 (H-22), 1.25–1.29 (H23–29), 1.25–1.29 (H30), 1.25–1.29 (H31), 0.87 (H32), 7.18 (H33), 5.05 (H34), 1.39 (H35). Annonacinone: 13C NMR (CDCl3, 125 MHz): 174.08 (C1), 134.45 (C2), 25.31 (C3), 27.55 (C4), 28.97–29.92 (C5), 28.97–29.92 (C6– 7), 23.97 (C8), 42.99 (C9), 211.55 (C10), 42.91 (C11), 25.35 (C12), 25.47 (C13), 33.45 (C14), 74.15 (C15), 82.79 (C16), 29.01 (C17– 18), 82.89 (C19), 74.00 (C20), 33.70 (C21), 25.80 (C22), 28.97– 29.92 (23–29), 32.12 (C30), 22.89 (C31), 14.32 (C32), 149.15 (C33), 77.62 (C34), 19.42 (C35). 1H NMR (CDCl3, 500 MHz): 2.26 (H3), 1.52–1.58 (H4), 1.31 (H5), 1.31 (H6–7), 1.52–1.58 (H8), 2.37–2.43 (H9), 2.37–2.43 (H11), 1.52–1.58 (H12), 1.31 (H13), 1.38 (H14), 3.40 (H15), 3.79 (H16), 1.68; 1.98 (H17–18), 3.79 (H19), 3.40 (H20), 1.38 (H-21), 1.31 (H-21), 1.31 (H23–29), 1.31 (H30), 1.31 (H31), 0.89 (H32), 6.99 (H33), 4.99 (H34), 1.38 (H35). Scoparone: 13C NMR (CDCl3, 125 MHz): 161.4 (C2, C@O), 113.4 (C3), 111.4 (C4ª), 143.3 (C4), 108.0 (C5), 146.3 (C6), 152.8 (C7), 100.0 (C8), 150,0 (C8a) 56,4 (OCH3), 56.4 (OCH3). 1H NMR (CDCl3, 500 MHz): 6.23 (H3, d, J = 9.5 Hz), 7.59 (H4, d, J = 9.5 Hz), 6.83 (H5), 6.78 (H8), 3.90 (OCH3), 3.88 (OCH3). In preliminary evaluation, all compounds inhibited the promastigote growth in all Leishmania species in this study (Table 1). The results revealed that the activity response differs for each species, observing that each species of Leishmania tested with the studied compounds, presented different inhibition. In the evaluation of results, it was observed that the leishmanicidal activity was based in a dose-dependent response, the lower dose (3.123 lg/mL) of all compounds induced lower inhibitions and the higher dose (100 lg/mL) demonstrated the greater inhibitions in all species (Fig. 1). Pentamidine used as positive control in this study, control wells with DMSO did not affect the growth of parasites. The wide ranges of pharmacological properties of scoparone are evident in many previous works, such as anti-allergic effect,
Table 1 Leishmania spp. response to secondary metabolites isolated from Brazilian Northeastern plants and A. salina toxicity. Compounds (lg/mL)
Scoparone Corossolone Annonacinone Pentamidine
IC50 (lg/mL) ± SD L. donovani
L. mexicana
L. major
Toxicity A. salina
27.51 ± 0.97Aa 18.73 ± 0.82Ab 7.66 ± 0.77Ac 1.41 ± 0.24Ad
9.11 ± 0.25Cc 18.64 ± 0.79Ab 8.00 ± 1Ac 1.75 ± 0.34Ad
14.37 ± 0.98Bb 16.14 ± 1.13Ab 6.72 ± 0.37Ac 1.46 ± 0.5Ad
59.4a ± 8.34 7.09c ± 2.17 17.05b ± 3.46 –
Different capital letters indicate statistical differences among columns (species) and different lower case letters indicate significant statistical differences among lines (p < 0.001).
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Fig. 1. Leishmanicidal activity of corossolone (I), annonacinone (II) and scoparone (III), against promastigotes of L. donovani, L. major and L. mexicana. Promastigotes in a logarithimic phase were seeded at 1 106/well and incubated for 24 h with the isolated compounds, the number of living promastigotes was determined indirectly by optical density (OD – 620 nm) and correlated to the percentage of survival. Control wells contained DMSO or no additives, and Pentamidine was used as positive control. Each concentration was tested in triplicate in replicate experiments.
inhibiting anaphylaxis in rats (Choi and Yan, 2009), as a potent hepatoprotective (Bilgin et al., 2011) and inducing the release of dopamine (Yang et al., 2010). The leishmanicidal activity of several coumarins has been reported to inhibit parasite growth in vitro. Ferreira et al. (2010) reported the activity against Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis of nine coumarins; a second study showed a significant inhibition of L. major promatigotes in the presence of aurapten (Napolitano et al., 2004); a third study indicated significant inhibition against using coumarins such aurapten and umbelliprenin (Iranshahi et al., 2007). A lack of studies showing the antiparasitic activities of scoparone was found. Acetogenins are widely studied due to its anticancer (Chen et al., 2012b; D’Ambrosio et al., 2011; Tantithanaporn et al., 2011), antimicrobial (Parellada et al., 2011; Lima et al., 2011) and antiprotozoal (Boyom et al., 2009; Camacho et al., 2003; Grandic et al., 2004) properties. Vila-Nova et al. (2011) reported the effect of annonacinone and corossolone isolated from A. muricata against
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L. chagasi promastigote and amastigote forms, and analyzed the toxicity against murine macrophages cells. In Table 1, promastigotes of Leishmania species demonstrated different susceptibilities in relation to tested compounds; L. mexicana was more sensitive to scoparone followed by L. major and L. donovani (EC50 = 9.11 ± 0.25, 14.37 ± 0.98 and 27.51 ± 0.97 lg/mL, respectively). Comparing the three species within the same line, a similar resistance to corossolone and annonacinone was observed. The drug control Pentamidine was statically different from all the compounds tested. Comparing each species in relation to each compound (within the same column), L. donovani and L. major were more susceptible to annonacinone (EC50 = 7.66 ± 0.77 and 6.72 ± 0.37 lg/mL, respectively), and L. mexicana to annonacinone and scoparone (EC50 = 8 ± 1 and 9.11 ± 0.25 lg/mL, respectively). In general, annonacinone was the most active substance. Pentamidine was statically similar to each other in all Leishmania species. Compounds with anti-Leishmania-activity presenting an EC50 < 10 lg/mL demonstrates high inhibition activity, EC50 > 10 lg/ mL < 50 lg/mL moderate and EC50 > 50 lg/mL weak (Kayser et al., 2000). Then, acetogenin annonacinone (EC50 = 6.72 8.00 lg/mL) indicated high leishmanicidal activity, corossolone (EC50 = 16.14 18.73 lg/mL), and scoparone (EC50 = 9.11 27.51 lg/mL) presented moderate actions. Another study using A. muricata seed extracts against L. amazonensis, L. brasiliensis and L. donovani (Osorio et al., 2007) presented lower activity (EC50 = 63.2 98.6 lg/mL) than the acetogenins in this work. It can be explained by the fact that the extract can contain inferior concentrations of acetogenins. The emergence of drug resistance in some countries led to development of strategies to prevent it, and the introduction of new therapies are essential to prevent this resistance to overcome. Drugs developed from medicinal plants can be a great source of new compounds for protozoa diseases treatments with lower protozoa resistance. Researches for new drugs are encouraged by WHO as part of search for new and better drugs for leishmaniasis treatment (WHO, 2009). Herbal remedies are an ancient source of new drugs but many compounds are still unknown. Leishmaniasis is a neglected disease and its treatment has been the same for over 60 years, which induced resistance in this protozoa. The need of new drugs with lack of resistance from the parasite and available to the population lead us to the research for leishmanicidal compounds from plants such as A. muricata and P. floribundum. A coumarin, scoparone, and two acetogenins, annonacinone, and corossolone, were isolated and its leishmanicidal activity was tested in three different species of Leishmania (L. donovani, L. mexicana and L. major), confirming the antiparasitic potential of these compounds. Nevertheless different species present different susceptibilities in relation to the tested compounds. Further studies are necessary to reveal the pharmacological significance of antiparasitic compounds from A. muricata and P. floribundum.
Acknowledgments We are grateful to the CENAUREMN (Northeastern Center for the Application and Use of Nuclear Magnetic Resonance) of the Federal University of Ceará for the NMR spectra of the compounds, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), and Sistema Único de Saúde (PPSUS) for their financial support.
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Research Brief
Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone Nadja Soares Vila-Nova a, Selene Maia de Morais a,b,⇑, Maria José Cajazeiras Falcão b, Terezinha Thaize Negreiros Alcantara b, Pablito Augusto Travassos Ferreira b, Eveline Solon Barreira Cavalcanti b, Icaro Gusmão Pinto Vieira c, Cláudio Cabral Campello a, Mary Wilson d,e,f a
Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Avenida Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil Curso de Química, Centro de Ciências e Tecnologia, Universidade Estadual do Ceara, Avenida Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazil PADETEC – Parque de Desenvolvimento Tecnologico, Universidade Federal do Ceará, Av. Humberto Monte, 2977, Bairro Parquelandia Bloco 310, CEP 60.440-593 Fortaleza, Ceará, Brazil d Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA e Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA f VA Medical Center, Iowa City, IA 52242, USA b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Acetogenins were isolated from A.
"
" "
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muricata and scoparone from P. floribundum. All compounds inhibited the promastigote growth in the three species. Annonacinone presented high leishmanicidal activity. Corossolone, and scoparone demonstrated moderate leishmanicidal actions. Toxicity was tested using A. salina assay, corossolone was the most toxic compound.
a r t i c l e
i n f o
Article history: Received 3 September 2012 Received in revised form 12 November 2012 Accepted 26 November 2012 Available online 8 December 2012 Keywords: Leishmanicidal Acetogenin Coumarin Leishmania Artemia salina
a b s t r a c t Leishmaniasis is a zoonotic disease that can manifest itself in visceral and cutaneous form. The aim of this study was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied to compounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmania mexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form were used to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundum and two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities in relation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. major and L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetogenin annonacinone (EC50 = 6.72 8.00 lg/mL) indicated high leishmanicidal activity; corossolone
⇑ Corresponding author at: Curso de Química, Universidade Estadual do Ceará, Av. Paranjana, 1700, Campus do Itaperi, CEP 60740-903, Serrinha, Fortaleza, Ceará, Brazil. Tel.: +55 85 31019933. E-mail address: [email protected] (S.M. de Morais). 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.11.025
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(EC50 = 16.14 18.73 lg/mL) and scoparone (EC50 = 9.11 27.51 lg/mL) moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate these compounds for further studies aiming the development of new leishmanicidal agents. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Leishmaniasis is a tropical zoonotic disease caused by more than 20 protozoa species of Leishmania genus (Singh et al., 2012) manifesting itself in visceral and cutaneous form. The geographical distribution of leishmaniasis relate to the growth of sandfly vector, which are dominant in tropical and temperate regions (Roy et al., 2012). The chemotherapy used in the treatment of leishmaniasis is based on the use of drugs that are toxic heavy metals, known as antimoniates, among which the most used are meglumine antimoniate (GlucantimeÒ) and sodium stibogluconate (PentostanÒ). When this type of treatment is not effective, other medications such as pentamidine and amphotericin B and its lipossomal formulation, are also used. All these medications are administrated intravenously and/or intramuscular require clinical supervision or hospitalization due to the severity of side effects (Chan-Bacab and Pena-Rodríguez, 2001; Rath et al., 2003). In dogs, one of the main reservoirs, treatment is not a recommended measure in Brazil, since it does not diminish the importance of these animals as reservoir of the parasite. It only provides clinical remission and not a parasitological cure (Sessa et al., 1994; Alves and Bevilacqua, 2004; Ait-Oudhia et al., 2012). In the early 1990s, the World Health Organization (WHO) reported that 65–80% of the population of developing countries depended on medicinal plants as the only form of access to basic health care (Veiga et al., 2005). A vast number of Brazilian plants are able to produce secondary metabolites with antiparasitic activity; many of these metabolites have not been properly isolated and chemically or biologically evaluated. The Annonaceae family is a great producer of acetogenins that has shown promising results in the search for new drugs against various protozoa, revealing good potential as a source of agents for the treatment of leishmaniasis (Rocha et al., 2005). Annona muricata is a well-known plant in Brazil, the compounds there are most found in this plant are acetogenins and alkaloids (Vila-Nova et al., 2011; Tempone et al., 2005), and has antiprotozoal, antioxidant and anticancer properties (Rondon et al., 2011; Boyom et al., 2009; Lima et al., 2011; Chen et al., 2012a). Platymiscium floribundum is a regional plant in Ceará state used by the population as anti-inflammatory and the isoflavonoids isolated from this plant have demonstrated activity against cancer cells (Militão et al., 2006; Falcao et al., 2005), and the crude extract demonstrated antifungal and acetylcholinesterase activity (Cardoso-Lopes et al., 2008), pterocarpans isolated from the heartwood demonstrated antimitotic effect on sea urchin eggs (Militão et al., 2005), but a few of studies have been developed in the search of others chemotherapeutic values as well as isolating others compounds from this plant. Due to the shortcomings described above, there is a pressing need for new leishmanicidal treatments, and compounds from Northeastern Brazilian plants can be promising sources for future drugs. 2. Material and methods 2.1. Plants The heartwood and sapwood of the stem of P. floribundum and leaves of A. muricata were collected in the state of Ceará, Brazil.
Aerial parts of the two plants were deposited in the Prisco Bezerra Herbarium under the reference number 31052 and 43951, respectively. 2.2. Chromatographic procedures The active chemical constituents were isolated from plant extracts by silica gel (d 0.063–0.200 mm; 70–230 mesh) and Sephadex (LH-20) column chromatography and solvents for elution were petroleum ether, hexane, chloroform, ethyl acetate, acetone and methanol from VETEC (Brazil). The fractions collected in the columns were compared by thin layer chromatography (TLC), using silica gel (60 G F 254) on glass plates (3 cm 8 cm). The TLC plates were analyzed in a iodine chamber, under UV Light (at 312 and 365 nm), Vilbert Loumart, CN-15 LM model, and by spraying the reagent 2.5% vaniline in sulfuric acid diluted in ethanol (1:1), followed by heating in an oven at 100 °C. 2.3. Isolation of compounds and spectroscopic identification A. muricata seeds (2 kg) were triturated and left in contact with methanol for 1 week, then the solvent was filtered, evaporated to dryness and a light yellow solid was obtained (402 g). This material was chromatographed on a filtering silica gel column yielding two main compounds. The trunk heartwood of P. floribundum (800 g) was cold extracted using hexane, chloroform and ethanol successively. Respective extracts were obtained by elimination of solvents in a rotatory evaporator. The chloroform extract was introduced on the top of a glass chromatographic column, filled with silica gel, and the solvents hexane and ethyl acetate, in mixtures of increasing polarity, were used to elute the column. Fiftysix 10 mL fractions were collected and compared by TLC. This procedure conducted to the isolation of a unique compound, revealed by a simple spot on TLC plate. The structures of compounds were determined by spectroscopic analysis of infrared spectra, recorded on a FT-IR PerkinElmer 1000 spectrophotometer, values expressed in cm 1, and nuclear magnetic resonance spectra, recorded on a Bruker Avance DRX-500 spectrometer in CDCl3. 2.4. Leishmanicidal assay L. major, L. mexicana and L. donovani mCherry-Luciferaseexpressing (mCherry-Luc) species were generated with the integrating vector pIR1SAT, provided by Dr. Steve Beverley, as described (Thalhofer et al., 2010). The promastigote forms were cultured in hemoflagellate-modified MEM (HOMEM) medium, supplemented with 10% fetal bovine serum at 26 °C. For the leishmanicidal assay, an adapted methodology from Tempone et al. (2005) was used. The compounds were dissolved in DMSO at a concentration of 0.2% and diluted with HOMEM medium in 96-well microplates. The assay was performed at concentrations of 100, 50, 25, 12.5 and 6.25 lg/mL. Control wells contained DMSO or no additives. Each concentration was tested in triplicate in replicate experiments. Promastigotes at a logarithmic phase were counted in a Neubauer hemocytometer and seeded at 1 106/well. The plates were incubated at 26 °C for 24 h, and the viability of the promastigotes, based on their morphology, was observed under a
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light microscope. Pentamidine (Sigma–Aldrich, St. Louis, MO, USA) was used as the positive control drug. The optical density (OD) was determined in a Fluostar Omega (BMG Labtech) at 620 nm. The number of living promastigotes was determined indirectly by the optical density (OD – 620 nm) representing the percentage of survival. The EC50 values (±SD) were calculated using a nonlinear regression curve using the statistical software GhaphPad Prism 4.0. 2.5. Toxicity tests using Artemia salina A. salina (Artemiidae), also known as brine shrimp larva, is an invertebrate normally used as a safe, practical and economic substitute assay to determine toxicity of compounds from natural products (Barbosa et al., 2009). The toxicity tests were developed using A. salina assay by Vanhaecke et al. (1981) with modifications. Dried cysts of A. salina (15–20 g in 50 mL of seawater) were incubated in a hatcher (a mini-aquarium with two compartments, one at darkness and another illuminated) at 28–30 °C with strong aeration, under a continuous light regime during 24 h. A. salina cysts were placed for hatching in the dark compartment of the aquarium. The phototropic nauplii that migrated to the illuminated compartment were collected with a Pasteur pipette and placed in another flask, when then they were used in the toxicity bioassay. A solution is prepared containing the chemical compound and DMSO (1% v/v) in seawater. Each test consisted of exposing groups of 30 larvae aged 24 h to various concentrations of the toxic compound (10,000, 1000, 100, 10 and 1 ppm). The toxicity was determined after 24 h (nauplii in instar II/III) of exposure. Each concentration was tested in triplicate in replicate experiments. The numbers of survivors were counted, larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation. 3. Statistical analysis The data were tested for compliance with the requirements for carrying out the analysis of variance and normal distribution and homogeneity of variances among treatments. The ANOVA was then performed using the GLM procedure of SAS (2002) and Tukey’s test was used to compare means. For the toxicity assay using A. salina, the EC50 and its confidence intervals were calculated by Probit analysis using the software StatPlus 2009 Professional 5.8.4. 4. Results and discussion The assay of acute toxicity against A. salina has been used in a great diversity of medicinal plants research and its chemical compounds in order to determine their pharmacological assessment, including pursuing anticancer agents, which are those with high toxicity. Barbosa et al. (2009) demonstrated that the A. salina assay was efficient in the search for antileishmanial substances. The evaluation of plant extract toxicity by the brine shrimp bioassay, an LC50 value lower than 1000 lg/mL is considered bioactive (Meyer
et al., 1982). This test was used to find active compounds from plants in our laboratory, as well as to a toxicity screening of the isolated compounds. The assay demonstrated that A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic compound of all three (Table 1). The spectral data of the compounds isolated from A. muricata were the same of previous reported data (Vila-Nova et al., 2011) for the acetogenins, corossolone, and annonacinone. The compound isolated from P. floribundum was characterized as scoparone (6,7dimethoxycoumarin) by comparison of 1H and 13C-nuclear magnetic ressonance data with earlier described by Ma et al. (2006). Corossolone: 13C NMR (CDCl3, 125 MHz): 174.84 (C1), 131.31 (C2), 33.64 (C3), 70.01 (C4), 37.30 (C5), 29.20–29.92 (C6–7), 23.99 (C8), 42.79 (C9), 211.60 (C10), 42.88 (C11), 23.84 (C12), 25.42 (C13), 33.64 (C14), 74.32 (C15), 82.75 (C16), 28.96 (C17– 18), 82.89 (C19), 74.02 (C20), 33.59 (C21), 25.78 (C22), 29.20– 29.92 (C23–29), 32.11 (C30), 22.88 (C31), 14.32 (C32), 152.12 (C33), 78.21 (C34), 19.29 (C35). 1H NMR (CDCl3, 500 MHz): 2.39 (H3a), 2.50 H3b), 3.83 (H4), 1.25–1.29 (H5), 1.25–1.29 (H6–7), 1.55 (H8), 2.38 (H9), 2.38 (H11), 1.55 (H12), 1.25–1.29 (H13), 1.38 (H14), 3.49 (H15), 3.80 (H16), 1.64; 1.98 2 H18), 3.80 (H19), 3.40 (H20), 1.39 (H-21), 1.25–1.29 (H-22), 1.25–1.29 (H23–29), 1.25–1.29 (H30), 1.25–1.29 (H31), 0.87 (H32), 7.18 (H33), 5.05 (H34), 1.39 (H35). Annonacinone: 13C NMR (CDCl3, 125 MHz): 174.08 (C1), 134.45 (C2), 25.31 (C3), 27.55 (C4), 28.97–29.92 (C5), 28.97–29.92 (C6– 7), 23.97 (C8), 42.99 (C9), 211.55 (C10), 42.91 (C11), 25.35 (C12), 25.47 (C13), 33.45 (C14), 74.15 (C15), 82.79 (C16), 29.01 (C17– 18), 82.89 (C19), 74.00 (C20), 33.70 (C21), 25.80 (C22), 28.97– 29.92 (23–29), 32.12 (C30), 22.89 (C31), 14.32 (C32), 149.15 (C33), 77.62 (C34), 19.42 (C35). 1H NMR (CDCl3, 500 MHz): 2.26 (H3), 1.52–1.58 (H4), 1.31 (H5), 1.31 (H6–7), 1.52–1.58 (H8), 2.37–2.43 (H9), 2.37–2.43 (H11), 1.52–1.58 (H12), 1.31 (H13), 1.38 (H14), 3.40 (H15), 3.79 (H16), 1.68; 1.98 (H17–18), 3.79 (H19), 3.40 (H20), 1.38 (H-21), 1.31 (H-21), 1.31 (H23–29), 1.31 (H30), 1.31 (H31), 0.89 (H32), 6.99 (H33), 4.99 (H34), 1.38 (H35). Scoparone: 13C NMR (CDCl3, 125 MHz): 161.4 (C2, C@O), 113.4 (C3), 111.4 (C4ª), 143.3 (C4), 108.0 (C5), 146.3 (C6), 152.8 (C7), 100.0 (C8), 150,0 (C8a) 56,4 (OCH3), 56.4 (OCH3). 1H NMR (CDCl3, 500 MHz): 6.23 (H3, d, J = 9.5 Hz), 7.59 (H4, d, J = 9.5 Hz), 6.83 (H5), 6.78 (H8), 3.90 (OCH3), 3.88 (OCH3). In preliminary evaluation, all compounds inhibited the promastigote growth in all Leishmania species in this study (Table 1). The results revealed that the activity response differs for each species, observing that each species of Leishmania tested with the studied compounds, presented different inhibition. In the evaluation of results, it was observed that the leishmanicidal activity was based in a dose-dependent response, the lower dose (3.123 lg/mL) of all compounds induced lower inhibitions and the higher dose (100 lg/mL) demonstrated the greater inhibitions in all species (Fig. 1). Pentamidine used as positive control in this study, control wells with DMSO did not affect the growth of parasites. The wide ranges of pharmacological properties of scoparone are evident in many previous works, such as anti-allergic effect,
Table 1 Leishmania spp. response to secondary metabolites isolated from Brazilian Northeastern plants and A. salina toxicity. Compounds (lg/mL)
Scoparone Corossolone Annonacinone Pentamidine
IC50 (lg/mL) ± SD L. donovani
L. mexicana
L. major
Toxicity A. salina
27.51 ± 0.97Aa 18.73 ± 0.82Ab 7.66 ± 0.77Ac 1.41 ± 0.24Ad
9.11 ± 0.25Cc 18.64 ± 0.79Ab 8.00 ± 1Ac 1.75 ± 0.34Ad
14.37 ± 0.98Bb 16.14 ± 1.13Ab 6.72 ± 0.37Ac 1.46 ± 0.5Ad
59.4a ± 8.34 7.09c ± 2.17 17.05b ± 3.46 –
Different capital letters indicate statistical differences among columns (species) and different lower case letters indicate significant statistical differences among lines (p < 0.001).
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Fig. 1. Leishmanicidal activity of corossolone (I), annonacinone (II) and scoparone (III), against promastigotes of L. donovani, L. major and L. mexicana. Promastigotes in a logarithimic phase were seeded at 1 106/well and incubated for 24 h with the isolated compounds, the number of living promastigotes was determined indirectly by optical density (OD – 620 nm) and correlated to the percentage of survival. Control wells contained DMSO or no additives, and Pentamidine was used as positive control. Each concentration was tested in triplicate in replicate experiments.
inhibiting anaphylaxis in rats (Choi and Yan, 2009), as a potent hepatoprotective (Bilgin et al., 2011) and inducing the release of dopamine (Yang et al., 2010). The leishmanicidal activity of several coumarins has been reported to inhibit parasite growth in vitro. Ferreira et al. (2010) reported the activity against Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis of nine coumarins; a second study showed a significant inhibition of L. major promatigotes in the presence of aurapten (Napolitano et al., 2004); a third study indicated significant inhibition against using coumarins such aurapten and umbelliprenin (Iranshahi et al., 2007). A lack of studies showing the antiparasitic activities of scoparone was found. Acetogenins are widely studied due to its anticancer (Chen et al., 2012b; D’Ambrosio et al., 2011; Tantithanaporn et al., 2011), antimicrobial (Parellada et al., 2011; Lima et al., 2011) and antiprotozoal (Boyom et al., 2009; Camacho et al., 2003; Grandic et al., 2004) properties. Vila-Nova et al. (2011) reported the effect of annonacinone and corossolone isolated from A. muricata against
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L. chagasi promastigote and amastigote forms, and analyzed the toxicity against murine macrophages cells. In Table 1, promastigotes of Leishmania species demonstrated different susceptibilities in relation to tested compounds; L. mexicana was more sensitive to scoparone followed by L. major and L. donovani (EC50 = 9.11 ± 0.25, 14.37 ± 0.98 and 27.51 ± 0.97 lg/mL, respectively). Comparing the three species within the same line, a similar resistance to corossolone and annonacinone was observed. The drug control Pentamidine was statically different from all the compounds tested. Comparing each species in relation to each compound (within the same column), L. donovani and L. major were more susceptible to annonacinone (EC50 = 7.66 ± 0.77 and 6.72 ± 0.37 lg/mL, respectively), and L. mexicana to annonacinone and scoparone (EC50 = 8 ± 1 and 9.11 ± 0.25 lg/mL, respectively). In general, annonacinone was the most active substance. Pentamidine was statically similar to each other in all Leishmania species. Compounds with anti-Leishmania-activity presenting an EC50 < 10 lg/mL demonstrates high inhibition activity, EC50 > 10 lg/ mL < 50 lg/mL moderate and EC50 > 50 lg/mL weak (Kayser et al., 2000). Then, acetogenin annonacinone (EC50 = 6.72 8.00 lg/mL) indicated high leishmanicidal activity, corossolone (EC50 = 16.14 18.73 lg/mL), and scoparone (EC50 = 9.11 27.51 lg/mL) presented moderate actions. Another study using A. muricata seed extracts against L. amazonensis, L. brasiliensis and L. donovani (Osorio et al., 2007) presented lower activity (EC50 = 63.2 98.6 lg/mL) than the acetogenins in this work. It can be explained by the fact that the extract can contain inferior concentrations of acetogenins. The emergence of drug resistance in some countries led to development of strategies to prevent it, and the introduction of new therapies are essential to prevent this resistance to overcome. Drugs developed from medicinal plants can be a great source of new compounds for protozoa diseases treatments with lower protozoa resistance. Researches for new drugs are encouraged by WHO as part of search for new and better drugs for leishmaniasis treatment (WHO, 2009). Herbal remedies are an ancient source of new drugs but many compounds are still unknown. Leishmaniasis is a neglected disease and its treatment has been the same for over 60 years, which induced resistance in this protozoa. The need of new drugs with lack of resistance from the parasite and available to the population lead us to the research for leishmanicidal compounds from plants such as A. muricata and P. floribundum. A coumarin, scoparone, and two acetogenins, annonacinone, and corossolone, were isolated and its leishmanicidal activity was tested in three different species of Leishmania (L. donovani, L. mexicana and L. major), confirming the antiparasitic potential of these compounds. Nevertheless different species present different susceptibilities in relation to the tested compounds. Further studies are necessary to reveal the pharmacological significance of antiparasitic compounds from A. muricata and P. floribundum.
Acknowledgments We are grateful to the CENAUREMN (Northeastern Center for the Application and Use of Nuclear Magnetic Resonance) of the Federal University of Ceará for the NMR spectra of the compounds, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), and Sistema Único de Saúde (PPSUS) for their financial support.
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