Experimental Parasitology 133 (2013) 334–338

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Experimental Parasitology

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Research Brief

Different susceptibilities of Leishmania spp. promastigotes to the Annona muricataacetogenins annonacinone and corossolone, and the Platymiscium floribundumcoumarin 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, Brazilb Curso de Química, Centro de Ciências e Tecnologia, Universidade Estadual do Ceara, Avenida Paranjana, 1700, Campus do Itaperi, 60740-000 Fortaleza, Ceará, Brazilc PADETEC – Parque de Desenvolvimento Tecnologico, Universidade Federal do Ceará, Av. Humberto Monte, 2977, Bairro Parquelandia Bloco 310, CEP 60.440-593 Fortaleza,Ceará, Brazild Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USAe Department of Microbiology, University of Iowa, Iowa City, IA 52242, USAf VA Medical Center, Iowa City, IA 52242, USA

h i g h l i g h t s

" Acetogenins were isolated from A.muricata and scoparone from P.floribundum.

" All compounds inhibited thepromastigote growth in the threespecies.

" Annonacinone presented highleishmanicidal activity.

" Corossolone, and scoparonedemonstrated moderateleishmanicidal actions.

" Toxicity was tested using A. salinaassay, corossolone was the mosttoxic compound.

0014-4894/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.exppara.2012.11.025

⇑ Corresponding author at: Curso de Química, UniAv. Paranjana, 1700, Campus do Itaperi, CEP 60740-9Brazil. Tel.: +55 85 31019933.

E-mail address: selenemaiademorais@gmail.com (

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 September 2012Received in revised form 12 November 2012Accepted 26 November 2012Available online 8 December 2012

Keywords:LeishmanicidalAcetogeninCoumarinLeishmaniaArtemia 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 thisstudy was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied tocompounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata.Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmaniamexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form wereused to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundumand two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidalactivity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities inrelation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. majorand L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetog-enin annonacinone (EC50 = 6.72 � 8.00 lg/mL) indicated high leishmanicidal activity; corossolone

ll rights reserved.

versidade Estadual do Ceará,03, Serrinha, Fortaleza, Ceará,

S.M. de Morais).

N.S. Vila-Nova et al. / Experimental Parasitology 133 (2013) 334–338 335

(EC50 = 16.14 � 18.73 lg/mL) and scoparone (EC50 = 9.11 � 27.51 lg/mL) moderate activity. A. saline lar-vae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In con-clusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate thesecompounds for further studies aiming the development of new leishmanicidal agents.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Leishmaniasis is a tropical zoonotic disease caused by morethan 20 protozoa species of Leishmania genus (Singh et al., 2012)manifesting itself in visceral and cutaneous form. The geographicaldistribution 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 leishmaniasisis based on the use of drugs that are toxic heavy metals, knownas antimoniates, among which the most used are meglumine anti-moniate (Glucantime�) and sodium stibogluconate (Pentostan�).When this type of treatment is not effective, other medicationssuch as pentamidine and amphotericin B and its lipossomal formu-lation, are also used. All these medications are administrated intra-venously and/or intramuscular require clinical supervision orhospitalization due to the severity of side effects (Chan-Bacaband Pena-Rodríguez, 2001; Rath et al., 2003). In dogs, one of themain reservoirs, treatment is not a recommended measure in Bra-zil, since it does not diminish the importance of these animals asreservoir of the parasite. It only provides clinical remission andnot 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) re-ported that 65–80% of the population of developing countries de-pended on medicinal plants as the only form of access to basichealth care (Veiga et al., 2005). A vast number of Brazilian plantsare able to produce secondary metabolites with antiparasitic activ-ity; many of these metabolites have not been properly isolated andchemically or biologically evaluated. The Annonaceae family is agreat producer of acetogenins that has shown promising resultsin the search for new drugs against various protozoa, revealinggood potential as a source of agents for the treatment of leishman-iasis (Rocha et al., 2005). Annona muricata is a well-known plant inBrazil, the compounds there are most found in this plant are ace-togenins and alkaloids (Vila-Nova et al., 2011; Tempone et al.,2005), and has antiprotozoal, antioxidant and anticancer proper-ties (Rondon et al., 2011; Boyom et al., 2009; Lima et al., 2011;Chen et al., 2012a). Platymiscium floribundum is a regional plantin Ceará state used by the population as anti-inflammatory andthe isoflavonoids isolated from this plant have demonstrated activ-ity against cancer cells (Militão et al., 2006; Falcao et al., 2005), andthe crude extract demonstrated antifungal and acetylcholinester-ase activity (Cardoso-Lopes et al., 2008), pterocarpans isolatedfrom the heartwood demonstrated antimitotic effect on sea urchineggs (Militão et al., 2005), but a few of studies have been developedin the search of others chemotherapeutic values as well as isolatingothers compounds from this plant.

Due to the shortcomings described above, there is a pressingneed for new leishmanicidal treatments, and compounds fromNortheastern Brazilian plants can be promising sources for futuredrugs.

2. Material and methods

2.1. Plants

The heartwood and sapwood of the stem of P. floribundum andleaves of A. muricata were collected in the state of Ceará, Brazil.

Aerial parts of the two plants were deposited in the Prisco BezerraHerbarium under the reference number 31052 and 43951,respectively.

2.2. Chromatographic procedures

The active chemical constituents were isolated from plant ex-tracts by silica gel (d 0.063–0.200 mm; 70–230 mesh) and Sepha-dex (LH-20) column chromatography and solvents for elutionwere petroleum ether, hexane, chloroform, ethyl acetate, acetoneand methanol from VETEC (Brazil). The fractions collected in thecolumns were compared by thin layer chromatography (TLC),using silica gel (60 G F 254) on glass plates (3 cm � 8 cm). TheTLC plates were analyzed in a iodine chamber, under UV Light (at312 and 365 nm), Vilbert Loumart, CN-15 LM model, and by spray-ing 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 withmethanol for 1 week, then the solvent was filtered, evaporated todryness and a light yellow solid was obtained (402 g). This materialwas chromatographed on a filtering silica gel column yielding twomain compounds. The trunk heartwood of P. floribundum (800 g)was cold extracted using hexane, chloroform and ethanol succes-sively. Respective extracts were obtained by elimination of sol-vents in a rotatory evaporator. The chloroform extract wasintroduced on the top of a glass chromatographic column, filledwith silica gel, and the solvents hexane and ethyl acetate, in mix-tures of increasing polarity, were used to elute the column. Fifty-six 10 mL fractions were collected and compared by TLC. This pro-cedure conducted to the isolation of a unique compound, revealedby a simple spot on TLC plate.

The structures of compounds were determined by spectroscopicanalysis of infrared spectra, recorded on a FT-IR PerkinElmer 1000spectrophotometer, values expressed in cm�1, and nuclear mag-netic resonance spectra, recorded on a Bruker Avance DRX-500spectrometer in CDCl3.

2.4. Leishmanicidal assay

L. major, L. mexicana and L. donovani mCherry-Luciferase-expressing (mCherry-Luc) species were generated with theintegrating vector pIR1SAT, provided by Dr. Steve Beverley, asdescribed (Thalhofer et al., 2010). The promastigote forms werecultured in hemoflagellate-modified MEM (HOMEM) medium,supplemented with 10% fetal bovine serum at 26 �C. For the leish-manicidal assay, an adapted methodology from Tempone et al.(2005) was used. The compounds were dissolved in DMSO at a con-centration of 0.2% and diluted with HOMEM medium in 96-wellmicroplates. The assay was performed at concentrations of 100,50, 25, 12.5 and 6.25 lg/mL. Control wells contained DMSO or noadditives. Each concentration was tested in triplicate in replicateexperiments. Promastigotes at a logarithmic phase were countedin a Neubauer hemocytometer and seeded at 1 � 106/well. Theplates were incubated at 26 �C for 24 h, and the viability of thepromastigotes, based on their morphology, was observed under a

336 N.S. Vila-Nova et al. / Experimental Parasitology 133 (2013) 334–338

light microscope. Pentamidine (Sigma–Aldrich, St. Louis, MO, USA)was used as the positive control drug. The optical density (OD) wasdetermined in a Fluostar Omega (BMG Labtech) at 620 nm.

The number of living promastigotes was determined indirectlyby the optical density (OD – 620 nm) representing the percentageof survival. The EC50 values (±SD) were calculated using a nonlinearregression 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 aninvertebrate normally used as a safe, practical and economic sub-stitute assay to determine toxicity of compounds from naturalproducts (Barbosa et al., 2009). The toxicity tests were developedusing A. salina assay by Vanhaecke et al. (1981) with modifications.Dried cysts of A. salina (15–20 g in 50 mL of seawater) were incu-bated in a hatcher (a mini-aquarium with two compartments,one at darkness and another illuminated) at 28–30 �C with strongaeration, under a continuous light regime during 24 h. A. salinacysts were placed for hatching in the dark compartment of theaquarium. The phototropic nauplii that migrated to the illuminatedcompartment were collected with a Pasteur pipette and placed inanother flask, when then they were used in the toxicity bioassay.A solution is prepared containing the chemical compound andDMSO (1% v/v) in seawater. Each test consisted of exposing groupsof 30 larvae aged 24 h to various concentrations of the toxic com-pound (10,000, 1000, 100, 10 and 1 ppm). The toxicity was deter-mined after 24 h (nauplii in instar II/III) of exposure. Eachconcentration was tested in triplicate in replicate experiments.The numbers of survivors were counted, larvae were considereddead if they did not exhibit any internal or external movementduring several seconds of observation.

3. Statistical analysis

The data were tested for compliance with the requirements forcarrying out the analysis of variance and normal distribution andhomogeneity of variances among treatments. The ANOVA was thenperformed using the GLM procedure of SAS (2002) and Tukey’s testwas used to compare means. For the toxicity assay using A. salina,the EC50 and its confidence intervals were calculated by Probitanalysis 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 agreat diversity of medicinal plants research and its chemical com-pounds in order to determine their pharmacological assessment,including pursuing anticancer agents, which are those with hightoxicity. Barbosa et al. (2009) demonstrated that the A. salina assaywas efficient in the search for antileishmanial substances. The eval-uation of plant extract toxicity by the brine shrimp bioassay, anLC50 value lower than 1000 lg/mL is considered bioactive (Meyer

Table 1Leishmania spp. response to secondary metabolites isolated from Brazilian Northeastern p

Compounds (lg/mL) IC50 (lg/mL) ± SD

L. donovani L. mexic

Scoparone 27.51 ± 0.97Aa 9.11 ±Corossolone 18.73 ± 0.82Ab 18.64 ±Annonacinone 7.66 ± 0.77Ac 8.00 ±Pentamidine 1.41 ± 0.24Ad 1.75 ±

Different capital letters indicate statistical differences among columns (species) and d(p < 0.001).

et al., 1982). This test was used to find active compounds fromplants in our laboratory, as well as to a toxicity screening of the iso-lated compounds. The assay demonstrated that A. saline larvaewere less sensitive to the coumarin scoparone and acetogenincorossolone was the most toxic compound of all three (Table 1).

The spectral data of the compounds isolated from A. muricatawere the same of previous reported data (Vila-Nova et al., 2011)for the acetogenins, corossolone, and annonacinone. The compoundisolated from P. floribundum was characterized as scoparone (6,7-dimethoxycoumarin) by comparison of 1H and 13C-nuclear mag-netic 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 promas-tigote growth in all Leishmania species in this study (Table 1). Theresults revealed that the activity response differs for each species,observing that each species of Leishmania tested with the studiedcompounds, presented different inhibition. In the evaluation of re-sults, it was observed that the leishmanicidal activity was based ina dose-dependent response, the lower dose (3.123 lg/mL) of allcompounds 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, controlwells with DMSO did not affect the growth of parasites.

The wide ranges of pharmacological properties of scoparone areevident in many previous works, such as anti-allergic effect,

lants and A. salina toxicity.

ana L. major Toxicity A. salina

0.25Cc 14.37 ± 0.98Bb 59.4a ± 8.340.79Ab 16.14 ± 1.13Ab 7.09c ± 2.171Ac 6.72 ± 0.37Ac 17.05b ± 3.460.34Ad 1.46 ± 0.5Ad –

ifferent lower case letters indicate significant statistical differences among lines

Fig. 1. Leishmanicidal activity of corossolone (I), annonacinone (II) and scoparone(III), against promastigotes of L. donovani, L. major and L. mexicana. Promastigotes ina logarithimic phase were seeded at 1 � 106/well and incubated for 24 h with theisolated compounds, the number of living promastigotes was determined indirectlyby optical density (OD – 620 nm) and correlated to the percentage of survival.Control wells contained DMSO or no additives, and Pentamidine was used aspositive control. Each concentration was tested in triplicate in replicateexperiments.

N.S. Vila-Nova et al. / Experimental Parasitology 133 (2013) 334–338 337

inhibiting anaphylaxis in rats (Choi and Yan, 2009), as a potenthepatoprotective (Bilgin et al., 2011) and inducing the release ofdopamine (Yang et al., 2010). The leishmanicidal activity of severalcoumarins has been reported to inhibit parasite growth in vitro.Ferreira et al. (2010) reported the activity against Leishmania ama-zonensis, Leishmania infantum and Leishmania braziliensis of ninecoumarins; a second study showed a significant inhibition of L. ma-jor promatigotes in the presence of aurapten (Napolitano et al.,2004); a third study indicated significant inhibition against usingcoumarins such aurapten and umbelliprenin (Iranshahi et al.,2007). A lack of studies showing the antiparasitic activities ofscoparone was found.

Acetogenins are widely studied due to its anticancer (Chenet al., 2012b; D’Ambrosio et al., 2011; Tantithanaporn et al.,2011), antimicrobial (Parellada et al., 2011; Lima et al., 2011) andantiprotozoal (Boyom et al., 2009; Camacho et al., 2003; Grandicet al., 2004) properties. Vila-Nova et al. (2011) reported the effectof annonacinone and corossolone isolated from A. muricata against

L. chagasi promastigote and amastigote forms, and analyzed thetoxicity against murine macrophages cells.

In Table 1, promastigotes of Leishmania species demonstrateddifferent susceptibilities in relation to tested compounds; L. mexi-cana 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 ob-served. The drug control Pentamidine was statically different fromall the compounds tested. Comparing each species in relation toeach compound (within the same column), L. donovani and L. majorwere more susceptible to annonacinone (EC50 = 7.66 ± 0.77 and6.72 ± 0.37 lg/mL, respectively), and L. mexicana to annonacinoneand scoparone (EC50 = 8 ± 1 and 9.11 ± 0.25 lg/mL, respectively).In general, annonacinone was the most active substance. Pentam-idine 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 seedextracts against L. amazonensis, L. brasiliensis and L. donovani (Oso-rio 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 factthat the extract can contain inferior concentrations of acetogenins.

The emergence of drug resistance in some countries led todevelopment of strategies to prevent it, and the introduction ofnew therapies are essential to prevent this resistance to overcome.Drugs developed from medicinal plants can be a great source ofnew compounds for protozoa diseases treatments with lower pro-tozoa resistance. Researches for new drugs are encouraged byWHO as part of search for new and better drugs for leishmaniasistreatment (WHO, 2009).

Herbal remedies are an ancient source of new drugs but manycompounds are still unknown. Leishmaniasis is a neglected diseaseand its treatment has been the same for over 60 years, which in-duced resistance in this protozoa. The need of new drugs with lackof resistance from the parasite and available to the population leadus to the research for leishmanicidal compounds from plants suchas A. muricata and P. floribundum. A coumarin, scoparone, and twoacetogenins, annonacinone, and corossolone, were isolated and itsleishmanicidal activity was tested in three different species ofLeishmania (L. donovani, L. mexicana and L. major), confirming theantiparasitic potential of these compounds. Nevertheless differentspecies present different susceptibilities in relation to the testedcompounds. Further studies are necessary to reveal the pharmaco-logical significance of antiparasitic compounds from A. muricataand P. floribundum.

Acknowledgments

We are grateful to the CENAUREMN (Northeastern Center forthe Application and Use of Nuclear Magnetic Resonance) of theFederal University of Ceará for the NMR spectra of the compounds,and Conselho Nacional de Desenvolvimento Científico e Tecnológ-ico (CNPq), Fundação Cearense de Apoio ao DesenvolvimentoCientífico e Tecnológico (FUNCAP), and Sistema Único de Saúde(PPSUS) for their financial support.

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