Experimental Parasitology 143 (2014) 60–68

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Experimental Parasitology

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Antileishmanial activity and cytotoxicity of Brazilian plants

http://dx.doi.org/10.1016/j.exppara.2014.05.0040014-4894/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Departamento de Produtos Farmacêuticos, Faculdadede Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901Belo Horizonte, Minas Gerais, Brazil. Fax: +55 31 3409 6935.

E-mail address: roc2006@farmacia.ufmg.br (R.O. Castilho).

Tatiana G. Ribeiro a, Miguel A. Chávez-Fumagalli b, Diogo G. Valadares c, Juçara R. Franca a, Paula S. Lage b,Mariana C. Duarte b, Pedro H.R. Andrade d, Vivian T. Martins c, Lourena E. Costa b, Ana L.A. Arruda e,André A.G. Faraco a,e, Eduardo A.F. Coelho b,d, Rachel O. Castilho a,e,⇑a Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901 Belo Horizonte, Minas Gerais,Brazilb Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901Belo Horizonte, Minas Gerais, Brazilc Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901 Belo Horizonte, Minas Gerais, Brazild Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901 Belo Horizonte, Minas Gerais, Brazile Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 31270-901 Belo Horizonte, Minas Gerais, Brazil

h i g h l i g h t s

� Brazilian medicinal plants wereevaluated against L. amazonensis.� D. alata and J. cuspidifolia leaves had

high selectivity index.� These extracts or fractions had high

leishmanicidal activity and lowcytotoxicity.� The activity of D. alata and J.

cuspidifolia could be due to a directmechanism.� The compounds that could contribute

to the observed activity are discussed.

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 14 November 2013Received in revised form 24 April 2014Accepted 11 May 2014Available online 17 May 2014

Keywords:Dipteryx alataJacaranda cuspidifoliaMurine macrophageLeishmania amazonensisPromastigotesAmastigotes

a b s t r a c t

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance hasincreased the importance of discovering new therapeutic products. The present study aimed to investi-gate the in vitro leishmanicidal activity from 16 different Brazilian medicinal plants. Stationary-phasepromastigotes of Leishmania amazonensis and murine macrophages were exposed to 44 plant extractsor fractions for 48 h at 37 �C, in order to evaluate their antileishmanial activity and cytotoxicity, respec-tively. The most potent extracts against L. amazonensis were the hexanic extract of Dipteryx alata (IC50 of0.08 lg/mL), the hexanic extract of Syzygium cumini (IC50 of 31.64 lg/mL), the ethanolic and hexanicextracts of leaves of Hymenaea courbaril (IC50 of 44.10 lg/mL and 35.84 lg/mL, respectively), the etha-nolic extract of H. stignocarpa (IC50 of 4.69 lg/mL), the ethanolic extract of Jacaranda caroba (IC50 of13.22 lg/mL), and the ethanolic extract of J. cuspidifolia leaves (IC50 of 10.96 lg/mL). Extracts of D. alataand J. cuspidifolia presented higher selectivity index, with high leishmanicidal activity and low cytotox-icity in the mammalian cells. The capacity in treated infected macrophages using the extracts and/orfractions of D. alata and J. cuspidifolia was also analyzed, and reductions of 95.80%, 98.31%, and 97.16%,respectively, in the parasite burden, were observed. No nitric oxide (NO) production could beobserved in the treated macrophages, after stimulation with the extracts and/or fractions of D. alataand J. cuspidifolia, suggesting that the biological activity could be due to mechanisms other than macro-phage activation mediated by NO production. Based on phytochemistry studies, the classes of compounds

T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68 61

that could contribute to the observed activities are also discussed. In conclusion, the data presented inthis study indicated that traditional medicinal plant extracts present effective antileishmanial activity.Future studies could focus on the identification and purification of the antileishmanial compounds withinthese plants for analysis of their in vivo antileishmanial activity.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Leishmaniasis is a disease with a wide spectrum of clinical man-ifestations caused by different species of protozoa belonging to theLeishmania genus (Desjeux, 2004). The parasites are transmittedthrough the bite of an infected sandfly, and more than 20 Leish-mania species can cause this disease in humans (Grimaldi andTesh, 1993). Ninety-eight countries and 3 territories on 5 conti-nents have reported endemic leishmaniasis transmission, leadingto the classification of leishmaniasis as one of the six most impor-tant neglected tropical diseases by the World Health Organization(Committee WHOE, 2010). Approximately 0.2–0.4 million cases ofvisceral leishmaniasis (VL), and about 0.7–1.2 million of cases ofcutaneous leishmaniasis are registered annually, reporting theimportance of this disease (Alvar et al., 2012).

Historically, chemotherapy has been used in the treatment ofleishmaniasis, and it has been based on the use of pentavalentantimony compounds; however, the results obtained using theseproducts are considered unsatisfactory, mainly due to their hightoxicity and lack of efficacy in some cases of VL. Alternative treat-ments for leishmaniasis include amphotericin B and its lipid for-mulations, as well as paromomycin and miltefosine. However,the high costs, toxic side effects, and long treatment periodsrequired limit the clinical usefulness of these drugs (Sundaret al., 2012; Kedzierski et al., 2009; Murray et al., 2005). Therefore,the development of new and cost-effective alternative therapeuticstrategies for leishmaniasis has become a high priority (Frézardet al., 2009; Valadares et al., 2012a; Ribeiro et al., 2014).

In recent years, considerable attention has been given to study-ing plants in an attempt to search for new antileishmanial drugs(Weniger et al., 2001; Newman et al., 2007). The medicinal valueof plants lies in their active compounds, which produce a measur-able physiological action in the body (Basile et al., 2000; Cowan,1999). Identification of natural products and the wide variety ofseparation techniques available increase the probability of findingnovel products that in turn could lead to the development of newpharmaceutical compounds (Saklani and Kutty, 2008).

The present study investigated the in vitro antileishmanialactivity of 16 different Brazilian plant species (mainly from Cerra-do), in order to evaluate their activity against Leishmania amazon-ensis. Studies were carried out to establish their minimuminhibitory concentrations (IC50) in Leishmania, as well as their cyto-toxic effects on murine macrophages (CC50), to determine theselectivity index (SI) of each extract or fraction. The products thatpresented higher SI values were used in the treatment of macro-phages infected with L. amazonensis. The possible mechanism thatcould be involved in the elimination of parasites by infected cellswas also discussed.

2. Materials and methods

2.1. Plants and preparation of extracts

The extracts used in this study were obtained from a bank ofextracts at the Laboratório de Fitoquímica of Faculdade de Farmá-cia da UFMG (Belo Horizonte, Minas Gerais, Brazil). The geographiclocation of the plant collection site is shown in Fig. 1. The voucher

species were deposited in the UFMG Herbarium (BHCB), in the Uni-versidade Federal de Mato Grosso do Sul Herbarium (CGMS), or inthe Universidade Federal do Rio de Janeiro Herbarium (R). The vou-cher locations of the plants are shown in Table 1. About 1 kg ofleaves, roots, and stem barks of the plants were dried at 40 �C withforced air circulation. The powdered dry materials were extractedby maceration using hexane and ethanol successively at room tem-perature. The solvents were evaporated under reduced pressure toprovide a solid residue. Dry ethanolic extracts of Campomanesialineatifolia and Chrysobalanus icaco were suspended in water andsequentially partitioned using four solvents with different polari-ties to yield hexane, dichloromethane, ethyl acetate, and butanolicfractions. Licania tomentosa was extracted by maceration usingdichloromethane, ethanol, and ethyl acetate. The dry ethanolextract of Jacaranda cuspidifolia was suspended in water andsequentially partitioned with CHCl3/H2O (1:1). Dimethyl sulfoxide(DMSO) was used as the solvent at the concentration of 0.1% in thefinal test solution.

2.2. Phytochemical analysis

The presence of tannins, flavonoids, coumarins, quinones, alka-loids, triterpenes, steroids and saponins were evaluated in the hex-anic extract of D. alata and in the ethanolic extract and in thechloroformic fraction of J. cuspidifolia by thin layer chromatogra-phy (TLC) analysis and specific reagents (Wagner et al., 1984).

2.2.1. Determination of flavonoid contentThe total flavonoid content was determined using the method

adapted from Brazilian Pharmacopeia IV (Brazilian Pharmacopeia,2010). Briefly, an aliquot of 2 mL of the ethanol solution (20 lg/mL) of extracts and fraction of D. alata and J. cuspidifolia from eachsample was added to 1 mL of AlCl3�6H2O solution (2%, w/v). Thismixture was combined with methanolic acetic acid solution (5%,v/v) and, after 30 min; absorbances were read at k 425 nm. Theblank referred to the same solution without AlCl3�6H2O reagent.Tests were carried out in triplicate. The total flavonoid contentwas calculated from the calibration curve of quercetin standard.Results were expressed in terms of percentage (%).

2.2.2. Determination of phenolic contentThe quantitative phenolic content in samples was estimated

using the method adapted from Brazilian Pharmacopeia IV(Brazilian Pharmacopeia, 2010). Briefly, an aliquot of 2 mL of theethanol solution (20 lg/mL) of extracts and fraction of D. alataand J. cuspidifolia was added 2 mL of Folin–Denis reagent. This mix-ture was combined with Na2CO3 solution and, after 30 min, theabsorbances were read at k 715 nm. The blank referred to the samesolution without Folin–Denis reagent. Tests were carried out intriplicate. The total phenolic content was calculated from thecalibration curve of pyrogallol standard. Results were expressedin %.

2.3. Parasites and mice

L. amazonensis (IFLA/BR/1967/PH-8) was used in this study. Thestationary-phase promastigotes were grown at 24 �C in Schneider’s

Table 1Botanical data of the selected Brazilian plants.

Botanical name Family Popular name (Brazil) Voucher number Localities

Bowdichia virgiloides Kunth. Fabaceae Sucupira preta CGMS 11966 Campo Grande, MSCampomanesia lineatifolia Ruiz & Pav. Myrtaceae Gabiroba ou guabirabeira BHCB 150606 Belo Horizonte, MGCecropia pachystachya Trécul Cecropiaceae Imbaiba CGMS 11697 Campo Grande, MSChrysobalanus icaco L. Chrysobalanaceae Bajerú, guajerú, abajero, ajuru, ajuru-branco,

ariu, cajuru, goajuru, guajiru, guajuru, oajuruR 195.942 Cabo Frio, RJ

Diospyros hispida D.C. Ebenaceae Fruto-de-boi, olho-de-boi, caqui-do-mato,caquí-do-cerrado, caquí-bravo

CGMS 11964 Campo Grande, MS

Dipteryx alata Vog. Fabaceae Baruí, Baru CGMS 17488 Campo Grande, MSSyzygium cumini L. Myrtaceae Jamelão CGMS 11693 Campo Grande, MSEugenia uniflora L. Myrtaceae Pitanga CGMS 11694 Campo Grande, MSHymenaea courbaril L. Fabaceae Jatoba pequeno CGMS 11696 Campo Grande, MSHymenaea stignocarpa Mart. ex. Hayne Fabaceae Jatoba grande CGMS 11695 Campo Grande, MSJacaranda caroba Vell. Bignoniaceae Carobinha CGMS 11692 Campo Grande, MSJacaranda cuspidifolia Mart. Bignoniaceae Jacarandá, caroba, caiuá, caroba-branca,

pau-de-colher, dacarandá-de-minasCGMS 11923 Campo Grande, MS

Jacaranda ulei Bureau & K. Schum. Bignoniaceae Caroba CGMS 11972 Campo Grande, MSLicania tomentosa Benth. Chrysobalanaceae Oiti, goiti, oitizeiro e oiti-da-praia R195.941 Rio de Janeiro, RJMelancium campestre Naudin Cucurbitaceae Melancia-do-campo CGMS 4441 Campo Grande, MSVernonia phosphorea (Vell.) Monteiro Asteraceae Assa-peixe CGMS 11970 Campo Grande, MS

Fig. 1. Geographical location of the collection site of the studied plants.

62 T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68

medium (Sigma, St. Louis, MO, USA), supplemented with 10%heat-inactivated fetal bovine serum (FBS, Sigma), 20 mM L-gluta-mine, 200 U/mL penicillin, and 100 lg/mL streptomycin, at pH7.4. Stationary-phase promastigotes were prepared as describedpreviously (Coelho et al., 2003). Murine macrophages were col-lected from the peritoneal cavities of female BALB/c mice (8 weeksold), which were purchased from the Institute of Biological Sci-ences of UFMG. The Animal Use Committee of the UFMG approvedthe experimental protocols (code 182/2012).

2.4. In vitro antileishmanial activity

The inhibition of Leishmania growth was assessed by cultivatingstationary promastigotes of L. amazonensis (5 � 105 cells) in the

presence of a concentration range (1.56–200 lg/mL) of differentextracts or fractions in 96-well culture plates (Corning Life Sciences,Corning, NY, USA) for 48 h at 24 �C. Amphotericin B (AmpB, Cristá-lia, São Paulo, Brazil) was used as a positive control (10 lg/mL). Cellviability was assessed by measuring the reduction of 2 mg/mL of[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide](MTT; Sigma), to produce formazan. Absorbance was measuredusing a multi-well scanning spectrophotometer (LABTRADE, model660), at a wavelength of 570 nm. Background absorbance was sub-tracted from total absorbance to calculate formazan production.The results were expressed as the mean percentage reduction ofproduction induced by L. amazonensis, compared to that in thenon-treated control wells, and the 50% inhibitory concentration(IC50) was calculated as described (Valadares et al., 2012b).

T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68 63

2.5. In vitro cytotoxicity

The inhibition of macrophages viability was assessed in vitro bycultivating murine macrophages (5 � 105 cells) in the presence of aconcentration range (3.91–500 lg/mL) of different extracts or frac-tions in 96-well culture plates (Nunc), for 48 h at 24 �C. AmpB wasused as a positive control (10 lg/mL). Cell viability was assessed bymeasuring the reduction of 2 mg/mL MTT, and absorbance wasmeasured at 570 nm by using a multi-well scanning spectropho-tometer. Background absorbance was subtracted from totalabsorbance to calculate formazan production. The results wereexpressed as the mean percentage reduction of macrophage viabil-ity compared to that in the untreated control wells, and the 50%inhibitory concentration (CC50) was calculated as described(Valadares et al., 2012b). The SI was calculated using the ratiobetween CC50 and IC50 for all evaluated products.

2.6. Treatment of infected macrophages

Murine macrophages (5 � 105 cells) were plated on round glasscoverslips inside the wells of a 24-well culture plate (Nunc) in anRPMI 1640 medium supplemented with 20% FBS, 2 mM L-glutamine,200 U/mL penicillin, and 100 lg/mL streptomycin, at pH 7.4. After24 h of incubation at 37 �C in 5% CO2, stationary-phase promastig-otes of L. amazonensis were added to the wells (5 � 106 cells), andthe cultures were incubated for 24 h at 37 �C in 5% CO2. A previoustitration curve was performed to determine the minimum timeincubation of parasites with the macrophages, in order to obtainthe maximum of infection into the host cells (data not shown). Next,free parasites were removed by extensive washing with an RPMI1640 medium, and infected macrophages were quantified and trea-ted for 48 h with the extracts or fractions of D. alata and J. cuspidifoliain different concentrations (Table 4). AmpB was used as a positivecontrol (10 lg/mL). Cells were washed in RPMI 1640 and incubatedwith 4% paraformaldehyde for 15 min, at which time they weretreated with 70% ethanol in an ice-bath for 4 h and again washedthree times with sterile PBS. The percentage of the inhibition ofLeishmania intra-macrophage viability was determined by counting200 cells in triplicate.

2.7. Nitric oxide production

To visualize the macrophage activation via NO production aftertreatment with the extracts or fraction that presented higher SIvalues (>10.0), cells (5 � 105) were incubated alone in an RPMI1640 medium (background control) or separately stimulated withthe extracts and fraction of D. alata and J. cuspidifolia in differentconcentrations (3.91–500 lg/mL) or concanavalin A (ConA; 5 lg/mL), at 37 �C in 5% CO2 for 24, 48, and 72 h. Following incubation,100 lL of culture supernatant was mixed with an equal volume ofGriess reagent (Sigma). After an incubation of 30 min at room tem-perature, nitrite concentration was calculated using a standardcurve of known concentrations. Data were expressed as lM per4 � 105 cells (Valadares et al., 2011).

2.8. Statistical analysis

Statistical analyses were performed using GraphPad PrismTM(version 5.0 for Windows). The IC50 and CC50 and values were cal-culated from the mean percentage reduction of the promastigotesand macrophages, respectively, compared to that in the untreatedcontrols. The curves were determined by applying sigmoidalregression to the logarithm concentration–response data. All theresults are representative of three different experiments, per-formed in triplicate, with similar results.

3. Results and discussion

The current treatment options for leishmaniasis are consideredinadequate, because these drugs are associated with high toxicity,and high costs, and because parasite resistance to these drugs isincreasing (Tasdemir et al., 2006). In this context, strategies toidentify new products with lower toxicity and higher efficacy couldbe considered highly desirable. Phytotherapy has received consid-erable attention as an alternative to chemotherapy in parasitic dis-ease treatment (González-Coloma et al., 2011). Furthermore,natural products are potential sources of new and selective agentsfor the treatment of other tropical diseases caused by protozoans(Wright and Phillipson, 1990). A number of published studies havedemonstrated that many plant extracts exhibited leishmanicidalactivity (Estevez et al., 2007; Getti et al., 2009; Sharma et al.,2009; Singh et al., 2011; Zahir et al., 2012; Lage et al., 2013). Inthe present study, 16 plants selected from the Brazilian flora andobtained mainly from Cerrado region were evaluated. The antile-ishmanial activity of 44 plant extracts and fractions were investi-gated using stationary-phase promastigotes of L. amazonensis.The main focus was on plants traditionally used in Brazil for theiranti-inflammatory and antimicrobial properties, as well as for non-specific symptoms that could be frequently described as parasiticin origin. Table 2 describes the plants selected and their medicinaluses.

The parasite stage used in this study has been widely employedin preliminary tests to screening the plant extracts presentingantileishmanial activity (Weniger et al., 2001; González-Colomaet al., 2011; Gamboa-Angulo et al., 2012; García et al., 2012). Inthe present study, we examined the concentration-dependentinhibitory effects of different plant extracts on L. amazonensis andmurine macrophages, in order to evaluate their antileishmanialactivity and in vitro cytotoxicity, respectively. The control experi-ments were performed using DMSO at the same concentration(0.1%) as diluents to the plant extracts. This concentration of DMSOdid not affect parasites viability. The results of the antileishmanialand cytotoxicity assays, as well as the selectivity index, are shownin Table 3. This last parameter can be considered relevant, becausevalues higher than 10.0 could suggest a better safety of a productfor use in mammalian hosts (Lenta et al., 2007).

The butanolic fraction of C. lineatifolia showed good activityagainst L. amazonensis, with IC50 of 62.30lg/mL and SI of 8.3. Incontrast, the ethanolic extract, and the dichloromethane, ethyl ace-tate, and hexanic fractions obtained from this plant were less effec-tive and/or more toxic. These results are important, because thepresent study is the first to evaluate the usefulness of this plantin the treatment of leishmaniasis. This species is traditionallyemployed to treat dysentery, stomach problems, diarrhea, antimi-crobial, cystitis, and urethritis disorders (Dickel et al., 2007;Madalosso et al., 2012). The active butanolic fraction of C. lineatifo-lia is rich in phenolic compounds, mainly tannis, and the latter arepossibly responsible by antileishmanial activity (Madalosso et al.,2012). Tannins represent a group of phenolic metabolites, mainlypresent in higher plant species. Their presence in fairly high con-centrations has commonly been used to explain the curative andpalliative efficacy claimed for a variety of traditional herbal medi-cines, and the profound health benefits of certain foodstuffs(Haslam, 2007). This substance class exhibits a remarkably widerange of biological and pharmacological activities, includingantiviral, antimicrobial, antioxidant, and antitumor activities, andhas also been used to treat degenerative and cardiovascular dis-eases, and hypertension (Buzzini et al., 2008; Borges et al., 2010;Landete, 2011; Salminen et al., 2011). In vitro studies that evalu-ated the activity of polyphenolics against promastigotes and axenicamastigotes of L. donovani and L. major showed that the phenolic

Table 2Traditional uses described in the literature of the Brazilian plants.

Botanical name Uses Referencesa

Bowdichia virgilioides Kunth. Inflammatory disorders Barros et al. (2010), Silva et al. (2010), Thomazzia et al. (2010)Campomanesia lineatifolia

Ruiz & Pav.Dysentery, stomach problems, diarrhea, antimicrobial, cystitis,urethritis, and hepatic disorders

Madalosso et al. (2012), Dickel et al. (2007), Corrêa (1952), D’Ávila(1910), Moraes (1881)

Cecropia pachystachyaTrécul

Cardiotonic, antioxidant, hypoglycemic, and sedative effects Aragão et al. (2010), Consolini et al. (2006), Velázquez et al. (2003)

Chrysobalanus icaco L. Chronic diarrhea, dysentery, hemorrhage, leukorrhea, diabetes, HIVl; antiangiogenic; cytotoxic for K562 cells; inhibited proliferation ofMDR Lucena cells; and had genotoxic, antioxidant, and antibacterialactivities

Castilho and Kaplan (2011), Fernandes et al. (2003), Alves dePaulo et al. (2000), Pereira (1997), Gustafson et al. (1991), Prestaand Pereira (1987), Morton (1981)

Diospyros hispida D.C. Leprosy, skin eruptions, eye infections, infectious diseases,activities against Plasmodium falciparum, Cryptococcus neoformans,and Leishmania (L.) chagasi

Albernaz et al. (2010), Mallavadhani et al. (1998), Watt (1956)

Dipteryx alata Vog. Anti-rheumatic agent, tonic, and menstrual regulators Esteves-Pedro et al. (2012)Eugenia uniflora L. Diarrhea, hypertension, antimicrobial activity, antigiardial activity,

and anti-Trypanosoma activityVictoria et al. (2012), Lopes et al. (2012), Consolini et al. (1999)

Hymenaea courbaril L. Diarrhea, dysentery, intestinal colic, pulmonary weakness, andchronic cystitis, and antiviral activity

Cecílio et al. (2012), Panizza (1997), Lorenzi and Matos (2002)

Hymenaea stignocarpa Mart.ex. Hayne

Antidiarrheal, gastro-protective, and cicatrizing activities Orsi et al. (2012)

Jacaranda caroba Vell. Venereal diseases and general infections, as a depurative, ulcercicatrization, dyspepsia, antioxidant, anticholinesterase, and anti-MAO-A effects.

Di Stasi et al. (2002), Gachet and Schühly (2009) Brandão et al.(2012, 2008), Ferreres et al. (2013)

Jacaranda cuspidifolia Mart. Syphilis and gonorrhea, and antimycobacterial activity Arruda et al. (2011, 2012), Corrêa (1926)Jacaranda ulei Bureau & K.

Schum.No pharmacological activity found –

Licania tomentosa Benth. Anti-herpes simplex virus and anti-tumor activity. Fernandes et al. (2003), Miranda et al. (2002)Melancium campestre

NaudinNo pharmacological activity found –

Syzygium cumini L. Antidiabetic, anti-inflammatory, antiulcerogenic, cardioprotective,antidiarrheal, antimicrobial, antioxidant, and hepatoprotectiveactivities

Bag et al. (2012), Baliga et al. (2011), Khare (2004), Shafi et al.(2002), Chattopadhyay et al. (1998), Mukherjee et al. (1998),Bhuiyan et al. (1996), Shaikh et al. (1994)

Vernonia phosphorea (Vell.)Monteiro

No pharmacological activity found –

a References indicating the studies performed with each plant species.

64 T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68

compounds were active against both stages, but higher antileish-manial activity was observed against the amastigote forms(Kolodziej and Kiderlen, 2005).

The extracts and fractions of C. icaco had an IC50 of around 60 to70 lg/mL. Butanolic and ethyl acetate fractions showed SI values of8.1 and 7.7, respectively. There are no previous reports of theeffects of this plant on any species of Leishmania in scientific liter-ature. Phytochemical studies of extracts and fractions of C. icacoresulted in the purification of stigmasterol, sitosterol, and campes-terol from hexanic extract; pomolic acid from dichloromethane;and 7-O-methylkaempferol from butanolic fraction (Castilho andKaplan, 2011). The biological activity of plant extracts has beenattributed to different compounds belonging to diverse chemicalgroups, including alkaloids, flavonoids, phenyl propanoids, ste-roids, and terpenoids. Pomolic acid is a pentacyclic triterpenederivative of ursolic acid. These triterpene, oleanolic, and betulinicacids are abundant in the plant kingdom and possess antitumorand anti-inflammatory activities (Fernandes et al., 2003; Vechiaet al., 2009). Betulinic acid and its acyl derivatives at the C-28 posi-tion have been evaluated for its antiprotozoal activity, and showeda good leishmanicidal activity (Domínguez-Carmona et al., 2010).Quercetin diglycoside from Kalanchoe pinnata also showed aneffective antileishmanial activity (Muzitano et al., 2006).

The IC50 value of the hexanic extract of D. alata was 0.08 lg/mL,whereas its SI value was 129.0. The ethanolic extract was lesseffective and more toxic in the mammals cells (Table 3). An evalu-ation of the capacity of the hexanic extract in treating infectedmacrophages was performed. Cells were first pre-infected withstationary-phase promastigotes of L. amazonensis, and later treatedwith 0.08, 0.16, 3, and 5 lg/mL of hexanic extract of D. alata for48 h at 37 �C. The results showed that macrophages infected withL. amazonensis and later treated presented reductions in the

parasite burden in the order of 46.71%, 53.14%, 80.85%, and95.80%, respectively (Table 4). Macrophages represent the main-host phagocytic cells in leishmaniasis and play a relevant role inthe immunological control of intracellular parasitism through theproduction of oxygen derivative metabolites (Balaraman et al.,2004). Through the up-regulation of NO production within thecells, macrophages can trigger an intracellular killing mechanismof the internalized parasites (Mauël and Ransijn, 1997), so thepresent study aiming to investigate the NO production by macro-phages treated with the hexanic extract of D. alata. In the results,were observed no production of nitrite after 48 h of incubationwith the hexanic extract while the concanavalin A, demonstrateda high NO production, suggesting that the leishmanicidal activityobserved may be due to a mechanism other than NO productionby macrophages.

Tempone et al. (2001) and Valadares et al. (2011) showed thatsnake venom and an aqueous extract of Agaricus blazei presentedeffective antileishmanial activity without inducing NO productionby the infected macrophages. According to these data, togetherwith those described in this work, it can be speculated that othermechanisms than NO production may well be involved in the elim-ination of parasites without requiring macrophage activation viaiNOS expression. This finding suggests that the products can trans-pose the membrane of the macrophages, and reach the internalizedparasites through a direct effect of the hexanic extract of D. alataonto the parasites. To provide additional information concerningthe chemical compounds of this extract, a chemical characteriza-tion was performed. The phytochemical screening using thin layerchromatography (TLC) showed the presence of tannins, flavonoids,triterpenes and steroids, as well as the presence of lupeol andbetulinic acid was verified. Recently, Puebla et al. (2010) identi-fied some chemical constituents, such as triterpenoids (lupeol,

Table 3In vitro antileishmanial activity, cytotoxicity, and selectivity indices of the plant extracts tested. The results are expressed as mean ± standard deviation (SD) of three independentexperiments, which presented similar results.

Botanical name Part plant Extract/fractions (F)a IC50b CC50

c SId

Bowdichia virgiloides Kunth. Leaves Ethanolic NA – –Hexanic NA – –

Campomanesia lineatifolia Ruiz & Pav. Leaves Ethanolic 103.27 ± 14.19 627.12 ± 13.01 6.1Buthanolic F 62.30 ± 0.20 519.34 ± 35.16 8.3Dichloromethane F NA – –Ethyl acetate F 96.13 ± 0.29 613.37 ± 23.12 6.4Hexanic F 147.70 ± 0.49 502.69 ± 24.17 3.4

Cecropia pachystachya Trécul Leaves Ethanolic NA – –Hexanic NA – –

Chrysobalanus icaco L. Leaves Ethanolic 61.47 ± 0.57 379.13 ± 7.96 6.2Hexanic 62.28 ± 0.20 219.10 ± 4.17 3.5Buthanolic F 61.24 ± 0.44 498.22 ± 0.51 8.1Dichloromethane F 91.64 ± 0.39 617.38 ± 12.77 6.7Ethyl acetate F 77.30 ± 0.42 593.57 ± 22.31 7.7Hexanic F 130.20 ± 0.26 418.46 ± 31.18 3.2

Diospyros hispida D.C. Leaves Ethanolic NA – –Hexanic NA – –

Dipteryx alata Vog. Leaves Ethanolic 51.46 ± 7.14 351.96 ± 14.3 6.8Hexanic 0.08 ± 0.04 10.32 ± 2.64 129.0

Syzygium cumini Lam. Leaves Hexanic 31.64 ± 0.99 132.4 ± 0.78 4.0Eugenia uniflora L. Leaves Hexanic NA – –Hymenaea courbaril L. Leaves Ethanolic 44.10 ± 0.57 131.90 ± 12.28 3.0

Hexanic 35.84 ± 0.51 46.53 ± 8.13 1.3Hymenaea stignocarpa Mart. ex. Hayne Leaves Ethanolic 4.69 ± 0.77 34.84 ± 9.42 7.4

Hexanic 199.40 ± 0.39 33.51 ± 7.62 0.2Jacaranda caroba Vell. Leaves Ethanolic 13.22 ± 0.96 38.27 ± 2.24 2.9

Hexanic NA – –Roots Ethanolic NA – –

Jacaranda cuspidifolia Mart. Leaves Ethanolic 10.96 ± 0.76 763.29 ± 31.02 69.6Hexanic NA – –Chloroformic F 7.43 ± 1.15 528.61 ± 13.94 71.1

Stem bark Ethanolic NA – –Jacaranda ulei Bureau & K. Schum. Leaves Ethanolic NA – –

Hexanic NA – –Roots Hexanic NA – –

Licania tomentosa Benth. Leaves Dichloromethane NA – –Ethanolic NA – –Ethyl acetate NA – –

Melacium campestre Naudin Leaves Hexanic NA – –Ethanolic 120.37 ± 12.73 114.17 ± 3.22 0.9

Stem bark Hexanic 160.93 ± 21.07 321.17 ± 41.17 2.0Roots Ethanolic 92.65 ± 6.91 621.69 ± 33.19 6.7

Vernonia phosphorea Vell. Leaves Ethanolic NA NA –Hexanic NA NA –

Amphotericin B – – 0.08 ± 0.03 0.78 ± 0.21 10.0

NA – no activity (IC50 > 200 lg/mL).(–) – not calculate.

a (F) – fraction.b IC50 – 50% inhibitory concentration for Leishmania amazonensis.c CC50 – 50% inhibitory concentration for murine macrophages.d SI – selective index between murine macrophages and L. amazonensis.

T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68 65

lupenone, 28-hydroxylup-20(29)-en-3-one and betulin), isoflavo-noids, (afrormosin, odoratin and dipteryxin), one chalcone (isoli-quiritigenin) and one aurone (sulfuretin). Based on these data,we determined the content of flavonoids and total phenolic com-pounds. The total flavonoids and phenolic contents were of 0.8%and 4.0% in the hexanic extract, respectively. The seeds of D. alataare edible and nutritive, and the oil has medicinal properties; otherplant parts are popularly used as anti-rheumatic agents, tonics, andmenstrual regulators (Esteves-Pedro et al., 2012). The resultsobtained in this study are promising, because there are no previouspublished reports of any antileishmanial activity of this plant, andthere are many studies of the action of phenolics and terpenoids inLeishmania (Tasdemir et al., 2006).

Three species of Jacaranda genus were also assayed, and theresults showed that an ethanolic extract of leaves of J. cuspidifoliapresented an IC50 of 10.96 lg/mL, and an SI value of 69.9. To studythis ethanolic extract, it was fractionated in CHCl3/H2O (1:1). Thechloroformic fraction showed an IC50 of 7.43 lg/mL, and an SI

value of 71.1. The evaluation of the capacity of the ethanolicextract and the chloroformic fraction in treating infected macro-phages was also performed. The results showed that macrophagesthat were infected and later treated with 5, 10, 25, and 50 lg/mL ofethanolic extract or chloroformic fraction of J. cuspidifolia pre-sented reductions in the parasite burden in the order of 49.23%,72.50%, 86.48%, and 98.31%, respectively, for the ethanolic extractand of 69.35%, 84.76%, 93.17%, and 97.16%, respectively, for chloro-formic fraction (Table 4). The present study also aimed to investi-gate the NO production by macrophages treated with the ethanolicextract and chloroformic fraction of J. cuspidifolia, and it could beobserved that both evaluated products did not induce NO produc-tion, suggesting that the leishmanicidal activity observed may bedue to a mechanism other than NO production by macrophages.

The presence of tannins, flavonoids, alkaloids, steroids andsaponins was identified in the ethanolic extract and in thechloroformic fraction of J. cuspidifolia by TLC analysis with specificreagents. Based on these results, it was determined the content of

Table 4Reduction of infection in macrophages after the treatment with the hexanic extract of D. alata, ethanolic extract and chloroformic fraction of J. cuspidifolia. The results areexpressed as medium ± standard deviation (SD) of the percentages of the macrophages infected with L. amazonensis, and by reduction of the internalized parasites in the treatedcultures.

Extract or fraction Concentration(lg/mL)

Percentage of infected macrophagesafter treatmenta

Number of amastigotes per macrophageafter treatmentb

Reduction of internalizedparasites (%)c

Hexanic extract of D. alata 0.08 50.40 ± 5.86 7.31 ± 0.81 46.71 ± 4.180.16 44.32 ± 8.52 5.12 ± 1.21 53.14 ± 7.23

3 18.11 ± 11.48 4.23 ± 2.10 80.85 ± 9.165 3.97 ± 4.02 2.61 ± 0.19 95.80 ± 2.92

Ethanolic extract of J. cuspidifolia 5 48.01 ± 10.52 8.75 ± 3.17 49.23 ± 8.1510 26.00 ± 5.71 6.57 ± 2.13 72.50 ± 4.6525 12.79 ± 5.20 4.67 ± 3.18 86.48 ± 3.1750 1.59 ± 2.75 3.28 ± 0.78 98.31 ± 1.18

Chloroformic fraction of J. cuspidifolia 5 28.99 ± 10.75 5.71 ± 2.85 69.35 ± 7.8410 14.41 ± 6.82 2.18 ± 0.92 84.76 ± 4.3625 6.46 ± 9.33 1.46 ± 1.12 93.17 ± 8.1850 2.69 ± 3.90 1.08 ± 1.07 97.16 ± 2.17

a Percentage of infected macrophages.b Average number of amastigotes per macrophage after treatment.c Average reduction in percentage of internalized parasites were determined by counting 200 cell coverslips, in triplicate, as compared to the non-treated controls. In the

non-controls, the percentage of infected macrophages and the number of amastigotes per cell were of 94.57 ± 5.83 and 9.21 ± 0.86, respectively.

66 T.G. Ribeiro et al. / Experimental Parasitology 143 (2014) 60–68

flavonoids of 0.53% and 0.96% for ethanolic extract and chlorofor-mic fraction, respectively, and the total phenolic was of 1.88% inthe ethanolic extract and 2.63% in the chloroformic fraction. Thearilethanoids of cinnamoyl glucosides represent the main class ofcompounds of the chloroformic fraction and verbascoside wasisolated and identified and might contribute to the observedantileishmanial activity (Arruda et al., 2011). This species is tradi-tionally employed in microbial diseases (Table 2). Tasdemir et al.(2006) evaluated the verbascoside and other phenolics and itshowed in vitro leishmanicidal activity against L. donovani axenicamastigotes. This is the first time that potential activity has beenreported for this species.

Syzygium cumini, Hymenaea courbaril, and H. stignocarpapresented SI values between 0.2 and 4.0, indicating inadequateefficacy for consideration as an antileishmanial product (Lentaet al., 2007). Extracts prepared from Bowdichia virgilioides, Cecropiapachystachya, Diospyros hispida, Eugenia uniflora, J. caroba, J. ulei, L.tomentosa, and Vernonia phosphorea species did not show anyantileishmanial activity or toxicity into the mammalian cells.

The results of the present study showed that of the 44 extractsand fractions derived from 16 Brazilian plants, D. alata and J. cus-pidifolia presented promising results against L. amazonensis, andthey could be evaluated in vivo models for treatment of this dis-ease. In addition, these results contribute to the promotion of theuse of traditional medicine employing natural compounds withlow toxicity. Experiments are in progress to isolate and identifythe active constituents responsible for the antileishmanial activityof the more active plants, as well as to use them in a more refinedway for evaluation of the in vitro and in vivo efficacy.

Acknowledgments

This work was supported by Grants from Pró-Reitoria de Pesquisafrom Universidade Federal de Minas Gerais (Edital 01/2014), Institu-to Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (INCTNanoBiofar), FAPEMIG (CBB-APQ-00496-11, CBB-APQ-00819-12and CBB-APQ-01051-12), and CNPq (APQ-472090/2011-9 andAPQ-482976/2012-8). EAFC and AAGF are Grant recipient of CNPq.MACF is a Grant recipient of FAPEMIG/CAPES.

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