REVIEW ARTICLE published: 06 August 2012 doi: 10.3389/fmicb.2012.00283 Conventional therapy and promising plant-derived compounds against trypanosomatid parasites

Daniela Sales Alviano1, Anna Léa Silva Barreto2, Felipe de Almeida Dias 1, Igor de Almeida Rodrigues 3,4, Maria do Socorro dos Santos Rosa3, Celuta Sales Alviano1 and Rosangela Maria de Araújo Soares 2*

1 Laboratório de Estruturas de Superfície de Microrganismos, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil 2 Laboratório de Biologia de Protistas, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil 3 Laboratório de Quimioterapia Experimental para Leishmaniose, Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil 4 Instituto de Química Programa de Pós Graduação em Ciência de Alimentos, Centro de Ciências Matemáticas e da Natureza, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil

Edited by: Leishmaniasis and trypanosomiasis are two neglected and potentially lethal diseases that Mirian A. F.Hayashi, Universidade affect mostly the poor and marginal populations of developing countries around the world Federal de São Paulo, Brazil and consequently have an important impact on public health. Clinical manifestations such as Reviewed by: Octavio Luiz Franco, Universidade cutaneous, mucocutaneous, and visceral disorders are the most frequent forms of leishma- Catolica de Brasilia, Brazil niasis, a group of diseases caused by several Leishmania spp. American trypanosomiasis, Sergio Schenkman, Universidade or Chagas disease, is caused by Trypanosoma cruzi, a parasite that causes progressive Federal de São Paulo, Brazil damage to different organs, particularly the heart, esophagus, and lower intestine. African *Correspondence: trypanosomiasis, or sleeping sickness, is caused by Trypanosoma brucei and is character- Rosangela Maria de Araújo Soares, Laboratório de Biologia de Protistas, ized by first presenting as an acute form that affects blood clotting and then becoming Departamento de Microbiologia a chronic meningoencephalitis. The limited number, low efficacy, and side effects of con- Geral, Instituto de Microbiologia Prof. ventional anti-leishmania and anti-trypanosomal drugs and the resistance developed by Paulo de Góes, Centro de Ciências da parasites are the major factors responsible for the growth in mortality rates. Recent Saúde, Universidade Federal do Rio de Janeiro, Bloco I, Ilha do Fundão, research focused on plants has shown an ingenious way to obtain a solid and potentially rich 21941-590 Rio de Janeiro, Rio de source of drug candidates against various infectious diseases. Bioactive phytocompounds Janeiro, Brazil. present in the crude extracts and essential oils of medicinal plants are components of e-mail: [email protected] an important strategy linked to the discovery of new medicines. These compounds have proven to be a good source of therapeutic agents for the treatment of leishmaniasis and trypanosomiasis. This work highlights some chemotherapeutic agents while emphasizing the importance of plants as a source of new and powerful drugs against these widespread diseases.

Keywords: leishmaniasis, Chagas disease, sleeping sickness, Trypanosoma spp., Leishmania spp., chemotherapy, medicinal plants, phytotherapy

INTRODUCTION (v) no need for hospitalization; and (vi) no induction of resis- Leishmaniasis and trypanosomiasis are neglected tropical diseases tance. As described below, this ideal drug does not exist for caused by protozoans of the Trypanosomatid family, a diverse the treatment of these parasitoses, and it will take a long time group of flagellated parasites that show similar cellular structures before such a drug is available. Additionally, because the vac- and undergo morphological alterations during their life cycles. The cine approach has not produced satisfactory results in clinical human diseases caused by trypanosomatids such as leishmaniasis, trials, chemotherapy based on drugs that are not highly effec- African trypanosomiasis (or sleeping sickness), and American try- tive and cause side effects is the only treatment available for these panosomiasis (or Chagas disease) are transmitted by insects that maladies. affect 20 million people and cause 100,000 deaths per year, pri- Considering the low number and efficacy of drugs available for marily in the tropical and subtropical areas of the world. In these the treatment of these diseases as well as their side effects and the regions, half of a billion people are at risk of infection (Stuart et al., resistance developed by parasites, the research in phytosciences, 2008). mainly regarding the properties of bioactive phytocompounds in According to the World Health Organization (WHO), an ideal the crude extracts and essential oils of medicinal plants, may lead drug for the treatment of parasitic diseases should fulfill the fol- to the discovery of new medicines with appropriate efficiency that lowing requirements: (i) parasitological cure in all the phases are more accessible to the patients (Alviano and Alviano, 2009). of the disease; (ii) effective in single or few doses; (iii) low Overall this review aims to provide a better understanding of the cost for the patients; (iv) no collateral or teratogenic effects; recent developments of the phytosciences to treat leishmaniasis

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and trypanosomiasis neglected tropical diseases in terms of drug acute myocarditis and meningoencephalitis, which may be fatal in discovery and development in the world today. 2–8% of the cases in the absence of specific treatment (Moncayo and Ortiz Yanine, 2006; Develoux et al., 2009). LEISHMANIASIS, CHAGAS DISEASE, AND SLEEPING African trypanosomiasis or sleeping sickness occurs only in 36 SICKNESS countries in sub-Saharan Africa that contain vector insects of the Leishmaniasis is a generic term for diverse clinical manifesta- genus Glossina (tsetse flies). Sleeping sickness is a serious public tions including cutaneous, mucocutaneous, and visceral disorders health problem in these countries because 55 million people are at caused by species of the genus Leishmania. All Leishmania species risk of infection and there are an estimated 30,000 new cases annu- display similar morphologies and present two main developmen- ally. If left untreated, the disease is almost always fatal. Its etiologic tal stages throughout their life cycles. The extracellular replicative agents are two subspecies of Trypanosoma brucei, T. b. gambiense forms, the promastigotes, are found in the gut of the insect vec- in West Africa, and T. b. rhodesiense in East Africa, and the last tors, the phlebotomines sandfly. The amastigote is the obligatory is responsible for most severe form of the same disease. The try- intracellular form observed in the mononuclear phagocytic sys- panosomes of the brucei group primarily infect connective tissues. tem cells of vertebrate hosts. During blood feeding, the infected Hemolytic anemia occurs in the early stages of infection, followed sandflies expel, together with saliva, a number of infective pro- by lymph node, spleen, and liver hypertrophy and cardiovascular mastigotes found at the cardiac valve. Cutaneous leishmaniasis and endocrine disorders. In the final phase of the infection,the par- (CL), mucocutaneous leishmaniasis (MCL), and visceral leishma- asites reach the nervous system and cause inflammatory reactions niasis (VL) have a high impact in several countries of the world due that lead to the meningoencephalitis associated with the clinical to their morbidity and mortality rates. Many Leishmania species aspects of sleeping sickness (Gehrig and Efferth, 2008; Astelbauer act as etiological agents of cutaneous and MCL; one of the most and Walochnik, 2011). well known agents of the diffuse cutaneous form is Leishmania amazonensis and MLC forms is L. brasiliensis. On the other hand, CURRENT CHEMOTHERAPY AGAINST LEISHMANIASIS, L. donovani and L. chagasi are the only etiological agents of VL. CHAGAS DISEASE, AND SLEEPING SICKNESS AND IMPACT The CL is characterized by lesions on the face, hands, and/or feet. OF PARASITE RESISTANCE MCL involves the nasal, oral, and pharyngeal mucosa and causes Chemotherapy is the main tool used to control parasitic infections. difficulty in eating as well as an increased risk of secondary infec- The drugs available for these parasites control are often ineffective tions that have a significant mortality rate (Murray et al., 2005; and sometimes life-threatening, adverse side effects, and some of Piscopo and Mallia, 2007; David and Craft, 2009). The VL is char- these drugs require hospitalization (Croft et al., 2006; Ashutosh acterized by fever, weakness, and fatigue are worsened by anemia, and Goyal, 2007; Piscopo and Mallia, 2007). which is caused by a persistent inflammatory state, hypersplenism, The drugs available for leishmaniasis infections are the pen- and sometimes by bleeding. As the disease advances, splenomegaly tavalent antimonial formulations of sodium stibogluconate (Pen- and hepatomegaly can increase, causing abdominal distension and tostan) and N -methyl glucantime (Glucantime), which target pain. These VL symptoms often persist for a long time before the the amastigotes of both CL and VL. Of the various proposed patient either seeks medical care or dies from secondary infections, mechanisms of action for these two drugs, we can mention the severe anemia, or organ failure (Chappuis et al., 2007; Piscopo and production of ATP inhibition of the citric acid cycle and glycol- Mallia, 2007). ysis by converting the pentavalent antimony the active trivalent The life cycle of Trypanosoma cruzi,the etiological agent of Cha- antimony which is more active and more toxic, inhibition of oxi- gas disease, involves blood sucking by triatomine bugs that serve dation of fatty acids in amastigotes and induction of apoptosis as insect vectors and a mammalian host where the parasite under- and inhibition of the enzyme DNA topoisomerase (Berman et al., goes an obligate intracellular amastigote replicative form and an 1987; Frézard et al., 2009). Variations in the clinical responses to extracellular non-replicative trypomastigote form in the blood- these drugs have been a persistent problem in the treatment of stream. The epimastigote replicative form and a non-replicative leishmaniasis over the past 50 years due to intrinsic differences infective metacyclic trypomastigote form are also observed in the in species sensitivity and the development of resistance. When insect vector. This disease is mainly transmitted by insect vectors these drugs are ineffective or cannot be prescribed, treatment feces, but transmission may also occur by blood transfusion, organ with amphotericin B, pentamidine, or paromomycin is indicated. transplant, congenital and oral routes, and laboratory accidents. However, as none of these drugs are free of adverse effects, the The prevalence and incidence of Chagas disease and the associated search for alternative therapeutic agents is essential. The combi- mortality are constantly changing as a consequence of vector con- nation of amphotericin B liposome is an alternative employed to trol programs, rural-urban human migration, and changes in the reduce adverse effects, increasing the efficiency of the drug. As flu- socio-economic status of risk areas (Lewinsohn, 2003; Moncayo conazole and ketoconazole, azoles which initially were designed and Ortiz Yanine, 2006). for the treatment of fungal infections, have been used for treat- Despite the fact that most people infected with the parasite ing CL. Miltefosine has been recently approved as the first oral never appear to become symptomatic, Chagas disease presents drug for VL. It yields cure rates of approximately 98% and is in two stages: the acute stage, which appears shortly after the used to treat cases of resistance to antimoniates; however, it has infection, and the chronic stage, which may last several years. been shown to induce parasite resistance in vitro (Croft et al., In the acute phase, fever, lymphadenopathies, hepatomegaly, and 2006; Santos et al., 2008; Goto and Lindoso, 2010; Tiuman et al., splenomegaly may appear, as well as more severe problems, such as 2011).

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Nifurtimox (Nif; Bayer 2502) and benznidazole (Bz; RO 7- the first stage of sleeping sickness by T. b. rhodesiense but can 1051) have been used for the treatment of Chagas disease since cause allergic reactions and some undesirable effects in the uri- the end of 1960. The mechanism of action of Nif is based on a nary tract. In their final stages, both forms of infections are treated partial deficiency in the ability of T. cruzi to detoxify free radi- with melarsoprol, a drug derived from arsenic. This drug has many cals. Nif leads to the formation of nitro anion radicals that in turn side effects, the most dramatic of which is reactive encephalopathy, produce highly toxic reduced oxygen metabolites. However, Nif is which can be fatal, and kills up to 5% of patients. It is supposed no longer commercially available. Instead of producing oxidative that the mechanism of action is the interaction of the drug with a damage, the Bz mechanism of action might involve covalent mod- transporter specific for adenosine reducing the absorption of these ifications of parasite proteins, lipids, and DNA by nitro reduction nucleoside by the parasite and by the arsenic binding to glycerol intermediates (Coura and de Castro, 2002). The likelihood of cur- 3-phosphate dehydrogenase, resulting in inhibition of glycolysis, ing Chagas disease with Nif and Bz varies according to the phase and low levels of ATP (Denise and Barret, 2001). Another option of the disease, the period of treatment, the dosage, and the age is eflornithine, which is less toxic but only effective against T. b. of the patient. Usually, satisfactory results are achieved when the gambiense infections. A combination treatment with nifurtimox patients are treated in the acute phase, in recent chronic infec- and eflornithine was recently (2009) introduced, but is not effec- tion, in congenital infection, and after laboratory accidents. The tive for T. b. rhodesiense (Wilkinson and Kelly, 2009; Astelbauer major limitation of these compounds is the low efficacy in the and Walochnik, 2011; Jacobs et al., 2011). treatment of patients in the chronic phase of the disease (Coura One major drawback to the treatment of leishmaniasis, Cha- and de Castro, 2002). gas disease, and sleeping sickness is the emergence of resistance Treating infections with Nif may lead to collateral effects such to current chemotherapeutics. Due to their high toxicity, drugs as psychic alterations, anorexia, excitability, sleepiness, and diges- usually used for the treatment of these diseases have to be admin- tive manifestations (nausea, vomiting, and diarrhea). Doses of Bz istered in low doses, allowing drug resistance to develop. Because for Chagas disease treatment may cause hypersensitivity leading to there are few drugs available for the treatment of these para- dermatitis with cutaneous eruptions and generate neuropatholo- sitoses, resistance has a considerable impact on the control of these gies such as paresthesia and polyneuritis of the peripheral nerves. maladies. However, the most serious reaction to Bz is the depression of bone The primary mechanism generally observed in parasite resis- marrow that leads to thrombocytopenic purpura and agranulocy- tance is a decrease in the drug concentration within the parasite tosis. Due to these characteristic side effects, Nif and Bz should not cell. The drug level may be lowered by a variety of mechanisms, be used by elderly or pregnant patients or by patients presenting including decreased uptake, increased efflux, and inhibition of any severe disease associated with Chagas disease, such as cardiac, drug activation and inactivation of active drug by the metabolism. respiratory, renal, or hepatic insufficiency, systemic infection, and In recent years, a large-scale increase in clinical resistance of Leish- neoplasia (Coura and de Castro, 2002). mania to pentavalent antimonials has been reported. The mech- The use of nanotechnology as liposomes, antibody conjugates, anisms of resistance of Leishmania spp. and T. cruzi against the and nanoparticles as drug carriers can overcome anatomical barri- chemotherapeutics developed in the field are not elucidated, and ers and take the drug directly to the site of action, reaching the tar- most of our knowledge stems from work on laboratory mutants get microorganisms, and reducing their side effects. Alternatively, (Nogueira et al., 2006; Ashutosh and Goyal, 2007). amphotericin B can be used as a second line treatment in Cha- The resistance mechanisms in T. b. rhodesiense and T. b. gambi- gas disease using nano-drug delivery systems (Yardely and Croft, ense,recently reviewed by Gehrig and Efferth(2008),are frequently 1999; Romero and Morilla, 2010). The E1224 azole compound, mediated by a reduced net drug uptake. Another drug resistance a prodrug transformed into ravuconazole in vivo, discovered and mechanism for melarsoprol is the overexpression of efflux pumps developed by Esai Pharmaceuticals had preclinical and clinical (Mäser et al., 2003). phase I studies completed. This promising compound for the treat- A study using RNA interference in T. brucei showed the loss of ment of Chagas disease, has been tested in phase II clinical studies function of a nitroreductase and an amino acid transporter nec- in adult patients in Bolivia [Drugs for Neglected Diseases Inicia- essary for activation of the prodrug nifurtimox and acquiring of tive (DNDi), 2012]. Furthermore, the ravuconazol was evaluated eflornithine, respectively, showing the mechanisms of resistance extensively in animal models (Diniz et al., 2010). to these two drugs (Baker et al., 2011). The treatment for sleeping sickness depends on the stage of the disease. The drugs used in the first stage of the disease have PLANTS AS PROMISING SOURCES OF ANTI-LEISHMANIAL a low toxicity and are easy to administer. Therefore, early iden- AND ANTI-TRYPANOSOMAL COMPOUNDS tification results in an increased potential for curing the disease. Considering the lack of vaccines, the toxicity of the chemother- Treatment success in the second stage of disease depends on a apies, the side effects of the treatment, and the resistance of the drug that can cross the blood-brain barrier to reach the parasite. parasites to the drugs, there is an evident need to discover drugs Such drugs are toxic and complicated to administer. There are four that can be used as therapeutics for leishmaniasis, sleeping sick- drugs typically used for the treatment of sleeping sickness. Early ness, and Chagas disease. In recent years, many pharmacologically infections are treated with pentamidine and suramin. Pentami- active and microbicidal compounds derived from plants have been dine is used for the treatment of the first stage of T. b. gambiense discovered (Cowan, 1999; Alviano and Alviano, 2009; Izumi et al., sleeping sickness. It has few undesirable effects and is generally 2011), indicating that phytoscience is important in the search of well tolerated by patients. Suramin is used for the treatment of novel compounds with a potential to control these diseases.

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Table 1 | Plants and identified antiprotozoal bioactive phytocompounds.

Scientific name Major components Effects Parasites Reference

Drymis brasiliensis Polygodial Cell proliferation (promastigotes), Cell L. amazonensis, Corrêa et al.(2011) viability (trypomastigotes), L. braziliensis, mitochondrial changes, nuclear L. chagasi, L. major, changes, plasma membrane damages Trypanosoma cruzi (promastigotes) Baccharis retusa Sakuranetin Cell proliferation (promastigotes), Cell L. amazonensis, L. Grecco et al.(2012) viability (trypomastigotes), activity in braziliensis, L. chagasi, intracellular amastigotes L. major, T. cruzi Ocimum gratissimum Eugenol Cell lysis e proliferation L. amazonensis Ueda-Nakamura et al. (promastigotes), mitochondrial Trypanosoma brucei (2006), Abiodun et al.(2012) changes, stimulation of NO production rhodesiense in macrophages Croton cajucara Linalool, acetyl Mitochondrial changes L. amazonensis, T. cruzi Rosa et al.(2003), Campos aleuritolic acid (promastigotes), activity in intracellular et al.(2010) amastigotes. Cell viability (trypomastigotes), activity in intracellular amastigotes, mitochondrial, and kinetoplast changes (epimastigotes) Piper claussenianum Trans-nerolidol Cell proliferation, activity in intracellular L. amazonensis Marques et al.(2010, 2011) amastigotes, increased NO production in infection macrophages, decreased arginase activity of the parasite Lippia alba Geranial, neral, geraniol, Cell proliferation (epimastigotes) and T. cruzi Escobar et al.(2010) and intracellular amastigotes activity trans-β-caryophyllene Lippia origanoides Oxygenated Cell proliferation (promastigotes) L. chagasi Escobar et al.(2010) monoterpenes Moringa stenopetala Benzyl-isothiocyanate Cell proliferation (bloodstream forms) Trypanosoma brucei Nibret and Wink(2010) brucei Hagen abyssinica Ledol Cell proliferation (bloodstream forms) T. b. brucei Nibret and Wink(2010) Leonotis ocymifolia Caryophyllene oxide Cell proliferation (bloodstream forms) T. b. brucei Nibret and Wink(2010) Cupania cinerea Cupacinoside, taraxerol Cell proliferation (bloodstream forms) T. b. rhodesiense Gachet et al.(2011) Kola acuminata Proanthocyanidin Cell proliferation (bloodstream forms T. brucei (clone221a) Kubata et al.(2005) and procyclic forms), cell lysis and changes in plasma membrane Salvia hydrangea Salvadione, perovskone Cell proliferation (bloodstream forms) T. b. rhodesiense Farimani et al.(2011) Carlina acaulis Carlina oxide Cell proliferation (bloodstream forms) T. b. brucei Herrmann et al.(2011) (polyacetylene) Syzygium aromaticum Eugenol Cell proliferation and viability T. cruzi Santoro et al.(2007) (epimastigotes and bloodstreams trypomastigotes) and loss of nuclear content, and masses of condensed chromatin (trypomastigotes) Ocimum basilicum Linalool Cell proliferation and viability T. cruzi Santoro et al.(2007) (epimastigotes and bloodstreams trypomastigotes), cytoplasmic extraction and nuclear alteration (epimastigotes) Achillea millefolium Chamazulene Cell proliferation and viability T. cruzi Santoro et al.(2007) (epimastigotes and bloodstream trypomastigotes)

(Continued)

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Table 1 | Continued

Scientific name Major components Effects Parasites Reference

Zanthoxylum chiloperone Chantin-6-one Cell lysis (bloodstream T. cruzi Ferreira et al.(2011) trypomastigotes), anti amastigotes activity and in vivo activity in infected mice Centaurea salmantica Cynaropicrin Cell proliferation (bloodstream T. b. rhodesiense, T. b. Zimmermman et al.(2012) trypomastigotes), In vivo activity gambiense (reduction of parasitemia) Lippia sidoides Thymol Cell proliferation, accumulation of lipid L. amazonensis Medeiros et al.(2011) droplets, wrinkled, or ruptured membranes and the loss of cytoplasm (promastigotes) Cymbopogon citratus Citral Cell proliferation, ultrastructural L. infantum, L. major, Machado et al.(2012) alterations like mitochondrial and L. tropica kinetoplast swelling and disruption of nuclear membrane, loss of mitochondrial membrane potential and other alterations Ambrosia scabra Psilostachyin C Cell proliferation (epimastigotes and T. cruzi, L. amazonensis, L. Sülsen et al.(2011) promastigotes), ultrastructural mexicana changes, anti amastigotes activity, In vivo activity (reduction of parasitemia) Chamomilla recutita (−) α-bisabolol Cell proliferation (promastigotes) L. infantum Morales-Yuste et al.(2010) Xanthium strumarium Xanthatin Cell proliferation (bloodstream forms), T. b. brucei Nibret et al.(2011) mitochondrial membrane potential reduction, trypanothione reductase inhibition Saussurea costus Sesquiterpene lactones Cell proliferation (bloodstream forms) T. b. rhodesiense Julianti et al.(2011) Piper aduncum 20,60-dihydroxy-40- Cell proliferation (promastigotes), L. amazonensis Torres-Santos et al.(1999) methoxychalcone mitochondrial damage, anti-intracellular amastigote activity Piper rusbyi Kavapyrone, Flavokavain Cell proliferation (promastigotes) L. amazonensis, Flores et al.(2007) In vivo activity (lesion size reduction) L. braziliensis, L. donovani Piper auritum Safrole Cell proliferation (promastigotes), L. major, L. mexicana, Monzote et al.(2010) anti-intracellular amastigote activity L. braziliensis, L. donovani Piper regnellii Eupomatenoid-5 Cell proliferation (promastigotes and L. amazonensis Vendrametto et al.(2010), axenic amastigotes), anti-intracellular Trypanosoma cruzi Pelizzaro-Rocha et al.(2011) amastigotes Ultrastructural alteration and lipoperoxidation in the cell membrane (epimastigotes and bloodstream forms) Aframomum sceptrum Sceptrumlabdalactone Cell proliferation (promastigotes and L. donovani T. b. brucei Cheikh-Ali et al.(2011) B bloodstream forms)

In fact, promising results have been obtained by our group ANTI-LEISHMANIAL COMPOUNDS and others in the search of plant-derived crude extracts, Essential oils are known to possess a wide variety of hydropho- essential oils, and compounds with activity against path- bic compounds with antimicrobial potential. The ability to dif- ogenic microorganisms, including Leishmania spp., T. cruzi fuse across cell membranes certainly gives to those molecules (Alviano and Alviano, 2009; Izumi et al., 2011), and T. brucei some advantage in targeting cellular components, being a valuable (Gehrig and Efferth, 2008). Useful antiprotozoal phytocom- research option for the search of bioactive compounds (Bakkali pounds can be divided into several categories summarized in et al., 2008). The Ocimum gratissimum essential oil and eugenol, Table 1. its major component, was tested on the growth, viability, and

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FIGURE 1 | Effects of linalool-rich essential oil (15.0 ng/ml) extracted swelling (C,D), and the gross alterations in the organization of the from C. cajucara on the promastigote stage of L. amazonensis, as nuclear and kinetoplast chromatins (C,D). In the presence of the observed by transmission electron microscopy. (A) Control parasites; essential oil, the parasites were completely destroyed after 30 min of (B–E) parasites treated for 5 (B), 10 (C), 15 (D), and 30 (E) min, showing treatment (E). N, nucleus; K, kinetoplast, F,flagellum. Bars, 1 µm. Image promastigotes with different degrees of damage. Note the disruption of reproduced with permission from © Rosa et al.(2003) American Society the flagellar membranes [arrowheads in (B,C)], the mitochondrial for Microbiology. ultrastructural alterations of the amastigote and promastigote with L. amazonensis. Concomitantly, the association indexes were forms of L. amazonensis,as well as on the interaction of these flagel- drastically lower in the latter conditions, compared to the control lates with mouse peritoneal macrophages, concomitant with nitric system (Ueda-Nakamura et al., 2006). oxide production stimulation by the infected macrophages. Sig- The linalool-rich essential oil extracted from the leaves of Cro- nificant mitochondrial alterations occurred at the ultrastructural ton cajucara,has effects on L. amazonensis parasites,on the interac- level of the parasite, such as remarkable swelling, disorganiza- tion of these flagellates with mouse peritoneal macrophages and on tion of the inner membrane, and an increase in the number of nitric oxide production by the infected macrophages. The median cristae after treatment of parasites with O. gratissimum essen- lethal doses and absolute lethal doses of the essential oil and tial oil. However, mouse macrophages were unaffected under the linalool-rich essential oil from C. cajucara for promastigotes and same conditions. In addition, nitric oxide production was dra- amastigotes were very low. Mitochondrial swelling and alterations matically stimulated when mouse peritoneal macrophages were in the organization of the nuclear and kinetoplast chromatins were treated with 150 µg/ml essential oil,both before and after infection observed by electron microscopy when L. amazonensis parasites

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FIGURE 2 | Structure of cupacinoside and taraxerol. Reprinted (adapted) with permission from © Gachet et al.(2011) American Chemical Society.

FIGURE 3 | Effects of Kola acuminate proanthocyanidin (50 µg) on Cells showing disintegrated cell membranes and loss of cytoplasmic contents bloodstream form trypanosomes observed by scanning and caused by the effect of the drug. (F) TEM of a trypanosome cell confirming transmission electron microscopy. (A) Control parasites showing the the necrotic process of cell membrane disintegration and loss of cytoplasmic normal cell morphology and membrane integrity. (B,C) Parasites treated for contents that led to cell death. Scale bar: 1 µm. Image reproduced with 6 h showing morphological changes and the rounding up of the cells. (D,E) permission from © Kubata et al.(2005) Elsevier.

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FIGURE 4 | Structure of salvadione and perovskone. Reprinted (adapted) with permission from © Farimani et al.(2011) American Chemical Society. were treated with the essential oil from C. cajucara (Figure 1). agents of sleeping sickness. Cupacinoside and taraxerol showed The viability of mouse macrophages was unaffected by the same IC50 values <10 µM against T. b. rhodesiense, with taraxerol concentrations. When the macrophages were pre-treated with C. exhibiting only low cytotoxicity against rat skeletal myoblast cell cajucara essential oil, as well as when the macrophages were pre- line (L-6 cells; Gachet et al., 2011). infected with the parasites and then treated with the essential oil, A proanthocyanidin isolated from Kola acuminate seeds was the association indexes were 50% lower than those for the control able to inhibit the proliferation and cause the lysis of Try- system. In addition, the macrophages pre-treated with C. caju- panosoma brucei bloodstream forms in vitro. The in vivo effect cara essential oil produced twice the amount of nitric oxide as the was trypanostatic and prolonged the survival of infected ani- untreated macrophages (Rosa et al., 2003). mals that were treated with this substance. Additionally, it was Piper species have been reported to have activity against Leish- not toxic to the human epidermoid carcinoma cells (KB 3-1), but mania parasites (Torres-Santos et al., 1999; Flores et al., 2007; it caused ultrastructural changes in parasites, such as rupture of Sarkar et al., 2008). Our group recently showed that the leaf essen- the plasma membrane and vesicles and the formation of multi- tial oil from Piper claussenianum was able to inhibit the growth vesicular bodies in lysosome-like organelles, when the parasites of L. amazonensis promastigotes with an IC50 of 0.0038% (Mar- were treated for 6 h with the compound (Figure 3; Kubata et al., ques et al., 2010). Trans-nerolidol is a major component of this 2005). essential oil. We also examined the effect of the essential oil on Salvadione and perovskone (Figure 4), two new triterpenoids the interaction between parasites and host cells and its activity with rare carbon skeletons isolated from aerial parts and flowers against intracellular amastigotes. The IC50 concentration used of Salvia hydrangea were used in the treatment of leishmania- against promastigotes yielded a 31% reduction in the percent- sis in Iran. These compounds were tested in vitro against T. b. age of infected macrophages and led to a 17% increase in the rhodesiense and exhibited moderated potency with IC50 values production of NO by macrophages. The activity of the enzyme 4.33 and 15.92 µM, respectively and good selectivity index for L-6 arginase may be able to modulate nitric oxide production by cells (Farimani et al., 2011). macrophages by inhibiting nitric oxide synthase (iNOS). Pro- A study using essential oils of cloves (Syzygium aromaticum), mastigotes were grown in the presence of the IC50 of the essential basil (Ocimum basilicum), and a yarrow (Achillea millefolium) oil and showed a 62% reduction in arginase activity. (Marques and the main constituents, eugenol and linalool showed activ- et al., 2011). Monzote et al.(2010) described the anti-leishmanial ity on T. cruzi bloodstream trypomastigotes and epimastigotes activity of the essential oil from Piper auritum. In that study, essen- forms. The essential oils inhibited epimastigotes proliferation, tial oil inhibited the growth of promastigotes in all species of caused trypomastigotes lysis and ultrastructural changes in both Leishmania used, with IC50 values between 12.8 and 63.3 µg/mL. forms, mainly in the nucleus. In epimastigotes forms was observed In addition,piper-oil inhibited the growth of intracellular amastig- shrinkage of the nuclear material with separation of the nuclear otes of L. donovani at non-toxic concentrations (Monzote et al., membrane and in trypomastigotes forms loss nuclear material 2010). and masses of condensed chromatin appeared (Santoro et al., 2007). ANTI-TRYPANOSOMIAL COMPOUNDS Acetyl aleuritolic acid, a terpene isolated from the methano- One new diterpene glycoside, cupacinoside, and one known com- lic extract of stem bark of C. cajucara showed significant try- pound, taraxerol (Figure 2) from n-hexane, and dichloromethane panocidal effect in trypomastigotes of a strain isolated from wild extracts from the bark of Cupania cinerea (Sapindaceae) have reservoirs (GLT291), genotype TCI as clone Dm28c. was also shown significant in vitro activity against one of the etiologic effective against intracellular amastigotes and did not show a

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significant effect on proliferative epimastigotes. This compound CONCLUSION was also able to inhibit the activity of trypanothione reductase, At a time when there is an urgent need for the efficient treatment an important enzyme in the regulation of redox balance and of leishmaniasis, Chagas disease, and sleeping sickness, this review defense against oxidative stress in this parasite (Campos et al., shows that plant-derived extracts and compounds exhibit signif- 2010). Xanthatin, a sesquiterpene lactone, was described by Nibret icant antiprotozoal activities in vitro and emphasizes the impor- et al.(2011) as strong trypanocidal agent with an IC 50 value of tance of phytoscience in the search for novel anti-leishmanial and 2.63 µg/mL. According to the authors, it seems that the biological anti-trypanosomal therapeutic agents. activity of the compound is a result of its effect in inducement of apoptosis in trypanosomes as evidenced by a reduction in ACKNOWLEDGMENTS mitochondrial membrane potential. Furthermore, xanthatin was This work was supported by Conselho Nacional de Desenvolvi- able to inhibit the two key enzymes involved in the inflammatory mento Científico e Tecnológico (CNPq), Coordenação de Aper- process, cyclooxygenase and 5-lipoxygenase, which can be very feiçoamento de Pessoal de Nível Superior (CAPES), and Fundação interesting in diseases that cause this kind of response (Nibret Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de et al., 2011). Janeiro (FAPERJ).

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Kinetoplastids: related pro- Conflict of Interest Statement: The istry and biological activity of essen- oxide dismutase-A (TcFeSOD-A) tozoan pathogens, different diseases. authors declare that the research was tial oils from Piper claussenianum enzyme in Trypanosoma cruzi pop- J. Clin. Invest. 118, 1301–1310. conducted in the absence of any com- (Piperaceae). Nat. Prod. Commun. 5, ulation with in vitro-induced resis- Sülsen, V., Frank, F. M., Carzola, S. I., mercial or financial relationships that 1837–1840. tance to benznidazole. Acta Trop. Barrera, P., Freixa, B., Vila, R., Sosa, could be construed as a potential con- Marques, A. M., Barreto, A. L. S., 100, 119–132. M. A., Malchiodi, E. L., Muschi- flict of interest. Curvelo, J. A. R., Romanos, M. T. Pelizzaro-Rocha, K. J., Veiga-Santos, etti, L. V., and Martino, V. S. V., Soares, R. M. A., and Kaplan, P., Lazarin-Bidóia, D., Ueda- (2011). Psilostachyin C: a natural Received: 15 January 2012; accepted: 18 M. A. C. (2011). 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Agents Rodrigues, Rosa, Alviano and Soares. Moncayo, A., and Ortiz Yanine, M. I. Rosa, M. S., Mendonça-Filho, R. R., Chemother. 43, 1234–1241. This is an open-access article distributed (2006). An update on Chagas dis- Bizzo, H. R., Rodrigues, I. A., Ueda-Nakamura, T., Mendonça-Filho, under the terms of the Creative Com- ease (human American trypanoso- Soares, R. M. A., Souto-Padrón, T., R. R., Morgado-Díaz, J. A., Kore- mons Attribution License, which per- miasis). Ann. Trop. Med. Parasitol. Alviano, C. S., and Lopes, A. H. hisa Maza, P., Prado Dias Filho, B., mits use, distribution and reproduction 100, 663–677. (2003). Antileishmanial activity of Aparício Garcia Cortez, D., Alviano, in other forums, provided the original Monzote, L., García, M., Montalvo, a linalool-rich essential oil from D. S., Rosa, M. S., Lopes, A. H., authors and source are credited and sub- A. M., Scull, R., and Miranda, M. Croton cajucara. Antimicrob. Agents Alviano, C. S., and Nakamura, C. ject to any copyright notices concerning (2010). Chemistry, cytotoxicity and Chemother. 47, 1895–1901. V. (2006). Antileishmanial activity any third-party graphics etc.

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