Introduction

Leishmaniasis is a group of infectious diseases, with clinical significance and epidemiological diversity (Alvar et al. 2012). It is caused by intracellular protozoa of the genus Leishmania (Grimaldi et al. 1991). This important disease with epidemiological impacts worldwide especially affects developing countries. Currently, leishmaniasis is present in 98 countries on five continents and affects about 12 million people. Furthermore, it is estimated that 310 million people are at risk of contracting this disease with an annual incidence of 1.3 million new cases (WHO 2014). Despite enormous efforts by scientists researching various aspects of Leishmania parasites, the disease remains an important public health problem (Alvar et al. 2012; Bañuls et al. 2007).

The Leishmania genus comprises 35 species, with about 20 species known to be pathogenic to humans (Fraga et al. 2013). These parasites are transmitted to vertebrate hosts by phlebotomine vectors (Kato et al. 2010; Lainson and Shaw 2005). Nearly 20,000–40,000 deaths annually are associated with leishmaniasis worldwide (Alvar et al. 2012; WHO 2014). In Brazil, Leishmania species are agent of visceral (Leon et al. 1992), cutaneous, and mucosal clinical presentation forms and diffuse leishmaniasis due to Leishmania amazonensis (Reithinger et al. 2007; Brito et al. 2012). There is an expansion of the geographic distribution of L. amazonensis, and this species was associated with unusual clinical manifestations at new areas of transmission (Azeredo-Coutinho et al. 2007). Furthermore, co-infection between HIV and Leishmania has been reported in 35 countries, mainly in Spain, Italy, France, and Portugal (Alvar et al. 2008). Approximately 8.5 % of Brazilian patients with HIV are estimated to be co-infected with Leishmania (Lindoso et al. 2014).

Leishmaniasis is a typical example of a re-emerging tropical disease, due to the diversity of animal reservoirs and the difficulty of vector control. This neglected disease needs urgent investment by the pharmaceutical industry and attention by research institutes (Cavalli and Bolognesi 2009; Ameen 2010). The current chemotherapy of leishmaniasis involves parenteral administration of drugs such as pentavalent antimonials, pentamidine, and amphotericin B. However, these drugs have a number of disadvantages, including patient hospitalization and its associated high costs (Singh and Sivakumar 2004). Moreover, these drugs exhibit significant side effects (González et al. 2009). Despite the descriptions of over 25 new substances against Leishmania species, few are effectively used in humans (Palumbo 2009) and, in most cases, are used in parenteral form (Singh and Sivakumar 2004). The oral administration of drugs such as miltefosine and ketoconazole has been used with varying effects (González et al. 2009). In addition, most of the available drugs for the treatment of leishmaniasis are expensive, require a long period of use, and become inefficient, requiring the development of new therapies (Palumbo 2009). Furthermore, the lack of efficient vaccines and the high toxicity of treatment contribute to the spread of the disease (El-On 2009). Considering the inefficiency of current drugs and the fact that some varieties of Leishmania are resistant to these treatments, new drugs are being researched in order to find a more selective and effective therapy with fewer side effects. Therefore, our research group conducted studies on new therapeutic agents (Charret et al. 2009, 2013; Marra et al. 2012).

The literature has reported several studies about biological activities of extracts from marine algae (Shalaby 2011). These also have exhibited appreciable anticoagulant, anti-inflammatory, antitumoral, antiparasitic, antibacterial, and antiviral activities (Mayer et al. 2009). In recent years, the interest in the study of marine algae as a source of pharmacologically active compounds has increased worldwide, but there are few investigations that focus on the anti-leishmanial activity of seaweeds (Soares et al. 2012). Thus, the major aim of this work was to evaluate the potential activity against Leishmania amazonensis by natural substances isolated from marine algal species such as Caulerpa racemosa (Forsskål) J.Agardh (Chlorophyta, Bryopsidales), Osmundaria obtusiloba C. Agardh R. E. Norris (Rhodophyta, Ceramiales), Stypopodium zonale (J.V. Lamouroux) Papenfuss, Dictyota ciliolata Sonder ex Kützing, and Dictyota menstrualis (Hoyt) Schnetter, Hörning & Weber-Peukert (Phaeophyceae, Dictyotales), obtained from different geographical regions of Brazil.

Material and methods

Preparation of the extracts

Algae were collected along the coastline of Brazil (Table 1) with a license from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA, license number 10594-3). Extractions were performed with solvents of low and medium polarity, resulting in 10 extracts identified by abbreviations. The extracts from five marine algae (C. racemosa, O. obtusiloba, S. zonale, D. ciliolata, and D. menstrualis) were evaluated. The diterpenes pachydictyol A and isopachydictyol A were obtained from the dichloromethanic extract from D. menstrualis.

Table 1 Seaweeds species collected, abbreviation code, solvent used, and collection sites at different geographical regions of Brazil
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Isolation of the mixture pachydictyol A and isopachydictyol A

Air-dried D. menstrualis (95 g) was extracted in 100 % dichloromethane (CH2Cl2) exhaustively at room temperature, yielding 5 g of crude extract, which was subjected to silica gel (Merck; 0.015–0.045 mm), eluted with CH2Cl2/EtOAc (4:1), CH2Cl2/EtOAc (1:1), EtOAc, and 100 % acetone. Fractions 2 and 3 contain a crude mixture of diterpenes isomers pachydictyol A/isopachydictyol A (55 mg). Gel-column chromatography with 100 % hexane furnished 10 fractions. Fractions 3–10 yielded 45 mg of the pure mixture of the diterpenes pachydictyol A/isopachydictyol A (3:1). Pachydictyol A and isopachydictyol A were identified by comparison of physical and spectroscopic data with reported values (Durán et al. 1997) and comparison with authentic molecules.

Animals

Male BALB/c mice, 8 weeks old, were purchased from the animal facility of the Núcleo de Animais de Laboratório (NAL-UFF). The animals were used in therapeutic assay to obtain peritoneal macrophages for cytotoxicity assay. All experiments were conducted according to a protocol approved by the Ethics Committee for Use of Laboratory Animals (Permit Number: 318/13).

Parasites

Leishmania amazonensis strain MHOM/BR/77LTB0016 amastigotes were obtained from mouse popliteal lymph nodes (Bernardino et al. 2006). The amastigotes differentiated into promastigotes after 3 days in culture with Schneider’s insect medium (Sigma, USA) at 26 °C, supplemented with 10 % (v/v) inactivated fetal bovine serum (Cultilab, São Paulo, Brazil).

Anti-leishmania assays

Algal crude extracts and the purified product within a concentration range of 40–320 μg mL−1 were tested against L. amazonensis promastigotes (in the early stationary phase) starting with a concentration of 1×107 parasites mL−1 (Bernardino et al. 2006). Assays were performed in triplicate in 96-well microtiter plates. Dimethylsulfoxide (DMSO, Sigma) and the reference drug pentamidine (Sanofi-Aventis, Brazil) were used as controls. After 24 h, tests were carried out with 3-[4,5-dymethilthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, MTT (Sigma, USA), and after 4 h, plates were read using 545-nm filters (Mikus and Steverding 2000).

Cytotoxicity assays

In vitro evaluation of the cytotoxic activity of natural products on macrophages was performed according to the technique described by Bernardino et al. (2006). Briefly, peritoneal macrophages from BALB/c mice were obtained after washing with cold DMEM (Gibco, USA) medium. The peritoneal cells were cultured in 96-well plates at a concentration of 3×105 cells per well and maintained at 37 ° C in 5 % CO2. After 24 h, all extracts and purified products (Table 1) in different concentrations (20, 40, 80, and 160 μg mL−1) were added to the macrophages culture. DMSO, culture medium, and pentamidine were used as controls. Incubation for 24 h was conducted with MTT (5 mg mL−1; Sigma) as previously noted. Then, by regression analysis, the CC50/24 h was determined (Bernardino et al. 2006).

Evaluation of in vivo activity

Superior values of selectivity index (SI), which corresponds to the ratio between the values of CC50/24 h and EC50/24 h were used as criteria for the selection of the extract to be evaluated in vivo. BALB/c mice were inoculated subcutaneously in the left-hind footpad with 1 × 106 promastigotes of L. amazonensis. Two weeks after infection, BALB/c mice were treated orally daily with an alcoholic algal extract of O. obtusiloba (OO-Et/Buz) (5 or 20 mg kg−1). Similarly, the positive control group was treated daily with ketoconazole (50 mg kg−1), and the negative control group received DMSO. The treatment was performed for 28 consecutive days. Lesion thicknesses were evaluated weekly by measuring the diameters of both rear feet with a direct-reading dial caliper. The size of lesions in millimeters was calculated by subtracting the diameter of the uninfected foot from that of the infected foot.

Parasite quantification

A determination of the number of parasites in the lymph nodes was estimated by a modified limiting-dilution assay (Buffet et al. 1995). Popliteal lymph nodes of the infected footpads were removed and used to prepare a cell suspension in phosphate-buffered saline. After centrifugation the pellet was resuspended in Schneider’s insect medium, pH 7.2. The suspension was then serially diluted in 12-fold dilutions, incubated at 26 °C for 7 days, and monitored in an inverted microscope for the presence or absence of promastigotes. The parasite-burden per gram in the corresponding organ was calculated according to the method used by Pourrajab et al. (2012). To assay body weight, mice were weighed before, during, and at the end of the experiment to compare the weight of the treated and untreated mice.

Statistical analysis

All experiments were repeated at least three times. Data obtained were analyzed using Student’s t test, and p ≤ 0.05 were considered significant. For comparison of more than two groups, a one-way ANOVA was performed. If a significant main effect or association was identified (p ≤ 0.05), the respective group means were compared using Dunnett’s multiple comparison test. Data were analyzed using GraphPad Prism 5.0 software (Graph Prism Inc., USA).

Results

This study employed in vitro and in vivo tests to evaluate initial anti-leishmania activity against promastigote forms of L. amazonensis, its toxicity against mammalian cells, and after that, the therapeutic activity of Brazilian marine algae extracts. The extract from the brown alga S. zonale (Sz-EtOAc-Hex/Mar) inhibited 50 % of the parasitic activity at dilutions ten times less concentrated than that of pentamidine. Similarly, other brown algae extracts from D. menstrualis (DM-EtOAc/SPSP, DM-EtOAc/Buz, and DM-Hex/Buz) and D. ciliolata (DC-EtOAc/Angra and DC-Hex/Angra) showed high activity against L. amazonensis at low concentrations. In addition, the extract from the red alga O. obtusiloba (OO-Et/Buz and OO-EtOAc/Buz) and a mixture of isomers pachydictyol A and isopachydictyol A isolated from D. menstrualis showed potential anti-leishmania activity. In contrast, the two extracts from the green alga C. racemosa showed no significant activity against L. amazonensis (Table 2).

Table 2 Effect of the algae extracts and purified product of D. menstrualis on Leishmania and macrophages and their respective selectivity indexes (SI)
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In addition, a cytoxicity assay of the crude extract of S. zonale (Sz-EtOAc-Hex/Mar), D. ciliolata (DC-EtOAc/Angra, and DC-Hex/Angra), D. menstrualis (DM-EtOAc/SPSP, DM-EtOAc/Buz, and DM-Hex/Buz), and C. racemosa collected from Buzios (CR-EtOAc/Buz) and the diterpene isomers mixture pachydictyol A and isopachydictyol A showed moderate cytotoxicity on mammalian cells similar to pentamidine. However, extracts of O. obtusiloba (OO-EtOAc/Buz and OO-Et/Buz) and C. racemosa (CR-EtOAc/SPSP) in the concentration tested were not cytotoxic to murine peritoneal macrophages (p ≤ 0.05) (Table 2).

The selectivity index (SI) was determined to be a secure indicator of the therapeutic use of a new potential compound. Among the marine macroalgae evaluated, a total of eight extracts obtained from S. zonale, D. menstrualis, D. ciliolata, and O. obtusiloba had a selectivity index better than pentamidine. The best result was observed in assays with an extract from S. zonale, which showed an SI about 15 times higher than pentamidine. This extract was also found to be about 40 times less toxic to murine peritoneal macrophages compared to the promastigote forms of L. amazonensis that were affected by the extract. Additionally, extracts of Dictyota (DM-EtOAc/SPSP, DM-EtOAc/Buz, DM-Hex/Buz, DC-EtOAc/Angra, and DC-Hex/Angra) and O. obtusiloba (OO-Et/Buz and OO-EtOAc/Buz) showed a selectivity index better than pentamidine. However, the mixture of isomers pachydictyol A and isopachydictyol A and C. racemosa extracts showed low SI values (Table 2).

The extract of O. obtusiloba (OO-Et/Buz) was considered promising and was used for the therapeutic evaluation in mice subcutaneously infected with L. amazonensis. At the second week post-infection, BALB/c mice showed small cutaneous lesions initially characterized by mild edema, erythema, and a progressive increase of lesions in all animal groups. However, at 5 weeks post-infection, a significant increase in the diameters of cutaneous lesions in the untreated animals was observed in relation to animals treated with O. obtusiloba extract (Fig. 1). After that, both groups treated with 5 mg kg−1 of alcoholic extract of O. obtusiloba and those treated with 20 mg kg−1 of the extract had similar skin-lesion progressions compared to the group treated with ketoconazole, a reference drug. Treatment with 5 or 20 mg kg−1 of the alcoholic extract of O. obtusiloba or ketoconazole decreased the parasite load observed in lymph nodes in comparison to the untreated animals (Fig. 2).

Fig. 1
figure 1

BALB/c mice infected with L. amazonensis after treatment with alcoholic extract of Osmundaria obtusiloba (OO 5 mg or OO 20 mg kg−1) or ketoconazole (50 mg kg−1). Lesion sizes were measured at the indicated times post-infection (mean ± standard deviation). The black arrow indicates the start of the oral treatment. *p < 0.05). These results are representative of one experiment (n = 6 per group)

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Fig. 2
figure 2

Parasite load (number of parasites per mg) in popliteal lymph node of BALB/c mice infected with L. amazonensis after treatment with alcoholic extract of Osmundaria obtusiloba (OO 5 mg or OO 20 mg kg−1) or ketoconazole (50 mg kg−1). These results are representative of one experiment (n = 3 per group)

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Discussion

Leishmaniasis has been considered a neglected disease, despite its high rates of mortality and morbidity (Alvar et al. 2012; WHO 2014). The drugs used in leishmaniasis treatment have serious side effects (Ameen 2010), and the search for anti-leishmanial activity among natural products such as algae may be useful in the development of new drugs.

Marine organisms constitute a promising source of biologically active compounds. However, few studies have researched marine algae for their potential for the development of leishmanicidal drugs (Soares et al. 2012). Among ten algal extracts examined, this study evaluated S. zonale to demonstrate the best selectivity index, approximately 15 times better than pentamidine. Given these considerations, further in vivo studies will be necessary in order to elucidate the role of S. zonale extract in Leishmania infection. The presence of meroditerpenes, in particular atomaric acid isolated from S. zonale that has been selected for antiviral activity (Soares et al. 2007), could also be contributing to anti-leishmanial activity since some terpenes can inhibit target enzymes from Leishmania (Gray et al. 2006). Some inhibitors of kinases are cytotoxic to L. amazonensis (Becker and Jaffe 1997). We are not aware of any study thus far of the anti-leishmanial action of S. zonale, but there are reported studies concerning its antiviral (Mendes et al. 2011), antitumoral, and antioxidant (Penicooke et al. 2013) activities.

The literature also does not report anti-leishmanial activity of D. menstrualis and D. ciliolata. A comparison of the activity from extracts obtained from D. menstrualis (DM-EtOAc/SPSP, DM-EtOAc/Buz, and DM-Hex/Buz) from different sites showed no significant difference in their inhibitory effect on L. amazonensis promastigotes. In our study, all Dictyota extracts showed better anti-leishmanial activity compared to the reference drug, pentamidine. It is possible that the superior inhibitory effects of the algal extracts could be related to the presence of α, β-unsaturated dialdehydes such as xeniane, dichotomane, and cycloxeniane diterpenes isolated from these algae in previous studies (Cavalcanti et al. 2007; De-Paula et al. 2008). In other studies, best values of anti-leishmanial activity were observed with extracts obtained from Dictyota caribaea (Freile-Pelegrin et al. 2008). Diterpenes isolated from brown algae Dictyota pfaffii and D. menstrualis have potentially important biological activities such as antiviral (Abrantes et al. 2010), antimalarial, and antibacterial (Jongaramruong and Kongkam 2007) effects. In addition, diterpenoids of D. menstrualis have demonstrated anti-HIV activity (Pereira et al. 2005). More recently, dolabelladienetriol, a diterpene isolated from Brazilian D. pfafii, was shown to be effective against promastigote forms of L. amazonensis (Soares et al. 2012). In the present study, the mixture of diterpene isomers pachydictyol A and isopachydictyol A was less active than pentamidine as well as the extracts from D. menstrualis. This might be due to the presence of different molecules found in the crude extract that may develop a synergism to anti-leishmanial activities. In addition, the hexanic extract of D. ciliolata (DC-Hex/Angra) showed better anti-leishmanial activity than the ethyl acetate extract (DC-EtOAc/Angra). These results can be explained by the greater abundance of diterpenes in hexanic extract, in particular of α, β-unsaturated dialdehydes and prenylated guaiane diterpenes (Cavalcanti et al. 2007; De-Paula et al. 2008).

The extract of C. racemosa was considerably less active compared to those described in the literature (Süzgeç-Selçuk et al. 2011). Additionally, a sample of the green alga C. racemosa (CR-EtOAc/SPSP) collected in Pernambuco State inhibited the growth of parasites about three times more effectively than a sample (CR-EtOAc/Buz) collected in Rio de Janeiro State (Table 2). The difference in EC50 values found was possibly directly related to the fact that these algae have been collected from different regions, as differing environmental conditions influence the production of metabolites. In previous studies, extracts from Brazilian C. racemosa resulted in the isolation of caulerpin, a bis-indole alkaloid that has an anti-inflammatory effect (Rocha et al. 2007; de Souza et al. 2009).

Several studies have demonstrated the important antimicrobial activities of C. racemosa, O. obtusiloba, S. zonale, D. menstrualis, and D. ciliolata (Mayer et al. 2009; Soares et al. 2007; Mendes et al. 2011; Freile-Pelegrin et al. 2008; Abrantes et al. 2010; Süzgeç-Selçuk et al. 2011). Although the activities of many of these seaweeds are notable, the targets of their potential products are unknown. We investigated several alternative therapies for the treatment of leishmaniasis and we have identified several promising natural substances, especially algal species such as O. obtusiloba, S. zonale, D. ciliolata, and D. menstrualis, which showed excellent selectivity indices, and which hold promise for in vivo tests.

In the present study, extracts of the red algae O. obtusiloba (OO-Et/Buz and OO-EtOAc/Buz) showed interesting anti-leishmanial effects and were not cytotoxic to murine peritoneal macrophages (240–198 μg mL−1, respectively). In addition, SI of extracts showed their security to the host cells and specificity to the parasite. The alcoholic extract (OO-Et/Buz) and the ethyl acetate extract were about ten times less toxic to peritoneal macrophages than pentamidine was. Studies by de Souza et al. (2012) showed that a lipid extract of O. obtusiloba exhibited moderate toxicity toward Vero cells (72 μg mL−1).

The present study was conducted to evaluate the therapeutic activity of an alcohol extract of O. obtusiloba in BALB/c mice infected with L. amazonensis. A highly significant reduction in the size of cutaneous lesions was observed in animals treated with 5 and 20 mg kg−1 of O. obtusiloba alcohol extract compared to the untreated infected group (P ≤ 0.05), (Fig. 1). We observed an important reduction of parasite load in the popliteal lymph nodes obtained from the infected treated group. The parasite load in the lymph nodes of infected mice treated with 20 mg kg−1 extract (0.05 × 1010 parasites) was approximately 40 times smaller than in the untreated infected group (2.13 × 1010 parasites) and approximately 20 times smaller compared to the group treated with ketoconazole (1.12 × 1010 parasites). In addition, compared to the untreated infected control group, no signs of apparent toxicity were observed with no significant changes in body weight varying from 25 to 34 g between animals receiving ketoconazole or even in those treated with different concentrations of alcoholic extract of O. obtusiloba.

Thus, the treatment of the murine model of L. amazonensis infection showed the ability to control the dissemination of parasites to the draining lymph nodes as well as the evolution of cutaneous lesion, indicating a potential therapeutic use of the alcoholic extract of O. obtusiloba. The probable activity of O. obtusiloba extracts could be related to the presence of bromophenols in these extracts with important antimicrobial activity (Liu et al. 2011). Until now, no leishmanicidal effects of O. obtusiloba have been described but other biological activities have been reported, such as potent antiviral activity verified in assays with HSV-1 and HSV-2 viruses (de Souza et al. 2012). In a recent review of the natural products isolated from the genus Osmundaria (Osako and Teixeira 2013), the authors describe the presence of halogenated substances, such as bromophenols and fimbriolides in various species. These molecules may be responsible for the observed activity.

Upon comparison of these data, O. obtusiloba is the algal species with satisfactory activity in both in vitro toxicity and in vivo therapeutic tests against L. amazonensis. This fact should encourage further studies of these algae, as new anti-leishmanial products and on the characterization of molecules and mechanism of action in relation to their anti-leishmanial activity.