Antiprotozoal and molluscicidal activities of five Brazilian plants

Leishmaniasis, Chagas’ disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider’s Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 μg/mL, showed 90.4 ± 11.52 and 88.9 ± 2.20% growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 μg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100% lethal to snails at 200 μg/mL and showed an LD50 of 143 μg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs. Correspondence


Introduction
The general floristic research on diversity in the Upper Paraná River floodplain, area of Porto Rico (PR, Brazil), carried out by Souza et al. (1), demonstrated high floristic heterogeneity characterized by forest for-mations.This fact is probably related both to the diversity of environments, which include dry and flooded areas like swamps, lakes and ponds located between the forest domain of Paraná State and the savanna of Mato Grosso do Sul and São Paulo States, and to anthropic disturbance.In their survey of vascular plants the authors registered 117 families and 652 species.Many of these plants are used in folk medicine, and most of them have not been studied scientifically.
In the last few years, much research has been carried out to evaluate the effectiveness and safety of the use of plants or their metabolites for the prevention or treatment of diseases.Screening of plant extracts represents a continuous effort to find new bioactive molecules or extracts.Approximately 20% of the plants in the world or their extracts have been submitted to pharmacological or biological tests (2).
American tegumentary leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people, primarily those with the worst living conditions (3).Plants with antiprotozoal or molluscicidal activities can be useful tools to control these diseases.
American tegumentary leishmaniasis, whose etiological agents are flagellate protozoa of the genus Leishmania (family Trypanosomatidae), affects skin and mucous membranes and can cause disfiguring lesions (4).The etiological agent of Chagas' disease is Trypanosoma cruzi, also a flagellate protozoan belonging to the family Trypanosomatidae.This parasitic disease may cause mainly cardiomyopathy and megasyndromes (3).Clinical data demonstrate that the current drugs for the treatment of these diseases present several side effects and do not always provide the desired cure levels (3,(5)(6)(7).
Schistosomiasis is an important endemic disease in Brazil.Its etiological agent, the helminth Schistosoma mansoni (family Schistosomatidae), requires snails of stagnant or slow-flowing fresh water to complete its developmental cycle, with the species Biomphalaria glabrata (family Planorbidae) being the most important vector of S. mansoni in Brazil.The severity of the clinical forms causes much concern and can lead to death (3).The synthetic molluscicides that have been used are very expensive, can be toxic to other organisms, and can lead to deleterious long-term effects on the environment and to the development of resistance by vector snails (8,9).Thus, other alternatives need to be evaluated.

Plant material
Vegetative samples of the five species were collected from riparian vegetation on banks in different areas of the Upper Paraná River floodplain: C. podantha (leaves) and H. gardneriana (aerial parts) from the Ivinhema River, municipal district of Jatei (MS, Brazil), M. arenosa (aerial parts) from Garças' Lake, municipal district of Bataiporã (MS, Brazil), N. falcifolia (leaves) from the Baía River, municipal district of Taquaruçu (MS, Brazil), and P. elegans (leaves) from Figueira's Pond, municipal district of Porto Rico (PR, Brazil).Voucher specimens of each species were added to the collection of the HNUP Herbarium (Nupelia, Universidade Estadual de Maringá, PR, Brazil; Registration Numbers 1281, 2844, 1834, 1421, and 463, respectively).

Extraction
The following amounts of dried plant parts were powdered with a knife mill: 200 g C. podantha, 450 g H. gardneriana, 600 g M. arenosa, 580 g N. falcifolia, and 395 g P. elegans.Crude extracts were obtained by extraction with absolute ethanol at room temperature, and the solvent was removed by rotary evaporation under reduced pressure, producing 15.8, 32.7, 71.0, 64.0, and 55.8 g of ethanolic extracts, respectively.

Culture and maintenance of the parasites
Leishmania (Viannia) braziliensis promastigotes, strain MHOM/BR1987/M11272, were grown at 25ºC in Schneider's Drosophila medium supplemented with 10% (v/v) heat-inactivated fetal calf serum.Cells were harvested in the late log phase (day 3 of culture), resuspended in fresh medium, counted in a Neubauer chamber and adjusted to a concentration of 4 x 10 6 promastigotes/mL.T. cruzi epimastigotes, strain Y, were cultivated in liver infusion tryptose medium (10) containing 10% fetal calf serum and incubated at 28ºC for 96 h.Both parasite strains were maintained through weekly transfers in the respective medium.

Anti-leishmanial activity
The assay was conducted as described by Araujo et al. (5) and Ferreira et al. (11).Growth inhibition of L. (V.) braziliensis promastigotes was evaluated with the ethanolic extract of the plants at concentrations ranging from 0.125 to 320 µg/mL.The extracts were dissolved in DMSO (the highest concentration used was 1.6%, v/v) added to the promastigote cultures (day 3 of culture) at 4 x 10 6 promastigotes/mL, and incubated at 25ºC.After 24 h, the surviving parasites were counted in a Neubauer chamber and their number was compared with that of controls grown in the presence of DMSO only.All tests were done in triplicate and pentamidine isethionate (0.7 µg/mL; May & Baker Lab., London, UK) was used as reference drug.The LD 50 /24 h values were determined by linear regression analysis using statistical error limits up to 10%.

Anti-Trypanosoma cruzi activity
The growth inhibition of T. cruzi epimastigotes was evaluated with the ethanolic extract of the plants (10, 100, or 1000 µg/mL).The extracts were dissolved in DMSO, added to the culture medium and the parasites (1 x 10 6 epimastigotes/mL) were cultivated for 96 h at 28ºC.Growth was evaluated by counting in a Neubauer chamber and compared to that of controls grown in medium containing only DMSO (1.0%).All tests were done in duplicate and benznidazole (10 µg/mL; N-benzyl-2-nitro-1-imidazolacetamide; Roche Pharmaceuticals, Rio de Janeiro, RJ, Brazil) was used as reference drug.

Maintenance of the snails
The snails, B. glabrata, SUCEN strain, were maintained in aquaria with potable water, air circulation, aquatic plants, and Poecilia reticulata fishes, at room temperature.Mature snails, relatively uniform in age and size (shell diameter: 10-12 mm), were used for the tests.

Molluscicidal activity
The acute toxicity on B. glabrata was evaluated by a rapid screening procedure (9,12).The ethanolic extracts were dissolved in 100 µl DMSO at the concentration of 400, 300, or 200 µg/mL and then added to glass beakers containing 100 mL of water from the aquaria.Two snails were placed in each container and maintained in a well-aerated place at room temperature.After 24 h the snails were placed on a Petri dish and their heartbeats were checked using a stereomicroscope.For all extracts that were 100% lethal at the concentration of 200 µg/mL, the concentrations of 150, 100, and 50 µg/mL were also tested.For these assays, ten snails were used, with a 50-mL volume of aquarium water per snail.To confirm mortality the snails were transferred to vessels containing only distilled water and their condition was re-evaluated 24 h later.A control was carried out with DMSO.All tests were performed in duplicate and niclosamide (5 µg/ mL; Bayluscide ® , Bayer AG, Leverkusen, Germany) was used as reference drug.

Results and Discussion
Several natural compounds have been identified for the treatment of leishmaniasis and research on plants and their metabolites can contribute to overcoming the drug resistance of Leishmania parasites (7).Among the plant species evaluated here, N. falcifolia presented the best results regarding antileishmanial activity, with the ethanolic leaf extract displaying an LD 50 of 138.5 µg/mL and 65.6 ± 5.4% growth inhibition of the promastigote forms of L. (V.) braziliensis at the highest concentration tested, 320 µg/mL (Table 1).Extracts of H. gardneriana (aerial parts) and C. podantha (leaves), which also demonstrated reasonable potency, presented an LD 50 of 237 and 271 µg/mL, respectively.No growth inhibition was obtained at drug concentrations lower that 40 µg/mL.The medium containing DMSO did not affect the growth of the protozoa.
Ethanolic extracts of C. podantha and M. arenosa (aerial parts) inhibited the growth of epimastigote forms of T. cruzi even at very low concentrations (10 µg/mL), presenting 90.4 ± 11.52 and 88.9 ± 2.20% growth inhibition of this protozoan, respectively.On the other hand, extracts of H. gardneriana, N. falcifolia, and P. elegans (leaves) showed similar activities only when a concentration of 1000 µg/mL was used (Table 2).The medium containing 1.0% DMSO did not affect the growth of the protozoa.Benznidazole, used as the positive control against T. cruzi at 10 µg/mL, showed 80% growth inhibition (data not shown).
The best results in terms of molluscicidal activity (Table 3) were obtained with the ethanolic extract of M. arenosa, which induced 100 and 60% snail mortality at concentrations of 200 and 150 µg/mL, respectively, with an LD 50 of 143 µg/mL.The N. falcifolia extract was 100% lethal to the snails at the concentration of 200 µg/mL, but mortality was not obtained at lower concentrations.Control assays with DMSO showed no effect on the snails.Niclosamide at 5 µg/ mL was used as positive control against B. glabrata and showed 100% lethality (data not shown).
Although the literature indicates that ideal concentrations of plant extracts are below 100 µg/mL for molluscicidal activity (8), the results obtained for M. arenosa, LD 50 of 143 µg/mL, justify the continuation of its study.This plant is native to the area and the extract was obtained from regenerating parts of the plant, factors that can be considered of importance.
The genus Nectandra is well represented in the Brazilian flora, with several species presenting many benefits to man.They have been used in popular medicine for the relief of pain, arthritis, rheumatism and diarrhea, and also as antifungals.Pharmacological studies have demonstrated the antitumoral activity of N. rigida Nees, the antimalarial activity of N. cuspidata Nees and the vascular and antimalarial activities of N. salicifolia Nees (13)(14)(15)(16)(17).In our study, N. falcifolia leaves presented good results regarding their antiprotozoal activity against promastigote forms of L. (V.) braziliensis.
Some species of the genus Melochia have been used in folk medicine, such as M. corchorifolia L. (dysentery, abdominal swellings and water-snake bites), M. umbellata (Houtt.)Stapf (deobstruent) and M. pyramidata L. (bronchitis and cough) (14,22,23).The extract obtained from the aerial parts of M. arenosa demonstrated molluscicidal effects and activity against T. cruzi epimastigotes that can be explored in the future.
Continuous research for new drugs with high activity and reduced adverse effects is very important, especially considering that in Brazil parasitic diseases constitute a serious public health problem.Screening the biodiversity of the tropical forests can reveal new phytotherapeutic drugs, but studies of this type are just beginning.The biodiversity existing in the Brazilian flora is a potential source of many new bioactive molecules (7).The biological properties of the five plant species studied here, C. podantha, H. gardneriana, M. arenosa, N. falcifolia, and M. arenosa had not been evaluated until the present study.The results obtained here in biological assays with these plant species that occur naturally in the studied area demonstrate that the local flora presents a great medicinal potential.The more active plant extracts were prioritized for fractionation and identification of their active components, a work that is already under way.