Antileishmanial activity of some Brazilian plants , with particular reference to Casearia sylvestris

Leishmaniasis is a complex of diseases caused by Leishmania protozoa which treatment is restricted to a limited number of drugs that exhibit high toxicity, collateral effects and are often costly. There are a variety of tropical plants distributed in Brazil, and for many poor people the therapy for several diseases is based mainly on the use of traditional herbal remedies. In this work, the cytotoxic activity of 17 plant methanol extracts was evaluated on several Leishmania species and murine macrophages. Among them, the extract of Casearia sylvestris, Piptocarpha macropoda, Trembleya parviflora, Samanea tubulosa and Plectranthus neochilus showed a promissing leishmanicidal activity, exhibiting IC50 values below of 20 μg/mL against at least one species of Leishmania. Casearia sylvestris showed the most expressive activity against all promastigote forms of Leishmania species (IC50 values of 5.4 μg/mL, 5.0 μg/mL, 8.5 μg/mL and 7.7 μg/ mL for L. amazonensis, L. braziliensis, L. chagasi and L. major, respectively), being more effective than the reference drug miltefosine. In spite of the cytotoxic effect on macrophages (CC50 value of 5.2 μg/mL), C. sylvestris exhibited a strong inhibition against intracellular amastigotes of L. braziliensis (IC50 value of 1.3 μg/ mL). Further studies, including bio-guided fractionation will be conducted to identify the active compounds.


INTRODUCTION
Leishmaniasis is a vector borne disease caused by protozoa parasites of the genus Leishmania (WHO 2010).According to the World Health Organization, leishmaniasis is considered a major Neglected Tropical Disease with expressive economic, social, and political impacts.Leishmaniasis is distributed in more than 90 countries, with an annual incidence of 1.5 to 2.0 million cases, and 350 million people under the risk of infection (WHO 2013).
Leishmaniasis comprises a complex of clinical manifestations including ulcerative skin lesions, destructive mucosal inflammation, and disseminated visceral infection in its most severe form (Murray LUCIANA M.R. ANTINARELLI, NÍCOLAS C. PINTO, ELITA SCIO and ELAINE S. COIMBRA et al. 2005).These clinical manifestations occur due to a complex interaction between the parasite and the immune response of the mammalian host.
Current chemotherapeutic agents for the treatment of all clinical manifestations of leishmaniasis are pentavalent antimonials compounds (e.g., sodium stibogluconate and meglumine antimoniate) and amphotericin B, which unfortunately are considerably toxic (Croft and Olliaro 2011, Tempone et al. 2011, Singh and Sundar 2012).Furthermore, these drugs exhibit several limitations, including high cost and the need for daily parenteral administration (Singh and Sundar 2012).More recently the antitumor drug miltefosine was introduced as the first and still the only oral therapeutic option for the treatment of visceral leishmaniasis in India (Sundar et al. 2002, Dorlo et al. 2012).
There are a variety of tropical plants distributed in Brazil and for many poor people the therapy for several diseases is based mainly on the use of traditional herbal remedies (Lorenzi and Matos 2002).In most cases, these plants are used without any scientific base.In the last years, several works showed the leishmanicidal effect of some Brazilian plant extracts or essential oils (Santin et al. 2009, Albernaz et al. 2010, Alviano et al. 2012, Brito et al. 2013).In a previous work, we reported the antileishmanial activity of extracts of 20 plants from the Brazilian flora (Braga et al. 2007).Due to our continuous search for new alternatives for the treatment of leishmaniasis, this study aimed to investigated the leishmanicidal activity of 17 plant methanolic extracts against four Leishmania species and murine macrophages.

PLANT MATERIAL
Specimens of 17 species were collected in Juiz de Fora, Minas Gerais, Brazil.A voucher specimen was deposited at the Herbarium Leopoldo Krieger (CESJ) of the Federal University of Juiz de Fora.The dried parts of the plants (50 g each) were powdered and macerated with methanol (3x 200 ml) for five days at room temperature.After evaporation of the solvent under reduced pressure, the respective methanolic extracts were obtained.All extracts were kept dried in tightly stoppered bottles under refrigeration (4 °C) until used for the biological tests.
Antipromastigote assay: The antileishmanial activity was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) method based on tetrazolium salt reduction by mitochondrial dehydrogenases (Mossman 1983).Briefly, promastigotes from a logarithmic phase culture were suspended to yield 2 millions of cells/mL (L.amazonensis) or 3 millions of cells/mL (L.chagasi, L. braziliensis and L. major) after Neubauer chamber counting.ANTILEISHMANIAL ACTIVITY OF BRAZILIAN PLANTS The screening was performed in 96-well microtiter plates maintained at 25 °C.The analyses were made in duplicate.The extracts were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as a stock solution.Parasites were exposed to increasing concentration of the extracts solutions (at minimum five serial dilutions) for 72h at 25 °C.Controls containing 0.5% DMSO and medium alone were also included.The viability of promastigotes was obtained by measuring the absorbance at 570 nm (Multiskan MS microplate reader, LabSystems Oy, Helsink, Finland).Amphotericin B (Cristalia, São Paulo, Brazil) and miltefosine (Cayman Chemical Company, Michigan, USA) were used as reference drugs.
Antiamastigote assay: Inflammatory macro phages were obtained from the peritoneal cavity of BALB/c mice previously inoculated with 3% thioglycollate medium (Sigma Chemical Co; St. Louis, MO, USA).After 72h, the peritoneal exudate was collected by washing with cold Hank´s Balanced Sal Solution (Sigma Chemical Co; St. Louis, MO, USA) (Silva et al. 2012).Briefly, peritoneal macrophages added at 2x10 6 cells/mL to coverslips (13 mm diameter) previously arranged in a 24-well plate in RPMI 1640 medium (Cultilab, Campinas, São Paulo, Brazil) supplemented with 10% inactivated FBS, and allowed to adhere at 37 °C in 5% CO 2 .Adherent macrophages were infected with L. braziliensis (MRHO/BR/75/M2903) or L. amazonensis (IFLA/Br/67/PH8) promastigotes in the stationary growth phase using a 1:10 ratio at 33 °C for 4h.After this time, the no phagocytosed promastigotes were removed washing twice in sterile Phosphate Buffered Saline (PBS) and the test samples were added at nontoxic concentrations to the macrophages (5.0, 1.0 and 0.1 µg/mL) and maintained at 33 ºC 5% CO 2 for 72h.Cells were washed, fixed with absolute ethanol, and stained with Giemsa.Cells were then dehydrated in acetone followed by a gradient acetone-xylol (9:1; 1:1; 1:9) and finally xylol.The slides were mounted with Canada balsam for parasite counting at a optical microscopy (1000x magnification).At least 100 infected cells were counted and the results were expressed as Index infection, obtained multiplying the percentage of infected cells by mean number of amastigotes per cell.Miltefosine was used as reference drug.All procedures were performed in agreement with the Ethical Principles in Animal Research and according to protocols approved by the "Pró-Reitoria de Pesquisa/UFJF -Ethical Committee for Animal Research" (#016/2012-CEEA).
CYTOTOXICITY ON MAMMALIAN CELLS Murine macrophages were obtained and cultured as described before.Briefly, the inflammatory peritoneal macrophages were used for cytotoxicity assay in a concentration of 2x10 6 cells/mL in 96-well culture plates in RPMI 1640 medium supplemented with 10% inactivated FBS, at 37 °C and 5% CO 2 atmosphere.After 24h, the adherent macrophages were incubated with the extracts in a serial dilution, in duplicate at each concentration for 72h at 37 °C and 5% CO 2 atmosphere.The viability of the macrophages was determined with the MTT assay using a multiwall scanning spectrophotometer (Multiskan EX microplate reader), as described above, and was confirmed by comparing the morphology with the control (macrophages incubated in RPMI 1640 medium supplemented with 10% inactivated FBS), via light microscopy.Dose response curves were plotted (values expressed as percentage of control optical density) and the values were expressed as CC 50 values (50% cytotoxicity concentration).All procedures were performed in agreement with the Ethical Principles in Animal Research and according to protocols approved by the "Pró-Reitoria de Pesquisa/UFJF -Ethical Committee for Animal Research" (#015/2012-CEEA).

STATISTICAL ANALYSIS
For Leishmania and murine macrophages assays, the IC 50 or CC 50 values, respectively, were carried out and the 95% confidence intervals were included, LUCIANA M.R. ANTINARELLI, NÍCOLAS C. PINTO, ELITA SCIO and ELAINE S. COIMBRA calculated by Litchtfiet and Wilcoxon method using the Probit analysis, and the graphs were plotted by the program GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA).One-way ANOVA followed by Dunnett post test were used.Differences were regarded as significant when p < 0.0001 (***) and p < 0.001 (**).

RESULTS AND DISCUSSION
In the present work, 17 methanolic plant extracts were evaluated for antileishmanial activity against promastigote forms of Leishmania and citotoxicity against murine macrophages.In addition the efficacy of the most promising plant extract against intracellular amastigotes of Leishmania sp was also evaluated.Table I shows the botanical name, local name, voucher specimen number and popular uses of the tested plants.
Table II shows the extracts effect on promastigote forms of different Leishmania species: three from the New World (L.braziliensis, L. chagasi and L. amazonensis) and one species from the Old World (L.major) (Santos et al. 2008).Due to this, a different sensitivity of theses parasites to the tested extracts was expected.Previous in vitro studies have also shown differences in sensitivity of Leishmania species to different reference drugs, including pentavalent antimonials, amphotericin B (Minodier and Parola 2007), miltefosine (De Morais-Teixeira et al. 2011) and crude or purified plant extracts (Braga et al. 2007, Fabri et al. 2012a, b).Among the 17 methanolic extracts tested, 11 showed leishmanicidal activity against at least one promastigote forms of Leishmania species with IC 50 values ranging from 5.0 to 88.3 µg/mL.Interestingly, in a general biological evaluation, the tested extracts were more active against L. braziliensis, responsible for cutaneous and mucocutaneous leishmaniasis; and against L. chagasi promastigote forms, the causative agent of fatal visceral leishmaniasis in the American continent (Cruz et al. 2009).Promastigotes of L. amazonensis were less sensitive to the extracts assayed.
Phytochemical screening of most of the extracts assayed in this study was previously reported and a variety of secondary metabolites was related to these extracts such as alkaloids, triterpens, sterols, tannins, saponins and flavonoids (Scio et al. 2012).In this work, among the extracts assayed, Casearia sylvestris, Piptocarpha macropoda, Trembleya parviflora, Samanea tubulosa and Plectranthus neochilus showed strong leishmanicidal activity, exhibiting IC 50 values below of 20 µg/mL, for at least one species of Leishmania.Biological activity of C. sylvestris, P. macropoda and S. tubulosa could be due to the presence of secondary metabolites such as alkaloids, sterols, tannins and flavonoids (Scio et al. 2012).C. sylvestris showed remarkable activity against promastigotes of all Leishmania species, with IC 50 below 10 µg/ mL, being more effective than the reference drug miltefosine (Table II), which is currently the only oral drug available for the treatment of visceral leishmaniasis (De Morais-Teixeira et al. 2011).So, this plant extract was selected for further analysis against intracellular amastigote forms, since this form of parasite is found in the mammalian host and is important for human disease.Amastigotes of L. braziliensis and L. amazonensis were used as model in order to determine the antileishmanial property of C. sylvestris against intracellular forms of parasite (Fig. 1 and Table III).The effectiveness of C. sylvestris extract on the infection rate of the infected macrophages and the intracellular replication of the amastigote forms was determined by the infection index.Figure 1 shows the dose-dependent effect of this extract on amastigotes of L. amazonensis and L. braziliensis.Amastigotes of L. braziliensis were most sensitive to the treatment with C. sylvestris extract and the inhibition of infection index was 62.1%; 64.0% and 65.0% (0.5 µg/mL; 1.0 µg/mL and 5.0 µg/mL, respectively).Furthermore, IC 50 value for this extract against L. braziliensis amastigotes was very low (1.3 µg/mL), and highlighted the activity of extract against intracellular form of the

TABLE II Effect of plant extracts in promastigotes of
Leishmania species and murine macrophages.a Data are IC 50 values in μg/mL and 95% confidence intervals are in brackets. 1L.a.= Leishmania amazonensis; 2 L.m.= L. major; 3 L. b.= L. braziliensis and 4 L.c.= L. chagasi.These data represent the average of three independent experiments.AmB (amphotericin B) and miltefosine were used as reference drug.The highest concentration used of DMSO was 0.1% (v/v), which is not toxic to the parasites.parasite (Table III).In addition, Table III furnishes information about the selectivity and specificity of C. sylvestris extract.Regarding selectivity, when this value is greater than 1, the extract is more selective against the parasite than macrophages, otherwise the extract is more toxic for macrophages (Tempone et al. 2010).As can be observed, besides the toxic effect on macrophages, when the extract ANTILEISHMANIAL ACTIVITY OF BRAZILIAN PLANTS was assayed against amastigotes, it was much more destructive to parasite than to the host cells, Regarding the cytotoxicity effects on macrophages, C. sylvestris and P. macropoda were the most cytotoxic to mammalian cells (Table II).Cytotoxicity tests with natural products are important because they represent a potential source for the isolation of compounds for the development of new antiprotozoal agents (Brenzan et al. 2007).

Promastigotes
Casearia sylvestris, which is popularly named "erva-de-lagarto", "língua-de-tiú", cafezinho-domato", "corta-lengua", is distributed throughout Brazil (Ferreira el al. 2011).Pharmacological studies have highlighted the promising bioactive potential of extracts and compounds isolated from C. sylvestris, especially its antitumor (Ferreira et al. 2010), anti-ulcer (Da Silva et al. 2008), antifungal (Alves et al. 2000), antibacterial (Chiappeta et al. 1983), antioxidant (Menezes et al. 2004) and  cruzi (Mesquita et al. 2005).Phytochemical studies have attributed the high antitumor activity of C. sylvestris to the numerous secondary metabolites, especially diterpenes, which are likely based on its larger hydrophobicity and facility to penetrate across cell membrane and interact with intracellular targets (Ferreira et al. 2010).Secundary metabolites of C. sylvestris, such as alkaloids, sterols, saponins, tannins and flavonoids may be associated with significant activity against Leishmania sp.However more investigations are necessary to determine the activity of each extract component separately and in combination to ensure whether they act alone or synergistically.

CONCLUSION
In conclusion, the methanolic extracts of Casearia sylvestris and Piptocarpha macropoda showed significant leishmanicidal activity, specially C. sylvestris extract, which may be a potential source of active compounds for the development of novel therapeutic agents to treat leishmaniasis.
being four-fold more selective against the parasite.De Muylder et al. (2011) established a cut-off regarding the specificity of compounds between these two stages of the parasite Leishmania sp.Specificity value > 2 was the cutoff point chosen to define a compound as being more active against the intracellular amastigote stage; while a specificity value < 0.4 indicated a more active compound against promastigotes; compounds with specificity values between 0.4 and 2 were considered as being active against both stages.It is interesting to point out that the extract was more specific for intracellular form of L. braziliensis (specificity index= 3.8) and this result reinforces the potential leishmanicidal activity of C. sylvestris.Previously, Mesquita et al. (2005) demonstrated the antileishmanial potential of C. sylvestris against promastigotes forms of L. donovani, of which extracts displayed a significant activity with IC 50 values ranging from 0.1 to 4.9 µg/mL depending on the plant parts used.

Figure 1 -
Figure1-Effect of Casearia sylvestris extract on L. braziliensis and L. amazonensis infected macrophages.Peritoneal macrophages previously infected with L. braziliensis or L. amazonensis promastigotes in the stationary growth phase were exposed to the compounds for 72h and the results were expressed as the infection index (percentage of infected macrophages x mean number of parasites per cells).The test considered the mean of two assays performed in duplicate.Differences were regarded as significant when p < 0.0001 (***) and p < 0.001 (**).

Table I
shows the botanical name, local name, voucher specimen number and the popular uses of the plants tested.
PREPARATION OF PLANT EXTRACTS ANTILEISHMANIAL ACTIVITY OF BRAZILIAN PLANTS