Antiprotozoal Activity of Quinonemethide Triterpenes from Maytenus ilicifolia (Celastraceae)

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas’ disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC50) values of less than 0.88 nM. Both compounds showed IC50 lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas’ disease.


Introduction
The World Health Organization (WHO) estimates that more than one billion people suffer from neglected tropical diseases. Leishmaniasis is a widespread disease, affecting 12 million people around the world with about 1-2 million estimated new cases occurring every year [1]. About 8-10 million people are infected with Trypanosoma cruzi (the parasite that causes Chagas' disease), mostly in Latin America [2].
For more than 60 years, pentavalent antimonials, Sb (V) compounds, have been the first-line chemotherapy against leishmaniasis, but the emergence of resistant strains has limited their usefulness. Alternatively, amphotericin B, pentamidine isethionate, miltefosine and paramomycin are available, but their use is limited due to toxicity or high cost of treatment [3]. Benznidazole is the only drug manufactured for the treatment of Chagas' disease, but is only effective during the acute phase of the disease [4,5]. Searching for new drugs with high and specific activity is very important, especially in countries where these parasitic diseases constitute a serious public health problem [6].
The existing Brazilian biodiversity is a potential source of many unknown bioactive molecules [7] that may be studied in search for natural products with leishmanicidal and trypanocidal activities.
The friedo-nor-oleanane derivatives are members of a small group of triterpenoid natural products known as quinonemethides and are considered as chemotaxonomic indicators of the Celastraceae family [8]. They accumulate in the stem bark and root barks of some family species [9]. Many biological activities have been reported for members of this class, including antitumor [10], antioxidant [11,12] and trypanocidal [13]. Here, we present the effect of two quinonemethide triterpenes, maytenin (1) and pristimerin (2) [14], on the viability of Leishmania promastigotes and intracellular amastigotes and of T. cruzi epimastigotes. These compounds were isolated from the roots of the Brazilian plant Espinheira santa.

Maytenin (1) and Pristimerin (2): Leishmanicidal, Trypanocidal and Cytotoxic Activities against Extracellular Parasites and Macrophages
New potential leishmanicidal and trypanocidal drugs are generally screened in vitro against promastigote and epimastigote forms [15]. Although the most clinically relevant stages of Trypanosomatidae parasites are intracellular amastigotes and trypomastigotes, extracellular promastigotes and epimastigotes have the obvious advantage of being easier and cheaper to handle in the large-scale requirement of high throughput screening methods. Furthermore, promastigotes and epimastigotes have common metabolic machinery and pathways, and potential therapeutic targets effective against these extracellular forms may be relevant against the intracellular ones [16]. One of the most commonly used methods in drug screening for parasitic diseases is the microscopic counting of viable parasites, which is time-consuming, labor-intensive, and dependent on the ability of the observer [17]. For this reason, we decided to carry out a colorimetric viability assay using the tetrazolium salt MTT, which is a rapid, simple, and reliable method for evaluation of leishmanicidal and trypanocidal activities [15].
The quinonemethide triterpenes are among the secondary metabolites of interest since many of them exhibit a broad range of biological activities. In the present study we observed that compounds 1 and 2 ( Figure 1) showed potent in vitro activity against Leishmania amazonensis and Leishmania chagasi promastigotes and Trypanosoma cruzi epimastigotes. As reported in Table 1, the IC 50 values obtained for compounds (1) and (2) were 0.09 nM and 0.05 nM for L. amazonensis promastigotes and 0.46 nM and 0.41 nM for L. chagasi promastigotes, respectively. The IC 50 values for T. cruzi epimastigotes were 0.25 nM and 0.30 nM, respectively. The two compounds showed potent activity when compared to the positive controls pentamidine for L. amazonensis (IC 50 = 6.75 nM) and L. chagasi (IC 50 = 4.0 nM) and benznidazole for T. cruzi (IC 50 = 31.20 μM). The selectivity index (SI), a relevant characteristic for defining hit compounds [18], was calculated for compounds 1 and 2 by dividing their cytotoxic activity against murine macrophages (LC 50 ) by their leishmanicidal and trypanocidal activities (SI = LC 50 /IC 50 ). Macrophages were employed for SI calculation since they are the main host cell for Leishmania and because they were used for the intracellular experiments. Using the LC 50 values of 21.25 nM and 9.71 nM, which were found for murine macrophages treated with compounds 1 and 2, respectively, we obtained SIs for L. amazonensis and L. chagasi of 243.65 and 46.61 for 1 and 193.63 and 23.85 for 2, as shown in Table 1. For T. cruzi epimastigotes the SIs were 85 for 1 and 332.37 for 2. These results indicate that both compounds have good selectivity for trypanosomatid extracellular forms. It is interesting to note that L. amazonensis presented much higher sensitivity (five and eight times higher) to both compounds when compared to L. chagasi. Variations in sensitivity to several drugs among promastigotes of different species have also been reported in previous studies. Given the known biochemical and molecular differences between Leishmania species, it is not surprising that there is variation in intrinsic sensitivity to several drugs [19]. In fact, sensitivities to miltefosine varied among promastigotes of Leishmania donovani, Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania mexicana, and Leishmania panamensis. L.donovani was the most sensitive species, whereas L. major was the least sensitive species [20]. On the other hand, promastigotes of L. major were the most sensitive to tamoxifen [21].

Leishmanicidal Activity of Compounds 1 and 2 on Intracellular Parasites
Subsequently, we analyzed whether the quinonemethide triterpenes 1 and 2 had antiprotozoal activity against L. amazonensis and L. chagasi intracellular amastigotes. When infected macrophages were treated with compounds (1) and (2) (Table 1). Contrarily to the observed for promastigote forms, amastigotes of L. amazonensis were more resistant to both compounds when compared to L. chagasi (Table 1), although the differences were less pronounced for the intracellular forms than for the promastigotes. Variations in susceptibility to drugs among Leishmania species have been previously reported using amastigote-macrophage model. L. donovani and Leishmania braziliensis amastigotes were found to be three to fivefold more sensitive to sodium stibogluconate than L. major, L. tropica, and L. Mexicana [19]. Using the same model, L. mexicana amastigotes were the least sensitive to amphotericin B among six species, possibly due to variations in the type and quantity of sterols, the targets of this drug [20].

General Methods
TLC analyses were performed using Merck silica gel 60 (230 mesh) and precoated silica gel 60 PF 254 Analytical HPLC-DAD was performed on a Varian Pro Star 240, with a diode array ultraviolet detector, and the separation was achieved using a reverse phase in a Phenomenex C 18 column (250 mm × 4.6 mm). Preparative HPLC was performed on a Varian Prep-Star 400 system using a reverse phase Phenomenex C 18 (250 mm × 21.2 mm) preparative column. 1D ( 1 H, 13 C, DEPT 90° and DEPT 135°) and 2D gHMQC, gHMBC and 1 H-1 H gCOSY) NMR experiments were recorded on a Varian INOVA 500 spectrometer (11.7 T) at 500 MHz ( 1 H) and 125 MHz ( 13 C), respectively, with CDCl 3 as internal reference. Positive-ion low-resolution mass spectra were acquired on an MS ultrOTOFQ-ESI-TOF instrument (Bruker Daltonics, Raleigh, NC, USA), using MeOH-H 2 O (1:1) as solvent and a cone voltage of 40 V.

Plant Material
Three-years-old root barks of Maytenus ilicifolia was collected in district of Juruce, municipality of Jardinopolis (São Paulo State, Brazil, at 21°04'18.8'' S; 47°44'08.8'' W) in March 2010. The plant was identified by Rita Maria de Carvalho. A voucher specimen (HPM-BR 0059) has been deposited in the Herbarium of the University of Campinas, São Paulo, Brazil.

Extraction and Isolation of Quinonemethide Triterpenes from Maytenus ilicifolia
Roots (270.00 g) were frozen in liquid N 2 and mechanically pulverized in a mortar. Afterwards, the material was submitted to extraction in ultrasonic bath using dichloromethane. Solvent was removed by distillation under reduced pressure, providing a dichloromethane extract (4.9 g). An aliquot of 1.0 g of crude extract was fractionated by five 5 fractions were obtained (F1-5). The extract was then fractionated by prep. TLC and eluted with hexane-EtOAc (8:2), whereupon five fractions were obtained. After being analyzed by HPLC-DAD, fractions F2 (280 mg) and F3 (240 mg) were submitted to preparative HPLC eluted with MeOH-H 2 O (9:1, flow rate: 10 mL min −1 ; detection: 254 nm) and MeOH-H 2 O (8:2, flow rate: 12 mL min −1 ; detection: 254 nm), respectively, generating 1 (59.0 mg) and 2 (20.6 mg). Compounds 1 and 2 were characterized by comparing their 1 H-NMR, 13 C-NMR, ES-MS, and the assignments were based on 2D-NMR experiments, including gHMQC and gHMBC, and were compared with literature values [22].

Promastigotes
Cultured promastigotes at the end of the exponential growth phase were seeded at 8 × 10 6 parasites/mL in 96 well flat-bottom plates (Costar ® ). Maytenin and pristimerin were dissolved in DMSO (the highest concentration was 1.4%, which was not hazardous to the parasites, as previously accessed), added to parasite suspension to final concentrations between 1.6 µg/mL and 100 µg/mL and incubated at 28 °C for 72 h. The assays were carried out in triplicate. Pentamidine isethionate purchased from Sigma-Aldrich (St Louis, MO, USA) was used as reference drug (a 16.7 mg/mL stock solution was prepared in sterile deionized water) and added to parasite suspension to final concentrations between 1.6 and 100 µg/mL. Leishmanicidal effects were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrasodium bromide (MTT) method with modifications [23]. Absorbances were read at 490 nm. The drug concentration corresponding to 50% of parasite growth inhibition was expressed as the inhibitory concentration (IC 50 ).

Cytotoxicity on Macrophages
The cytotoxicity was evaluated on mouse peritoneal macrophages. Peritoneal macrophages were collected from BALB/c mice and were seeded in 96 well flat-bottom plates (Costar ® ) at a density of 1 × 10 5 cells/well (100 µL/well) in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum, 25 mM HEPES and 2 mM L-glutamine and incubated for 24 h at 37 °C in a 5% CO 2 -air mixture. The medium was then removed and the cells were treated with different concentrations of quinonemethides triterpenes. Positive (with pentamidine) and negative (without drugs) controls were included in each experiment. The plates were incubated under the same conditions for 24 h. Subsequently, the MTT colorimetric assay was carried out as described above. Absorbance was read in a 96-well plate reader (Robonik ® ) at 595 nm. The 50% lethal concentration (LC 50 ) was determined by logarithm regression analysis of the obtained data. The cytotoxicity for macrophages and for promastigotes was compared using the selectivity index (SI), which was determined as the ratio between LC 50 for macrophages and IC 50 for promastigotes.

Amastigotes
Peritoneal macrophages from BALB/c mice were plated at 3 × 10 5 cells/well on coverslips (13 mm diameter) previously arranged in a 24-well plate in RPMI 1640 medium supplemented with 10% inactivated FCS, and allowed to adhere for 4 h at 37 °C in 5% CO 2 . Adherent macrophages were infected with promastigotes in the stationary growth phase using a ratio of 5:1 (parasites:cell) at 37 °C in 5% CO 2 for 4 h. After that time, the non-internalized parasites were removed by washing, and infected cultures were incubated in RPMI medium or treated with different concentrations of quinonemethide triterpenes for 24 h. The cells were then fixed in a methanol solution and stained with Giemsa. The infection index was determined by multiplying the percentage of infected macrophages by the mean number of amastigotes per infected cell. The concentration that caused a 50% decrease in the infection index compared to the control was determined by regression analysis of the log-transformed data. All experiments involving mice were performed as indicated by the ethical committee of Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara (protocol number CEUA/FCF/Car n24/2012).

Evaluation of Trypanocidal Activity
All experiments were performed with T. cruzi Y strain epimastigote forms. The parasites were grown axenically at 28 °C in LIT medium supplemented with 10% fetal calf serum and harvested during the exponential growth phase (7-day-old culture forms). It was added 1 × 10 7 parasites/mL to each well of a 96-well microplate and the same volume of LIT medium was used as control. Compounds were dissolved in dimethyl sulfoxide (DMSO) and further added to the microplate for final concentrations from 1 μg/mL to 100 μg/mL. The plates were maintained at 28 °C for 72 h and MTT was performed as described above. MTT assay was prepared as described elsewhere [15]. Absorbance of samples was read at 595 nm. All assays were conducted in triplicate. The drug concentration corresponding to 50% of parasite growth inhibition was expressed as the IC 50 and was determined from sigmoidal regression of the concentration-response curves after 72 h of incubation (Benznidazole was employed as positive control). For the statistical analysis Probit´s method was employed [24].

Conclusions
The results herein presented suggest that the quinonemethide triterpenes 1 and 2 should be further considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease.