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Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae).

Introduction

Leishmaniasis comprises several diseases caused by protozoan parasites of the Leishmania genus, which are transmitted by sand-flies. This parasite has been endemic in 88 countries of four continents (Paloque et al., 2012) and has caused serious public health problems. The infection, manifests as cutaneous, mucocutaneous or visceral leishmaniasis (Vendrametto et al., 2010). The World Health Organization (WHO) considers leishmaniasis to be one of the most serious and most neglected diseases worldwide and recommends meglumine antimoniate as the first-choice treatment. This drug requires a long treatment period (Croft et al., 2006; WHO, 2015), is highly toxic and can cause serious side effects (Rodrigues et al., 2009). Second-line drugs include pentamidine and amphotericin B; however have also showed highly toxic effects. Recently, miltefosine, an alkylphosphocholine compound, was approved for visceral Leishmania infections, but teratogenic and gastrointestinal side effects have been reported (Porwal et al., 2009). Thus, there is an urgent need for safer and more efficient compounds for the treatment of leishmaniasis.

In the last years, there has been a growing interest in the therapeutic use of medicinal plants and natural products for the prevention and treatment of parasitic diseases (Ibrahim et al., 2014; Izumi et al., 2012; Batista et al., 2009; Camacho et al., 2003). Among them is Croton cajucara Benth. (Euphorbiaceae) popularly known as "sacaca" which is a plant found in the Amazonian region with a safe history of use in folk medicine. Both the bark and the leaves of C. cajucara are popularly used in teas and pills for the treatment of several diseases, including diabetes, diarrhea, stomachaches, fevers, hepatitis and malaria (Maciel et al., 2007). C. cajucara has been shown to possess anti-genotoxicity, anti-atherogenic, anti-tumor, anti-ulcerogenic, hypoglycemic, hypolipidemic, anti-estrogen, anti-inflammatory and anti-nociceptive activities (Maciel et al., 2000; 2006). The leaves of C. cajucara contain steroids and flavonoids, both as major compounds and its bark is a rich source of terpenes, such as trans-dehydrocrotonin (DCTN) and crotonin (CTN), both the clerodane-type 19-nor-diterpenes and the triterpene acetyl aleuritolic acid (AAA) (Maciel et al., 1998; 2000; 2003).

Crude methanol extract of the bark of C. cajucara and its isolated terpenes, DCTN, CTN and AAA (Fig. 1) were conducted against Trypanosoma cruzi. In these assays, the crude extract was more effective than the isolated clerodanes DCTN or CTN on trypomastigote while the trypanocidal effect of the triterpene AAA was against epimastigotes as well as on intracellular amastigotes (Campos et al., 2010).

These early results indicating the effectiveness of terpenes against T. cruzi led us to investigate the anti-leishmanial activity of C. cajucara terpenes (DCTN, CTN and AAA) obtained from the bark of this Croton species, against promastigotes, axenic and intracellular amastigotes of Leishmania amazonensis. Furthermore, the effects of DCTN and CTN on the trypanothione reductase enzyme were also investigated.

Material and methods

Plant material

The stem bark of C. cajucara was collected in the Para state (Amazonian region of Brazil) and identified by Nelson A. Rosa. A voucher specimen (no. 247) has been deposited in Herbarium of the Museu Paraense Emilio Goeldi (Belem- Brazil). In addition, the isolated tested samples were compared with voucher specimens.

Preparation of extracts. Terpenes isolation

The extraction of the powdered bark was carried out as previously reported (Maciel et al., 1998; 2003). Hexane followed by MeOH was used for extraction in a Soxhlet apparatus. After evaporation of the solvent, the hexane extract was filtered over a silica gel chromatography column, affording three fractions; A, B and C. According to the previously reported methodologies fractions B and C after submission to chromatography on a silica gel column eluted with mixtures of hexane-C[H.sub.2][Cl.sub.2]-MeOH with increasing polarity give the terpenes AAA (0.06%), CTN (0.02%) and the major DCTN (0.7%). The MeOH extract was to the filtered over a silica gel chromatography column and eluted with hexane-EtOAc at different ratios of increasing polarity, led also isolation of the compounds AAA (0.02%), and DCTN (0.2%). Quantitative purity of the tested isolated compounds was assessed by [sup.1]H and [sup.13]C 1D and 2D-NMR analyses and also by comparison with data previously reported (Maciel et al., 1998; 2003) and elemental analysis (DCTN anal. calc, for [C.sub.20][H.sub.26][O.sub.4]: C 72.79; H 7.93, found C 72.74; H 7.89. CTN anal. calc, for [C.sub.20][H.sub.28][O.sub.4]: C 72.26; H 8.49, found C 72.32; H 8.47. AAA anal. calc, for [C.sub.32][H.sub.50][O.sub.4]: C 77.06; H 10.10, found C 77.01; H 10.16).

Parasite culture

L amazonensis promastigotes MHOM/BR/77/LTB0016 strains were grown at 25[degrees]C in the Schneider medium from Sigma-Aldrich (St. Louis, MO, USA) supplemented with 20% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM of L-glutamine, penicillin at 100 U/ml and streptomycin at 100 mg/ml from Sigma-Aldrich, at pH 7.2. Cells were harvested in the late log phase, resuspended in fresh medium, counted in Neubauer's chamber, and adjusted to a final concentration of 4 x [10.sup.6]/ml. This strain has been characterized by molecular and immunological techniques (Temporal et al., 2002).

Promastigotes assays

The assays were carried out in 96-well plates in a volume of 180 [micro]l/well. The terpenes were added to a parasite culture in a concentration ranging from 150 to 9.38 [micro]g/ml solubilized in DMSO (the highest percentage used was 1.6%, v/v, which was not hazardous to the parasites). After 24 h incubation at 26[degrees]C, the remaining parasites were counted in a Neubauer chamber, and the percentage of inhibition was calculated and compared to the controls (DMSO without the drugs and with the parasites alone). The [IC.sub.50] [+ or -] SD values were calculated by linear regression from these percentages of inhibition x log [dose] using statistical error limits up to 10%. All tests were conducted in triplicate for each concentration, and three independent assays were performed. The drug pentamidine isethionate (May & Baker Lab., England) was used as reference drug.

Axenic amastigotes assays

The axenic amastigotes were obtained by promastigote culture in the Schneider medium with a pH of 7.2. After 3 days, the culture was centrifuged for 10 min at 3000 g and was resuspended in the Schneider medium at pH 7.5, and after 5 days in the log phase, the medium was changed and centrifuged in the same conditions as above. The parasite concentration was adjusted at 5 x [10.sup.5] parasites/ml using the Trypan blue dye (0.1% PBS) to determine the parasite viability, and the sample was then resuspended in the Schneider medium at pH 5.5 supplemented with 20% FCS. All procedures were completed in an ice bath and were incubated at 26[degrees]C for 10 days. At this point, the procedure was repeated in the same conditions as day 5 and was incubated at 32[degrees]C. The amastigote culture at the 16 day was ready to use and was then heat shocked for use in the appropriate assays (Castro-Pinto et al., 2004). The [IC.sub.50] [+ or -] SD values were calculated by linear regression from these percentages of inhibition x log [dose] using statistical error limits up to 10%.

Intracellular amastigotes assays

Murine peritoneal macrophages were isolated from peritoneal space of BALB/c mouse with iced RPMI-1640 medium (SigmaAldrich), supplemented with 1 mM L-glutamine, 1 M HEPES, penicillin G (105 IU-I) and streptomycin sulfate (0.10 g [L.sup.-1]). The concentration was adjusted to 2 x [10.sup.-6] macrophage/ml and incubated in a LAB-TEK chamber at 37[degrees]C and 5% C[O.sub.2] for 1 h. Non-adherent cells were removed and stationary-phase L. amazonensis promastigotes were added at a 3:1 parasite/macrophage ratio. The cultures were incubated for further 4 h, and free parasites were removed. The chambers were washed, and the monolayers were incubated with DCTN for further 24, 48 and 72 h. The cultures were fixed in methanol and stained with Instant Prov (Newprov, Curitiba, Brazil) hematological dyer, and examined under microscopy. The number of intracellular amastigotes was assessed by counting amastigotes in at least 100 macrophages per each sample and percentage of reduction of infected macrophages. The results were expressed by infection index (IF) using the following equation:

IF = % infected cells x amastigotes number/ total number of macrophages

The [IC.sub.50] [+ or -] SD values were obtained by linear regression using GraphPad Prism 5.0 using statistical error limits up to 10%. All tests were conducted in duplicate for each concentration, and two independent assays were performed.

Soluble fraction preparation of L. amazonensis

The soluble fraction (SF) was obtained from infective promastigotes of the L. amazonensis culture (Schneider medium with FCS 10%). The parasites were removed from the medium by centrifugation at 500 g/10 min (Sorvall Biofuge Stratos, Loughborough, LE, UK). The pellet was resuspended in PBS at a pH of 7.2 and was then centrifuged two more times under the same conditions. Finally, 40 mM HEPES (Sigma-Aldrich) and 1 mM EDTA (Sigma-Aldrich) was added to the final buffer. The material was lysed in a Dounce-type homogenizer and centrifuged at 12,500 g/15 min. The supernatant was considered to be the soluble fraction containing trypanothione reductase (TryR) (Castro-Pinto et al., 2007). The whole preparation of FS was carried out at 8-12[degrees]C to avoid damage to the enzyme. The protein concentration of FS was assessed using a p,Quant spectrophotometer (BiotekInstrument Inc., Winooski) at 260 and 280 nm. The concentration was expressed according to the following equation (Johnstone and Thorpe, 1982):

Protein concentration (mg/ml) = (optical density at 280 nm x 1.5) - (optical density at 280 nm x 0.75). The sample was stored at -70[degrees]C until the assays were performed.

Trypanothione reductase assay

The ability of terpenes (AAA, DCTN and CTN) to inhibit TryR activity was assessed using the equivalent of 1 mg/ml of soluble protein fraction. The terpenes, at 1 mM, were incubated with the soluble fraction for 6 min, and after 40 mM HEPES pH 7.5,1 mM EDTA, and 100 [micro]M NADPH were added, plus 100 mM trypanothione disulfide (T[(S).sub.2], Bachem), to optimize and direct the reaction to NADPH consumption by TryR. The control contained all reagents with no T[(S).sub.2] addition. The assay was initiated in the spectrophotometer (Shimadzu Corporation, Japan) at 340 nm to check the NADPH consumption. All reactions were performed at 25[degrees]C in a total volume of 300 [micro]l. The inhibition percentage was calculated based on optical density decrease (Gonzalez et al., 2005; Castro-Pinto et al., 2004, 2007; Castro et al., 2008).

Statistical analysis

Each experiment was repeated thrice, each time in quadruplicate. Significance was determined using non-paired Student's t test and one-way ANOVA. The differences were considered to be significant when p < 0.05.

Results and discussion

The C. cajucara clerodane diterpenes DCTN, CTN and triterpene AAA were evaluated for anti-leishmanial effects against L amazonensis. Promastigotes showed a higher sensitivity to clerodane diterpene DCTN with [IC.sub.50] = 12.07 dt 0.06 [micro]g/ml at 24 h of culture (Table 1). The three terpenes were then assayed with L amazonensis axenic amastigotes in 24 h of culture, showing a similar activity than against promastigotes. The most active compound was the DCTN with [IC.sub.50] = 19.98 [+ or -] 0.05 [micro]g/ml. The assays with axenic amastigotes showed a higher activity of DCTN with [IC.sub.50] = 12.50 [+ or -] 0.08 and 5.63 [+ or -] 0.07 [micro]g/ml in culture for 48 and 72 h, respectively.

Thus, after these promising results, the cytotoxicity and activity of nor-clerodane diterpene DCTN against amastigotes was evaluated in mouse peritoneal macrophage cells. Pentamidine, an anti-parasitic agent, was used as positive control and was also assayed in the same culture conditions. The macrophage cells in the presence of DCTN were not killed up to concentration of 100 [micro]g/ml after 24, 48 and 72 h of culture, in accordance with previous reports for T. cruzi infected macrophage cells (Campos et al., 2010). Additionally, DCTN has been shown to have no genotoxic or cytotoxic effects (Agner et al., 1999). The treatment of macrophage infected culture revealed that after 24, 48 and 72 h of culture; DCTN is still at non-toxic concentrations and reduced the infection with [IC.sub.50] = 0.47 [+ or -] 0.026 [micro]g/ml, [IC.sub.50] = 0.28 [+ or -] 0.06 [micro]g/ml and [IC.sub.50] = 0.16 [+ or -] 0.01 [micro]g/ml, respectively. Table 2 illustrates the [IC.sub.50] values of the effect on the proliferation of intracellular amastigotes of L amazonensis and the drug concentration required to kill all macrophage cells after 24, 48 and 72 h of treatment with DCTN and with pentamidine. The results allow us to observe that although the treatment with DCTN started with 0.47 [micro]g/ml (24 h), after 72 h a lower concentration (0.16 [micro]g/ml) was needed. Most importantly, no toxicity was observed, as compared to pentamidine (100% macrophage killed, Table 2).

Several natural products have showed to be as selective trypanothione reductase inhibitors in previous studies involving parasites of trypanosomatid class (Gallo et al., 2008; Campos et al., 2010; Macari et al., 2011). The enzyme trypanothione reductase is an important drug target in trypanosomatids because it is essential to the survival of these parasites and is involved in oxidative stress protection (Fairlamb, et al., 1985; Schmidt et al., 2002; Khan, 2007). This enzyme is NADPH dependent and catalyzes a thiol metabolism based on trypanothione, which has a part in regulated redox balance (Muller et al., 2003) and also in triggering several events responsible for oxygen reactive species neutralization (Schmidt et al., 2002). Thus, in the present study we evaluated the effect of DCTN, CTN and AAA on the trypanothione reductase of L. amazonensis. The trypanothione reductase inhibitory activity was assessed by NADPH consumption in 240 nm spectrophotometric lecture that works as trypanothione reductase co-factor of fraction soluble from L amazonensis promastigotes culture (Castro-Pinto et al., 2004, 2007; Castro et al., 2008). The obtained results of trypanothione reductase assays with the terpenes showed that these compounds interfere in the system trypanotione/trypanothione reductase (Fig. 2). The tested C. cajucara natural products were able to inhibit the enzyme activity (p < 0.01) when compared to control and to control with addition of the substrate trypanothione; however, the terpene DCTN was more effective. Future investigations of these terpenes on glutathione/glutathione reductase system present in mammal cells should be performed.

Conclusion

Clerodane diterpenes from C. cajucara showed promising in vitro antileishmanial effects against L amazonensis. DCTN clerodane diterpene presented the best profile against promastigotes, axenic amastigotes and intracellular amastigotes; furthermore, this diterpene did not present macrophage toxicity up to the assayed concentration. In addition, the action on trypanothione reductase enzyme revealed a possible mechanism of action. This is an important finding that could lead to the development of a new therapeutic agent against leishmaniasis.

http://dx.doi.org/ 10.1016/j.phymed.2015.08.012

ARTICLE INFO

Article history:

Received 23 February 2015

Revised 22 August 2015

Accepted 23 August 2015

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors thank Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) for financial support and fellowships.

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Gerson S. Lima (a,b), Denise B. Castro-Pinto (c), Gerzia C. Machado (c), Maria A.M. Maciel (d,e), Aurea Echevarria (a), *

(a) Departamento de Quimica, Instituto de Ciencias Exatas, Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ, Brazil

(b) Biomanguinhos, FIOCRUZ, Rio de Janeiro, RJ, Brazil

(c) Laboratorio de Bioquimica de Tripanossomatideos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil

(d) Universidade Potiguar Laureate International Universities, Programa de Pos-graduacao em Biotecnologia, Campus Salgado Filho, Natal-RN, Brazil

(e) Universidade Federal do Rio Grande do Norte, Instituto de Quimica, Campus Lagoa Nova, Natal-RN, Brazil

Abbreviations: Anal, calc., analysis calculated; FCS, fetal calf serum; [IC.sub.50], halfmaximal inhibitory concentration; [IC.sub.50] [+ or -] SD, half-maximal inhibitory concentration [+ or -] standard deviation; BALB/c, albino mouse laboratory-bred strain of the house mouse; IF, infection index; SF, soluble fraction; TryR, trypanothione reductase; T(S)2, trypanothione disulfide.

* Corresponding author. Tel./fax: +55 21 2682 2872.

E-mail address: echevarr@ufrrj.br, echevarr@hotmail.com (A. Echevarria).

Table 1

[IC.sub.50] values (compound concentration required to kill 50%
[+ or -] SD (a) of the par-asite) of nor-clerodane diterpenes trans-
dehydrocrotonin (DCTN) and cro-tonin (CTN), the triterpene
acetylaleuritolic acid (AAA) against Leishmania amazonensis
promastigotes and axenic amastigotes in 24 h of culture.

Compound          [IC.sub.50] ([micro]g/ml)

                  Promastigotes         Axenic amastigotes

DCTN              12.07 [+ or -] 0.06   19.98 [+ or -] 0.05
AAA                41.7 [+ or -] 0.02   41.44 [+ or -] 4.83
CTN                48.0 [+ or -] 0.09   58.25 [+ or -] 19.2
pentamidine (b)    23.1 [+ or -] 0.04   24.09 [+ or -] 0.03

(a) SD = standard deviation.

(b) Positive control.

Table 2

[IC.sub.50] values (compound concentration required to kill
50% [+ or -] SD of the intracellular amastigotes of Leishmania
amazonensis) of DCTN (trans-dehydrocrotonin) and pentamidine, and
toxic concentration to kill all macrophages in 24,48 and 72 h of
culture.

Compound      Treatment (h)   [IC.sub.50] ([micro]g/ml)
                              Intracellular amastigotes

DCTN          24              0.47 [+ or -] 0.03
              48              0.28 [+ or -] 0.06
              72              0.16 [+ or -] 0.01
pentamidine   24              0.34 [+ or -] 0.03
              48              0.25 [+ or -] 0.01
              72              0.21 [+ or -] 0.01

Compound      [LC.sub.50] ([micro]g/ml)   % Macrophage   SI (a)
              Macrophages                 killed

DCTN          >100                          0            212.8
              >100                          0            357.1
              >100                          0            625.0
pentamidine   37.50 [+ or -] 0.02         100            110.3
              18.75 [+ or -] 0.02         100             75.0
               9.38 [+ or -] 0.01         100             44.7

(a) SI = selectivity index (LCS0 (macrophages)/[IC.sub.50]
(intracellular amastigotes).
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Author:Lima, Gerson S.; Castro-Pinto, Denise B.; Machado, Gerzia C.; Maciel, Maria A.M.; Echevarria, Aurea
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Nov 15, 2015
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