Parasitol Res DOI 10.1007/s00436-013-3659-x ORIGINAL PAPER Treatment with triterpenic fraction purified from Baccharis uncinella leaves inhibits Leishmania (Leishmania ) amazonensis spreading and improves Th1 immune response in infected mice Eduardo Seiji Yamamoto & Bruno Luiz S. Campos & Márcia Dalastra Laurenti & João H.G. Lago & Simone dos Santos Grecco & Carlos E. P. Corbett & Luiz Felipe D. Passero Received: 23 September 2013 / Accepted: 16 October 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract The current medications used to treat leishmaniasis have many side effects for patients; in addition, some cases of the disease are refractory to treatment. Therefore, the search for new leishmanicidal compounds is indispensable. Recently, it was demonstrated that oleanolic- and ursolic-containing fraction from Baccharis uncinella leaves eliminated the promastigote and amastigote forms of Leishmania (Leishmania ) amazonensis and L . (Viannia ) braziliensis without causing toxic effects for J774 macrophages. Thus, the aim of the present work was to characterize the therapeutic effect of the triterpenic fraction in L . (L .) amazonensis infected BALB/c mice. Oleanolic- and ursolic acidcontaining fraction was extracted from B. uncinella leaves using organic solvents and chromatographic procedures. L. (L .) amazonensis -infected BALB/c mice were treated intraperitoneally with triterpenic fraction during five consecutive days with 1.0 and 5.0 mg/kg of triterpenic fraction, or with 10.0 mg/kg of amphotericin B drug. Groups of mice treated with the triterpenic fraction, presented with decreased lesion size and low parasitism of the skin—both of E. S. Yamamoto : B. L. S. Campos : M. D. Laurenti : C. E. P. Corbett : L. F. D. Passero (*) Laboratório de Patologia de Moléstias Infeciosas, Departamento de Patologia da Faculdade de Medicina, da Universidade de São Paulo, Av. Dr. Arnaldo, 455Cerqueira César 01246-000 São Paulo, Brazil e-mail: felipepassero@yahoo.com.br J. H. Lago Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, 09972-270 Diadema, São Paulo, Brazil S. d. S. Grecco Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-180 Santo André, São Paulo, Brazil which were associated with high amounts of interleukin-12 and interferon gamma. The curative effect of this fraction was similar to amphotericin B-treated mice; however, the final dose, required to eliminate amastigotes, was lesser than amphotericin B. Moreover, triterpenic fraction did not cause microscopic alterations in liver, spleen, heart, lung, and kidney of experimental groups. This work suggests that this fraction possesses compounds that are characterized by leishmanicidal and immunomodulatory activities. From this perspective, the triterpenic fraction can be explored as a new therapeutic agent for use against American Tegumentar Leishmaniasis. Introduction Leishmaniasis is an infectious disease caused by a protozoal parasite belonging to the Leishmania genus (Silveira et al. 2009). In the New World, there are two subgenera, and different species are capable of infecting humans and animals, causing visceral and cutaneous leishmaniasis (Rittig and Bogdan 2000; Campos et al. 2008). This diversity among species has led to the development of a variety of cutaneous lesions, ranging from a single ulcer that is able to heal spontaneously, to numerous lesions that affect the cutis and mucosa, and which do not spontaneously heal, consequently requiring specific treatments. Although different species cause the disease and induce distinct lesion phenotypes, the specific treatment used to target the disease is based on pentavalent antimonials and, as the second-line form of the drug, amphotericin B (Santos et al. 2008; Sundar and Chakravarty 2013). Pentavalent antimony is likely reduced in vivo to trivalent antimonials, which is more toxic for promastigote and amastigote forms of the parasite (Frézard et al. 2009). Parasitol Res Although this treatment has long been used, the mechanism of action is still regarded as controversial, and some studies have indicated that different biochemical pathways of the parasite can be affected. It is possible that the inhibition of ATP detected in Leishmania sp. at the intracellular level can be a direct result of antimony interference in glycolysis. Moreover, some alterations in the transporters of nucleosides may also result from the binding of antimony to the ribose moiety, forming stable complexes with adenine and thus interfering with the nucleoside transporters (Aït-Oudhia et al. 2009). Antimonials also seem to interfere with enzymes associated with DNA replication, such as DNA topoisomerase (Walker and Saravia 2004). Amphotericin B is a polyene macrolide antibiotic, which is recommended when antimonial treatment fails. This antibiotic has a great affinity for ergosterol, the major cell membrane sterols present in Leishmania parasites (Saha et al. 1986). Thus, amphotericin B forms a complex with membrane sterols and alters their permeability through the uncontrolled loss of ions by forming barrel-shaped transmembrane pores, leading to cell lysis (Mishra et al. 2007). In spite of the undeniable leishmanicidal activity observed among both drugs, patients frequently present with many side effects during treatment. These side effects include local pain and systemic effects such as nausea, vomiting, weakness, myalgia, abdominal colic, diarrhea, skin rashes, hepatotoxicity, nephrotoxicity, and cardiotoxicity (Rath et al. 2003); each of these undesired effects leads patients to abandon treatment. Therefore, it is crucial that compounds with lesser toxicity profiles and stronger leishmanicidal effects be found. Plants possess diverse secondary metabolites that display a great array of pharmacological activity, with the potential to be used against leishmaniasis (Brenzan et al. 2007; Passero et al. 2013). The Baccharis genus (Asteraceae) is characterized by a broad array of microbicidal activity (Muelas-Serrano et al. 2000), which can be associated with the diversity of its compounds, such as triacontane derivatives, diterpenoids, triterpenoids, flavonoids, and chromenes (Bohlmann et al. 1979; Labbe et al. 1986; Zdero et al. 1988; Jarvis et al. 1991; Verdi et al. 2005). Studies have shown that the flavonoids, lactone and triterpenoids, from Baccharis dracunculifolia and Baccahris retusa (Silva et al. 2009; Grecco et al. 2012) were active against different forms of promastigote and amastigote Leishmania sp. pointed out for the chemotherapeutical potential of compounds present in this genus. Recently, Passero et al. (2011) demonstrated that the fraction containing the triterpenes (oleanolic and ursolic acids) purified from B . uncinella was not toxic for the J774 macrophages and presented activity against the amastigote forms of Leishmania (Leishmania ) amazonensis and Leishmania (Viannia ) braziliensis . Furthermore, the incubation of infected macrophages treated with high triterpene fraction concentrations increased the production of nitric oxide, suggesting that in addition to having a direct effect on the amastigote forms, these triterpenes triggered a possible immunostimulatory mechanism within the infected cells. Thus, the aim of the present work was to analyze the therapeutic effect of this fraction on the treatment of experimental American Tegumentar Leishmaniasis caused by L. (L.) amazonensis. Materials and methods General Silica gel (230–400 mesh; Merck Corporation, Whitehouse Station, NJ, USA) was used for column chromatographic separation, while silica gel 60 PF254 (Merck Corporation) was used for analytical (0.25 mm) thin layer chromatography (TLC). All chemicals employed were of analytical reagent grade. Nuclear magnetic resonance (NMR) spectra (500 MHz) were recorded on a Bruker model DRX-500 NMR spectrometer (Bruker Corporation, Rheinstetten, Germany) with chloroform-d (CDCl3) as a solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) were recorded in parts per million (ppm) relative to TMS. Plant material Aerial parts of Baccharis uncinella DC. were collected in the “Campos de Altitude” region of the Campos do Jordão, São Paulo, SP, Brazil, in June 2005. The plant was authenticated by Dr Oriana A. Fávero, and a voucher specimen was deposited at the herbaria of the Prefeitura Municipal de São Paulo (PMSP) with the reference number PMSP8983. Extraction and fractionation The dried and powdered aerial parts of B. uncinella (430 g) were exhaustively extracted with MeOH (5×1 L) at room temperature, and the resulting solutions were filtered and concentrated under vacuum to obtain the MeOH extracts (25.1 g). This crude extract was dissolved in MeOH/H2O (1:2 v/v; 500 mL) and partitioned with n-hexane. After the parts were dried over Na2SO4 and the solvent was evaporated under reduced pressure, 18 g of the n-hexane phase were obtained. Part of this material (8.3 g) was subjected to SiO2 column chromatography and was eluted with increasing amounts of EtOAc in n-hexane to give 77 fractions, which were then pooled together in six groups (I–VI) after TLC analysis. Group IV was composed of a triterpenic fraction Parasitol Res (715 mg), which were characterized as oleanolic and ursolic acids. Animals and parasites Eight-week-old male BALB/c mice obtained from the Animal Facility of the School of Medicine of São Paulo University (FMUSP), Brazil, were maintained in the laboratory during the experiments, in accordance with the institutional guidelines regarding the welfare of experimental animals and with the approval of the Animal Ethics Committee of São Paulo University (322/12). L . (L.) amazonensis (MHOM/BR/1973/M2269) parasites were isolated from a patient in Pará state, north of Brazil, who presented with anergic diffuse leishmaniasis. The parasite was identified using monoclonal antibodies and isoenzyme electrophoretic profiles at the Leishmaniasis Laboratory of the Evandro Chagas Institute (Belém, Pará state, Brazil). This parasite was grown in Roswell Park Memorial Institute-1640 medium—RPMI 1640 (Gibco®, Life Technologies, Carlsbad, CA, USA), supplemented with 10 % heat-inactivated fetal bovine serum, 10 μg/mL of gentamicin, and 1,000 U/mL of penicillin (R10) at 25 °C. Promastigote forms in the stationary phase of growth were used. Infection and treatment BALB/c mice were infected subcutaneously into the hind footpad with 106 promastigote forms of L. (L.) amazonensis. Fifteen days after infection, the groups of mice were treated intraperitoneally during five consecutive days with 1.0 mg/kg and 5.0 mg/kg of triterpenic fraction purified from B. uncinella. In addition, one group was treated with 10 mg/kg of amphotericin B using the same protocol of triterpene treatment. Control mice (infected) were injected intraperitoneally only with vehicle solution (phosphate-buffered saline; PBS). Healthy mice were injected subcutaneously and intraperitoneally with PBS. At 5 weeks postinfection (PI), animals were killed, and the skin parasitism, cellular immune response, and histological alterations of the internal organs (heart, lung, liver, spleen, and kidneys) were analyzed. Parasite load The parasite load in the skin lesion was determined using the quantitative limiting-dilution assay, as previously described (Passero et al. 2010). Briefly, the infected footpad fragments from the different groups were aseptically excised and were homogenized in Schneider’s medium. The cellular suspensions were subjected to 12 serial dilutions with four replicate wells. The number of viable parasites was determined based on the highest dilution that promastigotes could be grown after 10 days of incubation at 25 °C. Analysis of cellular immune response The popliteal lymph nodes of all groups were collected and macerated, and the cells were counted under Trypan blue exclusion. The cell suspensions (2×105/well) were cultured in R10 under stimulation with 5.0 μg of whole antigen of L. (L.) amazonensis or 1.0 μg of concanavalin A, which was used as a positive control. After 72 h, the supernatants of the different groups were collected, and the amounts of interleukin (IL)-4, IL-12, and interferon gamma (IFN-γ) (BD, Franklin Lakes, NJ, USA) were quantified by sandwich enzyme-linked immunosorbent assay in accordance with the manufacturer’s recommendations. Statistical analysis The results were expressed as the mean ± standard deviation of three independent experiments and the nonparametric Mann-Whitney U test was used to compare lesion size, parasite load and cytokine production between the groups. Differences were considered statistically significant at a 5% significance level (p < 0.05). GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA,USA) was used to analyze the results. Differences were considered statistically significant when p< 0,05. Results Nuclear Magnetic Resonance The n-hexane phase of the MeOH extract from the aerial parts of B . uncinella was subjected to several chromatographic steps to yield a fraction that was analyzed by TLC, revealing one homogeneous spot. Its 1H-NMR spectrum (CDCl3) displayed several overlapping signals at δ2.3–0.8, which were characteristic of aliphatic hydrogens, as well as two signals at δ 3.3 and 2.6, which were characteristic of allylic H-18 hydrogen of oleanane/ursane triterpenoids (Kontogianni et al. 2009). Analyzing the integration values of these signals, it is possible to establish that the proportion of oleanane/ursane is 6:1. The 13C and DEPT 135° NMR spectra of the mixture revealed differences in the chemical shifts of the olefinic carbons C-12 (δ123.2/125.8) and C-13 (δ144.8/138.0), as well as differences in the signals attributed to the methyl groups C-18 (δ42.6/52.7), C-19 (δ47.3/38.7), and C-20 (δ 31.4/38.4). Therefore, comparisons of these NMR data with those reported in the literature (Seebacher et al. 2003) allowed for the identification of oleanolic and ursolic acids in the triterpenic fraction from the aerial parts of B. uncinella. Parasitol Res Lesion size, parasite load, and histological alterations The infected group presented with an increased lesion size between 2 and 5 weeks PI, although mice treated with 5.0 mg/ kg of the triterpenic fraction showed smaller sized lesions from 2 to 5 weeks PI (P <0.05), while mice treated with 1.0 mg/kg of the triterpenic fraction presented with a moderate lesion size from 2 to 5 weeks PI (P <0.05). Treatment with amphotericin B also inhibited the growth of cutaneous lesions in mice (Fig. 1). The parasitism in mice treated with 1.0 and 5.0 mg/kg of the triterpenic fraction and 10 mg/kg of amphotericin B presented with lesser skin parasitism when compared to the infected and untreated group. In addition, the group treated with 5.0 mg/kg of the triterpenic fraction presented with a significantly reduced parasite load in the skin compared to groups treated with 1.0 mg/kg of the triterpenic fraction and amphotericin B (Fig. 2). In addition, treatment with 1.0 and 5.0 mg/kg of the triterpenic fraction did not alter the architecture of the liver, heart, lungs, and kidneys when compared to infected mice as showed in Fig. 3. In addition, it was verified that the treatment with 1.0 and 5.0 mg/kg of the triterpenic fraction increased the regions of white pulp when compared to infected infected mouse spleen, suggesting an immunomodulatory action of triterpenic acids. Analysis of immune response in BALB/c mice BALB/c mice treated with 1.0 and 5.0 mg/kg of the triterpenic fraction presented with significant increases in IL-12 (Fig. 4b) and IFN-γ (Fig. 4a) levels as compared to mice treated with amphotericin B and the control groups (P <0.05). The levels of IL-4 (Fig. 4c) observed in the triterpenic fraction-treated groups were similar to those of infected mice; however, Fig. 1 Lesion developments in mice subjected to treatment with 1.0 and 5.0 mg/kg of the triterpenic fraction purified from B. uncinella leaves. *P <0.05 compared to the lesion size of mice treated with the triterpenic fraction and amphotericin B. **P <0.05 compared to the lesion size of mice treated with amphotericin B and 5.0 mg/kg of the triterpenic fraction Fig. 2 Analysis of the parasite load in the skin of the different experimental groups. *P <0.05 compared to skin parasitism of the infected group; •P <0.05 compared to skin parasitism of the groups treated with 1.0 mg/kg of triterpene and amphotericin B treatment with amphotericin B caused a decrease in the levels of IL-4 compared to infected mice (P <0.05). Discussion A previous study conducted by Passero et al. (2011) demonstrated that a fraction containing oleanolic and ursolic acids was not cytotoxic for J774 macrophages, and it eliminated the intracellular amastigote of L. (L.) amazonensis and L . (V.) braziliensis with the participation of nitric oxide. In the present manuscript, the therapeutic efficacy of this fraction was evaluated in vivo using a murine model of American Tegumentar Leishmaniasis. The group of infected mice that received treatment with 1.0 or 5.0 mg/kg of the triterpenic fraction demonstrated an inhibition of lesion size, which was accompanied by the elimination of skin parasitism. This effect can be associated with the direct effect of oleanolic and ursolic acids on the promastigote and amastigote forms of L. (L.) donovani, L. (L .) amazonensis , and L . (V.) braziliensis , as previously indicated (Peixoto et al. 2011; Passero et al. 2011; Musayeib et al. 2013). Although these studies examined the antileishmanial activity of triterpenes in vitro, none of the work to date has characterized the therapeutic potential of these triterpenes in leishmaniasis in vivo. Therefore, oleanolic and ursolic acids triterpenes possess therapeutic effects, which were similar to those found after administering amphotericin B. Although similar protective effects had been detected in the triterpene- and amphotericin B-treated groups, the total dose administrated to decrease parasitism was different, as exemplified by the fact that in order to inhibit skin parasitism to 86 %, a total dose of 1.25 mg of amphotericin B needed to be injected in mice, while to inhibit approximately 93 % of the parasitism, a total dose of 0.625 mg of the triterpene fraction was required (in mice treated with 5.0 mg/kg of triterpenic Parasitol Res Fig. 3 Histological analysis of spleen, liver, heart, lung, and kidney of mice submitted to the treatment with triterpenic fraction. Magnification: ×20 fraction). These findings indicated that the most effective or selective compounds used to treat cutaneous leishmaniasis will demand lower concentrations and/or fewer cycles of drug administration. In addition, mice treated with the triterpenic fraction did not present with any alterations to their heart, lungs, spleen, liver, or kidneys. This result was expected, as previous works have demonstrated that oleanolic and ursolic acids are nontoxic among experimental animals and humans (Liu 1995; Liu 2005). In addition, these triterpenes have been used as a component in a number of agents, serving Fig. 4 IFN-γ (a ), IL-12 (b ), and IL-4 (c ) levels produced by mononuclear cells from infected mice, as well as from mice receiving 1.0 mg/kg of the triterpenes, 5.0 mg/kg of the triterpenes, and amphotericin B. *P <0.05 compared to the cytokine production of the infected group; •P <0.05 compared to the cytokine production of the groups treated with 1.0 and 5.0 mg/kg of the triterpenes Parasitol Res different purposes in Asiatic countries (reviewed in Liu 1995), highlighting the possibility that this class of compounds might be of interest in a number of drug developments (Ferreira et al. 2010). Studies in the area of oncology also demonstrated that oleanolic and ursolic acids are highly selective for cancer cells when compared to normal cells (Lúcio et al. 2011; Sannigrahi et al. 2012), reinforcing the absence of toxicity of these triterpenes to mammals. In leishmaniasis, an elevation in the Th1 cytokines is essential for eliminating intracellular amastigotes and for developing resistance, as IL-12 can trigger the differentiation and activation of the subset of CD4+ T-lymphocytes that secrete IFN-γ, which, in turn, activate the infected macrophages, consequently eliminating intracellular parasites (Rocha et al. 2007; Ota et al. 2008; Kaur et al. 2008). After treatment with the triterpene fraction, high amounts of both of these cytokines could be detected, suggesting that in addition to the direct effect they exert on Leishmania parasites, these compounds could modulate immune cells to produce high concentrations of these beneficial cytokines (Masihi 2003). It is possible that these triterpenes, once administered in infected mice, can alter some immunological pathways and increase Th1 cytokines. Conversely, treatment with amphotericin B did not stimulate the production of IL-12 and IFN-γ; however, diminished concentrations of IL-4 were verified after treatment. The IL-4 cytokine is associated with the Th2 immune response and, consequently, with disease progression (Nakaya et al. 2011; Passero et al. 2012; Carvalho et al. 2012). Therefore, the low levels of IL-4 observed among amphotericin B-treated mice may be important in reducing the pathology, which was mediated by the amastigote forms. In triterpene-treated and infected groups, similar levels of IL-4 have been found; however, the presence of this cytokine did not interfere with the production of IL-12 and IFN-γ. In addition to their leishmanicidal effect, these data indicate that oleanolic and ursolic acids can beneficially modulate the immunological response of BALB/c mice. 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