Leishmanicidal activity of the alkaloid-rich fraction from Guatteria latifolia

Experimental Parasitology 172 (2017) 51e60

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Leishmanicidal activity of the alkaloid-rich fraction from Guatteria latifolia ~o d, C. Ferreira a, 1, C.L.A. Passos a, 1, D.C. Soares a, K.P. Costa c, M.J.C. Rezende c, A.Q. Loba A.C. Pinto c, L. Hamerski b, *, E.M. Saraiva a, ** es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Instituto de Microbiologia Paulo de Go Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d ^nica, Departamento de Biologia Geral, Universidade Federal Fluminense, Universidade Federal Fluminense, Nitero i, RJ, Brazil Setor de Bota a

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


The crude branches extract (GCE), subfractions 1 and 2 (GF1, GF2) of G. latifolia have anti-Leishmania amazonensis activity.
GCE, GF1 and GF2 decrease NO production in IFN-g and LPS-stimulated macrophages.
GF2 decrease TNF-a production in IFN-g and LPS-stimulated macrophages.
GCE, GF1 and GF2 affected the cell cycle of promastigotes without changes in the mitochondrial membrane potential.
Puterine, oxoputerine and lysicamine are presents in GF1 and, in the GF2 only oxoputerine and lysicamine are present.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 March 2016 Received in revised form 12 December 2016 Accepted 18 December 2016 Available online 21 December 2016

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania and includes cutaneous, mucocutaneous and visceral clinical forms. The drugs currently available for leishmaniasis treatment are pentavalent antimonials, amphotericin B and miltefosine, which present high toxicity, elevated cost and development of parasite resistance. The natural products constitute an important source of substances with leishmanicidal potential. Here we evaluated in vitro the anti-Leishmania amazonensis activity of crude extracts of branches, leaves and fruits of Guatteria latifolia. The branch extract (GCE) exhibited promising leishmanicidal activity against promastigotes (IC50 51.7 mg/ml), and was submitted to fractionation guided by in vitro assays. Among the seven subfractions obtained, GF1

Keywords: Guatteria latifolia

es, Universidade * Corresponding author. Instituto de Microbiologia Paulo de Go Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Bloco D, sala D1-44, Ilha do Fund~ ao, Rio de Janeiro, RJ, 21941-902, Brazil. ** Corresponding author. Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Bloco H, Sala SS-23, ~o, Rio de Janeiro, RJ, 21941-902, Brazil. Ilha do Funda E-mail addresses: [email protected] (L. Hamerski), [email protected] (E.M. Saraiva). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.exppara.2016.12.014 0014-4894/© 2016 Published by Elsevier Inc.

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and GF2 were the most actives against promastigotes with IC50 25.6 and 16 mg/ml, respectively. Since GCE, GF1 and GF2 were not toxic for macrophages, next, we tested their effect on intracellular amastigotes, and the IC50 values obtained were, respectively 30.5, 10.4 and 7.4 mg/ml, after 24 h treatment. The selectivity index for GCE, GF1 and GF2 were >6.5, >19.2 and > 27, respectively. Additionally, GCE, GF1 and GF2 affected the division pattern of the promastigotes by increasing 6.7, 9.4 and 7-fold the cells in SubG0/G1 phase, and decreasing 1.6, 2.5 and 1.8-fold the cells in G0/G1 phase, respectively. To assess the GCE and GFs capacity to modulate microbicidal mechanisms of macrophages, nitric oxide (NO) and TNF-a production were tested. Our results indicated that at the IC50s GCE, GF1 and GF2 decreased NO production of infected macrophages stimulated with IFN-g and LPS, besides, only GF1 decreased the production of TNF-a. Our data warrant further studies of GCE, GF1 and GF2 to identify active compounds against Leishmania parasites. © 2016 Published by Elsevier Inc.

1. Introduction Leishmaniasis is a public health problem that affected 98 countries with approximately 1.7 million cases worldwide each year (Alvar et al., 2012). The drugs currently available for leishmaniasis treatment are pentavalent antimonials, amphotericin B and its lipid formulations, pentamidine and miltefosine (Croft et al., 2006; Ouellette et al., 2004). However, these drugs need to be administered by intravenous infusion and the treatment has poor patient compliance because many of them require daily systemic administration for long periods. These drugs are toxic and they have side effects such as chills, fever, thrombophlebitis, myocarditis, nephrotoxicity and ultimately death (Andrews et al., 2014; Singh et al., 2014; Croft et al., 2006; Ouellette et al., 2004). Miltefosine is the first oral drug approved for treatment of leishmaniasis in India. However, it has been reported, its low efficacy against nchezcutaneous leishmaniasis, besides its teratogenicity (Sa ~ ete et al., 2009; Sindermann and Engel, 2006). Moreover, the Can emergence of drug-resistant strains of Leishmania sp. is rapidly increasing worldwide. Therefore, there is an urgent need for new ncheztherapies against leishmaniasis (Andrews et al., 2014; Sa ~ ete et al., 2009; Sindermann and Engel, 2006). Can Different studies have demonstrated that natural products are a promising source to search for novel drugs for the treatment of neglected tropical diseases such as leishmaniasis. Actually, leishmanial activity were already reported for genera (families) such as Peschiera (Apocynaceae) (Delorenzi et al., 2001), Pourouma (Moraceae) (Torres-Santos et al., 2004), Kalanchoe (Crassulaceae) (Muzitano et al., 2006), Baccharis (Asteraceae) (Tempone et al., 2008), Physalis (Solanaceae) (Guimar~ aes et al., 2009), Piper (Piperaceae) (Ferreira et al., 2011), Copaifera (Fabaceae) (Soares et al., 2013), Croton (Euphorbiaceae) (Lima et al., 2015), Lippia (Verbenaceae) (Funari et al., 2016), and Serjania (Sapindaceae) (Passos et al., 2017), among several others (reviewed by Singh et al., 2014; Oliveira et al., 2016; Ullah et al., 2016). Among the Annonaceae species many secondary metabolites displaying antileishmanial activity were isolated (Siqueira et al., 2015; Montenegro et al., 2003; Vila-Nova et al., 2011). The family Annonaceae has about 135 genus and 2500 species of plants. In Brazil, the Guatteria genus is the most abundant with 88 species, from which 47 are endemic ~o et al., 2012; Loba ~o and Mello-Silva, 2007). Although (Loba considered endangered G. latifolia is a prevalent species in Minas ~o and Mello-Silva, 2007), Gerais and in Rio de Janeiro states (Loba for which any biological or chemical studies have been done. Previous phytochemical investigations of other species of this genus have revealed significant biological activities such as antimicrobial (Costa et al., 2010), antitumor (Fontes et al., 2013), antioxidant and antiparasitic (Mahiou et al., 2000). In the present investigation, we report that a bioassay-guided

fractionation of crude branches extract of G. latifolia led to the isolation of alkaloids rich fractions. In vitro activity of these fractions was assessed against promastigote and intracellular amastigote forms of Leishmania amazonensis, as well as for some macrophage toxicity and microbicidal mechanisms.

2. Materials and methods 2.1. Ethics statement All animal experiments were performed in strict accordance with the Brazilian animal protection law (Lei Arouca number 11.794/08) of the National Council for the Control of Animal Experimentation (CONCEA, Brazil). Animals were housed in a temperature-controlled room (24  C), with a 12 h light-dark cycle, with food and water ad libitum in mini-isolators (Alesco Brasil). This study protocol was approved by the Committee for Animal Use of the Universidade Federal do Rio de Janeiro (Permit Number: 128/ 15).

2.2. Plant material Guatteria latifolia was collected on October 27, 2011, in Itatiaia National Park, at Serra da Mantiqueira, Rio de Janeiro state. Species ~o (Uniidentification was performed by Dr. Adriana Quintella Loba versidade Federal Fluminense, Rio de Janeiro, Brazil). The voucher specimen (number RB 518836) was deposited in the herbarium of ^nico of Rio de Janeiro. the Jardim Bota

2.3. Extracts and fractionation procedures Branches, leaves and fruits were dried at 40  C for 48 h, and then extracted with ethanol and water 9:1 (v/v) for six days with five solvent changes (first 48 h extraction was followed by four 24 h extractions). The ethanol/water extracts obtained after evaporation of solvent in vacuum were stored at 70  C until analyzed. Since only the crude branches extract (GCE) showed leishmanicidal activity, this extract (3.0 g) was suspended in 400 ml of distilled water using ultrasound (Bransone1510) for 15 min. The aqueous solution was partitioned with the same volume of n-butanol (Tedia, Brazil) for three times. The n-butanol (m ¼ 1400 g) phases was chromatographed over C-18 (reverse phase) column. Gradient elution was carried out with methanol/water (10e100% of methanol) and provided seven subfractions (GF1 to GF7). The subfractions GF1 (335.5 mg), GF2 (384.0 mg), GF3 (372.2 mg) and GF5 (134.2 mg) were tested for leishmanicidal activity. The subfractions GF4, GF6 and GF7 were not tested because of the low mass obtained.

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2.4. Liquid chromatography-mass spectrometry (LC-MS) analysis Liquid chromatography-mass spectrometry (LC-MS) analysis was carried out on an LCeMS/MS system consisting of a Shimadzu LC-20AD equipped with a Shimadzu UV/VIS SPD-M20A DAD detector (Shimadzu Corporation, Kyoto, Japan) coupled to an microTOF II -ESI-TOF Mass Spectrometer (Bruker Daltonics, Bremen, Germany) using a phenyl-hexyl column (5 mm, 250  4.6 mm; Luna, Phenomenex, USA) at 30  C. The mobile phase consisted of acetonitrile and 0.05% acetic acid aqueous solution with isocratic elution 27% ACN. The flow rate was set at 0.7 ml/min, the injection volume was 20 ml and the detection wavelength was set at 254 nm1. The ion spray voltage was set at 4.5 KV for positive ionization, and the heater gas temperature was 310  C. Nitrogen was used as a nebulizing gas (50 psi), auxiliary gas (60 psi) and curtain gas (10 psi). The working solutions of fractions (1.0 mg/ml) were prepared by dissolving each one in methanol and filtered in a 0.45 mm cellulose filter.

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promastigotes using 2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]2H-tetrazolium-5-carboxanilida (XTT, Sigma) method as described (Ferreira et al., 2011). Briefly, promastigotes were cultivated in medium containing 1, 10, 50 and 100 mg/ml of GCE and GFs for 48 and 96 h at 26  C and then incubated with XTT activated with phenazine methosulfate (PMS, Sigma) for 3 h. The reaction product was read at 450 nm in a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices). 2.7. Cytotoxicity for host macrophages Mice peritoneal macrophages adhered in 96-well plates were treated with the GCE and GFs for 24 h, and cell viability was determined using 1 mg/ml XTT (2,3-Bis[2-Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxinilide inner salt, Sigma) and 200 mM PMS (phenazine methosulfate). After 3 h incubation, the reaction product was read at 450 nm. Results are expressed in percentage of viable cells compared to untreated control (Scudiero et al., 1988; Roehm et al., 1991).

2.5. Parasite culture L. amazonensis (WHOM/BR/75/Josefa) promastigotes were cultured at 26  C in Schneider's insect medium (Sigma), 10% fetal calf serum (Gibco, MD, US.) and 20 mg/ml of gentamycin (ScheringPlough, Rio de Janeiro, Brazil). 2.6. Anti-promastigote activity Initially, crude fruits, leaves and branches extracts were tested at 50 mg/ml for the screening of the leishmanicidal activity in

2.8. Anti-amastigote activity Mice peritoneal macrophages obtained after stimulation with 3% thioglycollate for 3 days were harvested in RPMI 1640 medium (LGC Biotec, Brazil) and cultured inside 24-well plates for 2 h adherence at 35  C, 5% CO2. Non-adherent cells were removed, and macrophages were incubated overnight as above. Adhered macrophages were infected with L. amazonensis promastigotes (stationary growth phase) at a 10:1 parasite/macrophage ratio during 1 h at 35  C, 5% CO2. Free parasites were washed out with 0.01 M

Fig. 1. Effect of Guatteria latifolia and subfractions on Leishmania amazonensis. Promastigotes were cultured with 50 mg/ml of crude extracts of branches, leaves, fruits or 1% DMSO (vehicle), during 48 h and 96 h (A), and for 48 h with 1, 10, 50 and 100 mg/ml of (B) GCE, (C) GF1, (D) GF2, (E) GF3 or (F) GF5, and parasite viability checked by the XTT assay. Results were expressed as the percentage of promastigotes survival compared to the untreated control (CTRL, 100%) and are shown as the mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001, compared to control. GCE e G. latifolia crude extract, GF e G. latifolia subfractions.

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Fig. 2. Safety for macrophages of Guatteria latifolia (GCE) and subfractions (GFs). Peritoneal macrophages were incubated for 24 h with the indicated concentrations of (A) GCE, (B) GF1, (C) GF2, (D) GF3, (E) GF5 and 1% DMSO (vehicle) at 37 C/5% CO2, and cell viability was evaluated by XTT assay. Data from 3 independent experiments are expressed as % of viable cells in relation to control.

phosphate buffered saline (PBS), and the cultures maintained for 24 h at 35  C, 5% CO2. Infected macrophage cultures were treated with different concentrations of the GCE and GFs for an additional 24 h at 35  C, 5% CO2. Cultures were then PBS washed and incubated with 0.01% sodium dodecyl sulfate for 10 min followed by 1 ml of Schneider's medium supplemented with 10% FCS, and maintained at 26  C for 2 days. The relative intracellular load of viable L. amazonensis amastigotes was measured, after promastigote transformation, using Alamar Blue (Invitrogen), according to the manufacturer's instructions. After 4 h of incubation, the fluorescence was read at 540/610 nm excitation/emission in a SpectraMax Paradigm (Molecular Devices). 2.9. Selectivity index calculation The selectivity index (SI) was calculated by dividing the CC50 value obtained in macrophages by the IC50 in L. amazonensis intracellular amastigotes as described (Passos et al., 2015). 2.10. Nitric oxide production Thioglycollate-stimulated peritoneal macrophages obtained as above (106 cells/well in 24-well plates) were activated with 0.1 mg/ ml LPS þ 0.5 mg/ml IFN-g (both from Sigma) or left untreated for 24 h at 37  C, 5% CO2. Cultures were then treated overnight with 30.5 mg/ml of the GCE and 10.4 mg/ml of GF1 and 7.45 mg/ml of GF2. Nitrite concentrations in the culture supernatants were determined by the Griess method as described (Ferreira et al., 2011).

2.11. Nitric oxide-trapping capacity A cell-free system with a NO donor was used to test the capacity of GCE and CFs to trap NO. SNAP (s-nitroso n-acetyl DLpenicillamine, Sigma) liberates nitric oxide in solution, which is then transformed to nitrite in the medium. The addition of a NO scavenger to the SNAP solution results in nitrite decay in the supernatant. Using this protocol, 30.5 mg/ml of the GCE and 10.4 mg/ml of GF1 and 7.45 mg/ml of GF2 were incubated with 1 mM SNAP. Rutin (1 mM, Sigma), a known NO scavenger, was used as a positive control. After 6 h of incubation, the nitrite concentration was determined using the Griess method as described (Ferreira et al., 2011). 2.12. Cytokine production Thioglycollate stimulated peritoneal macrophages cultured in 24-well plates were infected with L. amazonensis promastigotes at a 10:1 parasite/macrophage ratio as described above. Next, the cells were activated with IFN-g and LPS as described above or left untreated and then incubated with 30.5 mg/ml GCE, 10.4 mg/ml GF1, 7.45 mg/ml GF2. TNF-a production was evaluated by ELISA according to the manufacturer's instructions (Ebioscience, CA, US), in assays performed in triplicate (Ferreira et al., 2014). 2.13. Cell cycle analysis Promastigotes were incubated in Schneider's complete medium, with or without 51.7 mg/ml GCE, 25.6 mg/ml GF1, 16 mg/ml GF2 and

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Fig. 3. Leishmanicidal effect of Guatteria latifolia (GCE) and subfractions (GFs) on intracellular amastigotes. Leishmania-infected macrophages were left untreated or were treated with (A) GCE, (B) GF1, (C) GF2, (D) GF3 and (E) GF5 at the indicated concentrations. After 24 h at 5% CO2 35  C, cultures were washed, fed with Schneider's medium and cultured at 26  C. The macrophage parasite load was evaluated after 48 h in culture by measuring the transformed promastigotes using the XTT assay. The results represent the mean ± SEM of 3 experiments performed in triplicate. *P < 0.05, **P < 0.001, ***P < 0.0001, compared to control.

12.23 mg/ml Miltefosine for 48 h. The cells were washed with PBS, fixed in 70% (v/v) ice-cold methanol/PBS for 1 h at 4  C, washed again once with PBS and then incubated in PBS supplemented with 10 mg/ml propidium iodide (PI) and 20 mg/ml RNAse at 37  C for 45 min, according to Ambit et al. (2008). For each sample, 10,000 events were collected on a BD FACScalibur (Becton and Dickson) and analyzed using CellQuest software.

leaves and fruits extracts at 50 mg/ml against L. amazonensis promastigotes. Our results have shown that only the crude branches extract (GCE) was active with 54 and 37% of viable parasites after 48 and 96 h treatment, respectively (Fig. 1A). Next, we tested the IC50s of GCE and its fractions, which were 51.7 mg/ml for GCE, 25.6 mg/ml for GF1 and 16 mg/ml for GF2 after 48 h treatment (Fig. 1BeD). GF3 and GF5 were not active against the parasites (Fig. 1EeF).

2.14. Measurement of the mitochondrial membrane potential (DJm)

3.2. Host cell toxicity

Promastigotes were treated or not treated with 51.7 mg/ml GCE, 25.6 mg/ml GF1, 16 mg/ml GF2 for 24 h and then incubated with a JC1 solution (5 mg/ml, Sigma) for 20 min at 37  C according to the manufacturer's instructions. DJm was measured in 96-well opaque plates using 490/530 nm excitation/emission (JC-1 monomers) and 525/590 nm excitation/emission (J-aggregates) in a SpectraMax Paradigm (Molecular Devices), as described (Ferreira et al., 2011).

In the mammalian host Leishmania is an intramacrophage parasite, thus it is important to check the safety of GCE and GFs on these host cells. The XTT method was used to evaluate macrophage viability, and our results indicated that GCE, GF1 and GF2 were not toxic for macrophages even at the highest concentrations tested (Fig. 2). Dimethyl Sulfoxide (DMSO) was used as the samples diluent, it was not toxic to the cells too. 3.3. Anti-amastigote activity

3. Results

Following, we assessed the anti-amastigote activity after 24 h treatment of Leishmania-infected mice peritoneal macrophages with different concentrations of GCE and GF-fractions. Our results had shown IC50s values of 30.5, 10.4 and 7.4 mg/ml, respectively for GCE, GF1 and GF2. Similarly, to the promastigotes, GF3 and GF5 were also not effective for amastigotes (Fig. 3). The selectivity index (ratio of CC50 on peritoneal macrophages/IC50 on amastigotes) for GCE, GF1 and GF2 were >6.5, >19.2 and > 27, respectively.

3.1. Anti-promastigote activity

3.4. Nitric oxide (NO) production

2.15. Statistical analysis Data were analyzed using Student's t-test when comparing two groups or one-way ANOVA for more than two groups using the software GraphPad Prism. P values of less than 0.05 were considered significant.

Initially, we screened the leishmanicidal potential of branches,

Since NO is a potent leishmanicidal mediator produced by

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Fig. 4. Effects of Guatteria latifolia (GCE) and subfractions (GFs) on nitric oxide (NO) production, and GF1 on decreased TNF-a production by murine peritoneal macrophages. Leishmania-infected macrophages (105) were stimulated or not with LPS and IFN-g and incubated in the presence or absence of 30.5 mg/ml of GCE, 10.4 mg/ml of GF1 and 7.4 mg/ml of GF2. (A) NO production was evaluated after 48 h of treatment by the Griess method. (B) Scavenger effect analysis was performed in a cell-free system by incubating SNAP solution (1 mM) as the NO donor with the same concentrations of GCE and GF1 and GF2 used in the NO test. Rutin (1 mM), a NO scavenger, was used as positive control, and RPMI medium served as negative control. Nitrite levels were determined by the Griess method. (C) The production of TNF-a was determined by specific ELISA after 48 h treatment. The data represent the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.0001.

macrophages, we next tested if GCE and GFs could modulate its activity. Thus, Leishmania-infected macrophages, activated or not by LPS/IFN-g, were treated with GCE 30.5 mg/ml, 10.4 mg/ml GF1 and 7.45 mg/ml GF2 and NO measured by the Griess assay. Our results indicated that GCE, GF1 and GF2 were unable to induce NO in nonactivated macrophages (Fig. 4). Moreover, GCE, GF1 and GF2 decreased NO production induced by LPS/IFN-g stimulated and infected macrophages by 86.6%, 96.8% and 97.6%, respectively (Fig. 4A). To exclude a possible NO scavenging effect, GCE, GF1 and GF2, were incubated with S-nitroso N-acetyl-DL-penicillamine (SNAP) as a NO donor. The addition of 7.4 mg/ml GF2 to the SNAP solution did not reduce NO levels, indicating that GF2 was not able to scavenge NO (Fig. 4B). However, 30.5 mg/ml GCE and 10.4 mg/ml GF1 decreased NO levels by 28.9% and 16.0%, indicating a NO scavenging effect (Fig. 4B). Rutin, the positive scavenger control, decreased 57.7% NO levels liberated in the SNAP solution.

3.5. Production of cytokines Tumor necrosis factor (TNF)-a is a macrophage activating cytokine, which contributes to parasite killing mechanisms. Thus, the ability of GCE, GF1 and GF2 to modulate TNF-a production by Leishmania-infected macrophages, activated or not by LPS/IFN-g was evaluated. Our results showed that neither the extract nor the fractions were able to modulate TNF-a production in Leishmaniainfected macrophages (Fig. 4C). LPS/IFN-g activation of these

macrophages induced a 5.7-fold increase in TNF-a production compared to unstimulated cells, and similarly to the non-activated cells, GCE and GF2 did not affect this cytokine production in activated macrophages. Actually, GF1 (7.4 mg/ml) treatment of LPS/IFNg activated infected-macrophages decreased 46% TNF-a production (Fig. 4C).

3.6. Cell cycle Because GCE, GF1 and GF2 did not stimulate NO or TNF-a production by macrophages, it seems that the effect of the extracts was independent of macrophage activation, thus, we analyze a direct effect on the parasites. Initially, we evaluate the cell cycle of promastigotes treated with 51.7 mg/ml GCE, 25.6 mg/ml GF1 and 16 mg/ ml GF2 during 48 h. Our results showed an increase of 6.7, 9.4 and 7-fold respectively, of cells in Sub-G0/G1 phase, and a decrease of 1.6, 2.5 and 1.8-fold respectively, of cells in G0/G1 phase. CGE, GF1 and GF2 decrease 3.7, 4.7 and 3.8-fold, respectively the cells in Hyperploidies phase. Moreover, GF1 and GF2 decrease 5.4 and 1.6fold the cells in G2/M phase, respectively (Fig. 5). DMSO (1%) did not alter the cell cycle of the parasite. Miltefosine 30 mM (12.2 mg/ ml) used as a control increased 4.0-fold the percentage of cells in Sub-G0/G1 phase (Fig. 5).

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Fig. 5. Extract of Guatteria latifolia (GCE) and subfractions (GFs) affect Leishmania amazonensis promastigote cell cycle. Promastigotes were cultured or not with 51.7 mg/ml GCE, 25.6 mg/ml GF1, 16 mg/ml GF2 and 12.2 mg/ml Miltefosine for 48 h, and cell cycle phases analyzed by flow cytometry after DNA staining with PI. (A) Untreated parasites control, (B) 1% DMSO, (C) 51.7 mg/ml GCE, (D) 25.6 mg/ml GF1, (E) 16 mg/ml GF2 and (F) 12.2 mg/ml Miltefosine, of one representative experiment. (G) Results shows as mean ± SD from 3 independent experiments. *P < 0.05, ***P < 0.0001.

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3.8. Liquid chromatography-mass spectrometry (LC-MS): identification of alkaloids

Fig. 6. Analysis of the mitochondrial membrane potential of L. amazonensis parasites treated with extract of Guatteria latifolia (GCE) and subfractions (GFs). The mitochondrial membrane potential (DJm) was measured by JC-1 assay after treating promastigotes with 51.7 mg/ml GCE, 25.6 mg/ml GF1, 16 mg/ml GF2 and 12.2 mg/ml Miltefosine for 24 h. Results represent the mean ± SEM of 3 independent experiments. *P < 0.05, in relation to control.

Next, we assayed the chemical identification of the compounds present in GF1 and GF2. Accordingly, the analysis of the 1H, COSY, HMBC and HSQC NMR spectra for GF1 and GF2 revealed signal values similar to those reported for aporphine alkaloids. The LC-MS of GF1 (2 mg/ml) gave ions peaks at m/z ¼ 296.1306 [MþHþ] (tR ¼ 6.8 min) (Fig.S1); m/z ¼ 306.0776 [MþHþ] (tR ¼ 21.8 min) (Fig.S2), m/z ¼ 292.0990 [MþHþ] (tR ¼ 25.3 min) (Fig.S3). The peaks were characterized as puterine (C18H18NO3, [MþHþ], m/z calc. 296.1281) (Roblot et al., 1983), oxoputerine (C18H12NO4, [MþHþ], m/z calc. 306.0760) (Cortes et al., 1985; Santos et al., 2015) and lysicamine (C18H14NO3, [MþHþ], m/z calc. 292.0968) (Park et al., 1991; Costa et al., 2009), respectively. The LC-MS of GF2 (4 mg/ ml) gave ions peaks at m/z ¼ 306.0760 [MþHþ] (tR ¼ 21.4 min) (Fig.S4), m/z ¼ 292.0977 [MþHþ] (tR ¼ 24.9 min) (Fig.S5). The peaks were characterized as, oxoputerine (C18H12NO4, [MþHþ], m/z Calc.

Fig. 7. Chromatograms of Guatteria latifolia subfractions. (A) GF1 and (B) GF2 (27% CAN, Phenomenex Luna, phenyl-hexyl, 4.2  250 mm, 0.7 ml/min, 254nm-1). Chemical structures of (C) puterine, (D) lysicamine and (E) oxoputerine identified in the subfractions.

3.7. Parasite mitochondrial alterations Leishmania is a single mitochondrial parasite and the maintenance of the mitochondrial transmembrane potential is essential for its survival. Thus, we assessed the parasite mitochondrial toxicity of GCE and GFs by the reduction of the mitochondrial membrane potential (DJm) using the JC-1 assay. Our data indicated that GCE, GF1 and GF2 did not change the DJm of promastigotes (Fig. 6). Miltefosine (12.2 mg/ml) used as a positive control reduce 1.7-fold the DJm in comparison of untreated cells (Fig. 6).

306.0760) and lysicamine (C18H14NO3, [MþHþ], m/z Calc. 292.0968), respectively. The compounds in mixtures in the fractions GF1 and GF2 were established through independent data set from the 1D and 2D NMR experiments and the values are in good agreement with those reported in the literature for these compounds. Liquid chromatography, using High Performance Liquid Chromatography (HPLC) analysis indicated a higher concentration of lysicamine in GF1, and a 3:2 lysicamine:oxoputerine proportion in GF2 (Fig. 7).

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4. Discussion

5. Conclusion

Leishmaniasis is the second most prevalent neglected infection caused by a protozoan to which no vaccine is available and the drugs used for its treatment present important side effects and are expensive for most of the needed population (Andrews et al., 2014). Plants represent a good source to search new therapeutic agents for leishmaniasis treatment (Singh et al., 2014; Ndjonka et al., 2013). Here, we studied Guatteria latifolia, a species which is quite frequent in Itatiaia National Park (Rio de Janeiro, Brazil), occurring ~o and Mello-Silva, 2007). in the forest edge and along rivers (Loba Anti-Leishmania activity has already been reported for some Guatteria species, thus, leishmanicidal activity tested only in promastigotes, has been reported for three alkaloids (isoguattouregidine, argentinine and coreximidine) isolated from G. foliosa (Mahiou et al., 1994) and for three others (puertogaline A and B, and sepeerine) isolated from G. boliviana (Mahiou et al., 2000). Among the alkaloids isolated from leaves of G. dumetorum and G. amplifolia, cryptodorine and xylopine, respectively, were the most active against promastigotes of L. mexicana and L. panamensis (Correa et al., 2006, Montenegro et al., 2003). However, the root bark extract from G. schomburgkiana and the methanol extract of the aerial parts from G. amplifolia were inactive against promastigotes from different Leishmania species (Fournet et al., 1994; Weniger et al., 2001). As far as we are aware, our study was the first to ascribe a biological activity to G. latifolia, as we demonstrated that its branch crude extract (GCE), as well as its GF1 and GF2 subfractions, present a potent effect against L. amazonensis promastigotes and intracellular amastigotes, these last form of the parasite that is responsible for maintaining the infection in the vertebrate host. Moreover, GCE and both GFs present low toxicity to the host cells, an important property to be considered. The GCE fraction decreased NO production Aporphine Leishmania-infected macrophages stimulated with IFN-g/LPS, a property that is exacerbated by both subfractions. However, it is possible that the NO reduction observed with GCE and GF1 treatment, could be partially due to its NO scavenger ability. Besides, GF1 also reduces TNF-a production in activated infected-macrophages. All these results suggested that the leishmanicidal effect of GCE and GFs seems to be directly on the parasite rather than through macrophage activation. Although the mechanism of parasite death was not fully explained, GCE and GFs affected the parasite cell cycle, increasing the number of cells in the SubG0/G1 phase and decreasing the number of cells in the G0/G1 phase, reinforcing the idea of a direct effect on the parasite. We also observed a small decrease in the mitochondrial membrane potential, which need to be reevaluated with the isolated compounds present in the active subfractions. The LC-MS analysis shows puterine, oxoputerine and lysicamine in GF1 and GF2 present only oxoputerine and lysicamine. It has been shown that lysicamine has no significant anti-leishmanial activity (Montenegro et al., 2003), thus, the leishmanicidal activity observed in GF2 could be attributed for oxoputerine. Antileishmanial activity for different aporphine and oxoaporphine alkaloids was already reported, and this activity is more significant when the D ring was substituted (Montenegro et al., 2003; Santos et al.,2015; Silva et al., 2012). Taken together, our present study showed that GCE, GF1 and GF2 from Guatteria latifolia branches possess anti-leishmanial activity. Our data warrant further studies of GCE, GF1 and GF2 to identify active compounds against Leishmania parasites. Overall, this study contributes to aggregate value to G. latifolia, which is among the endangered species.

The presented work shows that the crude extract of branches from G. latifolia and its derived subfractions GF1 and GF2 may be used to search new compounds against L. amazonensis. Importantly, this is the first report to describe biological activities of G. latifolia, that are its leishmanicidal property and ability to modulate macrophages NO and TNF-a production. Further studies are required for the isolation of the effective's compounds. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments We thank Dr. Ana L. Leandrini de Oliveira [Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography, UCSD, USA] for critical reviewing the manuscript. This work was supported by Conselho Nacional de Desenvolvimento Cientígico, Fundaça ~o Carlos Chagas Filho de Amparo  fico e Tecnolo a ~o de AperfeiçoaPesquisa do Estado do Rio de Janeiro, Coordenaça mento de Pessoal de Nível Superior. The authors would like to ~o Paulo, thank Dr. Norberto Peporine Lopes (Universidade de Sa ^ncias Farmace ^uticas de Ribeira ~o Preto) for LC-MS Faculdade de Cie analysis and HRESIMS spectra. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.exppara.2016.12.014. References lez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., Den Boer, M., Alvar, J., Ve 2012. The who leishmaniasis control team. Leishmaniasis Worldwide and Global Estimates of its Incidence. PLoS One 7, e35671. Ambit, A., Fasel, N., Coombs, G.H., Mottram, J.C., 2008. An essential role for the Leishmania major metacaspase in cell cycle progression. Cell Death Differ. 15, 113e122. Andrews, K.T., Fisher, G., Skinner-Adams, T.S., 2014. Drug repurposing and human parasitic protozoan diseases. Int. J. Parasitol. Drugs Drug Resist 4 (2), 95e111. Correa, J.E., Rios, C.H., Castillo, A.D., Romero, L.I., Ortega-Barria, E., Coley, P.D., Kursar, T.A., Heller, M.V., Gerwick, W.H., Rios, L.C., 2006. Minor alkaloids from Guatteria dumetorum with antileishmanial activity. Planta Med. 72 (3), 270e272. , A., 1985. Alcaloïdes des Annonacees, LVIII. Alcaloïdes Cortes, D., Ramahatra, A., Cave des Ecorces de Guatteria schomburgkiana. J. Nat. Prod. 48, 254e259. Costa, E.V., Marques, F.A., Pinheiro, M.L.B., Braga, R.M., Maia, B.H.L.N.S., 2009. First report of alkaloids in the genus Guatteriopsis (Annonaceae). Biochem. Syst. Ecol. 37, 43e45. Costa, E.V., Pinheiro, M.L., Barison, A., Campos, F.R., Salvador, M.J., Maia, B.H., Cabral, E.C., Eberlin, M.N., 2010. Alkaloids from the bark of Guatteria hispida and their evaluation as antioxidant and antimicrobial agents. J. Nat. Prod. 73 (6), 1180e1183. Croft, S.L., Seifert, K., Yardley, V., 2006. Current scenario of drug development for leishmaniasis. Indian J. Med. Res. 123, 399e410. Delorenzi, J.C., Attias, M., Gattass, C.R., Andrade, M., Rezende, C., da Cunha Pinto, A., Henriques, A.T., Bou-Habib, D.C., Saraiva, E.M., 2001. Antileishmanial activity of an indole alkaloid from Peschiera australis. Antimicrob. Agents Chemother. 45, 1349e1354. Ferreira, C., Soares, D.C., Barreto-Junior, C.B., Nascimento, M.T., Freire-De-Lima, L., Delorenzi, J.C., Lima, M.E., Atella, G.C., Folly, E., Carvalho, T.M., Saraiva, E.M., Pinto-Da-Silva, L.H., 2011. Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis. Phytochem. 72, 2155e2164. Ferreira, C., Soares, D.C., Nascimento, M.T., Pinto-Da-Silva, L.H., Sarzedas, C.G., Tinoco, L.W., Saraiva, E.M., 2014. Resveratrol is active and synergizes with Amphotericin B against Leishmania amazonensis. Antimicrob. Agents Chemother. 58 (10), 6197e6208. Fontes, J.E.N., Ferraz, R.P., Britto, A.C., Carvalho, A.A., Moraes, M.O., Pessoa, C., Costa, E.V., Bezerra, D.P., 2013. Antitumor effect of the essential oil from leaves of Guatteria pogonopus (Annonaceae). Chem. Biodivers. 10 (4), 722e729. ~ oz, V., 1994. Leishmanicidal and trypanocidal activFournet, A., Barrios, A.A., Mun ities of Bolivian medicinal plants. J. Ethnopharmacol. 41 (1e2), 19e37.

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