marine drugs

Article The Brown Alga zonale (): A Potential Source of Anti-Leishmania Drugs

Deivid Costa Soares 1, Marcella Macedo Szlachta 1, Valéria Laneuville Teixeira 2, Angelica Ribeiro Soares 3 and Elvira Maria Saraiva 1,* 1 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21.941-902, ; [email protected] (D.C.S.); [email protected] (M.M.S.) 2 Laboratório Produtos Naturais de Algas Marinhas (ALGAMAR), Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Niterói 24.210-150, Brazil; [email protected] 3 Grupo de Produtos Naturais de Organismos Aquáticos (GPNOA), Núcleo em Ecologia e Desenvolvimento Sócioambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro, Campus-Macaé, Rio de Janeiro 27.965-045, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-21-2560-8344 (ext. 160); Fax: +55-21-2562-6789

Academic Editor: Keith B. Glaser Received: 5 August 2016; Accepted: 1 September 2016; Published: 8 September 2016 Abstract: This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 µg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 µM (9 µg/mL) and 22.9 µM (10 µg/mL), and selectivity indexes of 8.4 µM and 11.5 µM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.

Keywords: marine natural products; meroditerpenes; Stypopodium zonale; leshmanicidal activity

1. Introduction Leishmaniasis comprises a wide spectrum of diseases that are characterized by cutaneous, mucosal, and visceral organ lesions. The form and morbidity of the disease are dependent upon both the Leishmania species and immunological status of the host [1]. Leishmaniasis affects all continents and approximately 0.2 to 0.4 million cases of visceral leishmaniasis and 0.9 to 1.2 million cases of cutaneous leishmaniasis occur annually, causing significant morbidity and mortality. Thus, leishmaniasis is recognized as one of the most neglected tropical diseases for which drug development has been stimulated by the Drugs for Neglected Diseases Initiative [2]. Currently, pentavalent antimonials, pentamidine, amphotericin B, and paromomycin are the drugs available for the treatment of leishmaniasis. However, all of these drugs exhibit toxicity, adverse side effects, and increased incidence of the emergence of drug-resistant strains, which reinforces the need to develop new approaches for leishmaniasis therapy [2,3]. Marine organisms have been studied as an important source of biologically-active secondary metabolites [4,5]. However, few studies have assessed the leishmanicidal activity of algae extracts [6–16].

Mar. Drugs 2016, 14, 163; doi:10.3390/md14090163 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 163 2 of 11

TheMar. brown Drugs 2016 algae, 14 of, 163 genus Stypopodium (Dictyotaceae) is widespread in both tropical and subtropical2 of 11 regions, and has been well recognized as a rich source of structurally-unique and biologically-active diterpenessubtropical of mixedregions, biogenesis and has been (meroditerpenoids) well recognized[ 16as– 23a ]rich. These source compounds of structurally exhibit‐unique interesting and pharmacologicalbiologically‐active activities, diterpenes such of as mixed antitumoral biogenesis [24 (meroditerpenoids)], insecticidal [25], [16–23]. and antiviral These [compounds23,26] effects, andexhibit also plays interesting an ecological pharmacological role by providing activities, such chemical as antitumoral defense against [24], insecticidal herbivory [25], [27]. and antiviral [23,26]Here, effects, we describe and also the plays anti-leishmanial an ecological role activity by providing of lipophilic chemical extract defense of againstStypopodium herbivory zonale [27].and Here, we describe the anti‐leishmanial activity of lipophilic extract of Stypopodium zonale and meroditerpenoid atomaric acid, the major compound isolated from the lipophilic extract of S. zonale. meroditerpenoid atomaric acid, the major compound isolated from the lipophilic extract of S. zonale. Additionally, the methyl ester derivative of atomaric acid was obtained by a methylation procedure Additionally, the methyl ester derivative of atomaric acid was obtained by a methylation procedure and tested against the same parasites. The extract of the compounds, atomaric acid, and its methyl and tested against the same parasites. The extract of the compounds, atomaric acid, and its methyl ester derivative, inhibited the growth of Leishmania amazonensis intracellular amastigotes in infected ester derivative, inhibited the growth of Leishmania amazonensis intracellular amastigotes in infected Stypopodium zonale macrophagesmacrophages and and exhibited exhibited low low toxicity toxicity for for the the host host cells. cells. These These findings findings characterize characterize Stypopodium aszonale a potential as a potential source of source substances of substances for the for development the development of drugs of drugs for leishmaniasis for leishmaniasis treatment. treatment. 2. Results 2. Results 2.1. Crude Extract Analysis and Structural Elucidation of Pure Compounds 2.1. Crude Extract Analysis and Structural Elucidation of Pure Compounds Specimens of Stypopodium zonale (J.V. Lamouroux) Papenfuss were collected in Búzios, Specimens of Stypopodium zonale (J.V. Lamouroux) Papenfuss were collected in Búzios, Rio de RioJaneiro de Janeiro State, State Brazil., Brazil. The dichloromethanic The dichloromethanic extract of extract Stypopodium of Stypopodium zonale (SZE) zonale was(SZE) analyzed was by analyzed both by1D both and 1D 2D and nuclear 2D nuclear magnetic magnetic resonance resonance (NMR) (NMR) spectroscopy. spectroscopy. Characteristic Characteristic signals signals for formeroditerpenoids meroditerpenoids were were observed observed for for the the major major compounds. compounds. SZE SZE was was fractionated fractionated by by SiO SiO2 2 chromatographychromatography to to yield yield atomaric acid acid (ATA), (ATA), identified identified as the as themajor major compound compound in the inextract. the extract.This Thisknown known meroditerpenoid meroditerpenoid and and its itsmethyl methyl ester ester derivative derivative AAE AAE (Figure (Figure 1) 1obtained) obtained by by a amethylation methylation procedureprocedure were were identified identified by by spectroscopy spectroscopy in in comparison comparison with previously reported reported data data [17,26]. [17,26 ].

(A) (B)

FigureFigure 1. 1.Chemical Chemical structure structure of of ( A(A)) Atomaric Atomaric acid (ATA) and ( (BB)) its its methyl methyl ester ester derivative derivative (AAE). (AAE).

Atomaric acid (ATA): 1H‐NMR (CDCl3, 300 MHz) δ: 0.93 (s, 3H, H‐19), 1.02 (s, 3H, H‐18), 1.15 (d, 1 Atomaric3H, J = acid6.9 Hz,(ATA): H‐20),H-NMR 1.26 (d, (CDCl1H, J = 314.4,, 300 H MHz)‐4a), 1.38δ: 0.93 (dd, (s, 1H, 3H, 6.0 H-19), e 12.0, 1.02 H‐7), (s, 1.49 3H, (m, H-18), 2H, H 1.15‐5), (d,1.51 3H, J =(m,6.9 1H, Hz, H H-20),‐8b), 1.57 1.26 (m, (d, 1H, 1H, HJ‐12a),= 14.4, 1.66 H-4a), (s, 3H, 1.38 H‐17), (dd, 1.68 1H, (s, 6.0 3H, e 12.0,H‐16), H-7), 1.73 (m, 1.49 1H, (m, H 2H,‐3), 1.74 H-5), (m, 1.51 (m,1H, 1H, H H-8b),‐8a), 1.81 1.57 (m, (m, 1H, 1H, H‐12b), H-12a), 1.88 1.66 (m, 1H, (s, 3H, H‐4b), H-17), 1.96 1.68 (m, 1H, (s, 3H, H‐9a), H-16), 2.22 1.73(s, 3H, (m, H 1H,‐7′), H-3),2.25 (d, 1.74 1H, (m, 0 1H,J = H-8a), 13.8, H1a), 1.81 (m, 2.26 1H, (m, H-12b), 2H, H‐13), 1.88 2.32 (m, (m, 1H, 1H, H-4b), H‐11), 1.96 2.39 (m, (m, 1H, 1H, H-9a), H‐9b), 2.22 2.84 (s, (d, 3H, 1H, H-7 J =13.8,), 2.25 H‐ (d,1b), 1H, J =3.7313.8, (s, H1a), 3H, 8 2.26′–OCH (m,3), 2H, 6.54 H-13), (d, 1H, 2.32 J = 3.00 (m, Hz, 1H, H H-11),‐4′), 6.69 2.39 (d, (m, 1H, 1H, J = H-9b),3.00 Hz, 2.84 H‐2’). (d, 13 1H,C‐NMRJ = 13.8, (CDCl H-1b),3) 0 0 13 3.73δ: (s,15.5 3H, (C‐ 820),–OCH 17.53 (C), 6.54‐7′), 17.6 (d, 1H, (C‐18),J = 3.0020.4 Hz,(C‐16, H-4 C‐),19), 6.69 20.6 (d, (C 1H,‐17),J =22.23.00 (C Hz,‐8), H-2’).23.3 (C‐9),C-NMR 24.9 (C (CDCl‐4, C‐ 3) δ: 15.512), 34.7 (C-20), (C‐1), 17.5 35.0 (C-7 (C‐03),), 17.6 32.7 (C-18),(C‐13), 36.1 20.4 (C (C-16,‐5), 38.5 C-19), (C‐6), 20.6 40.0 (C-17), (C‐2), 22.241.7 (C (C-8),‐7), 52.6 23.3 (C (C-9),‐11), 55.0 24.9 (8 (C-4,′‐ C-12),OCH 34.73), 112.8 (C-1), (C 35.0‐4’), 113.5 (C-3), (C 32.7‐2′), (C-13),122.2 (C 36.1‐10), (C-5), 125.6 (C 38.5‐6′), (C-6), 128.2 40.0(C‐3′ (C-2),), 133.3 41.7 (C‐15), (C-7), 147.5 52.6 (C (C-11),‐1′), 151.7 55.0 0 0 0 0 0 (8 -OCH(C‐5′), 3174.8), 112.8 (C‐14). (C-4’), 113.5 (C-2 ), 122.2 (C-10), 125.6 (C-6 ), 128.2 (C-3 ), 133.3 (C-15), 147.5 (C-1 ), 0 151.7 (C-5Methyl), 174.8 ester (C-14). of the Atomaric acid (AAE): 1H‐NMR (CDCl3, 300 MHz) δ: 0.93 (s, 3H, H‐19), 1.02 (s, 3H, H‐18), 1.15 (d, 3H, J = 8.0 Hz, H‐20),1 1.26 (m, 1H, H‐4a), 1.38 (m, 1H, H‐7), 1.49 (m, 2H, H‐5), 1.51 Methyl ester of the Atomaric acid (AAE): H-NMR (CDCl3, 300 MHz) δ: 0.93 (s, 3H, H-19), 1.02 (s, 3H, H-18),(m, 1H, 1.15 H (d,‐8b), 3H, 1.57J =(m,8.0 1H, Hz, H H-20),‐12a), 1.66 1.26 (s, (m, 3H, 1H, H‐17), H-4a), 1.68 1.38 (s, 3H, (m, H 1H,‐16), H-7), 1.73 (m, 1.49 1H, (m, H 2H,‐3), 1.74 H-5), (m, 1.51 1H, H‐8a), 1.81 (m, 1H, H‐12b), 1.88 (m, 1H, H‐4b), 1.96 (m, 1H, H‐9a), 2.22 (s, 3H, H‐7′), 2.26 (m, 2H, (m, 1H, H-8b), 1.57 (m, 1H, H-12a), 1.66 (s, 3H, H-17), 1.68 (s, 3H, H-16), 1.73 (m, 1H, H-3), 1.74 (m, H‐13), 2.32 (m, 1H, H‐11), 2.39 (m, 1H, H‐9b), 2.41 (d, 1H, J = 14.0, H1a), 2.84 (d, 1H, J = 14.0, H‐1b), 1H, H-8a), 1.81 (m, 1H, H-12b), 1.88 (m, 1H, H-4b), 1.96 (m, 1H, H-9a), 2.22 (s, 3H, H-70), 2.26 (m, 2H, 3.72 (s, 3H, 8′–OCH3), 3.65 (s, 3H, –COOCH3), 4.27 (sl, –OH), 6.54 (d, 1H, J = 3.00 Hz, H4′), 6.69 (d, 1H, H-13), 2.32 (m, 1H, H-11), 2.39 (m, 1H, H-9b), 2.41 (d, 1H, J = 14.0, H1a), 2.84 (d, 1H, J = 14.0, H-1b), J = 3.00 Hz, H‐2′). 13C‐NMR (CDCl3) δ: 15.7 (C‐20), 16.8 (C‐18), 17.9 (C‐7′), 20.4 (C‐16, C‐19), 20.7 (C‐ 3.72 (s, 3H, 80–OCH ), 3.65 (s, 3H, –COOCH ), 4.27 (sl, –OH), 6.54 (d, 1H, J = 3.00 Hz, H40), 6.69 (d, 3 3 Mar. Drugs 2016, 14, 163 3 of 11

0 13 0 1H, J = 3.00 Hz, H-2 ). C-NMR (CDCl3) δ: 15.7 (C-20), 16.8 (C-18), 17.9 (C-7 ), 20.4 (C-16, C-19), 20.7

(C-17),Mar. 22.2 Drugs (C-8), 2016, 14 23.3, 163 (C-9), 25.0 (C-4, C-12), 33.0 (C-13), 35.6 (C-1, C-3), 36.6 (C-5), 38.9 (C-6),3 of 11 40.6 0 0 0 (C-2),Mar. 41.9 Drugs (C-7), 2016, 51.4 14, 163 (–COOCH 3), 53.1 (C-11), 55.5 (8 –OCH3), 113.2 (C-4 ), 114.7 (C-2 ), 123.23 of 11 (C-10), 124.017), (C-6 22.20), 126.8(C‐8), (C-323.3 0(C),‐ 133.09), 25.0 (C-15), (C‐4, C 146.8‐12), 33.0 (C-1 (C0),‐13), 152.6 35.6 (C-5 (C‐01,), C 174.7‐3), 36.6 (C-14). (C‐5), 38.9 (C‐6), 40.6 (C‐2), 17),41.9 22.2 (C‐7), (C ‐51.48), 23.3 (–COOCH (C‐9), 25.03), 53.1 (C‐ 4,(C C‐11),‐12), 55.5 33.0 (8 (C′–OCH‐13), 35.63), 113.2 (C‐1, (CC‐‐3),4′), 36.6 114.7 (C (C‐5),‐2 38.9′), 123.2 (C‐6), (C 40.6‐10), (C 124.0‐2), 2.2. Anti-Leishmania41.9(C‐6 ′(C), 126.8‐7), 51.4 (C ‐(–COOCH3′ Activity), 133.0 (C of3),‐15), Stypopodium53.1 146.8 (C‐11), (C ‐55.51′), zonale 152.6 (8′–OCH Extract(C‐53′), 174.7113.2 (C(C‐‐14).4′), 114.7 (C‐2′), 123.2 (C‐10), 124.0 (C‐6′), 126.8 (C‐3′), 133.0 (C‐15), 146.8 (C‐1′), 152.6 (C‐5′), 174.7 (C‐14). Initially,2.2. Anti‐Leishmania we investigated Activity of the Stypopodium potential zonale leishmanicidal Extract effects of SZE on Leishmania amazonensis. Thus, promastigotes were treated with 10 or 50 µg/mL of SZE and parasite viability was evaluated. 2.2. AntiInitially,‐Leishmania we investigated Activity of Stypopodiumthe potential zonale leishmanicidal Extract effects of SZE on Leishmania amazonensis. Our findings showed that SZE inhibited 100% of promastigote growth two days after treatment Thus,Initially, promastigotes we investigated were treated the potentialwith 10 or leishmanicidal 50 μg/mL of SZEeffects and of parasite SZE on viabilityLeishmania was amazonensis evaluated.. at both concentrations that we assayed (Figure2). To test the safety of SZE in mammalian cells, Thus,Our findings promastigotes showed were that treatedSZE inhibited with 10 100% or 50 μofg/mL promastigote of SZE and growth parasite two viability days after was treatment evaluated. at macrophages were treated with different concentrations of SZE and cell viability was assessed Ourboth findings concentrations showed that that weSZE assayed inhibited (Figure 100% of2). promastigote To test the safetygrowth of two SZE days in mammalianafter treatment cells, at usingbothmacrophages the concentrations 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2 were treated that we with assayed different (Figure concentrations 2). ToH test-tetrazolium-5-carboxanilide of SZEthe safetyand cell of viability SZE in wasmammalian assessed inner using saltcells, (XTT) assay.macrophagesthe Treatment 2,3‐bis(2‐methoxy were with treated SZE‐4‐nitro up with to‐5‐ a sulfophenyl)different concentration concentrations‐2H‐tetrazolium of 50 µ ofg/mL SZE‐5‐ carboxanilideand was cell not viability toxic inner for was salt host assessed (XTT) cells assay.using (Figure 3). The anti-amastigotetheTreatment 2,3‐bis(2 with‐methoxy SZE activity up‐4‐ nitroto of a SZE‐concentration5‐sulfophenyl) was evaluated of‐2 H50‐tetrazolium μ ing/mLL. amazonensis was‐ 5not‐carboxanilide toxic-infected for host inner peritoneal cells salt (Figure (XTT) macrophages 3). assay. The treatedTreatmentanti for‐amastigote 24 with h with SZEactivity differentup toof aSZE concentration concentrations was evaluated of 50 μin of g/mLL. SZE. amazonensis was Our not results toxic‐infected for indicated hostperitoneal cells that(Figure macrophages SZE 3). inhibitedThe intracellularantitreated‐amastigote for amastigotes 24 hactivity with indifferent of a concentration-dependentSZE wasconcentrations evaluated inof L.SZE. amazonensis manner, Our results‐ withinfected indicated 42%, peritoneal 60% that and SZEmacrophages 95.2% inhibited inhibition at 0.001,treatedintracellular 1, andfor 24 10 amastigotes hµ g/mL,with different respectively,in a concentration concentrations while‐dependent amphotericin of SZE. manner, Our Bresults with (AMB) 42%,indicated at 60% 0.1 and µthatg/mL 95.2% SZE caused inhibitioninhibited a 24.5% intracellularat 0.001, 1, and amastigotes 10 μg/mL, in respectively,a concentration while‐dependent amphotericin manner, B (AMB) with 42%, at 0.1 60% μg/mL and 95.2% caused inhibition a 24.5% reduction in amastigote growth (Figure4). The IC 50 of SZE for amastigotes was 0.27 µg/mL. atreduction 0.001, 1, in and amastigote 10 μg/mL, growth respectively, (Figure 4).while The amphotericin IC50 of SZE for B amastigotes(AMB) at 0.1 was μg/mL 0.27 μcausedg/mL. a 24.5% reduction in amastigote growth (Figure 4). The IC50 of SZE for amastigotes was 0.27 μg/mL.

Figure 2. Effects of SZE against Leishmania amazonensis promastigotes. Promastigotes were treated Figure 2. Effects of SZE against Leishmania amazonensis promastigotes. Promastigotes were treated Figureonce with 2. Effects different of SZEconcentrations against Leishmania of SZE (openamazonensis squares promastigotes. and asterisks). Promastigotes 0.05% dimethyl were sulfoxide treated once with different concentrations of SZE (open squares and asterisks). 0.05% dimethyl sulfoxide once(DMSO) with (SZE different diluent; concentrations black circle) andof SZE parasites (open maintained squares and in asterisks).medium were 0.05% used dimethyl as controls sulfoxide (open (DMSO) (SZE diluent; black circle) and parasites maintained in medium were used as controls (open (DMSO)circle). Anti (SZE‐promastigote diluent; black activity circle) wasand parasitesestimated maintained by counting in viablemedium parasites were used over as a controlsperiod of (open four circle).circle).days. Anti-promastigote The Anti results‐promastigote from three activity activity experiments was was estimatedestimated are shown by by ascounting counting parasite viable numbers viable parasites parasites ± SEM. over over a period a period of four of four days.days. The The results results from from three three experiments experiments are are shownshown as as parasite parasite numbers numbers ± SEM.± SEM. 125

125100

10075

7550 % Viable cells % 5025 % Viable cells % 250 CTRL DMSO 0.001 1 10 50 0 SZE (g/mL) CTRL DMSO 0.001 1 10 50 SZE (g/mL) Figure 3. Safety of SZE against macrophages. Peritoneal macrophages were incubated for 24 h with FigureFigurethe 3. indicatedSafety 3. Safety of concentrations SZE of SZE against against of macrophages. macrophages. SZE or 1% DMSO Peritoneal (vehicle); macrophages macrophages cell viability were were was incubated assessed incubated for by 24 forthe h with 24XTT h with the indicatedtheassay. indicated Data concentrations from concentrations three independent of of SZE SZE or orexperiments 1% 1% DMSODMSO carried (vehicle); (vehicle); out cell in cell triplicateviability viability arewas wasexpressed assessed assessed byas %the by viable XTT the XTT assay.assay.cells Data compared Data from from three with three independent controls. independent experiments experiments carried carried out out in in triplicate triplicate are are expressed expressed as % viable cells compared cells compared with controls. with controls.

Mar. Drugs 2016, 14, 163 4 of 11 Mar. Drugs 2016, 14, 163 4 of 11

150 Mar. Drugs 2016, 14, 163 4 of 11

150100 ***

*** % Killing % 10050 *** *** *** *** % Killing % 500 *** CTRL AMB 0.1 0.001 1 10 *** SZE (g/mL) 0 FigureFigure 4. 4. Anti-amastigoteAnti‐amastigote activity activity ofCTRL SZE. of AMBPeritoneal SZE. 0.1 Peritoneal0.001 murine 1 macrophages murine10 macrophages infected with infectedLeishmania with amazonensis were treated with SZE at the indicated concentrations.SZE (g/mL) DMSO at 0.01% (vehicle) and Leishmania amazonensis were treated with SZE at the indicated concentrations. DMSO at 0.01% (vehicle) Amphotericin B [AMB] at 0.1 μM were used as controls. Amastigote growth was assessed 24 h after and AmphotericinFigure 4. Anti‐amastigote B [AMB] atactivity 0.1 µ Mof wereSZE. Peritoneal used as controls. murine macrophages Amastigote infected growth with was Leishmania assessed 24 h SZE treatment. The results from three experiments performed in duplicate are shown as the afteramazonensi SZE treatment.s were treated The results with SZE from at three the indicated experiments concentrations. performed DMSO in duplicate at 0.01% are(vehicle) shown and as the percentageAmphotericin of amastigotesB [AMB] at 0.1killing μM ±were SEM used compared as controls. with Amastigotean untreated growth control was (CTRL); assessed *** p24 < h0.0001 after percentage of amastigotes killing ± SEM compared with an untreated control (CTRL); *** p < 0.0001 SZEcompared treatment. with controls.The results from three experiments performed in duplicate are shown as the compared with controls. percentage of amastigotes killing ± SEM compared with an untreated control (CTRL); *** p < 0.0001 2.3. Leishmanicidalcompared with Activity controls. of Atomaric Acid (ATA) and Its Methyl Ester Derivative (AAE) 2.3. LeishmanicidalTo identify Activity the anti of‐ Atomaricleishmanial Acid active (ATA) compounds and Its Methyl in SZE, Ester we Derivative first tested (AAE) ATA, the major 2.3. Leishmanicidal Activity of Atomaric Acid (ATA) and Its Methyl Ester Derivative (AAE) Tocompound identify present the anti-leishmanial in this extract, along active with its compounds derivative AAE in SZE, on the we proliferation first tested of L. ATA, amazonensis the major (Figure 5). ATA and AAE at 50 μM inhibited promastigote growth by up to 86% and 100%, compoundTo present identify in the this anti extract,‐leishmanial along withactive its compounds derivative in AAE SZE, on we the first proliferation tested ATA, of theL. amazonensis major respectively, after three days with only a single treatment. (Figurecompound5). ATA andpresent AAE in atthis 50 extract,µM inhibited along with promastigote its derivative growth AAE on by the up proliferation to 86% and of 100%, L. amazonensis respectively, (Figure 5). ATA and AAE at 50 μM inhibited promastigote growth by up to 86% and 100%, after three days with only a single treatment. respectively, after three days with only a single treatment.

Figure 5. Effects of ATA and AAE against Leishmania amazonensis promastigotes. Promastigotes were treated once with ATA and AAE, and parasite viability was quantified daily. ATA 10 μM (open Figuresquare) 5. and Effects 50 μ ofM ATA (black and square); AAE against AAE 10 Leishmania μM (open amazonensis triangle) and promastigotes. 50 μM (black Promastigotes triangle); control were Figure 5. Effects of ATA and AAE against Leishmania amazonensis promastigotes. Promastigotes were treated(open circle); once withand ATADMSO and vehicle AAE, (blackand parasite circle). viabilityThe results was from quantified two experiments daily. ATA are10 μ shownM (open as treated once with ATA and AAE, and parasite viability was quantified daily. ATA 10 µM (open square) parasitesquare) andnumbers 50 μM ± SEM.(black square); AAE 10 μM (open triangle) and 50 μM (black triangle); control and 50(openµM circle); (black and square); DMSO AAE vehicle 10 (blackµM (open circle). triangle) The results and from 50 µ Mtwo (black experiments triangle); are controlshown as (open circle);parasiteTo and test DMSO thenumbers safety vehicle ± SEM.of ATA (black and circle). AAE on The host results cells, from we evaluated two experiments the dehydrogenase are shown asactivity parasite of numbersmacrophages± SEM. using the XTT method. We found that treatment of macrophages with ATA or AAE only Toaffected test the dehydrogenase safety of ATA activities and AAE at onhigh host concentrations cells, we evaluated (Figure the6A,B). dehydrogenase ATA was cytotoxic activity for of macrophages at 300 μM, affecting 64.5% of cells viability, while AAE at 200 μM affected the viability Tomacrophages test the safety using of the ATA XTT andmethod. AAE We on found host cells,that treatment we evaluated of macrophages the dehydrogenase with ATA or activity AAE of onlyof 60% affected of macrophages. dehydrogenase The CC activities50 values at for high macrophages concentrations treated (Figure with ATA6A,B). and ATA AAE was were cytotoxic 169.5 μ forM macrophages using the XTT method. We found that treatment of macrophages with ATA or AAE macrophages(75 μg/mL) and at 262.5300 μ μM,M affecting (209 μg/mL), 64.5% respectively of cells viability, (Table while 1). AAE at 200 μM affected the viability only affected dehydrogenase activities at high concentrations (Figure6A,B). ATA was cytotoxic for of 60%To of evaluate macrophages. the anti The‐amastigote CC50 values activity for macrophages of ATA and AAE, treated we with treated ATA infected and AAE macrophages were 169.5 with μM macrophagesthese(75 μg/mL) compounds. at and 300 262.5µM, We μ affecting foundM (209 that μ 64.5%g/mL), a 24 h of respectivelytreatment cells viability, with (Table ATA while 1). at 0.1, AAE 1, 10, at and 200 100µM μ affectedM killed the17%, viability 26%, of 60% of36%, macrophages. Toand evaluate 52% of amastigotes,the The anti CC‐amastigote50 respectivelyvalues activity for macrophages(Figure of ATA 7A). and AAE AAE, treated administered we treated with ATA infected at the and same macrophages AAE concentrations were 169.5with µM (75 µg/mL)thesekilled compounds. 20%, and 30%, 262.5 36%, WeµM andfound (209 62% µthatg/mL), of athe 24 amastigotesh respectivelytreatment with(Figure (Table ATA 7B). 1at). The 0.1, IC1, 10,50 values and 100 of μATAM killed and AAE17%, were26%, To2036%, μ evaluateM and (9 μ 52%g/mL) the of amastigotes, anti-amastigote and 23 μM (10 respectively μg/mL), activity respectively (Figure of ATA 7A). and (Table AAE AAE, administered 1). we treated at infectedthe same macrophagesconcentrations with thesekilled compounds. 20%, 30%, We 36%, found and 62% that of a 24the h amastigotes treatment with(Figure ATA 7B). at The 0.1, IC 1,50 values 10, and of 100 ATAµ Mand killed AAE 17%,were 26%, 36%,20 and μM 52% (9 μ ofg/mL) amastigotes, and 23 μM respectively (10 μg/mL), (Figurerespectively7A). (Table AAE administered1). at the same concentrations killed 20%, 30%, 36%, and 62% of the amastigotes (Figure7B). The IC 50 values of ATA and AAE were 20 µM (9 µg/mL) and 23 µM (10 µg/mL), respectively (Table1). Mar. Drugs 2016, 14, 163 5 of 11 Mar. Drugs 2016, 14, 163 5 of 11

Mar. Drugs 2016, 14, 163 5 of 11 Figure 6. Safety of ATA and AAE against macrophages. Peritoneal macrophages were incubated for Figure 6. Safety of ATA and AAE against macrophages. Peritoneal macrophages were incubated 24 h with the indicated concentrations of ATA (A), AAE (B), or 1% DMSO (vehicle); cell viability was for 24 h with the indicated concentrations of ATA (A), AAE (B), or 1% DMSO (vehicle); cell viability assessed by the XTT assay. Data from two independent experiments carried out in triplicate are was assessed by the XTT assay. Data from two independent experiments carried out in triplicate are expressed as % viable cells compared with controls; * p < 0.05, ** p < 0.01 compared with controls. expressed as % viable cells compared with controls; * p < 0.05, ** p < 0.01 compared with controls.

Table 1. In vitro leishmanicidal effect and cytotoxicity results for ATA and AAE.

Compound CC50 IC50 SI Figure 6. Safety of ATA and AAE against macrophages. Peritoneal macrophages were incubated for ATA 169.5 µM (75 µg/mL) 20.2 µM (9 µg/mL) 8.4 (8.3) 24 h with the indicatedAAE concentrations 262.5 µM (209 of ATAµg/mL) (A), AAE 22.9 (B),µ orM 1% (10 DMSOµg/mL) (vehicle); 11.5 cell (21) viability was assessed by the XTT assay. Data from two independent experiments carried out in triplicate are The selectivity index (SI) is defined as the ratio of CC50 on murine peritoneal macrophages to IC50 on expressedL. amazonensis as %intracellular viable cells amastigotes. compared with controls; * p < 0.05, ** p < 0.01 compared with controls.

Figure 7. Activity of ATA and AAE against intracellular amastigotes. Peritoneal murine macrophages infected with Leishmania amazonensis were treated with ATA (A) or AAE (B) at the indicated concentrations. DMSO at 0.01% (Vehicle) and Amphotericin B (AMB) at 1 μM were used as controls. Amastigote growth was assessed 24 h post‐SZE treatment. The results are from three and two experiments performed in duplicate for ATA and AAE, respectively, are shown as % of amastigotes killing ± SEM compared with untreated controls (CTRL); * p < 0.05 compared with controls.

Table 1. In vitro leishmanicidal effect and cytotoxicity results for ATA and AAE. Figure 7. Activity of ATA and AAE against intracellular amastigotes. Peritoneal murine macrophages Figure 7. ActivityCompound of ATA and AAE againstCC50 intracellular amastigotes.IC50 Peritoneal murineSI macrophages infected with Leishmania amazonensis were treated with ATA (A) or AAE (B) at the indicated infected with LeishmaniaATA amazonensis169.5 μM were(75 μg/mL) treated with20.2 μ ATAM (9 μ (Ag/mL)) or AAE8.4 ( B(8.3)) at the indicated concentrations. DMSO at 0.01% (Vehicle) and Amphotericin B (AMB) at 1 μM were used as controls. concentrations. DMSOAAE at 0.01%262.5 (Vehicle) μM (209 and μg/mL) Amphotericin22.9 μM B (AMB)(10 μg/mL) at 1 µM11.5 were (21) used as controls. Amastigote growth was assessed 24 h post‐SZE treatment. The results are from three and two AmastigoteThe selectivity growth index was(SI) is assessed defined 24as the h post-SZE ratio of CC treatment.50 on murine The peritoneal results are macrophages from three to and IC50 two on experiments performed in duplicate for ATA and AAE, respectively, are shown as % of amastigotes L.experiments amazonensis performed intracellular in duplicateamastigotes. for ATA and AAE, respectively, are shown as % of amastigotes killing ± SEM compared with untreated controls (CTRL); * p < 0.05 compared with controls. killing ± SEM compared with untreated controls (CTRL); * p < 0.05 compared with controls. As nitric oxide (NO) and reactive oxygen species (ROS) are potent leishmanicidal mediators, we Table 1. In vitro leishmanicidal effect and cytotoxicity results for ATA and AAE. studiedAs nitricwhether oxide ATA (NO) and and AAE reactive could oxygenmodulate species these (ROS) effector are molecules potent leishmanicidal in macrophages mediators, to kill weintracellular studied whether amastigotes.Compound ATA andTreatment AAE could withCC50 modulate ATA or these AAE effector ofIC 50uninfected molecules macrophagesSI in macrophages that towere kill stimulatedintracellular or amastigotes. not withATA IFN Treatment ‐γ resulted169.5 with μ inM no ATA(75 significant μg/mL) or AAE of changes20.2 uninfected μM (9in μ theg/mL) macrophages production8.4 (8.3) of that NO were (Figure stimulated 8A,C). AAE 262.5 μM (209 μg/mL) 22.9 μM (10 μg/mL) 11.5 (21) Byor notcontrast, with IFN- infectedγ resulted macrophages in no significant stimulated changes with in IFN the‐γ productionexhibited a of 75% NO and (Figure 73%8A,C). reduction By contrast, in NO productioninfectedThe macrophagesselectivity after treatment index stimulated (SI) withis defined ATA with as and IFN-the AAE ratioγ exhibited ofat CC10050 μ onM, a murine 75% respectively and peritoneal 73% (Figure reduction macrophages 8B,D). in NO to productionIC50 on L. amazonensis intracellular amastigotes. afterBy treatment analyzing with ROS ATA production, and AAE we at 100 foundµM, that respectively treatment (Figure of uninfected8B,D). macrophages with 100 μM ATABy increased analyzing ROS ROS by production, 82% compared we found with that untreated treatment controls of uninfected (Figure macrophages9A). Similar results with 100 wereµM As nitric oxide (NO) and reactive oxygen species (ROS) are potent leishmanicidal mediators, we ATAobserved increased in macrophages ROS by 82% treated compared with phorbol with untreated12‐myristate13 controls‐acetate (Figure (PMA),9A). a Similarclassical results ROS inducer were studied whether ATA and AAE could modulate these effector molecules in macrophages to kill (Figureobserved 9A). in macrophagesEvaluating infected treated macrophages, with phorbol 12-myristate13-acetatea similar increase was (PMA),observed a classical after treatment ROS inducer with intracellular amastigotes. Treatment with ATA or AAE of uninfected macrophages that were (Figure100 μM9 A).ATA Evaluating compared infected with untreated macrophages, controls, a similar although increase infected was observedmacrophages after were treatment unable with to stimulated or not with IFN‐γ resulted in no significant changes in the production of NO (Figure 8A,C). 100respondµM ATAto PMA compared stimulation with untreated (Figure controls,9A). We althoughalso observed infected modulation macrophages of wereROS unablelevels in to By contrast, infected macrophages stimulated with IFN‐γ exhibited a 75% and 73% reduction in NO macrophagesrespond to PMA treated stimulation with AAE. (Figure Thus,9A). treatment We also observedwith 100 μ modulationM AAE increased of ROS levelsROS production in macrophages by 62% production after treatment with ATA and AAE at 100 μM, respectively (Figure 8B,D). andtreated 31% with in infected AAE. Thus, and treatment uninfected with macrophages, 100 µM AAE respectively increased ROS (Figure production 9B). Both by 62%ATA and and 31% AAE in By analyzing ROS production, we found that treatment of uninfected macrophages with 100 μM reversedinfected andROS uninfected inhibition macrophages,induced by Leishmania respectively after (Figure PMA treatment9B). Both ATAof infected and AAE macrophages, reversed ROS and increasedATA increased ROS productionROS by 82% 1.3 compared‐fold compared with untreated to infected controls macrophages (Figure 9A).treated Similar with resultsPMA alonewere (Figureobserved 9A,B). in macrophages treated with phorbol 12‐myristate13‐acetate (PMA), a classical ROS inducer (Figure 9A). Evaluating infected macrophages, a similar increase was observed after treatment with

100 μM ATA compared with untreated controls, although infected macrophages were unable to respond to PMA stimulation (Figure 9A). We also observed modulation of ROS levels in macrophages treated with AAE. Thus, treatment with 100 μM AAE increased ROS production by 62% and 31% in infected and uninfected macrophages, respectively (Figure 9B). Both ATA and AAE reversed ROS inhibition induced by Leishmania after PMA treatment of infected macrophages, and increased ROS production 1.3‐fold compared to infected macrophages treated with PMA alone (Figure 9A,B).

Mar. Drugs 2016, 14, 163 6 of 11 inhibition induced by Leishmania after PMA treatment of infected macrophages, and increased ROS Mar. Drugs 2016, 14, 163 6 of 11 production 1.3-fold compared to infected macrophages treated with PMA alone (Figure9A,B). Mar. Drugs 2016, 14, 163 6 of 11

Figure 8. Nitric oxide (NO) production by macrophages is affected by ATA and AAE. Uninfected Figure 8. Nitric oxide (NO) production by macrophages is affected by ATA and AAE. Uninfected macrophages (A,C) and Leishmania infected‐macrophages (B,D) at a 10:1 ratio were stimulated or not macrophagesFigurewith 200 8. μ ( ANitricM,C IFN) andoxide‐γ andLeishmania (NO) were production incubatedinfected-macrophages in by the macrophages presence or absenceis (B affected,D) atof a eitherby 10:1 ATA 100 ratio and μM were AAE.ATA stimulated (UninfectedA,B) or 100 or not with 200macrophagesμM µAAEM IFN- (C,D (γA). ,andNOC) and production were Leishmania incubated was infected evaluated in‐ themacrophages after presence 48 h of(B or ,treatmentD absence) at a 10:1 using of ratio either the were Griess 100 stimulated µmethod.M ATA or Thenot (A ,B) or 100 µMwithresults AAE 200 from μ (CM, D threeIFN). NO‐γ independentand production were incubated experiments was in evaluated the that presence were after performed or absence 48 h of in of treatment duplicate either 100 are μ usingM shown ATA the (asA Griess ,theB) or mean 100 method. μM AAE (C,D). NO production was evaluated after 48 h of treatment using the Griess method. The The resultsnitrite fromconcentrations three independent ± SEM; ** p < experiments 0.01; * p < 0.05. that were performed in duplicate are shown as the results from three independent experiments that were performed in duplicate are shown as the mean mean nitrite concentrations ± SEM; ** p < 0.01; * p < 0.05. nitrite concentrations ± SEM; ** p < 0.01; * p < 0.05.

Figure 9. ROS production by macrophages is affected by ATA and AAE. Uninfected macrophages and macrophages infected with Leishmania amazonensis at a 10:1 ratio were stimulated or not with Figure 9. ROS production by macrophages is affected by ATA and AAE. Uninfected macrophages FigurePMA 9. ROS and production incubated in by the macrophages presence or absence is affected of 100 by μM ATA ATA and (A) AAE. or 100 Uninfected μM AAE (B macrophages) for 30 min. and andMacrophages macrophages were infected then stained with withLeishmania 50 μM amazonensis dihydrorhodamine at a 10:1 123 ratio (DHR). were Datastimulated represent or notmeans with ± macrophages infected with Leishmania amazonensis at a 10:1 ratio were stimulated or not with PMA and PMASEM ofand three incubated independent in the experimentspresence or performedabsence of in100 triplicate; μM ATA *** (A p)

3. Discussion In this present study, we showed a leishmanicidal activity of Stypopodium zonale extract (SZE) against Leishmania amazonensis, promastigotes, and intracellular amastigotes. SZE inhibited intracellular amastigotes growth in a time- and concentration-dependent manner. Currently, anti-promastigote activity has been demonstrated for algae extracts from various other species, such as Caulerpa sertularioides, Gracillaria corticata, Gracillaria salicornia, and Sargassum oligocystum, which inhibited the growth of L. major promastigotes [28]. However, there are no previously published reports of algae extracts for activity against intracellular amastigotes, as only tests of axenic amastigotes have been reported. Our results showed that SZE could kill intracellular amastigotes with an IC50 value of 0.27 µg/mL. Studies comparing the leishmanicidal activity of 32 algae species against axenic amastigotes reported that only four of those exhibited an IC50 value below 20 µg/mL, which suggests that SZE is one of the most active extracts that has been previously studied [13–15]. These data added, along with the low toxicity of SZE for host cells, demonstrates its potential as a source of molecules for leishmanicidal drug development. With an aim to identify the compounds responsible for the strong anti-leishmanial activity that we observed, SZE was fractionated and atomaric acid (ATA) was characterized as its major compound. This meroditerpene is a dominant compound in certain Stypopodium zonale populations [18], and it may be involved in important ecological interactions within the marine environment [27–30]. The biological activities of ATA have been previously reported [18,23,26], but its leishmanicidal activity was demonstrated for the first time in this present study. Herein, we also tested the methyl ester derivative of the atomaric acid (AAE), a semi-synthetic compound obtained by a usual chemical modification approach, to evaluate whether a less-polar version of ATA would show increased activity. Similar to SZE, both ATA and AAE showed activity against the promastigotes and intracellular amastigotes of L. amazonensis, along with low toxicity for host cells. The anti-Leishmania activities of ATA and AAE were comparable, showing IC50 values for intracellular amastigotes of 20 µM (9 µg/mL) and 23 µM (10 µg/mL), and selectivity indexes (SI) of 8.4 (8.3) and 11.5 (21), respectively, suggesting that derivatization of ATA could improve its activity by reducing its CC50 by 2.8-fold. SZE was 33- and 37-fold more active than ATA and AAE, respectively. This difference likely resulted from the association of these major compounds with minor substances present in SZE, suggesting a possible synergism among these compounds. The anti-leishmanial activity of substances isolated from algae has been previously reported for terpenes [14,16]. The halogenated sesquiterpenes, elatol and obtusol, which were isolated from the red alga Laurencia dendroidea, as well as the diterpene dolabelladienotriol obtained from the brown alga pfaffii (Dictyotaceae), were active against the promastigote and amastigote forms of L. amazonensis. Here, our findings suggested an anti-leishmanial activity for two meroditerpenes, ATA, and its derivate AAE, establishing the robust leishmanicidal potential of algal terpenes. We determined that ATA and AAE modulate macrophage activity by inhibiting NO production, which is an important mediator of Leishmania killing. ATA and AAE treatment reduced NO production in infected macrophages stimulated by IFN-γ. These data suggest that the Leishmania killing mediated by ATA and AAE occurred independently of NO production. Similarly, dolabelladienotriol, an algae-isolated substance with anti-amastigote activity, can also inhibit NO production [16]. In contrast to ATA and AAE, dolabelladienotriol can inhibit NO in both infected and uninfected macrophages that are stimulated or not with IFN-γ + LPS [16]. Recently, Kar and colleagues [31] showed that mouse splenocytes treated with fucoidan, a polysaccharide from the brown alga vesiculosus, increased ROS production and efficiently resolved L. donovani infection. The anti-amastigote activity of ATA and AAE could be explained at least in part because of the capacity of these molecules to stimulate ROS production in macrophages that are infected or not with Leishmania amazonensis. Together, our present findings show that Stypopodium zonale is an interesting source of natural products for drug discovery and the development of novel anti-protozoal agents. Atomaric acid and its methyl ester derivative, which exhibit leishmanicidal activity in vitro, may represent an attractive and safe candidate source for the development of drugs for the treatment of cutaneous leishmaniasis. Mar. Drugs 2016, 14, 163 8 of 11

4. Experimental Section

4.1. Seaweed Sampling The brown alga Stypopodium zonale was collected by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA), license number 45755 of 29/12/2014, by snorkeling at a depth of 2–3 m at Praia do Forno, Município de Búzios, Rio de Janeiro State (22◦450 S, 41◦520 W), Brazil, in February 2007; alga were identified by Dr. Lísia M. Gestinari (NUPEM-UFRJ Campus Macaé). A voucher specimen was deposited at the herbarium of the Universidade Federal do Rio de Janeiro (RFA 3823). Algae were washed in seawater to eliminate associated organisms and then were air-dried.

4.2. Extract Preparation and Procedures for Obtaining Meroditerpene Air-dried and powdered algal material (130.0 g dry weight) was successively extracted with dichloromethane (2 L × 3 times, at room temperature for three weeks). Solvent was removed by vacuum yielding 11.8 g lipophilic extract (SZE). The chemical profile of the major compounds in SZE was determined by 1H NMR (nuclear magnetic resonance, 300 MHz) spectroscopy in a Bruker Advance spectrometer. SZE (2.5 g) was chromatographed on a SiO2 flash column using an n-hexane-ethyl acetate (EtOAc) and methanol (MeOH) step gradient system; 20 fractions (F1–F20) were obtained. All fractions, i.e., F1–F20, were analyzed by thin layer chromatography using Kieselgel 60 F254 aluminum support plates (Merck, Rio de Janeiro, RJ, Brazil). Fraction F4 (0.152 g), which contained the major compound in SZE, was eluted with 20% EtOAc in n-hexane, re-chromatographed on a SiO2 column with 25% EtOAc in n-hexane and, finally, 0.0036 g of the meroditerpenoid atomaric acid (ATA) was isolated.

4.3. Preparation of the Methyl Ester of Atomaric Acid (AAE) To evaluate possible structural modifications on S. zonale compounds for effects on anti-leishmanial activity, the methyl ester of atomaric acid (AAE) was obtained after a methylation reaction of the extract. Briefly, 1.0 g of SZE was dissolved in a mixture of CHCl3–MeOH (4:1) and fresh diazomethane (CH2N2) in an excess of ethyl ether solution. After overnight magnetic stirring of the mixture, it was fractionated by silica gel vacuum liquid chromatography and eluted with increasing amounts of EtOAc in n-hexane. From the 12 fractions (F1–F12) that were obtained, F2 (0.2470 g) was chromatographed in a silica gel column and eluted with 15% EtOAc in n-hexane to yield 0.1360 g purified AAE.

4.4. Structural Elucidation Chemical structures of the purified compounds were established by a comparison of previously reported 1H NMR, 13C NMR, mass spectrometry, and infrared spectroscopy data [22,23], and meroditerpenes, atomaric acid (ATA), and its methyl ester derivative (AAE) were identified (Figure1).

4.5. 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, Rio de Janeiro, Brazil). The study protocol was approved by the Committee for Animal Use of the Universidade Federal do Rio de Janeiro (CEUA Permit Number: 128/15; CONCEA Protocol: 01200.001568/2013-87).

4.6. Parasite Culture L. amazonensis (WHOM/BR/75/Josefa) promastigotes were cultured at 26 ◦C in Schneider’s insect medium (Sigma, St. Louis, MO, USA), 10% fetal calf serum (FCS, Gibco, Frederick, MD, USA), and 20 µg/mL gentamycin (Schering-Plough, Rio de Janeiro, RJ, Brazil). Mar. Drugs 2016, 14, 163 9 of 11

4.7. Anti-Promastigote Activity The leishmanicidal properties of SZE, ATA, and AAE were evaluated by measuring promastigote viability. Stationary-phase promastigotes were treated with different concentrations of SZE, ATA, or AAE, and parasite survival was estimated by counting viable/motile forms in a hematocytometer during five days of culture at 26 ◦C. Data are expressed as the number of live parasites. As controls, promastigotes were maintained in culture medium and treated with the vehicle, dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA).

4.8. Anti-Amastigote Activity Mouse peritoneal macrophages obtained after stimulation with 3% thioglycolate for three days were harvested in RPMI 1640 medium (LGC Biotec, Cotia, SP, Brazil) and cultured in 24-well plates for ◦ 2 h until they were adherent at 35 C, 5% CO2. Non-adherent cells were removed, and macrophages were incubated overnight, as above, in RPMI with 10% FCS. Adherent macrophages were infected with L. amazonensis promastigotes (stationary growth phase) at a 10:1 parasite/macrophage ratio for ◦ 1 h at 35 C, 5% CO2. Free parasites were washed out with 0.01 M phosphate buffered saline (PBS), ◦ and cultures were maintained as above for 24 h at 35 C, 5% CO2. Infected macrophage cultures were ◦ treated with different concentrations of SZE, ATA, or AAE for an additional 24 h at 35 C, 5% CO2. The cultures were then washed in PBS, fixed and stained with Giemsa. The number of amastigotes per macrophage and the percentage of infected macrophages were determined by counting at least 200 cells in triplicate cultures. Infectivity index was obtained by multiplying the percentage of infected macrophages by the mean number of amastigotes per infected macrophage. Amphotericin B used as a positive control was from Cristália (Itapira, SP, Brazil). The results were expressed as the percentage of killing compared with untreated controls.

4.9. Cytotoxicity for Host Macrophages Mouse peritoneal macrophages that adhered to 96-well plates were treated with different concentrations of SZE, ATA or AAE for 24 h. Cell viability was determined using 1 mg/mL XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxinilide inner salt, Sigma) with 200 µM PMS (Phenazine methosulfate, Sigma). After 3 h incubation, the reaction product was quantified at 450 nm. Data are expressed as the percentage of viable cells compared with untreated controls [32].

4.10. Nitric Oxide Production Thioglycolate-stimulated peritoneal macrophages were obtained as described above (at 106 cells/well in 24-well plates) and were either activated with 200 ng/mL IFN-γ (eBioscience, ◦ San Diego, CA, USA) or left untreated. After incubation for 24 h at 35 C, 5% CO2, cells were treated with 100 µM ATA or AAE. Nitrite concentrations in culture supernatants were determined using the − Griess method. The reaction was read at 540 nm, and the concentration of NO2 was determined based on a standard curve of sodium nitrite. Data were expressed as the micromolar concentrations of nitrite [33].

4.11. Detection of Reactive Oxygen Species (ROS) Mouse peritoneal macrophages that adhered to 96-well opaque culture plates were infected or not with L. amazonensis promastigotes. At 24 h post-infection, cultures were treated with 100 µM ATA or AAE and stimulated or not with 1 µg/mL phorbol 12-myristate13-acetate (PMA, Sigma). Cells were stained with 50 µM dihydrorhodamine 123 (DHR 123, Life Technologies, Waltham, MA, USA), and ROS was measured immediately using 500/526 nm excitation/emission wavelengths.

Acknowledgments: This work was supported by Financiadora de Estudos e Projetos (FINEP), PETROBRAS, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Mar. Drugs 2016, 14, 163 10 of 11

Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We are also grateful to CNPq for providing Research Grants to Angelica Ribeiro Soares. We thank the NMR Laboratory (Instituto de Pesquisas de Produtos Naturais Walter Mors, UFRJ) for the analyses. Author Contributions: D.C.S., A.R.S., V.L.T. and E.M.S. conceived, designed the experiments, analyzed data and wrote the manuscript. D.C.S., M.M.S., A.R.S. performed the experiments. Conflicts of Interest: The authors declare that they have no conflict of interest.

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