V) complexes

European Journal of Medicinal Chemistry 90 (2015) 732e741

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Original article

Leishmanicidal activity of polysaccharides and their oxovanadium(IV/ V) complexes Alex Evangelista do Amaral a, Carmen Lúcia Oliveira Petkowicz a, ^ b, Marcelo Iacomini a, Glaucia Regina Martinez a, Ana Lucia Ramalho Merce Maria Eliane Merlin Rocha a, Silvia Maria Suter Correia Cadena a, Guilhermina Rodrigues Noleto a, * a b

, Curitiba, Parana , Brazil Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Parana , Curitiba, Parana , Brazil Departamento de Química, Universidade Federal do Parana

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 September 2014 Received in revised form 21 November 2014 Accepted 3 December 2014 Available online 5 December 2014

The parasites of the genus Leishmania cause a range of leishmaniasis diseases, whose treatment is impaired due to intramacrophage parasites living in the mammalian host. Immunostimulation has been considered an important strategy to leishmaniasis treatment. The immunomodulatory effects of the polysaccharides arabinogalactan (ARAGAL), galactomannan (GMPOLY), and xyloglucan (XGJ), as well as their oxovanadium (IV/V) complexes (ARAGAL:VO, GMPOLY:VO, and XGJ:VO) were evaluated on peritoneal macrophages. At 25 mg/mL of GMPOLY:VO and of XGJ:VO, and 10 mg/mL of ARAGAL:VO, nitric oxide (NO) production by the macrophages was not altered compared with the control group. All polymers increased the production of interleukins 1 beta and 6 (IL-1b and IL-6), but the oxovanadium complexes were more potent activators of these mediators. ARAGAL:VO 10 mg/mL, GMPOLY:VO and XGJ:VO 25 mg/mL led to an increase of 562%, 1054%, and 523% for IL-1b, respectively. For IL-6 at the same concentration, the levels increased by 539% and 794% for ARAGAL:VO and GMPOLY:VO, respectively. Polysaccharides and their oxovanadium complexes exhibited important leishmanicidal effects on amastigotes of Leishmania (L.) amazonensis. The native and complexed polymers reduced the growth of promastigote-form Leishmania by ~60%. This effect was reached at concentrations 12 times lower than that observed for Glucantime (300 mg/mL promoted an inhibition of ~60%). The 50% inhibitory concentration (IC50) values for the complexes were determined. XGJ:VO showed the lowest IC50 value (6.2 mg/mL; 0.07 mg/mL of vanadium), which for ARAGAL:VO was 6.5 mg/mL (0.21 mg/mL of vanadium) and 7.3 mg/mL (0.06 mg/mL of vanadium) for GMPOLY:VO. The upregulation of IL-1b and IL-6 release and downregulation of NO production by macrophages and the important leishmanicidal effect are essential to stablish their potential use against this pathology. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Polysaccharides Oxovanadium(IV/V) complexes Immunomodulators Leishmaniasis

1. Introduction Leishmania spp., an obligate intramacrophage protozoa, is transmitted to humans by different species of phlebotomine sandflies infected with the protozoan. After its internalization into the host's macrophages [1], they promote a range of diseases,

* Corresponding author. Departamento de Bioquímica e Biologia Molecular, Setor ^ncias Biolo gicas, Centro Polite cnico, Universidade Federal do Paran de Cie a, C.P. , Brazil. 19046, CEP 81531-980, Curitiba, Parana E-mail addresses: [email protected], [email protected] (G.R. Noleto). http://dx.doi.org/10.1016/j.ejmech.2014.12.003 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

namely leishmaniasis. The disease manifests in different forms (cutaneous, mucocutaneous, and visceral) according to the Leishmania species involved [2]. According to the World Health Organization (WHO), around 1.3 million new cases of leishmaniasis occur annually. The number of cases recorded and the distribution of leishmaniasis has grown since 1993, being a serious health problem in developing countries. The classical treatment relies on pentavalent antimonials (PVAs) with sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), which are glucose derivatives complexed with antimony salt [3,4]. Although these drugs are an example of clinically established metal-based drugs against a parasitic disease

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Fig. 1. Representative structures of polysaccharides ARAGAL (a), XGJ (b) and GMPOLY (c).

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[5], the main problem in treating leishmaniasis with the pentavalent antimonial drugs is the serious side-effects [4,6] and the development of resistance [7e9]. Nowadays, other drugs such as amphotericin B, pentamidine, and miltefosine are being used, but they all also trigger many side effects [6,10]. The intracellular nature of the parasite in the mammalian host hinders the access of the medications [11,12]. It has been proposed that the clinical cure could be obtained by immunomodulatory compounds that would be able to activate macrophages to for leishmanicidal activity [10,13]. In this sense, certain native and modified polysaccharides have been characterized as biological response modifiers for their immunomodulatory effects [14]. Some of these polymers trigger activation of macrophages for different activities like antitumor [15,16], antimicrobial and antileishmania [17e21]. In addition, it was shown that certain oxovanadium complexed polysaccharides exhibited potent leishmanicidal activity [17,21]. Vanadium offers interesting chemical and biological properties for the development of anti-parasitic drugs [22]. In the oxidation states ranging from (þIII) to (þV), vanadium is able to interact with a variety of ligands, including carbohydrates, to form biologically active complexes, stable in the physiological pH range [22,23]. The therapeutic effects exhibited by vanadium compounds also include the insulin-mimetic action [24], tumor growth inhibition [25], immunomodulation [26,27], and anti-parasitic action, particularly as potential agents for the treatment of leishmaniasis [27e30]. In view of the described above, the vanadium-polysaccharides complexes could offer excellent opportunities to find new drugs against leishmaniasis. Thus, the aim of this study was to produce complexes of oxovanadium(IV/V) with different polysaccharides previously reported as able to activate macrophage immune responses [14,17,31,32] and evaluate the effects of the native polysaccharides and their oxovanadium complexes on some functions of macrophages related with leishmanicidal effects and on Leishmania amastigote forms. 2. Materials and methods 2.1. Material Vanadyl sulfate (VOSO4$3H2O), lipopolysaccharides (LPS), N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT), sulfanilamide, and naphthyl ethylenediamine were obtained from Sigma Chemical Co. (St Louis, MO). Eagle's minimum essential medium (MEM) and fetal bovine serum were obtained from Cultilab (Brazil); bacteriological agar, bacteriological peptone, and beef extract were obtained from Himedia; and Glucantime® (Meglumine antimoniate) was obtained from Aventis. All other reagents were commercial products of the highest purity available and were supplied by Merck. Tissue culture materials were supplied by Corning or Nunc A/S. 2.2. Polysaccharides The arabinogalactan (ARAGAL) used in this study was isolated from the exudate of Anadenanthera colubrina trunks and was characterized by Delgobo et al. [33]. The heteropolysaccharide has a (1 / 3)-linked b-D-Galp main chain and many different side chains containing b-D-Galp-(1 / 6), a-Araf, a-Rhap, and b-GlcAp (Fig. 1a). Xyloglucan (XGJ) was isolated from seed cotyledons from Hymerio et al. [32]. XGJ has a naea courbaril as described by Rosa Glc:Xyl:Gal ratio of 3.7:2.5:1.0 and consists of a cellulose backbone which is partially substituted at O-6 by a-D-Xylp units. Some xylosyl residues are further substituted at O-2 by b-D-Galp (Fig. 1b). Galactomannan (GMPOLY) was obtained from the lichen Ramalina

celastri and was previously characterized by Miceno et al. [34]. GMPOLY has a (1 / 6)-linked a-D-Manp main chain of which ~44% is monosubstituted at O-4 by b-D-Galp and ~33% is disubstituted at O-4 and O-2 by b-D-Gal (Fig. 1c). 2.3. Oxovanadium solutions Solutions of 0.1 M and 0.01 M oxovanadium(IV/V) were prepared as previously described [35e37]. Vanadyl sulfate (VOSO4.3H2O) was dissolved in nitric acid (rather than in the usual  sulfuric acid procedure) to prevent SO2 4 and HSO4 interference in the equilibria studies, especially at more basic pH values. Under these conditions, vanadium(IV) VO2þ and vanadium(V) VO3þ can be present due to the oxidizing character of nitric acid. The resulting solutions were then standardized for metal concentration using permanganate titration [38], while hydrogen ion concentration (HNO3) was standardized using Gran's Plot [39]. All solutions were prepared with double-distilled, deionized, CO2-free water. 2.4. Potentiometric titration experiments The 0.1 mmol water soluble biopolymers were let to dissolve in a proper amount of water for at least 2e3 h before being transferred to the reaction cell for the potentiometric titration measurements. The mean molecular weight of each biopolymer studied was calculated taking into account the monomeric repeating sugar units, then their individual appropriate mass was weighted. All potentiometric titration experiments were performed in triplicate in a Metrohm Titrando 809 automatic burette with TIAMO integrated software (Switzerland) employing a previously standardized 0.1 M KOH aqueous solution and in a 0.100 M KCl adjusted ionic strength. The equilibrium constants were calculated using the software Hyperquad [40], and the database SC QUERY [41] was used to provide the previously calculated and reported hydrolysis and protonation constant for the complete calculations of the equilibrium constants of the biopolymers in the presence of vanadyl ion [17]. The metal solution was prepared using VOSO4.3H2O, 0.1 M e Aldrich, standardized against KMnO4, dissolved in 0.3 M HNO3 aqueous solution. The mineral acid was selected as such so that SO2 4 ions would not be present in the systems due to the gelling effect this anion produces in the presence of some biopolymers, and to avoid insoluble hydrolytic products being formed as the titrations (pH) progress. The potentiometric titration-obtained profiles enabled the calculations of the speciation distribution species employing the software HySS [42]. 2.5. Preparation of oxovanadium complexes Both ARAGAL:VO and XGJ:VO complexes at a 2:1 ligand to metal ratio were obtained by dissolving 0.4 g (2.5 mmol) of the polysaccharides in water (160 mL) and later mixing with 0.1 M VO2þ/ VO3þ (12.5 mL, 1.25 mmol). The mixture was titrated by adding 0.1 M and 0.01 M KOH pH 7.8, dialyzed against ultrapure water for 72 h and then lyophilized. The dialysis procedure was performed to obtain the solid complexes without free metal ions. GMPOLY:VO complex was previous characterized [17]. In the present study, GMPOLY:VO was prepared according to the literature [17], and dialyzed against ultrapure water for 72 h to remove any uncomplexed oxovanadium, and then lyophilized. The material obtained was analyzed using 51V nuclear magnetic resonance (51V NMR). To determine the vanadium concentration, 10 mg of complexes were dissolved in 0.1 M HNO3 and atomic absorption spectroscopy was used (SpectrAA model 220FS, India).

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The lyophilized complexes were dissolved in phosphatebuffered saline solutions (PBS), sterilized in a 0.22 mm membrane and frozen. These stock solutions were dissolved in culture medium suitable for biological activity experiments. 2.6.

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V nuclear magnetic resonance spectroscopy (51V NMR)

The samples were prepared by dissolving 30 mg of polysaccharide-oxovanadium complexes in 0.8 mL of either 0.1 or 1.0 M KOD to reach pH 7.8. KOD was added to 0.5 mL of 0.1 M VO2þ/ VO3þ solutions until pH reached 7.8, adjusting the final volumes to 1.5 mL. 51V NMR analysis was carried out using a Bruker DXR-400 Advance spectrometer with a 5 mm inverse (BBO) probe. 51V NMR spectra were obtained at 105.15 MHz with 0.5 s D1 and 0.1 s AQ, at 30  C. Chemical shifts are expressed in d (ppm) relative to that of the external standard VOCl3 (d ¼ 0). 2.7. Macrophage Isolation Albino Swiss mice (6e8 weeks old) were used as peritoneal macrophage donors. All legal recommendations of the Brazilian legislation (Law No. 6.638, 05 Nov. 1979) for animal handling procedures for scientific research were approved by the Animal Ethics Committee. The mouse peritoneal macrophages were collected by infusing the donors’ peritoneal cavity with 8e10 mL of chilled PBS. The cells were plated in culture medium (minimum essential medium-MEM, 5% fetal bovine serum and antibiotics) in 24- or 96well plates (4  105 cells/well). After 1e2 h of incubation at 37  C under 5% CO2 in a humidified incubator, non-adherent cells were removed by washing twice with PBS at 37  C [17]. 2.8. Leishmania culture Leishmania (Leishmania) amazonensis (designated MHOM/BR/ 73/M2269) promastigotes were cultured at 26e28  C in Evans' modified Tobie's medium (EMTM), and in the exponential phase they were transferred to and maintained in MEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mg/mL gentamicin.

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nitrite concentration was calculated from a standard NaNO2 curve (10e100 mM). The results are expressed as mmol per 5  105 cells. 2.11. Quantification of interleukins IL-1b and IL-6 Adherent macrophages (1  106 cells/well) were plated on a 24well tissue culture plate with standard medium in the absence (negative control) or presence of ARAGAL (10 mg/mL), GMPOLY and XGJ (25 mg/mL), or ARAGAL:VO (2.5, 5, and 10 mg/mL), GMPOLY:VO and XGJ:VO (5, 10, and 25 mg/mL). LPS (50 ng/mL) was used as a positive control. After 48 h at 37  C under 5% CO2, the supernatant from each culture was collected and maintained at 80  C until further utilization. IL-1b and IL-6 concentration in the supernatant were determined using an enzyme-linked immunosorbent assay (ELISA) as specified by the manufacturers (RayBiotech, Inc.). The results are expressed in pg/mL. No components of the culture medium showed immunoreactivity to the cytokines under study. 2.12. Measurement of leishmanicidal activity For leishmanicidal activity, adherent macrophages at concentration of 5  105/well (in 24-well plates) were infected with 2.5  106 log phase promastigotes for 12 h of incubation at 34  C under 5% CO2 in a humidified incubator. After 12 h of incubation, the preparation was washed three times with PBS to remove the uninternalized parasites. The infected macrophages were treated with polysaccharides and their complexes at different concentrations: ARAGAL (10 mg/mL), XGJ and GMPOLY (25 mg/mL), or ARAGAL:VO (2.5, 5, and 10 mg/mL), XGJ:VO and GMPOLY: VO (5, 10, and 25 mg/mL) for 48 h incubation at 37  C under 5% CO2 in a humidified incubator. Glucantime (300 mg Sb5þ/mL), being the drug of choice in leishmaniasis treatment, was thus included as a positive control for effects on the amastigote forms. After 48 h of incubation, the cells were scraped off, suspended in saline (0.4 mL), transferred to Tobie's and Evans' media (EMTM), and incubated at 24  C for 5e10 days. After that, cell densities (Leishmania promastigotes) were determined microscopically using a Neubauer hemocytometer [17]. 2.13. Statistical analysis

2.9. Measurement of cell viability Adherent macrophages were incubated for 48 h in the standard medium in the absence (control) or presence of varying concentrations of polysaccharide-oxovanadium complexes, 5, 10, 25, 50, and 100 mg/mL, which correspond to VO concentrations of 0.161, 0.322, 0.805, 1.61, and 3.22 mg/mL for ARAGAL:VO; 0.055, 0.104, 0.261, 0.522, and 1.044 mg/mL for GMPOLY:VO, and 0.059, 0.119, 0.297, 0.595, and 1.190 mg/mL for XGJ:VO. Cytotoxicity was evaluated using an MTT reagent as described by Reilly et al. [43].

The data were analyzed statistically by analysis of variance and Tukey's test to comparison the averages. Mean values ± SD were used, and the values were considered significant when p < 0.05. The IC50 values to oxovanadium complexes were calculated by nonlinear regression. 3. Results and discussion 3.1. Complexation of arabinogalactan and xyloglucan with Oxovanadium(IV/V)

2.10. Measurement of nitric oxide (NO) production To measure NO production, adherent macrophages (4  105 cells/well) were plated in a 96-well culture dish and incubated in the presence of polysaccharides and their oxovanadium complexes: ARAGAL (10 mg/mL), GMPOLY and XGJ (25 mg/ mL) or ARAGAL:VO (2.5, 5, and 10 mg/mL), GMPOLY:VO and XGJ:VO (5, 10, and 25 mg/mL). Controls without the polymers and with LPS (50 ng/mL) were performed. After 48 h, the NO production was indirectly assessed by measuring nitrite concentrations in the culture medium using the Griess reaction [44] with modifications. The isolated supernatants were mixed with equal volumes of Griess reagent and incubated at 25  C for 10 min. Absorbance was measured at 550 nm in a microplate reader. The

The use of metals as therapeutic agents for different applications has been of great interest to medicine. When complexed with different ligands, the use of metals in the physiological conditions is facilitated [5,45,46]. For example, the main drugs used to treat leishmaniasis more sixty years ago consisted of antimony (V) complexed with sodium gluconate (Pentostam) or Meglumine (Glucantime), respectively [3]. The interest in using polysaccharides with metals in medicine ranges from diagnosis to therapeutic agents for different pathologies [47,48]. In this study, the potentiometric titration method was used to study the interaction between oxovanadium(IV/V) with different polysaccharides aiming at their biological applications. The protonation constants for ARAGAL (11.10 ± 0.05) and XJG

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(11.20 ± 0.03) were determined in this study taking into account the O in primary unlinked C6 in the monomeric sugar units. Fig. 2a and b shows the experimental data for calculating the stability constants for the complexes ARAGAL:VO and XGJ:VO found in the equilibria studied at percentages above 10%, metal ion concentration considered as 100%. These constants also enabled the species distribution diagrams. The main species formed are summarized in Table 1 for maximum presence according to pH values. An analysis of the pH profiles of both systems with VO shows that ARAGAL binds to VO a little more strongly than XGJ, although both systems have high binding constants toward VO and the formation of different complexed species in both systems. The stability of all complexed species in all studied systems may be explained by the ability of this polysaccharides to form 3D structures and, by doing so, it can thus bind to the metal ion through any free eOH groups of eventually all sugar units, producing a high-stability constant. The hydroxo-complexed species are mostly present at physiological pH for ARAGAL:VO (~95%) and XGJ:VO (~90%), but for XGJ:VO there is also the presence of L2M species (~50%). The complexation of GMPOLY with VO was previously characterized and the binding constant of GMPOL:VO value was determined by Noleto et al. [17]. GMPOLY:VO exhibited a high amount (~95%) of L2M at physiological pH range [17], as did the GALMANA:VO and GALMAN-B:VO from Mimosa scabrella [49]. Complexation was confirmed by the 51V NMR spectroscopy since the present experimental conditions were suitable for investigating the oxovanadium(IV) (VO2þ) and oxovanadium(V) (VO3þ) mixture. The latter species can be detected by 51V NMR. Spectra of VO2þ/VO3þ, obtained at 30  C and pH 7.8, contained a single signal at d 559 ppm.

Fig. 2. (a) Potentiometric pH profile of ARAGAL in the presence or absence of VO. Simulated (d) and experimental ( ) profiles to ARAGAL:VO 2:1 ligand to metal ratio. (b) Potentiometric p[H] profile of XJG in the presence or absence of VO. Simulated (d) and experimental ( ) profiles to XGJ:VO 2:1 ligand to metal ratio. T ¼ 25  C, I ¼ 0.100 M KNO3.

⋄

⋄

Table 1 Stability constants for the systems ARAGAL:VO and XGJ:VO. Species

pH range

Maximum relative to VO % formation

ARAGAL:VO 2.0e5.0 60 ARAGAL: VOOH 4.0 95 e10.0 ARAGAL2:VOOH 9.0 40 e11.0 XGJ:VO2 (OH)2 3.0e8.5 90 XGJ:VO 2.0e4.0 45 XGJ2:VO 6.0 50 e10.0 GMPOLY:VO 3.0e6.5 70 4.5 95 GMPOLY2:VO e10.0

Binding constants (log K) 12.16 ± 0.020 7.73 ± 0.010 13.74 ± 0.030 9.49 ± 0.070 11.80 ± 0.100 9.15 ± 0.030 8.41 ± 0.007 [17] 7.03 ± 0.06 [17]

The ligand to metal ratio of 1:1, T ¼ 25.0  C, I ¼ 0.10 M KCl. The main species present according the pH value.

Fig. 3a shows the complexation of ARAGAL with VO. Besides the signal at d 559 ppm, five other signals with different intensities were observed at d 423.1,498.4, 514.0, 571.8, and 576.0 ppm, indicating the presence of five types of complexes formed by ARAGAL and VO. These results are in agreement with the vanadium concentration detected in the complexes. The complex ARAGAL:VO contained a higher amount of vanadium (3.2%), while GMPOLY:VO and XGJ:VO showed 1.0 and 0.8%, respectively. For ARAGAL, the 4,6 and 3,4-OH groups from (1 / 3)- or (1 / 6)-linked b-D-galactose units which are not substituted are probably the main sites of complexation for the LM species. In addition, the complexation for LM species could also take place with the 2,3-cis-diol from a-rhamnopyranose units present as a minor component in the side chains. The b-D-glucuronic acid, another minor component from ARAGAL side chains could also be involved in the complex's formation. The participation of the O atom of the pyranose ring and the carboxylic acid group has been reported in the formation of monomeric complex species with vanadyl at pH 3 [50]. XGJ:VO (Fig. 3b) showed two additional signals at d 571.8 and 576.0 ppm, which were similar to GMPOLY:VO (d 571.6 and 575.8 ppm) (Fig. 3c). These results indicate the occurrence of two types of complexes with oxovanadium ions for both XGJ and GMPOLY. Similar results were obtained for complexes formed with oxovanadium and galactomannans (GALMAN-A:VO and GALMANB:VO) from M. scabrella seeds [49]. According to the literature [51], no complexation took place with vanadate and b-D-glucopyranose units present in the main chain of XGJ, as was probably also the case with the trans-OH groups from a-D-xylopyranose units from xyloglucans. Thus, the complexation for LM species with XGJ should occur only with the 3,4- or 4,6-OH groups from terminal b-D-galactose units. Other complex species, such as L2M, which was observed for XGJ:VO, could be generated by involving pairs of OH groups of two different ligands as demonstrated by Adriazola et al. [21] for a seed galactomannan and oxovanadium complex. Terminal b-D-galactose units are also present in GMPOLY and are probably involved in the complexation for LML species as well as the mains-chain a-D-mannopyranose units that are not disubstituted and have free 2,3-cis-diol, although this site is not easily available due to some steric hindrance caused by the highly branched structure of the polymer. 3.2. Effects of polysaccharides and their Oxovanadium(IV/V) complexes on peritoneal macrophages The polysaccharides used in this study were previously

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Fig. 3.

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V NMR spectra of complexes of VO with ARAGAL (a), XGJ (b) and GMPOLY (c) at pH 7.8 using D2O.

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characterized as biological response modifiers for exhibiting immunomodulatory effects [17,31,32]. The possibility of achieving leishmanicidal effects by macrophage activation and the important anti-parasitic activities described for vanadium motivated the evaluation of the possible leishmanicidal effects of vanadium complexation for biopolymers. The effects of the polysaccharides complexed with oxovanadium(IV/V) on the viability of murine peritoneal macrophages were

evaluated 48 h after treatment with variable concentrations of complexes. Cell viability was calculated considering the control (medium) at 100%. Fig. 4a shows that ARAGAL:VO at 25, 50, and 100 mg/mL decreased cell viability by 34%, 94%, and 98%, respectively. GMPOLY:VO at 50 and 100 mg/mL decreased cell viability by 34% and 78%, respectively. Complex XGJ:VO at 50 and 100 mg/mL decreased cell viability by 18% and 49%, respectively. Therefore, subsequent experiments to evaluate the activation of macrophages

Fig. 4. Effects of ARAGAL:VO, GMPOLY:VO and XGJ:VO on macrophage viability and on nitric oxide production. Murine peritoneal macrophages were exposed for 48 h to the polysaccharides complexed with vanadium at the indicated concentrations. (a) Cell viability was determined by MTT assay. Culture medium was used as negative control, corresponding to 100% viability. The results are expressed as the mean ± SD (n ¼ 3, each experiment triplicate). *p < 0.05; **p < 0.01; ***p < 0.001 significant difference from negative control (medium). (b) Nitric oxide production was determined by nitrite dosage by the Griess method. The results are expressed as mean ± SD (n ¼ 3, each experiment in triplicate). ***p  0.001 significant difference from negative control (medium).

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by polymers and their complexes were carried out at concentrations with over 80% viability. The complexes (Fig. 4a) were more cytotoxic to macrophages than the uncomplexed polymers, since none of them was cytotoxic at equivalent concentrations [17,31,32]. Macrophages are the main host cells of Leishmania and also targets of activation to exert microbicidal effects. It has been proposed that to promote the activation of these cells, the release of mediators such as superoxide anion, nitric oxide, and proinflammatory interleukins, which are involved in the leismanicidal activity, could culminate in the death of parasites. It has been recently shown that the drugs used in leishmaniasis treatment exhibit immunomodulatory effects [52]. The effects of oxovanadium complexes on NO production by macrophages were determined 48 h after treatment, and the results are shown in Fig. 4b. The nitrite/nitrate concentration in the culture supernatant was not significantly altered by any complexes in concentrations of up to 25 mg/mL, when compared with the control group. For GMPOLY and XGJ, the complexation with vanadium eliminated the ability these polysaccharides have to stimulate macrophages for nitric oxide production (Fig. 4b). In the same conditions, GMPOLY increased the nitric oxide production by ~40% at 10 mg/mL in a dose-independent manner [17], while XGJ promoted a ~92% increase at 50 mg/mL [32]. Fig. 5 shows the effects of polysaccharides and their oxovanadium complexes on IL-1b, IL-6 production by macrophages after

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48 h of treatment. In Fig. 5a, it can be observed that 10 mg/mL ARAGAL promoted a 4.6-fold increase in IL-1b production, compared with the control group, while with 10 mg/mL ARAGAL:VO, the production increased 6.6 times. GMPOLY 25 mg/mL and GMPOLY:VO 25 mg/mL increased IL-1b production 7.7 and 11.5 times, respectively compared with the control group, with a significant difference between the complexed and uncomplexed forms of up to 1.5 fold. XGJ and XGJ:VO, at 25 mg/mL, both increased 5.0 and 6.2 times, respectively, the IL-1b level, compared with the control group. Regarding IL-6 production (Fig. 5b), ARAGAL and ARAGAL:VO, at 10 mg/mL, increased the IL-6 production by 4.0 and 6.4 times, respectively, compared with the control group. ARAGAL:VO increased IL-6 production 1.6 fold compared with ARAGAL. GMPOLY and GMPOLY:VO, at 25 mg/mL, increased IL-6 production by around 4.0 to 8.0 times, with a significant difference between the complexed and uncomplexed forms of 2 times. XGJ and XGJ:VO at 25 mg/mL increased IL-6 production by around 3.0 times. 3.3. Effects of polysaccharides and their oxovanadium(IV/V) complexes on intracellular amastigotes of Leishmania (L.) amazonensis The leishmanicidal effect of polysaccharides and their oxovanadium(IV/V) complexes on intracellular amastigotes of Leishmania

Fig. 5. IL-1b and IL-6 production by peritoneal macrophages treated with the polysaccharides and their oxovanadium complexes. Murine peritoneal macrophages were exposed to the polysaccharides complexed with vanadium at the indicated concentrations or the controls. After 48 h, the IL-1b (a) and IL-6 (b) concentration in the supernatant were determined using an enzyme-linked immunosorbent assay (ELISA). The results are expressed in pg/mL and as the mean ± SD (n ¼ 3, each experiment triplicate). *p < 0.05; **p < 0.01; ***p < 0.001 and ns, not significant.

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(L.) amazonensis was indirectly evaluated by promastigote content, as described in Material and Methods. The results of these evaluations are shown in Table 2. ARAGAL (10 mg/mL) reduced the growth of Leishmania promastigote forms by 61%, whereas ARAGAL:VO at concentrations of 2.5, 5, and 10 mg/mL reduced the growth by 39%, 48%, and 67%, respectively, compared with the negative control. GMPOLY (25 mg/mL) showed an effect equivalent to the ARAGAL biopolymer. While the complex GMPOLY:VO reached ~72% activity at concentration of 10 mg/mL, XGJ (25 mg/mL) reduced the growth of Leishmania promastigote form by 59%, and XGJ:VO at concentrations of 5 and 10 mg/mL reduced the growth by 63% and 58%, respectively, compared with the control. An effect around 60% was obtained for macrophages treated with Glucantime (300 mg/mL), which was used as positive control for leishmanicidal activity, but at 12-fold concentration. The 50% inhibitory concentration (IC50) values for complexes were determined. For ARAGAL:VO, the IC50 value was 6.5 mg/mL, with values of 7.3 mg/mL and 6.2 mg/mL for GMPOLY:VO and XGJ:VO, respectively. The results of the present study suggest that the oxovandium(IV/V) complexes of polysaccharides were more potent than their uncomplexed biopolymers, since the complexes exhibited similar effect but at lower concentrations. 3.4. General considerations The activation of macrophages by polysaccharides aiming to potentiate their immunomodulatory effects [16,53], as well as the effect of vanadium compounds on Leishmania, have been evaluated [28e30]. In the present study, all polymers and their oxovanadium complexes exhibited potent leishmanicidal activity and activated macrophages by releasing some mediator involved in the leishmanicidal effect. The intensity of leishmanicidal effects was similar to Glucantime, the antimonial compound used as positive control, however at concentrations 12 times lower (Table 2). Since the infected macrophages were treated with the polymers and their complexes or the antimonial compound, the intracellular amastigotes would be killed by the effect of the biopolymers on macrophage activation. It is believed that the IL-1b and IL-6 levels released by macrophages in the presence of the compounds significantly contributed to the leishmanicidal effects, since the complexes promoted no effect on NO production, an important

Table 2 Effects of the polysaccharides and their oxovanadium complexes on intracellular amastigotes evaluated by the growth of promastigote forms.

Control Glucantime ARAGAL ARAGAL:VO

GMPOLY GMPOLY:VO

XGJ XGJ:VO

Concentration (mg/mL)

Leishmania (L.) amazonensis (% promastigote/mL)

e 300.0 10.0 2.5 5.0 10.0 25.0 5.0 10.0 25.0 25.0 5.0 10.0 25.0

101.9 40.5 40.0 62.5 53.2 33.5 42.8 48.2 28.9 34.9 42.2 81.2 44.1 38.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.2a 14.6b 6.9b 19.9a 28.3ª 15.6b 20.6b 8.5b 14.6c 3.4c 22.3b 6.8a 12.5c 10.7c

IC50 (ug/mL) e e e 6.5

molecule involved in the leishmanicidal activity (Fig. 4b). ARAGAL ~o does not activate the NO pathway as demonstrated by Moreta et al. [31], but increased the superoxide anion in mouse macrophages treated with the biopolymer. The superoxide anion is also important for leishmanicidal activity [54]. In previous studies, GMPOLY and XGJ increased the NO production by macrophages treated with them at 40 and 92%, respectively, and the release of superoxide anion was not simulated [17,32]. Thus, for GMPOLY and XGJ the leishmanicidal effect could be due to the NO and interleukins (IL-1b and IL-6) released by macrophages treated with these biopolymers. The possible effect of interleukin 12 (IL-12) cannot be ruled out, which is described to be also important for leishmanicidal effect and its production by macrophages in the presence of these biopolymers, however, these effects have not been evaluated yet. An expressive increase in IL-12 release has been observed in macrophages treated with sodium antimony gluconate (SAG) and others antileishmanial drugs [51]. According to the IC50 values for leishmanicidal activity of polysaccharide-oxovanadium complexes, XGJ:VO was shown to be a little more potent by exhibiting a lower (6.2 mg/mL) IC50 value than ARAGAL:VO and GMPOLY:VO at 6.5 and 7.3 mg/mL IC50 values, respectively (Table 2). For other oxovanadium-complexed galactomanann (GALMAN-A:VO2þ/ 3þ VO ), a higher IC50 ¼ 74.4 mg/mL for the complex was found, which contained 0.58 mg/mL more of vanadium [21] than GMPOLY:VO. The activation of macrophages for the production of mediators involved in the leishmanicidal effects, as well as the important antileishmanial activity observed, shows the need to investigate the mechanism of action and to evaluate the effect of these compounds in vivo. 4. Conclusion The results showed a varied set of results regarding the inflammatory response in peritoneal macrophages treated with an arabinogalactan (ARAGAL), a galactomannan (GMPOLY), a xyloglucan (XGJ), and their oxovanadium complexes. The increase in interleukins (IL-1b and IL-6) released by macrophages treated with the compounds suggests an intensive Th1 response, which is important for these cells to eliminate intracellular amastigotes of Leishmania. All polymers and their complexes exhibited important leishmanicidal activity (~60e70%). Since immunochemotherapy is viewed as an effective tool to treat leishmaniasis, the results of the present study indicate the promising potential of these polysaccharides for the development of new therapeutic alternatives against leishmaniasis. Studies to evaluate the effects of polysaccharides and their complexes with vanadium in cutaneous leishmaniasis are in progress in our laboratory. Conflict of interest The authors declare that there are no conflicts of interest.

e 7.3

e 6.2

The results are expressed as mean ± SE (n ¼ 3, each experiment in triplicate). a, p < 0.05; b, p < 0.01 e c, p < 0.001, significant difference from negative control (medium). The IC50 values to inhibition of Leishmania by oxovanadium complexes were calculated by nonlinear regression considering three concentrations (2.5, 5.0 and10.0 to ARAGAL:VO; 5.0, 10.0 and 25.0 to GMPLY:VO and XGJ:VO).

Acknowledgments This investigation was supported by the Brazilian research funding agencies CNPq 472651/2012-9, CAPES, and Fundaç~ ao Arauc aria. References [1] S. Kamhawi, Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 22 (2006) 439e445. [2] R. Reithinger, J. Dujardin, H. Louzir, C. Pirmez, B. Alexander, S. Brooker, Cutaneous leishmaniasis, Lancet Infect. Dis. 7 (2007) 581e596. € Ericsson, U. Hellgren, Handbook of Drugs for [3] Y.A. Abdi, L.L. Gustafsson, O. Tropical Parasitic Infections, second ed., Taylor & Francis, London, 2003.

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