Metals and inorganic compounds have been used in medical therapies since the sixteenth century (Sadler, 1991; Abdel-Rahman, 2013; Abdel-Rahman, 2016)    . In 1969, the antitumor activity of metallic complexes containing cisplatin was discovered (Katsaros and Anagnostopoulou 2002)  enabling the development of new antitumor drugs such as complexes involving organic metals and/or inorganic platinum, ruthenium and rhodium (PAULA et al. 2005; Waxman and Anderson 2001)   . Gallori and colleagues suggest that compounds containing ruthenium can bind to the DNA molecule, and that this interaction can alter the cell cycle, causing cell death through mechanisms such as apoptosis (Gallori et al. 2000)  . Evidence also suggests that several ruthenium complexes interact with specific proteins. One such application is the use of red ruthenium, which is traditionally used as a cytological stain for electron microscopy. In this case, binding of the compound occurs in the anionic sites of calcium-binding proteins (Clarke 2003; Suriano et al. 2005)   . It was observed that several compound Ru2+ and Ru3+ amine ligands tend to interact selectively the carbonyl and imino sites of biomolecules that do not protonam at neutral pH. This leaves the pairs of electrons (of Nitrogen) available to coordinate bond with metal ions. Accordingly, ruthenium complexes often bind to proteins and imidazole nitrogen purine nucleotide (Clarke et al. 1978; Gupta et al. 2011)   . Vilanova-Costa investigated the action of the compound cis-[RuCl2(NH3)4], which showed antitumor activity in vitro on human tumor cell lines and in vivo on a tumor mouse strain (Sarcoma-180 (S-180) (Pereira et al. 2014; Vilanova-Costa et al. 2014; Vilanova-Costa et al. 2015)    . However, despite its pharmacological potential, the activity of ruthenium against leishmaniasis has not been extensively studied, even though the formation of granulomas that is characteristic of chronic leishmaniasis is similar to that observed in solid tumors (Arrais-Silva et al. 2006)  . Works that demonstrate the action of ruthenium against L. Mexicana promastigotes indicate the possible therapeutic action of the compounds of ruthenium (Navarro et al. 2006)  . Using in vitro models, Navarro (Navarro et al. 2006)  also demonstrated that some compounds containing the metal ruthenium decrease the L. Mexicana promastigotes forms by between 36% and 49% after treatment for 48 hours at a dose of 10μM, with loss of movement, fission of parasitic forms and vacuolation abundant in the host cell. The present study reports the synthesis of [Ru(Cl)3(H2O)2(gly)] and its characterization by UV-VIS analysis, IR, TG/DTG and TG/DTA. A biological analysis of the effectiveness of the ruthenium compound in treating an experimental model of leishmaniasis was also carried out.
Materials: For synthesis of analytical grade complex compounds, RuCl3∙3H2O and glycine (NH2CH2COOH) were used. Distilled water, ethyl ether (C4H10O) and absolute alcohol (C2H6O) were used as solvents.
Synthesis: For the synthesis of compound [Ru(Cl)3(H2O)2(gly)], 300.0 mg (1.440 mmol) of RuCl3∙3H2O was solubilized in 9.0 mL of distilled water, then 1000 mg (13.32 mmol) of glycine was added to the mixture under constant stirring, protected from light. After solubilization, the solution was allowed to reflux for 4 hours. The solution was held under ambient cold for 24 hours for precipitation. The precipitates were then filtered, washed with ethanol, ether and dried under reduced pressure. The solution was cooled again to obtain a higher yield of the precipitate. The solid was again washed with ethanol, ether and dried under reduced pressure.
Cell culture and parasites: Primary mouse macrophages were obtained from normal BALB/c mice by peritoneal lavage (Barbiéri et al. 1993; Giorgio et al. 1998)   . Leishmania Amazonensis (MHOM/BR/73/M2269) amastigote forms were isolated from active skin lesions of BALB/c mice as described previously (Barbiéri et al. 1993)  . The parasites were suspended in RPMI 1640 medium and used immediately after isolation.
Macrophage infection and assessment of intracellular parasites: Peritoneal macrophages were infected with L. Amazonensisamastigotes (5:1 parasites/host cell) for 1 h, as previously described (Colhone et al. 2004)  . After the interaction period, the cultures were washed to remove extracellular parasites and fresh medium was added to the cell culture and incubated at 35˚C, 5% of CO2 for 24 h, as previously described (Degrossoli and Giorgio 2007)  . Alternatively, different concentrations of RuCl3∙3H2O and glycine were added to culture of infected cells. Intracellular parasite destruction was assessed by morphological examination. Briefly, in order to evaluate the percentage of infected macrophages and the number of amastigotes per macrophage, cells cultured on coverslips were stained with Giemsa 0.6% (Giorgio et al. 1998)  . Intracellular amastigotes, which are exclusively localized in parasitophorous vacuoles (Chang 1980)  , were examined microscopically at a magnification of 1000×. About 600 cells were counted per triplicate coverslip (Linares et al. 2000)  .
Cytotoxicity assay: The cytotoxicity of ruthenium to macrophages was tested using the MTT viability assay, after incubation of peritoneal-derived macrophages with 50 to 400 µg/mL of ruthenium for 24 hours. The formation of formazan was measured by adding 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Molecular Probes, Eugene, OR, USA) and incubating the cultures at 37˚C in the dark. After 4 h the medium was removed, 200 uL of DMSO (dimethyl sulphoxide) was added per well and the absorbance was measured using an ELISA reader at 540 nm (LabsystemsMultiskan). As a negative control, cells were also incubated with the highest concentration of DMSO used for ruthenium solubilization (0.01%).
Data analysis: The results were expressed as mean ± standard deviation (SD). To compare the average values of the parameters, analysis of variance (ANOVA) followed by multiple comparisons of Tukey test were used, with a statistical significance threshold of 5% (p < 0.05). The BioEstat version 5.0 software was used for all analyses (Ayres et al. 2007)  .
Instrumental Methods: For analysis of UV-VIS and IR for the compound of ruthenium equipment of the company Perkin Elmer®, model Lambda 25 UV-VIS analysis for the region of visible and UV-Spectrum-100 for the mid-infrared region were used. For the analysis of TG/DTG and TG-DTA, 6.6204 mg of the compound [Ru(Cl)3(H2O)2gly] was placed in aα-alumina crucible and 90 uL of the sample was heated to 800˚C at a heating rate of 20˚C/min under an atmosphere of dry air a flow rate of 100 mL/min; the SDT 2960 TA Instruments® equipment was used for this analysis.
3. Results and Discussion
Ultraviolet visible analysis: Figure 1 shows the UV-Vis spectrum of the compound [Ru(Cl)3(H2O)2(gly)] in water indicating the band 290 nm. The literature shows that ruthenium-glycine compounds exhibit absorption in the 290 nm region, little intense, as characterized as LMCT-type interactions (Load Transfer Ligand Metal) between the oxygen of the carboxyl group (glycine) and metallic ion (ruthenium) (Yeh and Taube 1980)  . the band found in the ultraviolet region at 230 nm, can be attributed to internal transitions of the ligand (IL), and were very similar in intensity to electronic transitions π - π* free ligand (Bento and Tfouni 1988)  .
Analysis in infrared medium region: Table 1 highlights the values of the vi-
Figure 1. UV-vis spectrum of compound [Ru(Cl)3(H2O)2(gly)] 2 × 10−4 mol∙L−1 (290 nm ε = 1685 L∙cm−1∙mol−1) and glycine (194 nm ε = 13 129 L∙cm−1∙mol−1).
Table 1. IR experimental for groups identified in the analyses.
νa: antisymmetrical stretching, νs: symmetrical stretching, νsh: sholder stretching, δa: asymmetric bending, δs: symmetrical bending, ρw: wagging, ρr: rocking.
brational groups and their respective wave numbers, when comparing the experimental vibrational spectra of free glycine and synthesized [Ru(Cl)3(H2O)2(gly)].
The analysis of vibrational modes in the IR spectrum of glycine ligand, the carboxylate ion (COO−) shows a peak at 1664 cm−1, indicating their ionized form. The literature shows that compounds with the glycine ligand with bidentate characteristics decrease significantly the distance between the symmetric and asymmetric vibrational modes of the carboxylate ion. However, the increase in value of the distance between the vibrational modes symmetric and asymmetric carboxylate ion indicates that this group will form a monodentate connection with the metallic center (Kitamura et al. 2014; NAKAMOTO 1978)   . When analyzing the data obtained for the compound [Ru(Cl)3(H2O)2(gly)], when compared to the free ligand, we observed an increase in the difference between the symmetric and asymmetric displacement of the carboxylate ion, which indicates the formation of a monodentate connection between the carboxylate ion (glycine) and Ru3+ metal (Figure 2).
The data also show the presence of peaks relating to the bending group ( ) in δδ ( ) 1571 cm−1 and δσ ( ) 1490 cm−1 indicating the zwitterionic state of the glycine present in the complex. The displacement observed in the symmetric stretch of the -C-N group, in the compound [Ru(Cl)3(H2O)2(gly)] compared to the free ligand (glycine), did not change significantly, νs (-C-N)free ligand
Figure 2. Variation of the group Δ(νaCOO− - νs COO−), compound Glycine and complex [Ru(Cl)3(H2O)2(gly)].
892 cm−1 and νs (-C-N)compound 889 cm−1. Vibrations related to bending and twisting group (CH2) glycine in the complex suffered small variations in the region of δ (CH2) 1436 cm−1 and ρω (CH2) 1334 to 1322 cm−1 as compared to free glycine. Specifically, in the regions ν (CH2) 2869 cm−1 and δσχ (CH2) 2167 cm−1, it was possible to observe significant vibrational changes in relation to the free ligand, which may be attributed to intermolecular interaction between the and COO− groups.
TG/DTG and TG/DTA curves: The TG/DTG and TG/DTA curves shown in Figure 3 demonstrate the thermal decomposition of the complex in five consecutive steps. The first stage indicates the output of the ligand H2O (TG(experimental) = 12.18%; TG(theoretical) = 11.32%) with an endothermic peak at 115˚C. Studies with compounds of ruthenium complexed to ligands water, amine and glycine were obtained results similar to those observed in this work as the output water temperature ligand (Kohata et al. 1985)  . The following steps indicate thermal decomposition to 490˚C with formation of the residue RuClO. (TG(experimental)= 41.11%; TG(theoretical) = 40.81%). The last four steps are characterized by exothermic peaks at 230˚C, 307˚C, 440˚C and 463˚C due to oxidative decomposition of glycine monoxide and carbon dioxide.
Based on data obtained by infrared and thermal analysis, we propose a specific structure for the complex [Ru(Cl)3(H2O)2(gly)], as shown in Figure 4.
Effect of ruthenium and glycine compound treatment cell viability
In order to analyze the effects of the ruthenium-glycine compound in an experimental model of leishmaniasis, cytotoxicity on the infection of macrophages had their effects analyzed. Cell viability was assessed using the MTT assay. The negative control cells (CLT) showed 100% cell viability. No cell toxicity was ob-
Figure 3. (a) TG/DTG curve; (b) TG-DTA curve. Compound: [Ru(Cl)3(H2O)2(gly)].
served for the ruthenium compound at doses between 50 and 400 µg/mL (Figure 5).
Effect of [Ru(Cl)3(H2O)2(Gly)] compound and free glycine ligand in experimental infection of murine macrophages infected with Leishmaniaamazonensis.
Two controls (untreated cells and cells treated with DMSO) were used in this analysis. As expected, these controls showed no significant changes in cell infection percentages.
Figure 4. Structural representation for the compound Ru(Cl)3(H2O)2(gly)]. Image obtained by GaussView 5.0 program.
Figure 5. Effect of compound III Ruthenium and Ruthenium on cell viability of murine peritoneal macrophages. Cell viability was determined by MTT test. Peritoneal macrophages of mice were exposed for 24 hours to the compounds III and Ruthenium Ru in different concentrations. As a positive control was used Glucantime® in the concentration of 300 µg/mL. After 4 h incubation with MTT, this was removed and added DMSO. As a negative control was used only RPMI culture. The absorbance was measured at 492 nm. 100% viability corresponds to determined viability for the negative control. Results are expressed as mean ± DP. Results Ruthenium (n = 7, each experiment being in five replications). *Significant difference from the negative control when p < 0.05; **p ˂ 0.01.
When cultures of macrophages were infected by the parasite L. Amazonensis and treated with 50 µg/mL of the compound, there were no changes in the percentage of infection when compared to control. In contrast, treatment with concentrations of 100 and 400 µg/mL was able to reduce the infection of macrophages by 31% at both doses examined. Similarly, a 49% reduction in the percentage of infected cells was found when macrophages were treated with 400 µg/mL [Ru(Cl)3(H2O)2(gly)] compound (Figure 6(a)). The parasite load was evaluated using the number of parasites per cell. The control conditions showed a parasitic load of 2.0 amastigotes per cell (Figure 6(b)). When macrophages were treated with the compound at a range of concentrations of 200 mg/mL to 400 mg/mL, the number of parasites per cell fell from 1.5 to about 3 per cell (Figure 6(b)).
Figure 6. Effect of compound Ruthenium in murine peritoneal macrophages infected with L. amazonensis amastigotes. Peritoneal macrophages of mice were incubated for 1 h with L. amazonensis amastigotes in the ratio 5:1 then exposed for 24 h at 37˚C and 5% CO2 to ruthenium at the indicated concentrations. As a negative control were used macrophages withdrawn from the peritoneum of Balb/c only grown in culture medium RPMI 1640 and control treated with DMSO at 0.1%. (a) The average number of infected macrophages adhered to the glass coverslips treated with (50 µg /mL to 400 µg/mL) and stained with Giemsa, was obtained by counting by optical microscopy. The values obtained by the average percentage of infected macrophages in independent events made in triplicate; (b) Average number of amastigotes determined in cultured macrophages when exposed to ruthenium treatment for 24 h with the indicated concentrations and counted MO. The result is Mean ± DP in triplicate. The significance of the difference among treatment is indicated in the figure (*p ≤ 0.05; **p ≤ 0.01).
To investigate the effects of isolated glycine ligand, macrophages were treated with different doses of the ligand in a concentration range of 100 mg/mL to 750 mg/mL. When glycine doses below 500 mg/mL were used there were no significant changes in the percentage of infection, as compared to the control (Figure 7(a)). Under the same experimental conditions, a dose of 750 mg/mL glycine reduced the percentage of infected macrophages by 44% (Figure 7(b)).
Thermal analysis allowed the determination of the stoichiometry of the ruthe-
Figure 7. Effect of Glycine in murine peritoneal macrophages infected with L. amazonensis amastigotes. Macrophages were incubated for 1 h with L. amazonensis amastigotes in the ratio 5: 1, and then exposed for 24 h at 37˚C and 5% CO2 glycine at the indicated concentrations. As a negative control were used peritoneal macrophages removed from BALB/c mice cultured alone in RPMI 1640 culture medium (a) The number of infected macrophages adhered to the glass coverslips treated with glycine (100 µg mL to 750 µg/ml) and stained with Giemsa, they were obtained by counting by optical microscopy. The values obtained by the average percentage of infected macrophages in independent events done in quadruplicate; (b) Average number of amastigotes determined in cultured macrophages when exposed to treatment with glycine for 24 h and counted at the indicated concentrations to MO. The result is Mean ± DP in triplicate. The significance of the difference among treatment is indicated in the figure (*p ≤ 0.05; **p ≤ 0.01).
nium-glycine compound [Ru(Cl)3(H2O)2(gly)], however, it cannot determine the correct isomerism compound. With the analysis of the compound in the infrared region, it was possible to determine the shape of connection between the glycine ligand and metal ruthenium. It is observed that the glycine carboxylate group coordinated to the metal by only one of the oxygens, monodentate connection, thus assisting the determination of the formulation observed on thermal analysis. Spectroscopy in the UV-visible region reveals that the compound exhibits charge transfer processes LMCT between the metal and the ligand glycine. Treatment of cells infected with L. Amazonensis with the compound at concentrations of 100 and 400 µg/mL was able to reduce the infection of macrophages by 31%. The parasitic burden, evaluated by the number of parasites per cell, also showed a 50% decrease when macrophages were treated with the compound at concentrations of 200 µg/mL to 400 µg/mL.
Mato Grosso research support foundation (FAPEMAT), Federal University of Mato Grosso (UFMT).
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