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Química Nova

Print version ISSN 0100-4042

Quím. Nova vol.35 no.7 São Paulo  2012

http://dx.doi.org/10.1590/S0100-40422012000700017 

ARTIGO

 

Evaluation of antimicrobial activity and toxic potential of extracts and triterpenes isolated from Maytenus imbricata

 

 

Vanessa G. RodriguesI,*; Lucienir P. DuarteI; Grácia D. F. SilvaI; Fernando C. SilvaI; Jefferson V. GóesI; Jacqueline A. TakahashiI; Lúcia P. S. PimentaI; Sidney A. Vieira FilhoII

IDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte - MG, Brasil
IIDepartamento de Farmácia, Escola de Farmácia, Universidade Federal de Ouro Preto, Rua Costa Sena, 171, 35400-000 Ouro Preto - MG, Brasil

 

 


ABSTRACT

The phytochemical study of hexane/ethyl ether (1:1) extract of the roots of M. imbricata, Celastraceae, resulted in the isolation and characterization of six known triterpenes: 11α-hydroxylup-20(29)-en-3-one, previously isolated from this species besides, 3β,11α-di-hydroxylup-20(29)-ene, 3,7-dioxofriedelane, 3-oxo-29-hydroxyfriedelane, tingenone and 6-oxo-tingenol. The chemical structures of these triterpenes were established by spectrometric data (IR, 1H and 13C NMR) and through comparison with literature data. The hexane/ethyl ether (1:1), ethyl acetate and methanol extracts, and 11α-hydroxylup-20(29)-en-3-one, tingenone and 6-oxo-tingenol, showed antimicrobial properties on in vitro assays. All extracts and triterpenes, except 3β,11α-di-hydroxylup-20(29)-ene, presented toxicity demonstrated by the larvicidal effect test using Artemia salina.

Keywords: Maytenus imbricata; Celastraceae; pentacyclic triterpenes.


 

 

INTRODUCTION

From different species of the genus Maytenus, various groups of secondary metabolites have been found, such as triterpenes,1 sesquiterpenes,2 phenolic glycosides,3 alkaloids,1 flavonoids,4,5 and tannins,5 among others. Members of this genus are important not only in terms of experimentally observed biological activities,6 but also because they are used in folk medicine for gastric diseases,7 and as antiseptic, anti-asthmatic, anti-tumor,8 antiviral,9 and anti-inflammatory agents.10 In a recent review, Niero et al.11 provided an adequate description of the ethnopharmacological, chemical and pharmacological knowledge about species of the genera Maytenus, with particular emphasis on those growing in Brazil. Many species of this genus are effective medicines, and represent promising sources of bioactive substances of medicinal interest.

The indiscriminate use of antibiotics has induced an increase in the incidence of infectious diseases caused by pathogenic microorganisms that have acquired resistance to several antibiotics currently used in clinical treatments. This scenario has led to a continuous, urgent search for new antimicrobial compounds, especially those bearing different chemical structures and having specific mechanisms of action.12 In this context, the compounds isolated from plants have emerged as a promising alternative. Indeed, around 50% of drugs approved by the Food and Drug Administration (FDA) between 1981 and 2006 were of natural origin.13

It has been reported that the molecular diversity of natural products is higher than those derived from chemical synthesis processes. Thus, the biological assays of natural products represent a continuing source of new structural models for compounds with antimicrobial properties and are an important alternative for reducing the incidence of infectious diseases.14

According to the literature, compounds that present brine shrimp (Artemia salina) toxicity, in general also have cytotoxic properties against cells of solid tumors found in humans. This bioassay has being considered as adequate for initial screening of bioactive molecules, paving the way for subsequent tests of greater complexity such as Aedes aegypti larvicidal assays or for testing the response of cancer cells to anti-cancer drugs.15

Maytenus imbricata Mart, ex. Reissek is a shrub or subshrubs found in Cerrado regions (rupestrian fields) of Minas Gerais and Bahia States, Brazil. Leaves, twigs and stems of this species were subjected to biological assays. The CHCl3, EtOAc and EtOH extracts of leaves, the hydroalcoholic extract of roots, the EtOAc extract of stems and epicatechin, isolated from M. imbricatadant activity. The 3,4-seco-friedelan-3-oic acid, isolated from leaves, presented inhibitory activity of ATP synthesis raising possibilities for its potential use in the future development of natural herbicides.16

In this paper, the phytochemical study of the roots of Maytenus imbricata and isolation of the pentacyclic triterpenes (PCTT) 11α-hydroxylup-20(29)-en-3-one (1), 3β,11α-di-hydroxylup-20(29)-ene (2), 3,7-dioxofriedelane (3), 3-oxo-29-hydroxyfriedelane (4), tingenone (5) and 6-oxo-tingenol (6) (Figure 1) were reported.

 

 

The extracts, and some constituents from roots, of M. imbricata were submitted to in vitroerties against the bacteria Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium, Escherichia coli and the fungus Candida albicans. The cytotoxic effect of these constituents was screened through their larvicidal effect on Artemia salina.

 

EXPERIMENTAL

Plant material

Roots of Maytenus imbricata (Celastraceae) were carefully collected so as to prevent damage to the specimen. The collection area is located on Morro de Santana, Ouro Preto municipality, Minas Gerais, Brazil. The plant material was identified by the botanists Dr. R. M. de C. Okano, Departamento de Botânica of the Universidade Federal de Viçosa (UFV) and M. C. T B. Messias, Departamento de Botânica of the Universidade Federal de Ouro Preto (UFOP). A voucher specimen (Nº 27780) was deposited in the collection of the Herbarium of Departamento de Botânica, UFV, Brazil.

General procedures

Purification processes by column chromatography (CC) were carried out using silica gel 60 (0.063-0.200 mm) as the stationary phase. Organic solvents or mixtures of increasing polarity were used as mobile phases. Silica gel 60 GF (Merck) was used to perform analytical [(TLC), 0.25 mm] or preparative [(PTLC), 0.75 mm] thin layer chromatographic processes.

The 1H (400 or 200 MHz) and 13C (100 or 50 MHz3) NMR spectra were obtained on a Bruker Avance DRX-400 or DPX-200 spectrometer, operating at 300 K. The chemical shifts (δ pressed in units of ppm, using TMS as a reference (δ H = δ C = 0) and the coupling constants (J) were expressed in Hz. CDCl3 was used as the solvent for the samples.

The IR spectra of constituents in KBr discs were obtained on a Shimadzu IR408 spectrometer and the results reported in reciprocal centimeters (cm-1) in the range 400-4000 cm-1.

The melting point (mp) ranges were determined on an MQAPF-302 apparatus (Microquímica Equipamentos Ltda, Brazil).

Extraction and isolation of compounds

The collected roots of M. imbricata were powdered in a mill. The powder (1.5 kg) was then submitted to extractions in a Soxhlet apparatus with hexane-ethyl ether (1:1), ethyl acetate, and finally with methanol. During extraction with hexane/ethyl ether (1:1) a solid was formed and separated by filtration giving the hexane/ethyl ether solid material [HES (22.2 g)]. The solvent of the filtrated product was removed in a rotatory evaporator, giving the hexane/ethyl ether (1:1) extract [HEE (16.1 g)]. During extraction using ethyl acetate, another solid was formed and separated by filtration, giving the ethyl val was ethyl acetate extract [EAE (21.2 g)]. Finally, the methanol extract [ME (176.7 g)] was obtained. A total of 2 L of hexane/ethyl ether (1:1), 2 L of ethyl acetate and 2 L of methanol were used in the extraction processes.

The extract HEE (3.0 g) was submitted to silica gel (300.8 g) CC eluted with hexane-EtOAc. Three hundred fifty fractions of 100 mL each were obtained and grouped according to the similar profiles observed in the chromatoplates. Fractions 14 to 19 produced a white crystalline solid (12.3 mg), 0.41% yield and mp 247.7-250.1 ºC. This solid was identified as 3,7-dioxo-friedelane (3). The fractions 44-49 provided a white solid in the form of flakes (63.1 mg), 2.10% yield, mp 154.2-158.8 ºC, subsequently identified as 11α-hydroxylup20(29)-en-3-one (1). Fractions 84-95 produced a white crystalline solid (7.0 mg), 0.23% yield, mp 146.9-148.4 ºC which was identified as 3-oxo-29-hydroxyfriedelane (4). Fractions 107-128 produced an amorphous orange solid (183.3 mg) which was subjected to silica gel CC eluted with Hex, EtOAc and MeOH, pure or in mixtures of increasing polarity, providing 240 fractions of 10 mL each. Solvent evaporation from fractions 211 to 235 (eluted with Hex/EtOAc 6:4) produced an orange solid (64.4 mg), 2.15% yield, mp 145.0-147.9 ºC, which was identified as tingenone (5).

Fractions 205-207 produced an amorphous orange solid (146.7 mg) which was submitted to silica gel CC (47.3 mg) eluted with Hex, EtOAc and MeOH, pure or in mixtures of increasing polarity, thdrawn, fractions 1 to 15 (eluted with Hex/EtOAc 9:1) yielded an orange solid (25.3 mg), which showed three spots when analyzed by TLC. These fractions (1-15) were submitted to silica gel PTLC eluted with Hex/EtOAc 7:3. The less polar compound (Rf ~ 0.9) was isolated as a white solid (11.0 mg), 0.37% yield, mp 212.9 218.6 ºC and was identified as 3β,11α-di-hydroxylup-20(29)-ene (2). Fraction 76 yielded a light yellow solid (8.0 mg), 0.27% yield, mp 218.7-221.4 ºC that was identified as 6-oxo-tingenol (6). These terpenes were characterized through their NMR spectral data and also by comparing with published data.

The extract HES (2.0 g) was submitted to silica gel (107.9 g) CC eluted with Hex, EtOAc and MeOH, pure or in mixtures of increasing polarity, providing 114 fractions of 25 mL each that were grouped according to the similar profiles observed in the chromatoplates.

Fractions 33-42 produced an amorphous brown solid (55.0 mg) which was subjected to silica gel CC eluted with Hex, EtOAc and MeOH, pure or in mixtures of increasing polarity, providing 117 fractions of 3 mL each. After solvent evaporation from fractions 33 to 47 (eluted with Hex/EtOAc 2:8), a white solid in the form of flakes was obtained (14.0 mg), 0.7% yield and mp 154.2-158.8 ºC. This solid was identified as 11α-hydroxylup-20(29)-en-3-one (1).

Fractions 66-85 produced an orange solid (314.0 mg), 15.7% yield, mp 145.0-147.9 ºC, which was identified as tingenone (5).

Fractions 94-102 produced an amorphous brown solid (300.9 mg) which was subjected to silica gel CC eluted with Hex, EtOAc and MeOH, pure or in mixtures of increasing polarity, providing 294 fractions of 3 mL each. Solvent evaporation from fractions 121 to 133 (eluted with EtOAc) produced a light yellow solid (7.9 mg), 0.4% yield, mp 218.7-221.4 ºC which was identified as 6-oxo-tingenol (6).

The extracts EAE, EAS and ME have yet to be submitted to phytochemical studies. However phytochemical prospection17 allowed the detection of the presence of alkaloids, flavonoids and catechin.

Biological assays

Antimicrobial activity

To evaluate antibacterial and antifungal activity, the microdilution method was used to determine the average minimal inhibitory concentrations inhibiting the growth of 50% (MIC50) and 90% (MIC90) of the microorganisms.18 All extracts and the triterpenes 1, 5 and 6 were tested against Salmonella typhimurium (ATCC 13311), Escherichia coli (ATCC 25723), Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778) and Candida albicans (ATCC 18804) . The bacteria and the fungus were maintained in brain heart infusion (BHI) culture medium, at 7 ºC. An initial duplicate screening was carried out using disposable microplates with all samples being tested at a concentration of 100.00 µg/mL. In this step, all extracts and the triterpenes 1, 5 and 6 showed antimicrobial activity. Subsequently, these were tested at concentrations of 250.00, 125.00, 62.50, 31.30, 15.60, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24 and 0.12 µg/mL to determine the minimal inhibitory concentration (MIC50 and MIC90). All 12 microdilution assays were performed in duplicate. Chloramphenicol (MIC50 = 0.24µg/mL and MIC90 = 15.60 µg/mL) was used as a positive control for bacteria and miconazole (MIC50 = 3.32 µg/mL and MIC90 = 600.48 µg/mL) for C. albicans. The inocula of bacteria and fungus used in experiments contained 4.16 x 103 cells/mL. At the end of incubation time (24 h), the plates were analyzed using a Microplate TP-Reader (Thermoplate, Brazil).

Evaluation of toxic potential through Artemia salina larvicidal activity

The cysts of Artemia salina for the test were acquired in Belo Horizonte City, MG, Brazil. The A. salina cysts (10 mg) were added to 100 mL of synthetic marine salt solution (38.0 g/L) previously prepared using deionized water, and maintained under artificial light at 28 ºC. Larvae hatching occurred after 24 h of incubation time. Lapachol, a compound with proven anti-A. salina effect, was used as the control. The extracts and terpenes 1, 3, 4, 5 and 6 isolated from M. imbricata were submitted to larvicidal assays, at concentrations ranging from 1000.00 to 0.06 µg/mL, to determine their toxic potential. The concentration range was chosen based on the highest concentration that showed 100% mortality and at the concentration which induced no death of A. salina. The samples were dissolved in DMSO and their concentration ranges were HES and HEE (15.62 0.48 µg/mL), EAS, EAE and ME (1,000.00-62.50 µg/mL), terpene 1 (31.25-1.95 µg/mL), 3 and 4 (125.00-7.81 µg/mL), 5 (0.97-0.06 µg/mL) and 6 (500.00-31.25 µg/mL). Terpene 2 was not submitted to larvicidal assays against A. salina because it was used in another test and an insufficient amount remained for testing.

 

RESULTS AND DISCUSSION

Phytochemical study

Through the phytochemical study of HEE it was possible to isolate the known pentacyclic triterpenes 1, 2, 3, 4, 5 and 6 (Figure 1). The chemical structures of these constituents were identified based on IR, 1H and 13C NMR spectral data, and by comparing with literature data.

Compound 1 was previously isolated through phytochemical study of the aerial part of M. imbricata.19 In this paper, the isolation of terpenes 2, 3, 4, 5 and 6 from M. imbricata is reported for the first time.

The profile of IR spectra of compounds 1 to 5 were in accordance with the published data for pentacyclic triterpenes.20-23

The 1H NMR spectrum of 1 disclosed signals of 7 methyl groups [δ 1.69 (3H), δ 1.09 (3H), δ 1.07 (6H), δ 1.06 (3H), δ 0.98 (3H) and δ 0.80 (3H)], of methine [triple doublet at δ 3.91 (H11ax-ax, J = 10.8 Hz; and H11ax-eq, J = 4.8 Hz )] and of olefin hydrogens (δ 4.72 and δ 4.60). These data, together with the signals at δ 109.95 (C29) and δ 150.22 (C20) observed in the 13C NMR spectrum, suggested that 1 was a PCTT of lupane series,21 which was then compared with the literature.22

Seven methyl signals [δ 1.69 (3H), δ 1.26 (3H), δ 1.04 (9H), δ 0.96 (3H) and δ 0.79 (3H)], together with the signals of methine hydrogen [δ 3.93 (H11ax-ax, J = 10.8 Hz and H11ax-eq, J = 4.8 Hz) and of methine hydrogen H3 (δ 3.21), were observed in the 1H NMR spectrum of 2.21 Two singlet signals (δ 4.72 and δ 4.59) were attributed to H29 of terminal double bond. The signals of carbons [δ 109.95 (C29) and δ 150.25 (C20)] confirmed the double bond and suggested 2 as member of the PCTT lupane series.21 The NMR spectral data of 2 were in accordance with literature data.21

The signals in the 1H NMR spectrum of 3 at δ 0.77; δ 0.88; δ 0.91; δ 0.96; δ 1.00; δ 1.07 and δ 1.18 were attributed to seven methyl groups, and a doublet at δ 0.88 was associated to another methyl group (C23). These signals are commonly related to PCTT of the friedelane series.21 The 13C NMR spectrum of 3 revealed signals at δ 211.12 and at δ 210.62 corresponding to ketone carbonyls. The chemical shift values found in the 13C NMR spectrum of terpene 3 were compared with literature data.21

The signals at δ 0.73; δ 0.87; δ 1.03; δ 1.04; δ 1.05 and δ 1.22 observed in the 1H NMR spectrum of 4 were associated to 6 methyl groups, and a doublet signal at δ 0.88 to another methyl group (C23), while a doublet at δ droxylated carbon. The 13C NMR spectrum disclosed signals at δ 6.83 assigned to methyl carbon (C23) of PCTT friedelane,21 at δ 213.18 attributed to carbonyl (C3) and at δ 74.78 assigned to hydroxylated carbon (C29). The NMR data of terpene 4 were in accordance with published data.21

The profile of the IR spectrum, especially in the region between 1500-1708 cm-1, characteristic of C=O and C=C bonds, suggested compound 5 as a quinone methide PCTT. 20 The signals observed in the 1H NMR spectrum of 5 at δ 0.98; δ 1.01; δ 1.35; δ 1.51 and at δ 2.23 corresponded to 5 methyl groups, a doublet signal at δ 1.00 was attributed to another methyl group (C30); and a doublet at δ 2.92 (H22) and a multiplet at δ 2.51 were associated to hydrogen linked to C20. These spectral data, together with the doublet signal at δ 7.03 and at δ 6.38; and a singlet at δ 6.55, confirmed 5 as being a quinone methide triterpene.21 The carbon signals at δ 178.43 (C2) and at δ 213.58 (C21) were attributed to carbonyl carbons. The chemical shift values found in the 13C NMR spectrum of terpene 5 were consistent with the literature data.23

The IR spectrum of 6 revealed bands at 1592 and 1458 cm-1 that were associated to the C=C bond of aromatic compound.20 Six methyl signals [singlets at δ 2.63; δ 1.58; δ 1.38; δ 1.00 and δ 0.99, as well as a doublet at δ 0.99 (C30)], were identified in the 1H NMR spectrum of 6. Two singlets, at δ 6.86 (H1) and at δ 6.25 (H7), confirmed the aromatic and olefin hydrogens, respectively. The carbon signals at δ 187.94 (C6) and at δ 214.69 (C21) were associated to carbonyls. The chemical shift values found in the 13C NMR spectrum of terpene 6 were consistent with the literature data.24

Antimicrobial activity

The level of resistance or sensitivity of the bacteria and fungus to the samples was determined by the presence or absence of growth. The results of MIC50 of the extracts and triterpenes from M. imbricata subjected to antimicrobial assays are shown in Table 1.

In accordance with the results, it was found that all samples showed MIC50 within the concentration range used in the experiments. The solid HES, extract HEE, and terpene 5 showed better inhibition of S. aureus growth. Azithromycin is a macrolide antibiotic that suppresses the biosynthesis of protein, retards bacterial growth, or causes death of microorganisms, when tested against S. aureus presented a MIC50 of 4.0 µg/mL.25 This represents a lower activity than that found for HES, HEE, and for terpene 5, which showed a lower value for MIC50. All extracts and terpenes 5 and 6 induced a high rate of growth inhibition of C. albicans (Table 1). Fluconazole is an antifungal used to treat infectious diseases caused by fungus. In assays with C. albicans, fluconazole showed a MIC50 of 1.1 µg/mL.26 The MIC50 value found using HES, HEE and terpene 5, indicated its higher activity against C. albicans, evidencing their potential as antifungal agents.

The extract from leaves of Maytenus ilicifolia showed no activity against Salmonella sp.27 However, in this work, it was verified that all extracts as well as the compounds 1, 5 and 6, obtained from roots of M. imbricata were active against Salmonella typhimurium.

As shown in Table 2, higher antimicrobial activity was found for the extract HEE against S. aureus (MIC90 = 0.12 µg/mL) and for ME against C. albicans (MIC90 = 31.30 µg/mL).

Vancomycin, an antibiotic highly effective against Gram-positive bacteria, has being adopted as a first choice for the treatments of infectious diseases caused by S. aureus. This is the main pathogen associated to hospital-acquired infections. In assays in vitro against S. aureus, vancomycin presented a value higher (MIC100 = 1.5 µg/mL)28 value than that found for HEE (MIC90 = 0.12 µg/mL).

Neomycin is an antibiotic used to prevent or treat skin infections caused by bacteria, including S. aureus. Jain et al.29 tested neomycin against S. aureus, and found MIC100 = 190.0 µg/mL, a value higher than the MIC90 of HES, HEE and compound 5. These authors also tested the ethanol extract from roots of Maytenus senegalensis against S. aureus and found MIC100 = 1250.0 µg/mL. This value is higher than the MIC90 found for the extracts HES, HEE, EAS and for terpenes 1, 5 and 6 (Table 2).

The methanol extract (ME) was active against B. cereus. This bacterium showed high sensitivity when treated with ME extract. In addition, this extract also presented activity against E. coli and C. albicans.

Considering the results of the in vitro assays with bacteria and Candida albicans, a high antibacterial and antifungal activity was attributed to the extracts and terpenes isolated from roots of M. imbricata.

Evaluation of cytotoxic potential

The toxicity assays using Artemia salina, a marine microcrustacean, were carried out according to the methodology described by Pimenta et al..30 The experiments were performed in triplicate and the lethal concentration of sample necessary to induce 50% death (LC50) of brine shrimps, was established using the Probit Method, a parametric statistical procedure with a 95% confidence interval. Extracts of plants or compounds submitted to assays with A salina are considered active when the LC50 is less than 1000.0 µg/mL.31 The evaluation of toxicity using A. salina was performed with the extracts HES, HEE, EAS, EAE and ME, and compounds 1, 3, 4, 5 and 6, followed by the respective determination of LC50 (Table 3).

 

 

The extracts HES and HEE and all terpenes showed high toxicity, evidenced by their low LC50 values (Table 3). Tingenone (5) is a pentacyclic triterpene with known cytotoxic properties.32 In the present work, this terpene showed higher toxicity when compared to the other samples of M. imbricata tested. This terpene is the main constituent found in the extracts HES (40%) and HEE (9.4%). Thus, it is possible to assign the high cytotoxicity observed for these two extracts to compound 5. Using Artemia franciscana, Macari et al.33 studying Maytenus guyanensis found LC50xane extract of barks. Based on this LC50, these authors considered this extract as being a potential larvicide product. Through the assay with A. salina, it was found that, except for the extract ME, all other samples presented values of LC50 lower than 363 µg/mL. Based on these results can be established that substances from M. imbricata also represent potential larvicide agents. Bouzada et al.,34 using a similar methodology with A. salina, found LC50 > 250 µg/mL for the methanol extract from leaves of Maytenus ilicifolia. On the other hand, ethanol extract of Maytenus obtusifolia was considered of low toxicity to the larvae of A. salina, with LC50 greater than 1000 mg/mL.35 The values of this average lethal dose (LC50) are high when compared with those found for HES and HEE in the present work (Table 3). Since the extracts and terpenes showed good activity against A. salina it is possible to conclude that they also have potential antitumor, pesticide, trypanosomicide and/or molluscicide activities.15

 

CONCLUSION

The triterpenes 11α-hydroxylup-20(29)-en-3-one, 3β,11α-dihydroxylup-20(29)-ene, 3,7-dioxofriedelane, 3-oxo-29-hydroxyfriedelane, tingenone and 6-oxo-tingenol were isolated from roots of Maytenus imbricata. All extracts, and the terpenes 11α-hydroxylup20(29)-en-3-one, tingenone and 6-oxo-tingenol, were active against S. typhimurium, E. coli, S. aureus, B. cereus and C. albicans. The extracts HES and HEE and the terpenes 1, 3, 4, 5 and 6 resentative larvicidal effects against Artemia salina. The results of this work indicate the promising potential of this plant as a larvicide, or as a source of drugs with antimicrobial or antitumor properties.

 

SUPPLEMENTARY MATERIAL

The Figures 1S-21S present spectra of IR, 1H and 13C NMR of compounds isolated of hexane/ethyl ether (1:1) extract of the roots of M. imbricata. The Tables 1S-6S present comparison of 13C NMR data of compounds and of literature. This supplementary material is available free of charge at http://quimicanova.sbq.org.br, as PDF file.

 

ACKNOWLEDGMENTS

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) by financial support.

 

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Recebido em 3/10/11; aceito em 26/2/12; publicado na web em 2/7/12

 

 

* e-mail: vanessa.greg@yahoo.com

 

 

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