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Chemical constituents from Sidastrum paniculatum and evaluation of their leishmanicidal activity

Abstract

Sidastrum paniculatum (L.) Fryxell, Malvaceae, is popularly known in Brazil as “malva-roxa” or “malvavisco”. The species is found mainly in Northeast region where it is used by locals to treat spider bites and bee stings. Aiming to identify the chemical compounds from S. paniculatum secondary metabolism and to contribute to the chemotaxonomic knowledge of Malvaceae family, a phytochemical study of S. paniculatum was carried out. Besides that, the isolated compounds were evaluated for antileishmanial activity against promastigotes of Leishmania braziliensis. By using chromatographic techniques the study resulted the isolation of eight compounds: 3-oxo-21β-H-hop-22(29)-ene; sebiferic acid; sitosterol 3-O-β-d-glucopyranoside/stigmasterol 3-O-β-d-glucopyranoside; phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein. The structure of all isolated compounds was elucidated by spectroscopic analysis, including two-dimensional NMR techniques. In addition, the isolated compounds phaeophytin a; 132(S)-hydroxyphaeophytin a; 132(S)-hydroxy-(173)-ethoxyphaeophorbide a and 7,4′-di-O-methylisoescutellarein exhibited antileishmanial activity against promastigotes of L. braziliensis.

Keywords
Sidastrum paniculatum ; Malvaceae; Leishmania braziliensis ; Antileishmanial activity

Introduction

Sidastrum paniculatum (L.) Fryxell, Malvaceae, is popularly known in Brazil as “malva roxa” or “malvavisco”. The species is wide spread in Northeast region, especially in Paraiba and Pernambuco states (Baracho, 1995Baracho, G.S.O., (Bacharelado em Biologia) 1995. Gênero Sidastrum Baker F. (Malvaceae) em Pernambuco e Paraíba, Brazil. Monografia. Universidade Federal da Paraíba, João Pessoa, PB.). The Sidastrum genus comprises eight species with neotropical distribution, occurring from Mexico to Argentina. Previous phytochemical studies on Sidastrum species have reported the presence of steroids, phenolic acids, flavonoids and phaeophytins (Gomes et al., 2011aGomes, R.A., Ramirez, R.R.A., Maciel, J.K.S., Agra, M.F., Souza, M.F.V., Falcão-Silva, V.S., Siqueira-Junior, J.P., 2011a. Phenolic compounds from Sidastrum micranthum (A St.-Hil.) fryxell and evaluation of acacetin and 7,4′-di-O-methylisoscutellarein as modulator of bacterial drug resistance. Quim. Nova 34, 1385-1388.). From S. paniculatum were previously isolated steroids, a feruloyl derivative and a glycosil flavonoid (Cavalcante et al., 2010Cavalcante, J.M.S., Nogueira, T.B.S.S., Tomaz, A.C.A., Silva, D.A., Agra, M.F., Carvalho, P.R.C., Ramos, S.R.R., Nascimento, S.C., Gonçalves-Silva, T., Souza, M.F.V., 2010. Steroidal and phenolic compounds from Sidastrum paniculatum (L.) Fryxell and evaluation of cytotoxic and anti-inflammatory activities. Quim. Nova 33, 846-849.). Many species from Malvaceae family are used in folk medicine to treat diseases, S. paniculatum leaves are traditionally used as topical treatment to spider bites and bee stings. Sidastrum micranthum leaves are used to prepare a tea to treat asthma and bronchitis (Gomes et al., 2011bGomes, R.A., Agra, M.F., Souza, M.F.V., 2011b. Constituintes químicos de Sidastrum micranthum (Malvaceae). In: 34ª Reunião Anual da Sociedade Brasileira de Química, Florianópolis, Brazil.). Sida rhombifolia is used in Indian folk medicine against hypertension, diabetes and gout (Chaves et al., 2013Chaves, O.S., Gomes, R.A., Tomaz, A.C., Fernandes, M.G., Graças Mendes Jr., L., Agra, M.F., Braga, V.A., Souza, M.F.V., 2013. Secondary metabolites from Sida rhombifolia L. (Malvaceae) and the vasorelaxant activity of cryptolepinone. Molecules 18, 2769-2777.). Hibiscus esculentus and Malva neglecta are used in popular medicine to treat ulcer and stomachache. In fact, these plants have showed to possess an effective gastroprotective effect against ulcers induced by ethanol in rats (Gürbüs et al., 2003Gürbüs, I., Üstün, O., Yesilada, E., Sezik, E., Kutsal, O., 2003. Antiulcerogenic activity of some plants used as folk remedy in Turkey. J. Ethnopharmacol. 88, 93-97., 2005Gürbüs, I., Özkan, A.M., Yesilada, E., Kutsal, O., 2005. Antiulcerogenic activity of some plants used in folk medicine of Pinarbasi (Kayseri, Turkey). J. Ethnopharmacol. 101, 313-318.). The methanol extract of Herissantia crispa, species rich in phenolic compounds, showed antidiarrhoeal and antiulcerogenic activity on the HCl/ethanol-induced gastric lesions (Lima et al., 2009Lima, I.O., Costa, V.B.M., Matias, W.N., Costa, D.A., Silva, D.A., Agra, M.F., Souza, M.F.V., Lima, E.O., Batista, L.M., 2009. Biological activity of Herissantia crispa (L.) Brizicky. Rev. Bras. Farmacogn. 19, 249-254.). Other relevant pharmacological activities of Malvaceae species are well reported such as anti-inflammatory, antioxidant and antibacterial activities (Silva et al., 2006Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S., Matias, W.N., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes Químicos e atividade antioxidante de Sida galheirensis ULBR (Malvaceae). Quim. Nova 29, 1250-1254.; Falcão-Silva et al., 2009Falcão-Silva, V.S., Silva, D.A., Souza, M.F.V., Siqueira-Júnior, J.P., 2009. Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae). Phytother. Res. 23, 1367-1370.; Costa et al., 2009Costa, D.A., Matias, W.M., Lima, I.O., Xavier, A.L., Costa, V.B.M., Diniz, M.F., Agra, M.F., Batista, L.M., Souza, M.F.V., Silva, D.A., 2009. First secondary metabolites from Herissantia crispa L. (Brizicky) and the toxicity activity against Artemia salina Leach. Quim. Nova 32, 48-50.; Silva et al., 2005Silva, D.A., Chaves, M.C.O., Costa, D.A., Morais, M.R.R., Nóbrega, F.B.P., Souza, M.F.V., 2005. Flavonoids from Herissantia tiubae. Pharm. Biol. 43, 197-200.; Karou et al., 2005Karou, D., Savadogo, A., Canini, A., Yameogo, S., Montesano, C., Simpore, J., Colizzi, V., Traore, A.S., 2005. Antibacterial activity of the alkaloids from Sida acuta. Afr. J. Biotechnol. 4, 1452-1457.; Ghosal et al., 1975Ghosal, S., Chauhan, R., Mehta, R., 1975. Chemical constituents of Malvaceae: alkaloids of Sida cordifolia. Phytochemistry 14, 830-832.; Oliveira et al., 2012Oliveira, A.M.F., Pinheiro, L.S., Pereira, C.K.S., Matias, W.N., Gomes, R.A., Chaves, O.S., Souza, M.F.V., Almeida, R.N., Assis, T.S., 2012. Total phenolic content and antioxidant activity of some Malvaceae family species. Antioxidants 1, 33-34.).

Leishmanicidal activity against Leishmania major has also been reported for Malvaceae species (Rocha et al., 2005Rocha, L.G., Almeida, J.R.G.S., Macedo, R.O., Barbosa-Filho, J.M., 2005. A review of natural products with antileishmanial activity. Phytomedicine 12, 514-535.). The leishmaniasis is caused by more than 20 species of protozoan parasite that belongs to Leishmania genus and has great epidemiological and clinical diversity (Kamhawi, 2006Kamhawi, S., 2006. Phlebotomine sand flies and Leishmania parasites: friends or foes?. Trends Parasitol. 22, 439-445.; Mishra et al., 2009Mishra, B.B., Singh, R.K., Srivastava, A., Tripathi, V.J., Tiwari, V.K., 2009. Fighting against leishmaniasis: search of alkaloids as future true potential anti-leishmanial agents. Mini Rev. Med. Chem. 9, 107-123.). Leishmania (Viannia) braziliensis is endemic in Latin America and is the causative agent of mucocutaneous disease in the Américas (Brelaz et al., 2012Brelaz, M.C., Oliveira, A.P., Almeida, A.F., Assis-Souza, M., Brito, M.E., Pereira, V.R., 2012. Antigenic fractions of Leishmania(Viannia) braziliensis: the immune response characterization of patients at the initial phase of disease. Parasite Immunol. 34, 183-239.). The current chemotherapeutic arsenal consists of pentavalent antimony, pentamidine, various formulations of the antibiotic amphotericin B, and recently miltefosine (Croft and Olliaro, 2011Croft, S.L., Olliaro, P., 2011. Leishmaniasis chemotherapy - challenges and opportunities. Clin. Microbiol. Infect. 17, 1478-1483.). These drugs are widely prescribed despite their toxicity, high cost and difficult administration (Croft et al., 2006Croft, S.L., Sundar, S., Fairlamb, A.H., 2006. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 19, 111-126.). All these facts show the urgent need for the research and development of new leishmanicidal compounds, including from natural sources (Santos et al., 2008Santos, D.O., Coutinho, C.E., Madeira, M.F., Bottino, C.G., Vieira, R.T., Nascimento, S.B., Bernardino, A., Bourguignon, S.C., Corte-Real, S., Pinho, R.T., Rodrigues, C.R., Castro, H.C., 2008. Leishmaniasis treatment - a challenge that remains: a review. Parasitol. Res. 103, 1-10.).

In order to increase the knowledge about S. paniculatumphytoconstituents and aiming to contribute to the chemotaxonomic knowledge of Malvaceae family, the species S. paniculatum was submitted to a phytochemical study. In addition, isolated compounds were evaluated for antileishmanial activity against promastigotes of L. braziliensis.

Materials and methods

General experimental procedures

The isolation of chemical constituents was performed on glass chromatographic columns using Silica (ASTM, 230–400 mesh, Merck) or Sephadex LH-20 as stationary phase. TLC were performed on silica gel PF254 plates and the spots were visualized under UV light (254 and 366 nm) and by exposure to iodine vapor and vanillin–sulphuric acid reagent. Isolated compounds were identified by Infrared (IR), Perkin-Elmer FT-IR-1750 and Shimadzu – Prestige 21 model using KBr discs; and 1D and 2D NMR analysis (1H 500 MHz, 13C 125 MHz – Varian and 1H 400 MHz, 13C 100 MHz – Bruker) using deuterated chloroform, DMSO or pyridine. Melting points were measured in a MQAPF-302 apparatus (Microquimica Equipamentos Ltda).

Plant material

The aerial parts of Sidastrum paniculatum (L.) Fryxell, Malvaceae, were collected in Pedra da Boca Park, located in Araruna city, Paraiba/Brazil, in June 2008 (SISBIO Authorization Number 46923-2). The plant was identified by Prof. Dr. Maria de Fátima Agra, and a voucher specimen (JPB 6051) was authenticated and deposited at Lauro Pires Xavier Herbarium (CCEN/UFPB).

Extraction and isolation

The plant material was dried in an oven at 40 °C for 72 h. After that, it was grounded in a mechanical mill, yielding 3.7 kg of a powder which was submitted to maceration with ethanol during three consecutive days. This process was repeated in order to maximize the extraction of chemical constituents. A rotary evaporator was used to concentrate the extract resulting 250 g of crude ethanol extract (CEE). The CEE was partitioned with n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc) and n-butanol. From this process were obtained 82 g of n-hexane phase (HP), 32 g of CHCl3 phase (CP), 3 g of EtOAc phase (EAP), 10 g of n-butanol phase (BP) and 110 g of hydroalcohol phase (HAP).

HP (7 g) was subjected to column chromatography with silica gel (210 g of silica gel packed in a glass column with 4 cm of diameter) and eluted with hexane, dichloromethane and methanol resulting 25 fractions which were analyzed and combined by analytical TLC. The 05/06 fraction yielded a precipitate that was washed with hexane to yield 20 mg of colorless crystals (1). The 10/12 fraction, a colorless solid, was pure when analyzed by TLC, being labeled as compound 2 (12 mg). The fraction 20/22 showed a white powder soluble in pyridine, the powder was washed with hexane resulting the mixture of the compounds 3 and 4 (30 mg). The fractions 13/18 (900 mg) were rechromatographed in silica column (27 g of silica gel in glass column with 2 cm of diameter) resulting the isolation of the compounds 5 (50 mg), 6 (15 mg) and 7 (19 mg).

An aliquot (2 g) of EAP was chromatographed in Sephadex column (Sephadex gel length of 30 cm) using methanol as solvent. The fractions were analyzed by TLC, joined, and the chromatography was repeated to isolate the compound 8 (20 mg).

In vitro activity against L. braziliensis

Promastigotes of L. braziliensis (MHOM/BR/87/BA125) were obtained from Dr. Valéria de Matos Borges (Centro de Pesquisa Gonçalo Moniz/FIOCRUZ/BA). The parasites were maintained in vitro in Schneider's medium, supplemented with 10% FBS and 2% human urine. Stock solutions of compounds and pentamidine (reference leishmanicidal drug) were prepared in DMSO immediately before use. The cytotoxicity of compounds isolated from S. paniculatum and pentamidine against promastigotes was determined. Stationary phase L. braziliensis promastigotes were plated in 96-well vessels (Nunc) at 1 × 105 cells per well, in Schneider's medium, supplemented with 10% FBS and 2% human urine. Each compound solution was added at the following concentrations: 0.01 µM, 0.1 µM, 1 µM, 10 µM and 100 µM.

Cells were also cultured in a medium free of compounds or vehicle (basal growth control) or with DMSO 0.1% (vehicle control). After 48 h, extracellular load of L. braziliensis promastigotes was estimated by counting the promastigotes in Schneider's medium in a CELM automatic cell counter (model CC530) (Rangel et al., 1996Rangel, H., Dagger, F., Hernandez, A., Liendo, A., Urbina, J.A., 1996. Naturally azole-resistant Leishmania braziliensispromastigotes are rendered susceptible in the presence of terbinafine: comparative study with azole-susceptible Leishmania mexicanapromastigotes. Antimicrob. Agents Chemother. 40, 2785-2791.).

Results and discussion

Structure elucidation of isolated compounds

By using typical chromatographic procedures the compounds 1-8 were isolated from the aerial parts of S. paniculatum.

The IR spectra of 1 suggested the presence of olefinic, methyl, methylene, methynic hydrogens and carbonyl by showing bands at 3050; 2943–2862 and 1708 cm-1, respectively. The 1H NMR showed seven methyl singlets, one of those presenting characteristic chemical shift of proton bonded to sp2 carbon (δH 1.72). Olefinic protons were found as a broad singlet at δH 4.76 indicating the presence of isopropenyl group. The 13C NMR showed 30 carbons and the isopropenyl group and the carbonyl were confirmed by the signals at δC 148.64, δC 110.06 and δC 220.51. HMQC and HMBC correlations showed that the carbons are consistent with a hopane-type skeleton with a carbonyl at position C-3. Comparisons with the literature led to identify the compound 1 as 3-oxo-21β-H-hop-22(29)-ene (David et al., 2004David, J.P., Meira, M., David, J.M., Guedes, M.L.S., 2004. Triterpenos e ferulatos de alquila de Maprounea guianensis. Quim. Nova 27, 62-65.; Sousa et al., 2012Sousa, G.F., Duarte, L.P., Alcântara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M., Takahashi, J.A., 2012. New triterpenes from Maytenus robusta: structural elucidation based on NMR experimental data and theoretical calculations. Molecules 17, 13439-13456.).

Compound 2 showed IR bands at 1708, 2943, 2862 and 3435 cm-1 suggesting presence of hydroxyl, double bond and carbonyl groups in the molecule. The 1H NMR spectra showed a triterpenoid profile and the presence of two isopropenyl groups was indicated by the presence of vinyl methyls (δH 1.77 and δH 1.73) and exomethylenes protons at δH 4.76 (2H), δH 4.80 (1H) and δH 4.87 (1H). The 13C NMR of compound 2 confirmed the triterpenoid skeleton and the two terminal double bonds were confirmed by the presence of the signals δC 148.65, δC 147.83, δC 113.00 and δC 110.09. The 2D correlations were carefully analyzed thus the compound 2 was identified as sebiferic acid, a 3,4-seco-hopane-type triterpene (Sousa et al., 2012Sousa, G.F., Duarte, L.P., Alcântara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M., Takahashi, J.A., 2012. New triterpenes from Maytenus robusta: structural elucidation based on NMR experimental data and theoretical calculations. Molecules 17, 13439-13456.; Pradhan et al., 1984Pradhan, B.P., De, S., Nath, A., Shoolery, J.N., 1984. Triterpenoid acids from Sapium sebiferum. Phytochemistry 23, 2593-2595.). Triterpenes were isolated from some Malvaceae species, such as S. micranthum, Herissantia tiubae, Sida acuta and Abutilon pakistanicum(Gomes et al., 2011bGomes, R.A., Agra, M.F., Souza, M.F.V., 2011b. Constituintes químicos de Sidastrum micranthum (Malvaceae). In: 34ª Reunião Anual da Sociedade Brasileira de Química, Florianópolis, Brazil.; Silva et al., 2009bSilva, D.A., Falcão-Silva, V.S., Gomes, A.Y.S., Costa, D.A., Lemos, V.S., Agra, M.F., Braz-Filho, R., Siqueira-Junior, J.P., Souza, M.F.V., 2009b. Triterpenes and phenolic compounds isolated from the aerial parts of Herissantia tiubae and evaluation of 5,4',-dihydroxy-3,6,7,8,3'-pentamethoxyflavone as a modulator of bacterial drug resistance. Pharm. Biol. 47, 279.; Chen et al., 2007Chen, C., Chao, L., Pan, M., Liao, Y., Chang, C., 2007. Tocopherols and triterpenoids from Sida acuta. J. Chin. Chem. Soc. 54, 41-45.; Ahmed et al., 1990Ahmed, Z., Kazmi, S.N.H., Malik, A., 1990. Pakistanol, a new pentacyclic triterpene from Abutilon pakistanicum. J. Nat. Prod. 53, 1342-1344.). Hopane triterpenes and 3,4-seco-triterpenes have been recently reported from Wissadula periplocifolia (Teles et al., 2014Teles, Y.C.F., Gomes, R.A., Oliveira, M.S., Lucena, K.L., Nascimento, J.S., Agra, M.F., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2014. Phytochemical investigation of Wissadula periplocifolia (L.) C. Presl and evaluation of its antibacterial activity. Quim Nova 37, 1491-1495.).


The structural assignment of the mixture of compounds 3 and 4 was performed based on spectral analysis and comparisons with the literature (Kojima et al., 1990Kojima, H., Sato, N., Hatano, A., Ogura, H., 1990. Sterol glucosides from Prunella vulgaris. Phytochemistry 29, 2351-2355.; Rashed et al., 2014Rashed, K., Ciric, A., Glamoclija, J., Calhelha, R.C., Ferreira, I.C.F.R., Sokovic, M., 2014. Antimicrobial and cytotoxic activities of Alnus rugosa L. aerial parts and identification of the bioactive components. Ind. Crops Prod. 59, 189-196.). The data are in good agreement with those reported and the structures were identified as the mixture of glucosyl steroids: sitosterol 3-O-β-d-glucopyranoside (3) and stigmasterol 3-O-β-d-glucopyranoside (4). These steroids are widely spread in plants, being important component of vegetable cell wall and membrane. They have been previously reported from many Malvaceae, such as W. periplocifolia, S. rhombifolia, Sida galheirensis, H. crispa and Bakeridesia pickelli (Teles et al., 2014Teles, Y.C.F., Gomes, R.A., Oliveira, M.S., Lucena, K.L., Nascimento, J.S., Agra, M.F., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2014. Phytochemical investigation of Wissadula periplocifolia (L.) C. Presl and evaluation of its antibacterial activity. Quim Nova 37, 1491-1495.; Chaves et al., 2013Chaves, O.S., Gomes, R.A., Tomaz, A.C., Fernandes, M.G., Graças Mendes Jr., L., Agra, M.F., Braga, V.A., Souza, M.F.V., 2013. Secondary metabolites from Sida rhombifolia L. (Malvaceae) and the vasorelaxant activity of cryptolepinone. Molecules 18, 2769-2777.; Silva et al., 2006Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S., Matias, W.N., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes Químicos e atividade antioxidante de Sida galheirensis ULBR (Malvaceae). Quim. Nova 29, 1250-1254.; Costa et al., 2007Costa, D.A., Silva, D.A., Cavalcanti, A.C., Medeiros, M.A.A., Lima, J.T., Cavalcante, J.M.S., Silva, B.A., Agra, M.F., Souza, M.F.V., 2007. Chemical constituents from Bakeridesia pickelii Monteiro (Malvaceae) and the relaxant activity of kaempferol-3-O-beta-D-(6"-E-p-coumaroyl) glucopyranoside on guinea-pig ileum. Quim. Nova 30, 901-903., 2009Silva-Filho, A.A., Resende, D.O., Fukui, M.J., Santos, F.F., Pauletti, P.M., Cunha, W.R., Silva, M.L.A., Gregório, L.E., Bastos, J.K., Nanayakkara, N.P.D., 2009. In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D.C. (Asteraceae). Fitoterapia 80, 478-482.).


The substances 5, 6 and 7 were isolated as dark green amorphous solid, and showed quite similar IR spectra. The 1H and 13C NMR indicated that the compounds are chlorophyll derivatives. Comparisons with the literature data led to identify the compounds as phaeophytin a (5), 132-hydroxy-phaeophytin a (6) (Nogueira et al., 2013Nogueira, T.B., Nogueira, R.B., Silva, D.A., Tavares, J.F., Lima, E.O., Pereira, F.O., Fernandes, M.M., Medeiros, F.A., Sarquis, R.S.F., Braz Filho, R., Maciel, J.K.S., Souza, M.F.V., 2013. First chemical constituents from Cordia exaltata Lam and antimicrobial activity of two neolignans. Molecules 18, 11086-11099.) and 132(S)-hydroxy-173-ethoxyphaeophorbide a (7) (Silva et al., 2009aSilva, M.S., Tavares, J.F., Queiroga, K.F., Agra, M.F., Barbosa-Filho, J.M., Almeida, J.R.G.S., Silva, S.A.S., 2009a. Alcaloides e outros constituintes de Xylopia langsdorffiana (Annonaceae). Quim. Nova 32, 1566-1570.), previously reported from Malvaceae species W. periplocifolia, S. rhombifolia and S. galheirensis (Teles et al., 2014Teles, Y.C.F., Gomes, R.A., Oliveira, M.S., Lucena, K.L., Nascimento, J.S., Agra, M.F., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2014. Phytochemical investigation of Wissadula periplocifolia (L.) C. Presl and evaluation of its antibacterial activity. Quim Nova 37, 1491-1495.; Chaves et al., 2013Chaves, O.S., Gomes, R.A., Tomaz, A.C., Fernandes, M.G., Graças Mendes Jr., L., Agra, M.F., Braga, V.A., Souza, M.F.V., 2013. Secondary metabolites from Sida rhombifolia L. (Malvaceae) and the vasorelaxant activity of cryptolepinone. Molecules 18, 2769-2777.; Silva et al., 2006Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S., Matias, W.N., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes Químicos e atividade antioxidante de Sida galheirensis ULBR (Malvaceae). Quim. Nova 29, 1250-1254.). These compounds are formed from chlorophyll degradation by action of enzymes such as Mg-dechelatase and chlorophyllase. Additional structural modifications can occur and recently new structures of chlorophyll derivatives are being reported (Silva et al., 2010Silva, T.M.S., Camara, C.A., Barbosa-Filho, J.M., Giulietti, A.M., 2010. Feoforbídeo (Etoxi-Purpurina-18) isolado de Gossypium Mustelinum (Malvaceae). Quim. Nova 33, 571-573.). Researchers are interested in understanding if they belong to the secondary metabolism of plants once they have showed to possess many biological activities (Teles et al., 2014Teles, Y.C.F., Gomes, R.A., Oliveira, M.S., Lucena, K.L., Nascimento, J.S., Agra, M.F., Igoli, J.O., Gray, A.I., Souza, M.F.V., 2014. Phytochemical investigation of Wissadula periplocifolia (L.) C. Presl and evaluation of its antibacterial activity. Quim Nova 37, 1491-1495.).


The compound 8 was isolated as yellow powder. By analyzing its 1H and 13C NMR, besides bidimensional NMR spectra, it was possible identify the compound 8 as 5,8-dihydroxy-7,4′-dimethoxy-flavone (7,4′-di-O-methylisoescutellarein), previously isolated from Sidastrum micrathum (Gomes et al., 2011bGomes, R.A., Agra, M.F., Souza, M.F.V., 2011b. Constituintes químicos de Sidastrum micranthum (Malvaceae). In: 34ª Reunião Anual da Sociedade Brasileira de Química, Florianópolis, Brazil.). Flavonoids are considered a characteristic group of constituents from family. Flavones, flavonols, their methyl and sulphur derivatives, and glucosyl flavonoids, have been reported from Malvaceae species (Chaves et al., 2013Chaves, O.S., Gomes, R.A., Tomaz, A.C., Fernandes, M.G., Graças Mendes Jr., L., Agra, M.F., Braga, V.A., Souza, M.F.V., 2013. Secondary metabolites from Sida rhombifolia L. (Malvaceae) and the vasorelaxant activity of cryptolepinone. Molecules 18, 2769-2777. Gomes et al., 2011aGomes, R.A., Ramirez, R.R.A., Maciel, J.K.S., Agra, M.F., Souza, M.F.V., Falcão-Silva, V.S., Siqueira-Junior, J.P., 2011a. Phenolic compounds from Sidastrum micranthum (A St.-Hil.) fryxell and evaluation of acacetin and 7,4′-di-O-methylisoscutellarein as modulator of bacterial drug resistance. Quim. Nova 34, 1385-1388.; Silva et al., 2006Silva, D.A., Silva, T.M.S., Lins, A.C.S., Costa, D.A., Cavalcante, J.M.S., Matias, W.N., Souza, M.F.V., Braz-Filho, R., 2006. Constituintes Químicos e atividade antioxidante de Sida galheirensis ULBR (Malvaceae). Quim. Nova 29, 1250-1254.; Cavalcante et al., 2010Cavalcante, J.M.S., Nogueira, T.B.S.S., Tomaz, A.C.A., Silva, D.A., Agra, M.F., Carvalho, P.R.C., Ramos, S.R.R., Nascimento, S.C., Gonçalves-Silva, T., Souza, M.F.V., 2010. Steroidal and phenolic compounds from Sidastrum paniculatum (L.) Fryxell and evaluation of cytotoxic and anti-inflammatory activities. Quim. Nova 33, 846-849.; Costa et al., 2007Costa, D.A., Silva, D.A., Cavalcanti, A.C., Medeiros, M.A.A., Lima, J.T., Cavalcante, J.M.S., Silva, B.A., Agra, M.F., Souza, M.F.V., 2007. Chemical constituents from Bakeridesia pickelii Monteiro (Malvaceae) and the relaxant activity of kaempferol-3-O-beta-D-(6"-E-p-coumaroyl) glucopyranoside on guinea-pig ileum. Quim. Nova 30, 901-903., 2009Costa, D.A., Matias, W.M., Lima, I.O., Xavier, A.L., Costa, V.B.M., Diniz, M.F., Agra, M.F., Batista, L.M., Souza, M.F.V., Silva, D.A., 2009. First secondary metabolites from Herissantia crispa L. (Brizicky) and the toxicity activity against Artemia salina Leach. Quim. Nova 32, 48-50.; Silva et al., 2005Silva, M.S., Tavares, J.F., Queiroga, K.F., Agra, M.F., Barbosa-Filho, J.M., Almeida, J.R.G.S., Silva, S.A.S., 2009a. Alcaloides e outros constituintes de Xylopia langsdorffiana (Annonaceae). Quim. Nova 32, 1566-1570.; Nawwar and Buddrus, 1981Nawwar, M., Buddrus, J., 1981. A gossypetin glucuronide sulphate from the leaves of Malva sylvestris. Phytochemistry 20, 2446-2448.).


3-oxo-21β-H-hop-22(29)-ene (1): mp 168–170 °C; IR: 2943, 2862, 1708 cm-1. 1H NMR (500 MHz, CDCl3, δ ppm): 2.08 (m, H-1), 2.24 (m, H-2), 1.95 (m, H-5), 1.10 (m, H-6), 1.10 and 2.01 (m, H-7), 1.42 (m, H-9), 1.10 (m, H-11), 1.42 (m, H-12), 1.32 (m, H-13), 1.32 (m, H-15), 1.62 (m, H-16), 1.39 (m, H-17), 1.60 (m, H-19), 1.81 (m, H-20), 2.66 (m, H-21), 1.02 (s, H-23), 1.00 (s, H-24), 0.73 (s, H-25), 1.14 (s, H-26), 0.86 (s, H-27), 0.68 (s, H-28), 4.76 (bs, 2H-29), 1.72 (s, H-30). 13C NMR (125 MHz, CDCl3, δ): 31.59 (C-1), 33.78 (C-2), 220.51 (C-3), 46.82 (C-4), 47.32 (C-5), 20.57 (C-6), 34.17 (C-7), 41.65 (C-8), 43.30 (C-9), 36.17 (C-10), 21.96 (C-11), 24.27 (C-12), 49.72 (C-13), 42.66 (C-14), 33.55 (C-15), 21.37 (C-16), 55.14 (C-17), 44.82 (C-18), 41.53 (C-19), 27.30 (C-20), 46.24 (C-21), 148.64 (C-22), 29.36 (C-23), 19.57 (C-24), 23.29 (C-25), 22.08 (C-26), 17.06 (C-27), 15.96 (C-28), 110.06 (29), 25.04 (C-30) (David et al., 2004David, J.P., Meira, M., David, J.M., Guedes, M.L.S., 2004. Triterpenos e ferulatos de alquila de Maprounea guianensis. Quim. Nova 27, 62-65.; Sousa et al., 2012Sousa, G.F., Duarte, L.P., Alcântara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M., Takahashi, J.A., 2012. New triterpenes from Maytenus robusta: structural elucidation based on NMR experimental data and theoretical calculations. Molecules 17, 13439-13456.).

3,4-seco-21β-H-hop-22(29)-en-3-oic acid (sebiferic acid) (2): mp 178–180 °C; IR: 1708 cm-1; 2941 cm-1, 3435 cm-1. 1H NMR (500 MHz, CDCl3, δ): 2.25 (m, H-1), 1.85–2.44 (m, H-2), 2.04 (m, H-5), 1.39 (m, H-6), 1.00 (m, H-7), 1.72 (m, H-9), 1.48 (m, H-11), 1.43 (m, H-12), 1.40 (m, H-13), 1.41 (m, H-15), 1.51 (m, H-16), 1.43 (m, H-17), 1.60 (m, H-19), 1.78 (m, H-20), 2.67 (m, H-21), 4.87 (s, H-23), 1.77 (s, H-24), 0.82 (s, H-25), 1.03 (s, H-26), 0.97 (s, H-27), 0.71 (s, H-28), 4.76 (bs, H-29), 1.73 (s, H-30). 13C NMR (125 MHz, CDCl3, δ): 30.14 (C-1), 30.07 (C-2), 180.07 (C-3), 147.83 (C-4), 46.88 (C-5), 22.83 (C-6), 26.70 (C-7), 41.35 (C-8), 43.21 (C-9), 37.94 (C-10), 22.49 (C-11), 24.30 (C-12), 49.72 (C-13), 42.99 (C-14), 33.49 (C-15), 21.60 (C-16), 55.86 (C-17), 44.76 (C-18), 42.01 (C-19), 27.31 (C-20), 46.41 (C-21), 148.65 (C-22), 113.00 (C-23), 26.78 (C-24), 25.20 (C-25), 17.02 (C-26), 16.39 (C-27), 16.21 (C-28), 110.09 (29), 25.01 (C-30) (Sousa et al., 2012Sousa, G.F., Duarte, L.P., Alcântara, A.F.C., Silva, G.D.F., Vieira-Filho, S.A., Silva, R.R., Oliveira, D.M., Takahashi, J.A., 2012. New triterpenes from Maytenus robusta: structural elucidation based on NMR experimental data and theoretical calculations. Molecules 17, 13439-13456.; Pradhan et al., 1984Pradhan, B.P., De, S., Nath, A., Shoolery, J.N., 1984. Triterpenoid acids from Sapium sebiferum. Phytochemistry 23, 2593-2595.).

Sitosterol 3-O-β-d-glucopyranoside(3)/Stigmasterol 3-O-β-d-glucopyranoside(4): The 1H and 13C NMR spectral data are consistent with published data (Kojima et al., 1990Kojima, H., Sato, N., Hatano, A., Ogura, H., 1990. Sterol glucosides from Prunella vulgaris. Phytochemistry 29, 2351-2355.; Rashed et al., 2014Rashed, K., Ciric, A., Glamoclija, J., Calhelha, R.C., Ferreira, I.C.F.R., Sokovic, M., 2014. Antimicrobial and cytotoxic activities of Alnus rugosa L. aerial parts and identification of the bioactive components. Ind. Crops Prod. 59, 189-196.).

Phaeophytin a (5): The 1H and 13C NMR spectral data are consistent with published data (Nogueira et al., 2013Nogueira, T.B., Nogueira, R.B., Silva, D.A., Tavares, J.F., Lima, E.O., Pereira, F.O., Fernandes, M.M., Medeiros, F.A., Sarquis, R.S.F., Braz Filho, R., Maciel, J.K.S., Souza, M.F.V., 2013. First chemical constituents from Cordia exaltata Lam and antimicrobial activity of two neolignans. Molecules 18, 11086-11099.).

132(S)-hydroxy-phaeophytin a(6): The 1H and 13C NMR spectral data are consistent with published data (Nogueira et al., 2013Nogueira, T.B., Nogueira, R.B., Silva, D.A., Tavares, J.F., Lima, E.O., Pereira, F.O., Fernandes, M.M., Medeiros, F.A., Sarquis, R.S.F., Braz Filho, R., Maciel, J.K.S., Souza, M.F.V., 2013. First chemical constituents from Cordia exaltata Lam and antimicrobial activity of two neolignans. Molecules 18, 11086-11099.).

132(S)-hydroxy-(173)-ethoxyphaeophorbide a (7): The 1H and 13C NMR spectral data are consistent with published data (Silva et al., 2009aSilva, M.S., Tavares, J.F., Queiroga, K.F., Agra, M.F., Barbosa-Filho, J.M., Almeida, J.R.G.S., Silva, S.A.S., 2009a. Alcaloides e outros constituintes de Xylopia langsdorffiana (Annonaceae). Quim. Nova 32, 1566-1570.).

5,8-dihydroxy-7,4′-dimethoxy-flavone (7,4′-Di-O-Methylisoescutelarein) (8): 1H NMR (500 MHz, DMSO-d6, δ): 6.87 (s, H-3), 6.56 (s, H-6), 8.12 (d, J = 8.6 Hz, H-2′), 7.13(d, J = 8.6 Hz, H-3′), 7.13 (d, J = 8.6 Hz, H-5′), 8.12 (d, J = 8.6 Hz, H-6′), 3.90 (s, OCH3-7), 3.86 (s, OCH3-4′), 12.44 (s, OH-5). 13C NMR (125 MHz, DMSO-d6, δ): 164.0 (C-2), 103.6 (C-3), 182.9 (C-4), 153.6 (C-5), 96.2 (C-6), 154.8 (C-7), 126.8 (C-8), 145.0 (C-9), 104.4 (C-10), 123.5 (C-1′), 129.0 (C-2′), 115.1 (C-3′), 162.9 (C-4′), 162.9 (C-5′), 129.0 (C-6′), 56.9 (OCH3-7), 56.1 (OCH3-4′) (Gomes et al., 2011aGomes, R.A., Ramirez, R.R.A., Maciel, J.K.S., Agra, M.F., Souza, M.F.V., Falcão-Silva, V.S., Siqueira-Junior, J.P., 2011a. Phenolic compounds from Sidastrum micranthum (A St.-Hil.) fryxell and evaluation of acacetin and 7,4′-di-O-methylisoscutellarein as modulator of bacterial drug resistance. Quim. Nova 34, 1385-1388.).

Leishmanicidal activity

The protozoan parasite L. braziliensis is the causative organism for the clinically important disease cutaneous and mucocutaneous leishmaniasis (Gonzalez et al., 2009Gonzalez, U., Pinart, M., Rengifo-Pardo, M., Macaya, A., Alvar, J., Tweed, J.A., 2009. Interventions for American cutaneous and mucocutaneous leishmaniasis. Cochrane Database Syst. Rev. CD004834.). The current drugs for Leishmania infections are inadequate due to low efficacy and high toxicity, and the problem is further compounded by the increasing prevalence of drug-resistant parasites. Current biomedical research always has its focus on the search for newer intervention strategies and natural compounds have been used as novel treatments for parasitic diseases (Amato et al., 2008Amato, V.S., Tuon, F.F., Bacha, H.A., Neto, V.A., Nicodemo, A.C., 2008. Mucosal leishmaniasis: current scenario and prospects for treatment. Acta Trop. 105, 1-9.; Ndjonka et al., 2013Ndjonka, D., Rapado, L.N., Silber, A.M., Liebau, E., Wrenger, C., 2013. Natural products as a source for treating neglected parasitic diseases. Int. J. Mol. Sci. 14, 3395-3439.).

The antileishmanial activity of compounds 3-8 was assessed against extracellular promastigotes of L. braziliensis as causative agent of cutaneous leishmaniasis. As a parameter for antileishmanial activity, the maximum leishmanicidal effect and IC50 value were used.

The obtained results showed that the mixture of phytosterols 3/4 was inactive against promastigote forms of L. braziliensis (Table 1). The obtained result is in agreement with previous reports which demonstrated that stigmasterol and sitosterol 3-O-β-d-glucoside were inactive against Leishmania donovani (Graziose et al., 2012Graziose, R., Rojas-Silva, P., Rathinasabapathy, T., Dekock, C., Grace, M.H., Poulev, A., Lila, M.A., Smith, P., Raskin, I., 2012. Antiparasitic compounds from Cornus florida L. with activities against Plasmodium falciparum and Leishmania tarentolae. J. Ethnopharmacol. 142, 456-461.; Kirmizibekmez et al., 2011Kirmizibekmez, H., Atay, I., Kaiser, M., Brun, R., Cartagena, M.M., Carballeira, N.M., Yesilada, E., Tasdemir, D., 2011. Antiprotozoal activity of Melampyrum arvense and its metabolites. Phytother. Res. 25, 142-146.; Camacho et al., 2002Camacho, M.R., Phillipson, J.D., Croft, S.L., Marley, D., Kirby, G.C., Warhurst, D.C., 2002. Assessment of the antiprotozoal activity of Galphimia glauca and the isolation of new Nor-secofriedelanes and Nor-friedelanes. J. Nat. Prod. 65, 1457-1461.). Morever, Torres-Santos et al. verified that stigmasterol and sitosterol were not active against promastigotes and intracellular amastigotes of Leishmania amazonensis (Torres-Santos et al., 2004Torres-Santos, E.C., Lopes, D., Oliveira, R.R., Carauta, J.P., Falcao, C.A., Kaplan, M.A., Rossi-Bergmann, B., 2004. Antileishmanial activity of isolated triterpenoids from Pourouma guianensis. Phytomedicine 11, 114-120.).

Table 1
Leishmanicidal effect of compounds isolated from S. paniculatum against promastigotes of L. braziliensis.

Data are reported as means ± S.E.M. Differences with **p< 0.01, *p < 0.05 were considered significant in relation to DMSO 0.1% group. NA: When the compound is not active until 100 µM. (a) IC50 is the concentration required to give 50% inhibition, calculated by linear regression analysis from the Kcvalues at employed concentrations (100, 10, 1, 0.1 and 0.01 µM). (b) EM is the maximum effect of treatment.

On the other hand, it has been reported the use of porphyrin compounds for their ability to inhibit Leishmania (Hörtensteiner et al., 1998Hörtensteiner, S., Wüthrich, K.L., Matile, P., Ongania, K.H., Krautler, B., 1998. The key step in chlorophyll breakdown in higher plants: cleavage of pheophorbide alpha macrocycle by a monooxygenase. J. Biol. Chem. 273, 15335-15339.; Sakata et al., 1990Sakata, K., Yamamoto, K., Ishikawa, H., Yagi, A., Etoh, H., Ina, K., 1990. Chlorophyllone-A, a new pheophorbide-A related compound isolated from Ruditapes philippinarum as an antioxidative compound. Tetrahedron Lett. 31, 1165-1168.). Previous works revealed that phaeophytins possess potent cytotoxic activities (Cheng et al., 2001Cheng, H.H., Wang, H.K., Ito, J., Bastow, K.F., Tachibana, Y., Nakanishi, Y., Xu, Z.H., Luo, T.Y., Lee, K.H., 2001. Cytotoxic pheophorbide-related compounds from Clerodendrum calamitosumand C. crytophyllum. J. Nat. Prod. 64, 915-919.). In this study, the three phaeophytin-related compounds phaeophytin a (5), 132-hydroxy-(132-S)-phaeophytin a (6) and 132(S)-hydroxy-173-ethoxyphaeophorbide a (7) exhibited leishmanicidal activity against promastigotes of L. braziliensis, especially 132-hydroxy-(132-S)-phaeophytin a (6), showing that the addition of hydroxyl group in 132-position increased efficacy, but not potency. In addition, the importance of heme in Leishmania sp. metabolisms justifies considering the potential of porphyrins and their precursors and derivatives as potential antiparasitic agents by interfering with heme metabolism (Abada et al., 2013Abada, Z., Cojean, S., Pomel, S., Ferrié, L., Akagah, B., Lormier, A.T., Loiseau, P.M., Figadère, B., 2013. Synthesis and antiprotozoal activity of original porphyrin precursors and derivatives. Eur. J. Med. Chem. 67, 158-165.). Heme is one of many indispensable nutrients that must be scavenged from the phagolysosomal compartment by Leishmania (Naderer and McConville, 2008Naderer, T., McConville, M.J., 2008. The Leishmania-macrophage interaction: a metabolic perspective. Cell Microbiol. 10, 301-308.). In metazoa, heme biosynthesis occurs through 8 highly conserved chemical reactions, resulting in the insertion of ferrous iron into the protoporphyrin IX ring (Hamza and Dailey, 2012Hamza, I., Dailey, H.A., 2012. One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans. Biochim. Biophys. Acta 1823, 1617-1632.). However, several unicellular organisms, such as Leishmania lack several of the enzymes necessary for a complete heme-biosynthetic pathway (Korený et al., 2013Korený, L., Oborník, M., Lukeš, J., 2013. Make it, take it, or leave it: heme metabolism of parasites. PLoS Pathog. 9, e1003088.). Heme not only is a crucial component of cytochromes in the respiratory chain, but also functions as an essential cofactor for hemoproteins, such as the ones involved in the biosynthesis of polyunsaturated fatty acids and sterols (Tripodi et al., 2011Tripodi, K.E., Menendez-Bravo, S.M., Cricco, J.A., 2011. Role of heme and heme-proteins in trypanosomatid essential metabolic pathways. Enzyme Res. 2011, 873230.).

The flavone 5,8-dihydroxy-7,4′-dimethoxyflavone (8) showed activity against L. braziliensis. The structure antileishmanial activity relationship of flavones has been described and it has been shown that this class of flavonoids, consists of potential antiprotozoal agents. The presence of OH groups on the flavone benzochromone skeleton enhances the leishmanicidal potential. Particularly important positions are C-5, C-7, and C-8 (Silva-Filho et al., 2009Silva-Filho, A.A., Resende, D.O., Fukui, M.J., Santos, F.F., Pauletti, P.M., Cunha, W.R., Silva, M.L.A., Gregório, L.E., Bastos, J.K., Nanayakkara, N.P.D., 2009. In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D.C. (Asteraceae). Fitoterapia 80, 478-482.). On the other hand, the replacement of the OH groups, either on A or B rings, by methoxyl groups was demonstrated to decrease the antiprotozoal activity (Tasdemir et al., 2006Tasdemir, D., Kaiser, M., Brun, R., Yardley, V., Schmidt, T.J., Tosun, F., Rüedi, P., 2006. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies. Antimicrob. Agents Chemother. 50, 1352-1364.). Numerous natural compound screens have successfully identified novel treatments for parasitic diseases (Ndjonka et al., 2013Ndjonka, D., Rapado, L.N., Silber, A.M., Liebau, E., Wrenger, C., 2013. Natural products as a source for treating neglected parasitic diseases. Int. J. Mol. Sci. 14, 3395-3439.). Extracts obtained from plants and pure compounds, such as certain types of flavonoids, have been reported to possess significant antiprotozoal activity with no side effects (Muzitano et al., 2006Muzitano, M.F., Tinoco, L.W., Guette, C., Kaiser, C.R., Rossi-Bergmann, B., Costa, S.S., 2006. The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata. Phytochemistry 67, 2071-2077.; Fonseca-Silva et al., 2011Fonseca-Silva, F., Inacio, J.D., Canto-Cavalheiro, M.M., Almeida-Amaral, E.E., 2011. Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis. PLoS ONE 6, e14666.). While the mechanism of action of the 5,8-dihydroxy-7,4′-dimethoxyflavone to kill these parasites is not yet known, biochemical experiments with other flavones provide initial insights. Among naturally occurring flavonoids, the flavones quercetin, luteolin and baicalein are reported as topoisomerase II inhibitor (Austin et al., 1992Austin, C.A., Patel, S., Ono, K., Nakane, H., Fisher, L.M., 1992. Site specific DNA cleavage by mammalian DNA topoisomerase II induced by novel flavone and catechin derivatives. Biochem. J. 282, 883-889.; Mittra et al., 2000Mittra, B., Saha, A., Chowdhury, A.R., Pal, C., Mandal, S., Mukhopadhyay, S., Bandopadhyay, S., Majumder, H.K., 2000. Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis. Mol. Med. 6, 527-541.). Therefore, we will continue this study to evaluate the inhibitory effects of theses analogs on topoisomerase of Leishmania as well as other validated chemotherapeutic target.

Conclusion

The phytochemical investigation of S. paniculatum led to isolation of eight compounds, being two triterpenes: 3-oxo-21β-H-hop-22(29)-ene (1) and sebiferic acid (2); a mixture of steroids: sitosterol 3-O-β-d-glucopyranoside/stigmasterol 3-O-β-d-glucopyranoside (3/4); three chlorophyll derivatives: phaeophytin a (5); 132-hydroxy-(132-S)-phaeophytin a (6); 132-hydroxy-(132-S)-phaeophorbide a (7); and a flavone: 7,4′-di-O-methylisoescutelarein (8).

The studied species S. paniculatum has demonstrated quite similar group of chemical constituents to S. micranthum, S. galheirensis, H. tiubae and W. periplocifolia, showing a related chemotaxonomic profile of these species.

The antileishmanial activity against promastigotes of L. braziliensis of compounds 5-8 is being here reported for the first time.

Acknowledgments

The authors thank CAPES, MCT, FINEP, FAPEAL (Pronem 20110722-006-0018-0010), CNPQ (479822/2013-1), CNPQ (404344/2012-7), INCT-INOFAR/CNPq (573.564/2008-6) for the joint funding of this research project. The authors thank several colleagues working at the Federal University of Paraiba and Federal University of Alagoas for their constructive criticism and assistance in carrying out this project.

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Publication Dates

  • Publication in this collection
    Jul-Aug 2015

History

  • Received
    02 Aug 2014
  • Accepted
    10 Feb 2015
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