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Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi.

ABSTRACT

The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by [sup.1]H and [sup.13]C--nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L. infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to pentamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. Conclusion: This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy.

Keywords:

Sterols

Triterpenes

Leishmania infantum chagasi

Introduction

In Northeastern Brazil there are many places where Leishmaniasis is endemic with several lethal cases among humans and dogs. A change was observed in relation to the urbanization of this disease, which in the past occurred only in the countryside but nowadays affects large urban centers (Lindoso and Goto 2006). The search for new leishmanicidal compounds is constant since the drug arsenal used to treat this illness is not sufficient and displays undesirable side effects.

Several compounds isolated from plants, such as terpenoids, sterols, flavonoids, alkaloids, phenolics and naphthoquinones have been studied to evaluate their effects on Leishmania spp. (Cham-Bacab and Pena-Rodriguez 2001). Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone caused different susceptibilities on Leishmania spp. promastigotes (Vila-Nova et al. 2013). Some cycloartane-type triterpene glycosides, isolated from the genus Astragalus, have exhibited antitumor, immunodepressant, antiviral, and leishmanicidal activities (Ozipek et al. 2005). Cycloartane-type triterpenes can be found in the fruit peel of Musa spp. (Imam and Akter 2011).

In our continuous search for leishamanicidal compounds M. paradisiaca fruit peel, which is used in folk medicine to combat leishmaniasis (Weigel et al. 1994), was selected to isolate cycloartane-type triterpenes and tested against L infantum chagasi, in attempt to confirm the popular use of this dischargeable material.

Materials and methods

Plant material

A bunch of unripe "silver" bananas (M. paradisiaca) was acquired in the local market in Fortaleza, Ceara, Brazil.

Preparation of ethanol extract of banana peel

The peel of unripe banana fruit was removed, minced and placed to dry at 40[degrees]C for a period of one week. Subsequently, this material was ground and placed in commercial ethanol (70%) for one week. Then, the solution was filtered and the solvent ethanol was removed in a rotary evaporator, yielding the ethanol extract of banana peel (EEBP). Subsequently, the EEBP was subjected to a filtering column using solvents of increasing polarity level, hexane, chloroform, ethyl acetate, methanol and water. The solvent of each organic phase was evaporated to obtain the extracts (Pizzolatti et al. 2003).

Isolation of the compounds by chromatographic methods

The EEBP was subjected to column chromatography on silica gel ([delta] 0.063-0.200 mm; 70-230 mesh). The columns had varying lengths and sizes in proportion to the sample and eluents of P.A. grade were used in order of increasing polarity: hexane, chloroform, ethyl acetate and methanol. Thin layer chromatography (TLC) was performed using GF 254 silica gel 60 on glass slides, forming the chromatographic plate. The development of the TLC plates was made by spraying with vanillin 2.5% solution of perchloric acid in ethanol (1:1) followed by heating in an oven at about 100[degrees]C.

A silica gel column chromatography of ethanol extract (60 g) of the unripe banana peel was performed. The solvents were used in mixtures of increasing polarity, starting with hexane then hexane/chloroform (1:1), chloroform, chloroform/ethyl acetate (1:1), ethyl acetate/methanol (1:1) and methanol. The fractions (500 ml) were collected and solvents were eliminated in a rotary evaporator. TLC plates sprayed with vaniline/[H.sub.2]S[O.sub.4] reagent were used to compare the obtained fractions and compounds were isolated. The hexane/chloroform fraction (6.6g), which contained triterpenes by Liberman-Buchard test (Matos 2009), was rechromatographed using the same mixtures of solvents, collecting 70 ml x 10 ml fractions and a compound C1 was obtained from hexane, with hexane/chloroform compound C2 was obtained, with chloroform/ethyl acetate C3 and from ethyl acetate/methanol, compound C4 was separated.

Structural determination of the isolated compounds

The chemical structures of the purified compounds were confirmed by spectroscopic analysis of the nuclear magnetic resonance spectra recorded on a Bruker Avance DRX-300 spectrometer, in CD[Cl.sub.3].

HPLC-Finger print of hexanexhloroform (1:1) extract and constituents of M. paradisiaca

HPLC instrument: Shimadzu system with a SPD-10A VP detector, SCL-10A VP controller; LC-10A VP bomb, software LC solution, column: CLC ODS (m) 15 cm, running time: 30 min, flow rate: 1 ml/min, detector: 212 nm, mobile phase: 100% methanol grade HPLC, loop of injection: 20 [micro]l.

Cultivation of L. infantum chagasi

Promastigotes of L infantum chagasi strain IOCL2272 (Department of Pathology/UFC) were grown in Schneider (Sigma[R]) medium supplemented with gentamicin 40 mg/ml, 5% human male sterile urine and 10% fetal calf serum (FCS) (Cultilab[R]). Cultures were maintained in a BOD incubator at 23.6[degrees]C and transplanting was done every three to four days.

Amastigotes of L. infantum chagasi strain IOCL2272 were cultured with the murine monocyte cell line RAW 264.7 (Sigma-Aldrich[R]) in 96 well microplates. Cells were counted in a Neubauer chamber and plated at a density of 1 x [10.sup.6] cells/well. Promastigotes were added at a ratio 10:1 parasites/cell. Cells were cultured in Dulbecco's medium (Cultilab[R]) with 5% FCS, sodium bicarbonate, and 40 mg/ml gentamicin. Bottles were left open and cultivated under glass with 5% C[O.sub.2] at 37[degrees]C. After 24 h, the amastigotes inside the monocyte cells were observed under inverted microscope.

Assays on L infantum chagasi promastigotes

The methodology used to evaluate the leishmanicidal activity of sterols from M. paradisiaca against promastigote and amastigote of L infantum chagasi 10CL2272 was based on Tempone et al. (2005). The sterols and triterpenes were dissolved in dimethylsulfoxide (DMSO) and diluted in Schneider medium (Sigma[R]). The final concentration of DMSO did not exceed 1%. L infantum chagasi were placed in the Schneider (Sigma[R]) medium supplemented (gentamicin 40 mg/ml, 5% human male sterile urine and 10% fetal calf serum (FCS) (Cultilab[R]) and 10% PBS) with different concentrations of the drug and the parasite in the absence of the drug (control). The experiments were performed in 96-well plates with compounds at concentrations of 100, 50, 25,12.5 and 6.25 [micro]g/ml. Promastigotes of L. infantum chagasi were used at [10.sup.6] parasites/well in logarithmic phase. The reference medicine used was pentamidina. Negative controls were performed with 106 parasites in Schneider (Sigma[R]) medium supplemented without compounds. The promastigotes were incubated at 26[degrees]C for 48 h. Afterwards cell viability was examined based on the conversion of soluble tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl] 2,5 diphenyltetrazolium bromide) (Sigma[R]) into insoluble formazan by mitochondrial enzymes. Twenty [micro]l of MTT [5 mg/ml] per well were added to the culture, and then kept for 4 h at 26[degrees]C. Thereafter, 100 [micro]l of a solution of sodium dodecyl sulfate (SDS) 10%: isopropyl alcohol (1:1) was added. After 15 min stirring a reading was made of the optical density in a spectrophotometer at 570 nm. Assays were performed in triplicate.

Assays on L. infantum chagasi amastigotes

The medium was removed by aspiration, and the drugs were dissolved in DMSO, diluted in RPMI and were added at concentrations of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 [micro]g/ml in the microtiter plate containing confluent layer of cells with amastigotes. The in situ immunoassay to determine the [EC.sub.50] ELISA was performed according to Piazza et al. (1994). Before starting the tests, the infected cells were stained and parasites observed under a microscope. When more than 50% of infected cells in a microscopic field were observed, the parasite load was considered appropriate to perform the test. Briefly, after 18 h incubation with the compounds, the microplate wells were incubated with 0.01% saponin (Sigma-Aldrich[R]) in 1 x PBS plus 1 % bovine serum albumin (Sigma-Aldrich[R]) for 30 min at 37[degrees]C. The blocking reaction was made with lx PBS plus 5% skimmed milk (Nestle[R]) for 30 min at 37[degrees]C. The microplates were washed and dried three times, then the serum from immunized rabbits diluted 1:500 in 1 x PBS was added, followed by 3% skimmed milk 0.05% in Tween 20 (PBSLT) plus 10% FCS, and the plates were kept at 37[degrees]C overnight. The conjugated anti-rabbit IgG (Sigma-Aldrich[R]) was diluted 1:10,000 in PBSLT and after further washing, orthophenylenediamine chromogen (OPD) (Sigma-Aldrich[R]) was added. In order to stop the reaction 4 N chloric acid (Novaquimica[R]) was added, and the reading was performed using a 570 nm filter in a microplate reader. The positive control for this assay was 40 [micro]g/ml amphotericin B (Sigma-Aldrich[R]) and the negative control was the Dulbecco medium alone (Cultilab[R]). The rabbits were immunized against promastigotes of L. infantum chagasi and the serum was obtained after 30 days of immunization.

Cytotoxicity to mammalian cells

RAW 264.7 and [LLC-MK.sub.2] cell were cultured in 96-well plates at a concentration of [10.sup.6] cells per well in RPMI medium containing 10% of modified PBS at 37[degrees]C and 5% C[O.sub.2]. After 48 h, the medium was changed and incubated with the extracts at different concentrations under the same conditions. After 24 h, cell viability was determined by MTT assay. 20 [micro]l of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 3 h at 37[degrees]C and 5% C[O.sub.2]. Subsequently, the medium was aspirated and the culture was added 100 [micro]l of DMSO. The absorbance was determined with a spectrophotometer at a wavelength of 570 nm.

Statistical analysis

The [EC.sub.50] values, drug concentration able to inhibit 50% of parasites, with a confidence interval of 95% were calculated using a non-linear regression curve using the statistical software GraphPad Prism 5.0. The entire experiment was performed in triplicate.

Results

The structures of isolated compounds were elucidated by [sup.1]H and [sup.13]C NMR, including uni- and bidimensional techniques, and also compared with literature data.

The chemical composition of the lipophilic extracts of unripe pulp and peel of banana fruit 'Dwarf Cavendish' (Musa acuminata) was determined by gas chromatography-mass spectrometry. In banana peel, sterols represent about 49-71% of the lipophilic extract with two triterpene ketones (31-norcyclolaudenone and cycloeucalenone) as the major components (Oliveira et al. 2008).

Previous studies reported the isolation of several triterpenes such as cyclomusalenol, cyclomusalenone, cyclartanol, stigmast-7-en-3-ol, 24-methylenecycloartanol, stigmast-7-methylene, lanosterol and beta-amyrin. Cycloartane triterpenes such as 3-epicycloeucalenol, 3-epicyclomusalenol, 24-methylenepollinastanone, 28-norcyclomusalenone, 24-oxo-29-norcycloartanone have been also isolated from the fruit peel of M. sapientum (Imam and Akter 2011).

The [sup.1]H NMR spectrum of C1 showed a doublet at [delta] 4.69 (2H, J = 15.4 Hz, geminal coupling) characteristic of a terminal double bond. A double doublet at [delta] 0.4 and [delta] 0.6 (J = 3.75 Hz) corresponds to a cyclopropene ring present in cycloartane-type triterpenes, common compounds isolated from banana peel. The absence of signals characteristic of hydroxymethyne groups (~[delta] 3.2) and the presence of a signal in [delta] 213 in the 13 C NMR spectrum suggests a C = 0 group at C-3 position. The terminal double bond was also confirmed due to peaks at 150.2 ppm (carbon without hydrogen) and 109.1 ppm (a terminal [sp.sup.2] carbon linked to two hydrogens). These data in comparison with previous reports of Ragasi et al. (2007) characterize C1 as 31-norcidolaudenone.

The [sup.1]H NMR spectrum of C2 also showed a doublet at [delta] 4.69 (J = 15.81 Hz) of a terminal double bond, which is confirmed in [sup.13]C NMR spectrum by the peaks in [delta] 156.9 (carbon without hydrogen) and 106.0 ppm (a terminal [sp.sup.2] carbon). A cyclopropene methylene is represented by two double doublets at [delta] 0.4 and [delta] 0.6 (J = 2.25 Hz). Several methyl groups between [delta] 0.7 and [delta] 0.9 characterize a triterpene skeleton and [sup.13]C NMR spectrum confirms this information and the presence of a ketone group at [delta] 213. The comparison of overall peaks of ]H and [sup.13]C NMR spectra with literature data led to identification of C2 as cycloeucalenone (Khuonghuu et al. 1975).

C3 fraction showed absorption peaks characteristics of a mixture of stigmasterol and [beta]-sitosterol (Chaturvedula and Prakash, 2012), already mentioned in Musa species (Oliveira et al. 2008).

C4 also showed peaks of the cyclopropene moiety at [delta] 0.3 and [delta] 0.6, a terminal double bond represented by a doublet at [delta] 4.7 (J = 18 Hz) and several singlets between [delta] 0.8 and [delta] 1.0, which correspond to methyl groups of triterpenes. The analysis of [sup.13]C NMR spectrum of C4 suggested the structure of 24-methylenecycloartanol based also on the following peaks: C-1 (32.09). C-2 (29.86), C-3 (79.02), C-4 (39.64), C-5 (47.32), C-6 (21.07), C-7 (28.25), C-8 (48.04), C-9 (20.02), C-10 (26.13), C-11 (26.13), C-12 (34.09), C-13 (45.46), C-14 (48.97), C-15 (33.02), C-16 (26.61), C-17 (52.36), C-18 (18.20), C-19 (29.63), C-20 (35.69), C-21 (18.20), C-22 (34.09), C-23 (31.77), C-24 (156.80), C-25 (33.97), C-26 (22.02), C-27 (22.15), C-28 (19.46), C-29 (25.61), C-30 (15.38), C-31 (106.5). These data were similar to those of previous report (Barla et al. 2006).

The chemical structures of M. paradisiaca tested compounds are shown in Fig. 1.

The relative percentage of main constituents of the hexane:chloroform (1:1) extract was achieved by HPLC analysis of total extract and constituents (Fig. 2A). The major compound is 31-norcyclolaudenone (85.22%), followed by cycloeucalenone (9.38%), stigmasterol (3.74%), 24-methylenecycloartanol (1%) and under the conditions of HPLC chromatographic analysis betasitosterol appear with the same retention time of cycloeucalenone at 15.11 min. In the HPLC analysis of the mixture stigmasterol and [beta]-sitosterol (Fig. 2B), the relative percentage of these two steroids was established, then [beta]-sitosterol percentage in relation to initial hexane:chloroform (1:1) extract was possible to measure as 0.66%.

In the leishamanicidal assays, cycloeucalenone showed no activity against the promastigote form of L. infantum chagasi, whereas 31-norcyclolaudenone, the mixture stigmasterol + [beta]-sitosterol and 24-methylene-cycloartanol presented leishmanicidal activities with [EC.sub.50] values of 39.29 [micro]g/ml, 14.35 and 16.55 [micro]g/ml, respectively (Table 1). These activities were similar to the control pentamidine with [EC.sub.50] value of 23.71 [micro]g/ml.

In relation to amastigote assay, the mixture stigmasterol + [beta]-sitosterol, 24-methylene-cycloartanol and cycloeucalenone with [EC.sub.50] values of 126.5, 98.75 and 185 [micro]g/ml, respectively, had statistical similarity to the standard drug Amphotericin B with [EC.sub.50] 32.23 (14.30-72.62).

The sterols and triterpenes isolated from M. paradisiaca were tested against RAW 264.7 cells and LLC-MK2 (Table 2). Pentamidine, the control drug, presents higher toxicity then compounds from banana peel.

Discussion

The peel, sap and fruit of M. paradisiaca are used in folk medicine in the treatment of leishmaniasis in an endemic area of leishmaniasis in Northwestern Ecuador (Weigel et al. 1994). The main compounds present in Musa spp. are sterols and cycloartane-type triterpenes (Oliveira et al. 2008). Numerous benefits have been attributed to the phytosterols, such as being anti-inflammatory, anti-carcinogenic, and protective of immune systems (Quilez et al. 2003). Ozipek et al. (2005) reported the leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides isolated from the lower stem parts of Astragalus oleifolius. These compounds showed notable growth inhibitory activity against Leishmania donovani with [IC.sub.50] values ranging from 13.2 to 21.3 [micro]g/ml.

A major obstacle in the treatment of leishmaniasis are the side effects caused by the drugs currently available that have high toxicity, including nephrotoxic potential of amphotericin B, second choice drug in Brazil (WHO 2010). Singh et al. (2008) considered compounds with [EC.sub.50] values of mammalian cells above 250 [micro]g/ml to be safe, so cycloartane-type triterpenes and sterols from M. paradisiaca are safe compounds against tested cells.

Banana peels are used in many recipes to prepare cakes, biscuits and are additioned in banana sweets preparations, so the population already uses it without suffering from any toxic effects (Oliveira et al. 2009). The banana peel due to its leishmanicidal constituents with low toxicity could be a useful source for the development of a product to be used in Leishmaniasis treatment.

Conclusion

The cycloartane-type triterpenes and the mixture of steroids sitosterol and stigmasterol display leishmanicidal action against L. infantum chagasi strain and low toxicity to RAW 264.7 and LLC-[MK.sub.2] cells. The presence of leishmanicidal compounds in banana peel partially supports its ethnopharmacological use.

ARTICLE INFO

Article history:

Received 24 January 2014

Received in revised form 3 April 2014

Accepted 11 May 2014

Acknowledgement

The authors thank to Ceara State Government Foundation for Scientifical and Technological Development (FUNCAP) and Brazilian Counsel for Scientifical and Technological Development (CNPq) for financial support.

References

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Imam, M.Z., Akter, S., 2011. Musaparadisiaca L. and Musasapientum L.: a phytochemical and pharmacological review. J. Appl. Pharm. Sci. 1, 14-20.

Khuonghuu, F., Sangare, M., Chari, Vm, Bekaert, A., Devys, M., Barbier, M., Lukacs, G., 1975. C-l 3 nuclear magnetic-resonance spectral analysis of cycloartanol and related compounds. Tetrahedron Lett. 22, 1787-1790.

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Matos, F.J.A., 2009. Introducao a Fitoquimica Experimental. Edicoes UFC, Fortaleza, Brasil, 150 pp.

Oliveira, L., Freire, C.S.R., Silvestre, A.J.D., Cordeiro, N., 2008. Lipophilic extract from banana fruit residues: a source of valuable phytosterols. J. Agric. Food Chem. 56, 9520-9524.

Oliveira, L.F., Borges, S.V., Nascimento, J., Cunha, A.C., Jesus, T.B., Pereira, P.A.P., Pereira, A.G.T., Figueiredo, L.P., Valente, W.A.. 2009. Utilizafao de casca de banana na fabricaqao de doces de banana em massa--avaliafao da qualidade--the use of banana preserves--evaluation of quality. Alim. Nut. Araraquara 20, 581-589.

Ozipek, M., Donmez, A.A., Cali?, I., Brun, R., Riiedi, P., Tasdemir, D., 2005. Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius. Phytochemistry 66, 1168-1173.

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Quilez, J., Garcla-Lorda, P., Salas-Salvado, J., 2003. Potential uses and benefits of phytosterols in diet: present situation and future directions. Clin. Nutr. 22, 343-351.

Ragasi, C.Y., Martinez, A.T., Chua.J.E.Y., Rideout, J.A., 2007. A triterpene from Musa errans. Philipp. J. Sci. 136, 167-171.

Singh, N., Kumarn, R., Gupta, S., Dube, A., Lakshmi, V., 2008. Antileishmanial activity in vitro and in vivo of constituents of sea cucumber Actinopyga lecanora. Parasit. Res. 103, 351-354.

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A.A.S. Silva (a), S.M. Morais (a,b),*, M.J.C. Falcao (b), I.G.P. Vieira (b), L.M. Ribeiro (b), S.M. Viana (c), M.J. Teixeira (c), F.S. Barreto (d), C.A. Carvalho (e), R.P.A. Cardoso (e), H.F. Andrade-Junior (e)

(a) Programa de Pos Graduacao em Ciencias Veterinarias, Universidade Estadual do Ceara, Ceara, Brazil

(b) Curso de Quimica, Universidade Estadual do Ceara, Ceara, Brazil

(c) Departamento de Patologia e Medicina Legal, Universidade Federal do Ceara, Ceara, Brazil

(d) Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara, Ceara, Brazil

(e) Institute de Medicina Tropical de Sao Paulo, Universidade de Sao Paulo, Sao Paulo, Brazil

* Corresponding author at: Addess: Curso de Qulmica, Universidade Estadual do Ceara. Avenida Dr. Silas Munguba, 1700, CEP: 60714-903, Fortaleza-CE, Brazil, Tel.:+55 +85 31019645.

E-mail address: selenemaiademorais@gmail.com (S.M. Morais).

http://dx.doi.org/10.1016/j.phymed.2014.05.005

Table 1
Leishmanicidal activity of triterpenes and sterols isolated
from the fruit peel of M. paradisiaca.

                         [EC.sub.50] (([micro]g/ml)

Compounds              Promastigote    Amastigote

31-Norciciolaudenone   39.29 (34.44-   200.7 (75.63-
  (C1)                 44.82) (a)      532.8) (a)

Cycloeucalenone        >100 *          185.0 (78.49-
  (C2)                                 436.2) (a,b)

Stigmasterol +         14.35 (10.38-   126.5 (64.97-
  [beta]-sitosterol    19.84) (a)      246.2) (a,b)
  (C3)
24-Methylene-          16.55 (11.13-   98.75 (47.34-
  cycloartanol         24.60) (a)      206.0) (a,b)
  (C4)
Pentamidine            23.71 (18.44-   --
                       30.50) (a)
Amphotericin B         --              32.23 (14.30-
                                       72.62)b
(-) Not evaluated.

(a) Similar letters means no statistical difference.

Not compared.

Table 2
Cytotoxicity in mammalian cell of sterols
isolated from the fruit peel of Musa
paradisiaca.

                       [EC.sub.50] ([micro]g/ml) cell mammalian

Drug                   Cell RAW        Cell [LLC-MK.sub.2]

31-Norciclolaudenone   197.7 (111.1-   697.5 (359.0-
                       337.8) (a)      1355.0) (a)

Cycloeucalenone        241.8 (170.7-   610.5(344.1-
                       342.6) (a)      1083) (a)

Stigmasterol +         124.5 (77.76-   377.3(199.9-
  [beta]-sitosterol    199.4) (a)      712.1) (a)

24-Methylene-          505.6 (155.0-   2758 (249.1-
  cycloartanol         1649) (a)       30,533) (a)

Pentamidine            17.9 (2.4-      --
                       25.8) (b)

(a) Similar letters means no statistical difference.
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Author:Silva, A.A.S.; Morais, S.M.; Falcao, M.J.C.; Vieira, I.G.P.; Ribeiro, L.M.; Viana, S.M.; Teixeira, M
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
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