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Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

ARTICLE INFO

Keywords: Calophyllum brasiliense (-) mammea A/BB derivatives Antileishmanial activity Morphological and ultrastructural alterations Flow cytometry

ABSTRACT

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy8-(2-methylbutanoy1)-6-(3-methylbuty1)-4-phenyl-chroman-2-one (3), the most active antileishmanial with [LD.sub.50] of 0.9 [micro]M), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Introduction

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. Several species of this parasite are human pathogens, and are responsible for one of three clinical forms of the disease, visceral, cutaneous, and mucosal, Leishmania amazonensis causes cutaneous leishmaniasis, which in anergic patients may lead to diffuse leishmaniasis (Desjeux 2004). Leishmaniasis is a significant cause of morbidity and mortality in some tropical and subtropical regions and in underdeveloped countries, directly affecting about 2 million people annually (WHO 2001a, b; Croft and Coombs 2003).

Unfortunately, the drugs utilized in the treatment of leishmaniasis (pentavalent antimonials, amphotericin B and pentamidine) are limited to some extent by their toxicity to the patients, requirement for intravenous administration, require long-term treatment, lack of efficacy, and high cost, and are prone to stimulate drug resistance (WHO 2001a,b; Croft and Coombs 2003; Croft et al. 2005). Taking into account the side effects and the resistance that pathogenic protozoa develop against the drugs currently used to treat leishmaniasis, more attention should be given to extracts and biologically active compounds isolated from plant species commonly used in herbal medicine, as sources of new chemotherapeutic compounds with better activity and fewer side effects. It is estimated that about 25% of all modern medicines are directly or indirectly derived from medicinal plants. Extensive studies have shown that plant extracts and chemically defined molecules of natural origin possess antileishmanial activity (Camacho et al. 2003; McConville and Handman 2007).

Calophyllum brasiliense Camb. (Clusiaceae) has been used in folk medicine for the treatment of rheumatism, varicose veins, hemorrhoids, and chronic ulcers (Correa 1978). C. brasiliense has proved to be a rich source of bioactive compounds, including coumarins (Ito et al. 2003; Reyes-Chilpa et al. 2004), xanthones (Sartori et al. 1999; (to et al. 2002), triterpenoids (Reyes-Chilpa et al. 2004), and biflavonoids (Da Silva et al. 2001). The methanol extract from the heartwood of a congener C. kunstleri showed potent leishmanicidal activity (Takahashi et al. 2004), and xanthones from the heartwood of C. brasiliense have exhibited activity against Trypanosoma cruzi (Abe et al. 2004). Compounds isolated from C brasiliense have displayed high cytotoxic activity against some tumor cell lines (Kimura et al. 2005; Ito et al. 2006), mainly the coumarin mammea A/BB (Reyes-Chilpa et al. 2004; Ruiz-Marcial et al. 2007). We have previously shown that the extracts, fractions, and mainly coumarin (-) mammea A/BB isolated from C. brasiliense leaves show significant molluscicidal activity against the snail Biomphalaria giabrata (Gasparotto-Junior et al. 2005) and potent in vitro and in vivo antileishmanial activity against L. amazonensis and L. braziliensis (Brenzan et al. 2007, 2008a; Honda et al. 2010). We also demonstrated antileishmanial activity of (-) mammea A/BB derivatives against L. amazonensis (Brenzan et al. 2008b). The coumarin mammea A/BB isolated from C brasiliense leaves, showed trypanocidal effects in vitro against T. cruzi (Reyes-Chilpa et al., 2008). Considering these effects, in our most recent study, we validated a method for quantitative analysis of the biologically active compound (-) mammea A/BB in C. brasiliense extracts by high performance liquid chromatography (HPLC) (Brenzan et al. 2010).

In the present stuay, we describe the morpnhological and ultrastructural alterations in L. amazonensis treated with natural, synthetic and derivative coumarins of (-) mammea A/BB, observed by different microscopic techniques. In addition, we report the drug target of (-) mammea A/BB and the derivative 5,7-dihydroxy-8-(2-methylbutanoy1)-6-(3-methylbuty1)-4-phenyl-chroman-2-one (3) in the parasite.

Materials and methods

Plant extraction and purification of the compounds

The natural compounds (-) mammea A/BB and (-) mammea BBB was isolated, purified and identified as described in our previous studies (Gasparotto-Junior et al. 2005; Brenzan et al. 2008b, 2010). Calophyllum brasiliense leaves were collected on Cardoso Island, July 2000, in the State of Sao Paulo, Brazil. A voucher specimen (SP 363818) is deposited and authenticated at the Herbarium of the Institute de Botanica de Sao Paulo, Sao Paulo, Brazil. The extract was prepared by exhaustive maceration in ethanol-water (90:10) at room temperature, filtered, and con-centrated under vacuum at 40 [degrees]C to obtain an aqueous extract and a dark-green residue, it was dissolved with dichloromethane and the organic solvent was completely removed at room temperature, yielding dichloromethane extract. Subsequently, this extract was chrornatographed in a vacuum silic-agel column with hexane, hexane-dichloromethane (50:50), dichloromethane; dichloromethane-ethyl acetate (90:10 to 50:50); ethyl acetate and methanol and methanol-water (90:10). Next, the hexane fraction was rechromatographed on a silica-gel column with different mixtures of solvents to obtain the compound (-) mammea A/BB (1) and (-) mammea B/BB (2) (Fig. 1). The procedure details, which led us to obtain the compound (-) mammea A/BB (1) and (-) mammea B/BB (2) was described in our previous studies (Gasparotto-Junior et al. 2005; Brenzan et al. 2008b, 2010). Subsequently, these pure compounds were tested by antiprotozoal activity.

Derivatives

The derivatives 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3); 7-hydroxy-5-methoxy-8-(2-methylbutanoy1)-6-(3-methylbut-2-en-l-y1)-4-phenylcoumarin (4); 5,7-dimethoxy-8-(2-methylbutanoy1)-6-(3-methylbut-2-en-l-y1)-4-phenylcoumarin (5); 5,7-dimethoxy -841-methoxy-2-methylbuty1)-643-methylbut-2-en-1-y1)-4-phenylcoumarin (6) and 5,7-dihydroxy-4-phenylcournarin (7) were obtained from the compound (-) mammea A/BB (1) by hydrogenation and methoxylation reactions, as described in our previous work (Brenzan et al. 2008b).

Structure elucidation

The structure of the isolated compounds and derivatives were identified by chromatography-mass spectrometry (Shimadzu CG/MS 17 A QP 5000 mass spectrometer, DB5 column (30 in; 0.32 [micro]m)): nuclear magnetic resonance (Balker DRX-400; Gemini 300 (7.05T); Varian), [H.sup.1] NMR (300 and 200 MHz), [C.sup.13] NMR (75.5 MHz) and COSY (300 MHz) using deuterated solvent (CD[Cl.sub.3]) and [a.sub.D] analysis (Perkin-Elmer polarimeter at 20 C at 589 rim) using dichloromethane as described previously by Gasparotto-Junior et al. (2005) and Brenzan et al. (2008b).

Parasites

Promastigote forms of L. amazonensis (MHOM/BR/75/Josefa), originally isolated from a human case of diffuse cutaneous leishmaniasis, has been maintained by weekly transfers in Warren's medium (brain-heart infusion plus haemin and folic acid) supple-mented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco Invitrogen Corp., New York, USA) at 25 [degrees]C in a tissue flask. Axenic amastigote forms of L. amazonensis were obtained by in vitro transformation of infective promastigotes (Ueda-Nakamura et al. 2001), and maintained by weekly transfers in Schneider's Insect medium (Sigma Chemical Co., St. Louis, Missouri, USA) at pH 4.6, supplemented with 20% heat-inactivated FBS at 32 [degrees]C in a tissue flask.

Antiprotozoal activity

This assay was evaluated as described in our previous study Brenzan et al. (2008b). Promastigote forms of L. amazonensis in log phase of growth ([10.sup.6] parasites/m1) were grown on a 24-well plate in Warren's medium supplemented with 10% heat-inactivated FBS in the absence or in the presence of different concentrations of compounds (1-7(150.0-0.3 [micro]M) at 25 [degrees]C, in order to evaluate parasite survival. In all tests, 0.5% dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, Mo., USA), a concentration that was used to dissolve the highest dose of the samples but was proven to have no effect on cell proliferation, and the medium alone were used as controls. Amphotericin B was used as the reference drug; it was assayed at concentrations of 1.0-0.025 [micro]M for promastigote forms. The percentage of inhibition was determined daily after promastigote forms were counted in a haemocytometer (Improved Double Neubauer). Each experiment was performed in triplicate on three different occasions, and the results were expressed as percentage of inhibition in relation to the control cultured in medium alone. The 50% lethal dose ([LD.sub.50]) was determined by logarithm regression analysis of the data obtained. The means and standard deviations of at least three experiments were determined. Statistical analysis of the differences between mean values obtained for experimental groups was done by means of Student's t test. p values of 0.05 or less were regarded as significant.

Scanning electron microscopy

In order to evaluate the morphological changes, promastigote forms of L. amazonensis were treated with the [DL.sub.50] (7.4; 30.1; 0.9; 2.4; 15.1; 1.9 and 60.2 [micro]M) of the compounds (-) mammea A/BB (1), (-) mammea BBB (2), and (-) mammea A/BB derivatives (3-7) (Brennan et al. 2008b). After 72 h of incubation promastigotes were fixed with 2.5% glutaratdehyde during 2h at room temperature. After fixation, the cells were placed on a specimen support with poly-L-lysine dehydrated in graded ethanol, critical-point dried in CO2, coated with gold, and observed in a Shimadzu SS-550 scanning electron microscope.

Transmission electron microscopy

To assay the ultrastructural changes induced by the compound (3), promastigote forms were treated with [LD.sub.50] (0.9 [micro]M) of this compound. After 72 h of treatment the cells were fixed with 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide and 0.8% potassium ferrocyanide, and 5 mM calcium. The postfixed cells were dehydrated in acetone and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a Zeiss 900 transmission electron microscope.

Flow cytometry analysis

Mitochondrial membrane potential was measured in L. amazonensis axenic amastigotes using Rhodamine 123 (Rh 123) reagent following the protocol of manufacturer. Briefly, leishmanial cells (5 x [10.sup.6] parasites/ml) after treatment in the absence or in the presence of the compounds (1) (123.2 [micro]M and 246.3[micro]M) and

(3) (121.9 [micro]M and 244.0 [micro]M) for 3h at 32 [degrees]C were harvested and washed with PBS. After that, the cells were incubated with Rh 123 (5 mg/ml for 30 min at 37 [degrees]C), and washed twice with PBS. Analysis for mean fluorescence intensity was done using FACSCalibur equipped with the CellQuest software. A total of 10,000 events were acquired in the region established as that corresponding to the parasites.The compound Carbonyl Cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control.

Results

Structure elucidation

The compounds (-) mammea A/BB (1), (-) mammea B/BB (2); 5,7-di hyd roxy-84 2-methylbutanoy1)-6-( 3-methyl-buty1)-4-phenyl-chroman-2-one (3); 7-hydroxy-S-methoxy-8-(2-methylbutanoy1)-6-(3-methylbut-2-en-1-yl)-4-phenylcouma-rin (4); 5,7-dimethoxy-8-(2-methylbutanoyl)-6-(3-methylbut-2-en-1-y1) 4 phenylcoumarin (5); 5,7-dimethoxy-8-(1-methoxy-2-methylbuty1)-6-(3-methylbut-2-en-l-y1)-4 phenylcoumarin (6); 5,7-dihydroxy-4-phenylcoumarin (7) were identified, and the spectral data were described in our previous study (Gasparotto-Junior et al. 2005; Brenzan et al. 2008a,b) (Fig. 1).

Antiprotozoal activity

The antileismanial activity of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) against Leishma-nia amazonensis was described in our previous study Brenzan et al. (2008b) (Table 1).

Table 1
Antileishmanial activity against promastigote forms of Leishmania
umazonensis of the compounds (Brenzan et al. 2008b).

Compound        Prornastigote
                [LD.sub.50]([micro]M)

(1)              7.4 [+ or -] 0.30
(2)              30.1 [+ or -] 3.5
(3)               0.9 [+ or -] 0.1
(4)               2.4 [+ or -] 0.4
(5)              15.1 [+ or -] 1.0
(6)               1.9 [+ or -] 0.2
(7)              60.2 [+ or -] 3.5
Amphotericin B  0.063 [+ or -] 0.1

Values represent the mean [+ or -] S.D. of at least three experiments
performed in triplicate.


Scanning electron microscopy

Promastigote forms treated with compounds (1-7) revealed notable morphological changes compared with the control cells, which showed the typical morphology, with elongated body and flagellum (Fig. 2A). Promastigotes treated with [LD.sub.50] (7.4, 30.1, 0.9, 2.4, 15.1, 1.9 and 60.2 pi.M) of compounds (1-7) showed changes in the size and shape of cells, as observed by light microscopy. The treated cells showed a rounded appearance (Fig. 2B-K and M, N), triangular shape (Fig. 2L), significant cell shrinkage (Fig. 2G and H), multiflagellated cells (Fig. 2B, F, E, L and K), flagellum reduced in size or rolling (Fig. 2B, F, K, 0, G and H), plasma membrane with significant alterations (Fig. 2C, D, F, J, M, N, and P), and aberrant multiseptation of the cell body (Fig. 2E and F). The main morphological alterations were observed in promastigotes treated with compound (3), which showed significant cell shrinkage and a rounded appearance of the cells (Fig. 2G and H), in accordance with the alterations observed by light microscopy.

Transmission electron microscopy

In order to obtain information about the action of the most active antileishmanial compound (3) on L. amazonensis, the cells were analyzed by transmission electron microscopy. The photomicrographs (Fig. 3) of promastigotes untreated or treated with L1350 (0.9 RM) of compound (3) revealed that the treated parasites sustained different degrees of damage after 72 h of incubation. Control cells showed a normal ultrastructure, with normal plasma membrane, nuclei, mitochondrion, kinetoplast, flagellum and flagellar pocket (Fig. 3A), In contrast, the promastigotes treated with compound (3) showed significant ultrastructural alterations (Fig. 3B-D). Among the alterations were the appearance of mitochondria! swelling, with reduction of the density of the mitochondria! matrix (Fig. 3B and D) and the presence of vesi-cles inside the mitochondrion (Fig. 3B), indicating damage and significant change in this organelle. Other important ultrastructural alterations induced by compound (3) were the abnormal chromatin condensation (Fig. 3B and D) and alterations in the nuclear envelope (Fig. 3D). In addition, the compound (3) also induced intense atypical cytoplasmic vacuolization (Fig. 3C) and the appearance of autophagic vacuoles (Fig. 3C).

Flow cytometry analysis

The mitochondrial transmembrane potential was studied by using the fluorescent probe Rh 123 by flow cytometry analysis. Histograms of total Rh123 fluorescence (Fig. 4) demonstrated that the treatment with compound (1) decreased the parasite mitochondrial membrane potential ([DELTA] [PSI] m) by 30.3% and 55.0% when treated with 123.2 paM and 246.3 RM, respectively (Fig. 4B and C). In addition, compound (3) decreased the parasite mitochondrial membrane potential (.6.@m) by 87.3%, and 85.3% when treated with 121.9 RM and 244.0 [micro]M, respectively (Fig. 4D and E). Similarly, a decrease in membrane potential values was also observed following treatment with the standard drug carbonyl cyanide m-chlorophenylhydrazone (CCCP) (70.3%) at 200 RM for 3 h at 32[degrees]C Untreated cells maintained their membrane potential (7.7%) (Fig. 4A).

Discussion

In our preceding studies, we have shown that the extracts, fractions, and mainly coumarin (-) mammea A/BB isolated from leaves of C. brasiliense show potent in vitro and in vivo antileish-manial activity against L. amazonensis and L braziliensis (Brenzan et al. 2007, 2008a,b; Honda et al. 2010). We also demonstrated the structure-activity relationship of coumarin (-) mammea A/BB isolated from C brasiliense leaves; for this, we evaluated the antileishmanial activity of natural (1-2), synthetic (7) and derivatives of (-) mammea A/BB (3-6), against promastigote and intracellular amastigote forms of L amazonensis (Brenzan et al. 2008b). This study showed that the compounds (3), (4) and (6) were more biologically active than the compound (-) mammea A/BB (1). Compounds (1), (3), (4) and (6) were active not only against promastigote forms, but also against intracellular amastigote forms. Interestingly, compound (3) showed the highest antileishmanial activity of all (Brenzan et al. 2008b). This study revealed that several aspects of the chemical structure of compounds (1-7) were important for their antileishmanial activity. We emphasize that the C. brasiliense extract, the compound (-) mammea A/BB and derivatives of this compound proved to be safe (Brenzan et al. 2008a,b; Honda et al. 2010). Continuing studies of the antileishmanial properties of these compounds, in the present study we describe the extensive morphological and ultrastructural alterations in L. amazonensis treated with natural (1-2), synthetic (7) and derivatives of (-) mammea A/BB (3-6), using scanning and transmission electron microscopy.

By scanning electron microscopy, morphological changes were observed, including cell shrinkage and rounded appearance of the cells, in accordance with the alterations observed by light microscopy. We also report significant ultrastructural changes induced in promastigotes of L. amazonensis treated with [LD.sub.50] (0.9 [micro]M) of derivative (3); considering their highest antileishmanial activity against promastigotes and intracellular amastigotes of L. amazonensis observed in our previous study (Brenzan et al. 2008b).

In addition, the ultrastructural changes in mitochondria of parasites led us to examine the possible mechanisms of action of the (-) mammea A/BB (1) and derivative (3) using flow cytometry. This assay was performed with the natural compound (-) mammea A/BB (1) and derivative (3), to compare the effects caused by the natural compound (-) mammea A/BB (1) and its more active derivative (3) on the mitochondrial transmembrane potential. Histograms of total Rh 123 fluorescence revealed that compound (3) sharply decreased the parasite mitochondrial membrane potential (A (Pm), more intensely than compound (-) mammea A/BB (1). Similar decrease in membrane potentials was also observed in L amazonensis amastigotes treated with copaiba oil (Santos et al. 2012).

Previous studies have demonstrated ultrastructural changes in mitochondria! morphology of promastigote forms of L amazonensis treated with natural leishmanicidal agents, such as a dihydroxy-methoxychalcone from Piper aduncum inflorescences (Torres-Santos et al. 1999); a purified indole alkaloid, coronaridine, from the stem of Peschiera australis (Delorenzi et al. 2001); linalool-rich essential oil from Croton cajucara (Rosa et al. 2003); eugenol-rich essential oil from Ocimum gratissimum (Ueda-Nakamura et al. 2006); (-) mammea A/BB coumarin from C. brasiliense leaves (Brenzan et al. 2007, 2008a, b); citral-rich essential oil from Cymbopogon citratus (Santin et al. 2009) and eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens (Vendrametto et al. 2010). These compounds showed significant changes in mitochondrial morphology that appear to precede the loss of cell viability, confirming the importance of this organelle for the parasite's viability.

The trypanosomatid mitochondrion is the first organelle affected after treatment with different sterol biosynthesis inhibitors (Rodrigues et al. 2007). The sterol biosynthesis inhibitors (Rodrigues et al. 2002; Lorente et al. 2004) and the specific inhibitor of squalene synthase (Rodrigues et al. 2005) also cause mitochondria! swelling in L. amazonensis. The mitochondria! changes appear to be caused by a decrease in endogenous sterols, which are essential for the maintenance of the cellular and normal structural organization of the mitochondria! membrane in trypanosomatids (Rodrigues et al. 2001, 2007). Biochemical studies have shown that, contrary to what is known for mammalian cells, there are large amounts of endogenous and exogenous sterols in the mitochondrial membranes of trypanosomatids (Lazardi et al. 2001; Rodrigues et al. 2001). This indicates that the mitochondrion of trypanosomatids is an important target in leishmaniasis chemotherapy, because it is responsible for respiration and oxidative phosphorylation in most eukaryotes and also in some parasitic protozoa (Rodrigues and Souza 2008).

Sterol biosynthesis inhibitors also induced ultrastructural changes in other organelles, such as the nucleus and plasma membrane, and promoted the formation of autophagic vacuoles. All these changes are most probably related to the marked alterations in the lipid composition of the membranes of different trypanosomatids. The absence of essential lipids should lead to significant differences in the physical and chemical properties of the parasite's lipid membranes, inducing the disorganization and eventual destruction of these macromolecutar structures (Rodrigues et al. 2007).

The abnormal chromatin condensation and alterations in the nuclear envelope were also observed in promastigotes treated with compound (3). Similar ultrastructural changes have been reported for T. cruzi epimastigotes treated with inhibitors of sterol biosyn-thesis, which cause abnormal chromatin condensation, suggesting that this alteration is correlated with changes in the nuclear structure and cell division (Lazardi et al. 1991; Vivas et al. 1996). Moreover, nuclear envelope dilation in tripomastigotes of T. cruzi treated with naphthofuranquinones was also observed (Menna-Barreto et al. 2009).

Ultrastructural analysis also showed that compound (3) induced intense atypical cytoplasmic vacuolization and the appearance of autophagic vacuoles in L. amazonensis promastigotes. Similar alterations were found when L. amazonensis promastigotes were treated with inhibitors of sterol biosynthesis (Santa-Rita et al. 2005; Rodrigues et al. 2007; Rodrigues and Souza 2008), yangambin, a lignan obtained from Ocotea duckei (Monte-Neto et al. 2011) and eupomatenoid-5, neolignan isolated from Piper regnellii (Vendrametto et al. 2010), which induces cell death by autophagy. The intense cytoplasmic vacuolization and autophagic-like vacuoles suggest recycling of abnormal membrane structures, indicating a process of intracellular remodeling (Rodrigues and Souza 2008).

In conclusion, the results of the present study show that natural products represent an unparalleled source of molecular diversity in drug discovery and the development of novel antipro-tozoal agents. In this context, the compounds (-) mammea A/BB (1) and 5,7-dihydroxy-8-(2-methylbutanoy1)-6-(3-methylbuty1)-4-phenyl-chroman-2-one (3) are potential candidates for further research to develop new antiprotozoal drugs, considering their significant antileishmanial activity that may act in depolarization of the mitochondria] membrane potential cells, leading to death of the parasite.

Acknowledgments

This study was supported through grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq, Capacitacao de Aperfeicoamento de Pessoal de Nivel Superior - CAPES, Financiadora de Estudos e Projetos - FINER and Programa tie Pos-graduacao em Ciencias Farmaceuticas da Universidade Estadual de Maringa.

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(*.) Corresponding author. Tel.: +55 44 3261 4840; fax: +55 44 3261 4999. E-mail address: dagcortez@uem.br (D.A.G. Cortez).

M.A. Brenzan (a), A.O. Santos (b), C.V. Nakamura (b), B.P. Dias Filho (b), T. Ueda-Nakamura (b), M.C.M. Young (c), A.G. Correa (d), J. Alvim Junior (d), J.A. Morgado-Diaz (e), D.A.G. Cortez (f), *

(a.) Pos-doutoranda em Ciencias Farmaceuticas, Departamento de Farmacia e Farmacologia, Universidade Estadual de Marines, Av. Colombo 5790, 87020-900 Maringa, PR, Brazil

(b.) Laboratorio de Inovacao Tecnologica no Desenvolvimento de Farmacos e Cosmeticos, Departamento de Ciencias Basicas da Saude, Universidade Estadual de Maringa, PR, Brazil

(c.) Instituto de Botanica de Sao Paulo, SP, Brazil

(d.) Laborarorio de Quimica, Universidade Federal de Sao Carlos, SP. Brazil

(e.) Instituto Nacional de Cancer, RI Brazil

(f.) Pos-Graduacao em Ciencias Farmaceuticas, Departamento de Farmacia e Farmacologia, Universidade Estadual de Maringa, Maringa, PR, Brazil

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doi:10.1016/j.phymed.2011.10.008
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Author:Brenzan, M.A.; Santos, A.O.; Nakamura, C.V.; Filho, B.P. Dias; Ueda-Nakamura, T.; Young, M.C.M.; Cor
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Mar 1, 2012
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