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Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits.

ABSTRACT

Infections by protozoans of the genus Leishmania are the major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which exert renal and cardiac toxicity. Thus, there is a strong need for safer and more effective treatments against leishmaniasis. The present study was designated to evaluate, by a bioguided assay, the leishmanicidal activity of extracts (hexane, ethyl-acetate and ethanolic) and molecules both obtained by means of extraction from pericarps of Garcinia brasiliensis fruits. The hexane extract presented the best activity on the extracellular (promastigotes) and intracellular (amastigotes) forms of Leishmania (L.) amazonensis, when compared to the other extracts. Based on these findings, this extract was fractionated by silica gel column chromatography, affording nine fractions then resulting in three purified prenylated benzophenones--7-epi-clusianone (1), garciniaphenone (2) and guttiferone-a (3). They showed significant activity on Leishmania (L) amazonensis, and little toxicity for mammalian cells. Structure-activity relationships were evaluated showing that the [1C.sub.50] value displayed is dependent of prenyl groups and phenolic hydroxyls number, and inversely proportional to the hydrophobicity. Our results are promising, showing that these compounds are biologically active on Leishmania (L.) amazonensis.

[c] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Garcinia brasiliensis

Leishmanicidal

Benzophenones

Leishmania

Introduction

Leishmaniasis is a group of tropical diseases caused by a number of species of protozoan parasites belonging to the genus Leishmania. This ailment affects around 12 million people in 80 countries and it is estimated that there are about two to three million new cases each year. It is also considered that presently there is a population of 350 million people under risk of infection (WHO 1997).

Despite a many research achievements, first-line chemotherapy is still based on pentavalent antimonials developed more than 70 years ago, which are toxic and prone to drug resistance. In some trials, alternative pharmaceutical formulations have been used to reduce the toxicity of these drugs (Frezard et al. 2000). Second-line drugs, such as Amphotericin B, are more toxic and Amphotericin B's lipid formulation is too expensive for routine use in underdeveloped countries (Murray 2001). Thus, there is a strong need for safer, cheaper and more effective treatments against leishmaniasis.

Research on antiprotozoal drugs of medicinal plant origin is a multidisciplinary task which involves researchers and students in the fields of botanics, phytochemistry, parasitology, pharmacology and medicine (Billo et al. 2005). From the mid-eighties, date when more formal and constant research on natural metabolites with leishmanicidal and antiprotozoal activity was initiated (Chan-Bacab and Pena-Rodriguez 2001), at nowadays, many natural products have been reported to show antiprotozoal activity, including naphthoquinones (Kayser et al. 2000), lignans (Sauvain et al. 1996), triterpenoids (Sauvain et al. 1996), neolignans (Barata et al. 2000), alkaloids (Delorenzi et al. 2001), chalcones (Torres-Santos et al. 1999) and benzophenones (Fumiko et al. 2004).

Members of genus Garcinia are rich and valuable source of bioactive compounds (Monache et al. 1984; Almeida et al., 2008). Recently, Garcinia species have received considerable attention from a pharmacological point of view because some them produce potent inhibitors of reverse transcriptase of human immunodeficiency virus type 1 (Gustafson et al. 1992). In addition, extracts and isolated molecules from stem bark of G. lucida shows potent tripanocidal activity (Fotie et al, 2007). In order to find new drugs against leishmaniasis, we have studied extracts and natural molecules from Brazilian plants such as Garcinia brasiliensis (syn. Rheedia brasiliensis), commonly known as 'bacupari', is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor diseases treatments (Correa M.P. 1978) Previous works have reported the presence of the biflavonoids, volkensiflavone, fukugetin (Botta et al. 1984) and prenylated xanthones in methanolic extracts of C. brasiliensis roots (Morton 1987).

How part of our continuous study of Garcinia species, we offer to do the bioguided-assay fractionation and to perform the leishmanicidal activity against both promastigote and amastigote Leishmania forms.

Materials and methods

Plant material

Garcinia brasiliensis fruits were collected from trees grown under controlled conditions at the herbarium of the University of Vicosa (latitude 20[degrees]45'14" south and longitude 42[degrees] 52'55" west), Minas Gerais, Brazil, where its voucher specimen is deposited (number VIC2604). To obtain the extracts, the pericarp fruits dried and powdered were treated with solvents in growing polarity gradient since hexane until ethyl-acetate and ethanol, at room temperature, using the soxhlet equipment for 24 h in each extraction. Each extract concentration was obtained under reduced pressures, which were finally chromatographed as described by Derogis et al. (2008).

General experiments procedures

The UV-vis spectrum was obtained on a spectrophotometer (Shimadzu-model 2550) double beam. Each of nine fractions (1 mg) from hexane extract was dissolved in 10 ml of ethanol. The sample solution was scanned from 700 to 200 nm. GC-MS analysis were obtained utilizing a Shimadzu GCMS-QP5050A equipment connected to an ion trap detector operating in Electron Impact mode at 70 eV with scan range of 29-400 Daltons, as described by Martins et al. (2008). The high performance liquid chromatography (HPLC) analysis of hexane extract was performed in Shimadzu LC-100 equipment using a C18 column Shimadzu CLC-ODS (250 - 4,6 mm) with a 5-[micro]m particle size. The suitable gradient was achieved using MeOH:acetic acid 0,1%, pH 3.80, (70:30 v/v) to MeOH 100% for 5 min, with a solvent flow rate of 1.2 ml/min, at 30 [degrees] C, [lambda] = 254 nm, and the injection volume of 20 [micro]l at concentration of 1 mg/ml. Class VP-LC10 software was used for data collection. Theirs Log P values were determined using the program QikProp. version 3.0, Schrodinger, LLC, New York, NY, 2008.

Extraction and isolation procedures

Dried and ground pericarps from fruits (1 kg) were extracted with solvents in crescent polarity, n-hexane, ethyl-acetate and ethanol, in this sequence, in soxhlet equipment, for 24 h with each solvent. The extracts were concentrated under reduced pressures using rotary evaporator; and then dried under vacuum, yielding 57.14g, 105.44g and 253.06g, of hexanic, ethyl-acetate and ethanolic extracts, respectively. These were used for the assays. After the evaluation of activity, the hexanic extract displayed the better leishmanicidal activity. After the analyze using thin layer chromatography (TLC) the hexane extract was chromatographed on silica gel (230-400 mesh) column (8 x 100 cm) eluted with crescent polarity mixtures of n-hexane/ethyl-acetate and ethyl-acetate/ethanol to give 32 fractions. These fractions were then pooled in nine groups according to their similarities in TLC. Each group was analyzed by spectrophotometer and characterized in relation their leishmanicidal activity in established concentrations against promastigote forms for to obtain a spectroscopic relationship among the fractions. The fractions were, so, rechromato-grafed on silica gel (230-400 mesh) column (8 x 100 cm) eluted with crescent polarity mixtures of n-hexane/ethyl-acetate and ethyl-acetate/ethanol to obtain the three prenylated benzophenones (Fig. 1)--7-epiclusianone (1), garciniaphenone (2) and guttiferone-a (3) that were established by uni- and bidimensional nuclear magnetic resonance (NMR) and comparison with literature values (Gustafson et al. 1992; Derogis et al. 2008). The HPLC chromatogram of the hexane extract showed the presence of three benzophenones with retention times 14.128, 13.176 and 12.832 min, respectively for (1), (2) and (3).

[FIGURE 1 OMITTED]

Leishmanicidal activity against promastigotes

Promastigotes of Leishmania (L.) amazonensis (MHOM/BR/ 71973/M2269) were grown on a 24-wells plate in Schneider's Drosophila medium (Sigma, USA) supplemented with 10.0% (v/v) heat-inactivated fetal bovine serum and 1.0% penicillin (10000 UI/ ml)/streptomycin (10.0 mg/ml) (Sigma, USA). Cells were harvested in the log phase, resuspended in fresh medium, counted in Neubauer's chamber and adjusted to a concentration of 1 x [10.sup.6] cells/mL. The fractions of hexane extract at 5.0 [micro]g/ml and the crude extracts and isolated compounds in the range of 0.05 to 100.0 [micro]g/ml were added to promastigote cultures, at 1 x [10.sup.6] cells/ ml, solubilized in dimethylsulfoxide (DMSO) (the concentration used was 0.6%, v/v in all wells) and incubated at 25[degrees]C. After 72 h of incubation, the surviving parasites were counted in a Neubauer's chamber and compared with controls, with just DMSO in concentration of 0.6% v/v, for the determination of 50.0% inhibitory growth concentration ([IC.sub.50]). All tests were performed in triplicate on three different occasions and Amphotericin B (Eurofarma) was used as the reference drug.

Leishmanicidal activity against amastigotes

Murine peritoneal macrophages were maintained in RPMI 1640 medium (Sigma, USA) supplemented with 10.0% heat-inactivated fetal bovine serum at 37[degrees]C in 5.0% [CO.sub.2]. Cells were cultured in 24-well plates chamber on the glass slides of 13 mm (Nunc, USA) to a cell density of 8 x [10.sup.5] cells per well and infected with late log-phase promastigotes at a multiplicity of infection of 10:1 (parasite/macrophage) incubated at 37 [degrees]C in 5.0% [CO.sub.2] during 24 h. Nonphagocytosed promastigotes were removed by washing, and the drug dilutions (in the range of 0.05 to 100 [micro]g/ml) were administered solubilized in DMSO at the concentration of 0.6% v/v. After 72 h, chamber slides were fixed in absolute methanol, stained with 10.0% Giemsa and examined under an oil immersion objective of the light microscope. At least 200 macrophages were counted per well for calculating the percentage of infected cells. The percent inhibition was calculated in relation to the control only with DMSO, for the determination of [IC.sub.50] value (Tripathi et al. 2006). All tests were performed in triplicate on three different occasions and Amphotericin B (Eurofarma) was used as the reference drug.

Cytotoxicity evaluation

For the cytotoxicity assay a suspension of 8 x [10.sup.5] murine peritoneal macrophages, in RPMI 1640 medium, supplemented with 10.0% heat-inactivated fetal bovine serum and 1.0% penicillin (10000 UI/ml)/streptomycin (10mg/ml) were added to each well in 24-well plates, on the glass slides of 13 mm. The plates were incubated in a 5.0% [CO.sub.2] air mixture at 37 [degrees]C to adhesion of the cells. After 24 hs, the nonadherent cells were removed by washing with the RPMI 1640 medium. Thus, several concentrations of extracts and purified compound (in the range of 0.05 to 100.0 [micro]g/ ml) were added to the wells containing the cells. These substances were solubilized in DMSO at the final concentration of 0.6% v/v and the plates were incubated for more 72 h. Then, the nonadherent cells were removed by washing with the RPMI 1640 medium and 50.0 [micro]l of the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well at concentration of 5.0 mg/ml, followed incubation for more four hours, as described by Mossman (1983).

After this, the medium was retired and 600.0 [micro]l of DMSO was added to each well and it was homogenized for 15 min. Next, the absorbance of each individual well, minus the control value, was calculated in according to the next formula at 570 nm.

% inhibition = ([[OD.sub.control] - [OD.sub.drugs]]/[[OD.sub.control] x 100])

Each experiment was performed in triplicate on three different occasions, and the percentage of viable cells was calculated in relation to controls cultured in the medium with just DMSO at the concentration of 0.6% v/v. The 50.0% cytotoxicity concentrations ([CC.sub.50]) were determined and the security factors (SF) established by the ratio between the values of [CC.sub.50] and [IC.sub.50] for amastigote forms.

Statistical analysis

The leishmanicidal activity was expressed as growth inhibition. Statistical analysis was performed using nonlinear regression to obtain the values of [IC.sub.50] (concentration that inhibits growth by 50.0% of promastigotes) and [CC.sub.50] (cytotoxic concentration for 50.0% of macrophages) these values followed by variance analyses and Tukey's test. Differences were significant when the p value was lower than 0.05.

Results and discussion

There is no vaccine available against leishmaniasis. Drug resistance, variable efficacy, toxicity, parenteral administration, and requirement for long courses of administration are the main drawbacks of current leishmanicidal drugs (Croft et al. 2006). There is an urgent need for new drugs for the treatment of these diseases, which mainly affect people in developing countries.

Plants are an important source of therapeutic agents in the search for new and selective agents for the treatment of tropical diseases caused by protozoan. Several studies have shown that natural products represent a diverse source of compounds in drug discovery and in the development of novel antiprotozoal agents (Chan-Bacab and Pena-Rodriguez, 2001).

In recent years, natural products of different biosynthetic origins and several groups of compounds have been isolated and have shown activity on different species of Leishmania (Torres-Santos et al. 1999; Ferreira et al. 2004; Fotie et al. 2007; Singh et al. 2008). The leishmanicidal activity of plant extracts have been attributed to compounds belonging to diverse chemical groups, such as isoquinoline alkaloids, indole alkaloids, quinones and terpenes (Araujo et al. 1998). In special, benzophenones isolated from Guttiferae family plants have exhibited a prominent tripanocidal (Abe et al. 2004) and leishmanicidal activity (Lenta et al. 2007).

As a part of our screening program in search for bioactive molecules from Brazilian plants, we have investigated the G. brasiliensis for leishmanicidal activity. The hexane extract exhibited [IC.sub.50] values of 1.43 [micro]g/ml and 10.66 [micro]g/ml on promastigotes and intracellular amastigotes, respectively. Ethyl-acetate and ethanol extracts were unsatisfactory on both stages of the parasite (Table 2). Thus, the bioactive hexane extract was chromato-graphed on a silica gel column and pooled in nine fractions according to their similarities in TLC. These fractions, after evaluated on promastigotes forms of L. (L.) amazonensis (Fig. 2) showed an important activity and a correlation with the presence of majors constituents of hexane extract.

[FIGURE 2 OMITTED]
Table 2

Leishmanicidal activity of the extracts and isolated compounds, their
effects on peritoneal macrophages murine and security factor in
relation to amastigota.

Plant material   Promastigotes  Amastigotes   Macrophages  Security
                  [IC.sub.50]       (a)           (b)       factor
                   ([mu]g/ml)   [IC.sub.50]   [CC.sub.50]    (SF)
                                ([mu]g/ml)    ([mu]g/ml)

Hexane extract    1.43 *        10.66 *       35.91 *      3.37

Acetate-ethyl    32.50 *        > 70.00       > 100.00     nd
extract

Ethanolic        22.93 *        > 100.00      > 70.00      nd
extract

7-epiclusianone   3.33          1.63 *        19.13 *      11.74

Garciniaphenone   5.04          > 70.00       > 80.00      Nd

Guttiferone-a    18.12 *        2.93 *        10.71 *      3.66

Amphotricin B     2.81 *        5.68 *        nd           nd

nd = not determined.

(a) Concentration for decrease of 50% infected macrophages in treated
vs nontreated wells.

(b) Ototoxicity concentration for 50% macrophages.
SF = ratio between [CC.sub.50] and [IC.sub.50].

* p < 0.05.


These nine fractions from hexane extract were evaluated in relation to theirs individual contributions for the leishmanicidal activity, have been considered your relative mass and percent of inhibition at 5.0 [micro]g/ml. These data were compared with the hexane extract crude and compound (1), the main constituent of this. The cumulative values of fractions individual contributions on the growth inhibition is less than the value of crude extract, suggesting a synergic effect.

The fraction two represent almost 50.0% of crude extract, and have as the main constituent the molecule (1). This fraction shown, at 5.0 [micro]g/ml, an inhibition of 69.23% on promastigote forms and this purified compound shown, at the same concentration, an growth inhibition of 70.10%, confirming that the compound (1) is the majoritary constituent of fraction II. In relation to the crude hexanic extract, the growth inhibition at 5.0 [micro]g/ml was 77.38%. If the compound (1), the main constituent of the hexanic extract on overall, was responsible for the growth inhibition action of hexanic extract, the activity of extract was be about two time smaller than the growth inhibition of compound (1). Thus, this result indicates the existence of a synergism among the constituents of extract. The crude extract have more activity than isolates one-to-one or even indicated the presence of a minoritary constituent, that not was identified but have a large activity (Table 1).
Table 1

Individual contributions for leishmanicidal activity of hexane extract
fractions.

FR            I      II      III     IV     V      VI     VII     VIII

RM          3.308  47.895  13.145   5.022  4.884   1.684  19.049  1.951

Is         84.44   69.23   49.30   67.00   7.87   18.98   18.18  16.73

RM x        2.793  33.158   6.480   3.365  0.384   0.320   3.463  0.326
[I.sub.5]

FR          IX     [[SIGMA]I.sub.5]  [I.sub.(1)-5)]  [I.sub.[Hx-5]]

RM          3.061

Is         46.35

RM x        1.419      51.708            70.100          77.380
[I.sub.5]

FR: number of the fraction; RM: relative mass in relation to all hexane
extract; [I.sub.5]: percent of growth inhibition to 5 [micro]g/ml;
[SIGMA][I.sub.5]: cumulative value of percent fractions growth
inhibition to 5 [micro]g/ml; [I.sub.[(1)-5]]: percent of growth
inhibition for (1) to 5 [micro]g/ml; [I.sub.[Hx-5]]: percent of growth
inhibition for hexane extract to 5 [micro]g/ml.


The chromatogram of Fig. 3 shown that the three benzophenones, presented in the hexane extract, being the compound (1) the more lipophilic them (2) and (3) with retention times of 14.128, 13.176 and 12.832 min, respectively (Fig. 3).

[FIGURE 3 OMITTED]

Chemical analysis of hexane extract identified 7-epiclusianone as its major constituent (20.2%), followed by garciniaphenone (6.3%) and guttiferone-a (2.1%). After analysis by spectrophotometer, was possible to set up an espectroscopic profile among the fractions (Fig. 4).

[FIGURE 4 OMITTED]

The fraction one (an oil fraction) obtained from hexane extract fractionally showed the best activity on promastigote forms of Leishmania (Fig. 2). After analysis by gas chromatography-mass spectrometry (GC-MS) (Fig. 5) was possible identify a complex mixture of sesquiterpenes compounds, similar to described by Martins et al. (2008).

[FIGURE 5 OMITTED]

Due to the great variability of compounds identify, and difficult for isolate pure compounds from this fraction, at the moment, the fraction one do not gone evaluated in relation the activity on amastigote forms or toxicity.

The leishmanicidal activity of the isolated compounds from the bio-guided assay was compared with the extracts and pharmaceutical formulation amphotericin B, as shown in Table 2. The isolated polyprenylateds benzophenones--(1), (2) and (3)-showed significant activity on promastigote and the compounds (1) and (3) on amastigote forms of L. (L) amazonensis when compared with [IC.sub.50] values of 2.81 [micro]g/ml and 5.68 [micro]g/ml of amphotericin B, a positive control, in relation to promastigotes and amastigotes, respectively. Thus, these substances were more potent than the reference compound and showing a satisfactory security factor; which turn them into potential drugs for the treatment of leishmaniasis, while the compound (2) showed good activity on promastigotes, but did not do on amastigotes (Table 2).

Furthermore, the toxicity of extracts and isolated compounds on murine peritoneal macrophages were evaluated, obtaining the security factor (SF) from the relation between the toxicity on macrophages and leishmanicidal activity on amastigote forms. These SF were satisfactory, showing that the hexanic extract and its isolated compounds display selectivity to amastigotes in relation to mammalian cells (Table 2).

In the absence of any suitable lead or drug candidates for an effective leishmanicidal drug from natural resources, the results of the present study indicate the possible development of a leishmanicidal drug from a natural source.

According to literature data (Marcucci et al. 2001), most of the biological activities of natural products in propolis extracts, such as antimicrobial, trypanocidal and antitumoral are associated mainly to its prenylated compounds, being this true maybe for benzophenones. It was reported that the biological activity of a compounds might be increased by the increasing number of prenyl residues attached (Aga et al. 1994). In our elucidation of structure activity relationship, we determined that the activity increase proportionally the number of prenyl groups, in other letters, proportionally to lipophylicity. How described by Urzua et al. (2008), the lipophilicity was shown to be an important variable that might be an important factor in the biological activity. Thus, the compound (1), the more lipophylic compound was the more active on both forms of protozoan Leishmania, due to the presence of four prenyl groups. The compound (3) was the second best, because same having five prenyl groups, it have two phenol hydroxyls that increase a hydrophobic character of the molecule. The compound (2), show just three prenyl groups, thus, is the more hydrophobic than (1) (Table 3); which justify your weak biological activity on amastigote forms of Leishmania, maybe, due to your difficult in across the barrier represented by the macrophage and to reach the amastigote form.

[TABLE 3 OMITTED]

In conclusion, the results obtained from the present study suggest that hexane extract and the polyprenylateds benzophenones isolated from G. brasiliensis possess interesting leishmanicidal activity and could provide lead molecules for development of potent drugs for the leishmaniasis treatment, showing strong activity on promastigote and amastigote forms of L. (L.) amazonensis (MHOM/BR/71973/M2269) and little citotoxicity on mammal cells, with great SF. These important results make them, important and potential new compounds for development of new drugs against leishmaniasis, but a further detailed evaluation about their mechanism of action is still needed.

Acknowledgements

The authors thank Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support.

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I.O. Pereira (a), * M.J. Marques (b), A.L.R. Pavan (b), B.S. Codonho (b), C.L. Barbieri (c), L.A. Beijo (d), A.C. Doriguetto (d), E.C. D'Martin (a), M.H. dos Santos (a)

(a) Department of Pharmacy, Laboratory of Phytochemistry and Medicinal Chemistry, Federal University of Alfenas, MG, Brazil

(b) Department of Biological Sciences, Laboratory of Molecular Biology, Federal University of Alfenas, MG, Brazil

(c) Department of Immunology, Microbiology and Parasitology, Federal University of Sao Paulo, SP, Brazil

(d) Department of Exacts Sciences, Federal University of Alfenas, MG, Brazil

* Corresponding author. Tel.: +55 3532991109; fax: +55 3532991067.

E-mail address: ivan.farma@bol.com.br (I.O. Pereira).

doi: 10.1016/j.phymed.2009.07.020
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Author:Pereira, I.O.; Marques, M.J.; Pavan, A.L.R.; Codonho, B.S.; Barbieri, C.L.; Beijo, L.A.; Doriguetto,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Apr 1, 2010
Words:4676
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