Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.20 no.3 Curitiba June/July 2010
Atividade leishmanicida do extrato de Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae
Ezequias Pessoa de Siqueira*,I; Elaine Maria de Souza-FagundesII; Marcos Eduardo Guerra SobralIII; Tânia Maria de Almeida AlvesI; Ana RabelloI; Carlos Leomar ZaniI
ILaboratório de Química de Produtos Naturais, Centro de Pesquisas Rene Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima, 1715, 30190002 Belo Horizonte-MG, Brazil
IIDepartamento de Biofísica e Fisiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270901 Belo Horizonte-MG, Brazil
IIIDepartamento de Botânica, Instituto de Ciêncais Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Caixa Postal 486, 31270-901 Belo Horizonte-MG, Brazil
Crude ethanolic extracts from Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, was fractioned by Gel Permeation Chromatography, using SephadexTM LH-20 gel. Sixteen fractions were obtained and were supplied to cytotoxicity in vitro assay against Leishmania (L.) amazonensis amastigota cells. It was observed eight cytotoxic fractions against Leishmania (L.) amazonensis amastigota cells at range of 19 to 29 µg.mL-1. Two of them were not citotoxic against human peripheral blood mononuclear cell, with a great potential to isolation of more selective leishmanicidal substances.
Keywords: Blepharocalyx salicifolius, Myrtaceae, Leishmania (Leishmania) amazonensis, gel permeation chromatography, citotoxicity.
O extrato etanólico bruto de Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, foi fracionado por meio de Cromatografia de Permeação em Gel, utilizando-se SephadexTM LH-20. Dezesseis frações foram obtidas e foram submetidas ao ensaio de citotoxicidade in vitro contra células amastigotas de Leishmania (Leishmania) amazonensis. Verificou-se atividade citocida contra células amastigotas de Leishmania (L.) amazonensis em oito frações, a uma concentração de 19 a 29 µg.mL-1. Duas destas frações apresentaram baixa toxicidade para células mononucleares de sangue periférico humano, com grande potencial de isolamento de substâncias leishmanicidas mais seletivas.
Unitermos: Blepharocalyx salicifolius, Myrtaceae, Leishmania (Leishmania) amazonensis, cromatografia de permeação em gel, citotoxicidade.
Leishmaniasis is caused by parasitic protozoa of the genus Leishmania. Humans are infected via the bite of phlebotomine sandflies, which breed in forest areas, caves, or the burrows of small rodents. There are four main types of the disease: cutaneous, diffuse cutaneous, mucocutaneous and visceral. If left untreated, the disease can have a fatality rate as high as 100% within two years. It is an endemic disease in 88 countries, mainly developing countries (Afghanistan, Bangladesh, Bolivia, Brazil, India, Iran, Nepal, Peru, Saudi Arabia, Sudan, Syria). Brazil has an important epidemiologic status, where all kinds of leishmaniasis were reported. (WHO, 2003).
The usual therapies against leishmaniasis do not have high efficacy, are extremely toxic and the pharmaceutical industries do not have economic refund to research new drugs in developing countries. According to Pecoul et al. (1999) only thirteen new drugs against tropical diseases were commercialized since 1975 to 1997 in a total of 1233 new drugs. Due to geographic broad dispersion, mainly in poorest countries, a great number of patients and the lack of therapeutic arsenal, the development of new drugs that can be used against leishmaniasis are very important.
In this work, preliminary studies were carried out to investigate the antiparasitic activity of fractions from Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, against amastigotes of Leishmania (L.) amazonensis and the toxicity against normal peripheral blood mononuclear cells (PBMC).
MATERIAL AND METHODS
Collection of the vegetal material and crude extract production
Leaves of Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, specie were collected at Parque Estadual do Rio Preto, Minas Gerais State, Brazil, in November 2006. Exsiccates of the species were committed at UFMG Herbarium. The leaves were dried using an oven at 35 ºC for two weeks. The leaves were grounded and conditioned in ethanol (analytical grade) for two months. The crude extract was obtained after filtration and evaporation of the solvent.
Gel permeation chromatography system
The Gel permeation chromatography system was constituted by a glass column of 50 mm diameter and 250 mm length coupled in series to the two other similar columns of 50 mm diameter and 480 mm length, filled with SephadexTM LH-20 (GE Healthcare, USA.) gel. The system was pumped by means of a P-500 (Pharmacia, USA.) pump. It was used distilled ethanol as mobile phase pumped at 2 mL.min-1. The fractions were collected by SF 2120 (Advantec, JP) collector, it was used 120 tubes of 22 mL.
Thin layer chromatography
Thin layer chromatography was developed using 20x20 cm2 HF254 (Merck) plates. Ethyl acetate, hexane, dichloromethane and methanol were used as solvents. The chromatograms were revealed by means of vanillin or NP/PEG.
Procedure for determinations of hydrodynamics parameters of the gel permeation chromatography system
Void volume (V0) and inclusion volume (Vi) were determined using an ethanolic solution of polyvidone 20 kDa (Sigma-aldrich) at 1 mg/mL and benzophenone (Merck) at 1 mg.mL-1, respectively. Twenty ml of the mixture (1:1 v/v) were pumped at 5 mL.min-1, using ethanol as solvent. The column systems were coupled to UV/VIS detector SPD 10-A (Shimadzu, JP). The data were analyzed using Class LC-10 software (Shimadzu, JP).
Chromatography of the Blepharocalyx salicifolius raw extract
Twenty ml of the ethanolic solution at 187 mg.mL-1 were separated by gel permeations chromatography, performing 3.74 g of raw extract. Ethanol was used as mobile phase at 2.0 mL.min-1. The fractions were analyzed and grouped according to thin layer chromatography chemical profile.
Assays using amastigotes of Leishmania (L.) amazonensis
Promastigotes of Leishmania (L.) amazonensis (strain IFLA/BR/196/PH-8) were obtained from lesions of infected hamsters. The parasites were grown at 26 ºC in pH 7.2 Schneider's medium, and then stimulated to differentiate into the amastigote forms by rising the temperature (32 ºC), and lowering the pH (6.0) of the Schneider's medium. After 7 d under these conditions, 90% of the promastigotes were transformed into amastigote-like forms, verified by means of microscope, and then used in the bioassays. Amastigote density was adjusted to 1x 108 parasites per mL, and 90 µL added to each well of 96-well plates. Solutions at 200 µg.mL-1 in DMSO (1% in water) were performed for each fraction and then ten microliters of the solution were added to each well of 96-well plates. The plates were incubated at 32 ºC for 72 h, and then the cells viability was determined using the MTT (methyl thiazolyl tetrazolium) assay (Teixeira et al., 2002). The results are expressed as percent inhibition in relation to the controls without drug. Amphotericin B at 0.2 µg.mL-1 (Fungison® Bristol-Myers Squibb B, Brazil) was used as a positive drug control. All assays were performed in triplicate.
Proliferation assay with human Peripheral Mononuclear Blood Cells (PBMC)
Peripheral blood mononuclear cells (PBMCs) were prepared using the protocol previously described by Gazzinelli et al. (1983). PBMCs were obtained from healthy adult volunteers of both sexes by centrifugation of heparinized venous blood over a Ficoll/Hypaque cushion. Mononuclear cells were collected from the interphase after Ficoll separation and washed three times in RPMI-1640 before further processing. The cell suspensions were adjusted to 1.5x 106 cells.mL-1. All cultures were carried out in RPMI-1640 medium, supplemented with 5% (v/v) heat-inactivated, pooled AB sera and 2 mM L-glutamine. An antibiotic/antimicotic solution containing 1000 U penicillin.mL-1, 1000 mg streptomycin.mL-1 and 25 mg fungisone.mL-1 was added to control fungal and bacterial contamination (complete medium). The cell proliferation and viability were determined by the MTT assay (Jiang & Xu, 2003). Briefly, the cells are incubated with the extracts fractions at 20 µg.mL-1 and controls stimulated with 2.5 µg.mL-1 Phytohemagglutinin (PHA, or phytohemagglutinin). After 72 h, 20 µL of MTT solution (5 mg.mL-1 in RPMI filtered through 0.2 µm pore size filter) were added to the plates and incubated at 37 ºC for 4 h. The supernatant was carefully removed and 200 µL of HCl 0.04 N in isopropanol was added to dissolve the formazan crystals. The absorbance was measured at 590 nm with a microplate reader (VersaMax Tunable, Molecular Devices).
The hydrodynamics parameters V0 and Vi were obtained as 630 mL and 2125 mL, respectively, as showed at Figure 1. None specie was detected before V0. Between V0 and Vi the separations occurred mainly by molecular exclusion. After Vi, adsorption was the main process of separation. These statements could be observed by means of the chemical profile obtained by thin layer chromatography. Data not showed.
As we know that before V0 none of the species can be detected (hydrodynamics parameters), the collections of the samples started after elution of 600 mL of ethanol. It was collected 190 fractions of 20 mL. By means of thin layer chromatography, the chemical profile was performed and sixteen new groups were formed according to its chemical profile. It was recovered 3.04 g (yield 81%). It was noticed that more polar substances were eluted after the fraction 94. After the fraction 124, all material eluted was recovered in a unique fraction (F16), as showed at Figure 2.
Results of leishmanicidal assay
It was verified that the fractions F2, F3, F4, F7, F9, F11, F12 and F13 were the more active, showing inhibition of the growing against amastigota of Leishmania (L.) amazonensis (in vitro assay) higher than 80% at 200 µg.mL-1. The crude extract and the fractions F8 and F16 showed proliferative effect. As showed in Figure 3.
Concentration that inhibits 50% of the growing (IC50) was performed in vitro assays for the more active fractions against amastigota cell of Leishmania (L.) amazonensis. These values were obtained by means of nonlinear regression from data obtained at 100 µg.mL-1 to 0.2 µg.mL-1 by means of successive dilutions. The results are presented in Table 1.
Impact of crude extracts and fractions on the viability of Leishmania (L.) amazonensis and on the proliferation of human PBMC
The crude extracts and fractions were evaluated about their antileishmania potential and their cytotoxity activity against normal cells using the PBMC proliferation PHA-induced assay. The results on the Table 2 show that the fractions F2, F3, F4, F7, F9, F11, F12 and F13 were the more active against amastigota of Leishmania (L.) amazonensis. Only the F11 and F12 fractions did not show a significant inhibitory effect on PBMC proliferation when compared with control (DMSO, 0.01%).
Several studies has been carried out using natural products as a source to obtain species that can be used as a target in treatment of leishmaniasis (Rao et al., 2004; Ayres et al., 2007; Dube et al., 2007), mainly from vegetal source (Rocha et al., 2005; Muzitano et al., 2006; Braga et al., 2007; Brenzan et al., 2007; Soares et al., 2007). These studies look at to increase the therapeutic arsenal, because there is a lack of effective and safety medicines.
The botany family Myrtaceae has a broad dispersion. It has 133 genera and about 3800 species (Wilson et al., 2001). Several studies present the results about isolation and biological activities of substances from Myrtaceae, as example the cytotoxic actions against tumoral cells (Balunas et al., 2006; Werka et al., 2007), anti-inflammatory action (Lima et al., 2007; Jiang & Xu, 2003), antioxidant action (Marzouk et al., 2007), bactericidal (Schmidt et al., 2006) and fungicidal (Woollard et al., 2008) activities. Thus, species from this family has been important targets to isolation of actives substances and developing of new drugs.
Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, popularly knew as guamirim, cambuim, murta, is a exemplar of Myrtaceae family. There are few studies about biological activity of Blepharocalyx salicifolius. Limberger et al. (2001) has studied the biological effects of the leaves infusions as bactericidal agent, anti-inflammatory, antinociceptive and antispasmodic. The results showed a great bactericidal effects against S. aureus and E. coli species and fresh leaves had antispasmodic effects.
Preliminary studies carried out at Laboratory of Chemistry of the Natural Products, Fiocruz, revealed that fractions F11 and F12 from Blepharocalyx salicifolius ethanolic extracts were active against Leishmania (L.) amazonensis amastigotas cells and had low toxicity against mononuclear cells of human peripheral blood.
Despite the fact that the raw extract did not show leishmanicidal activity, it was observed that eight fractions (50% of the total fractions) were active against Leishmania (L.) amazonensis at range of 19 to 29 µg.mL-1.
In the raw extract, the actives compounds could be at minor concentration diluted between hundreds of others compounds besides several compounds can have antagonic biological effects each other. The purification process increase the individual chemical characteristic for each species, thus the fraction seems to be more effective than the raw extract, as example, the fraction F12 had 86% of inhibition effect and 25 mg (0,67% of the total mass) whereas the F16 was not active (20% of the total mass).
The usual drugs used on leihsmaniasis therapy present several adverse effects as nephrotoxicity, hepatotoxicity and low efficacy (Soto et al., 2001). Besides the fractions F11 and F12 present activity against amastigota Leishmania (L.) amazonensis cells, they were not toxic to blood human cells. In the present study, we used cell proliferation of mononuclear cells (lymphocytes and monocytes) to assess the immunotoxic potential of these fractions and the crude extract because these in vitro models are currently used for pre-screening of immunotoxic potential, as a strategy. In this context, if the cells are viable (80% or greater) basic functionality can be determined assessing cell proliferation using mitogens such as plant lectin (PHA) especially for T cells (Carfi et al., 2007). Compounds may affect lymphocytes which are the primary effectors and regulators of acquired immunity, essential for anti-parasitic response as observed to leishmania infections. In this disease, the immunity is dependent on T lymphocytes CD4+ positive with the Th1 type cytokine profile such as IFN-γ that activates the macrophages to kill the parasites (Narayan et al., 2009). Therefore, the low toxicity observed to actives fractions F11 and F12 strongly suggest that Blepharocalyx salicifolius has a great potential to isolation of the actives substances against Leishmaniasis and with low toxicity.
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Received 9 March 2009; Accepted 6 October 2009