Research in Microbiology 155 (2004) 136–143 www.elsevier.com/locate/resmic
Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae) Ricardo R. Mendonça-Filho a,b,∗ , Igor A. Rodrigues a , Daniela S. Alviano a , André L.S. Santos a , Rosangela M.A. Soares a , Celuta S. Alviano a , Angela H.C.S. Lopes a , Maria do Socorro S. Rosa a a Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ),
Rio de Janeiro, RJ, 21941-590, Brazil b Instituto de Química, Programa de Pós-Graduação em Ciência de Alimentos, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, 219491-590, Brazil Received 27 March 2003; accepted 26 November 2003 First published online 28 November 2003
Abstract The available therapy for leishmaniasis, which affects 2 million people per annum, still causes serious side effects. The polyphenolicrich extract from the husk fiber of Cocos nucifera Linn. (Palmae) presents antibacterial and antiviral activities, also inhibiting lymphocyte proliferation, as shown by our group in previous works. In the present study, the in vitro leishmanicidal effects of C. nucifera on Leishmania amazonensis were evaluated. The minimal inhibitory concentration of the polyphenolic-rich extract from C. nucifera to completely abrogate parasite growth was 10 µg/ml. Pretreatment of peritoneal mouse macrophages with 10 µg/ml of C. nucifera polyphenolic-rich extract reduced approximately 44% the association index between these macrophages and L. amazonensis promastigotes, with a concomitant increase of 182% in nitric oxide production by the infected macrophage in comparison to nontreated macrophages. These results provide new perspectives on drug development against leishmaniasis, since the extract of C. nucifera at 10 µg/ml is a strikingly potent leishmanicidal substance which inhibited the growth of both promastigote and amastigote developmental stages of L. amazonensis after 60 min, presenting no in vivo allergenic reactions or in vitro cytotoxic effects in mammalian systems. 2003 Elsevier SAS. All rights reserved. Keywords: Leishmania amazonensis; Cocos nucifera; Leishmanicidal activity; Nitric oxide; Phagocytosis; Poliphenolic compounds; Medicinal plants
1. Introduction Leishmaniasis is a group of infectious diseases caused by different species of protozoan parasites of the genus Leishmania, which affect about 2 million people per annum. These parasites have a digenetic life cycle that includes an extracellular promastigote form in the sandfly vector and a nonflagellated intracellular amastigote stage within the mononuclear phagocytes of vertebrate hosts. Leishmaniasis presents a broad clinical spectrum ranging from asymptomatic and self-healing to cutaneous and/or visceral infections, causing significant morbidity and mortality. L. amazonensis is one of the major agents of diffuse cuta* Corresponding author.
E-mail address:
[email protected] (R.R. Mendonça-Filho). 0923-2508/$ – see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.12.001
neous leishmaniasis, which is usually unresponsive to treatment known to date [12,17,18,31]. In addition, visceralization of Leishmania strains that are classically restricted to cutaneous leishmaniasis has often been observed in patients with Leishmania-HIV co-infection [4,29]. Clinical reports indicate that a large proportion of the cases are becoming unresponsive to chemotherapy. Variable efficacy, toxicity, requirement for long courses of parenteral administration, resistance or a combination of these factors has been reported [11,14]. Although there are several drugs on trial for the chemotherapy of human leishmaniasis, many of them are new formulations of old drugs. Pentavalent antimonials are still the first choice among drugs used for the treatment of leishmaniasis, despite their cardiac and renal toxicity. Another disadvantage in the prescription of these drugs is its restriction to systemic use. In view of the present clinical scenario it is desirable that new drugs be developed [2,10].
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Most people living in developing countries are almost completely dependent on traditional medical practices for their primary health care needs and higher plants are known to be the main source of drug therapy in traditional medicine [1,35]. The recognition and validation of traditional medical practices and the search for plant-derived drugs could lead to new strategies for leishmaniasis control. The fruit of Cocos nucifera Linn. (Palmae) var. typical A has a fiber husk rich in polyphenolic compounds, whose decoction has been used against arthritis and diarrhea in the popular medicine of Northeast Brazil [16]. Catechins are polyphenols that are a form of flavonoids with several phenol groups. Catechin, epicatechin and epicatechin-(4 →2)phloroglucinol units are present in high amounts in the polyphenolic molecules found in C. nucifera [16]. These groups can capture prooxidants and free radicals, which confer upon them potent antioxidant characteristics [27]. Recent studies reported that catechins are a powerful inhibitor of cellular growth [9,34,40], presenting anticancer [7,19], antimutagenic [5], antibacterial, antiviral [32,39] and antiinflammatory [8] activities. Antibacterial and antiviral activities have already been observed by our research group using the polyphenolicrich extract from husk fiber of C. nucifera [16]. In the present work, we have extended the antimicrobial study of C. nucifera Linn. (Palmae), investigating its effects on L. amazonensis parasites, on the interaction of these flagellates with peritoneal mouse macrophages and on nitric oxide (NO) production by infected macrophages.
2. Materials and methods 2.1. Plant material Cocos nucifera Linn. (Palmae) var. typical A, commonly known as “Olho-de-Cravo”, was collected in Aracaju, State of Sergipe (Brazil), and authenticated by Dr. Benedito Calheiro Dias, Centro de Pesquisas do Cacau, Bahia (Brazil), where a voucher specimen is deposited. 2.2. Extraction and fraction isolation The husk fiber of coconut (414 g) was dried in the sun, finely ground and the powder soaked for 3 h in 6 l of boiling distilled water. The extract was filtered and lyophilized, yielding 21 g of crude water extract. 500 mg of the lyophilized crude water extract was dissolved in water (1 l) and partitioned with ethyl acetate. Thin-layer chromatography (silica; solvent system: AcOEt/AcOH/HCO2 H/H2 O 100:11:11:27) was used to evaluate the purification. Spots were visualized by spraying vanillin–sulfuric acid reagent. Ethyl acetate extract was also used for HPLC analysis and the contents indicated high concentrations of polyphenolic compounds such as catechins and epicatechins. Comparison
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with catechin standards confirmed these results, as previously described by Ezquenazi et al. [16]. This polyphenolicrich extract from C. nucifera was used in all parts of the present study. 2.3. Parasite culture Promastigote forms of Leishmania amazonensis (Raimundo strain MHOM/BR/76/Ma-5) were maintained by weekly transfers in brain heart infusion (BHI) medium supplemented with 10% fetal bovine serum (FBS) at 26 ◦ C. The parasites were maintained infective by periodical hamster footpad inoculations [37]. 2.4. Minimum inhibitory concentration (MIC) evaluation L. amazonensis promastigotes (106 parasites/ml) were incubated at 26 ◦ C for 120 h in fresh BHI medium, supplemented with 10% FBS, in the absence or in the presence of several concentrations (1, 2.5, 5, 7.5, 10, 15, 20 µg/ml) of the polyphenolic-rich extract of C. nucifera (cell growth was determined daily by assessment of visible turbidity) in order to evaluate the MIC, as described by Olliaro and Bryceson [33]. The MIC was considered the lowest concentration of the polyphenolic-rich extract that prevented the growth of L. amazonensis in vitro. 2.5. Peritoneal mouse macrophages Thioglycolate-elicited peritoneal macrophages from female Swiss mice (6–8 weeks of age) were collected in cold PBS (150 mM NaCl; 20 mM phosphate buffer, pH 7.2) and allowed to adhere to coverslips placed in 24-well culture plates for 30 min at 37 ◦ C in a 4% CO2 atmosphere [30]. Nonadherent cells were then removed and the adhering macrophages washed twice with PBS and cultured for 24 h in RPMI culture medium (GIBCO BRL, Gaithersburg, MD, USA), supplemented with 10% FBS. For obtaining the peritoneal mouse macrophages, mice were previously killed according to all federal guidelines and institutional policies. 2.6. Purification of amastigotes Intact living L. amazonensis promastigotes in the stationary growth phase were added to the plate wells containing cultured peritoneal mouse macrophages. Free promastigotes were removed after 2 h incubation and the infected macrophages were kept for 48 h under the conditions described above. Free amastigotes were then collected from the supernatants of the culture plate wells [37]. The amastigotes were washed twice in cold PBS and then resuspended in RPMI culture medium for antileishmanial activity assays [33].
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2.7. Antileishmanial activity
2.9. Cytotoxity assay
Promastigotes and amastigotes of L. amazonensis (106 parasites/ml) were incubated in RPMI culture medium in the absence or in the presence of 10 and 20 µg/ml of polyphenolic-rich extract from C. nucifera at 37 ◦ C, in order to evaluate parasite survival and cell morphology, under optical microscopy at 10 min intervals. Alternatively, after 30 min of parasite growth in the presence of the polyphenolic-rich extract from C. nucifera (10 µg/ml), cells were centrifuged, and washed three times in PBS prior to resuspension in new culture medium without the plant extract, in order to evaluate the leishmanicidal or leishmanistatic effect. Growth was determined by counting the cells during a 120 h period of incubation in vitro.
Parasite viability was assessed before and after incubations with 10 and 20 µg/ml of polyphenolic-rich extract of C. nucifera, by evaluation of the motility (promastigote form) and by the trypan blue method (promastigote and amastigote forms) [6], using a hemocytometer chamber. Cytotoxicity of the polyphenolic-rich extract for macrophages was measured by the neutral red uptake assay as described [37]. Briefly, macrophages were cultivated in 96-well microtiter plates (150 µl containing 105 cells/ml in EAGLEMEM medium/well) at 37 ◦ C in a humidified 5% CO2 atmosphere. The medium was supplemented with L-glutamine (0.10 g/l), Hepes (2.38 g/l), penicillin G (105 IE/l), streptomycin sulfate (0.10 g/l) and 4% FBS. After 24 h of incubation, 50 µl of polyphenolic-rich extract at 10 or 20 µg/ml were added to the cell cultures. 50 µl EAGLE-MEM medium were added to the control cells. After further incubation for 48 h, control and treated cells were washed three times with PBS (pH 7.2). 100 µl of neutral red solution (0.3% in EAGLE-MEM) were added to each well. After 3 h incubation at 37 ◦ C in this solution, the cells were then washed three times with PBS. 100 µl of a solution containing 1% acetic acid and 50% ethanol were added to the wells and the optical density of the supernatants was measured at 540 nm. Cell viability was determined using the following formula: [100 − (L2/L1) × 100], where L1 is the percentage of viable control cells and L2 is the percentage of viable treated cells, as previously described [13].
2.8. Infection of macrophages and NO production Peritoneal mouse macrophages were obtained as described above. The parasites and/or the macrophages were either not treated or treated with 10 and 20 µg/ml of polyphenolic-rich extract from C. nucifera, 20 min prior to the macrophage–parasite interaction. The adherent cultured macrophages and the free parasites were washed once and resuspended in fresh RPMI culture medium. Dead parasites were removed from the medium by centrifugation (1000 g for 5 min), and intact living L. amazonensis promastigotes in the stationary growth phase were then added to the macrophage culture plate wells. The parasite– macrophage interaction studies were performed at 37 ◦ C for 90 min using parasites and/or macrophages pretreated with the polyphenolic-rich extract, or macrophages that had already been infected with the parasites for 24 h, followed by treatment for 20 min with the polyphenolic-rich extract, washed twice with PBS and followed by an additional incubation for 90 min. In the last system (post-treatment system), all promastigotes had already differentiated into amastigotes before treatment with the extract. A ratio of 10 promastigotes to 1 macrophage was used for both infection assays [25]. After the interaction assays were done, the coverslips were fixed and Giemsa-stained, and the percentage of infected macrophages was determined by counting 600 cells in triplicate coverslips. The association indices were determined by multiplying the percentage of infected macrophages by the mean number of parasites per infected cell. Association indices were considered as the number of parasites that actually infected the macrophages. The supernatants from control (macrophages alone in culture) and L. amazonensis-infected macrophages nontreated or treated with 10 and 20 µg/ml of polyphenolic-rich extract from C. nucifera were analyzed for their nitrite contents by the Griess reaction, as previously described [23]. The absorbance at 550 nm was measured, and the concentration of nitrite was calculated using a linear regression of a standard curve, as described [26].
2.10. In vivo primary dermic irritation, cumulative dermic irritation and ocular irritation allergy tests For each irritation test 4 young rabbits (1.5–2 kg) were used. To perform the dermic tests we previously selected two different shaved and gently scraped regions (right and left), approximately 6 mm in diameter, on the rabbits’ backs. In the primary irritation test we used a small volume (0.04 ml) of sterile polyphenolic-rich extract from C. nucifera (100 µg/ml). The back regions were observed after application of the extract and during the subsequent 7 days. In the cumulative dermic irritation tests we added 0.5 ml of polyphenolic-rich extract from C. nucifera (100 µg/ml) daily after the first application. For the ocular irritation test, we dropped 0.1 ml of the polyphenolic-rich extract (100 µg/ml) into both eyes and observed the immunological reaction 24, 48 and 72 h after the application. 2.11. Statistical analysis All experiments were performed in triplicate. The mean and standard error 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 considered significant.
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3. Results 3.1. Inhibition of parasite growth The polyphenolic-rich extract of C. nucifera was capable of completely abrogating the cellular growth of L. amazonensis promastigote forms, and the MIC value was 10 µg/ml. 3.2. Antileishmanial activity Fig. 1 shows the time course of the viability of L. amazonensis promastigotes and amastigotes in the absence or in the presence of polyphenolic-rich extract of C. nucifera. 10 and 20 µg/ml of polyphenolic-rich extract was able to kill 100%
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of both developmental stages of the parasite after 60 min (Fig. 1). On the other hand, mouse macrophages were unaffected by 10 and 20 µg/ml of C. nucifera polyphenolicrich extract, presenting high cell viability levels (98–100%) (data not shown). The effect of polyphenolic-rich extract on the growth of L. amazonensis promastigotes was irreversible as determined by transfer of 30-min-treated parasites with 10 µg/ml of C. nucifera polyphenolic-rich extract to drugfree medium; even after five days in axenic culture, no increase in the cell number was observed (data not shown). Corroborating this result, microscopic observations showed complete lysis of promastigote cells after treatement of parasites with 10 or 20 µg/ml of C. nucifera polyphenolic-rich extract for 60 min (Fig. 2). The same inhibition profile and morphological alterations were observed in the amastigote forms (data not shown). 3.3. Infection of macrophages
Fig. 1. Time course of the viability of promastigote and amastigote forms of L. amazonensis in the absence or in the presence of polyphenolic-rich extract from husk fiber of C. nucifera Linn. (Palmae). Cell viability (%) of both parasite stages was calculated by [100 − (L2/L1) × 100], where L1 is the percentage of viable control cells and L2 is the percentage of viable treated cells. (!) L. amazonensis promastigotes (control); (") L. amazonensis amastigotes (control); (e) L. amazonensis promastigotes + C. nucifera polyphenolic-rich extract at 10 µg/ml; (a); L. amazonensis amastigotes + C. nucifera polyphenolic-rich extract at 10 µg/ml; (2) L. amazonensis promastigotes + C. nucifera polyphenolic-rich extract at 20 µg/ml; (1) L. amazonensis amastigotes + C. nucifera polyphenolic-rich extract at 20 µg/ml. The values represent means of three independent experiments that were performed in triplicate. Bars represent standard errors.
Fig. 3 shows the effects of polyphenolic-rich extract on the L. amazonensis-macrophage interaction. The parasites and/or the peritoneal mouse macrophages were either not treated or treated with two different concentrations (10 and 20 µg/ml) of C. nucifera polyphenolic-rich extract, 20 min prior to the macrophage-parasite interactions. When macrophages were pretreated with 10 or 20 µg/ml of the polyphenolic-rich extract, the association indices were respectively 44 and 69% lower in comparison to the control system (where both nontreated macrophages and parasites were analyzed). When parasites were pretreated with 10 or 20 µg/ml of the polyphenolic-rich extract, the association indexes were, respectively, 32 and 56% lower as compared to the control system, which were approximately the same results obtained when both macrophages and parasites were pretreated with the polyphenolic-rich extract. When the macrophages were preinfected with L. amazonensis for 24 h and then treated with 10 or 20 µg/ml of the polyphenolicrich extract for 20 min, followed by an additional incubation for 90 min, the association indexes were, respectively, 45 and 61% lower in comparison to the control system (where only macrophages and parasites interacted for 24 h).
Fig. 2. Microscopic observations of the time course viability of promastigote forms of L. amazonensis when incubated in the absence (A) or in the presence of 10 µg/ml (MIC concentration) of polyphenolic-rich extract from C. nucifera for 30 min (B) and 60 min (C). Note the increase in cell volume (arrows in B) and complete lysis of the parasite cell (C). Scale bar 1 µm.
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Fig. 3. Effects of polyphenolic-rich extract from husk fiber of C. nucifera Linn. (Palmae) on the L. amazonensis–macrophage interaction. The parasites and/or the peritoneal mouse macrophages were either not treated or treated with 10 and 20 µg/ml of C. nucifera polyphenolic-rich extract 20 min prior to macrophage–parasite interactions. The adherent cultured macrophages and the free parasites were washed once and resuspended in fresh culture medium. Dead parasites were removed from the medium by centrifugation (1000 g for 5 min) and intact living L. amazonensis promastigotes were then added to the macrophage culture well plates. Association indices were determined by light microscopy, counting 600 cells in triplicate coverslips after 90 min of interaction. Each bar represents the mean ± standard error of at least three independent experiments performed in triplicate. Association indices of assays performed using pretreated macrophages and/or pretreated parasites with C. nucifera polyphenolic-rich extract are significantly different from the association index of control (nontreated) macrophages. (P) parasites; (M() macrophages.
Fig. 4. Effects of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae) on NO production by peritoneal mouse macrophages. The parasites and/or the macrophages were either not treated or treated with 10 or 20 µg/ml of C. nucifera polyphenolic-rich extract 20 min prior to macrophage-parasite interactions. The adherent cultured macrophages and the free parasites were washed once and resuspended in fresh culture medium. Dead parasites were removed from the medium by centrifugation (1000 g for 5 min) and intact living L. amazonensis promastigotes were then added to the macrophage culture well plates. The supernatants from control and L. amazonensis-infected macrophages were collected and the nitrite concentration of each system was determined by the Griess reaction as described in Section 2. Each bar represents the mean ± standard error of at least three independent experiments performed in triplicate. (P) parasites; (M() macrophages.
bation for 90 min, the NO production was similar to that obtained when both macrophages and parasites were pretreated. 3.5. In vivo allergy tests
3.4. NO production Peritoneal mouse macrophages were either noninfected or infected with L. amazonensis and then their culture supernatants were evaluated for nitrite contents. The parasites and/or the macrophages were either not treated or treated with 10 or 20 µg/ml of the polyphenolic-rich extract 20 min prior to the macrophage-parasite interactions. Fig. 4 shows that noninfected macrophages that were treated with 10 or 20 µg/ml of the polyphenolic-rich extract produced 25 and 100% more NO, respectively, in relation to the macrophages alone in culture. Infected macrophages produced 5% more NO than noninfected macrophages. When infected macrophages were pretreated with 10 or 20 µg/ml the polyphenolic-rich extract, the NO production was respectively 182 and 353% higher than the control infected macrophages. When parasites were pretreated with 10 or 20 µg/ml of the polyphenolic-rich extract, the NO production was, respectively, 59 and 300% higher than the control infected macrophages. In addition, the same results were obtained when both macrophages and parasites were pretreated with 10 or 20 µg/ml of the polyphenolic-rich extract. When the macrophages were preinfected with L. amazonensis for 24 h and then treated with 10 or 20 µg/ml of the polyphenolic-rich extract, followed by an additional incu-
None of the tested animals showed any dermic or ocular allergic reactions to the polyphenolic-rich extract and thus, in light of these results, this extract was considered nonallergic.
4. Discussion There is a general lack of effective and inexpensive chemotherapeutic agents for treating parasitic protozoan diseases that occur mainly in the developing world. One such disease is leishmaniasis. Pentavalent antimonial drugs are the first-line treatment for leishmaniasis in most affected areas, with amphotericin B and pentamidine being used as alternative drugs [2]. These agents are not active orally and require long-term parenteral administration. They also have serious side effects and are expensive [5]. In addition, resistance to these compounds has become a severe problem. Therefore, new drugs are urgently required. In this sense, new drugs of herbal origin discovered through ethnopharmacological studies have shown interesting results. Our laboratory has initiated and developed original investigations of alternative compounds for the growth control of some microorganisms, including bacteria, fungi and protozoa [16,37]. In the course of screening for leish-
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manicidal compounds from Brazilian plants, we have found that the linalool-rich essential oil from the leaves of Croton cajucara displayed in vitro activity at 15 ng/ml against promastigote and amastigote forms of L. amazonensis, leading to a complete disruption of nuclear and kinetoplast chromatin, followed by cell lysis [37]. Moreover, several natural compounds with antileishmanial activity have been investigated in various laboratories and correspond to the following groups: alkaloids, terpenes, quinones, lactones, coumarins, acetogenins of annonaceae, chalcones, tetralones, lignans and saponins [13]. In the present work, we describe a novel pharmacological activity obtained from polyphenolic-rich extract of coconut husk fiber. An extensive range of popular medicinal utilization of this plant has been reported [15]. Our initial observation that the crude extract of C. nucifera strongly inhibited the in vitro growth of L. amazonensis promastigotes (data not shown) prompted us to perform a bioassay-guided fractionation of the antileishmanial activity. The ethyl acetate extract from the crude extract, rich in polyphenolic compounds, showed inhibitory activity against both developmental stages (promastigote and amastigote) of L. amazonensis. An irreversibility of the effect on promastigotes was tested by incubating the parasites with the polyphenolic-rich extract for 30 min, followed by culture in fresh medium, suggesting a metabolic injury that could not be reversed. These results were corroborated by lysis of promastigote cells. Catechins and tannins [20,22,24] naturally found in C. nucifera have also been selected and are currently being tested as antiaddiction therapy [36], and the varying catechin compositions of the polyphenolic-rich extract (catechin, epicatechin and epicatechin-(4 → 2)-phloroglucinol) could explain the different antimicrobial activities of C. nucifera toward distinct classes of organisms such as herpes simplex virus type I, Staphylococcus aureus [16] and Leishmania. However, the polyphenolic-rich extract from C. nucifera did not show any effect on the viability or on the morphology of mouse macrophages (data not shown). New drug therapy regimes have taken advantage of the knowledge obtained from studies on Leishmania–macrophage interaction [3]. Concerning infection of peritoneal mouse macrophages by L. amazonensis, when macrophages were pretreated with C. nucifera polyphenolic-rich extract as well as when the macrophages were preinfected with the parasites and then treated with 10 or 20 µg/ml of polyphenolicrich extract for 20 min, association indices were lower in comparison to control systems (where both nontreated macrophages and parasites were used) (Fig. 3). The phagocytic activity of macrophages was altered during pretreatment with the polyphenolic-rich extract from C. nucifera, which suggests an alteration in the biochemical process of phagocytosis, such as some modification in the signal transduction process and/or activation of macrophage killing mechanisms. In order to sustain a chronic infection, parasites must subvert macrophage-accessory cell activities and ablate the de-
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velopment of protective immunity [3]. The macrophage is armed with antimicrobial mechanisms that intracellular organisms must evade in order to survive. During the process of leishmanial infection the microbicidal interactions between parasites and host cells occur in two stages. First, during initial phagocytosis of promastigotes, the macrophages can undergo an oxidative response stimulated by the phagocytic event that produces superoxide (O− 2 ) as part of the respiratory burst of human and murine macrophages. Second, once infection with amastigote is established, the quiescent macrophage can be activated to potentially kill intracellular Leishmania. A second antileishmanial oxidant produced by activated macrophages is NO that is most relevant to killing established intracellular amastigotes [21]. Although there is strong evidence that NO plays an important role in murine leishmaniasis, it remains controversial as to whether NO plays a role in antileishmanial responses of human macrophages. Thus, in order to determine whether detectable NO was produced during infection of murine macrophages with L. amazonensis, we measured nitrite produced from NO released into culture supernatants of infected macrophages as well as the modulation of NO expression during the interaction in the presence of polyphenolic-rich extract from C. nucifera. We demonstrated that macrophages pretreated with 20 µg/ml of polyphenolic-rich extract from C. nucifera produced twice the amount of nitrite, reflecting NO production, in comparison to nontreated macrophages (Fig. 4). Previous reports showed that interferon-gamma (IFN-γ ) is necessary for maximal stimulation of leishmanicidal activity and transcription of inducible NO synthase (iNOS) by murine macrophages [21]. Our results suggest that polyphenolic compounds probably induce synthesis of NO by murine macrophages, which enhances the potential killing mechanisms of these cells. The exact molecular mechanisms through which these radicals promote intracellular killing of Leishmania have yet to be fully defined. A previous work showed that L. chagasi promastigotes are susceptible to killing by sulfo-NONOate, a compound that spontaneously generates NO in vitro at 25 ◦ C [21]. NO also inhibits Leishmania cysteine proteinase activity, a known virulence factor of this microorganism, leading to lethal metabolic inhibition through irreversible chemical modification of reactive residues of cysteine [38], as well as blocking the differentiation process from amastigotes to promastigotes [28]. Collectively, these results strengthen the idea that reactive nitrogen intermediates are essential to the control of intracellular microorganisms by murine macrophages, exerting static and/or lytic effects on both parasitic developmental stages. How polyphenols influence the augmentation of NO is completely unknown. Polyphenolic compounds such as catechins could trigger the signaling pathway or aberrant signaling that culminates in the expression of iNOS. Studies in our laboratory are in progress along this line. Additionally, we know that human macrophages are incapable of generating NO even in the presence of stimulatory cytokines such
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as INF-γ or TNF-α. Therefore, we are also investigating the possible participation of the polyphenolic-rich extract obtained from C. nucifera in stimulation of NO synthesis by human macrophages. Our results reveal a novel pharmacological activity against L. amazonensis and suggest that this polyphenolicrich extract may be useful for topical application in wound healing, in addition to its nonallergenic properties. The results presented herein provide motivation for further exploration of polyphenolic compounds, particularly as antileishmanial agents. Laboratory synthesis and the possibility of modifying the chemical structure of C. nucifera polyphenolic-rich extract constitute important advantages for the development of new antileishmanial therapies.
Acknowledgements We thank Mr. José Soares de Souza “Juca” for the C. nucifera collecting. We also thank Mr. Paulo C. Miguel and Ms. Soraya Alves da Silva for technical assistance. This work was performed with the financial support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ) and Fundação Universitária José Bonifácio (FUJB).
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