Antileishmanial effect of fruit seeds from Platonia insignis against macrophage-internalized amastigote forms of Leishmania amazonensis

ARTÍCULO ORIGINAL

 

Antileishmanial effect of fruit seeds from Platonia insignis against macrophage-internalized amastigote forms of Leishmania amazonensis

 

Efecto de las semillas del fruto de Platonia insignis contra las formas amastigotes de Leishmania amazonensis en el interior de los macrófagos

 

 

Ana Karina Marques Fortes LustosaB1,2
Érika Alves Bezerra1,3
Klinger Antonio da Franca Rodrigues3
Layane Valéria Amorim3
José de Sousa Lima Neto4
Bruno Quirino Araújo2
Joaquim Soares da Costa-Júnior5
Anderson Nogueira Mendes3
Fernando Aécio de Amorim Carvalho3
Daniel Dias Rufino Arcanjo1,3*
Antônia Maria das Graças Lopes Citó1,2

1 Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Federal do Piauí, Teresina, Piauí. Brasil.
2 Departamento de Química, Universidade Federal do Piauí, Teresina, Piauí. Brasil.
3 Núcleo de Pesquisas em Plantas Medicinais, Universidade Federal do Piauí, Teresina, Piauí. Brasil.
4 Coordenação do Curso de Farmácia, Teresina, Piauí, Brasil.
5 Departamento de Química, Instituto Federal do Piauí, Teresina, Piauí, Brasil.

 

 


ABSTRACT

Introduction: Leishmaniasis is an infectious parasitic disease with clinical manifestations which compromise skin tissue, mucosas and viscera. Previous studies have found that the fruit seeds of Platonia insignis Mart. (Clusiaceae) have a marked effect against promastigote forms of Leishmania amazonensis.
Objective: Evaluate the antileishmanial activity of an extract obtained from Platonia insignis fruit seeds (known as BBI) against L. amazonensis amastigotes inside macrophages.
Methods: Upon BBI incubation (0.78 to 50 mg/ml), estimation was made of the number of infected macrophages and parasites. Evaluation was performed of BBI cytotoxicity by the MTT assay (12.5 to 400 µg/ml) in non-infected murine macrophages, and of hemolytic activity in human O+ erythrocytes. Phagocytic capacity, lysosomal volume and nitrite concentration were also evaluated to determine the participation of immunomodulatory mechanisms.
Results: BBI reduces infection in macrophages (CI50=3.95 mg/ml). Treatment of murine peritoneal macrophages increases nitrite production, phagocytic capacity and lysosomal volume, confirming the involvement of immunomodulation in the activity of BBI against leishmaniasis. BBI displayed low toxicity for murine macrophages at a mean cytotoxic concentration (CC50) of 73.26 mg/ml, and hemolytic activity below 5.0% at the highest concentration assayed (400 µg/ml).
Conclusions: P. insignis fruit seed extract is a promising alternative to treat leishmaniasis, as well as to manufacture drugs with a potential application in the treatment of this disease.

Key words: leishmaniasis; nitrite; plant oils; protozoan infections; phagocytosis.


RESUMEN

Introducción: La leishmaniosis es una enfermedad parasitaria infecciosa con manifestaciones clínicas que dañan el tejido cutáneo, las mucosas y las vísceras. Estudios previos han demostrado que las semillas del fruto de Platonia insignis Mart. (Clusiaceae) tienen un efecto notable contra las formas promastigotes de Leishmania amazonensis.
Objetivo: Evaluar la actividad antileishmania del extracto de las semillas del fruto de Platonia insignis, conocido como BBI, contra las formas amastigotas de L. amazonensis. en el interior de los macrófagos.
Métodos: Después de la incubación del extracto BBI (0,78 en 50 mg/mL) se calculó el número de parásitos y de macrófagos infectados. Mediante el ensayo del MTT se evaluó la citotoxicidad del BBI (12,5 a 400 µg/mL) en macrófagos murinos no infectados, así como la actividad hemolítica en los eritrocitos humanos de grupo sanguíneo O Rh+. Se evaluaron la capacidad fagocítica, el volumen lisosomal y la concentración de nitrito para investigar la intervención de los mecanismos inmunomoduladores.
Resultados: El extracto BBI reduce la infección de los macrófagos (la concentración de la inhibición (IC50) es igual a 3,95 mg/mL). El tratamiento de los macrófagos peritoneales murinos aumenta la producción de nitrito, la capacidad fagocítica y el volumen lisosomal, lo cual confirma la participación de la inmunomodulación en el efecto del BBI contra la leishmaniosis. El extracto de las semillas del fruto de Platonia insignis ha demostrado baja toxicidad para los macrófagos murinos con concentración citotóxica media (CC 50) de 73,26 mg/mL, así como una actividad hemolítica menor que 5,0 % en la más alta concentración comprobada (400 µg/mL).
Conclusiones: El extracto de las semillas del fruto de P. insignis es una alternativa prometedora para la fabricación de fármacos para el tratamiento de la leishmaniosis.

Palabras clave: leishmaniosis; nitrito; aceites vegetales; protozoo; infecciones; fagocitosis.


 

 

INTRODUCTION

Leishmaniasis is a parasitic disease caused by protozoa of the Leishmania genus and considered as a public health problem due to affect millions of people worldwide. It is estimated that about 2 million of new cases arise each year, in addition to over 10 million cases.1,2 For the treatment of leishmaniasis, antimony compounds with high toxicity by parenteral administration are widely used. However, the treatment is not effective in many cases.3,4 Therefore, researchers worldwide are searching for new antileishmanial drugs in order to find more effective and safer alternatives. On this demand, natural products have demonstrated to provide essential and alternative requirements instead of conventional treatments, contributing not only to reduce the side effects and toxicity, but also the costs with expensive treatments.5

The species Platonia insignis Mart. (Clusiaceae), popularly known as "bacuri", is a large tree which can reach up to 40 m of height and 2.0 m of diameter. It is widespread in Amazon Rainforest as well as distributed in Marajó Island, State of Pará, Brazil, as well as in the states of Piaui, Mato Grosso, Amazonas, Tocantins, and Maranhão.6,7 The seeds oil is obtained by decoction of the seeds, and is extensively used in Brazilian traditional medical practices for the topical treatment of dermatological diseases, such as burns, skin wounds, eczemas and herpes.(8,9) Furthermore, the seeds decoction is also used to treat diarrhea.10,11

The seeds extract from P. insignis provide a high content of fatty acids, diterpenes, alcohols and long-chain hydrocarbons.12 Moreover, previous studies of this research group has reported the isolation and identification of 1,3-distearoyl-2-oleoylglycerol (TG-1), a triacylglicerol with wound healing properties(13), and garcinielliptone FC (GFC), a polyprenylated benzophenone with vasorelaxant,14 anticonvulsant,15 antioxidant,16 and antipromastigote effects.17 Furthermore, the compounds a- and g-mangostin were identified by derivatized CG-MS as the major compounds of the seeds extract from P. insignis. Interestingly, this extract induces a cytotoxic effect against promastigote forms of L. amazonensis.18 However, the intracellular amastigote forms of L. amazonensis are the life stage responsible for clinical manifestations of leishmaniasis, and the effects of P. insignis against these forms remain still unknown.

Thus, the leishmanicidal activity of P. insignis seeds extract (BBI) against macrophage-internalized amastigote forms of Leishmania amazonensis and underlying mechanisms as well as its cytotoxicity in mammalian cells were investigated in this study.

 

MATERIALS AND METHODS

Drugs and chemicals

Schneider's and RPMI 1640 media, penicillin, streptomycin, bovine serum (FBS), MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide), Zymosan, neutral red and Griess reagent (1 % Sulfanilamide in H3 PO4 10 % (v/v) in Milli-Q water) were purchased from Sigma Chemical (St. Louis, MO, USA). The seeds oil from P. insignis (BBI) was donated from Amazon Oil Indústria e Comércio Ltda. (Belém, PA, Brazil), Lot No. MBA-006/10. The stock solution of BBI (80 mg/mL) was prepared in DMSO and diluted to the appropriated concentrations during the experiment.

Parasites and mice

Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) were used in the experimental protocols. Parasites were grown in Schneider's medium, supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 26 °C. Macrophages were collected from peritoneal cavities of male and females BALB/c mice (4-5 weeks old), maintained under controlled temperature (24±1 °C) and light/dark cycles of 12/12 h.19 All experimental protocols were previously approved by the Animal Experimentation Ethics Committee from Federal University of Piauí (CEEAPI No. 76/2010).

Leishmanicidal activity of BBI on L. amazonensis amastigote forms

Murine macrophages (5×105 cells/500 µl of medium) were incubated at 37 °C and 5 % CO2 for 2 h to cell adhesion. The medium was replaced with 500 µL of supplemented RPMI-1640 (10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin) and incubated for 15 h. The RPMI-1640 supplemented medium was aspirated and fresh medium containing L. amazonensis promastigote forms (in stationary phase) was added in the proportion of 10 promastigotes per macrophages in each well. After 4 h of incubation (5 % CO2, 37 °C), the medium was aspirated in order to remove free promastigotes and 1 mL of RPMI-1640 was added containing BBI (0.78 - 50.0 µg/ml) in the experimental groups and 0.2 % DMSO as negative control. Three independent experiments were performed in triplicate for each concentration. After 48 h, the coverslips were removed, fixed in methanol and stained with Giemsa. For each coverslip, 300 cells were evaluated, and the numbers of infected macrophages and parasites per macrophage were determinate.20

Cytotoxicity determination

The cytotoxicity assessment was performed by MTT assay. In a 96-well plate, 100 µL of supplemented RPMI 1 640 medium (as described above) and approximately 1×105 macrophages per well was added. The cells were incubated for 4 h to adhesion with two washes with supplemented RPMI medium to remove cells that did not adhere. Then, BBI was incubated for 48 h in triplicate at concentrations of 400, 200, 100, 50, 25 and 12.5 µg/mL. Next, 10 µL of MTT diluted in PBS was added at final concentration of 5 µg/mL, and then incubated during 4 h (37 °C, 5 % CO2). Afterwards, the supernatant was discarded and 100 µL of DMSO added to each well. The plate was stirred for 30 min at room temperature to complete dissolution of the formazan, and the samples were read at 550 nm (Biotek ELx800 microplate reader).21,22

Hemolytic activity

Human erythrocytes from O+-type blood were diluted in phosphate-buffered saline (PBS) (in mM: KH2PO4, 1.5; Na2HPO4, 8.1; NaCl, 136.9; KCl, 2.6) at pH 7.2 to a final concentration of 5 % of cells. Then, 80 µL of cell suspension and 20 µL of BBI (400, 200, 100, 50, 25, 12.5, 6.25 and 3.12 µg/mL) was added in a 96-well plate, and the resulting suspension was incubated at 37 °C during 1 h. Thereafter, the hemolysis process was stopped by adding 200 of PBS, the suspensions were centrifuged at 1 000 g for 10 min and the absorbance of free hemoglobin in the supernatant was measured at 550 nm.2

Lysosomal compartment assay

Peritoneal macrophages (5×105) were plated and incubated with BBI (100, 50, 25, 12.5, 6.25 and 3.12 µg/mL). After 24 h of incubation in an incubator at 37 °C and 5 % CO2 was added to 10 µL of neutral red solution 2 % DMSO and returned to incubate for 30 min. The supernatant was discarded, carried by washing the wells with 0.9 % saline at 37 °C and added 100 µL of extraction solution (glacial acetic acid 1 % v/v, ethanol 50 % v/v dissolved in twice-distilled water) present to solubilize the neutral red inside the secretion of lysosomal vesicles. The absorbances were read at 550 nm.23

Phagocytosis test

Peritoneal macrophages (5×105) were plated and incubated with BBI (100, 50, 25, 12.5, 6.25 and 3.12 µg/ml). After 24 h of incubation in an incubator at 37°C and 5 % CO2 was added to 10 µl of zymosan colored solution and incubated for 30 min at 37 °C. After the procedure, 100 µl of Baker's fixer was added to stop the phagocytosis process and 30 min later, the plate was washed with 0.9 % saline to remove the zymosan and neutral red do not phagocytosed by macrophages. The supernatant was removed and 100 µL of the extraction solution was added after solubilization in Kline stirrer. The absorbances were measured at 550 nm.24

Nitric oxide (NO) production

The production of NO in macrophages infected by L. amazonensis was determined indirectly by measurement of nitrite by Griess' method.2 Briefly, 5×105 peritoneal macrophages per well were incubated at 37 °C and 5 % CO2 for 4 h to cell adhesion. Next, 5×106 promastigotes, in stationary phase, were added with the half part of the groups for 30 min. Thereafter, the macrophages were incubated with BBI (100, 50, 25, 12.5, 6.25 and 3.12 µg/ml) of LPS (2 µg/ml) for 24 h and then supernatant was collected and mixed with the same volume of Griess reagent and nitrite level was measured at 540 nm. For determining the concentration of nitrite, a standard curve prepared with sodium nitrite at concentrations of 1, 5, 10, 25, 50, 75, 100, and 150 μM diluted in the culture medium.

Statistical analysis

All assays were performed in triplicate and in three independent experiments. The statistical analysis of results was performed using GraphPad Prism 5.0 program. The values are expressed as mean ± standard deviation. For multiple concentrations of parametric data was used One-way Analysis of Variance followed by Bonferroni's Pos-test. The analysis was considered significant when p< 0.05.

 

RESULTS

Evaluation of in vitro antileishmanial effect of BBI

The murine macrophages infected with L. amazonensis were treated with different concentrations of BBI. A concentration-response effect was demonstrated by the reduction in the number of internalized amastigotes within macrophages (Fig. 1A) Likewise, the percentage of infection in macrophages has reduced at concentrations of 25 and 50 µg/mL of BBI (Fig. 1B). The inhibitory concentration (IC50) in 48 h of exposure to BBI was 3.95 µg/ml (Table).

 

Cytotoxicity assay and hemolytic activity

The results of the cytotoxicity of BBI in murine peritoneal macrophages are presented in Fig. 2A. The concentration of 73.26 µg/ml of BBI was able to reduce by 50 % the viability of macrophages (CC50). The selectivity index (SI) was calculated by the CC50:IC 50 ratio and to evaluate the security level of BBI in macrophages, and BBI has demonstrated to be 18.55 times more selective for the parasites than macrophages (Table). Besides, the cytotoxicity of BBI for human erythrocytes was very low, only inducing 5 % of hemolysis at concentration of 400 µg/ml (Fig. 2B).

Lysosomal compartment assay and phagocytosis test

The activation of macrophages was evaluated by increasing the number and size of endosomal/lysosomal compartments and increased phagocytic capacity of macrophages. As shown in Fig. 3A, BBI promoted an increase in neutral red retention of the secretory vesicles of macrophages at concentrations of 50, 25, 12.5, 6.25 and 3.12 µg/ml. Similarly, BBI stimulated phagocytosis of zymosan particles at concentrations of 25, 12.5 and 6.25 µg/mL (Fig. 3B).

Nitric oxide (NO) production

The determination of NO production was performed by nitrite measurement in infected and non-infected peritoneal macrophages treated with BBI. The treatment of macrophages with BBI was capable of stimulating a concentration-dependent increase of NO production in non-infected macrophages in all tested concentrations. Interestingly, the BBI-induced NO increase at concentrations from 12.5 to 100 µg/mL was markedly higher in comparison with the respective non-infected groups (Fig. 4). Besides, there was no difference in NO production between non-infected and infected negative groups.

 

DISCUSSION

Tropical diseases caused by trypanosomatids such as leishmaniasis are considered neglected diseases, and still remain to be a serious worldwide public health problem. Considering the current situation of leishmaniasis therapy and its high toxic potential drugs for humans, the searching for new safe antileishmanial compounds becomes a necessity increasingly urgent.5

On the other hand, many studies have been conducted in order to characterize the action of new drugs25-27 and extracts or derivatives of herbal compounds,28-30 as performed in this study. In this sense, the evaluation of leishmanicidal activity against macrophage-internalized amastigote forms is the most efficient way to assess the antileishmanial activity due to the better correlation with the clinical condition, considering the amastigote form is the only form usually found in patients with leishmaniasis.31

The BBI was able to promote an efficient antileismanial effect against amastigote forms of L. amazonensis at IC50 of 3.95 μg/ml. These findings are relevant due to be the first report of antileishmanial effect of BBI against macrophage-internalized amastigote forms of L. amazonensis. Recently, the anti-amastigote effect of meglumine antimoniate (Glucantime©), the first choice drug for leishmaniasis treatment, was assessed against intramacrophagic amastigotes by our research group. The reference drug meglumine antimoniate exhibited IC50 of 167.4 µg/ml, lower than BBI. Moreover, pentavalent antimonials are highly toxic drugs.2

The anti-amastigote effect of BBI was markedly higher than the previously observed for both P. insignis fractions and the isolated compound GFC.17,18 The difference in antileishmanial activity may due to the possible involvement of macrophages activation mechanisms in this effect. Therefore, the investigation of the involvement of immunomodulatory activity is strongly suggested.

It is well stated that natural products are able to activate macrophages by inducing the production of many cytokines, such as IFN-γ, IL-12, TNF-α and IL-10, reactive oxygen species (ROS) and nitric oxide (NO). Therefore, the phagocytic capacity and endosomal compartments which regulate the defense mechanisms of mammalian cells against intracellular parasites is marked increased.32,33

In the study, the phagocytic capacity and the lysosomal volume of murine macrophages were increased after treatment with the BBI. At concentrations of 50 and 100 μg/ml, this immunomodulatory effects were not observed, probably due to the cytotoxic effect at these concentrations. Besides, the highest BBI-induced phagocytic capability was observed at 12.5 μg/ml, and considering the IC50 of 3.95 μg/ml for BBI against macrophage-internalized amastigotes, the strong participation of immunomodulatory mechanisms is markedly reinforced.

Moreover, BBI also promoted an increase of NO production. Interestingly, this effect was higher in infected macrophages than in non-infected macrophages treated with same concentrations. The toxic activity of NO against amastigote forms of L. amazonensis has been classically demonstrated by studies demonstrating the resistance to treatment of cutaneous lesions in mice lacking the nitric oxide synthase (iNOS), treated with leishmanicidal drugs.34 Thus, these parameters are essential for the determination of macrophage activity, where internalization, disintegration and production of parasite antigens occur.35

The cytotoxicity evaluation of BBI in macrophages has demonstrated to have a slight decrease of cell viability, then suggesting a positive characteristic related to possible side effects. Likewise, in vitro cytotoxicity assays are common to macrophages in searching for effective drugs in the treatment of leishmaniasis. One valuable method comprises the yellow-colored and water-soluble compound known as MTT, which enters the cell through the plasma membrane and in contact with the superoxide produced by mitochondrial activity, is oxidized MTT-formazan, a purple and water-insoluble salt. Thus, the MTT oxidation is proportional to the mitochondrial activity and therefore cell viability.36 After determination of selectivity index between CC50 in macrophages and IC50 in internalized amastigotes, BBI demonstrated 18 times more selectivity to parasite then the macrophage. Moreover, the hemolytic activity assay is an important parameter in order to evaluate cytotoxicity, evaluating the potential damage caused on plasma membrane, then probably indicating in vivo cell damage.37 BBI promoted up to 5 % of hemolysis at high tested concentrations, reinforcing the safe application of this herbal extract in the treatment of leishmaniasis.

Thus, the present results demonstrated that BBI has leishmanicidal activity of the macrophage-internalized amastigote forms of L. amazonensis at non-toxic conditions for host cells, probably mediated by macrophage activation mechanisms. Therefore, the seeds oil from P. insignis is a promising precursor product for the formulation of new drugs with leishmanicidal activity, leading towards further studies of the evaluation of its efficacy in in vivo leishmaniasis experimental models.


ACKNOWLEDGEMENT

This work was supported by the Federal University of Piauí (UFPI, Brazil), Fundação de Amparo à Pesquisa do Estado do Piauí (FAPEPI, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).

 

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Recibido: 22/4/2017.
Aprobado: 8/5/2018.

 

 

Daniel Dias Rufino Arcanjo: Núcleo de Pesquisas em Plantas Medicinais, Universidade Federal do Piauí, Teresina, Piauí. Brasil. correo electrónico: daniel.arcanjo@ufpi.edu.br





Copyright (c) 2019 Ana Karina Marques Fortes Lustosa, Érika Alves Bezerra, Klinger Antonio Franca Rodrigues, Layane Valéria Amorim, José Sousa Lima-Neto, Bruno Quirino Araújo, Joaquim Soares da Costa-Júnior, Anderson Nogueira Mendes, Fernando Aécio Amorim Carvalho, Daniel Dias Rufino Arcanjo, Antônia Maria das Graças Lopes Citó

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