Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia ( Myrtaceae ) extract

Introduction: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. Methods: The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. Results: The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200μg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. Conclusions: The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.


INTRODUCTION
Malaria is endemic in 95 countries, with high prevalence in mainly tropical and subtropical countries situated south of the Sahara desert, in Southeast Asia, Latin America, and the Caribbean, particularly those located in the Amazon Basin region (1) .It is a parasitic disease, with four causative agents, all of which are protozoa belonging to the genus Plasmodium (Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum) (2) .P. falciparum is the most widely studied since it causes the highest number of deaths.Natural malaria transmission occurs through the bite of female mosquitoes from the Anopheles genus, with the most frequent species in Brazil being Anopheles darlingi (3) .
Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania.It is endemic in 98 countries, with an estimated 50,000 deaths per year, ranking ninth among infectious diseases.It presents high levels of endemicity, morbidity, and mortality in populations in Africa, Asia, the Middle East, the Mediterranean region, and Latin America (4) .These diseases are transmitted to humans and other mammals through the bite of the female insect Lutzomyia spp (5) .
Leishmaniasis may present diverse clinical manifestations, affecting the skin, mucous membranes, and internal organs.Cutaneous leishmaniasis may be caused by fourteen Leishmania species.Diffuse leishmaniasis is caused by Leishmania (Leishmania) amazonensis, the mucocutaneous form is mainly caused by Leishmania (Viannia) braziliensis, and visceral leishmaniasis is a parasitic disease involving the Leishmania donovani complex, caused by Leishmania (Leishmania) chagasi in Brazil (6) .
Given the increasing need to obtain and develop new drugs for the treatment and control of diseases for which there is still no treatment and/or satisfactory cure, natural plant products may be very useful (7) .In the Amazon, many plants are used for therapeutic purposes.These are sometimes the only source of medicine, particularly in isolated and distant places (8) .
Camu-camu (Myrciaria dubia McVaugh) is a medicinal plant native to the Amazon rainforest, known for its high levels of ascorbic acid, carotenoids, potassium, and polyphenols, such as flavonoids and ellagitannins (9) (10) (11) (12) .The main economic characteristic of this fruit is related to its high ascorbic acid content, which helps combat and prevent the formation of free radicals, making it useful against colds, pulmonary disorders, bladder irregularities, low immunity, and early or natural aging (13) (14) .
Myoda et al. (15) studied the effects of the methanol extract from the seeds and peel of M. dubia against the following microorganisms: Staphylococcus aureus, Escherichia coli, and Saccharomyces cerevisiae, proposing that the lipophilic and phenolic compounds present in the camu-camu extract are responsible for its antimicrobial activity.Given the phytotherapeutic potential of M. dubia, the objective of this study was to evaluate the action of the dichloromethanolic extract obtained from the leaves of M. dubia through bioassays against the protozoa Plasmodium falciparum and promastigote forms of Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi.

Collection of plant material
Samples of M. dubia were collected in August 2012, from along the banks of the river Javaés (09°32'S, 50°01'W), in the municipality of Pium, Tocantins, Brazil.The location of the samples was georeferenced by global positioning system (GPS), and the samples were preserved, identified, and registered (registration no.10.592) in the Herbarium at the Federal University of Tocantins, Porto Nacional Campus.

Continuous cultivation of the erythrocytic phase of the parasite Plasmodium falciparum
In the antimalarial activity assays, sanguineous forms of a chloroquine (CQ)-resistant Plasmodium falciparum clone W2 were used.Parasites were cultured in O-type Rh + human red blood cells with 2% hematocrit and 50mg/mL albumax (Gibco) under the conditions established by Trager and Jensen (17) .To this end, the parasites were grown in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) culture medium supplemented with 25mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) Hepes (Sigma-Aldrich), 21mM sodium bicarbonate (Sigma-Aldrich), 11mM glucose (Sigma-Aldrich) and 40μg/mL gentamicin, and then maintained at 37°C under a gaseous mixture containing 5% CO 2 , 5% O 2 and 90% balanced N 2 .The development of the parasites was monitored by daily observation of blood smears stained with Giemsa and analyzed using light microscopy.

Synchronization of parasites for use in in vitro tests
The cultures predominantly composed of rings (young trophozoites) used in chemotherapy trials were subjected to a synchronization process with sorbitol, as described by Lambros and Vanderberg (18) .

Preparation of the plates for chemotherapy trials
Synchronized parasite cultures were distributed in 96-well microtiter plates by adding 180μL of RPMI culture medium containing 0.05% parasitemia and 1.5% hematocrit to each well.Initially, 20µL of the extract, in triplicate, and in serial concentrations from 100 to 0.097μg/mL were added to the culture plate.The control wells (six per assay) contained infected red blood cells with no added test compounds (negative control).The standard antimalarial drug, artemisinin (ART), was tested in parallel as the positive control in all the experiments in serial dilutions from 50 to 0.7812ng/mL.
The plates were maintained for 72h under ideal conditions for the development of Plasmodium.After this period the plates were frozen at -80°C and thawed twice to lyse the erythrocytes (19) .

Anti-HRP II enzyme immunoassay
The anti-histidine-rich protein II (HRP II) enzyme immunoassay followed the protocol adopted by Noedl et al. (20) .In this test, the commercial monoclonal primary antibody (Mouse IgM anti-Plasmodium falciparum HRP II -Monoclonal) MPFM-55A (ICLLAB) was used at 1µg/mL to sensitize the plates (Maxysorp Nunc).After sensitization, 200µL of a blocking solution (2% PBS-BSA) was added to each well and the plate was incubated at room temperature for 2h.After washing thrice in PBS-Tween 20 (0.05% PBS-T), 100µL of hemolyzed P. falciparum culture samples was added to each well.The plate was incubated for 1h at room temperature in a humid chamber, then washed three times with PBS-T, and incubated with the secondary antibody MPFG55P (ICLLAB) (1:5.000) for 1h in the same conditions as mentioned above.Once again, the plate was washed thrice in PBS-T and the reaction was revealed using 100µL of 3.3', 5, 5'-tetramethylbenzidine (TMB) solution for 5 min.The reaction was stopped by adding 50µL per well of a 1M sulfuric acid solution.The optical density was determined using a microplate spectrophotometer (BIO-RAD Model 3550) with a wavelength of 450nm.Each assay was performed in triplicate, in three independent experiments.
Growth inhibition of 50% of the parasites (IC 50 ) was determined using dose-response curves, in non-linear regression function.The software Origin (OriginLab Corporation, Northampton, MA, USA) was used to determine the IC 50 value.According to Reynertson et al. (21) , samples with IC 50 less than 50μg/mL are considered very active, 50-100μg/mL moderately active, 100-200μg/mL slightly active and a sample with a value above 200μg/mL is considered inactive.

Cultivation of HepG2 cells
The human hepatoma (HepG2) cell lines were cultured according to Calvo-Calle et al. (22) .They were maintained in culture bottles supplemented with RPMI containing 5% fetal bovine serum (FBS) (Gibco/Invitrogen) and 40mg/L gentamycin (Schering-Plough).The cells were maintained in an incubator with 5% CO 2 at 95% humidity and 37 °C.After a confluence of about 80% is attained, the cells were subcultured, or used to perform cytotoxicity assays.

Cytotoxicity test
C y t o t o x i c i t y w a s d e t e r m i n e d u s i n g t h e M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method (23) .Cells were seeded at 2 × 10 4 /well and after 24h of treatment with the M. dubia extract (500-7.8µg/mL),20μL of MTT at a concentration of 5mg/mL in PBS (w/v) was added to each well.The plate was left for 4h in an incubator at 37°C.At the end of this period, the culture medium along with the excess MTT was discarded, and then 100μL of DMSO (Sigma-Aldrich) was added to each well.The optical density was read using a microplate spectrophotometer at a wavelength of 570nm.Cells with no added extract were used as negative control.
The 50% cytotoxic concentration (CC 50 ) of the cells was determined from nonlinear regression of the serial dilutions of the extracts using the program Origin.

Selectivity index
The selectivity index (SI) of the samples was evaluated by calculating the ratio of the CC 50 value and the IC 50 value.Values greater than 10 were considered to indicate lack of toxicity, while substances with values below 10 were considered toxic (24) .
The promastigote cultures were maintained in vitro using a parasitic aliquot in stationary growth phase, which was diluted in erythrosin B dye (0.04%).The concentration of the protozoa was estimated with the aid of a Neubauer chamber and adjusted to 1 × 10 6 promastigotes per mL.The parasites that were stained red were considered dead and those that were birefringent and mobile were considered alive.Only the living parasites were quantified.Consequently, the parasites were placed in RPMI/FBS culture medium and maintained at 24°C, and were subcultured every five days for up to eight passages.

Evaluation of the leishmanicidal potential of the crude extract of Myrciaria dubia using the resazurin method
Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi promastigotes were plated (1 × 10 6 parasites/ well) in 96-well plates and incubated with concentrations ranging from 200 to 1.56μg/mL of extract.Besides the control containing only parasites in culture medium, controls of the solvent 0.5% DMSO and the drug (3µg/mL pentamidine) were also used.The plate was incubated at 24°C for 72h.After this, 20µL of a 2mM resazurin solution was added to each well (25) .The plates were incubated for 4h, and the reduction of resazurin was determined by readings obtained from a fluorimeter (excitation wavelength of 485nm and emission wavelength of 590nm).The control value (RPMI blank) was subtracted from all obtained values.In all, three independent assays were performed and the samples were tested in triplicate.The results were expressed as growth inhibition (GI) rate according to the formula: % GI = 100 − [(A − a) / (B − a)] × 100.Where: A = Absorbance of treated cells; a = Absorbance of white cells and B = Absorbance of control cells.

Antiplasmodial activity
All assays were performed in triplicate and the results were expressed as the average of the repetitions.The M. dubia extract showed anti-P.falciparum (chloroquine-resistant strain W2) activity in an enzyme-linked immunosorbent assay (ELISA) using anti-HRPII monoclonal antibodies, involving a specific protein from the parasite, and the results of the bioassays are shown in Table 1.
The positive control used in the antiplasmodial bioassay was artemisinin, IC 50 = 6.77ng/mL (3.45).The compound used as the extract solvent (0.5% DMSO) showed no antiplasmodial, leishmanicidal, or cytotoxic effect, excluding the possibility of the solvent acting as an inhibitory agent (data not shown).
Regarding the cytotoxic activity of the extracts on the cell line derived from human hepatoma (HepG2), assessed by the ability of cells to metabolize tetrazolium salt (MTT) to formazan, the cytotoxic concentration for 50% of the cell population (CC 50 ) was calculated based on a dose response.CC 50 values above 500μg/mL were observed in all repetitions, indicating that the compounds present in the plant extract did not present toxicity.The SI, considering the IC 50 data for P. falciparum, was greater than 10, confirming that the extract is not toxic to HepG2 cells.

Leishmanicidal test
In the leishmanicidal tests, at a concentration of 200µg/mL, the M. dubia extract inhibited the growth of L. amazonensis and L. chagasi promastigotes up to 85% and 30%, respectively.This inference is based on the results obtained from the resazurin assay.The promastigote forms of L. braziliensis continued to proliferate in the presence of the extract (Figure 1).
The M. dubia extract presented greater inhibitory activity against the L. amazonensis strain than that against the other two strains of Leishmania, making it possible to calculate a dose response and IC 50 values (Table 1).The positive control used in the anti-leishmanial bioassays was pentamidine, possessing IC 50 value = 0.21µg/mL (0.09).

DISCUSSION
The results of this study show that M. dubia species could be added to the list of Amazonian plants with anti-P.falciparum W2 activity.Meneguetti et al. (26) reported that 76 species of plants distributed throughout 32 families in the Amazon presented antimalarial activity.Twenty six of the plant species mentioned previously showed in vitro activity against 3D7, W2, FcB1, K1, Dd2, D2, D6, F32, FcM29, and Nigerian strains of P. falciparum, particularly against P. falciparum and contained 66 active compounds.Borges et al. (27) reported the antioxidant, antibacterial, and antifungal properties of the leaf, bark, and fruit extracts from plants of the Myrciaria genus; these properties are attributable to the presence of several active components including phenolic compounds such as tannins, flavonoids, ellagic acid, and anthocyanins.
Yapu et al. (32) tested aqueous and ethanolic extracts from the peels of M. dubia cultivated in Peru against P. falciparum (FCR3 strain resistant to chloroquine), and obtained IC 50 values equal to 3 and 6µg/mL, respectively.These values are close to those of other species that have activity against P. falciparum, such as Remijia peruviana (IC 50 = 7.4µg/mL), Cinchona officinalis (IC 50 = 4.2µg/mL), and Pubescens cinchona (IC 50 = 1µg/mL), and to the value reported for M. dubia leaf extracts, 2.35 (1.05)μg/mL in this study.
Colarossi (33) analyzed the cytotoxicity of methanolic extracts from M. dubia seeds and pulp on Jurkat cells (immortalized human T lymphocytes).The CC 50 for the seed extract and pulp extract was >800μg/mL and 524.37μg/mL, respectively, indicating that the methanolic extract of M. dubia is non-toxic.
The leishmanicidal effect presented by P. edulis may be related to the presence of flavonoid and/or terpenoid compounds in the leaves of these plants (35) .The extract from the leaves of S. jambolanum exhibited a leishmanicidal effect, which could be explained by the presence of saponins and condensed and hydrolysable tannins in the leaves (36) .
The SIs obtained in this study for L. amazonensis, L. braziliensis, and L. chagasi were less than 10.This result does not indicate the absence of antiparasitic activity, rather it shows that a high dose would be required to achieve the desired therapeutic effect (37) , which is not feasible.The high IC 50 values observed in this study may also be related to the antagonistic effect of other compounds present in the mixture, or to an insufficient amount of the active component responsible for the desired effect.A more detailed analysis of the phytochemical profile of this extract could reveal the existence of purified compounds with potential anti-leishmanial action.
The lack of leishmanicidal activity in inhibition tests against L. braziliensis may be attributed to changes in the genetic sequences responsible for promoting variability between species and phenotypically determining the resistance of these organisms, consequently affecting the efficacy of some compounds (38) .L. braziliensis is the most prevalent species in humans and is found in all endemic areas in the country.It is considered the most resistant species to diverse treatments (39) .Genomic plasticity can be observed in the Leishmania genus, which allows for the adaptation of the parasite to diverse environments, including different vertebrate hosts.This plasticity may influence their resistance to therapeutic drugs (40) .
Meneguetti et al. (41) found 32 species of plants in the Brazilian Amazon with leishmanicidal activity distributed throughout 18 botanical families.Of these, 27 presented action against the species L. amazonensis, 6 against L. braziliensis, and 3 against L. chagasi.
Among the species studied for activity against L. amazonensis, the extracts with the optimal activity were the hexane and the methanolic ones from the leaves of the species Lacistema pubescens, with IC 50 values of 3.5μg/mL and 3.9μg/ mL, respectively.Lacistema pubescens possesses antioxidant properties, probably due to the phenolic compounds detected in this species (42) , just like M. dubia which possesses these same chemical properties (11) (12) .
Many plants have substances belonging to the compound classes alkaloids, terpenes, lignans, chalcones, flavonoids, and sesquiterpene lactones, all described in the literature as having effective leishmanicidal and antiparasitic activity (43) (44) (45) (46) (47) (48) .Flavonoids constitute one of the most abundant and widely distributed secondary metabolites in plants.The leishmanicidal activity of this class of metabolites has been observed in L. peruviana, L. braziliensis (49) , and L. infantum (50) .
The action mechanism of flavonoids on Leishmania species was proposed by Mittra et al. (51) , who concluded that flavonoids cause apoptosis of the parasite by inducing cleavage of the kinetoplast.Cruz et al. (52) demonstrated that flavonoids, in addition to altering the arrangement of mitochondrial deoxyribonucleic acid (DNA), also inhibit the enzyme arginase, which participates in the parasite's polyamine biosynthesis.Fonseca-Silva et al. (53) demonstrated that treatment of L. amazonensis with quercetin, a flavonoid most commonly found in plants, increases the production of reactive oxygen species (ROS), promotes mitochondrial dysfunction in the parasite, and inhibits cell growth.
Meslin et al. (55) reported the involvement of terpenoids in the activation of programmed cell death in Plasmodium species, and in the interference of one or more substances in the process of cell differentiation of the parasite, which has proven to be an extremely complex process, with fine-tuned regulation of gene expression, resulting in a turnover of proteins and destruction of organelles present in Plasmodium, as well as other species of pathogenic protists (56) .
To the best of our knowledge, this is the first study to evaluate the action of the dichloromethanolic extract obtained from the leaves of M. dubia through bioassays against P. falciparum and promastigote forms of L. amazonensis, L. braziliensis, and L. chagasi.This fact highlights the importance of this first report, since the results were satisfactory and contribute to the study of medicinal plants, and the biodiversity of the Amazon.
This study showed that M. dubia may be considered an important source for the development of novel drugs for diffuse cutaneous leishmaniasis and malaria treatment, since its extract was active against the parasite P. falciparum, presented moderate activity against the promastigote forms of L. amazonensis, and was non-toxic to HepG2 cells in in vitro tests.However, fractionation of the M. dubia extracts, and in vivo testing may help in the elucidation and characterization of the chemical compounds responsible for the inhibitory action of these protozoa.

TABLE 1
Evaluation of the dichloromethanolic extract from Myrciaria dubia in relation to the IC 50 (growth inhibition of 50% of the parasites), CC 50 (cytotoxic concentration for 50% of the HepG2 cells), and SI (selectivity index).