Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis

Journal Pre-proof Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis Caroline Gomes Macedo, Maria Yasmin Fonseca, Antônia Djane Caldeira, Santana Pinto de Castro, Wallace Pacienza Lima, Maria Paula Gonçalves Borsodi, Adilson Sartoratto, Milton Nascimento da Silva, Claudio Guedes Salgado, Bartira Rossi Bergmann, Kelly Christina Ferreira Castro PII:

S0014-4894(19)30086-4

DOI:

https://doi.org/10.1016/j.exppara.2020.107847

Reference:

YEXPR 107847

To appear in:

Experimental Parasitology

Received Date: 24 February 2019 Revised Date:

11 June 2019

Accepted Date: 24 January 2020

Please cite this article as: Macedo, C.G., Fonseca, M.Y., Caldeira, Antô.Djane., Pinto de Castro, S., Lima, W.P., Gonçalves Borsodi, M.P., Sartoratto, A., Nascimento da Silva, M., Salgado, C.G., Bergmann, B.R., Ferreira Castro, K.C., Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis, Experimental Parasitology (2020), doi: https:// doi.org/10.1016/j.exppara.2020.107847. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis Caroline Gomes Macedoa,b , Maria Yasmin Fonsecab , Antônia Djane Caldeirab, Santana Pinto de Castrob, Wallace Pacienza Limac,d, Maria Paula Gonçalves Borsodic, Adilson Sartorattoe, Milton Nascimento da Silvaf, Claudio Guedes Salgadog, Bartira Rossi Bergmannc, Kelly Christina Ferreira Castroa,b* a

Programa de Pós-Graduação em Biociências, Universidade Federal do Oeste do Pará, Santarém, PA, 68040-255, Brazil. b Laboratório de Pesquisa e Desenvolvimento de Produtos Naturais e Bioativos (P&DBio), Universidade Federal do Oeste do Pará, 68040-255, Santarém, PA, Brazil. c Laboratório de Imunofarmacologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, RJ, Brazil. d Escola de Ciências da Saúde, Universidade do Grande Rio, 25071-202, Duque de Caxias, RJ, Brazil e Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA), Universidade Estadual de Campinas, 13148-218, Campinas, SP, Brazil. f Programa de Pós-graduação em Química, Universidade Federal do Pará, 66075 750, Belém, PA, Brazil. g Laboratório de DermatoImunologia (LDI), Universidade Federal do Pará, 67200-000, Belém, PA, Brazil.

Kelly Christina Ferreira Castro Corresponding author. Laboratório de Pesquisa e Desenvolvimento de Produtos Naturais e Bioativos (P&DBio), Universidade Federal do Oeste do Pará, Rua Vera Paz, s/n (Unidade Tapajós), Bairro Salé, 68040-255, Santarém, PA, Brazil. e-mail: [email protected]

ABSTRACT

Leishmaniasis is an infectious disease that has high endemicity and is among the six parasitic diseases of higher occurrence in the world. The current treatments are limited due to their toxicity, treatment resistance and high cost which have increased the search for new substances of natural origin for its therapy. Based on this, an in vitro biological and chemical investigation was carried out to evaluate the potential of Piper marginatum against Leishmania amazonesis. P. marginatum leaves were collected to obtain the essential oil (EO) and the ethanolic extract (CE). The chemical profile of the CE and fractions was obtained by 1H NMR. The analysis of the EO chemical composition was performed by GC-MS. EO, CE and fractions were submitted to antileishmanial and cytotoxicity assays against macrophages. The chromatographic profiles of EO, CE and fractions showed the presence of phenolic compounds and terpenoids, having 3,4Methylenedioxypropiophenone as a major compound. All P. marginatum samples showed low toxicity to macrophages. The CE and the methanolic, hexane and ethyl acetate fractions had low cytotoxicity when compared to Pentamidine. All tested samples inhibited growth of L. amazonensis promastigotes. The antileishmanial activity of EO, CE and fractions were evaluated in macrophages infected with L. (L.) amazonensis and treated

with the concentrations 1, 10 and 100 µg/mL for 48h. All samples were active, but EO and CE showed superior activity against amastigote forms when compared to the promastigote forms of L. amazonensis. This work describes for the first time the antileishmanial activity of the species P. marginatum and its cytotoxicity against macrophages, suggesting that it can be an alternative source of natural products in the phytotherapeutic treatment of leishmaniasis.

Graphical abstract

Highlights • P. margintatum essential oil, ethanolic extract and fractions showed the presence of phenolic compounds and terpenoids. • They had a strong antileishmanial effect. • Low toxicity to macrophages. Keywords: Piper marginatum; Phenylpropanoids; Antileishmanial activity; Leishmania amazonensis.

Abbreviations: American Cutaneous Leishmaniasis (ACL); Selectivity Index: SI; Piper marginatum Essential Oil: EO; Piper marginatum Crude Extract: CE; Ethyl Acetate Fraction: EAF; Dichloromethane Fraction: DF; Hexane Fraction: HF; Residual Methanolic Fraction: MF; Nuclear Magnetic Resonance: NMR; 3-[4,5-dimethylthiazol-2yl]-diphenyl tetrazolium bromide: MTT; Gas Chromatography Coupled to Mass Spectrometry: GC-MS; Inhibitory concentration capable of killing 50% of a population: IC50; Refraction Index: RI.

1. Introduction

American cutaneous leishmaniasis (ACL) is an infectious parasitic disease that affects the skin structures in a localized or diffused manner. This disease is caused by different protozoan species from the genus Leishmania, and according to the World Health Organization (WHO), it has high endemicity, morbidity, mortality and capacity to produce deformities. ACL can be found in 88 countries. Of all its cases, 80% have occurred in four developing countries. Among these countries, Brazil has the highest prevalence with record cases in all regions with emphasis on the North, Northeast and Midwest which are considered endemic regions (ALVAR et al., 2012, WHO, 2017). In the North of Brazil, a progressive advance in the number of ACL cases has been noticed in the last four years. The region had 51% of the cases from the whole country in 2014 while the state of Pará alone registered 41% of them (Brazil, 2018). For all leishmaniasis forms, the first-choice treatment in Brazil is through the meglumine antimoniate (Glucantime®) provided exclusively by the Unified Health System (UHS). The drugs of second choice are amphotericin B and pentamidine. However, high cost, high toxicity, and the need for daily injections that usually cause adverse effects in patients, parasite resistance to these drugs and the time of treatment are limiting factors for their use (CROFT and COOMBS, 2003; RATH et al., 2003). In the absence of a vaccine and an effective treatment, there is an urgent need for developing potentially less toxic and cost-effective drugs. Chemical and biological studies of substances extracted from plants can be an attractive alternative in this search. The use of medicinal plants in Brazil has been growing due to the ease of access, low cost and its cultural compatibility. In addition, several plant species from Brazil remain without chemical studies although these species may have an important economic potential in the world context (RATES, 2001). The genus Piper from the family Piperaceae includes a large number of species popularly known as ‘papiroba’ which are widely used as food and especially in the traditional medicine for the treatment of various diseases. In the genus Piper, the chemical constituents belong to different classes of secondary metabolites, such as alkaloids, amides,

propenylphenols,

lignins,

neolignins,

terpenes,

steroids,

kavalactones,

piperolidines, chalcones, dihydrochalcones, flavones and flavonones which have important biological activities (PARMAR et al., 1997). Phenylpropanoids are a potentially active chemical group that occurs in Piper spp. where monolignoids such as apiol, myristicin,

eugenol, safrole, dimers of phenylpropanoids and dillapiole are included (BERNARD et al., 1995). Among the native species of the Brazilian Amazon, Piper marginatum Jacq, popularly known as ‘pimenta do mato’, stands out for its pharmacological potential, being widely used in folk medicine due to its healing properties. P. marginatum has a broad spectrum of antimicrobial and bactericidal activity (ARAÚJO et al., 2014; DUARTE et al., 2004; SÁNCHEZ et al., 2011; SILVA and BASTOS, 2007). The scarcity of reports about its essential oil and crude extracts leishmanicidal activity indicates the importance of evaluating its potential for use in the treatment of parasitic diseases. Biological studies performed with species of the genus Piper, such as P. regnellii, P. turbeculatum and P. aduncum, demonstrated their efficacy against L. amazensis, making this genus a very promising group (FERREIRA et al., 2010; NAKAMURA et al., 2006; TORRES-SANTOS et al., 1999). Based on this, the present study performed an in vitro biological and chemical investigation of P. marginatum against L. amazonensis evaluating its essential oil, ethanolic extract and phenylpropanoid rich fractions cytotoxicity, promastigote and amastigote activity.

2. Material and Methods

2.1 General experimental procedures

To obtain the essential oil and ethanolic extract, distilled water and ethanol 96% were used as solvent, respectively. For the essential oil chromatographic analysis, an aliquot of 1 µL was submitted to GC-MS analysis under the following conditions: GC-MS Agilent, model HP-6890 coupled to mass selective detector, capillary column HP-5MS (30m x 0.25 mm x 0.25 µm). Temperatures: injector = 220 °C, detector = 250 °C, column = 60 °C, 3°C. min-1, 240 °C (20 min) and carrier gas = He 1.0 mL/min. The EO chemical components were identified by calculating the retention indices (RI) and using the NIST library to compare the RIs calculated with the ones available in the literature (ADAMS, 2007). Hydrogen-1 Nuclear Magnetic Resonance (NMR) analysis of the ethanolic extract, hexane fraction and dichloromethane fraction was performed on a spectrometer - Varian Mercury 300 model, operating at 300 MHz of 1H. The samples were solubilized in CDCl3

from the brand TEDIA BRAZIL®. The coupling constants (J) were recorded in Hertz (Hz) and then compared to data present in the literature. For the biological tests, stock solutions of EO, CE, HF, DF, EAF and MF were used at concentrations of 20 mg/mL and dissolved in analytical grade DMSO.

2.2 Botanical material

P. marginatum leaves were collected in a natural forest area located in the Universidade Federal do Oeste do Pará (UFOPA), in the municipality of Santarém, Pará, Brazil, in November of 2015. The plant material was identified by Dr. Thaís Elias Almeida, and an exsiccate was deposited in the Herbarium of UFOPA under registration HSTM- nº 00370.

2.3 Preparation and obtaining of essential oil, ethanolic extract and fractions

The amount of 343g of dried and crushed leaves of P. marginatum was submitted to hydrodistillation via Clevenger for 4 hours to obtain the essential oil (EO). A mass of 83 g of dried and crushed leaves were subjected to extraction via Soxhlet to obtain the crude ethanolic extract (CE) followed by evaporation of the solvent under reduced pressure. The extraction yield was calculated, and EO and CE were stored under refrigeration of -10 °C. The amount of 2.5g of CE was solubilized in 75 mL of MeOH solution: H2O (80:20) v/v. This mixture was subjected to liquid-liquid partition, using solvents with increasing polarity, such as hexane, dichloromethane and ethyl acetate. The hexane (HF), dichloromethane (DF), ethyl acetate (EAF) and residual methanolic fractions (MF) were dried at room temperature and stored in an amber glass vial, duly identified and conditioned under refrigeration. For the biological tests, stock solutions of EO, CE, HF, DF, EAF and MF were used at concentrations of 20 mg/mL and dissolved in analytical grade DMSO. From the stock solutions serial dilutions were carried out to obtain the concentrations of 1; 10; 100 and 1000 µg / mL diluted in culture medium, with the aid of a vortex for a better dilution of the samples. These concentrations were used for the bioassays of cytotoxicity and leishmanicide.

2.4 Parasite cultivation

Promastigote forms of L. amazonensis (strain JOSEFA) were grown in medium 199 supplemented with 10% of fetal bovine serum (Cutilab). The parasites were kept in an incubator of biochemical oxygen demand (BOD) at 26ºC. The parasites were used in the stationary phase of growth.

2.5 Animals and Ethical Considerations

All procedures performed with mice from the vivarium of the Laboratory of Immunopharmacology were done according to the Ethics Committee for the Use of Experimental Animals (ECEA) of the Health Sciences Center of the Universidade Federal do Rio de Janeiro (UFRJ) under the number IBCCF 118 and carried out according to the international guidelines for the care and use of animals in laboratory.

2.6 Anti-promastigote activity

Aiming at an initial screening of the different components of the plant with the leishmanicidal activity, it was used by the direct direct cell counting method in the microscope. L. amazonensis promastigotes were incubated for 72h in the presence of different concentrations of EO, CE, HF, DF, EAF and MF (1, 10, 100 and 1000 µg/mL) at 26°C. The antileishmanial activity was determined by direct counting in a Neubauer chamber through optical microscopy of common light (CAMACHO et al., 2003; SCHMEDA-HIRSCHMANN and RAZMILIC, 1996). All concentrations were applied in triplicates, and the results represent the average percentage of cell viability ± standard deviation.

2.7 Anti-amastigote activity of L. L. amazonensis

For the experiment with intracellular amastigotes, bone marrow cells were removed from BALB/c mice and differentiated in vitro into macrophages BMDM as described by Marim et al. (2010). In this assay, 2.5 x 105 of macrophages BMDM/mL were plated in RPMI medium and left adhering for 24 hours in a 37°C incubator with 5% of CO2. Then, they were infected with promastigotes of L. amazonensis in a ratio of 5:1, incubated for 4 hours, and then washed and maintained for 24h. After the incubation period, infected macrophages were treated with different concentrations of EO, CE, HF, DF, EAF and MF of P. marginatum leaves (1, 10 and 100 µg/mL). As a positive control, Pentamidine was

used in the same concentration of the samples while the negative control had only culture medium for an additional 48h. The parasite survival was measured by direct counting as described in Delorenzi et al. (2001). The modulation of the immune response was assessed by the production of nitric oxide according to Green et al. (1982).

2.8 Evaluation of cytotoxicity

The assessment of macrophage cytotoxicity was performed by the direct method of 3-[4,5-dimethylthiazol-2yl]-diphenyl tetrazolium bromide (MTT). Macrophages BMDM (105 macrophages/well) were plated and then placed in a growth chamber at 37°C for adhesion with 5% of CO2 for 24 hours. After incubation, the cells were treated with varying concentrations of EO, CE, HF, DF, EAF and MF to be tested (0.1, 1, 10, 100 and 1000 µg /mL) for 48h. Pentamidine was used as the positive control at the same concentration of the samples. The negative control had only culture media. At the end of this period, the MTT colorimetric assay was performed to evaluate cell viability. The microplates were read at absorbance of 490 nm in SpectraMax M5 spectrophotometer. The tests were performed in triplicate. The toxicity and the activity against intracellular promastigote were compared using the selectivity index (SI) ratio (CC50 for macrophages / IC50 for parasite). 2.9 Statistical analysis

The data obtained was analyzed using GraphPad Prism version 5.0. The IC50 values were calculated using the sigmoid dose-response curve. Data were statistically analyzed by two-way ANOVA, and the results were considered significant when p<0.05.

3. Results

3.1 Essential Oil Analysis by Gas Chromatography Coupled to Mass Spectrometry (GC-MS) The chromatographic analysis (Table 1) of EO led to the identification of twentyeight

chemical

constituents

where

eight

were

monoterpenes

(23.14%),

fifteen

sesquiterpenes (41.59%) and five were phenylpropanoids (30.45%). From these, the substances 3,4-Methylenedioxypropiophenone (22.90%), δ-3-carene (10.19%), trans-

caryophyllene (9.67%) and spathulenol (6.89%) were the major components found in the EO. Table 1 - Chemical composition of P. marginatum EO with emphasis (*) to the major components Rt (min)

RI

Constituents

rel. %

5,39

933

α-pinene

2,59

6,49

977

β-pinene

1,83

6,84

991

β-mircene

0,69

7,52*

1013*

δ-3-carene*

10,19*

8,03

1028

Limonene

0,75

8,32

1037

cis-ocimene (β-Z-ocimene)

1,46

8,67

1047

trans-ocimene (β-E-ocimene)

1,55

10,66

1104

Linalool

4,08

18,20

1289

Safrole

1,10

21,84

1376

α-copaene

4,60

22,17

1384

β-bourbonene

1,81

22,49

1391

β-elemene

1,26

23,19

1409

Methyl eugenol

2,40

23,68*

1421*

trans-caryophyllene*

9,67*

23,97

1428

β-copaene

0,99

24,93

1452

α-humulene

0,64

26,09

1481

germacrene D

2,85

26,27

1485

β-selinene

1,07

26,71

1496

bicyclogermacrene

3,77

26,88

1501

α-muurolene

1,13

27,33

1512

myristicin

1,97

27,75

1523

δ-cadinene

1,15

28,62*

1545*

3,4-methylenedioxypropiophenone*

22,90*

29,33

1564

isoelemycin

2,08

30,08*

1583*

spathulenol*

6,89*

30,17

1585

caryophyllene oxide

2,48

32,23

1641

n.i

4,83

32,67

1653

α-eudesmol

1,88

32,94

1660

neo intermedeol

1,40

Legend: n.i= not identified; Rt =Retetion time; RI= Retention Index; rel. % = relative percentage

3.2 1H Nuclear Magnetic Resonance (NMR) analysis of the ethanolic extract, hexane and dichloromethane fractions

The 1H NMR spectrum of the CE, HF and DF samples showed typical signals of an ABX system which is characteristic of a 1,3,4-trisubstituted aromatic ring with signals centered in δ 7.56 (dd, J = 8.1 and 1.8 Hz, 1H), δ 7.44 (d, J = 1.8 Hz, 1H), and δ 6.85 (d, J = 8.1 Hz, 1H). These signals associated with the presence of a triplet in ẟ 1.30 (J = 7.5 Hz) and a quartet in ẟ 2.95 (J = 7.5 Hz), characteristics of a –C(O)CH2CH3 group, corroborate with the identification of the substance known as 3,4-methylenedioxypropiophenone.

3.3 Anti-promastigote activity The results showed that all the tested samples inhibited growth of L. amazonensis promastigotes, having HF and MF fractions as the most active and promising fractions with IC50 values of 0.99 µg/mL and 0.9 µg/mL, respectively (Figure 1-B). De Lima et al. (2012) classifies the samples as highly active when IC50 < 10 µg/mL, active when IC50 > 10 < 50 µg/mL, moderately active when IC50 > 50 < 100 µg/mL and inactive when IC50 > 100 µg/mL. Based on this classification, we can confirm that all tested samples were highly active, because of IC50 < 10 µg/mL (Table 4). The samples (EO and DF) presented lower activity (Figure 1-A and 1- B).

Figure 1 - Anti-promastigote activity. Around 2x105/ mL promastigotes of L. amazonensis were treated with different concentrations (0.1; 1; 10; 100; 1000 µg/mL) of Essential oil (EO), Ethanolic extract (CE) (A), Residual methanolic fraction (MF), Hexane fraction (HF), Ethyl acetate fraction (EAF) and Dichloromethane fraction (DF) (B) of the species Piper marginatum for 72 hours at 26ºC. Pentamidine was used as the positive control at the same concentrations of the material in study. The parasite viability was determined by direct counting in optical microscope in a Neubauer chamber with a dilution ratio of 1:2 in

formalin. The untreated control was maintained with medium and it was represented 100% viability.

3.4 Anti-amastigote activity

The samples activity against the amastigote forms is dose-dependent. In this case, the essential oil and crude extract were the most active with IC50 of 0.58 and 1.2 µg/mL. The fractions, differently, were more active on the promastigote forms (Figure 2- A and B). In Figure 2-C there was no significant production of nitrite by the macrophages which means that the antileishmanial effect could be caused by another microbicidal mechanism of the macrophage or by direct action of the compound on the parasite.

A

2000

EO CE Pentamidine

1500 1000 500

Amastigotes/100 M

Amastigotes/100 M

2500

0 1

0

10

100

1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100

Nitrite ( M)

Concentration ( g/mL)

Figure 2 - Anti-amastigote activity and oxide nitric production. BMDM macrophages infected with L. amazonensis were incubated for 48h at 37°C / 5% CO2 with different

concentrations (1; 10; 100 µg/mL) of essential oil (EO), crude ethanolic extract (CE) and fractions (MF, HF, EAF and DF) of Piper marginatum. The same concentrations were used for Pentamidine, the reference standard of the treatments. A) and B) Quantity of parasites counted by microscopy and expressed in amastigotes per 100 macrophages. C) Nitrite dosage to evaluate the modulation of the immune response (Griess reaction). Results expressed in average ± standard deviation (n = 3), * p <0.05.

3.5 Cytotoxicity evaluation

The tested samples showed low cytotoxicity to macrophages with concentrations ranging from 27.5 to 499 µg/mL and causing cytotoxic effects in 50% of the macrophages (Figure 3). In comparison to the reference standard Pentamidine, CE and the MF, HF and EAF fractions presented cytotoxic concentrations (CC50) ranging from 317 to 499 µg/mL which demonstrate lower cytotoxicity (CC50 Pentamidine: 160 µg/mL) and it was considered moderately toxic, according to De Lima et al. (2012). The Dichloromethane fraction and essential oil presented higher toxicity (CC50: 27,5 µg/mL and 34.5 µg/mL, respectively) and were toxic.

Figure 3 - Cell viability in macrophages non infected. The cells were treated with different concentrations (0.1; 1; 10; 100; 100 µg/mL) essential oil (EO), Crude ethanolic extract (CE) (A), Residual methanolic fraction (MF), Hexane fraction (HF), Ethyl acetate fraction (EAF) and Dichloromethane fraction (DF) (B) from the species Piper marginatum. The viability was determined by MTT method. The rest of the samples were moderately toxic.

In the present study, we evaluated the antileishmanial activity of P. marginatum. EO and CE showed superior activity against intracellular amastigote forms when compared to the results found for the anti-promastigote activity of L. amazonensis (Table 4). Analyzing the SI, the sample that presented the highest selectivity to the parasite of the host cell was CE. In the exception of EAF and DF, the samples tested showed CC50 higher than 20. Table 4 - IC50 values of growth inhibition of L. (L.) amazonensis promastigotes and amastigotes, CC50 of BMDM macrophages and calculus of the selectivity index of EO, CE and fractions, having Pentamidine as the reference substance for the antileishmanial activity. IC50 (µg/mL) Sample

CC50 (µg/mL) Non-infected

Amastigotes

EO

7.9 ± 0.14

0.58 ± 0.16

34.5 ± 0.1

59,5

NI

CE

3.1 ± 0.14

1.2 ± 0.19

499 ± 0.08

415,8

NI

MF

0.9 ± 0.11

15.5 ± 0.29

318 ± 0.1

20,5

NI

HF

0.99 ± 0.17

8.2 ± 0.19

457 ± 0.09

55,7

NI

EAF

1.7 ± 0.16

81.5 ± 0.21

317 ± 0.1

3,9

NI

DF

7.7 ± 0.19

17.4 ± 0.14

27,5 ± 0.1

1,6

NI

Pentamidine

1,4 ± 0,1

0.85 ± 0.13

160 ± 0.05

188,2

NI

LPS

ND

ND

ND

ND

60 ± 0,2

macrophages

SI

Nitrite

Promastigotes

(µg/ml)

Average ± Standard deviation (n = 3). LPS (1 µg/ml). SI= Selectivity Index (SI= CC50 of macrophages/IC50 of amastigotes) ND = Not determined NI= No induction

4. Discussion

The essential oil yield of P. marginatum was consistent with the information present in the literature. According to Andrade et al. (2008), the species P. marginatum can have essential oil yield ranging from 0.3% to 2% depending on the geographical location and the climatic conditions of the regions where the species is found. The phytochemical studies of P. marginatum leaf extracts were performed applying different extraction techniques (ARAÚJO et al., 2014; DUARTE et al., 2004; REIGADA et al., 2007) which make the comparison of our data with the information present in the literature difficult. The P. arboreum ethanolic extract obtained by the Soxhlet method had a yield of only 6.9% (FIGUEREDO et al., 2014). Differently, the P. marginatum yield in this study was 21.4% which is higher than that found for P. arboreum. This variability can be

influenced by environmental factors such as seasonality, temperature, water availability, soil nutrients and factors that stimulate or inhibit the production of the plants’ secondary metabolites (GOBBO-NETO and LOPES, 2007). In the Brazilian Amazon, the most common P. marginatum chemotype is the phenylpropanoid 3,4-Methylenedioxypropiophenone with concentrations ranging from 7.3 to 40.7% (ANDRADE et al., 2008) which confirms the results found in this study. Dill (2009) described that the substances found in the essential oils of Brazilian Piperaceae species vary according to the geographic region where the plant is collected. This means that the more tropical the climate of the region is, the higher is the incidence of sesquiterpenes and phenylpropanoids in the EOs composition. The hotter and drier the region is, the higher is the monoterpene content. Piper species are widely used in folk medicine to heal wounds, reduce swelling and skin irritations. Chemical studies of Piper species have already led to the isolation of active antiparasitic substances such as chalcones (Braga et al., 2007). The crude MeOH extract from leaves of P. marginatum was selected for bioactivity-guided phytochemical investigation, due to its potent activity observed against Cladosporium cladosporioides and Cladosporium sphaerospermum. Thus, some bioactive compounds were isolated, among them 3,4-methylenedioxypropiophenone, which presented higher antifungal activity (REIGADA et al., 2007). This data points out the importance of testing compounds produced by this species to evaluate their possible use in the treatment of leishmaniasis. Gilbert and Favoreto (2005) reported that substances such as alkaloids and chalcones are active against the parasite. Differently, phenolic compounds, terpenes and polysaccharides substances showed potential on the macrophage, the parasite host cell, stimulating lethal immunological response factors on Leishmania without damaging the macrophage. When comparing the tests with crude extracts of species from the genus Piper against L. amazonesis, the work of Nakamura et al. (2006) has a special approach. In this study, the authors observed that the hydroalcoholic extract of P. regnellii var. pallescens showed IC50 of 167 µg/mL whereas the ethyl acetate extract showed IC50 of 30 µg/mL on promastigote forms growth after 48h of culture. These IC50 values can be considered active and moderately active, accordingly. The results obtained in this study for all the samples of P. marginatum leaves were highly active since the IC50 found for the promastigote forms ranged from 0.9 to 7.9 µg/mL. Moreira et al. (2010) also tested the methanolic extract and the hexane, dichloromethane, ethyl acetate and n-butanolic fractions from P. cabralanum C.DC. leaves

against L. amazonensis promastigotes. However, only the methanolic extract and the hexane fraction were active against L. amazonensis with IC50 of 30 µg/mL and 25 µg/mL, respectively. A study performed by Torres-Santos et al. (1999) with dichloromethane extract of P. aduncum inflorescences against the promastigote forms of L. amazonensis showed that the dichloromethane extract significantly reduced the viability of the promastigote forms with an IC50 of 2.2 µg/mL. The bioguided fractionation allowed the purification of the substance 2',6'-dihydroxy-4'-methoxychalcone. Tests with this compound exhibited potential on the promastigote forms with IC50 of 0.5 µg/mL which suggest that it is the active principle present in the extract. Comparing this data with the results found for MF and HF obtained from fractionation of the CE (IC50 of 0.9 µg/mL), we can suggest that these fractions were as active as the substance isolated by Torres-Santos et al. (1999). Oliveira et al. (2013) emphasized that phytotherapics administered as plant extracts have some advantages over ‘active’ isolated substances. This includes their synergistic effect due to the presence of several substances which are often chemically and pharmacologically distinct, surpassing the biological activity of an isolated active principle. In relation to the low toxicity, active substances are present in smaller concentrations in plants, so the risks of adverse effects are also lower. The traditional use of several species as medicinal teas over centuries confirms this fact. However, some plants may cause toxic effects especially at doses higher than recommended. The main target of any chemotherapy for leishmaniasis is the intracellular amastigote form (CROFT; COOMBS, 2003). Torres-Santos et al. (1999) observed that the substance 2',6'-dihydroxy-4'-methoxychalcone isolated from P. aduncum inflorescences showed IC50 of 24 µg/mL against the intracellular amastigote form of L. amazonensis. One of the microbicidal mechanisms used by the macrophage is the production of nitric oxide, but there was no significant production of it. This means that the antileishmanial effect is caused by the direct action of the compound on the parasite or by another microbicidal mechanism of the macrophage without the activation of the nitric oxide synthase. The IC50 values of EO, CE, HF, DF, EAF and MF against the amastigote forms ranged from 0.58 to 81.5 µg/mL with emphasis to EO with IC50 of 0.58 µg/mL and CE with 1.2 µg/mL. The IC50 values of the fractions obtained from CE were considered less active in comparison to those found for EO and CE. The reduction in the biological activity of the fractions in comparison to the extract may be related to the probable loss of the synergic action of the compounds as previously demonstrated for other vegetal species against other protozoans. This proves that the interaction between two or more substances in an

extract may have a more potent biological effect than only using an individual compound (RASOANAIVO et al., 2011) An important criterion in the prospection of new compounds with antileishmanial action is that they are not toxic to mammalian cells (BHARGAVA and SINGH, 2012). Moura Carmo et al. (2012) when analyzing the cytotoxicity of P. duckei and P. demeraranum leaf essential oils against peritoneal macrophages found that both EOs were less toxic than Pentamidine. Nakamura et al. (2006) described that P. regnellii var. pallescens leaf hydroalcoholic extract and its fractions had low toxicity on macrophages. Monzote et al. (2010) tested the essential oil of P. auritum aerial parts, and the data indicated CC50 of 106.4 ± 3.4 µg/mL of peritoneal macrophages while amphotericin B had a value of 5.8 ± 0.5 µg/mL. The experiments of cytotoxicity against peritoneal macrophages showed that EO, CE and their fractions of P. marginatum had low toxicity with CC50 values varying from 27.5 to 499 µg/mL. The cytotoxicity results for P. marginatum are been reported for the first time. The analysis of the selectivity index (SI) showed that all the samples were more selective for the parasite than for the mammalian cells. According to Weniger et al. (2001), a SI ≥ 10 suggests high safety in the use of the tested product in mammalian cells because it indicates that the biological efficacy is not due to its cytotoxicity in these cells. Based on the results presented before, the sample CE can be considered as promising since it had a SI of 415.8 for L. amazonensis. When comparing the SI of the CE to the reference drug Pentamidine, the first one was found to be 2-fold more selective than Pentamidine. This study describes for the first time the antileishmanial activity of P. marginatum on L. amazonensis.

5. Conclusion

P. marginatum is a species that grows abundantly in all Brazilian regions, including the Amazon area. This plant can be an alternative source of natural products for the treatment of leishmaniasis, especially in the North of Brazil where this neglected disease is considered endemic and the population’s access to medical care is limited. P. margintatum essential oil, ethanolic extract and fractions showed the presence of phenolic compounds and terpenoids, having 3,4-methylenedioxypropiophenone as a major compound. The essential oil, ethanolic extract and fractions of P. marginatum had antipromastigote and anti-amastigote activity against Leishmania amazonensis with low

toxicity for macrophages. The ethanolic extract had the best selectivity index in comparison to the reference drug Pentamidine. This research is part of a continuous search for natural products with high activity and few side effects against neglected diseases such as leishmaniasis. P. marginatum can be considered an interesting candidate for further tests as a prototype drug for the treatment of leishmaniasis.

Acknowledgements

This research had the financial support of FAPESPA (Fundação de Amparo à Pesquisa do Estado do Pará).

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