Background: Leishmaniasis is a potentially fatal, neglected parasitic disease caused by different species of Leishmania sp. Natural products, especially from plants; represent a rich source for the screening of potential antiparasitic compounds.

Purpose and study design: In this study, we evaluated the leishmanicidal activity of thirteen plant extracts against the parasite Leishmania (Leishmania) amazonensis in vitro, the cytotoxic and hemolytic activity. The extracts with activity against the parasite, was determined the chemical constituents.

Results: The hexane extracts of Bidens sulphurea and Plectranthus neochilus were the most effective extracts against promastigote forms at 24h and 48h. The EC₅₀ (50% effective concentration) value obtained for these extracts against promastigote forms were calculated to be 84.26µg/mL and 46.32µg/mL in 24h, respectively. The EC₅₀ values against intracellular amastigotes were higher than 100µg/mL after 48h of incubation for both extracts. Regarding cytotoxicity in peritoneal macrophages, extracts of B. sulphurea showed CC₅₀ values (cytotoxicity concentration of 50% of cells) of 103.9 and 80.30µg/mL at 24 and 48h, respectively, whereas the CC₅₀ values for the P. neochilus extract were 66.95 and 34.39µg/mL at 24 and 48h, respectively. The extracts showed no significant hemolysis at the concentrations evaluated, and the CH₅₀ values were higher than 100µg/mL. The chemical constituent of the hexane extracts of B. sulphurea and P. neochilus and their activity against L. amazonensis has not been previously described.

Conclusion: Despite the unsatisfactory results against amastigotes forms, this study shows extracts obtained from botanical sources merit further study for their leishmanicidal properties.

Keywords: Leishmania (Leishmania) amazonensis, natural products, hexane plant extracts, leishmanicidal activity

VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; GC, gas chromatography; MS, mass spectrometry; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; EC_50, 50% effective concentration; CC_50, 50% cytotoxic concentration; HC_50, 50% hemolytic concentration; SD, standard deviation; SI, selectivity index

Leishmaniasis, one of the most important neglected tropical diseases, is endemic in 98 countries, with more than 12million cases and 350million people living in areas at risk of infection.^1,2 This disease is caused by an obligate intracellular protozoan of the genus Leishmania,³ and is broadly classified into three different forms: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis.⁴ In Latin America, Leishmania (Leishmania) amazonensis is responsible for the cutaneous diffuse form of the disease,⁵ that in some cases may also result in visceral leishmaniasis.^6,7 According to the Brazilian Ministry of Health, since 2005, the presence of L.(L.) amazonensis has been present in almost all Brazilian regions ⁵, thus raising concern about this infection. The first-line drugs for leishmaniasis treatment are sodium stibogluconate (Pentostan) and meglumine antimonite (Glucantime); amphotericin B and pentamidine are second-line drugs.² However, the current standard-of-care is unsatisfactory due to are expensive, potentially toxic and long-term treatment requirements, resulting in patient non-compliance.² Also, there are significant differences in the sensitivity of these species to standard drugs.^8,9

In the last decade, the scientific investigation of medicinal plants has received considerable attention in drug development against protozoan diseases.^10–12 In this context, the evaluation of plant-derived extracts and isolated natural compounds can result in potential leads for use against infectious diseases. Recently, it was demonstrated that the hexane extracts derived from plants of Asteraceae, Lamiaceae, Myrtaceae, and Verbenaceae families showed promising activity against cariogenic bacteria.¹³ As part of our ongoing interest in the antiparasitic activity of natural products and their derivatives, we evaluate here the leishmanicidal potential of thirteen selected plant-derived hexane extracts from the leaves of herbaceous or arbustive plant species (Table 1) against the parasite L. (L.) amazonensis.

Plant material and extraction

Specimens of thirteen species (Table 1) were collected in May 2010 at ¨Sítio 13 de Maio¨ (20°26’S 47°27’W 977m), localized near the city of Franca, State of São Paulo, Brazil and identified by Prof. Dr. Milton Groppo. Voucher specimens were deposited at the Herbarium of Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil. Leaves of each species were dried carefully in a circulating air oven (Quimis-Diadema, BR) at 40°C and ground in a knife mill (Tecnal - Piracicaba, BR).

The powdered leaves were extracted with hexane, as previously reported.¹³ Three extractions per species were retained, with each extraction lasting 15min. The samples were concentrated using a rotary evaporator under reduced pressure to provide the respective hexane extracts.

Gas Chromatography (GC) and gas chromatography mass spectrometry (GC-MS) analyses

Gas chromatography‒mass spectrometry (GC‒MS) analyses was carried out as previously reported.¹³ The chemical components of the hexane extract of Bidens sulphurea were identified on the basis of their retention indices relative to a homologous series of n-alkanes (C₈–C₄₀)¹⁴ on a Rtx-5MS capillary column under the same operating conditions and computer matching with the Wiley 7, NIST 08 and FFNSC 1.2 spectral libraries of the GC-MS system.

Animals

Male BALB/c mice were maintained under controlled conditions of temperature (22±20^oC), humidity (50±10%), and light–dark cycle. All the experiments were authorized by the University of Franca´s Ethics Committee for Animal Care (Approval number: 046/15). All animals were handled using good animal practice as defined by the University of Franca in concordance with Brazilian legislation.

Parasites

L.(L.) amazonensis (MHOM/BR/PH8) was routinely in M199 medium (Gibco, New York, USA) supplemented with heat-inactivated 10% fetal bovine serum (FBS), penicillin (10000 UI/mL) and streptomycin (10mg/mL) (Cultilab, Campinas, BR) at 25°C.

Anti-promastigote assay

A preliminary screening with promastigote forms was performed in the presence of 100mg/mL previously dissolved in dimethyl sulfoxide (DMSO) (Synth, Diadema, BR). The inhibition of cell growth was determined by counting cells with a haemocytometer (Global Glass, Porto Alegre, BR) after 24 and 48h of incubation at 24°C.¹⁵

The plant extracts that showed greater than or equal to 50% inhibition of cell growth during 48h were further evaluated at concentrations of 0.78 to 100µg/mL. Parasites incubated with amphotericin B (Eurofarma –São Paulo, BR) was used as a positive control and M199 medium (Sigma Aldrich – St Louis, EUA) with 0.1% DMSO served as negative control. The 50% effective concentration (EC₅₀) was calculated as described below. All tests were conducted in triplicate, and three independent assays were performed.

Anti-amastigote assay

To evaluate activity against intracellular amastigote forms, peritoneal macrophages cells were seeded (2×10⁵ cells/mL) into 24-wells plates containing glass coverslips (13mm). Non-adherent cells were removed, and the cells were infected with promastigote forms at a ratio 1:10 (macrophage/promastigote). Infected macrophages were incubated with the B. sulphurea, P. neochilus (6.25-100 and 3.12-50μg/mL) and amphotericin B (0.18-3μg/mL) for 48h at the same conditions described above. Parasites incubated in RPMI 1640 medium (Sigma Aldrich) with 0.1% DMSO served as the negative control. The number of amastigotes was determined by randomly counting 200 cells. The results were calculated using the negative control (0.1% DMSO) as representative of 100% cell survival. The 50% inhibitory concentration (EC₅₀) was calculated as described below. All tests were conducted in triplicate, and three independent assays were performed.

Cytotoxicity against peritoneal macrophages

The obtation of peritoneal macrophages as performed using previously reported protocol with slight modification.¹⁶ The macrophages (2x10⁵ cells) were incubated in the presence of a concentration range (0.78-100µg/mL) of Bidens sulphurea, Plectranthus neochilus and amphotericin B (0.002-1.56µg/mL) (Eurofarma) for 24 and 48 h. DMSO was used as a positive control (25%) and negative control was RPMI 1640 medium with 0.1% of DMSO. Cell viability was assessed by Trypan Blue exclusion (Inlab –Diadema, BR).¹⁷ The results were expressed as the mean percentage reduction of macrophage viability compared to that in the untreated control wells, and the 50% cytotoxic concentration (CC₅₀) was calculated as described below. All tests were conducted in triplicate, and three independent assays were performed.

Red blood cell lysis assay

The toxicity to red blood cells was determined as previously described with some modifications.¹⁸ Briefly, erythrocytes were incubated with B. sulphurea, P. neochilus and amphotericin B at room temperature for 30 min and hemolysis was determined by the hemoglobin release, quantitated by the absorbance of the supernatants at 415nm. The percentage of lysis was calculated in relation to total lysis. The negative control was erythrocytes with NaCl solution 0.9%, while the positive control used erythrocytes with water. The 50% hemolytic concentration (HC₅₀) was calculated as described below. All tests were conducted in triplicate, and three independent assays were performed.

Statistical analysis

Data represent the mean number (±SD) of three independent experiments performed in triplicate. The results were compared by analysis of variance, one-way ANOVA, followed by Dunnett's test to determine significance between the negative control group and treated groups. The EC₅₀, CC₅₀ and HC₅₀ were calculated using dose–response curves using GraphPad Prism 5 (GraphPad Software, San Diego, California, USA). The SI was calculated using the ratio of CC₅₀/EC₅₀.¹⁹

Several extracts and compounds isolated from plants have been investigated for their biological properties, including their leishmanicidal activit.^20,21 In the present study, thirteen hexane extracts from leaves of cultivable herbaceous or arbustive plant species (Table 1) were evaluated against L. (L.) amazonensis. The hexane extracts of these species were selected on the basis of previous reports in the literature or on their use as antimicrobial and antiparasitic activities in folk medicine.¹³

A preliminary screening of hexane extracts was performed at 100μg/mL against promastigote forms of L. (L.) amazonensis to select the most active extracts at higher concentrations. Five hexane extracts (Artemisia camphorata, Arrabidaea chica, Eclipta alba, Foeniculum vulgare, Lippia alba) showed no activity after 24h and activity lower than 25% in 48 h of treatment. Six extracts (Alternanthera brasiliana, Coreopsis lanceolata, Pelargonium graveolens, Stachytarpheta cayennensis, Senna occidentalis and Tagetes erecta) showed a percentage of inhibition of cell growth of less than 50% at 24 and 48h (Table 2). On the other hand, the hexane extracts of Bidens sulphurea and Plectranthus neochilus were the most effective extracts against promastigote forms of L. (L.) amazonensis at 24h and 48h; they showed a percentage of inhibition of cell growth higher than 90% after 48h (Table 2).

Another study demonstrated that ethanolic extracts from Artemisia kulbadica, Artemisia cininformes and Artemisia santolina had an EC₅₀ of 25.25 and 80μg/mL, respectively, against promastigotes forms of L. (L.) major after 24h of incubation.²² However, no study has demonstrated the effect of A. camphorata extracts against L. (L.) amazonensis. As described previously in the literature, the hexane extract of A. chica showed EC₅₀ values of 31.8µg/mL and 14.7µg/mL against L.(L). amazonensis e L. (L.) infantum at 120h, respectively.²³

In another study, the aqueous and ethanolic extract of E. alba inhibited 100% growth of L. (L.) donovani promastigotes at concentration of 0.5mg/mL.²⁴ Besides, study demonstrated that essential oils obtained from the L. alba species collected at different locations in Colombia showed different leishmanicidal activities against L.(L.) chagasi promastigotes, which suggest that different location may show changes in the chemical composition of the plant.²⁵ Maquiaveli and co-workers also reported that butanol fraction of the aqueous extract of S. cayennensis showed EC₅₀ values of 51.0 (72h) and 32.0 (24h) mg/mL against promastigote and amastigote forms, respectively.²⁶

To determine the EC₅₀ values of the hexane extracts of P. neochilus and B. sulphurea, promastigote forms of L. (L.) amazonensis were incubated with the hexane extracts for 24 and 48h. The activity of extracts has been classified as follows in the literature: highly active (EC₅₀ value <10µg/mL); active, (10<EC₅₀ <50µg/mL), moderately active (50 < EC₅₀ < 100µg/mL) and non-active (EC₅₀ >100 µg/mL).²⁷ Our results revealed that in 24 h the hexane extract of B. sulphurea showed an EC₅₀ value of 84.26 µg/mL (95% Confidence Interval (95% CI) 81.23-87.56µg/mL) (moderately active), while hexane extract of P. neochilus showed a value of 46.32µg/mL (95% CI-38.42-57.54µg/mL), considerate as moderate activity. In 48h, both extracts were considerate active, with EC₅₀ values of 40.37 (95% CI-29.64-55.64µg/mL) and 43.20µg/mL (95% CI-39.57-50.87µg/mL) for hexane extracts of B. sulphurea and P. neochilus, respectively. Amphotericin B showed an EC₅₀ value of 0.011µg/mL at 24 (95% CI-0.0058-0.019µg/mL) and 0.012µg/mL 48h (95% CI-0.0063-0.022µg/mL) (Table 3).

According to Tempone and co-workers, the methanol extracts of Aristolochia cymbifera, Plectranthus amboinicus, Plectranthus barbatus and Lippia alba showed EC₅₀ values of 45.14; 89.17; 54.46 and 62.67 μg/mL, respectively against L. (L.) chagasi at 48h.²⁸ In addition, the methanol extract of P. neochilus was inactive against Leishmania species.²⁸ Another study, Antinarelli and co-workers demonstrated that the methanolic extract of P. neochilus showed active against L. (L.) chagasi, but it did not show activity against L.(L.) amazonensis, L.(L.) major and Leishmania (Viannia) braziliensi.²⁰ Despite these results, it is interesting to notice that extracts methanolic and/or hexanic from the genus Plectranthus has showed values of EC₅₀ considered active or moderately active, because of that this genus should be better investigated about its antiparasitic activity.

Although promastigotes can be used for fast screenings of potential compounds, the clinically relevant form of the parasite is the amastigote form, which shows metabolic differences from the extracellular forms.^29,30 When the hexane extracts were evaluated against intracellular amastigotes, it was observed that after 48h of incubation with B. sulphurea the EC₅₀ value were 371µg/mL (95% CI-254-487µg/mL) and when incubated with P. neochilus the EC₅₀ were 141µg/mL (95% CI-90.09-192.4µg/mL), demonstrating that the hexane extracts have no activity against these parasitic forms. The EC₅₀ obtained after incubation with amphotericin B were 0.095 µg/mL (95% CI-0.07-0.12µg/mL) (Table 3).

An important criterion in the research of active compounds and extracts is to determine the absence of toxic effects on the host cells. In this study, the toxicity of hexane extracts of B. sulphurea and P. neochilus was evaluated on peritoneal macrophage. The hexane extract of B. sulphurea showed CC₅₀ value (50% cytotoxic concentration) of 103.9µg/mL and 80.30µg/mL (95% CI-99.46-128.98µg/mL and 73.15-88.15µg/mL, respectively) after 24 and 48h of incubation. Moreover, the hexane extract of P. neochilus showed CC₅₀ value of 66.95µg/mL and 34.39µg/mL (95% CI-59.55-75.27µg/mL and 28.21-41.93) at 24 and 48h, respectively. Amphotericin B was more toxic to mammalian cell than hexane extracts, showing CC₅₀ values of 4.29 and 2.98µg/mL (95% CI-3.25-6.77µg/mL and 1.41-3.87) in 24 and 48h, respectively (Table 4). However, the methanolic extract of P. neochilus presented a CC₅₀ value of 111μg/mL when incubated with peritoneal macrophages after 72hours of incubation.²⁰

According to one study, a selectivity index (SI) value greater than 10 can suggest better safety of the product for use in mammals.³¹ The hexane extract of B. sulphurea showed a SI of 1.23 and 1.98 in 24h and 48h, respectively. In addition, the hexane extract of P. neochilus showed a SI of 1.44 and 0.79 in 24h and 48h, respectively, Despite the low SI, the hexane extracts showed values close to those obtained by amphotericin B, with a SI of 390 and 248.3 in 24h and 48 h, respectively (Table 4).

Recently, the chemical composition of the hexane extract of P. neochilus was determined by gas chromatography–mass spectrometry (GC-MS). A total of thirteen compounds were detected, with predominance of sesquiterpenes (88.8%). The major constituents were identified as being spathulenol (46.1%), trans-caryophyllene (19.0 %), caryophyllene oxide (10.7%) and germacrene D (7.8%).¹³ According toAcebey and co-workers, the sesquiterpene spathulenol, isolated from an ethyl acetate extract of the bark of Hedyosmum angustifolium, did not show activity against L. (L.) amazonensis and L. (L.) infantum in vitro.³² This could indicate that some other compounds may be responsible for the activity of the extract. The other major constituents were already described in extracts or essential oils, but their isolated activity was not been described.^33,34 Thus, the activity of the isolates should be better investigated against parasites from genus Leishmania sp.

In our study, a total of fifteen compounds were detected on the B. sulphurea hexane extract and the major constituents were identified as being 2,4-bis(dimethylbenzyl)phenol (54.1%), (3-tert-butyl-5-hydroxymethyl-cyclohex-2-enyl)-methanol (8.1%), pulegol (7.3%) and (2-Dodecen-1-yl-succinic anhydride (7.2%) (Table 5). This is the first study describing the effects of the major constituents of B. sulphurea extract on protozoa.

The hexane extracts were evaluated in vitro in relation to the protozoan L. (L.) amazonensis and the results demonstrate a moderate leishmanicidal activity after 24 and 48 h of incubation. Despite the unsatisfactory results against amastigotes forms, this study shows extracts obtained from botanical sources merit further study for their leishmanicidal properties.

The authors are grateful to the National Council for Scientific and Technological Development, Brazil–CNPq and Centro Técnico Agropecuário (Centagro) for fellowships, and to the São Paulo Research Foundation, Brazil-FAPESP for financial support (Grant numbers 2013/11164-4). We also thank Jason Kim for the grammatical correction.

The authors have declared that there are no conflicts of interest.

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