Antileishmanial and cytotoxic activity of dillapiole n-butyl ether

ABSTRACT

Among the neglected diseases, American cutaneous leishmaniasis (ACL) still remains highly endemic in some tropical regions. The currently available drugs for treatment are highly toxic, prompting the search for new therapeutic options. The aim of this study was to evaluate the toxic potential of dillapiole n-butyl ether (DBE) against Leishmania amazonensis and L. guyanensis, as well as its toxicity on human peripheral blood mononuclear cells (PBMCs) in vitro. For cell cytotoxicity, concentrations of DBE that ranged from 7.8 to 500 µM were used for 48 and 72 h. For the evaluation of the antileishmanial activity, DBE was tested at concentrations of 0.28 to 18 µM for 24, 48, and 72 h. A value of 36 µM was used for the amastigote assay. The selectivity index (SI) was determined by dividing the CC₅₀/IC₅₀ (macrophages/promastigotes). DBE exhibited a CC₅₀ of 203.9 ± 0.5 µM in 72 h. DBE inhibited promastigote forms with an IC₅₀ of 3.0 µM for both Leishmania species for 72 h. The standard, Pentacarinat®, showed an IC₅₀ of 2.9 µM and 0.3 µM, respectively. The SI of DBE for both species was 67.9 for 72 h. DBE inhibited intracellular forms of L. amazonensis by 65.5% after 48 h. In molecular modeling, DLpOl-F showed two hydrogen bonds (SER418 and 421). DBE demonstrated promising in vitro antileishmanial potential.

KEYWORDS:
Leishmania; dillapiole analog; semi-synthesis; molecular modeling

RESUMO

Dentre as doenças negligenciadas, a leishmaniose cutânea americana (LCA) continua sendo altamente endêmica em algumas regiões tropicais. Os medicamentos disponíveis atualmente para o tratamento apresentam elevada toxicidade, o que torna a busca de novas opções terapêuticas necessária. O objetivo do estudo foi avaliar o potencial tóxico do éter n-butil dilapiol (EBD) contra Leishmania amazonensis e L. guyanensis e a sua toxicidade em células mononucleares do sangue periférico humano (CMSP) in vitro. Para a citotoxicidade celular, utilizou-se concentrações de 7,8 a 500 µM por 48 e 72 h. A atividade antileishmania do EBD foi testada em concentrações de 0,28 a 18 µM, por 24, 48 e 72 h. Para o ensaio em amastigotas usou-se um valor de 36 µM. O índice de seletividade (IS) foi determinado pela divisão CC₅₀/CI₅₀ (macrófagos/promastigotas). O EBD mostrou em células uma CC₅₀ de 203,9 ± 0,5 µM em 72 h. O EBD inibiu as formas promastigotas com uma CI₅₀ de 3,0 µM para ambas espécies de Leishmania por 72 h, já o padrão Pentacarinat® apresentou uma CI₅₀ de 2,9 µM e 0,3 µM, respectivamente. O IS do EBD para as duas espécies foi de 67,9 por 72 h. O EBD inibiu as formas intracelulares de L. amazonensis com 65,5% por 48 h. Na modelagem molecular, o DLpOl-F aparece com duas ligações de hidrogênio (SER418 e 421). O EBD demostrou um potencial antileishmania promissor in vitro.

PALAVRAS-CHAVE:
Leishmania; análogo do dilapiol; semissíntese; modelagem molecular

INTRODUCTION

Leishmaniases are anthropozoonoses caused by the protozoa of the genus Leishmania, which are transmitted through the blood supply of female sandflies (BRASIL 2017). They are considered a neglected public health problem since the pharmaceutical industry has little interest in investing in new drugs for the treatment of these diseases (WHO 2020). These diseases are divided into cutaneous, mucocutaneous and visceral leishmaniasis. The American cutaneous leishmaniasis (ACL) is distributed in 88 countries on four continents (the Americas, Europe, Africa and Asia), and about 1,5 million cases occur each year (WHO 2019). In 2019, more than 41,000 new cases of cutaneous and mucosal leishmaniasis were reported in the Americas, with Brazil having the highest concentration of cases at around 37% of the total (OPAS/OMS 2020).

The current treatment of this disease is based on N-methylglucamine antimonate (Glucantime®), and other drugs, such as pentamidine isethionate, miltefosine, amphotericin B and liposomal amphotericin B, constitute therapeutic options for cases of low efficiency of antimonate treatments (Carvalho et al. 2019). These existing drugs have serious drawbacks in terms of safety, resistance, stability and cost, as well as low tolerability, long duration of treatment and difficulty to administer (DNDi 2018). Therefore, new, more effective and more affordable alternatives are being sought for the treatment of leishmaniasis. One approach is to survey the chemical diversity of molecules found in natural sources and employ new technologies, such as the semi-synthesis technique, to discover new drugs (De Oliveira et al. 2012).

Piper aduncum L. (Piperaceae) is popularly known as matico and long pepper (Lorenzi and Matos 2008). It is widespread in South and Central America and grows naturally in the Amazon and the Atlantic Forest of Brazil (Santos et al. 2013). The essential oil extracted from its leaves is commonly characterized by a high concentration of phenylpropanoids such as dillapiole, which is its main component (De Almeida et al. 2009). This compound shows a number of biological activities, such as insecticidal (De Almeida et al. 2009; Volpe et al. 2016), larvicidal (Oliveira et al. 2013), antiparasitic (Gaínza et al. 2016), antifungal (De Almeida et al. 2009; Ferreira et al. 2016), and antileishmanial (Dal Picolo et al. 2014), and can be used as a natural chemical base to obtain various new molecules (Katz and Baltz 2016). Farah et al. (2010) demonstrated the antileishmanial activity of dillapiole and its isodillapiole analogue against Leishmania chagasi Chagas and Cunha 1937. Parise-Filho et al. (2012) demonstrated the antileishmanial activity of dillapiole and its analogs di-hydrodillapiole and isodillapiole against promastigote forms of Leishmania brasiliensisVianna 1911 and Leishmania (L.) amazonensisLainson and Shaw 1972. The analogs isodillapiole, dillapiole propyl ether and dillapiole isopropyl ether inhibited the promastigote forms of L. amazonensis and presented a non-toxic profile in J774 macrophages (Barros et al. 2021).

The design of antiparasitic drugs is based, especially, on the investigation of the biochemical pathways of the parasite and, when appropriate, as a leishmanolysin, or GP63, which is an important protease of the surface of Leishmania that is capable of hydrolyzing a wide variety of substrates both in the parasite and in the host (Shao et al. 2019). These are products that are involved in the adhesion and internalization of these parasites in host macrophages and have been related to the resistance of the parasite to lysis by the complement system and increased virulence of Leishmania (Rhaiem and Houimel 2016). In this context, molecular docking studies are predictive approaches for designing new drugs against parasite-specific targets (Reguera et al. 2014).

Among 15 dillapiole derivatives extracted from P. aduncum and evaluated for in vitro antileishmanial activity, dillapiole n-butyl ether (DBE) showed better results at the 50% inhibitory concentration (IC₅₀) (Barros et al. 2021). With the aim of further advancing the knowledge on the biological activity of DBE, the aim of this study was to evaluate the in vitro antileishmanial and cytotoxic activity of DBE obtained through semi-synthesis of dillapiole extracted from P. aduncum.

MATERIAL AND METHODS

Acquisition of plant material and DBE synthesis

Leaves of P. aduncum L. were taken from plants cultivated at Embrapa Amazônia Ocidental (Manaus, Amazonas state, Brazil) (2°53’36.2”S, 59°58’22.8”W) from plants identified in the herbarium of Instituto Nacional de Pesquisas da Amazônia (INPA) (voucher exsiccate # 10.480/2013, collector: E.S.Silva). The essential oil was extracted in the Laboratory of Medicinal Plants and Phytochemistry at INPA using the steam drag technique and dillapiole was isolated from the essential oil using a vacuum fractional distillation system according to the protocol of Pohlit et al. (2008).

Dillapiole was identified via (a) ¹H and ¹³C nuclear magnetic resonance (NMR) spectral data (Varian, Model INOVA 500 spectrometer) with internal standard tetramethylsilane (TMS) and the solvents used were chloroform (CDCl₃) deuterated, and (b) by means of mass spectrometry (gas chromatograph coupled to a mass spectrometer from Shimadzu, CGMS-QP2010, model AOC-20) with automatic injector at the Centro de Biotecnologia da Amazônia (CBA). (Supplementary Material, Figures S1-S4). DBE was prepared using semi-synthesis of 5.5 g of dillapiole obtained by an oxymercuration reaction (Pinto 2008), which was added to a 100 mL volumetric flask with tetrahydrofuran (5.0 mL, Moderna), suspension of mercury acetate (1.5 g, Fluka) and n-butanol (5.0 mL, Synth) and then homogenized in an ice bath for 72 h with stirring until the reaction occurred. The reaction product was analyzed using thin layer chromatography (TLC) plates using 20 x 20 cm size aluminum backed normal phase silica gel chromatoplates with MERCK fluorescence indicator F254, mobile phase hexane: ethyl acetate (9: 1) and alcoholic anisaldehyde solution developer. After obtaining the analog, a solution of sodium borohydride (2.0 g, Fluka), potassium hydroxide (7.0 g, Moderna) and n-butanol (5.0 mL) was added and the mixture was shaken. The solution was then filtered and washed according to the protocol of Tomar et al. (1979a, b). The DBE was purified in a silica gel chromatographic column 60 (70 - 230 mesh, Merck) and identified using spectroscopic methods (¹H and ¹³C NMR, Varian, Model INOVA 500 spectrometer) with internal standard tetramethylsilane (TMS) and the solvents used were chloroform (CDCl₃) deuterated (Supplementary Material, Figure S5-S7). The density of the DBE was determined by the average weight of the oil mass divided by its volume in triplicate.

Cell acquisition and cultivation

For the assay with human peripheral blood mononuclear cells (PBMC), 10 mL of peripheral blood were collected from two healthy volunteers. Each sample was processed individually, diluted in a conical tube (50 mL) with PBS 1X (1:1) and homogenized according to the protocol of Junior (2013). The project was approved by the human ethics committee of Universidade Federal do Amazonas (Amazonas, Brazil) (CEP/ UFAM, CAAE # 29406319.2.0000.5020). The J774 lineage macrophages were provided by Dr. Paulo Afonso Nogueira (Fiocruz Manaus). Cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum (FBS), 50 U mL^-1 penicillin and 50 µg mL^-1 streptomycin (Invitrogen).

Cell cytotoxicity tests

The macrophages were placed in 30 ml at a concentration of 10⁶ cells ml^-1 which were pre-cultured in 96-well plates containing RPMI 1640 medium supplemented with 10% SFBi and incubated at 37 °C with 5% CO₂. After 24 h, the cells adhered to the base of the wells and were treated with DBE for 48 and 72 h in triplicate (three plates) for each time. DBE diluted in 0.2% v/v dimethylsulfoxide (DMSO, Vetec®) was also used, standard positive controls using Pentacarinat® and Glucantime®, and a negative control containing 0.2% DMSO with cells (all controls in triplicate). The bioassay was incubated following Jaffe et al. (1984).

For the cytotoxicity test in J774 cells, macrophages were placed in 24 mL of SFBi at a concentration of 5 x 10⁶ cells mL^-1 adjusted in a Neubauer chamber and subjected to DBE concentrations from 7.8 to 500 µM for 48 hours in triplicate, Pentacarinat® Glucantime® and 0.2% DMSO were used as controls. To obtain the 50% cytotoxic concentration (CC₅₀) in cells, the colorimetric dose-response test (Alamar Blue®) was used according to Nakayama et al. (1997) with spectrophotometer reading at 590 nm.

Acquisition and maintenance of promastigotes

Leishmania (L.) amazonensis (MHOM/BR/2009/IM5584) and Leishmania (V.) guyanensisFloch 1954 (MHO/BR/75/M4147) were cryopreserved at the Leishmaniasis and Chagas Disease Laboratory at INPA. Subsequently, the samples were cultivated in RPMI 1640 medium supplemented with 10% of inactivated FBS and placed in an oven at 25 °C.

In vitro promastigote assay

The antileishmanial activity of DBE was evaluated via the inhibition of growth and the mortality of promastigote forms of L. (L.) amazonensis and Leishmania (V.) guyanensis during 24, 48 and 72 h, when incubated at 25 °C. For the bioassay, a Schneider culture medium supplemented with 10% inactivated FBS was used. Promastigotes were centrifuged and adjusted to 10⁶ mL^-1 of parasites. The concentrations tested ranged from 18 to 0.2812 µM for both species. The negative control was parasites plus medium and DMSO and the positive controls were Pentacarinat® and Glucantime®. Bioassays were made in triplicates and repeated twice. Parasite viability was quantified using the dose-response colorimetric test according to Nakayama et al. (1997) with spectrophotometer reading.

Amastigote-macrophage assay

After obtaining the PBMCs, they were placed in 100 mL at a cell concentration adjusted to 10⁶ mL^-1 in Schneider culture medium and supplemented with 10% SFBi, then in 24-well plates with a sterile glass coverslips prepared in triplicate (trhee plates) for each Leishmania species. Then, 1 mL of cell solution was added to each well, and the plates remained in an oven at 37 °C with 5% CO₂. After 7 days, the promastigote forms of L. (V.) guyanensis and L. (L.) amazonensis infected the cells in a 5:1 ratio (promastigotes: cells) and were incubated with DBE, Pentacarinat®, Glucantime® and dillapiole in the concentration of 36 µM, and cells plus DMSO (0.2%) for 24, 48 and 72 h (one plate for each time and Leishmania species). On each plate, trhee wells were assigned to DBE, Pentacarinat®, Glucantime®, dillapiole and DMSO, respectively. At the end of the test, the coverslips were stained with a panoptic kit, removed and analyzed under an optial microscope (1.000 x). Infectivity was determined by counting the presence/absence and number of amastigotes in 100 macrophages per coverslip/well in triplicate (three coverslips per treatment).

Cell selectivity index

To determine the selectivity of concentrations with antileishmanial activity against macrophages, the selective index (SI) was obtained by the ratio of the cellular CC₅₀ divided by the 50% inhibitory concentration (IC₅₀) for promastigotes, during 72 h using the following equation:

$\mathrm{SI}=\frac{{\mathrm{CC}}_{50}\mathrm{in\; macrophages}\left(\mathrm{cytotoxicity}\right)}{{\mathrm{IC}}_{50}\mathrm{against}\mathrm{Leishmania}}$

Molecular coupling of dillapiole and DBE

Theoretical computational studies to determine the molecular coupling were performed in the Laboratory of Theoretical and Computational Chemistry at UFAM (LQTC/UFAM), on a computer with 12 Intel^(R) Core^(TM) i5-10400F processing nuclei, 16 GB-RAM and a Linux MINT 20 operating system. The parent compound, dillapiole (DLpOI), and the derivative, dillapiole n-butyl ether (DLpOI-F), were structured in two dimensions (Figure 1) and then adjusted for approximate geometric conformation. Both procedures were performed using MarvinSketch (2017). The structures were optimized as isolated structures (in gas phase), using Gaussian software (Frisch et al. 2013), at DFT level with the B3LYP/3-21G method (optimizations together with vibrational frequency calculations to confirm the optimization quality).

Figure 1
Structure of DlpOl. The green circle marks the carbon where the structural differences occur (the DLpOI-F derivative was explored in S-isomeric form of the labeled carbon). This figure is in color in the electronic version.

After optimization, the structures were subjected to molecular docking (MDk) calculations via AutoDock-Vina (Trott et al. 2010), using an exhaustion value of 32 and seed of 1924167872, considering the sites in Protein Data Bank File (PDBF) structures obtained at the Research Collaboratory for Structural Bioinformatics - PDB (RCSB-PDB) (2021). Gp63 (PDB code: 1LML) (Schlagenhauf et al. 1998) was selected as a protein target for MDk. The PDBF underwent removal of oxygen from the water molecules, followed by the addition of polar hydrogens with the internal parameters of the AutoDock-Tools (Morris et al. 2009). The various centralizations of the boxes (CB) were defined after preliminary MDk of all PDBF, using DLpOl as a binder. In this step, the boxes with maximum dimensions were used to involve the entire protein region, and all positions in which there were couplings were selected as potential sites for further analysis. In the further analyses, the cubic dimensions of 20×20×20 Å (8000 ų) were chosen, since they fully met the sizes of the ligands.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism software (version 6.0) for the in vitro data. By means of a dose-response curve, the number of live cells used to calculate the CC₅₀ (50% cytotoxic concentration) and the viable parasites to calculate the IC₅₀ (inhibitory concentration for 50% of the live parasites) were obtained. The cytotoxicity in PBMC and inhibition of promastigote forms were analyzed with two-way analysis of variance (ANOVA) to compare the antileishmania activity among the experimental treatment (DBE) with standard drugs and dillapiole against different species of Leishmania, followed by Tukey’s post-hoc test for the pairwise comparison of the means of the different treatment groups, with 95% confidence to be considered significant. All variables that participated in the in vitro tests met the requirements for parametric analysis.

RESULTS

Acquisition and characterization of DBE

The dillapiole extracted from the P. aduncum presented NMR spectra with the following chemical shifts: NMR ¹H (CDCl₃; 500 MHz): δ 6.25, s (H-6); 3.31, dd (J= 2.0; 7.0 Hz, H-7); 5.91 m (H-8); 5.04 m (H-9a); 5.06 m (H-9b); 5.88 s (H-10); 3.76, s (OMe); 4.01 s (OMe). NMR ¹³C (CDCl₃; 125 MHz): δ 126 (C), 136 (C), 137.6 (C), 144.6 (C), 114.4 (C), 102.6 (CH), 33.8 (CH₂), 137.4 (CH), 115.5 (CH₂), 101.1 (CH₂), 61.2 (OCH₃), 59.9 (OCH₃) (Supplementary Material, Figure S1, S2 and S3). The dillapiole showed a molecular peak at m/z 222 (Supplementary Material, Figure S4).

The isolated DBE substance had a yield of 4.1 g, representing 73% of the theoretical yields of the reaction. DBE was a light-yellow oil with the molecular formula C₁₆H₂₄O₅, molecular weight of 296.35 g mol^-1 and density of 1.094 ± 0.06 g cm^-3.

The NMR spectra of DBE showed the following chemical shifts: NMR ¹H (CDCl₃; 500 MHz): δ 6.40 s (H-6); 2.83, dd (J= 6.0; 13.5 Hz, H-7a), , 2.53 dd (J= 6.5; 13.5 Hz, H-7b), 3.56 sext (J= 6.0; 13.0 Hz, H-8), 1.12 d (J= 6.0 Hz, H-9), 5.89 s (H-10); 3.48 m (H-11a); 3.38 m (H-11b); 1.51 m (H-12); 1.34 sext (J= 7,5; 15,0 Hz, H-13); 0.89 t (J= 7,5; 14,5 Hz, H-14); 3.77 s (OMe); 4.02 s (OMe). NMR ¹³C (CDCl₃; 125 MHz): 125.5 (C1), 136.1 (C2), 137.7 (C3), 144.9 (C4), 144.5 (C5), 103.9 (C6), 37.2 (C7), 76.2 (C8), 19.9 (C9), 101.3 (C10), 68.7 (C11), 32.4 (C12), 19.6 (C13), 14.1 (C14), 60.1 (OMe), 61.3 (OMe) (Supplementary Material, Figure S5, S6, and S7).

Cell cytotoxicity tests

After 48 h we obtained the following 50% cytotoxic concentrations (CC₅₀) in J774 macrophages: DBE (477.2 ± 0.8 µM), Pentacarinat® (22.8 ± 1.5 µM). For Glucantime® and dillapiole, it was not possible to obtain the CC₅₀ at the concentrations used (Table 1), because dillapiole was not toxic at these concentrations, and because Glucantime® is considered a prodrug. In peripheral blood mononuclear cells, DBE remained with a less toxic CC₅₀ than Pentacarinat® during 48 and 72 h in relation to DMSO 0.2%.

Table 1
The 50% cytotoxic concentrations (CC₅₀) (mean ± standard deviation of three replicates) of dillapiole n-butyl ether (DBE) and Pentacarinat® for human peripheral blood mononuclear cells (PBMC) and J774 macrophages, obtained through serial concentrations of 7.8 to 500 µM after 48 h (J774) and 48 and 72 h (PBMC). The mean CC₅₀ are represented in µM.

Test compound	J774	PBMCs
	48 h	48 h	72 h
DBE	477.2 ± 0.8a	350 ± 0.4a	203.9 ± 0.5a
Pentacarinat®	22.8 ± 1.5b	188.2 ± 1.8b	17.6 ± 0.8b
DMSO 0.2%	> 500	> 500	> 500

• Different superscript letters within the table indicate significant statistical differences according to the Tukey test. DMSO = dimethyl sulfoxide (negative control).

In vitro promastigote assay

In the test of DBE against L. (L.) amazonensis in different concentrations, we observed a reduction in the concentration of viable parasites, with a 50% inhibitory concentration (IC₅₀) of 3.0; 3.2 and 3.0 µM at 24, 48 and 72 h, respectively. IC₅₀ for Pentacarinat® was 3.4; 3.0 and 2.9 µM at 24, 48 and 72 h, respectively. For dillapiole and Glucantime® it was not possible to obtain the IC₅₀ at the concentrations used, because dillapiole did not inhibit 50% of viable parasites at these concentrations. The same occurred for Glucantime® when compared with DMSO 0.2% (Figure 2).

Figure 2
Evaluation of the antileishmanial activity of DBE (A) and dillapiole (B) against the promastigote forms of Leishmania (L.) amazonensis through the quantification of the viable forms of the parasite at 24, 48 and 72 h, Pentacarinat® and Glucantime® (positive controls) in µM and DMSO (0.2%) (negative control). The symbols indicate the mean of three replicates. This figure is in color in the electronic version.

The tests of DBE against L. (V.) guyanensis in different concentrations also showed parasitic inhibition with an IC₅₀ of 12.0; 2.0 and 3.0 µM at 24, 48 and 72 h, respectively. With Pentacarinat®, IC₅₀ was 8.0; 0.3 and 0.3 µM at 24, 48 and 72 h, respectively. For dillapiole and Glucantime®, it was not possible to obtain the IC₅₀ at the concentrations used since they did not inhibit 50% of the viable parasites when compared with DMSO 0.2%. DBE showed significant inhibition in the first three concentrations (p < 0.0001) and Pentacarinat® showed significant inhibition at all concentrations (p < 0.0001) (Figure 3).

Figure 3
Evaluation of the antileishmanial activity of DBE (A) and dillapiole (B) against the promastigotes forms of Leishmania (V.) guyanensis through the quantification of the viable forms of the parasites at 24, 48 and 72 h, Pentacarinat® and Glucantime® (positive controls) in µM and DMSO (0.2%) (negative control). The symbols indicate the mean of three replicates. This figure is in color in the electronic version.

Cell selectivity index

DBE showed less toxicity to PBMC than to L. amazonensis and L. guyanensis parasites, with an SI of 67.9 for both species, suggesting that DBE is less toxic than Pentacarinat® (Table 2).

Table 2
IC₅₀ values of dillapiole n-butyl ether (DBE) and Pentacarinat® from promastigotes of Leishmania spp. and CC₅₀ in human peripheral blood mononuclear cells (PBMC) for 72 h (mean ± standard deviation of three replicates). The means of IC₅₀ and CC₅₀ are represented in µM.

Test compound	IC50		CC50
	Leishmania amazonensis	Leishmania guyanensis	PBMCs
DBE	3.0 ± 1.0	3.0 ± 0.5	203.9 ± 0.5
Pentacarinat®	2.9 ± 3.0	0.3 ± 1.0	17.6 ± 0.8

• CI₅₀ = concentration capable of inhibiting the number of promastigotes in culture media by 50%; CC₅₀ = cytotoxic concentration for 50% of the cell layer; SD = standard deviation; PBMCs = peripheral blood mononuclear cells.

Amastigote-macrophage assay

After 48 h, DBE showed a significantly higher percentage of parasitic death of the amastigote forms of L. guyanensis when compared to Glucantime®, dillapiole, DMSO (0.2%) (p < 0.000.1), and Pentacarinat® (p = 0.0045) (Figure 4a). Against L. amazonensis, DBE caused the death of 65.5% of the intracellular forms, significantly higher than Pentacarinat® (p = 0.0011) and dillapiole (p < 0.0001) and did not differ significantly from Glucantime® (61%) (Figure 4b).

Figure 4
Evaluation of DBE, dillapiole, Pentacarinat®, Glucantime® (36 µM) against amastigotes of Leishmania (V.) guyanensis (A) and L. (L.) amazonensis (B) after 48 h. Columns are mean and bars the standard deviation of three replicates. The asterisks above the columns indicate significant differences according to a post-hoc Tukey test with 95% confidence. Dados considerados significativos **P < 0.01; ****P < 0.0001.

Molecular coupling of dillapiole and DBE

In most cases, the affinity energies in the MDk (E_MDk-Aff) for binding the derivative to GP63 show significant differences when compared to DLpOl. Such results suggest that the applied structural modification would affect the activity in biological organisms that are highly dependent on this target protein.

Considering the E_MDk-Aff values (Figure 5), the best centering for the DLpOl coupling would be at C01, and DLpOl-F would couple better in the C01 and C03 regions. In the centralizations C01, C02 and C03, the E_MDk-Aff value is higher for DLpOl-F, which indicates that this derivative has greater affinity in these positions, and only in C04 does DLpOl have greater affinity. At C01 (Figure 6), DLpOl appears with only one hydrogen bond, while DLpOl-F appears with two hydrogen bonds (one of them involving the oxygen of the derivatization), in addition to having more apolar interactions involving the alkyl groups. This difference in the number of interactions justifies the better E_MDk-Aff value of the derivative. This result also suggests the importance of increasing the chain, since new apolar interactions involving alkyls will be more highly favored. Thus, it is normal that this derivative would have better coupling with respect to DLpOl.

Figure 5
Best couplings of DLpOl-F (blue bars) were to almost all the PDBF-1LML coupling sites (except for C04), demonstrating that the derivation tends to enhance molecule-protein interactions (only M1 modes were considered). This figure is in color in the electronic version.

Figure 6
Depictions of surfaces with tendencies to hydrogen bond and the specific interactions for M1 couplings of DLpOl (A) and DLpOl-F (B) in the C01 region of PDBF-1LML. This figure is in color in the electronic version.

DISCUSSION

The NMR spectra of the DBE showed the chemical shifts as described in the literature and in the study by Tomar et al. (1979a,b). This demonstrated a viable reproductive process.

In this study, we evaluated the cytotoxic effect of DBE in contact with J774 macrophages for 48 h and obtained a CC₅₀ that was less toxic than Pentacarinat®. For Glucantime® and dillapiole, it was not possible to obtain the CC₅₀ at the concentrations used. Farah et al. (2010) demonstrated a viability in macrophages when exposed to dillapiole and its analogue isodillapiole, at a concentration of 50 µg mL^-1 after 72 h. This corroborates our study for 48 h. In PBMC cells, DBE showed a less toxic CC₅₀ than the standard drug (Pentacarinat®) for 48 h. After 72 h, CC₅₀ was 203.9 ± 0.5 µM for DBE and 17.6 ± 0.8 µM for Pentacarinat®, thus showing that DBE remained less toxic when compared to the negative control (DMSO 0.2%). Parise-Filho et al. (2012) observed a CC₅₀ of 22 µM for dillapiole using human 3T3 fibroblasts for 24 h. However, P. aduncum oil showed a high margin of safety of 240 mg kg^-1 and 120 mg kg^-1, with minimal toxic effects on biochemical and hematological parameters in vivo (Sousa et al. 2008). In our study, DBE presented a satisfactory safety margin in PBMCs, thus corroborating Barros (2018), who showed that peritoneal macrophages exposed to DBE exhibited a lower cytotoxic profile in vitro.

DBE showed a reduction in the concentration of viable parasites in promastigotes with an IC₅₀ of 3.0 µM against 2.9 µM for Pentacarinat® after 72 h. It was not possible to obtain the IC₅₀ for dillapiole and Glucantime®. One isodillapiole derivative showed antileishmanial activity at a concentration of 50 µg mL^-1 in promastigotes of L. chagasi for 72 h, which inhibited 96% of viable parasites (Farah et al. 2010). The inhibition of promastigotes and amastigotes of L. amazonensis was achieved with an IC₅₀ of 1.6 µM using a DBE derivative for 72 h (Barros 2018), likewise demonstrating the potential of DBE against L. amazonensis, with an IC₅₀ of 3.0 µM after 72 h, when using a new batch.

DBE showed parasitic reduction of the promastigote forms of L. (V.) guyanensis with an IC₅₀ of 3.0 µM, the same obtained for Pentacarinat® after 72 h. We found no publication using dillapiole analogs from P. aduncum with antileishmanial activity for the inhibition of L. guyanensis.

DBE was shown to be less toxic to PBMCs than to L. amazonensis and L. guyanensis parasites, with SI of 67.9 for both species, showing a less toxic profile than that of Pentacarinat®. In another study, DBE showed a SI of 76.5 and was more toxic to L. amazonensis than to peritoneal macrophages after 48 h (Barros 2018). This corroborates our findings since, for a prototype to be considered promising to become a drug, it must have an SI equal to or greater than 10 (Chiaradia 2007).

At 48 h, DBE showed a significantly higher performance against amastigote forms of L. guyanensis when compared to Glucantime®, dillapiole, and Pentacarinat®, as well as against amastigotes of L. amazonensis compared to Pentacarinat® and dillapiole. DBE inhibited amastigote forms of L. amazonensis by around 88% at a concentration of 4.5 µM when tested in infected peritoneal macrophages for 48 h (Barros 2018), which was corroborated by our results. We found no publications on dillapiole analogues against L. guyanensis, both in the promastigote and amastigote forms, thus our study is the first to describe the antileishmanial activity of DBE in this species, showing that DBE may be a molecule that has broad-spectrum activity for the treatment of cutaneous leishmaniasis.

The most important interactions in the formation of a protein-ligand complex are intermolecular interactions, such as Van der Waals forces, hydrophobic interactions, π-π, ionic or electrostatic interactions, covalent bonds (Guryanov et al. 2016) and, especially, the hydrogen bonds that are very important interactions for the biological system because they are responsible for maintaining the structure of proteins (Barreiro and Fraga 2015). Figure 5 compares the E_MDk-Aff values of DLpOl and DLpOl-F when these couple in four different regions of PDBF-1lML. Although these four regions are in the same protein, it is not appropriate to compare the results of one region with another. The important thing in the figure is that, for the same region (or centralization of the box), the compounds couple with different intensities. The best region for DLpOl coupling would be C01, since this is where this compound couples with more negative energy. DLpOl-F would couple with more negative energy in two regions, C01 and C03, with equal values of E_MDk-Aff. In the centralizations C01, C02 and C03, the E_MDk-Aff is more negative for DLpOl-F, indicating that this derivative has greater affinity in these regions, and only in C04 does DLpOl have greater affinity.

The crystalline form of leishmanolysin (PDBS-1LML), previously called GP63, was obtained from proteins that were purified from promastigote membranes and may be targeted for drugs for human leishmaniasis (Schlagenhauf et al. 1998). Leishmanolysin can act before infection, ensuring that the maximum number of parasites is produced and released from the insect, or shortly after infection, facilitating binding and entry into target cells, in addition to cleaving MHC and macrophage activation factors (McMahon and Alexander 2004). This suggests a possible DBE mechanism of action.

CONCLUSIONS

DBE showed low toxicity in PBMCs and J774 lineage macrophages, and demonstrated an antileishmanial activity similar to the reference drugs against promastigote and amastigote forms of L. amazonensis and L. guyanensis. It also presented a more viable SI than Pentacarinat®. As for molecular coupling, DLpOl-F showed a better tendency to bind to the protein target (PDBS-1LML) when compared to DLpOl. This study demonstrated that DBE has promising antileishmanial activity in vitro, and can be explored as an leishmanicidal drug, after confirmatory activity tests in in vivo models.

ACKNOWLEDGMENTS

The authors are grateful for the financial support received from the following funding agencies: Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

REFERENCES

• Barros, A.M.C. 2018. Atividade antileishmania de um derivado do dillapiole obtido a partir de Piper aduncum L. (Piperaceae). Master´s dissertation, Universidade Federal do Amazonas, Brazil, 106p. (https://tede.ufam.edu.br/handle/tede/6746?mode=full)
  » https://tede.ufam.edu.br/handle/tede/6746?mode=full
• Barros, A.M.C.; Lima, E.S.; Pinto, A.C.S.; Simplicio, F.G.; Chaves, F.C.M.; Pereira, A.M.R.F. 2021. Potencial dos análogos do dillapiole para uso em doenças negligenciadas, com ênfase para leishmaniose cutânea: revisão literária. Brazilian Journal of Development, 7: 73198-73218.
• Barreiro, E.J.; Fraga, C.A.M. 2015. Química Medicinal: As Bases Moleculares da Ação dos Fármacos 3rd ed., ArtMed, Porto Alegre, 590p.
• Brasil. 2017. Ministério da Saúde. Manual de vigilância da leishmaniose tegumentar. Ministério da Saúde, Brasília. ( (https://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf ). Accessed on 16 May 2021.
  » https://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf
• Carvalho, S.H.; Frezard, F.; Pereira, N.P.; Moura, A.S.; Ramos, L.M.Q.C.; Carvalho, G.B.; et al 2019. American tegumentary leishmaniasis in Brazil: a critical review of the current therapeutic approach with systemic meglumine antimoniate and short-term possibilities for an alternative treatment. Tropical Medicine and International Health, 24: 380-391.
• Chiaradia, L.D.; Dos Santos, R.; Vitor, C.E.; Vieira, A.A.; Leal, P.C.; Nunes, R.J.; et al 2007. Synthesis and pharmacological activity of chalcones derived from 2,4,6-trimethoxyacetophenone in RAW 264.7 cells stimulated by LPS: Quantitative structure-activity relationships. Bioorganic & Medicinal Chemistry, 16: 658-667.
• Cunha, A.M.; Chagas, E. 1937. Nova espécie de protozoário do gênero Leishmania patogênica para o homem, Leishmania chagasi n. sp Hospital 11: 148-152.
• Dal Picolo, C.R.D.; Bezerra, M.P.; Gomes, K.S.; Passero, L.F.D.; Laurenti, M.D.; Martins, E.G.A.; et al 2014. Antileishmanial activity evaluation of adunchalcone, a new prenylated dihydrochalcone from Piper aduncum L. Fitoterapia, 97: 28-33.
• De Almeida, R.P.; Souto, R.N.P.; Bastos, C.N.B.; Da Silva, M.H.L.; Maia, J.G.S. 2009. Chemical variation in Piper aduncum and biological properties of its dillapiole-rich essential oil. Chemistry and Biodiversity, 6: 1427-1434.
• De Oliveira, A.; Mesquita, J.T.; Tempone, A.G.; Lago, J.H.G.; Guimarães, E.F.; Kato, M.J. 2012. Leishmanicidal activity of an alkenylphenol from Piper malacophyllum is related to plasma membrane disruption. Experimental Parasitology, 132: 383-387.
• DNDi. 2018. Drugs for Neglected Diseases initiative Planejamento das atividades em leishmaniose tegumentar na DNDi. InfoLEISH - Boletim informativo da redeLEISH-3rd ed. ( (https://www.dndial.org/wp-content/uploads/2018/07/InfoLeish_2018_PORT.pdf ). Accessed on 31 Jul 2023.
  » https://www.dndial.org/wp-content/uploads/2018/07/InfoLeish_2018_PORT.pdf
• Farah, D.; Parise-Filho, R.; Magri, F.; Bonardo, V.P.; Fiorino, P.; Farah, V.M.; Fonteles, M.; Delorenzi, J. 2010. Anti-promastigote activity of dillapiole and isodillapiole against Leishmania chagasi Planta Medica, 76: 480. doi: 10.1055/s-0030-1264778
  » https://doi.org/10.1055/s-0030-1264778
• Ferreira, R.G.F.; Monteiro, M.C.; Silva, J.K.; Maia, J.G.S. 2016. Antifungal action of the dillapiole-rich oil of Piper aduncum against dermatomycoses caused by filamentous fungi. British Journal of Medicine and Medical Research, 15: 1-10.
• Floch, H. 1954. Leishmania tropica guyanensis n. ssp., cause of cutaneous leishmaniasis in the Guiananas and Central America. Public Inst Pasteur Guyane Fr Inini, 15: 1-4.
• Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; et al 2013. Gaussian 09, Revision D.01; Gaussian, Inc., Wallingford, 182p.
• Gaínza, Y.A.; Fantatto, R.R.; Chaves, F.C.M.; Bizzo, H.R.; Esteves, S.N.; Chagas, A.C.S. 2016. Piper aduncum against Haemonchus contortus isolates: cross resistance and the research of natural bioactive compounds. Revista Brasileira de Parasitologia Veterinária, 25: 383-393.
• Guryanov, I.; Fiorucci, S.; Tennikova, T. 2016. Receptor-ligand interactions: Advanced biomedical applications. Materials Science and Engineering: C, 68: 890-903.
• Jaffe, C.L.; Grimaldi Jr., G.; Mcmahon-Pratt, D. 1984. The cultivation and cloning of Leishmania In: Morel, C.M. (Ed.). Genes and Antigens of Parasites A Laboratory Manual 2nd ed. Fundação Oswaldo Cruz, Rio de Janeiro, p.47-91.
• Junior, I.A.S. 2013. Avaliação da metaciclogênese in vitro de Leishmania (V.) braziliensis e Leishmania (L.) amazonensis Master´s dissertation, Universidade Federal de Goiás, Brazil, 140p. (http://repositorio.bc.ufg.br/tede/handle/tede/4640).
  » http://repositorio.bc.ufg.br/tede/handle/tede/4640
• Katz, L.; Baltz, R.H. 2016. Natural product discovery: past, present, and future. Journal of Industrial Microbiology & Biotechnology, 43: 155-176.
• Lainson, R.; Shaw, J.J. 1972. Leishmaniasis of the New World: taxonomic problems. British Medical Bulletin, 28: 44-48.
• Lorenzi, H.; Matos, F.J.A. 2008. Plantas Medicinais no Brasil: Nativas e Exóticas 2nd ed. Plantarum, Nova Odessa, 576p.
• MarvinSketch. 2017. ChemAxon, Version 17.27.0; ChemAxon Ltd., Cambridge Innovation Center, Cambridge, MA, USA. (https://www.covalencia.org/post/chemsketch-um-software-para-desenhar-estruturas-quimicas#:~:text=O%20ChemSketh%20%C3%A9%20um%20SE,de%).
  » https://www.covalencia.org/post/chemsketch-um-software-para-desenhar-estruturas-quimicas#:~:text=O%20ChemSketh%20%C3%A9%20um%20SE,de%
• McMahon-Pratt, D.; Alexander, J. 2004. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunological Reviews, 201: 206-24.
• Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; et al 2009. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. Journal of Computational Chemistry, 30: 2785-2791.
• Nakayama, G.R.; Caton, M.C.; Parandoosh, N.Z. 1997. Assessment of the Alamar Blue assay for cellular growth and viability in vitro Journal of Immunological Methods, 204: 205-208.
• Oliveira, G.L.; Cardoso, S.K.; Júnior, C.R.L.; Vieira, T.M.; Guimarães, E.F.; Figueiredo, L.S.; et al 2013. Chemical study and larvicidal activity against Aedes aegypti of essential oil of Piper aduncum L. (Piperaceae). Annals of the Brazilian Academy of Sciences, 85: 1227-1234.
• OPAS/OMS. 2020. Organização Pan-Americana da Saúde/Organização Mundial da Saúde. Leishmanioses. Informe Epidemiológico das Américas # 9. (file:///C:/Users/pc/Downloads/OPASCDEVT200041_por%20(4).pdf). Accessed on 30 Jul 2023.
• Parise-Filho, R.; Pasqualoto, K.F.M.; Magri, F.M.M.; Ferreira, A.K.; Silva, B.A.V.G.; Damião, M.C.F.C.B.; et al 2012. Dillapiole as antileishmanial agent: discovery, cytotoxic activity and preliminary SAR studies of dille analogues. Archiv der Pharmazie, 345: 934-944.
• Pinto, A.C.S. 2008. Desenvolvimento de substâncias semissintéticas e bioativas a partir de 4-nerolidilcatechol e dillapiole Doctoral thesis, Universidade Federal do Amazonas, Brazil, 296p.
• Pohlit, A.M.; Pinto, A.C.S.; Cavalcanti, B.C.; Pessoa, C.Ó.; Silva, E.C.C.; Chaves, F. C. M.; Tadei, W. P. 2008. Biopharmaceuticals synthesis, In: Brazilian Network on Green Chemistry Awareness, Responsibility and Action. CARIOCA, José Osvaldo Beserra. p. 357
• Reguera, R.M.; Calvo-Álvarez, E.; Álvarez, V.R.; Balaña-Fouce, R. 2014. Target-based vs. phenotypic screenings in Leishmania drug discovery: A marriage of convenience or a dialogue of the deaf? International Journal of Parasitology: Drugs and Drug Resistance, 4: 355-357.
• Rhaiem, R.B.; Houimel, M. 2016. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library. Acta Tropica, 159: 9-11.
• Santos, M.L.; Magalhães, C.F.; Rosa, M.B.; Santos, D.A.; Brasileiro, B.G.; Carvalho, L.M.; et al 2013. Antifungal activity of extracts from Piper aduncum leaves prepared by different solvents and extraction techniques against dermatophytes Trichophyton rubrum and Trichophyton interdigitale Brazilian Journal of Microbiology, 44: 1275-1278.
• Schlagenhauf, E.; Etges, R.; Metcalf, P. 1998. The crystal structure of the Leishmania major surface proteinase leishmanolysin (gp63). Structure, 6: 1035-1046.
• Shao, S.; Sun, X.; Chen, Y.; Zhan, B.; Zhu, X. 2019. Complement evasion: An effective strategy that parasites utilize to survive in the host. Frontiers in Microbiology, 10: 532. doi: 10.3389/fmicb.2019.00532
  » https://doi.org/10.3389/fmicb.2019.00532
• Sousa, P.J.C.; Barros, C.A.L.; Rocha, J.C.S.; Lira, D.S.; Monteiro, G.M.; Maia, J.G.S. 2008. Avaliação toxicológica do óleo essencial de Piper aduncum L. Revista Brasileira de Farmacoginosia, 18: 10-13.
• Tomar, S.S.; Maheshwari, M.L.; Mukerjee, S.K. 1979a. Síntese e atividade sinérgica de alguns sinergistas de piretro de dilapiol. Agricultural and Biological Chemistry, 43:1479-1483.
• Tomar, S.S.; Maheshwari, M.L.; Mukerjee, S.K. 1979b. Síntese e atividade sinérgica de sinergistas de piretro à base de dilapiol. Journal of Agricultural and Food Chemistry, 27: 547-550.
• Trott, O.; Olson, A.J. 2010. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31: 455-461.
• Vianna, G. 1911. Sobre uma nova espécie de Leishmania (Nota preliminar). Brazil Med 25: 411.
• Volpe, H.X.L.; Fazolin, M.; Garcia, R.B.; Magnani, R.F.; Barbosa, J.C.; Miranda, M.P. 2016. Efficacy of essential oil of Piper aduncum against nymphs and adults of Diaphorina citri Pest Management Science, 72: 1242-1249.
• WHO. 2019. World Health Organization. Leishmaniosis. ( (http://www.who.int/leishmaniasis/en/ ). Accessed on 20 Jul 2023.
  » http://www.who.int/leishmaniasis/en/
• WHO. 2020. World Health Organization. Fact Sheet: leishmaniasis. ( (https://www.who.int/news-room/fact-sheets/detail/leishmaniasis ). Accessed on 30 Jul 2023.
  » https://www.who.int/news-room/fact-sheets/detail/leishmaniasis

• CITE AS:

  Barros, A.M.C.; Pinto, A.C.S.; Simplicio, F.G.; Chaves, F.C.M.; Silva, N.G.; Oliveira, K.M.T.; Lima, E.S.; Franco, A.M.R. 2023. Antileishmanial and cytotoxic activity of dillapiole n-butyl ether. Acta Amazonica 53: 285-293.

SUPPLEMENTARY MATERIAL

(only available in the electronic version)

Barros et al. Antileishmanial and cytotoxic activity of dillapiole n-butyl ether

Figure S1
¹H NMR spectrum (CDCl₃; 500 MHz) of dillapiole and enlargement of spectrum regions.

Figure S2
¹³C NMR spectrum (CDCl₃; 125 MHz) of dillapiole

Figure S3
DEPT 135 spectrum (CDCl₃; 125 MHz) of dillapiole.

Figure S4
MS/MS spectrum of the ion at m/z 222 in the dillapiole.

Figure S5
¹H NMR spectrum (CDCl₃; 500 MHz) of dillapiole n-butyl ether (DBE) and enlargement of spectrum regions.

Figure S6
¹³C NMR spectrum (CDCl₃; 125 MHz) of dillapiole n-butyl ether (DBE)

Figure S7
DEPT spectrum (CDCl₃; 125 MHz) of dillapiole n-butyl ether (DBE).

Data availability

The data that support the findings of this study are not publicly available.

Edited by

ASSOCIATE EDITOR:

Emiliano Barreto

Publication Dates

• Publication in this collection
  13 Nov 2023
• Date of issue
  Oct-Dec 2023

History

• Received
  18 Apr 2023
• Accepted
  01 July 2023