Antiprotozoal Compounds from Urolepis hecatantha (Asteraceae)

The dewaxed dichloromethane extract of Urolepis hecatantha and the compounds isolated from it were tested for their in vitro activity on Trypanosoma cruzi epimastigotes and Leishmania infantum promastigotes. The extract of U. hecatantha showed activity against both parasites with IC50 values of 7 µg/mL and 31 µg/mL, respectively. Fractionation of the dichloromethane extract led to the isolation of euparin, jaceidin, santhemoidin C, and eucannabinolide. The sesquiterpene lactones eucannabinolide and santhemoidin C were active on T. cruzi with IC50 values of 10 ± 2 µM (4.2 µg/mL) and 18 ± 3 µM (7.6 µg/mL), respectively. Euparin and santhemoidin C were the most active on L. infantum with IC50 values of 18 ± 4 µM (3.9 µg/mL) and 19 ± 4 µM (8.0 µg/mL), respectively. Eucannabinolide has also shown drug-like pharmacokinetic and toxicity properties.


Introduction
Chagas' disease and leishmaniasis are protozoan parasitic diseases caused by Trypanosoma cruzi and different species of the genus Leishmania and transmitted by infected bloodsucking triatomine bugs and phlebotomine sandflies, respectively.
ey are both classified as neglected tropical diseases by the World Health Organization (WHO) [1].
Chagas' disease or American Trypanosomiasis is a potentially life-threatening disease that affects 6 to 7 million people worldwide. It is estimated that about 30000 new cases occur annually and more than 12000 deaths per year are attributed to this parasitosis [2]. Chagas' disease was historically linked to poor rural areas of Latin America where the insect vector is present. In recent years, the disease has spread to cities and nonendemic areas due to migrations of infected people and nonvectorial transmission of the parasite, turning Chagas' disease into a global public health problem [3]. Nifurtimox and benznidazole are the only drugs currently available for Chagas' disease treatment. Both drugs are effective in the acute stage of the infection and vertical transmission prevention, but their efficacy diminishes in the chronic phase. Besides, frequent adverse events lead to high rates of treatment discontinuation [4]. erefore, the development of new trypanocidal drugs for Chagas' disease treatment is needed.
Leishmaniasis has three clinical forms: cutaneous, mucocutaneous, and visceral also known as kala-azar. Although cutaneous leishmaniasis is the most common form, visceral leishmaniasis is the most severe form [5]. According to the WHO, more than one billion people are at risk of infection. It is estimated that 30000 new cases of visceral leishmaniasis and more than one million new cases of cutaneous leishmaniasis occur annually [5]. e chemotherapy of leishmaniasis is based on the use of sodium stibogluconate, meglumine antimoniate, pentamidine, amphotericin B, paromomycin, and miltefosine. ese drugs are toxic and have other limitations such as the route of administration, length and cost of treatment, and emergence of drug resistance [6].
Natural products play an important role in the drug discovery process. One of the most relevant examples is artemisinin, a sesquiterpene lactone isolated from Artemisia annua currently used for malaria treatment [7]. Several natural products with promissory activity against pathogenic protozoa have been reported [8,9].
Urolepis hecatantha (DC) R. M. King and H. Rob. (syn. Eupatorium hecatanthum (DC) Baker) is the only species of the monotypic genus Urolepis (Asteraceae) [10]. e ethnomedical uses of the aerial parts of U. hecatantha by indigenous groups of northeast Argentina have been reported [11][12][13]. e fresh aerial parts are chewed as antitussive [11], while the infusion or decoction of the aerial parts is used topically for gangrene and ulceration treatment [12]. is species has been employed also as an analgesic for teeth pain treatment [13]. e isolation of flavonoids, terpenoids, and benzofuran derivatives from a collection of U. hecatantha from Bolivia has been reported [14].
In this work, we report the isolation of four compounds from U. hecatantha from Argentina and the evaluation of their in vitro activity on Trypanosoma cruzi and Leishmania infantum. e toxicity and pharmacokinetic properties of the compounds were also estimated.

Plant Materials.
e aerial parts of U. hecatantha (DC.) R. King and H. Robins (Asteraceae) were collected in Buenos Aires province, Argentina, in March 2018. e plant material was identified and deposited at the Herbarium of the Faculty of Pharmacy and Biochemistry, University of Buenos Aires (BAF 16100).

Extraction and Fractionation Procedures.
Grounded dried flowers and leaves of U. hecatantha (300 g) were extracted thrice at room temperature with dichloromethane (4.5 L, 6 h). Filtrates were joined and concentrated on a rotary evaporator at 40°C under reduced pressure to give 42 g of crude extract (DE). e crude extract was suspended in ethanol (147 mL) at 60°C, diluted with distilled water (63 mL), and filtered under vacuum. e filtrate was extracted successively with hexane (3 × 60 mL) and dichloromethane (3 × 60 mL). Dichloromethane subextracts were joined and taken to dryness on a rotary evaporator to yield 15 g of dewaxed extract. Dewaxed extract (DDE) was fractionated by silica gel column chromatography (60 × 5 cm, 220 g, 230-400 mesh) and eluted with a gradient of dichloromethane (CH 2 Cl 2 ) and increasing amounts of ethyl acetate (EtOAc): 100% CH 2 Cl 2 , CH 2 Cl 2 : EtOAc (9 : 1), , and 100% EtOAc. Fractions (5 × 200 mL) of each solvent ratio (A 1−5 to K 1−5 ) were collected. All column chromatography fractions were monitored by thin-layer chromatography using silica gel 60 F 254 plates and anisaldehyde sulphuric as spraying reagent.

Compounds Isolation.
Fractions A 5 (100% DCM) and B 1−5 (DCM: EtOAc 9: 1) were pooled and the solvent was evaporated on a rotary evaporator. e residue obtained was transferred to a small vial with a minimum amount of ethyl acetate and the solution was left at room temperature overnight. From this solution, yellow acicular crystals of compound 1 (euparin, 28 mg) were obtained.
Fractions D 2−5 , eluted with CH 2 Cl 2 : EtOAc (7 : 3), and the fraction E 1 , eluted with CH 2 Cl 2 : EtOAc (6 : 4), showing a similar profile on TLC, were reunited and brought to dryness on a rotary evaporator. e residue was suspended in a minimum amount of dichloromethane and purified by preparative TLC using silica gel plates. e plates were developed using toluene: EtOAc : formic acid (6 : 4 : 1) as a mobile phase. After drying, plates were analyzed under UV light where a deep green fluorescent band (Rf � 0.6) was observed. e fluorescent band was scraped out and extracted with methanol. After solvent evaporation, a yellow powder (10 mg) identified as jaceidin was obtained.
e residue was dissolved with a minimum amount of ethyl acetate and allowed to stand at room temperature for 24 hours. From this solution, pure crystals of compound 3 (santhemoidin C, 120 mg) were obtained. Fractions I 3-5 , eluted with CH 2 Cl 2 : EtOAc (2 : 8), and fractions J 1-3 , eluted with CH 2 Cl 2 : EtOAc (1 : 9), were combined and brought to dryness on a rotary evaporator. e residue was fractionated by silica gel column chromatography (50 × 3 cm, 150 g, 230-400 mesh) and eluted isocratically with a 2 : 1 mixture of CH 2 Cl 2 : EtOAc. Twenty fractions of 50 mL each were collected. See spectra in supplementary material. Assignments were made by H-H COSY, HSQC, and HMBC experiments.

Spectrometric Analyses.
e isolated compounds were identified by proton nuclear magnetic resonance ( 1 H-NMR) and carbon nuclear magnetic resonance ( 13 C-NMR), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), correlated spectroscopy (COSY) (Bruker Advance 600) (600 MHz in CDCl 3 ), and electron impactmass spectrometry (EI-MS). e purity of santhemoidin C (3), estimated by 1 H-NMR, was >95%. Likewise, the purity estimated for euparin (1) and eucannabinolide (4), also by 1 H-NMR, was 97.5% and ca. 94%, respectively (see the corresponding 1 H-NMR spectra in supplementary material). Jaceidin sample was analyzed by TLC using CH 2 Cl 2 : EtOAc as a solvent and a 10% solution of antimony (III) chloride in chloroform as spray reagent. A single spot was observed under long-wave UV light. It was identified by its mp 131-135°C ("Jaceidin," Human Metabolome Database, HMDB0033819) and by chromatographic analysis with an authentic sample and confirmed by UV spectroscopy with shift reagents.

Antiparasitic Activity Assay.
For the anti-Trypanosoma and anti-Leishmania activity assays, we followed the methods described by Aguilera et al., 2019 [15].
For the in vitro anti-Trypanosoma cruzi activity, epimastigotes of the Tulahuen 2 strain (genotype TcVI) grown in an axenic medium (BHI-Tryptose) were used. Cells from a 5-7-day-old culture were inoculated in a fresh culture medium to give an initial concentration of 10 6 cells/mL. e absorbance at 600 nm of the cells in culture was measured every day. On day five, the medium was inoculated with different doses of the compounds (25-0.05 µM) from a stock solution in dimethylsulfoxide (DMSO) (DMSO concentration in the culture medium never exceeded 0.4%). Control parasites were cultivated in medium with 0.4% DMSO v/v. Benznidazole was used as a positive control. At five days, the absorbance of the culture was measured and compared to the control and the IC 50 values were calculated for each compound using OriginLab 8.5 ® sigmoidal regression. Each experiment was done in duplicate, and each concentration was tested in triplicate.
Leishmania infantum (MHOM/BR/2002/LPC-RPV) was obtained from Fiocruz (Collection of Oswaldo Cruz Foundation, Rio de Janeiro, Brazil). Promastigotes were cultured as described [16] with some modifications at 28°C in an axenic medium (BHI-Tryptose supplemented with: FBS 10%, hemine 2 × 10 −5 mg/mL, glucose 3.0 × 10 −4 g/mL, streptomycin 2.0 × 10 −4 g/mL, ampicillin 1.3 × 10 −4 g/mL) as a low-cost alternative for Leishmania spp. culture. Assays were performed in 96-well plastic plates using 2 × 10 6 promastigotes per well. Compounds were dissolved in DMSO. Different serial dilutions (25-0.05 µM) of the compounds with a final volume up to 200 µL were added. After 48 h at 28°C, 20 µL of a 2 mM resazurin solution was added, and the oxidation-reduction was quantified at 570 and 600 nm. e resazurin solution was prepared at 2.5 mM in phosphatebuffered solution (PBS), pH 7.4, and filtered through 0.22 µm before use. Resazurin sodium salt was obtained from Sigma-Aldrich (St. Louis, MO, USA) and stored at 4°C protected from light. Glucantime was used as a positive control. e efficacy of each compound was estimated by calculating the IC 50 values using OriginLab 8.5® sigmoidal regression. Each antiproliferative experiment was done in duplicate, and each concentration was tested in triplicate.

Cytotoxicity Assay.
e cytotoxicity of the dewaxed dichloromethane extract of U. hecatantha and the isolated compounds was evaluated according to the method described by Aguilera et al., 2019 [15]. e J774.1 murine macrophages (ATCC, USA) were grown in a DMEM culture medium containing 4 mM L-glutamine and supplemented with 10% FCS. Cells were seeded in a 96-well plate (5.00 × 10 4 cells in 200 µL culture medium) and incubated at 37°C in a 5% CO 2 atmosphere for 48 h, to allow cell adhesion before drug testing. Afterwards, cells were exposed for 48 h to the compounds (25-400 µM) or the vehicle for control (medium with 0.4% DMSO v/v), and additional control (cells in medium) were used in each test. Cell viability was then assessed by measuring the mitochondria-dependent reduction of MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. For this purpose, MTT in sterile PBS (containing 0.2% glucose), pH 7.4, was added to the macrophages to achieve a final concentration of 0.1 mg/ mL, and the cells were incubated at 37°C for 3 h. After removing the medium, formazan crystals were dissolved in 180 µL of DMSO and 20 µL of MTT buffer (0.1 M glycine, 0.1 M NaCl, 0.5 mM EDTA, pH 10.5), and the absorbance at 560 nm was measured. e CC 50 was defined as the drug concentration at which 50% of the cells were viable, relative to the control (no drug added), and was determined using OriginLab 8.5 ® sigmoidal regression (% of viable cells compared to the logarithm of the compound concentration). Tests were performed in triplicate.

Toxicity and Pharmacokinetic Properties.
e toxicity and pharmacokinetic properties of the compounds were estimated with the open-access SwissADME software (http://www.swissadme.ch), a tool that allows the prediction of different pharmacokinetic parameters such as water solubility, gastrointestinal absorption, skin penetrability, lipophilicity, bioavailability, and so forth and Evidence-Based Complementary and Alternative Medicine 3 T. E. S. T (Toxicity Estimation Software Tool). e software input uses the SMILES codes of the molecules, which were generated with the ChemBioOffice 2010 program.

Statistical Analysis.
e statistical analysis was performed using Origin software package version 7.0. e statistical significance of the difference between the data pairs was evaluated by analysis of variance (one-way ANOVA), followed by the Tukey test. Statistical differences were considered significant at p < 0.05.
Euparin and eucannabinolide have been previously isolated from U. hecatantha collected in Bolivia [14]. e presence of santhemoidin C and jaceidin in this species is reported for the first time.
Euparin (1) and santhemoidin C (3) were the most active on L. infantum with IC 50 values of 18 ± 4 µM (3.9 µg/mL) and 19 ± 4 µM (8.0 µg/mL), respectively. e flavonoid jaceidin (2) and the sesquiterpene lactone eucannabinolide (4) were less active (IC 50 > 25 µM). Glucantime showed an IC 50 value of 26 ± 9 µM. e cytotoxic effect of compounds 1-4 was assayed on mammalian cells. e terpenoid compounds, santhemoidin C (3) and eucannabinolide (4), showed CC 50 values of >15 µM and 15 µM, respectively. Euparin (1) and jaceidin (2) presented CC 50 values > 25 µM. Taking in consideration that selectivity is a relevant characteristic for defining hit molecules, selectivity indexes (SI) of the compounds were calculated. e most active compound against T. cruzi, eucannabinolide (4), showed a SI value of 1.5. e differences in activity between T. cruzi and L. infantum for compound 4 compared to compound 3 are remarkable. Both are germacranolides with the same molecular formula (C 22 H 28 O 8 ) but differ in the stereochemistry of the C4-C5 double bond: lactone 3 is a germacrolide (a trans, trans-germacranolide) while lactone 4 is a heliangolide (a trans, cis-germacranolide) [22]; they also differ in the location of the acetyl group which in lactone 3 esterifies the hydroxyl group of the 4,5dihydroxytigloyloxy ester residue at C-8, while, in lactone 4, it esterifies the hydroxyl group at C-3 of the heliangolide skeleton. ese differences should be expected to strongly affect conformation, electronic distribution, and hydrogen bonding interactions. erefore, these stereochemical and positional changes could increase the activity of compound 4 on T. cruzi and decrease it for L. infantum. Santhemoidin C, euparin, and jaceidin did not display selectivity on T. cruzi epimastigotes. None of the compounds showed selectivity against L. infantum promastigotes.
e pharmacokinetic characteristics and toxicity of the compounds play an important role in the drug discovery process.
ese properties are influenced in part by the physicochemical properties of drugs. In this sense, the mutagenicity, the oral rat LD 50 , the Log P, solubility, gastrointestinal (GI) absorption, skin permeation (Log Kp), and the blood-brain barrier (BBB) permeant were predicted (Table 1). Euparin (1), santhemoidin C (3), and eucannabinolide (4) showed no mutagenicity and LD 50 values > 600 mg/kg with high GI absorption. Santhemoidin C and eucannabinolide presented log P values lower than 2, satisfying the criteria established by Lipinski [23] and showed the best skin permeation. Compounds 2-4 showed no BBB permeation.
Eucannabinolide has shown activity against Trypanosoma brucei rhodesiense trypomastigotes (IC 50 � 1.1 ± 0.1 µM) and has tested its cytotoxicity on mammalian cells (L6-cell line from rat-skeletal myoblasts; CC 50 � 7.8 ± 0.8 µM) [24]. is sesquiterpene lactone was also active when it was loaded onto polylactic acid nanoparticles with a free drug equivalent IC50 value of 3.32 µM [25]. Eucannabinolide has also exhibited cytotoxic activity against tumour cell lines [26] and anti-inflammatory activity [27]. Compound 4 has also shown drug-like pharmacokinetic and toxicity properties (Table 1). No reports about the biological activities of santhemoidin C have been found. Antibacterial and antiviral activity [28,29]    Evidence-Based Complementary and Alternative Medicine and protective effect on human lymphocytes against chromosomal damage [30] have been reported for jaceidin. Euparin has shown antipoliovirus activity [31] and cytotoxic activity against liver carcinoma cells [32]. is is the first time that the activity against T. cruzi and L. infantum of these compounds has been reported.

Conclusions
In this study, the activity against T. cruzi and L. infantum of the dichloromethane extract of U. hecatantha and the isolation of four compounds, euparin, jaceidin, santhemoidin C, and eucannabinolide, are reported. is is the first communication describing the isolation of santhemoidin C and jaceidin from U. hecatantha. e activity of the isolated compounds against T. cruzi and L. infantum is being reported for the first time. e sesquiterpene lactone eucannabinolide was the most active compound against T. cruzi and could be considered for further studies.

Data Availability
e data used to support the findings of this study are included within the article.

Disclosure
is investigation is part of the activities carried out within the "Research Network Natural Products against Neglected Diseases" (ResNet NPND): http://www.resnetnpnd.org/.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.