Trypanocidal Activity of Four Sesquiterpene Lactones Isolated from Asteraceae Species

The sesquiterpene lactones eupatoriopicrin, estafietin, eupahakonenin B and minimolide have been isolated from Argentinean Astearaceae species and have been found to be active against Trypanosoma cruzi epimastigotes. The aim of this work was to evaluate the activity of these compounds by analyzing their effect against the stages of the parasites that are infective for the human. Even more interesting, we aimed to determine the effect of the most active and selective compound on an in vivo model of T. cruzi infection. Eupatoriopicrin was the most active against amastigotes and tripomastigotes (IC50 = 2.3 µg/mL, and 7.2 µg/mL, respectively) and displayed a high selectivity index. This compound was selected to study on an in vivo model of T. cruzi infection. The administration of 1 mg/kg/day of eupatoriopicrin for five consecutive days to infected mice produced a significant reduction in the parasitaemia levels in comparison with non-treated animals (area under parasitaemia curves 4.48 vs. 30.47, respectively). Skeletal muscular tissues from eupatopicrin-treated mice displayed only focal and interstitial lymphocyte inflammatory infiltrates and small areas of necrotic; by contrast, skeletal tissues from T. cruzi infected mice treated with the vehicle showed severe lymphocyte inflammatory infiltrates with necrosis of the adjacent myocytes. The results indicate that eupatoriopicrin could be considered a promising candidate for the development of new therapeutic agents for Chagas disease.


In Vitro Anti-T. cruzi Activity
The trypanocidal effect of the STLs was assessed on bloodstream trypomastigotes by counting the remaining live parasites in a Newbauer chamber after incubation with each compound. The four STLs displayed trypanocidal activity on the bloodstream form of T. cruzi with 50% inhibitory concentration (IC50) values of 7.2 ± 0.3 µg/mL for eupatoriopicrin, 28.9±4.1 µg/mL for estafietin, 11.9 ± 4.5 µg/mL for eupahakonenin B and 7.7 ± 0.4 µg/mL for minimolide ( Figure 2). The IC50 of the reference drug benznidazole was 16.4 ± 2.3 µg/mL.

In Vitro Anti-T. cruzi Activity
The trypanocidal effect of the STLs was assessed on bloodstream trypomastigotes by counting the remaining live parasites in a Newbauer chamber after incubation with each compound. The four STLs displayed trypanocidal activity on the bloodstream form of T. cruzi with 50% inhibitory concentration (IC 50 ) values of 7.2 ± 0.3 µg/mL for eupatoriopicrin, 28.9±4.1 µg/mL for estafietin, 11.9 ± 4.5 µg/mL for eupahakonenin B and 7.7 ± 0.4 µg/mL for minimolide ( Figure 2). The IC 50 of the reference drug benznidazole was 16.4 ± 2.3 µg/mL.
The effect of the compounds on the intracellular form of T. cruzi was determined by a spectrophotometric method. Eupatoriopicrin was the most active STL on T. cruzi amastigotes with an IC 50 value of 2.3 ± 0.5 µg/mL. The IC 50 values for minimolide, eupahakonenin B and estafietin were 9.2 ± 0.8, 32.2 ± 4.6 and 26.9 ± 4.3 µg/mL, respectively ( Figure 3). Benznidazole presented an IC 50 of 3.5± 0.6 µg/mL. The trypanocidal effect of the STLs was assessed on bloodstream trypomastigotes by counting the remaining live parasites in a Newbauer chamber after incubation with each compound. The four STLs displayed trypanocidal activity on the bloodstream form of T. cruzi with 50% inhibitory concentration (IC50) values of 7.2 ± 0.3 µg/mL for eupatoriopicrin, 28.9±4.1 µg/mL for estafietin, 11.9 ± 4.5 µg/mL for eupahakonenin B and 7.7 ± 0.4 µg/mL for minimolide ( Figure 2). The IC50 of the reference drug benznidazole was 16.4 ± 2.3 µg/mL.  The effect of the compounds on the intracellular form of T. cruzi was determined by a spectrophotometric method. Eupatoriopicrin was the most active STL on T. cruzi amastigotes with an IC50 value of 2.3 ± 0.5 µg/mL. The IC50 values for minimolide, eupahakonenin B and estafietin were 9.2 ± 0.8, 32.2 ± 4.6 and 26.9 ± 4.3 µg/mL, respectively ( Figure 3). Benznidazole presented an IC50 of 3.5± 0.6 µg/mL. Figure 3. Inhibition of T. cruzi amastigotes by eupatoriopicrin, estafietin, eupahakonenin B and minimolide. Vero cells (5 × 10 3 cells/well) were seeded in 96-well plate and infected 24 h later with transfected trypomastigotes expressing β-galactosidase. After 24 h of co-culture, plates were washed, and compounds were added at 0-100 µg/mL in 150 µl medium. On day six post-infection, the reaction was developed by the addition of CPRG (100 mM) and Nonidet P-40 (1%). Plates were incubated for 6 h and the absorbance was read at 570 nm. Infected, non-treated cells were used as 100% infection control. The percentage of inhibition was calculated as 100 − {[(Absorbance of treated infected cells)/(Absorbance of untreated infected cells)] × 100}.

In Vivo Assay
Since eupatoriopicrin was the most active and selective STL against T. cruzi trypomastigotes and amastigotes, this compound was selected for evaluation on an in vivo model of Chagas disease. Balb/c mice were infected with T. cruzi trypomastigotes (K98 strain) and treated with benznidazole, eupatoriopicrin or the vehicle (DMSO) as control. Each compound was administered for five consecutive days since day 11 post infection (day in which parasites became detectable by microhemotocrit analysis). Mice receiving the STL presented lower parasitaemia levels, as compared to untreated animals ( Figure 5A). Parasitaemia was monitored till day 48 post-infection when parasites in mice blood became undetectable. T. cruzi infected mice treated with eupatoriopicrin showed an important decrease in the area under the parasitaemia curve (AUC) regarding the control group (AUC: 4.48 ± 1.36 and 30.47 ± 7.15, respectively; p < 0.001) ( Figure 5B). Similar results were observed for the reference drug, Benznidazole which showed a 34.8 times reduction in the AUC with respect to the control (AUC: 0.87 ± 0.77).

In Vivo Assay
Since eupatoriopicrin was the most active and selective STL against T. cruzi trypomastigotes and amastigotes, this compound was selected for evaluation on an in vivo model of Chagas disease. Balb/c mice were infected with T. cruzi trypomastigotes (K98 strain) and treated with benznidazole, eupatoriopicrin or the vehicle (DMSO) as control. Each compound was administered for five consecutive days since day 11 post infection (day in which parasites became detectable by microhemotocrit analysis). Mice receiving the STL presented lower parasitaemia levels, as compared to untreated animals ( Figure 5A). Parasitaemia was monitored till day 48 post-infection when parasites in mice blood became undetectable. T. cruzi infected mice treated with eupatoriopicrin showed an important decrease in the area under the parasitaemia curve (AUC) regarding the control group (AUC: 4.48 ± 1.36 and 30.47 ± 7.15, respectively; p < 0.001) ( Figure 5B). Similar results were observed for the reference drug, Benznidazole which showed a 34.8 times reduction in the AUC with respect to the control (AUC: 0.87 ± 0.77).

Histopathological Analysis
The anti-T. cruzi activity of the STL eupatoriopicrin was also analyzed on the chronic phase of the parasite infection. Samples of heart and skeletal (quadriceps) muscles from the experimental mice were taken and stained with hematoxylin-eosin for the histological examination. No significant differences were observed in the architecture of the hearts form the different experimental animals. Small foci of interstitial or perivascular lymphocyte infiltrate without damage to adjacent myocytes were observed in both control and treated mice ( Figure 6A-C). By contrast, significant changes were observed when skeletal muscle from the experimental mice were analyzed. The skeletal muscle from T. cruzi infected mice treated with the vehicle (DMSO) showed interstitial coalescent lymphocyte inflammatory infiltrates with necrosis of the adjacent myocytes. By contrast, muscle tissues from eupatopicrin-treated mice displayed only focal and interstitial lymphocyte inflammatory infiltrates and small areas of necrotic tissue ( Figure 6D-F).

Histopathological Analysis
The anti-T. cruzi activity of the STL eupatoriopicrin was also analyzed on the chronic phase of the parasite infection. Samples of heart and skeletal (quadriceps) muscles from the experimental mice were taken and stained with hematoxylin-eosin for the histological examination. No significant differences were observed in the architecture of the hearts form the different experimental animals. Small foci of interstitial or perivascular lymphocyte infiltrate without damage to adjacent myocytes were observed in both control and treated mice ( Figure 6A-C). By contrast, significant changes were observed when skeletal muscle from the experimental mice were analyzed. The skeletal muscle from T. cruzi infected mice treated with the vehicle (DMSO) showed interstitial coalescent lymphocyte inflammatory infiltrates with necrosis of the adjacent myocytes. By contrast, muscle tissues from eupatopicrin-treated mice displayed only focal and interstitial lymphocyte inflammatory infiltrates and small areas of necrotic tissue ( Figure 6D-F).

Discussion
In a previous study, we reported the anti-T.cruzi activity of the STLs eupatoriopicrin, estafietin, eupahakonenin B and minimolide against T. cruzi epimastigotes [8]. The four sesquiterpene lactones have been isolated from S. maimarensis, S. alpina, S. gilliesi and M. minima, respectively. The

Discussion
In a previous study, we reported the anti-T.cruzi activity of the STLs eupatoriopicrin, estafietin, eupahakonenin B and minimolide against T. cruzi epimastigotes [8]. The four sesquiterpene lactones have been isolated from S. maimarensis, S. alpina, S. gilliesi and M. minima, respectively. The compounds were identified by spectroscopic methods. The four compounds have previously shown significant activity on T. cruzi epimastigotes. Although the epimastigote stage of the parasite is non-infective, it is easy to cultivate and, therefore, useful for a preliminary screening test. Here, we decided to evaluate the effect of the STLs on the bloodstream and intracellular forms of T. cruzi. All the tested compounds exerted trypanocidal activity against trypomastigotes, with IC 50 values ranging from 7.2 to 28.9 µg/mL. Besides, when the compounds were tested on amastigotes, eupatoriopicrin showed the highest inhibitory effect (IC 50 = 2.3 ± 0.5 µg/mL).
In order to determine the selectivity of these compounds an in vitro cytotoxicity assay was carried out on Vero cells. The STLs rendered SI values ranging from 6.8 to 12.9 on bloodstream tripomastigotes. Eupatoriopicrin also displayed significant selectivity for intracellular amastigotes in comparison with the other tested compounds (SI = 40.6). Eupatoriopicrin has also been found to be active against Trypanosoma brucei rhodesiense, with an IC 50 of 0.43 µg/mL but with low selectivity [15]. The trypanocidal activity of estafietin on the infective and intracellular forms of T. cruzi has been previously described by our group [19]. This compound was included in the in vitro assays carried out in this work in order to compare its activity with that of the other three STLs. Eupatoriopicrin was the most active and selective compound, therefore it was selected for in vivo assessment in T. cruzi infected mice.
The clinical outcome of Chagas disease is highly variable, mainly because of the heterogeneity of its etiologic agent [20]. Trypanosoma cruzi is a monophyletic but heterogeneous group conformed by several Discrete Typing Units (DTUs) named TcI to TcVI [21]. The in vitro trypanocidal efficacy of the STLs was evaluated against two highly virulent T. cruzi strains, RA and Tulahuen, which belong to TcII-DTU and TcVI-DTU, respectively. By contrast, for the in vivo study we selected the K98 clone (TcI-DTU) because it does not present lethality during the acute phase [22]. This model mimics better the human infection, in which a small percentage (1-2%) of patients die from acute Chagas [23], and most of the patients progress to a chronic infection [24].
The administration of 1 mg/kg/day of eupatoriopicrin for five consecutive days to T. cruzi-infected mice induced a significant reduction in the parasitaemia levels in comparison with non-treated control animals. More interestingly, this reduction was similar to that achieved with the reference drug, benznidazole. Eupatoriopicrin showed a potent effect, causing a greater decrease in the number of circulating parasites. Although reductions in the parasitaemia levels have been reported for others sesquiterpene lactones, scarce studies have investigated their performance to prevent tissue damages during the chronic phase of the parasite infection [25][26][27][28]. Importantly, we found that the treatment of infected animals with eupatoriopicrin was also effective to reduce the cardiac and mainly the skeletal damage associated to T. cruzi infection.

Sesquiterpene Lactones Isolation
Fifty grams of dried aerial parts of each plant species were extracted twice at room temperature with dichloromethane (DCM) (500 mL, 3 min.). Filtrates were concentrated on a rotatory evaporator at 40 • C under reduced pressure. S. maimarensis and S. gilliesii crude extracts were dissolved in warm ethanol (140 mL), the solution was placed in a dropping funnel, water (60 mL) was added (ratio ethanol-water 70:30 v/v) and the mixture was extracted three times with hexane (3 × 50 mL) and then with DCM (3 × 50 mL). DCM sub-extracts were reunited and evaporated to yield dewaxed extract, rich in STLs.
S. alpina and M. minima crude extracts and S. maimarensis and S. gilliesii dewaxed extracts were fractionated by silica gel column chromatography (50 × 4.5 cm, 180 g, 230-400 mesh) and eluted isocratically with dichloromethane-ethyl acetate mixtures: 1:2 for S. maimarensis and S. gilliesii, 9.5:0.5 for S. alpina and 2:1 for M. minima. Column chromatography eluates were monitored by thin layer chromatography (TLC) using STL standards. Fractions showing a single spot corresponding to the desired compounds were pooled and dried in a rotary evaporator. Fractions enriched in the desired compound were also pooled and rechromatographed as above to recover additional amounts of lactone.
The STL eupatoriopicrin precipitated from S. maimarensis fractions as white crystals. The crystals were washed twice with a 7:3 mixture of ethyl ether-ethyl acetate. Recrystallization from n-heptane-ethyl acetate yielded needles mp 158-160 • C [9]. Eupahakonenin B was obtained as a greenish gum when S. gilliesi fractions showing a single spot on TLC with R f identical to a reference standard [10] were reunited and evaporated in rotavapor. Less pure fractions were reunited and rechromatographed under the same conditions described above in order to increase the amount of the isolated compound. The rechromatographed lactone was isolated as a colorless gum. Estafietin and minimolide were purified by column chromatography from crude extracts of S. alpina and Mikania minima, respectively (See supplementary materials). In both cases, STLs-rich fractions were pooled, the solvent was evaporated in a rotary evaporator and the residue was suspended in a minimum volume of warm heptane-ethyl acetate (1:2) to near complete dissolution and then kept in the refrigerator overnight. Both STLs precipitated as white needles and were recrystallized from heptane-ethyl acetate; estafietin, mp 104-105 • C [11]; minimolide, mp 112-113 • C [18].
The purity of the compounds was determined by HPLC using a RP-C18 column (250 × 4.6 mm -5 µm mesh) and a water: acetonitrile gradient (35% to 95% acetonitrile in 30 min. for eupatoriopicrin, minimolide and eupahakonenin B and 45% to 95% acetonitrile in 30 min. for estafietin. A flow rate of 1 mL/min was used. The diode array detector was set at 210 nm. The purity of the sesquiterpene lactones determined by HPLC was: eupatoriopicrin: 94.6%, minimolide: 99.8%, estafietin: 93.3% and eupahakonenin B: 93.9%. Further recrystallization of eupatoriopicrin, estafietin and eupahakonenin B yielded analytical samples with a purity >98.2% in all three cases. The identity of the compounds was confirmed by UV, IR, NMR and MS spectroscopy.

Parasites
Trypanosoma cruzi bloodstream trypomastigotes from RA and K98 strains [22] as well as transfected trypomastigotes expressing β galactosidase [26], were obtained from infected CF1 mice by cardiac puncture at the peak of parasitaemia on day 15 post infection.
Transfected trypomastigotes (Clone C4 of the Tulahuen strain) that express the Escherichia coli beta-galactosidase gene were kindly provided by Dr. Buckner [29].

Activity Assay on Bloodstream T. Cruzi Trypomastigotes
Mouse blood containing trypomastigotes from the RA T. cruzi strain was diluted with an RPMI medium to a cell density of 1.5 × 10 6 parasites /mL. Parasites were then seeded in duplicate in a 96-well microplate with increasing concentrations of each STL (0-100 µg/mL). Additional controls in which parasites were incubated with the solvent were also included. After 24 h incubation at 4 • C, parasites were diluted 1/10 with lysis buffer and the remaining live trypomastigotes were counted in a Neubauer chamber. The percentage of live trypomastigotes was calculated as {[(live parasites after incubation)/(live parasites in untreated wells)] × 100}.

Activity Assay on Intracellular T. Cruzi Amastigotes
A 96-well plate was seeded with the non-phagocytic Vero cell line at 5 × 10 3 per well in 100 µL of culture medium and incubated for 2 h at 37 • C in a 5% CO 2 atmosphere. Cells were washed and infected with transfected Tulhauen bloodstream trypomastigotes expressing β-galactosidase (MOI:1/10 cells/parasites). After 24 h of co-culture, the plate was washed twice with PBS to remove extracellular parasites and each compound and benznidazole was added at 0-100 µg/mL per well in 150 µL of fresh complete RPMI medium without phenol red. Non-treated cells (100% infection) and non-infected cells (0% infection) were used as controls. After five days, cells were lysed with 1% Nonidet P-40 and chlorophenol red-β-d-galactopyranoside (CPRG) (100 µM) was added as β-galactosidase substrate. After 4-6 h incubation at 37 • C, the absorbance was measured at 570 nm in a microplate reader. The percentage of inhibition was calculated as 100 − {[(absorbance of treated infected cells)/(absorbance of untreated infected cells) × 100}.

Cytotoxicity
Cytotoxicity was determined by the MTT method. Briefly, Vero cells (5 × 10 5 c/mL) were settled at a final volume of 150 µl in a flat-bottom, 96-well microplate and cultured at 37 • C in a 5% CO 2 atmosphere in the presence of increasing concentrations of the pure compounds and the reference drug benznidazole. Cells incubated in absence of the STLs were used as a 100% viability control. After 24 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added at a final concentration of 1.5 mg/mL. Plates were incubated for two additional hours at 37 • C. The purple formazan crystals were completely dissolved by adding 150 µl of ethanol and the absorbance was read at 595 nm in a microplate reader. Results were expressed as the ratio between the absorbance in the presence and absence of the compound multiplied by 100.

In Vivo Trypanocidal Activity
Inbred Balb/c mice (male, eight weeks old, and 26 g average weight) were nursed at Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires. Mice were infected with 3 × 10 5 T. cruzi trypomastigotes of the K98 strain by intraperitoneal route. Parasitaemia was measured weekly. Blood samples were diluted 1:5 in lysis buffer (0.75% NH 4 Cl, 0.2% Tris, pH 7.2) and parasites were counted in a Neubauer chamber.
Mice were divided in groups of five animals each and the drugs were administered by the intraperitoneal route (1 mg/kg of body weight/day) for five consecutive days after infection (11 to 15 dpi). Eupatoriopicrin and benznidazol were diluted with DMSO and the concentration was adjusted with 0.1M phosphate buffered saline (pH 7.2). A vehicle was employed as a negative control.

Histopathological Analysis
Samples of heart and skeletal (quadriceps) muscles were dissected at 100 dpi and fixed with 4% formalin in PBS. The fixed tissue was embedded in paraffin, sectioned and stained with haematoxylin and eosin. Sections were analyzed at 200 and 400× magnifications. Inflammatory infiltrates were qualitative graded as focal, multiple non-confluent, multiple confluent and multiple diffuse infiltrate [30]. The presence of necrotic areas was evaluated as well.

Ethic Statement
Animal experiments were approved by the Review Board of Ethics of Universidad de Buenos Aires, Facultad de Medicina, Argentina (no. 2943/2013) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council.

Statistical Analysis
Results are presented as means ± SEM. The GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA) was employed to carry out calculations. Parasitaemia levels was analyzed by the Dunett's test. The statistical significance was determined by one-way analysis of variance (ANOVA) performed with the GraphPad Prism 6.0 software. Comparisons were referred to the control group. p values <0.05 were considered significant.

Conclusions
Eupatoriopicrin, estafietin, eupahakonenin B and minimolide showed an in vitro anti-T. cruzi activity against the infective and intracellular forms of T. cruzi. Besides, the STL eupatoriopicrin, isolated from Stevia maimarensis, was also effective in an in vivo model of Chagas disease, reducing the parasiteamia and the tissue damage of the skeletal muscle. Even more important, the high anti-parasite activity of this STL against strains of T. cruzi with different genetic backgrounds signals eupatoriopicrin as a promising candidate for further studies in the search of novel therapeutic agents for Chagas disease.