Anti-trypanosomal activity of (8-hydroxymethylen)-trieicosanyl acetate against infective forms of Trypanosoma cruzi

Abstract

The activity of an (8-hydroxymethylen)-trieicosanyl acetate compound obtained from chloroform extracts of Senna villosa (Mill.) H.S. Irwin & Barneby (Leguminosae) against Trypanosoma cruzi was evaluated in vivo. Oral doses of 2.1, 8.4, and 33.6 µg/g were tested for 28 days in BALB/c mice infected with T. cruzi. Reduced parasitemia levels of 70.5%, 73.8%, and 80.9%, respectively, were observed. A significant reduction in amastigote nests was detected in the cardiac tissue of treated animals at doses of 8.4 and 33.6 µg/g. The LD₅₀ of (8-hydroxymethylen)-trieicosanyl acetate was impossible to determine because none of the animals died, even at oral doses of 5000 µg/g; consequently, it was impossible to determine the acute oral toxicity in vivo.

Keywords::

• American trypanosomiasis
• antiprotozoal
• BALB/c mice
• natural products
• Senna villosa

Introduction

Chagas’ disease (American trypanosomiasis) is the most important endemic parasitic disease in Latin America (Tarleton et al., 2007), with an estimated 15 million persons infected and 28 million at risk of infection in 21 countries of the American continent (WHO, 2007). The disease is caused by Trypanosoma cruzi, a flagellated protozoan, and it is widely distributed from Mexico to Argentina (Milei et al., 2009).

Benznidazole (N-benzyl-2-nitro-1-imidazole-acetamide) and nifurtimox [3-methyl-4(5-nitrofurfurylidene-amino)-tetrahydro-(1,4)-thiazine-1,1-dioxide] are currently the first-line drugs employed to treat Chagas’ disease (Castro et al., 2006). However, benznidazole is not readily available in Latin America and nifurtimox is no longer produced. Allopurinol (Paulino et al., 2005; Nakajima-Shimada et al., 1996; Berens et al., 1982), gentian violet, ketoconazole (Lira et al., 2001; Santa-Rita et al., 2005), and itraconazole (Apt et al., 1998; Apt et al., 2005; Coronado et al., 2006) are also used to treat Chagas’ disease, but they are associated with numerous toxic effects.

Traditional Mexican medicine uses a great variety of plants to combat infectious diseases, one of which is Senna villosa (Mill.) Irwin & Barneby (Leguminosae), commonly known as “salché”. It is widely distributed in the south of Mexico and in the Caribbean, and grows at an altitude range of 10 to 1600 m above sea level. The antimicrobial, antifungal, and antiprotozoal properties of S. villosa have been previously reported (Mena et al., 1997; Flores, 2001; Guzman et al., 2004).

A recent study described the structure of (8-hydroxymethylen)-trieicosanyl acetate, a compound isolated from the chloroform extract of S. villosa that was tested against epimastigote and trypomastigote forms of Trypanosoma cruzi in vitro. This compound exhibited significant activity against both forms of the parasite at concentrations of 3.3 and 6.6 µg/mL (Guzman et al., 2008).

The present study evaluated the in vivo antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate against infective forms (blood trypomastigotes and amastigotes) of T. cruzi.

Materials and methods

Plant material

Senna villosa was collected from the rural community of Komchen, a town 17 km away from Merida, Yucatan, Mexico, from July through September 2005, 2006, and 2007. The plant species was authenticated by Salvador Flores-Guido and a voucher (10284) was deposited at the herbarium of the Universidad Autonoma de Yucatan (UADY). Leaves were separated and dried in the shade at room temperature.

Extraction and isolation of (8-hydroxymethylen)-trieicosanyl acetate

Dried and powdered leaves (500 g) were mixed with CHCl₃ (3 L). The mixture was refluxed for 4 h and filtered (Guzman et al., 2004). The solvent was evaporated, and the residue was chromatographed on silica gel (70-230 mesh) and eluted with hexane, while increasing the polarity with ethyl acetate. The (8-hydroxymethylen)-trieicosanyl acetate component was obtained from fraction 5 and recrystallized from chloroform (Guzman et al., 2008).

Parasites and experimental animals

Trypomastigotes of T. cruzi strain H4 were used. The selected strain is capable of yielding a mortality rate of 50% in mice after 30 days of inoculation, and demonstrates a particular tropism towards cardiac tissue (Barrera-Perez et al., 2001). BALB/c mice were maintained on a 12:12 h light-dark cycle and had access to food and water ad libitum. Each mouse was IP-inoculated with 5 × 10⁴ trypomastigotes of T. cruzi.

Acute oral toxicity

The acute oral toxicity of (8-hydroxymethylen)-trieicosanyl acetate was evaluated in BALB/c mice as directed by the Organization for Economic Co-Operation and Development (OECD, 2001). The evaluated doses were 175, 550, and 1750 µg/g. The toxicity evaluation ended when the highest dose evaluated (5000 µg/g) was administered to three mice and none died.

In vivo antiprotozoal activity against trypomastigotes of T. cruzi

The antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate was evaluated at three doses: 2.1, 8.4, and 33.6 µg/g (n = 10 mice in each group). The compound was resuspended in 50 µL phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO4, and 1.4 mM KH₂PO4, pH 7.4) per mouse, and administered orally every 24 h for 28 days.

As a negative control, a group of infected mice received only 50 µL orally of the vehicle (PBS). A positive control group included infected mice treated orally with allopurinol (8.5 µg/g) diluted in 50 µL of PBS every day for the entire duration of the study.

Mice were examined every four days for 28 days after the time of infection to estimate the degree of parasitemia (blood trypomastigote counts) in all mice included in the assay. Treated animals were compared to the control groups, and the day at which each mouse died was recorded. All procedures were conducted in accordance with the internationally accepted principles for laboratory animal use and care.

Antiprotozoal activity against amastigote nests of T. cruzi

To determine the activity of (8-hydroxymethylen)-trieicosanyl acetate against the intracellular amastigote form of T. cruzi, cardiac tissue samples from treated and untreated mice were collected and fixed in formaldehyde (10%).

Paraffin-embedded tissue sections were stained with hematoxylin-eosin (HE) and examined under a light microscope. Four non-consecutive slides from the heart of each mouse were examined in a blinded fashion. The number of amastigote nests was quantified in 100 zones for each heart.

Statistical analyses

Data are expressed as means ± SEM. Statistical analyses were performed using the Student’s t-test (p <0.05), and ANOVA followed by Tukey’s multiple comparison test was used to compare more than two groups.

Results

Results of the acute oral toxicity evaluation

There were no signs of lethal toxicity at any of the evaluated doses of the compound (oral administration). After administration of the highest dose (5000 µg/g), slight lethargy was observed in 66% (n = 3, as indicated by OECD, 2001) of the treated mice, but they demonstrated a complete recovery 24 h later.

In vivo antiprotozoal activity against trypomastigotes of T. cruzi

In vivo evaluation of (8-hydroxymethylen)-trieicosanyl acetate at doses of 2.11, 8.4, and 33.6 µg/g demonstrated antiprotozoal activity against blood forms of T. cruzi, but none of the doses (even at 33.6 µg/g) resulted in complete elimination of the parasites from the bloodstream. At doses of 2.11 and 8.4 µg/g, a reduction in parasitemia of 75.8% and 75.9%, respectively, was observed in comparison to untreated mice. The highest activity of the compound (80.9%) was obtained at a dose of 33.6 µg/g (p < 0.05) (Figure 1). At day 28, 175.2, 156.2, and 114 × 10⁵ parasites/mL were detected in animals treated at doses of 2.1, 8.4, and 33.6 µg/g, respectively, compared to 597.5 × 10⁵ parasites/mL in the untreated mice.

Figure 1.  Parasitemic curves of BALB/c mice infected with 50,000 trypomastigotes of the H4 strain of T. cruzi and treated by the oral route with (8-hydroxymethylen)-trieicosanyl acetate at doses of 2.11, 8.4, and 33.6 µg/g and allopurinol (8.5 µg/g) for 28 days. Values represent means + SD (*p<0.05).

Antiprotozoal activity against amastigote nests of T. cruzi

An increased number of amastigote nests was observed in the cardiac tissue of the negative control mice (untreated). Animals treated with allopurinol or with the lowest dose evaluated (2.1 µg/g) also demonstrated a greater amount of amastigote nests in the cardiac tissue (Figure 2), whereas mice treated with higher doses (8.4 and 33.6 µg/g) displayed fewer amastigote nests (p < 0.05).

Figure 2.  Effect of (8-hydroxymethylen)-trieicosanyl acetate on the number of amastigote nests observed in BALB/c mice infected with T. cruzi and treated at doses of 2.11, 8.4, and 33.6 µg/g (**p<0.05).

Mortality

Mortality was lower (20% for 10 mice, p < 0.05) in the group treated with (8-hydroxymethylen)-trieicosanyl acetate at all doses evaluated (2.1, 84, and 33.6 µg/g) compared to mice treated with allopurinol (70% for 10 animals) and to untreated mice (50%) (Figure 3).

Figure 3.  Effect of (8-hydroxymethylen)-trieicosanyl acetate on the mortality rate of BALB/c mice after 28 days of infection with T. cruzi and treated at doses of 2.11, 8.4, and 33.6 µg/g (* p<0.05).

Discussion

The results obtained in the present study demonstrate that (8-hydroxymethylen)-trieicosanyl acetate does not produce signs of oral acute toxicity, even when high doses (5000 µg/g) are used.

The absence of acute oral toxicity in treated mice was similar to the findings obtained in studies performed by Guzman et al. (2008) using (8-hydroxymethylen)-trieicosanyl acetate in Vero cells, in which no cytotoxic activity was observed during the evaluation. Considering that the ideal active antiprotozoal compound should be innocuous to mammalian host cells (Schmeda-Hirschmann et al., 2001), (8-hydroxymethylen)-trieicosanyl acetate appears to be promising as a new anti-trypanosomal agent. However, it will be necessary to evaluate the probable long-term toxicity of the compound in animal models to determine whether it is safe.

Treatment o f infected mice with (8-hydroxymethylen)-trieicosanyl acetate revealed antiprotozoal activity against T. cruzi trypomastigotes. The antiprotozoal activity was observed at all doses evaluated (2.1, 8.4 and 33.6 µg/g) (Figure 1), but the antiprotozoal activity against the amastigote forms was only observed when higher doses (8.4 and 33.6 µg/g) were used (Figure 2). The antiprotozoal activity against T. cruzi found in the present study is similar to the in vitro activity previously reported by Guzman et al. (2004, 2008).

The antiprotozoal activity of the methanol, aqueous, and chloroform extracts of S. villosa against T. cruzi have also been previously reported in vitro (Guzman et al., 2004). In another study, (8-hydroxymethylen)-trieicosanyl acetate displayed antiprotozoal activity against the epimastigote and trypomastigote forms of T. cruziin vitro (Guzman et al., 2008). At all three doses evaluated, the antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate under in vivo conditions was not able to completely eliminate trypanosomes from the bloodstream. However, it is remarkable that the mice treated with the highest dose evaluated (33.6 µg/g, see Figure 1) exhibited a significant reduction in parasitemia, demonstrating the strongest antiprotozoal activity in comparison with untreated and allopurinol-treated mice.

These results suggest that (8-hydroxymethylen)-trieicosanyl acetate has promising potential as an active compound, as it is apparently not toxic and possesses superior antiprotozoal activity against T. cruzi [3.4 times more active against blood trypomastigotes than allopurinol (8.5 µg/g) in infected mice treated for 28 days at a similar dose (8.4 µg/g)]. S. villosa also demonstrated efficient in vitro antiprotozoal activity against Entamoeba histolytica and Giardia lamblia (IC₅₀ of 133 and 32 µg/mL, respectively). Thus, S. villosa should be considered as a plant with significant potential for antiprotozoal activity (Calzada et al., 2006).

The (8-hydroxymethylen)-trieicosanyl acetate demonstrated effectiveness against the epimastigote form. However, even when the parasitemia level was reduced using a dose of 2.1 µg/g, a large number of amastigote nests were found in the cardiac tissue (Figure 2). This probably implies that the trypomastigotes activated an evasion mechanism upon exposure to a hostile environment in the bloodstream due to the presence of (8-hydroxymethylen)-trieicosanyl acetate. These adverse conditions could induce parasite internalization, promoting greater tissue invasion, differentiation of the parasites to amastigote forms, and greater multiplication in cardiac tissue. On the other hand, treatment of infected mice with a low dose of (8-hydroxymethylen)-trieicosanyl acetate may not permit good bioavailability of the compound in cardiac tissue, making this tissue an ideal place to escape the antiprotozoal activity. However, when the dose of (8-hydroxymethylen)-trieicosanyl acetate was increased, the number of amastigote nests decreased significantly.

The H4 strain of T. cruzi can produce cardiac lesions with a mortality of up to 50% thirty days post-inoculation, and produces a high parasitemia level in infected mice (Barrera-Perez et al., 2001), which is similar to the mortality rate observed in untreated mice in the present study (Figure 3). But even with the high virulence of the H4 strain, treatment of infected mice with (8-hydroxymethylen)-trieicosanyl acetate at a dose of 33.6 µg/g revealed a significant reduction in the number of trypomastigotes, which resulted in about 50% lower parasitemia over the course of the study (Figure 1).

Mortality in mice treated with (8-hydroxymethylen)-trieicosanyl acetate was lower compared to negative control mice (Figure 3). These results are similar to those obtained in other studies reporting the trypanocidal activity of plant extracts and compounds isolated from natural sources under in vivo conditions (Asuzu & Chineme, 1990; Cunha et al., 2006). The reduction in mortality could be associated with the bloodstream parasitemia level and the amount of amastigote nests in the cardiac tissue. Marin-Neto et al. (2007) described tissue damage in the heart and gastroenteric tract during the acute phase of Chagas’ disease, showing a clear relationship between the level of parasitemia in the bloodstream and that in target organs. The concentrated presence of the parasite in cardiac tissue induces an intensive inflammatory process, myocarditis, and is correlated with the severity of clinical heart failure (Higuchi et al., 1987). Tissue damage and clinical progression are worse, and survival is decreased in patients with Chagas’ heart damage compared with patients presenting non-inflammatory forms of dilated cardiomyopathy (Freitas et al., 2005). This would probably explain the results obtained for (8-hydroxymethylen)-trieicosanyl acetate-treated mice, which displayed a reduced number of cardiac amastigote nests and a slight inflammatory process, leading to a lower mortality rate.

The drugs most commonly used to treat Chagas’ disease, such as nifurtimox, benznidazole, and allopurinol, induce only limited antiprotozoal activity, mainly during the acute phase of the disease. In addition, these drugs produce undesirable side effects in patients, such as anorexia, weight loss, nausea, diarrhea, and others (Fournet & Muñoz, 2002). In the present study, secondary effects attributable to allopurinol were clearly observed in treated mice (mainly anorexia, weight loss, and diarrhea), as previously described (Horiuchi et al., 2000). Mortality in this group was higher compared to mice treated with (8-hydroxymethylen)-trieicosanyl acetate and untreated mice (negative control) (Figure 3). During acute infection of mice with T. cruzi, a significant increase in the levels of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) occurs (Dantas et al., 2006), inducing hepatotoxicity. For this reason, the probability of developing hepatic insufficiency increases in infected and allopurinol-treated mice, leading to higher mortality.

Although allopurinol exhibits considerable antiprotozoal activity against amastigotes in the Vero cell line (Nakajima-Shimada et al., 1996; Paulino et al., 2005), the in vivo results demonstrated only a slight reduction of the number of amastigote nests present in mice treated with allopurinol compared to untreated mice (p <0.05). When mice were treated with 8.4 and 33.6 µg/g of (8-hydroxymethylen)-trieicosanyl acetate, a significant reduction (p<0.05) of amastigote nests was observed in the cardiac tissue (Figure 2). The lack of in vivo activity by allopurinol may be due to the low incorporation of the drug into the vertebrate stages of many T. cruzi strains (Avila & Avila 1981; Avila et al. 1981) and inadequate pharmacokinetic properties (Urbina, 1999). Moreover, Horiuchi et al. (2000) found that normal mice that were administered high doses of allopurinol showed abnormal pyrimidine metabolism along with renal toxicity, which complicates the course of Chagas’ disease in infected animals.

The antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate observed against both parasite forms (trypomastigote and amastigote) in infected mice may be responsible for the reduction of amastigote nests observed in infected mice, resulting in reduced mortality (Figure 3). Biotransformation of (8-hydroxymethylen)-trieicosanyl acetate is apparently non-toxic, and, consequently, better results were observed using this compound compared to mice treated with allopurinol. It will be necessary to evaluate the pharmacodynamics of (8-hydroxymethylen)-trieicosanyl acetate in animal models, as well as the antiprotozoal activity of the compound when treating infected mice for longer periods of time, at higher doses, when using different T. cruzi strains, and during the chronic phase of Chagas’ disease.

Declaration of interest

We gratefully acknowledge PROMEP (Programa de Mejoramiento del Profesorado) for financial support: “Actividad in vivo del compuesto aislado de las hojas de Senna villosa contra las formas de tripomastigote y amastigote”, Registration number: CIRB-05-022.

We gratefully acknowledge CONACYT (Consejo Nacional de Ciencia y Tecnologia) for financial support (reference number 164935) of the PhD studies at UAM-X.