International Scholarly Research Notices

International Scholarly Research Notices / 2015 / Article

Research Article | Open Access

Volume 2015 |Article ID 924670 | 7 pages | https://doi.org/10.1155/2015/924670

Toxicity and Loss of Mitochondrial Membrane Potential Induced by Alkyl Gallates in Trypanosoma cruzi

Academic Editor: Tzi Bun Ng
Received08 Sep 2014
Revised18 Dec 2014
Accepted22 Dec 2014
Published28 Jan 2015

Abstract

American trypanosomiasis or Chagas disease is a debilitating disease representing an important social problem that affects, approximately, 10 million people in the world. The main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease. In this context, the search for trypanocidal substances isolated from plants, synthetic or semi synthetic molecules, is an important strategy. Here, the trypanocidal potential of gallates was assayed in epimastigotes forms of T. cruzi and also, the interference of these substances on the mitochondrial membrane potential of the parasites was assessed, allowing the study of the mechanism of action of the gallates in the T. cruzi organisms. Regarding the preliminary structure-activity relationships, the side chain length of gallates plays crucial role for activity. Nonyl, decyl, undecyl, and dodecyl gallates showed potent antitrypanosomal effect (IC50 from 1.46 to 2.90 μM) in contrast with benznidazole (IC50 = 34.0 μM). Heptyl gallate showed a strong synergistic activity with benznidazole, reducing by 105-fold the IC50 of benznidazole. Loss of mitochondrial membrane potential induced by these esters was revealed. Tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential, at IC50 value.

1. Introduction

Chagas disease (or American trypanosomiasis) was recognized by World Health Organization as one of thirteen neglected tropical diseases in the world [1]. It was estimated that over 10 million people are infected, and Latin America was considered the area of the highest prevalence [2]. This parasitic disease is caused by Trypanosoma cruzi (family, Trypanosomatidae and order, Kinetoplastida), a hemoflagellate protozoa [3], which can be found on several strains with different mechanisms of pathogenesis, immunogenicity, treatment response, and epidemiology [4]. The development of infection includes an acute phase which lasts up to six months after infection, an indeterminate phase with no symptoms, and a chronic phase in which approximately 30% of patients show clinical evidence of heart disease or megavisceras [5].

The current chemotherapy uses benznidazole, a nitroimidazole derivative, which is orally administered in acute phase and short-term chronic phase [6, 7]. The usefulness of this drug is limited by its narrow therapeutic window and due to serious side effects, such as anorexia, nausea, headache, paresthesia, peripheral neuropathies, dermatitis, central nervous system (CNS) depression, and maniac symptoms [7, 8]. However, several parasite subpopulations with different host tissue’s tropism contribute to the low clinical efficacy of this drug [9]. Therefore, there are efforts for the discovery and design of new therapeutic compounds to treat Chagas disease, due to the complexity of this disease.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a precursor of hydrolysable tannins in the biosynthesis that occurs in plants [10] and its natural and semisynthetic derivatives have been associated with a wide variety of biological activities, such as antiproliferative [1113], chemopreventive [14], antihemolytic [15], antioxidant [16], and anti-inflammatory [17] activities. However, the main interest in gallic acid and in its derivatives has been associated with their antimicrobial properties. It has been shown that n-octyl gallate possesses fungicidal activity against Saccharomyces cerevisiae and Zygosaccharomyces bailii in any stage of their growth [18, 19]. Leal and coworkers reported the potent fungitoxicity of n-nonyl gallate against yeasts, dermatophytes, and hialohyphomycetes [20]. Lauryl gallate showed antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) [21, 22].

Thus, the aim of this study was to investigate the antitrypanosomal activity of gallic acid and its esters against epimastigote forms of Trypanosoma cruzi. Further analysis involving interactions generated by combinations between alkyl gallates and benznidazole was also carried out. In addition, the apoptosis-inducing activity by alkyl gallates was studied through the parasite mitochondrial membrane potential assay.

2. Material and Methods

2.1. Compounds

The compounds tested in this study were synthesized as described previously [14, 15].

2.2. Parasites

Epimastigote forms of Trypanosoma cruzi of Y strain, described by Silva and Nussenzweig (1953) [23], which is considered a standard strain of type I, were grown in LIT media (liver infusion tryptose media) at 28°C [24].

2.3. Cytotoxicity Assay Using MTT

The cytotoxicity assay with epimastigotes T. cruzi was performed using the MTT colorimetric method as described by Muelas-Serrano et al. (2000) [25] with modifications described in Cotinguiba et al. (2009) [26]. The parasites were treated with different concentrations of the substances for 72 hours and then, the parasite viability was estimated by measuring the absorbance at 595 nm. The concentration versus response curve was constructed to enable the determination of IC50. An equation that describes the curve was obtained using Origin 7.0 software. Means and standard deviations were calculated. ANOVA and Tukey’s tests were carried out when necessary. Differences in means were treated as significant when . Benznidazole was used as positive control (IC50 = 34.0 M).

2.4. Mitochondrial Membrane Potential Assay

The influence of the treatments with the gallates in the mitochondrial membrane potential was assessed. The parasites were treated with the substances during 72 hours using, as standard concentration, the IC50 previously determined by the MTT assay. The percentage of parasite cells which suffered loss in the mitochondrial membrane potential due to the treatment with the tested compounds was measured by flow cytometry using JC-1 dye (-tetrachloro--tetraethylbenzimidazolcarbocyanine iodide, BD MitoScreen kit) according to the manufacturer’s instruction [27, 28]. A FacsCanto I cytometer was used, and the data were recorded and analyzed on the software of the equipment. Pentamidine was used as positive control, at 58.7 M for 24-hour treatment.

3. Results and Discussion

The antitrypanosomal activity of a homologous series of alkyl gallates, gallic acid, and other analogues was evaluated against epimastigote forms of Trypanosoma cruzi. This study was carried out in order to correlate chemical characteristics of the molecules with the observed activity, including the importance of free hydroxyl groups and side chain length. Table 1 summarizes the IC50 values of these compounds.


CompoundRIC50

Gallic acid (1)H0.89>100b
Methyl gallate (2)CH30.92>100b
Ethyl gallate (3)CH2CH31.27>100b
Propyl gallate (4)CH31.73>100b
Butyl gallate (5)CH32.13>100b
Pentyl gallate (6)CH32.53>100b
Hexyl gallate (7)CH32.92>100b
Heptyl gallate (8)CH33.3237.3 ± 0.9
Octyl gallate (9)CH33.7223.0 ± 5.3
Nonyl gallate (10)CH34.112.90 ± 0.1
Decyl gallate (11)CH34.511.50 ± 0.3
Undecyl gallate (12)CH34.901.46 ± 0.0
Dodecyl gallate (13)CH35.302.13 ± 0.2
Tetradecyl gallate (14)CH36.0917.6 ± 1.8
Benznidazolec34.0

aValues described by Rosso and coworkers (2006) [16].
bPercentage of inhibition at 100 M.
cPositive control.

Gallic acid (1) was found to be totally inactive against T. cruzi (IC50 > 100 M) and its derivatives up to hexyl gallate (27) exhibited similar IC50 values (IC50 > 100 M). Heptyl gallate (8, IC50 = ) showed weak activity which increased in the case of longer carbon chain derivatives up to undecyl gallate (12, IC50 = ). However, the increase in the carbon chains, becoming bigger than undecyl gallate, led to lower activity, as shown for compounds 13 and 14 (Table 1). Such results indicate that the 3,4,5-trihydroxyphenyl moiety appeared to be necessary but not sufficient for antiprotozoal activity. On the other hand, Kubo et al. (2001) [18] verified that the length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity of the gallates.

Among the thirteen tested gallates (214) esterified with linear alcohols from C1 to C14, best results were observed for esters with chain lengths ranging from 9 to 12, which present values of 4.11−5.30, as reported previously by Rosso et al. (2006) [16].

n-Undecyl gallate (12), the most active derivative, with IC50 of 1.46 M (), was twenty-three times more potent than benznidazole, used as positive control (IC50 = 34.0 M). Such results indicate that esterification led to appearance of antitrypanosomal activity, suggesting that a free carboxyl group was not crucial for protozoal death. In contrast, esters with values lower than 3.32 or higher than 0.92 exhibited lower potency than the positive control.

Altogether, there is a clear and positive correlation among IC50 values, alkyl chain length and its contribution to the lipophilicity, which are in agreement with previous studies of this homologous series [19, 29]. Furthermore, the trypanocidal potential increased with the increase in the number of carbons found on the side chain until reaching the maximum activity, in this case at n-undecyl gallate (12), and longer carbon chains showed lower trypanocidal activity, reaching a cutoff at n-tetradecyl gallate (14). If the homologs longer than 14 might show antitrypanosomal activity, their IC50 values will be superior at 17.6 M, representing low potency, not corroborating for further chemical or biological investments.

In previous studies, the cutoff phenomenon was observed for alkanols, which was correlated with their amphipathic properties. In the case of alkyl gallates, their amphiphilicity appeared to be dependent on the presence of two features: hydrophilic phenolic hydroxyls and hydrophobic alkyl chain. Fujita and Kubo (2002) [19] reported the antifungal activity of gallates, suggesting that these compounds possessed amphiphilic capacity and surfactant properties and they might act disrupting the fluidic bilayer membrane, leading to fungal death.

The synergistic effects of the combination using gallic acid and its esters with benznidazole, the antichagasic drug, were also evaluated. The definitions and mathematical determination of the formula for synergism were used as described by Hellmann et al. (2010) [30]. Briefly, the fractional inhibitory concentration (FIC) index was calculated by using the formula FIC index = . In this formula, , , and were IC50 of gallate with benznidazole, IC50 of gallate alone, and IC50 of benznidazole alone, respectively. From this calculation, a FIC index ≤ 0.5 was considered evidence of synergistic effect, as this value can be generated only if the concentrations of both compounds in combination do not exceed one-quarter of the IC50 of either drug tested alone. A FIC index > 4.0 indicates antagonism. When tested alone, some gallates (27) have not led to a significant number of epimastigotes toxicity, even at the highest concentration tested (100 M). Therefore, to calculated FIC index for these compounds, the IC50 was arbitrarily defined as 50 M (Table 2).


CompoundIC50
(gallates alone)
IC50
(gallates with benznidazole)
FIC indexInteraction type

Gallic acid (01)294a17.42.017I
Methyl gallate (02)271a15.31.786I
Ethyl gallate (03)252a10.21.193I
Propyl gallate (04)235a8.901.043I
Butyl gallate (05)221a7.470.878A
Pentyl gallate (06)208a6.020.708A
Hexyl gallate (07)196a2.280.257S
Heptyl gallate (08)37.30.640.084S
Octyl gallate (09)6.490.620.165S
Nonyl gallate (10)0.860.440.560A
Decyl gallate (11)0.460.240.548A
Undecyl gallate (12)0.470.320.716A
Dodecyl gallate (13)0.720.510.765A
Tetradecyl gallate (14)6.440.580.155S
Benznidazoleb8.84

aHypothetical IC50 value used for calculating of FIC index.
bPositive control.
I, A, and S mean indifferent interaction, antagonism, and synergism, respectively.

The interactions between compounds 114 and benznidazole were highly variable, indicating dependence on the gallates chain length. Association between gallic acid (1) and short esters (24) with benznidazole was indifferent. Additive interaction was observed for inactive short esters (5 and 6) and most potent long esters, which exhibited FIC index values ranging from 0.560 to 0.765. Interestingly, medium chain length gallates (79) and the long chain length ester 14 with FIC index values bellow 0.257 exhibited synergism. The most significant synergistic effect was observed for heptyl gallate (8, FIC = 0.084), which reduced by 105-fold the concentration of benznidazole necessary to inhibit cell growth in 50% (IC50 of M).

Synergism was observed at M level, evidencing a strong antitrypanosomal activity and the potential of medium and long chain alkyl gallates as prototypes for further studies and development of novel therapeutic agents. It is also worth mentioning that the significant results from the association of benznidazole and alkyl gallates, which enabled the use of smaller doses of benznidazole, extended dosing intervals between consecutive administrations and short-term treatment leading to fewer and less intense side effects.

Few reports have been found on the antimicrobial synergistic effect against trypanosomatidae protozoa. Urbina and coauthors (1988, 1995, 2002, 2009) [3134] described systematic studies involving antifungal drugs and their associations as trypanocidal combined agents for treatment of Chagas disease, such as azoles (ketoconazole and posaconazole) with mevinolin, terbinafine, aspirin, and amiodarone. Recently, the interaction between benznidazole and parthenolide was investigated against T. cruzi, which evidenced synergistic and additive activities against epimastigotes and trypomastigotes, respectively.

Additionally, trypanocidal gallates (814) were investigated for their influence on the mitochondrial membrane potential. Considering that the loss of mitochondrial membrane potential is a typical characteristic of apoptotic cells [35], it was used to elucidate a possible mode of parasite death. In living cells, JC-1 dye crosses the plasmatic membrane as monomers, penetrating into the mitochondria. This process is controlled by the membrane potential of this organelle. The membrane of healthy mitochondria is polarized and JC-1 is rapidly internalized, increasing the concentration gradient, which may lead to its aggregation. In the flow cytometer, the JC-1 monomers were detected in the FL-1 channel (FITCS), while its aggregates were observed in the red channel FL-2 (PE). On the other hand, injured mitochondria exhibits depolarized membrane and JC-1 remains in the cytoplasm as monomers. Then, we suggest that the cells which loss their mitochondrial membrane potential, which could be apoptotic cells, lose their fluorescence and could be captured in Q2 quadrant, whereas normal cells are detected in Q4 quadrant. Figure 1 presents the flow cytometry graphs.

Figure 1 presents the flow cytometry analysis of n-undecyl gallate (12) on T. cruzi.

Among the tested gallates, compounds 8, 9, and 14 promoted important loss in the mitochondrial membrane potential, which maybe an apoptosis-inducing activity, with percentage of cells with reduction of mitochondrial membrane potential of 46.6, 43.6, and 53.2, respectively. The results are presented on Table 3.


CompoundNegative controlParasite treated with gallatesPositive control

Heptyl gallate (08)1.046.656.9
Octyl gallate (09)1.043.656.9
Nonyl gallate (10)1.05.956.9
Decyl gallate (11)1.09.056.9
Undecyl gallate (12)1.012.556.9
Dodecyl gallate (13)1.013.456.9
Tetradecyl gallate (14)1.053.256.9

Figure 1(a) shows that untreated epimastigotes (control) did not lose their mitochondrial membrane potential which can be observed due to the high fluorescence detected in FL-2 channel (PE). Similarly, it is observed that n-undecyl gallate (12) (Figure 1(b)) did not stimulate this influence in the treated cells since it did not induce a reduction of fluorescence as shown by the control, suggesting the maintenance of the membrane potential. In contrast, the cells treated with pentamidine (Figure 1(c)) show intense decrease in the fluorescence represented by PE, suggesting a loss of mitochondrial membrane potential which may indicate apoptosis of the parasites analyzed. The results of flow cytometry for n-undecyl gallate (12) are presented on Table 3.

The results showed that the gallate esters used in this study induced loss of the mitochondrial membrane potential and maybe apoptosis in the parasites. This indicates that one of the mechanisms of cell death induced by the gallates activity could be similar to the apoptosis. This can be visualized by comparing the results with pentamidine, an apoptosis-inducing drug [36], which led to different percentages of mitochondrial membrane potential relatively to the control. The treatment with some gallates, such as the substances 8, 9, and 14, showed a percentage of loss of mitochondrial membrane potential similar to the pentamidine treatment.

Pentamidine is a dicationic drug which has been used in the last 50 years for the treatment of African trypanosomiasis and antimony-resistant leishmaniasis and is known to cause changes in mitochondrial metabolism [37]. The drug that can cause a collapse in mitochondrial membrane potential leads to an imbalance in intracellular [38]. Electronic microscopy transmission studies with cells treated with pentamidine showed condensation and disruption of kinetoplast DNA core and collapse of mitochondrial membrane [39, 40].

Based on the results obtained by the flow cytometry and cytotoxicity analysis, it is possible to infer that other mechanisms of action, different from apoptosis, might be associated with the death of the parasites, since the substances that caused more significant dysfunction in the mitochondrial membrane potential (8, 9, and 14) are not the same substances that according to the cytotoxicity assay cause higher rates of parasites death (10, 11, 12, and 13). In addition, maintenance of cell viability might be associated with low affinity or steric hindrance of the molecular target due to its physical-chemical properties.

Another possible mechanism of action of the gallic acid esters would be similar to the results found by Abe et al. (2000) [41]. These authors showed that some gallates are potent inhibitors of the enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol [42] and, therefore, is essential for the survival of the microorganism.

Ergosterol is essential to the parasite because it is the most abundant sterol found in the membrane of lower eukaryotes and is therefore found in fungi and parasitic protozoa such as Leishmania and Trypanosoma [4346].

The inhibition of sterol biosynthesis is associated with the loss of cell membrane fluidity, which might lead to the parasite death through the same mechanism of action of commercial drugs, such as terbinafine [42].

Mitochondria are one of the first organelles to be affected after treatment of T. cruzi with ergosterol biosynthesis inhibitors, and even stronger effect may occur when an inhibitor of squalene epoxidase is used [3647].

Due to the results of our experiments that demonstrated that there are changes in the mitochondrial membrane potential of the parasites when treated with gallates, we suggest that these compounds cause the parasite death by more than one pathway, which probably includes loss of mitochondrial membrane potential, correlated with the induction of apoptosis and, also, interfering in the biosynthesis of ergosterol in the parasites.

The phenol moiety is often associated with cytotoxic effects due to the possible free radical or reactive oxygen species formation, which might be deleterious to the parasite metabolism.

4. Conclusions

This study demonstrated that gallic acid is not very toxic to T. cruzi epimastigotes. However its esterification yielding gallate esters with different side chain lengths afforded compounds with strong trypanocidal potential, which is probably also due to a change in the mitochondrial membrane potential and an interference in the biosynthesis of ergosterol in the parasites [48]. It is important to clarify that previous studies demonstrate a correlation between epimastigote and trypomastigote forms of T. cruzi, the last one the infective form. Thus, this research encourages the development of new research aiming to apply the gallates as an alternative to current therapy against Chagas disease.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors acknowledge the financial support from the State of São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)), CNPq, PADC-FCF-UNESP, and Coordination for the Improvement of Higher Education Personnel (Capes, Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior) to this research.

References

  1. P. J. Hotez, D. H. Molyneux, A. Fenwick et al., “Control of neglected tropical diseases,” The New England Journal of Medicine, vol. 357, no. 10, pp. 1018–1027, 2007. View at: Publisher Site | Google Scholar
  2. World Health Organization (WHO), 2010, http://www.who.int/mediacentre/factsheets/fs340/en/.
  3. H. B. Tanowitz, L. V. Kirchhoff, D. Simon, S. A. Morris, L. M. Weiss, and M. Wittner, “Chagas’ disease,” Clinical Microbiology Reviews, vol. 5, no. 4, pp. 400–419, 1992. View at: Google Scholar
  4. A. Prata, “Clinical and epidemiological aspects of Chagas disease,” The Lancet Infectious Diseases, vol. 1, no. 2, pp. 92–100, 2001. View at: Publisher Site | Google Scholar
  5. S. S. Estani, E. L. Segura, A. M. Ruiz, E. Velazquez, B. M. Porcel, and C. Yampotis, “Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas’ disease,” The American Journal of Tropical Medicine and Hygiene, vol. 59, no. 4, pp. 526–529, 1998. View at: Google Scholar
  6. J. D. Maya, B. K. Cassels, P. Iturriaga-Vásquez et al., “Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host,” Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, vol. 146, no. 4, pp. 601–620, 2007. View at: Publisher Site | Google Scholar
  7. J. R. Cançado, “Long term evaluation of etiological treatment of Chagas disease with benznidazole,” Revista do Instituto de Medicina Tropical de São Paulo, vol. 44, no. 1, pp. 29–37, 2002. View at: Publisher Site | Google Scholar
  8. D. A. Saúde-Guimarães and A. R. Faria, “Substâncias da natureza com atividade anti-Trypanosoma cruzi,” Revista Brasileira de Farmacognosia, vol. 17, no. 3, pp. 455–465, 2007. View at: Publisher Site | Google Scholar
  9. S. G. Andrade and J. B. Magalhães, “Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology,” Revista da Sociedade Brasileira de Medicina Tropical, vol. 30, no. 1, pp. 27–35, 1996. View at: Google Scholar
  10. A. Kanaujia, R. Duggar, S. T. Pannakal et al., “Insulinomimetic activity of two new gallotannins from the fruits of Capparis moonii,” Bioorganic and Medicinal Chemistry, vol. 18, no. 11, pp. 3940–3945, 2010. View at: Publisher Site | Google Scholar
  11. K. Dodo, T. Minato, T. Noguchi-Yachide, M. Suganuma, and Y. Hashimoto, “Antiproliferative and apoptosis-inducing activities of alkyl gallate and gallamide derivatives related to (-)-epigallocatechin gallate,” Bioorganic and Medicinal Chemistry, vol. 16, no. 17, pp. 7975–7982, 2008. View at: Publisher Site | Google Scholar
  12. C. Locatelli, R. Rosso, M. C. Santos-Silva et al., “Ester derivatives of gallic acid with potential toxicity toward L1210 leukemia cells,” Bioorganic and Medicinal Chemistry, vol. 16, no. 7, pp. 3791–3799, 2008. View at: Publisher Site | Google Scholar
  13. C. Locatelli, P. C. Leal, R. A. Yunes, R. J. Nunes, and T. B. Creczynski-Pasa, “Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: the relationship between free radical generation, glutathione depletion and cell death,” Chemico-Biological Interactions, vol. 181, no. 2, pp. 175–184, 2009. View at: Publisher Site | Google Scholar
  14. M. C. C. Morais, S. Luqman, T. P. Kondratyuk et al., “Suppression of TNF-α induced NFκB activity by gallic acid and its semi-synthetic esters: possible role in cancer chemoprevention,” Natural Product Research, vol. 24, no. 18, pp. 1758–1765, 2010. View at: Publisher Site | Google Scholar
  15. V. F. Ximenes, M. G. Lopes, M. S. Petrônio, L. O. Regasini, D. H. Siqueira Silva, and L. M. da Fonseca, “Inhibitory effect of gallic acid and its esters on 2,2′-azobis(2- amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione in erythrocytes,” Journal of Agricultural and Food Chemistry, vol. 58, no. 9, pp. 5355–5362, 2010. View at: Publisher Site | Google Scholar
  16. R. Rosso, T. O. Vieira, P. C. Leal, R. J. Nunes, R. A. Yunes, and T. B. Creczynski-Pasa, “Relationship between the lipophilicity of gallic acid n-alquil esters’ derivatives and both myeloperoxidase activity and HOCl scavenging,” Bioorganic and Medicinal Chemistry, vol. 14, no. 18, pp. 6409–6413, 2006. View at: Publisher Site | Google Scholar
  17. L. O. Regasini, D. C. Fernandes, I. Castro-Gamboa et al., “Chemical constituents of the flowers of Pterogyne nitens (Caesalpinioideae),” Quimica Nova, vol. 31, no. 4, pp. 802–806, 2008. View at: Publisher Site | Google Scholar
  18. I. Kubo, P. Xiao, and K. Fujita, “Antifungal activity of octyl gallate: structural criteria and mode of action,” Bioorganic and Medicinal Chemistry Letters, vol. 11, no. 3, pp. 347–350, 2001. View at: Publisher Site | Google Scholar
  19. K.-I. Fujita and I. Kubo, “Antifungal activity of octyl gallate,” International Journal of Food Microbiology, vol. 79, no. 3, pp. 193–201, 2002. View at: Publisher Site | Google Scholar
  20. P. C. Leal, A. Mascarello, M. Derita et al., “Relation between lipophilicity of alkyl gallates and antifungal activity against yeasts and filamentous fungi,” Bioorganic and Medicinal Chemistry Letters, vol. 19, no. 6, pp. 1793–1796, 2009. View at: Publisher Site | Google Scholar
  21. I. Kubo, P. Xiao, and K. Fujita, “Anti-MRSA activity of alkyl gallates,” Bioorganic and Medicinal Chemistry Letters, vol. 12, no. 2, pp. 113–116, 2002. View at: Publisher Site | Google Scholar
  22. I. Kubo, K.-I. Fujita, and K.-I. Nihei, “Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA),” Bioorganic & Medicinal Chemistry, vol. 11, no. 19, pp. 4255–4262, 2003. View at: Publisher Site | Google Scholar
  23. L. H. P. Silva and V. Nussenzweig, “Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco,” Folia Clínica et Biologica, vol. 20, pp. 191–208, 1953. View at: Google Scholar
  24. J. F. Fernandes and O. Castellani, “Growth characteristics and chemical composition of Trypanosoma cruzi,” Experimental Parasitology, vol. 18, no. 2, pp. 195–202, 1966. View at: Publisher Site | Google Scholar
  25. S. Muelas-Serrano, J. J. Nogal, R. A. Martínez-Díaz, J. A. Escario, A. R. Martínez-Fernández, and A. Gómez-Barrio, “In vitro screening of American plant extracts on Trypanosoma cruzi and Trichomonas vaginalis,” Journal of Ethnopharmacology, vol. 71, no. 1-2, pp. 101–107, 2000. View at: Publisher Site | Google Scholar
  26. F. Cotinguiba, L. O. Regasini, V. S. Bolzani et al., “Piperamides and their derivatives as potential anti-trypanosomal agents,” Medicinal Chemistry Research, vol. 18, no. 9, pp. 703–711, 2009. View at: Publisher Site | Google Scholar
  27. A. Cossarizza, M. Baccarani-Contri, G. Kalashnikova, and C. Franceschi, “A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1),” Biochemical and Biophysical Research Communications, vol. 197, no. 1, pp. 40–45, 1993. View at: Publisher Site | Google Scholar
  28. G. V. Kulkarni, W. Lee, A. Seth, and C. A. G. McCulloch, “Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts,” Experimental Cell Research, vol. 245, no. 1, pp. 170–178, 1998. View at: Publisher Site | Google Scholar
  29. F.-L. Hsu, H.-T. Chang, and S.-T. Chang, “Evaluation of antifungal properties of octyl gallate and its synergy with cinnamaldehyde,” Bioresource Technology, vol. 98, no. 4, pp. 734–738, 2007. View at: Publisher Site | Google Scholar
  30. J. K. Hellmann, S. Münter, M. Wink, and F. Frischknecht, “Synergistic and additive effects of epigallocatechin gallate and digitonin on Plasmodium sporozoite survival and motility,” PLoS ONE, vol. 5, no. 1, Article ID e8682, 2010. View at: Publisher Site | Google Scholar
  31. J. A. Urbina, K. Lazardi, T. Aguirre, M. M. Piras, and R. Piras, “Antiproliferative synergism of the allylamine SF 86-327 and ketoconazole on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi,” Antimicrobial Agents and Chemotherapy, vol. 32, no. 8, pp. 1237–1242, 1988. View at: Publisher Site | Google Scholar
  32. J. A. Urbina, J. Vivas, G. Visbal, and L. M. Contreras, “Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole,” Molecular and Biochemical Parasitology, vol. 73, no. 1-2, pp. 199–210, 1995. View at: Publisher Site | Google Scholar
  33. J. A. Urbina, J. L. Concepcion, S. Rangel, G. Visbal, and R. Lira, “Squalene synthase as a chemotherapeutic target in Trypanosoma cruzi and Leishmania mexicana,” Molecular and Biochemical Parasitology, vol. 125, no. 1-2, pp. 35–45, 2002. View at: Publisher Site | Google Scholar
  34. J. A. Urbina, “Ergosterol biosynthesis and drug development for Chagas disease,” Memórias do Instituto Oswaldo Cruz, vol. 104, no. 1, pp. 311–318, 2009. View at: Publisher Site | Google Scholar
  35. J. D. Ly, D. R. Grubb, and A. Lawen, “The mitochondrial membrane potential (Δψm) in apoptosis; an update,” Apoptosis, vol. 8, no. 2, pp. 115–128, 2003. View at: Google Scholar
  36. W. de Souza, M. Attias, and J. C. F. Rodrigues, “Particularities of mitochondrial structure in parasitic protists (Apicomplexa and Kinetoplastida),” International Journal of Biochemistry and Cell Biology, vol. 41, no. 10, pp. 2069–2080, 2009. View at: Publisher Site | Google Scholar
  37. M. Sands, M. A. Kron, and R. B. Brown, “Pentamidine: a review,” Reviews of Infectious Diseases, vol. 7, no. 5, pp. 625–634, 1985. View at: Publisher Site | Google Scholar
  38. A. E. Vercesi and R. Docampo, “Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ,” Biochemical Journal, vol. 284, no. 2, pp. 463–467, 1992. View at: Google Scholar
  39. S. L. Croft and R. P. Brazil, “Effect of pentamidine isethionate on the ultrastructure and morphology of Leishmania mexicana amazonensis in vitro,” Annals of Tropical Medicine and Parasitology, vol. 76, no. 1, pp. 37–43, 1982. View at: Google Scholar
  40. B. Hentzer and T. Kobayasi, “The ultrastructural changes of Leishmania tropica after treatment with pentamidine,” Annals of Tropical Medicine and Parasitology, vol. 71, no. 2, pp. 157–166, 1977. View at: Google Scholar
  41. I. Abe, T. Seki, and H. Noguchi, “Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters,” Biochemical and Biophysical Research Communications, vol. 270, no. 1, pp. 137–140, 2000. View at: Publisher Site | Google Scholar
  42. W. de Souza and J. C. F. Rodrigues, “Sterol biosynthesis pathway as target for anti-trypanosomatid drugs,” Interdisciplinary Perspectives on Infectious Diseases, vol. 2009, Article ID 642502, 19 pages, 2009. View at: Publisher Site | Google Scholar
  43. K. E. Bloch, “Sterol structure and membrane function,” CRC Critical Reviews in Biochemistry, vol. 14, no. 1, pp. 47–92, 1983. View at: Publisher Site | Google Scholar
  44. A. Arora, H. Raghuraman, and A. Chattopadhyay, “Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach,” Biochemical and Biophysical Research Communications, vol. 318, no. 4, pp. 920–926, 2004. View at: Publisher Site | Google Scholar
  45. E. Zinser, C. D. M. Sperka-Gottlieb, E.-V. Fasch, S. D. Kohlwein, F. Paltauf, and G. Daum, “Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae,” Journal of Bacteriology, vol. 173, no. 6, pp. 2026–2034, 1991. View at: Google Scholar
  46. N. Mbongo, P. M. Loiseau, M. A. Billion, and M. Robert-Gero, “Mechanism of amphotericin B resistance in Leishmania donovani promastigotes,” Antimicrobial Agents and Chemotherapy, vol. 42, no. 2, pp. 352–357, 1998. View at: Google Scholar
  47. K. Lazardi, J. A. Urbina, and W. de Souza, “Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi,” Antimicrobial Agents and Chemotherapy, vol. 34, no. 11, pp. 2097–2105, 1990. View at: Publisher Site | Google Scholar
  48. Z. J. Molina-Garza, A. F. Bazaldúa-Rodríguez, R. Quintanilla-Licea, and L. Galaviz-Silva, “Anti-Trypanosoma cruzi activity of 10 medicinal plants used in northeast Mexico,” Acta Tropica, vol. 136, no. 1, pp. 14–18, 2014. View at: Publisher Site | Google Scholar

Copyright © 2015 Rogério Andréo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

919 Views | 388 Downloads | 3 Citations
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19.