Note pubs.acs.org/jnp
In Vitro and in Vivo Effects of (−)-Epigallocatechin 3‑O‑gallate on Leishmania amazonensis Job D. F. Inacio, Marilene M. Canto-Cavalheiro, and Elmo E. Almeida-Amaral* Laboratório de Bioquímica de Tripanosomatideos, Instituto Oswaldo Cruz, Fundaçaõ Oswaldo Cruz, Pavilhão Leônidas Deane, Manguinhos, 21045-900, Rio de Janeiro, Brazil ABSTRACT: (−)-Epigallocatechin 3-O-gallate (1), the most abundant flavanol in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi. The present study reports the effects in vitro and in vivo of 1 on Leishmania amazonensis. L. amazonensisinfected macrophages treated with 1 exhibited a significant reduction of the infection index in a dose-dependent manner, with an IC50 value of 1.6 μM. Oral administration of 1 on L. amazonensis-infected BALB/ c mice (30 mg/kg/day) resulted in a decrease in the lesion size and parasite burden, without altering serological markers of toxicity. These data demonstrate the in vitro and in vivo leishmanicidal effects of compound 1.
L
eishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, results in extensive mortality and morbidity.1−3 Leishmania amazonensis, originally described in the Amazon region, occurs in many parts of Brazil and is the etiological agent of cutaneous or diffuse cutaneous lesions, but can also cause the complete clinical spectrum of leishmaniasis, including visceral infection. Treatment of leishmaniasis is based currently on pentavalent antimonials and amphotericin B, but these drugs present serious problems regarding side effects, variable efficacy, and cost.4−7 The lack of any affordable therapy has necessitated the development of new antileishmanial therapies. Natural products represent a major resource for the discovery of new molecules for the treatment of parasitic diseases.8,9 Several flavonoids have been reported to possess significant antiprotozoal activities.10,11 (−)-Epigallocatechin 3O-gallate (EGCG) (1), the most abundant flavanol constituent of green tea (Camellia sinensis (L.) Kuntze; Theaceae), is widely studied and has generated considerable interest as a biologically active compound with a wide range of potential therapeutic activities,12−15 including the treatment of T. cruzi infections.16 (−)-Epigallocatechin 3-O-gallate is a constituent of the FDAaprproved “botanical drug” sinecatechins, used as a topical treatment for perianal and genital warts.17 Considering that (−)-epigallocatechin 3-O-gallate (1) inhibits the proliferation of L. amazonensis promastigotes,18 the effects of 1 on the intracellular amastigotes of L. amazonensis were tested. L. amazonensis-infected macrophages were incubated in either the presence or absence of 1 (3, 6, and 12 μM) for 72 h. The treatment resulted in a dose-dependent inhibitory effect of the infection index (p < 0.001), with an IC50 value of 1.6 μM (Figure 1a). Compound 1 (12 μM) inhibited the growth of L. amazonensis by 83.6% after 72 h. The cytotoxic © 2013 American Chemical Society and American Society of Pharmacognosy
effect of 1 was evaluated, and a lack of toxicity was observed (Figure 1b). The IC50 value of 1 against murine macrophages was 436.3 μM, which correlates to a selectivity index of 129.4. According to Weniger et al., the biological efficacy of a test compound is not attributable to general cytotoxicity when the selectivity index is ≥10.19 The present results demonstrated a specific antileishmanial activity of (−)-epigallocatechin 3-Ogallate (1) against intracellular amastigotes of L. amazonensis. The antileishmanial potency of 1 was greater than miltefosine, which has already been used successfully for the treatment of New World leishmaniasis20 and has an IC50 value of 3.2 μM at 72 h for L. amazonensis and a selectivity index of 28.9.21 To assess the efficacy of (−)-epigallocatechin 3-O-gallate (1) in vivo, BALB/c mice were infected intradermally with 2 × 106 L. amazonensis promastigotes in the ear and treated orally with 1 (30 mg/kg/day). The oral administration of compound 1 promoted a significant reduction of the lesion size (p < 0.01) (Figure 2a) and effectively reduced the parasite burden (p < 0.001), as compared to the control group (Figure 2b). Received: July 30, 2013 Published: October 9, 2013 1993
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Figure 1. Susceptibility of intracellular amastigotes (a) and murine macrophages (b) to (−)-epigallocatechin 3-O-gallate (1). Macrophages were infected with L. amazonensis promastigotes for 3 h at 37 °C and then incubated in the presence or absence of 1 (3, 6, and 12 μM) for 72 h. The infection index was determined using light microscopy; at least 200 macrophages were counted on each coverslip in duplicate (panel a). Macrophages were incubated with the indicated concentration of 1 for 72 h, and cell viability was measured using the AlamarBlue assay (panel b). The values shown represent the means ± standard error of three independent experiments. In the control samples (absence of 1), a similar volume of vehicle (PBS) was added to the cells. The positive control for reduced cellular viability (disrupted cells) was obtained by adding 0.1% Triton X100 (T: 0.1% Triton X-100). [** indicates a significant difference relative to the control group (p < 0.01)].
Figure 2. In vivo leishmanicidal effect of (−)-epigallocatechin 3-O-gallate (1) using L. amazonensis-infected BALB/c mice. Mice were infected intradermally with 2 × 106 L. amazonensis promastigotes in the right ear. (a) Lesion development on the animals treated orally with 1 (30 mg/kg/ day; open square) or control group, treated orally with sterile PBS (vehicle of 1; closed square) once a day, five times a week. Inset: Lesion size at the end of the experiment (day 52) of the L. amazonensis-infected BALB/c mice untreated or treated with 1 (30 mg/kg/day) or meglumine antimoniate (30 mg/kg/day) once a day, five times a week. (b) Parasite burden of the L. amazonensis-infected BALB/c mice untreated or treated with 1 (30 mg/ kg/day) or meglumine antimoniate (30 mg/kg/day): Ear parasite loads were determined via a limiting dilution assay. (c−e) Parameters of toxicity for the kidneys and liver. At the end of experiment, the mice were euthanized and the serum samples were collected for colorimetric determination of creatinine (panel c), aspartate aminotransferase (AST) (panel d), and alanine aminotransferase (ALT) (panel e) concentrations, as parameters of toxicity for the kidneys and liver, respectively. Data are expressed as means ± standard errors, n = 5 ears. [*, **, and *** indicate significant differences relative to the control group (p < 0.05. p < 0.01. and p < 0.001, respectively)] (CTRL = control; antimonial = meglumine antimoniate; ip = intraperitoneal).
parameters of toxicity for the kidneys and liver, respectively, were observed in mice treated with compound 1 compared to untreated mice (control group). However further specific
Furthermore, no significant differences in the levels of serum creatinine (Figure 2c), aspartate aminotransferase (AST) (Figure 2d), and alanine aminotransferase (ALT) (Figure 2e), 1994
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following infection. (−)-Epigallocatechin 3-O-gallate (1) (30 mg/kg/ day) was diluted in PBS and administered orally once a day, five times a week, until the end of the experiment (day 52), when the animals were euthanized. A control group was treated orally with sterile PBS. Positive controls were treated with an intraperitoneal injection of meglumine antimoniate (30 mg/kg/day), a classic antileishmanial drug, once a day, five times a week, until the end of the experiment (day 52). Lesion sizes were measured twice per week using a dial caliper. This study was approved by the Animal Ethics Committee of the Oswaldo Cruz Foundation (license number LW-07/10). Parasite Load Quantification. Parasite load was determined using a quantitative limiting dilution assay as described previously.22 Briefly, infected ears were excised and weighed before being minced in Schneider’s medium with 10% fetal calf serum, and the resulting cell suspension was diluted serially. The number of viable parasites in each ear was estimated from the highest dilution at which promastigotes could be grown after seven days of incubation at 26 °C. Biochemical Values. Serum levels of aspartate aminotransferase, alanine aminotransferase, and creatinine in the infected BALB/c mice treated orally and intraperitoneally, as described above, were measured using laboratory colorimetric kits (Doles, Goiânia, Brazil). Statistical Analysis. All experiments were performed in three independent trials. The data were analyzed using Student’s t test or analysis of variance (ANOVA) followed by Bonferroni’s post-test in GraphPad Prism 5 (GraphPad Software). Results were considered to be significant when p ≤ 0.05. The data are expressed as means ± standard error.
toxicity studies, such as genotoxicity, remain to be performed. Taken together, these results have shown that compound 1 is orally active in mice, therefore obviating the need for the use of a parenteral route of administration. The decrease in lesion size and parasite load without compromising the overall health of the infected mice is encouraging and supports further studies of (−)-epigallocatechin 3-O-gallate (1) as a candidate for the chemotherapy of leishmaniasis. For example, studies should be conducted to determine the optimum dose and therapeutic regimen.
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EXPERIMENTAL SECTION
Test Compound and Reagents. (−)-Epigallocatechin 3-O-gallate (1) (purity ≥95%; lot 080M1690 V), Schneider’s Drosophila medium, fetal calf serum, and RPMI 1640 medium were obtained from Sigma Aldrich (St Louis, MO, USA). AlamarBlue was obtained from Invitrogen Molecular Probes (Leiden, The Netherlands). Other reagents were purchased from Merck (São Paulo, Brazil). Deionized distilled water was obtained using a Milli-Q system of resins (Millipore Corp., Bedford, MA, USA) and was used in the preparation of all solutions. Endotoxin-free sterile disposables were used in all experiments. Compound 1 was prepared in phosphate-buffered saline (PBS, pH 7.2). Parasites. The MHOM/BR/75/LTB0016 strain of L. amazonensis was used throughout this study. The strain was isolated from a human case of cutaneous leishmaniasis in Brazil. L. amazonensis promastigotes were grown at 26 °C in Schneider’s Drosophila medium (pH 7.2) supplemented with 10% (v/v) heat-inactivated fetal calf serum. Leishmania−Macrophage Interaction Assay. L. amazonensis promastigotes were washed with PBS, counted using a Neubauer chamber, and added to peritoneal macrophages at a multiplicity of infection (MOI) of 3.0. The macrophages were collected from Swiss mice (6−8 weeks old), plated in RPMI at 2 × 106 cells/mL (0.4 mL/ well) in Lab-Tek eight-chamber slides, and then incubated for 3 h at 37 °C in an atmosphere of 5% CO2. The free parasites were removed by successive washes with RPMI. L. amazonensis-infected macrophages were then incubated in the absence or presence of 1 (3, 6, and 12 μM) for 72 h. The percentage of infected macrophages was determined using light microscopy; a minimum of 200 cells on each coverslip were counted randomly in duplicate. The results were expressed as the infection index (% of infected macrophages × number of amastigotes/ total number of macrophages). The IC50 value was determined by logarithmic regression analysis using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Fundaçaõ Oswaldo Cruz. The relevant protocol was approved by the Committee on the Ethics of Animal Experiments of the Fundaçaõ Oswaldo Cruz (license number: LW-07/ 10). Pentamidine (12 μM) was used as reference drug. Viability Assay. Peritoneal macrophages (2 × 106 cells/mL) were allowed to adhere to 96-well tissue culture plates for 1 h at 37 °C in an atmosphere of 5% CO2. Nonadherent cells were removed by washing with RPMI-1640 medium. The adherent macrophages were then incubated with the indicated concentrations of 1 (3 to 3000 μM) for 72 h. The medium was then discarded, and the macrophages were washed with RPMI-1640, after which they were incubated with AlamarBlue (10% v/v) for 12 h at 37 °C in an atmosphere of 5% CO2. The absorbance was measured at 570 nm using a spectrophotometer, and the IC50 value was determined by logarithmic regression analysis using GraphPad Prism 5 (GraphPad Software). The selectivity index was determined as macrophage IC50/intracellular amastigote IC50, as previously described.19 Untreated peritoneal macrophages were lysed by the addition of 0.1% Triton X-100 as a positive control. In Vivo Infection in a Murine Model. BALB/c mice (five animals per group) were maintained under specific pathogen-free conditions. Stationary-phase promastigotes of L. amazonensis (2 × 106 cells in 10 μL of PBS) were inoculated intradermally in the right ear using a 271/2-gauge needle with the treatment beginning seven days
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AUTHOR INFORMATION
Corresponding Author
*E-mail: elmo@ioc.fiocruz.br. Tel: +55(21)3865-8131. Fax: +55(21)3865-8200. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Fundaçaõ Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ grant E-26/110.744/2013 for E.E.A.-A.); Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico/Programa Estratégico de Apoio à Pesquisa em Saúde (CNPq/PAPES grant 407590/2012-9 for E.E.A.-A.); and the Instituto Oswaldo Cruz/Fundaçaõ Oswaldo Cruz (IOC/Fiocruz).
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