Abstracts

Effects of seco-steroids purified from Physalis angulata L., Solanaceae, on the viability of Leishmania sp

Efeitos de seco-esteróides purificados de Physalis angulata L., Solanaceae na viabilidade de Leishmania sp

Elisalva T. Guimarães^I; Milena S. Lima^I; Luana A. Santos^I; Ivone M. Ribeiro^II; Therezinha B. C. Tomassini^II; Ricardo Ribeiro dos Santos^{I, III}; Washington L. C. dos Santos^I; Milena B. P. SoaresI, III, ^* * E-mail: milena@bahia.fiocruz.br, Tel. +55 71 3176 2260; Fax: +55 71 3176 2272.

^ICentro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão 121, 40296-710 Salvador-BA, Brazil

^IIFarManguinhos, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, 21041-050 Rio de Janeiro, RJ, Brazil

^IIICentro de Biotecnologia e Terapia Celular, Hospital São Rafael, Avenida São Rafael 2152,41253-190 Salvador, BA, Brazil

RESUMO

Physalis angulata L., Solanaceae, é uma erva anual utilizada na medicina popular em muitos países tropicais e subtropicais. Apesar dos extratos da P. angulata apresentarem uma grande variedade de substâncias, pouco é conhecido sobre a sua atividade farmacológica. Neste trabalho foi investigado a atividade antileishmania in vitro de seco-esteroides (fisalinas) purificados da P. angulata. O tratamento com as fisalinas B, F e G causou uma inibição concentração-dependente do crescimento de promastigotas de Leishmania amazonensis em cultura axênica, com valores de IC50 de 6,8, 1,4, e 9,2 μM respectivamente. A fisalina D foi menos ativa, com valores de IC50 de 30,5 μM. Foi também observada uma atividade leishmanicida em culturas de outras espécies de Leishmania (L. major, L. braziliensis e L. chagasi). Nossos resultados demonstram que as fisalinas inibem o crescimento dos promastigotas com o tratamento de espécies de Leishmania do Velho e do Novo Mundos e sugerem o potencial terapêutico destas moléculas na leishmaniose.

Unitermos:Leishmania sp., fisalinas, promastigotas, seco-esteroides.

ABSTRACT

Physalis angulata L., Solanaceae, is an annual herb commonly used in popular medicine in many tropical and subtropical countries. P. angulata extracts contain a variety of substances, but little is known about their pharmacological activities. In this work we investigated the in vitro antileishmanial activity of seco-steroids (physalins) purified from P. angulata. Addition of physalins B, F, and G caused a concentration-dependent inhibition in the growth of L. amazonensis promastigotes, being the IC50 values were 6.8, 1.4, and 9.2 μM, respectively. Physalin D was less active and had an IC50 value of 30.5 μM. Physalins were also active in cultures of other Leishmania species (L. major, L. braziliensis, and L. chagasi). Our results demonstrate the potent antileishmanial activity of physalins in cultures of Leishmania species of the New and Old Worlds and suggest the therapeutic potential of these seco-steroids in leishmaniasis.

Keywords:Leishmania sp., physalins, promastigotes, seco-steroids.

INTRODUCTION

Despite of the advances in the understanding of the immunology and molecular biology of leishmaniasis, the drugs available for the treatment of this illness still present today many adverse effects, such as acute toxicity and rapid development of drug resistance (Bryceson, 2001). The drugs most used in the treatment of leishmaniasis are pentavalent antimonials, which have been used for more than 50 years (Iwu et al., 1994; Carvalho et al., 2001). Some lipid-based formulations of the amphotericin B, such as AmBisome, are now indicated in the treatment of first-line against visceral leishmaniasis in developed countries (Larabi et al., 2003). The search of new chemotherapies is therefore a focus of research in this disease. The use of natural products in the treatment of a variety of diseases has also increased due to the considerable number of medicinal plants with proven biological activity applicable to the treatment of some diseases.

Physalis angulata L., Solanaceae, is an annual herb distributed in many countries located in tropical and subtropical regions of the world. This plant is widely used in popular medicine as a treatment for a variety of diseases, and has antitumoral activity already demonstrated (Lin et al., 1992; Chiang et al., 1992). In addition, this plant has compounds with anti-T. cruzi activity (Nagafuji et al., 2004; Abe et al., 2006). Physalin H, isophysalin B and others isolated of Physalis minima have shown leishmanicidal activity against the promastigotes of Leishmania major (Choudary et al., 2005; Choudary et al., 2007). We have investigated the activities of a group of seco-steroids isolated from P. angulata, known as physalins, and demonstrated the immunomodulatory activity of physalins in vitro and in vivo (Soares et al., 2003; Soares et al., 2006). In this work we investigated the antileishmanial effects of physalins B (1), D (2), F (3), and G (4), in vitro against promastigotes of Leishmania sp. Our results suggest that these drugs could be candidate for treatment of leishmaniasis.

MATERIALS AND METHODS

Parasites and drugs

L. amazonensis (MHOM/BR88/BA-125 Leila strain), L. major (MHOM/RI/-/WR-173), L. chagasi (MHOM/BR2000/Merivaldo2), and L. braziliensis (MHOM/BR88/BA-3456) promastigotes were cultivated in liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), 5% sterile urine and 50 μg/mL gentamycin (Sigma, St. Louis, MO, USA), pH 7.2, at 26 ºC until logarithmic phase. Physalins B (1), D (2), F (3), and G (4) were obtained from plant specimens collected in Belém do Pará, Brazil, as described before (Soares et al., 2003).

Antileishmanial assay

Promastigotes of L. amazonensis, L. major, L. chagasi, and L. braziliensis were grown in axenic cultures (seeding 2x 10⁶ in 5 mL) were incubated with 2, 5 or 15 μg/mL of physalins B, D, F and G. The parasites were observed and counted using a Neubauer chamber. Parasite growth inhibition was evaluated up to 5-7 days of treatment.

Statistical analyses

For comparing multiple groups with matched observations Friedman test was used followed by Dunns test for comparing selected groups. The inhibitory concentrations for 50% (IC50) of Leishmania growth, at fifth or seventh days of incubation, were calculated based in a nonlinear regression (curve fit) and the statistical analyses were made by one-way ANOVA and Newman-Keuls multiple comparison test using Prism version 4.00 for Windows, GraphPad Software (San Diego, CA). The critical level of significance was established for p<0.05.

RESULTS

Physalins B, G, and F, but not D, inhibit the growth of Leishmania spp.

To evaluate the anti-leishmania activity of physalins, promastigotes of L. amazonensis, L. major, L. braziliensis, and L. chagasi were incubated with different concentrations of the different physalins in axenic cultures. Treatment of parasites with physalin B resulted in a concentration-dependent inhibition on the proliferation of promastigotes (Figure 1). This drug reduced completely the promastigotes number since the first day of treatment when the concentration of 15 μg/mL was used. In addition, physalins F and G inhibited the growth of L. amazonensis and L. major, whereas addition of physalin D did not significantly alter the growth of these two parasites even at 15 μg/mL (Figure 2). Physalin F was more active against promastigotes than physalins B or G, and inhibited 100% the promastigotes when the smaller concentration was used. The estimation of IC50 values (Table 1) confirmed that physalin F was the most active (1.4 μM), having an activity comparable to the IC50 of amphotericin B (3.0 μM), a standard antileishmanial drug. Physalin D was the least active of the physalins tested, with an IC50 of 30.5 μM.

DISCUSSION

Previous studies have shown that physalin H, isophysalin B, 5 beta, 6 beta-epoxyphysalin B and others isolated of Physalis minima have in vitro antileishmanial activitiy against promastigotes of L. major (Choudhary et al., 2005; Choudhary et al., 2007). In the present study we demonstrated the antileishmanial activity of physalins B, F, and G isolated from Physalis angulata in vitro in axenic cultures of different Leishmania species, including L. amazonensis, L. braziliensis, L. chagasi (New World), and L. major (Old World).

Whereas physalins B, F, and G had potent antileishmanial activity, this was not found with physalin D, which had a high IC50 value. The lack of immunomodulatory/anti-inflammatory activity by physalin D, but not of physalins B, F, and G, has been described before (Soares et al., 2003; Soares et al., 2006). In addition, Jacobo-Herrera (2006) also found that physalins B and F, but not D, inhibited the activation of NF-kB, an important transcription factor in inflammatory responses, and suggested that the presence of a double bond and an epoxy ring between carbons 5 and 6 in physalins B and F, respectively, which are not present in physalin D, are critical for the anti-inflammatory activity.

Although some reports have demonstrated a lack of activity of physalin D, Januario (2002) reported an antimycobacterial activity of physalin D, in addition to physalin B. Magalhães (2006) also showed that both physalins B and D have antitumoral activity, although Chiang (1992) reported lack of activity of physalin D in human leukemia cells in vitro.

In conclusion, this study demonstrated an anti-leishmania action of physalins B, F, and G against different species, with IC50 values comparable to that of amphotericin B, a drug of reference for leishmaniasis treatment. The in vivo activity of physalin F has been demonstrated in a model of cutaneous leishmaniasis in BALB/c mice infected with L. amazonensis. These results suggest the use of physalins as an alternative drug in treatment of the cutaneous leishmaniasis.

ACKNOWLEDGEMENTS

This work was supported by FIOCRUZ/PDTIS, CNPq, Institutos do Milênio, IMSEAR, MCT, FINEP, RENORBIO, and BIOINOVA.

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