Elsevier

Journal of Ethnopharmacology

Volume 192, 4 November 2016, Pages 108-113
Journal of Ethnopharmacology

Stachytarpheta cayennensis extract inhibits promastigote and amastigote growth in Leishmania amazonensis via parasite arginase inhibition

https://doi.org/10.1016/j.jep.2016.07.044Get rights and content

Abstract

Ethnopharmacology relevance

Stachytarpheta cayennensis is a plant that is traditionally used to treat tegumentary leishmaniasis and as an anti-inflammatory agent.

Aim of the study

This study aimed to evaluate the action of S. cayennensis extracts on the Leishmania (Leishmania) amazonensis arginase enzyme.

Materials and methods: S. cayennensis was collected from the Brazilian Amazon region. Aqueous extracts were fractionated with n-butanol. The leishmanicidal effects of the n-butanolic fraction (BUF) were evaluated in L. (L.) amazonensis promastigotes and amastigotes. BUF was tested against recombinant arginase from both L. (L.) amazonensis and macrophage arginase. Promastigote cultures and infected macrophage cultures were supplemented with L-ornithine to verify arginase inhibition. NMR analysis was used to identify the major components of BUF.

Results

BUF showed an EC50 of 51 and 32 µg/mL against promastigotes and amastigotes of L. (L.) amazonensis, respectively. BUF contains a mixture of verbascoside and isoverbascoside (7:3 ratio) and is a potent L. (L.) amazonensis arginase inhibitor (IC50=1.2 µg/mL), while macrophage arginase was weakly inhibited (IC50>1000 µg/mL). The inhibition of arginase by BUF in promastigotes and amastigotes could be demonstrated by culture media supplementation with L-ornithine, a product of the hydrolysis of L-arginine by arginase.

Conclusions

Leishmanicidal effects of the S. cayennensis BUF fraction on L. (L.) amazonensis are associated with selective parasite arginase inhibition.

Introduction

Leishmaniasis is an anthropozoonosis that affects 12 million people worldwide. The disease is associated with malnutrition, immunodeficiency, ecological imbalance and poverty (WHO, 2016). Until recently, the treatment of leishmaniasis was based on a drug that was developed over a century ago.

Recently, scientists have begun to study plants used in traditional medicine to control Amazonian leishmaniasis in Brazil and Peru, and they have identified a variety of plants to treat leishmaniasis, typically in the form of powdered leaves (Moreira et al., 2002) or shoots (Kvist et al., 2006), in these countries. Extracts of the verbena Stachytarpheta cayennensis are often applied as an antileishmanial agent in the Ucayali Province in Peru (Kvist et al., 2006) and an area of endemic leishmaniasis in the Maranhão region of Brazil (Moreira et al., 2002); both sites are located within the Amazon region. In southern Brazil, S. cayennensis is also used as an anti-inflammatory, analgesic and diuretic agent, and its anti-inflammatory properties have been investigated by pharmacological testing (Alice et al., 1991). The Yanesha, a native ethnic group that inhabits the Peruvian Amazon, consumes the entire plant in the form of a tea for the treatment of an array of illnesses (Céline et al., 2009). The main bioactive compounds of S. cayennensis were identified as verbascoside, isoverbascoside and ipolamiide (Froelich et al., 2008, Penido et al., 2006).

Arginase enzymes play an essential role in the production of L-ornithine, which is used by the parasite to produce polyamines (Colotti and Ilari, 2011). Polyamines are involved in parasite growth and to synthesize the antioxidant trypanothione, which neutralizes free radicals, such as nitric oxide (NO) and reactive oxygen species (ROS) (Bocedi et al., 2010), that are produced by host cells as a defense response to infection by pathogens. Whereas inactivation of arginase in Leishmania reduced the infection rate, supplementation with L-ornithine restored parasite infectivity (Reguera et al., 2009). Arginase is synthesized in the cytoplasm and then transported to the glycosome, an organelle that is present in Leishmania. Mutating the arginase tag that normally transports arginase to the glycosome results in arginase retention in the cytoplasm and diminished infection, similar to an arginase gene knockout parasite (da Silva et al., 2012b). It has been shown that arginase can be inhibited by orientin, a flavonoid that is produced by Cecropia pachystachya and contains a catechol group (Cruz et al., 2013). Other flavonoids, both with and without catechol groups, can also inhibit arginase activity (Manjolin et al., 2013).

The overall goal of our research was to uncover the pharmacological mechanisms of action of medicinal plants and to improve the drug design for leishmaniasis control. In this present study, we demonstrate that antileishmanial activity of an enriched verbascoside/isoverbascoside extract from S. cayennensis inhibits Leishmania amazonensis arginase while showing weak inhibition of macrophage arginase.

Section snippets

Plant materials

Aerial parts of Stachytarpheta cayennensis (Rich.) Vahl. were collected in Formosa da Serra Negra, Maranhão, Brazil (6°22′35.5′′S 46°06′36.6′′W). The specimens were identified by the authors and deposited in the ESA herbarium at the Luiz de Queiroz College of Agriculture (ESALQ), University of Sao Paulo, under Voucher no. 132694.

Chemicals

Authentic standard verbascoside and isoverbascoside were obtained from PhytoLab GmbH & Co., Vestenbergsgreuth, Germany. Amphotericin B (Unianf) was obtained from União

Characterization of the primary components of BUF

The HPLC profile of BUF extracts revealed a major peak (90%) with a retention time (RT) of 23.5 min. A mixture of the compounds verbascoside and isoverbascoside (Fig. 1) was identified from the 1H NMR of BUF by comparisons with authentic samples. Duplication of signals for the caffeoyl moiety and the methyl group from rhamnose was essential to identify both compounds in the mixture. Based on the integral, we could also estimate a ratio of 7:3 for the compounds verbascoside and isoverbascoside,

Discussion

The aerial parts of the verbena of Stachytarpheta cayennensis are used in traditional medicine to treat tegumentary leishmaniasis in the Brazilian (Moreira et al., 2002) and Peruvian Amazon regions (Kvist et al., 2006). In this present study, we tested the antileishmanial activities of enriched verbascoside/isoverbascoside extract (BUF) of S. cayennensis and described its inhibitory effects on arginase from L. (L.) amazonensis. The compounds verbascoside and isoverbascoside were identified as

Conclusion

In conclusion, we have shown that butanolic enriched S. cayennensis extract (BUF) can inhibit the bioactivities of the arginase enzyme of L. (L.) amazonensis. The major constituents of BUF are the compounds verbascoside and isoverbascoside. Supplementation with L-ornithine could restore the growth of L. (L.) amazonensis promastigotes and partially restore macrophage infection. These results provide additional evidence that compounds that contain catechol groups can target arginase enzymes.

Acknowledgments

This study was supported by Grant #14/18642-1 from the São Paulo Research Foundation (FAPESP, Brazil). CCM received a fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq – 168248/2014-0).

References (24)

  • A. Bocedi et al.

    Trypanothione efficiently intercepts nitric oxide as a harmless iron complex in trypanosomatid parasites

    FASEB J.

    (2010)
  • G. Colotti et al.

    Polyamine metabolism in Leishmania: from arginine to trypanothione

    Amino Acids

    (2011)
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