Elsevier

Experimental Parasitology

Volume 166, July 2016, Pages 21-28
Experimental Parasitology

Full length article
Antileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis

https://doi.org/10.1016/j.exppara.2016.03.026Get rights and content

Highlights

  • The antileishmanial activity of a water extract of ginger was investigated.

  • A fraction was the responsible by this biological activity against L. amazonensis.

  • The main chemical constituents of the fraction were flavonoids and saponins.

  • A low toxicity and a null hemolytic activity were found.

  • The mechanism of action of this fraction was mainly due to a nitric oxide production.

Abstract

In recent years, considerable attention has been given to identify new antileishmanial products derived from medicinal plants, although, to date, no new effective compound has been recently applied to treat leishmaniasis. In the present study, the antileishmanial activity of a water extract from Zingiber officinalis Roscoe (ginger) was investigated and a purified fraction, named F10, was identified as responsible by this biological activity. The chemical characterization performed for this fraction showed that it is mainly composed by flavonoids and saponins. The water extract and the F10 fraction presented IC50 values of 125.5 and 49.8 μg/mL, respectively. Their selectivity indexes (SI) were calculated and values were seven and 40 times higher, respectively, in relation to the value found for amphotericin B, which was used as a control. Additional studies were performed to evaluate the toxicity of these compounds in human red blood cells, besides of the production of nitrite, as an indicator of nitric oxide (NO), in treated and infected macrophages. The results showed that both F10 fraction and water extract were not toxic to human cells, and they were able to stimulate the nitrite production, with values of 13.6 and 5.4 μM, respectively, suggesting that their biological activity could be due to macrophages activation via NO production. In conclusion, the present study shows that a purified fraction from ginger could be evaluated in future works as a therapeutic alternative, on its own or in association with other drugs, to treat disease caused by L. amazonensis.

Introduction

Leishmaniasis presents a high morbidity and mortality throughout the world, where 350 million people in 98 countries are at risk of contracting the infection (WHO, 2010). Moreover, approximately 1.0–1.5 million new cases of tegumentary leishmaniasis (TL), and 200,000 to 500,000 new cases of visceral leishmaniasis (VL) have been registered annually (Alvar et al., 2012). Several geographical regions in the world are endemic for multiple Leishmania species, which the case of the South America, where leishmaniasis are caused by at least eight different parasite species, each one with their own virulence and pathogenesis determinants, although many of them displaying common transmission areas (Grimaldi and Tesh, 1993, Reithinger et al., 2007). In addition, leishmaniasis has gained higher importance in HIV co-infected patients, as an opportunistic disease in areas where both infections are endemic (Desjeux, 2004, Alvar et al., 2008, Shafiei et al., 2014).

The treatment of leishmaniasis has been based on the administration of pentavalent antimonials. The parenteral injection of these drugs is still the first choice therapy, however, parasites resistance and side effects such as myalgia, arthralgia, anorexia, fever and urticarial; besides of the toxicity in the liver, heart, kidneys and spleen observed in the patients have limited their use in the clinical practice (Berman, 2003, Croft and Coombs, 2003, Oliveira et al., 2011). Amphotericin B (AmpB), a polyenic antibiotic with antileishmanial activity, presents high affinity for the ergosterol present in the parasites membrane. Liposomal AmpB is considered expensive; however, it is less toxic than free AmpB. Pentamidine is a dibenzamidine that interferes in the synthesis of Leishmania DNA, leading to the parasites death. Its adverse effects include hypotension, myalgia, abscess at the injection site and hypoglycemia. Miltefosine inhibits the phospholipid and sterol biosynthesis in the parasites, but it can cause toxicity to the gastrointestinal, hepatic, and renal systems; besides of being considered teratogenic, which restricts its use in pregnant women (Goto and Lindoso, 2010). Paromomycin is a broad spectrum aminoglycoside antibiotic, which was found to have an antileishmanial activity. The main side effects are ototoxicity and local pain upon injection (Croft et al., 2006, Lindoso et al., 2012, Seifert, 2011). In general, the high cost and/or the several side effects registered have limited the clinical use of these compounds (Kedzierski et al., 2009, Murray et al., 2005, Singh et al., 2012). Therefore, the development of new and cost-effective alternative therapeutic strategies to treat leishmaniasis could be considered a priority (Ribeiro et al., 2014a, Valadares et al., 2012a, Valadares et al., 2012b).

Natural products traditionally have played an important role in drug discovery, being the basis of most early medicine (Newman et al., 2003; Butler, 2005). In recent years, considerable attention has been given to secondary compounds derived from plants, in an attempt to search for new antileishmanial products (Dagnino et al., 2015, Schmidt et al., 2012). A variety of purification techniques have been employed to identify molecules in plants, thereby, supporting the development of new therapeutics (González-Coloma et al., 2012, Lage et al., 2013). Although several studies have used extracts and/or purified compounds presenting antileishmanial activity (Ribeiro et al., 2014b, Ribeiro et al., 2015, Rosa et al., 2010), an effective therapeutic to treat the disease have not been developed yet.

Ginger (Zingiber officinale Roscoe, Zingiberaceae) is one of the commonly used plant species around the world, being found mainly in the South-Eastern Asian countries. Ginger is a medicinal plant used in Chinese, Ayurvedic and Unani-Tibb medicine (Rong et al., 2009). Studies have been developed to evaluate the biological activity of ginger (Ajazuddin et al., 2014, Ding et al., 2013, Viljoen et al., 2014). Recently, the feasibility of using this plant to treat parasitic diseases has received considerable attention, since ginger can eliminate Angiostrongylus cantonensis (Lin et al., 2010a), Dirofilaria immitis (Merawin et al., 2010), Anisakis simplex (Lin et al., 2010b), Hymenolepis nana (Lin et al., 2014), Schistosoma mansoni (Aly and Mantawy, 2013), as well as act against gastrointestinal nematodes (Iqbal et al., 2006).

In this context, the present study evaluated the in vitro antileishmanial activity of a water extract derived from ginger, as well as its 25 purified fractions, using stationary-phase promastigotes and intracellular amastigotes of Leishmania amazonensis. After selecting the most active fraction against parasites, additional studies were employed to chemically characterize this fraction, as well as to evaluate its minimum Leishmania inhibitory concentration (IC50), and its cytotoxic effect on murine macrophages (CC50) or in human red blood cells. The antileishmanial effect on intra-macrophage parasite stages, besides of the mechanism of action based on the nitric oxide (NO) production, was also evaluated.

Section snippets

Chemicals and plant material

Reagents and solvents were obtained from commercial sources, and were used as described. The ginger rhizomes were obtained from a rural area of Belo Horizonte, Minas Gerais, Brazil; and the botanical identification was performed at the Department of Botanical, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Minas Gerais, Brazil.

Preparation of the water extract and purification of ginger fractions

The water extract of ginger was prepared by maceration of 500 g of fresh rhizome in 100 mL of a 10 mM Tris–HCl solution, pH 7.2, by using a

Antileishmanial activity, cytotoxicity and hemolytic activity of the water extract and F10 fraction from ginger

The inhibition of Leishmania viability using the ginger' water extract was evaluated using stationary-phase promastigotes of L. amazonensis. In the results, it was observed that the water extract was effective against the parasites, presenting an IC50 value of 125.5 μg/mL. Then, a gel filtration chromatography fractionation was performed, aiming to separate the biomolecules fractions based on differences in their size. This technique is usually applied for large biomolecules such as saponins or

Discussion

Natural products have been important sources of chemical diversity to discovery new pharmaceutical compounds over the past century (Firn and Jones, 2003). The interesting chemicals identified in these products are possibly derived from the phenomenon of biodiversity, in which the interactions among organisms and their environments formulate the diverse chemical entities allowing their survival and competitiveness (Lee, 2010). The therapeutic areas of infectious diseases have benefited from

Conclusion

The results presented here demonstrate the identification and first use of an antileishmanial and atoxic product, based on one purified fraction from ginger. This product was chemically evaluated, being composed mainly by flavonoids and saponins, and could be evaluated in future studies for the treatment of L. amazonensis infection in murine models.

Acknowledgments

This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-NanoBiofar), FAPEMIG (CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, APQ-482976/2012-8, and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and AAGF are grant recipient of CNPq.

References (74)

  • M. Ding et al.

    A systematic review of the evidence for topical use of ginger

    Explore (NY)

    (2013)
  • L.M. Garcez et al.

    Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: a model for vaccine trials

    Int. J. Parasitol.

    (2002)
  • Z. Iqbal et al.

    In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep

    J. Ethnopharmacol.

    (2006)
  • H.K. Kim et al.

    Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships

    Biochem. Pharmacol.

    (1999)
  • P.E. Kima

    The amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection and persist

    Int. J. Parasitol.

    (2007)
  • R.J. Lin et al.

    Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis

    Acta Trop.

    (2010)
  • D. Mandal et al.

    A triterpenoid saponin possessing antileishmanial activity from the leaves of Careya arborea

    Phytochemistry

    (2006)
  • N. Mascolo et al.

    Ethnopharmacologic investigation of ginger (Zingiber officinale)

    J. Ethnopharmacol.

    (1989)
  • H. Matsuda et al.

    Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

    Bioorg. Med. Chem.

    (2003)
  • L.T. Merawin et al.

    Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

    Res. Vet. Sci.

    (2010)
  • B.B. Mishra et al.

    Natural products: an evolving role in future drug discovery

    Eur. J. Med. Chem.

    (2011)
  • H.W. Murray et al.

    Advances in leishmaniasis

    Lancet

    (2005)
  • L.F. Oliveira et al.

    Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the New World

    Acta Trop.

    (2011)
  • R. Reithinger et al.

    Cutaneous leishmaniasis

    Lancet Infect. Dis.

    (2007)
  • T.G. Ribeiro et al.

    Antileishmanial activity and cytotoxicity of Brazilian plants

    Exp. Parasitol.

    (2014)
  • T.G. Ribeiro et al.

    Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds

    J. Ethnopharmacol.

    (2015)
  • X. Rong et al.

    A 35-day gavage safety assessment of ginger in rats

    Regul. Toxicol. Pharmacol.

    (2009)
  • F.N. Santos et al.

    Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune vaccine

    Vaccine

    (2007)
  • R. Shafiei et al.

    Emergence of co-infection of visceral leishmaniasis in HIV-positive patients in northeast Iran: a preliminary study

    Travel. Med. Infect. Dis.

    (2014)
  • Y. Shukla et al.

    Cancer preventive properties of ginger: a brief review

    Food Chem. Toxicol.

    (2007)
  • N. Singh et al.

    Leishmaniasis: current status of available drugs and new potential drug targets

    Asian pac. J. Trop. Med.

    (2012)
  • Y. Sivasothy et al.

    Essential oils of Zingiber officinalis var. rubrum Theilade and their antibacterial activities

    Food Chem.

    (2011)
  • D.G. Valadares et al.

    Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species

    Parasitol. Int.

    (2011)
  • D.G. Valadares et al.

    Prophylactic or therapeutic administration of Agaricus blazei Murill is effective in treatment of murine visceral leishmaniasis

    Exp. Parasitol.

    (2012)
  • A. Ajazuddin et al.

    Role of herbal bioactives as a potential bioavailability enhancer for active pharmaceutical ingredients

    Fitoterapia

    (2014)
  • J.F.T.K. Akoachere et al.

    Antibacterial effect of Zingiber officinale and Garcinia kola on respiratory tract pathogens

    East. Afr. Med. J.

    (2002)
  • J. Alvar et al.

    The relationship between leishmaniasis and AIDS: the second 10 years

    Clin. Microbiol. Rev.

    (2008)
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