Full length articleAntileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis
Graphical abstract
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.
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