Screening of Southeastern Brazilian Mikania. Species on Trypanosoma cruzi.

Abstract

Southeastern Brazilian Mikania. (Asteraceae) species were evaluated for trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi.at a concentration of 4000 µg/mL. Fourteen extracts were examined for in vitro. trypanocidal properties. Of total extracts, 92.9% (13 extracts) exhibited trypanocidal effects. The dichloromethane extract of Mikania camporum. B. Robinson and the methanol extract of Mikania micrantha. H. B. K. caused 100% lysis of the parasites.

Keywords:

• Asteraceae
• Chagas disease
• Mikania
• trypanocidal activity

Introduction

Trypanosoma cruzi. is the causative agent of Chagas disease, which occurs in South and Central America. It is endemic in 21 countries, with 16–18 million persons infected and 100 million people at risk (WHO, 2000). Human hosts are infected either by the triatomide insect vector bite, by blood transfusion, or by congenital transmission (Coura & Castro, 2002). The chronic phase of the disease occurs several years after infection, and the treatment is still a challenge, as the drug commercially available (benznidazole) possesses severe adverse effects, and its activity is limited to the acute phase of the disease (Garcia et al., 2005). Similarly, gentian violet is the only effective chemoprophylactic drug used in blood banks, despite imposed restrictions on its use because of undesirable effects such as coloring of the skin and possible mutagenic effects (Ribeiro et al., 1997). Therefore, effective new drugs for therapeutic treatment are clearly needed.

In this context, higher plants might be considered as a source of new potentially active compounds. For instance, Rojas de Arias et al. (1995), Chiari et al. (1996), Muelas-Serrano et al. (2000), and Taleb-Contini et al. (2004) demonstrated that species belonging to the Asteraceae family have potential trypanocidal activity. The genus Mikania. is the largest genus in the tribe Eupatorieae (Asteraceae), comprising nearly 450 species, which go under the name “guaco” (Herz, 1998). These species have been widely used in folk medicine to treat fever, rheumatism, flu, respiratory tract diseases, and against snakebite (Oliveira et al., 1984; Vilegas et al., 1997; Herz, 1998). This genus is a rich source of terpenoids, mainly sesquiterpene lactones and diterpenes (Herz, 1998), compounds with a variety of biological activities including trypanocidal activity (Picman, 1986; Fournet et al., 1993; Alves et al., 1995; Batista et al., 1999). Recently, we described the in vitro. trypanocidal properties of two isolated terpenoids from Mikania stipulacea. and Mikania hoehnei.. Both compounds, a diterpene and a sesquiterpene lactone, were active toward T. cruzi. tripomastigotes (Nascimento et al., 2004).

In view of these observations, the aim of this study was to determine the in vitro. trypanocidal activity of extracts from Mikania. species.

Materials and Methods

Plant material

The aerial parts of Mikania. species were collected in southeastern Brazil. Specification of species and their original habitats are listed in Table 1. Botanical identification was performed by Dr. Janie Garcia da Silva (Instituto de Biologia da Universidade Federal Fuminense-RJ) and Dr. Roberto Esteves (Instituto de Biologia da Universidade Estadual do Rio de Janeiro). Voucher specimens were deposited in the herbarium of the Departament of Biology, FFCLRP/USP, and were used for the authentication of the species (SPFR).

Table 1.. Mikania. species collected for in vitro. trypanocidal assay.

Species	Local	Voucher number	Extraction solvent	Crude extract
Mikania hoehnei. Robinson	Maringá-RJ	SPFR 04309	Hexane	M.h.H
			Ethyl acetate	M.h.Ea
			Ethanol	M.h.E
Mikania stipulacea. (M. Vahl) Willd	Maringá-RJ	SPFR 04123	Hexane	M.s.H
			Ethyl acetate	M.s.Ea
			Ethanol	M.s.E
Mikania cordifolia. (L. f.) Willd.	Campos de Jordão-SP	SPFR 05316	Dichloromethane methanol	M.c.D
			Methanol	M.c.M
Mikania camporum. B. Robinson	Campos de Jordão-SP	SPFR 7157	Dichloromethane	M.ca.D
			Methanol	M.ca.M
Mikania lasiandrae. DC.	Campos de Jordão-SP	SPFR 7158	Dichloromethane	M.l.D
			Methanol	M.I.M
Mikania micrantha. H. B. K.	Espírito Santo do Pinhal-SP	SPFR 7156	Dichloromethane	M.m.D
			Methanol	M.m.M

Preparation of plant extracts

The plants were dried carefully by forced air at 40°C and reduced to powder. M. stipulacea. and M. hoehnei. were extracted exhaustively at room temperature by maceration using hexane, ethyl acetate and ethanol successively to yield crude extracts. M. cordifolia., M. camporum., M. lasiandrae., and M. micrantha. were also subjected to exhaustive successive maceration with dichloromethane and methanol at room temperature.

Total evaporation of solvents, under reduced pressure, furnished the crude extracts (Table 1). The extracts so obtained were assayed against T. cruzi..

In vitro. trypanocidal assay

In vitro. trypanocidal activity of crude extracts was performed according to previously established protocols (Bastos et al., 1999). The bioassays were carried out using blood of Swiss albino mice in the peak of parasitemic infection (seventh day; 2 × 10⁶ trypomastigote forms/mL) after infection with the Y strain of T. cruzi.. The blood was obtained by cardiac puncture using heparin as anticoagulant in a 7:3 blood/anticoagulant ratio. Crude extract was mixed in microtiter plates (96 wells) with 5 µL DMSO (dimethylsulfoxide) and infected blood to give a final concentration of 4000 µg/mL. The plates were kept at 4°C for 24 h under stirring and the number of parasites determined according to Brener (1962). Negative and positive controls containing DMSO (2.5%) and gentian violet at 250 µg/mL, respectively, were run in parallel. The bioassays were performed in triplicate. The percentages of viable T. cruzi. trypomastigotes were determined using light microscopy. The results are shown as lysis percentages of T. cruzi. trypomastigote forms, which represent the trypanocidal activity. Statistical comparisons were performed by Kruskal-Wallis test.

Results and Discussion

Natural products from plants traditionally have provided many lead compounds in the search for new drugs and medicines (Borris, 1996).

Protozoal diseases are one of the biggest health problems, and Chagas disease is a human disease widely distributed in third-world countries (WHO, 2000). In the research about the trypanocidal effects of plants, we decided to verify the trypanocidal activity of M.hoehnei., M. stipulacea., M. cordifolia., M. camporum., M. lasiandrae., and M. micrantha., considering that extracts as well as pure compounds obtained from the Asteraceae family have been reported to possess significant trypanocidal activity (Chiari et al., 1996; Muelas-Serrano et al., 2000; Taleb-Contini et al., 2004).

In this study, the trypanocidal activity of 14 extracts of plants of the Mikania. genus was evaluated against the trypomastigote forms of T. cruzi. Y strain. Table 2 summarizes the results obtained from the crude extracts. Thirteen extracts (92.9%) showed activity (lysis % ≥ 50). The dichloromethane extract of M. camporum. (M.ca.D) and the methanol extract of M. micrantha. (M.m.M) were the most active ones (caused total lysis of the parasites). In earlier phytochemical studies, sesquiterpene lactones have been isolated from M. micrantha. (Becker et al., 1987).

Table 2.. Trypanocidal activity of the crude extracts of Mikania. species.

Crude extract	Lysis %±SD^a.,b,c
M.h.H	51.57±9.8^ns
M.h.Ea	79.69±2.7**
M.h.E	70.32±8.2**
M.s.H	94.53±3.6**
M.s.Ea	57.03±4.9**
M.s.E	67.97±4.9**
M.c.D	66.25±5.9**
M.c.M	76.37±5.9**
M.ca.D	100**
M.ca.M	62.87±16.3**
M.l.D	96.63±2.93**
M.l.M	34.60±15.7^ns
M.m.D	94.10±1.5**
M.m.M	100**

See Table 1 for definition of extract abbreviations.

Significance was set at 1% (α = 0,01); *p < 0.01; **p < 0.001; ns, not significant.

^a.Percent reduction of the parasite number in mice infected blood±SD.

^b.Trypanocidal activity of the positive control was the 100%.

^c.The negative control did not show any reduction of the parasite numbers (the trypanocidal activity was 0).

Four extracts exhibited a high percentage of lysis ranging between 79% and 97%: the ethyl acetate extract of M. hoehnei., M.h.Ea (79.69%), the hexane extract of M. stipulacea., M.s.H (94.53%), the dichloromethane extract of M. lasiandrae., M.l.D (96.63%), and the dichloromethane extract of M. micrantha. (94.10%). Of the extracts cited above, M.s.H and M.h.Ea were further investigated in order to isolate the compounds that could be associated with observed activities. From M.s.H, a novel diterpene of the ent.-kaurene type, ent.-9α.-hydroxy-15β.-E.-cinnamoyloxy-16-kauren-19-oic-acid was isolated, reducing the number of trypomastigotes by 62%, 63%, and 70% at 100, 250, and 500 µg/mL (Nascimento & Oliveira, 2001; Nascimento et al., 2004). The diterpenes of the ent.-kaurene type appear to be an interesting class of compounds, if we consider that other diterpenoids isolated from plants shown anti–T. cruzi. activities. In fact, Alves et al. (1995) showed that diterpene ent.-kaur-16-en-oic acid, isolated from M. obtusata., displayed trypanocidal activity against trypomastigotes of T. cruzi.. Already, the investigation of M.h.Ea revealed the presence of the known sesquiterpene lactone, 8β.-hydroxyzaluzanin D, which also proved to be active at 250 and 500 µg/mL (Chaves & Oliveira, 2003; Nascimento et al., 2004). Several sesquiterpene lactones have been reported to show trypanocidal activity. Results suggest the mechanism of action of this group of compounds relies on covalent bond formation of the α,β.-unsaturated γ.-lactone moiety with nucleophiles that are essential for the life of the parasite (Uchiyama et al., 2002).

All the other active extracts are undergoing a chemical investigation to identify the bioactive principle(s) present in M. cordifolia., M. camporum., M.. lasiandrae., and M. micrantha..

The species M. cordifolia. had already been tested by Chiari et al. (1996), Muelas-Serrano et al. (2000), and Rojas de Arias et al. (1995), however, different solvents and concentrations were used.

The results obtained in this study demonstrate that species of the Mikania. genus could contain active principles against T. cruzi., opening, in this way, new possibilities in the therapy of the disease caused by this parasite.

Acknowledgments

The authors would like to acknowledge Dr. Janie Garcia da Silva and Dr. Roberto Esteves for botanical identification of species and Dr. Diones Aparecida Dias e Dr. Noberto Peporine Lopes for collection of plant material. This work was supported by grants from FAPESP, CNPq, and CAPES.