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Trypanocidal activity of extracts from Brazilian Atlantic Rain Forest plant species.

Summary

The trypanocidal activity of crude hydro alcoholic extracts and several fractions of 13 plants from Brazilian Atlantic Rain Forest were tested in vitro against epimastigote and trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. Crude ethanol extracts with promising in vitro activity (D[L.sub.50] between 5-10 [micro]g/ml) against epimastigotes were fractionated by solvent partition and further tested against bloodstream form of the parasite. Activity against bloodstream parasites was observed in both dichloromethane and hexane fractions of Polygala sabulosa and P. paniculata.

Key words: Trypanocidal activity, Trypanosoma cruzi, Brazilian Atlantic Rain Forest plants

* Introduction

Chagas disease caused by the protozoan parasite Trypanosoma cruzi affects around 16-18 million people in Latin America countries where, at least, 25% of the population is nowadays living in areas of high transmission risk (WHO, 1997). During the last few years, T. cruzi transmission by triatomine vectors has been reduced and/or interrupted in several endemic areas due to the use of chemical insecticides for the control of the most important vector of the disease (Triatoma infestans). However, congenital transmission or transmission by infected blood transfusion are still public health problems in several American Countries (Schmunis, 1999). Moreover, original ecotopes of T. infestans have been occupied by distinct triatomine bug species, which are nowadays transmitting the parasite among humans and wild mammals (Schofield 1994).

During the last 30 years, the nitroheterocyclic drugs benznidazole (Rochagan[R], Roche) and nifurtimox (Lampit[R], Bayer) are the only choice for treatment of human Chagas disease. Both drugs are of limited efficacy during the chronic phase of the disease and present several undesirable effects (Rassi and Luquetti, 1992). Chemoprophylatic measures against Chagas disease are scarce, present low efficacy or are not adopted due their costs. Gentian violet (crystal violet) is a trypanocidal compound used to sterilize blood banked in highly endemic areas. Despite the efficiency of this measure, transfusion of blood containing crystal violet is not well accepted by clinicians and patients due to undesirable effects such as coloring of the skin (Ramirez et al., 1995).

Due the absence of alternative drugs for Chagas disease chemotherapy, the search for active substances of natural origin is of major importance since several available drugs, including semi-synthetic and synthetic derivates were originally isolated from natural compounds (Cragg et al., 1997; Soerjarto, 1996).

Despite the great biodiversity of the Atlantic Rain Forest in Brazil, the potential of the vegetal species as new drug sources is quite unexplored. Less than 20% of all plant species from this environment have been studied for the presence of biologically active substances (Elisabetsky and Costa-Campos 1996).

Plant derived compounds present a wide spectrum of biological activities against virus, cancer parasites. These compounds are mainly secondary metabolites such as alkaloids, flavonoids, terpenes, lignans and coumarins (Rates, 2001; Samuelsen, 2000).

Both in vitro and in vivo activities against malaria parasites (Plasmodium spp.), T. cruzi and Leishmania spp. were observed in crude extracts as well as in isolated compounds from several plant families (Mafezoli et al., 2000; Berger et al., 1998; Fournet et al., 1994, 1996; Gantier et al., 1996; Sepulveda-Boza and Cassels, 1996; Muelas-Serrano et al., 2000).

In the present work, in vitro activity of crude extracts from 13 plant species collected in the Atlantic Rain Forest of southern Brazil were studied against T. cruzi (Table 1). Furthermore, fractions isolated from active extracts were further tested in vitro against bloodstream trypomastigotes.

* Materials and Methods

Plant Collection and Identification The common names and geographical origin of plant species included in the present study are shown in Table 1. Plants collected at the Santa Catarina State, Brazil, were identified and deposited at the Departments of Botany of Universidade Federal de Santa Catarina and Universidade Federal do Parana, Brazil. The crude extract of Baccharis platypoda leaves was kindly supplied by Dr. Anselmo A. de Moraes from Universidade Federal Rural do Rio de Janeiro, Brazil. The Trichilia catigua bark sample was supplied by Laboratorio Catarinense, Joinville, Brazil.

Extraction and Fractionation Procedures

All extracts were prepared from dried samples (40[degrees]C for seven days), powdered and extracted with 80% ethanol during 14 days at room temperature. Following filtration, crude hydro alcoholic extracts were dried in vacuum and assayed for biological activity against T. cruzi epimastigotes. Those extracts with trypanocidal activity (D[L.sub.50] < 10 [micro]g/ml) were subjected to liquid-liquid partition using hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and water. After solvent removal, all fractions were submitted to biological assays against the parasite.

Trypanocidal Activity Assays

Epimastigote forms of T. cruzi Y strain (Silva and Nussenzweig, 1952) were cultivated in LIT medium supplemented with 10% fetal bovine serum (FBS) and harvested on the exponential growth phase. Parasites were washed once in sterile phosphate buffered saline (PBS) pH 7.2 and had their concentration adjusted to 5 x [10.sub.6] parasites/ml in the same medium.

Stock solutions of each extract or fraction were prepared in dimethylsulfoxide (DMSO) at 20 mg/ml and filtered in 0.45 [micro]m acetate membranes. Bioassays were performed in triplicate on 96 wells microtiter plates containing 200 [micro]l of the parasite suspension/well plus 500, 250, 100, 50 and 10 [micro]g/ml of each extract/fraction. As control, epimastigotes were incubated with 5% of DMSO, 10 [micro]g/ml of benznidazole (Rochagan[R]) or in LIT medium only. After incubation at 27[degrees]C for 72 hrs, trypanocidal activity was evaluated by determining the number of live parasites in Neubauer chambers.

Fractions obtained from active extracts were dissolved in DMSO and assayed in triplicate against T. cruzi blood trypomastigotes in 96 wells microtiter plates. Blood trypomastigotes were obtained from experimentally infected Swiss mice on the 7th day of infection with T. cruzi Y strain. After blood collection, the parasite number was determined in Neubauer chambers and the parasite concentration adjusted to 5 x [10.sup.5] parasites/mi in LIT medium. Two hundred [micro]l of the parasite suspension was incubated with 500 and 250 [micro]g/ml of each fraction at 4[degrees]C for 48 hrs. As control, the same number of parasites was incubated with 5% DMSO, with gentian violet (250 [micro]g/ml) or with LIT medium only. The trypanocidal activity was evaluated by determining the number of live trypomastigote forms as previously described (Brener, 1962). Activity was expressed as percentage of reduction of the parasite number calculated using the program Prism[R] v. 2.0 (GraphPad Software Incorporated, San Diego).

* Results and Discussion

The presence of trypanocidal activity was evaluated in crude extracts and fractions from 13 plant species from the Brazilian Atlantic Rain Forest. Belonging to 9 different families, these species extracts were tested in vitro against culture epimastigotes of T. cruzi (Table 2). Crude hydro alcoholic extracts from T. catigua, P. sabulosa, P. cyparissias, B. platypoda and E. jambolana revealed to be active against T. cruzi epimastigotes (D[L.sub.50] [less than or equal to] 10 [micro]g/ml).

Hexane, DCM, EtOAc and water fractions obtained from these active extracts were further tested against T. cruzi culture epimastigotes. Eleven out of 14 obtained fractions (78.5%) showed activity against T. cruzi epimastigotes in vitro (D[L.sub.50] [less than or equal to] 10 [micro]g/ml) (Table 3). The hexane fraction of P sabulosa and the aqueous fraction of P cyparissias revealed D[L.sub.50] of 1 and 2 [micro]g/ml, respectively.

As shown in Table 4, the DCM and aqueous fractions of Trichilia catigua extract and distinct fractions of three Polygala species extracts were tested in two concentrations (500 and 250 [micro]g/ml) against T. cruzi blood trypomastigotes. Our results indicate that DCM and aqueous fractions of P. sabulosa, EtOAc fraction of P. cyparissias, hexane fraction of P. paniculata and both DCM and aqueous fractions of T. catigua were active against bloodstream trypomastigotes, reducing the parasite number in vitro (58-100%). Since hemolysis was observed in the presence of P. sabulosa, P. cyparissias and P paniculata fractions, further studies are in progress to better address this problem prior in vivo studies (Table 4).

Using the trypanocidal activity of gentian violet as golden standard, similar activities were observed in the hexane and DCM fractions of P. paniculata and P. sabulosa at 250 [micro]g/ml (Table 3). No influence on the parasite number reduction was observed in the presence of DMSO or LIT medium.

Previous studies of carried out by our group have demonstrated the isolation of polyhydroxilated xanthones from EtOAc fraction of P. cyparissias (Pinheiro et al., 1998), prenylcoumarin and styrylpyrones from DCM fraction of P. sabulosa (Pizzolatti et al., 2000) and flavolignans from the EtOAc fraction of the T. catigua (Pizzolatti et al., 2002).

Also, the presence of xanthones and flavonoid glycosides in the aqueous fraction of P. cyparissias, sterols and coumarins in hexane fraction of P. sabulosa and phenyl-[gamma]-lactones and sterols in the DCM fraction of T. catigua were demonstrated (Pizzolatti et al., 1999).

Activity against T. cruzi has been demonstrated in both crude extracts and fractions of distinct plant species (Zani et al., 1995; Chiari et al., 1996; Berger et al., 1998; Mafezoli et al., 2000; Sepulveda-Boza and Cassels, 1996). These extracts and fractions have demonstrated distinct activities against T. cruzi epimastigotes and/or blood trypomastigotes.

Chiari et al. (1996) have observed activity against T. cruzi blood trypomastigotes in 23% of the ethanolic extracts obtained from 47 species of the Asteraceae family at a concentration of 12 mg/ml.

Studies carried out with species of the same family showed that 10% of the ethanolic extracts from 52 Asteraceae species reduced 70% of the trypomastigore number at a concentration of 250 [micro]g/ml (Zani et al., 1995). Recently Mafezoli et al. (2000) observed that 25% of the 32 extracts from 9 Rutaceae species showed trypanocidal activity in vitro. In this study, only 6 out of 25 fractions obtained from active extracts presented activity against T. cruzi blood trypomastigotes at a concentration of 2 mg/ml.

In the present study, both DCM fraction of P. sabulosa and the hexane fraction of P. paniculata showed activity against T. cruzi blood trypomastigotes at 250 [micro]g/ml. Despite this promising activity, further in vitro studies addressing cytotoxicity of these fractions to distinct cell lines are in progress.

Up to now, this is the first report of activity against T. cruzi in extracts and fractions obtained from Polygalaceae species. Furthermore, the presence of prenylcoumarin and styrilpyrones as major constituents of the active fractions of P. sabulosa, indicates that this species contains a new class of natural products, and is a promising source for active compounds against T. cruzi.
Table 1. Plant species used for screening.

Family Species Vernacular names

Euphorbiaceae Cronton celtidifolius Baill. Sangue-de-adave,
 pau-sangue
Anacardiaceae Schinus molle L. Anacauita, falso
 mole
Guttiferae Rheedia gardneriana Pl. Tr. Bacopari, magostao-
 amarelo
Compositae Spilanthes oleracea L. Agriao-do-Para
Polygalaceae Polygala sabulosa A. W. Bennett Timutu-pinheirinho
 Polygala paniculada L. Barba-de-sao-Pedro
 Polygala cyparissias ST. Pinheirinho-da-praia
 Hill and Moq.
Asteraceae Baccharis pseudotenuifolia DC Carqueja
 Baccharis platypoda DC Carqueja
Leguninosae Bauhinia forficata Link Pata-de-vaca
Myrtaceae Eugenia jambolana Lamark Jambolao
 Eugenia uniflora L. Pitanga
Meliaceae Trichilia catigua A. Juss. Catuaba, Catigua

Family Ethnomedical use

Euphorbiaceae Ulcer, rheumatism, inflammatory
 and skin diseases
Anacardiaceae Antirheumatic, skin disorders,
 cicatrizant, antimicrobial, bronchits
Guttiferae Kidney affections, Inflammations,
 infections, general pain
Compositae Toothache, stomatitis, throat
Polygalaceae General pain, parasitical diseases
 Parasitical diseases, Inflammations
 Kidney affections, parasitical
 diseases, topical anestetic
Asteraceae Disorders of the digestive tract
 Antiparasitic
Leguninosae Antiparasitic, diabetis
Myrtaceae Diabetis,
 Diabetis, disorders of the digestive tract
Meliaceae Mental tonic, sexual stimulant

Table 2. In vitro activity of extracts from Brazilian Atlantic Rain
Forest species against epimastigote forms of Trypanosoma cruzi.

Species Part Extract (a) D[L.sub.50]
 ([micro]g/ml) (b)

Croton celtidifolius Leaves EB 250
Croton celtidifolius Bark EB 250
Trichilia catigua Bark EB 10
 DCM 10
 EtOAc 10
 Aq NA
Schinus molle Leaves EB 50
Spilantes oleracea Aerial parts EB NA
Rheedia gardneriana Leaves EB NA
Polygala sabulosa Aerial parts EB 5
 Hex 1
 DCM 5
 EtOAc 10
 Aq NA
Polygala cyparissias Aerial parts EB 10
 Hex 250
 DCM 5
 EtOAc 5
 Aq 2
Polygala paniculata Aerial parts EB 50
Baccharis Aerial parts EB 50
pseudofenuifolia
Baccharis platypode Leaves EB 10
 Hex 10
 DCM 10
 EtOAc 10
Bauhinia forficata Leaves EB NA
Eugenia jambolana Leaves EB 5
Eugenia uniflora Leaves EB 50

(a) EB--crude hydro alcoholic extracts; Hex--hexane extract;
DMC--dichloromethane extract; EtOAc--ethyl acetate extract;
Aq--aqueous extract

(b) ND--Not determined; NA--No activity detected

Table 3. In vitro activity of two concentrations (250 and 500
[micro]g/ml) of Polyagala spp. and Trychilia catigua fractions
against blood trypomastigotes of Trypanosoma cruzi.

Species Fraction % of Reduction
 (500/250 [micro]g/ml)

Polygala sabulosa Hexane 32 (+)/16 (+)
 Dichloromethane 100 (+)
 Aqueous 63/58
Polygala cyparissias Dichloromethane 37/16
 Ethyl Acetate 79 (+)/47 (+)
 Aqueous 37/11
Polygala paniculata Hexane 100/90
 Butanol 21 (+)/0 (+)
 Aqueous 42/6
Trichilia catigua Dichloromethane 74/58
 Aqueous 58/32
Gentian violet Aqueous 100
DMSO 5% (250 [micro]g/ml) 0
Control LIT 0

(+) Presence of hemolysis


Acknowledgements

We want to thank Dr. A. Reis (UFSC) and Dr. O. A. Guimaraes (UFP) for identification of the plants.

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* Address

M. G. Pizzolatti, Departamento de Quimica, Laboratorio de Quimica de Produtos Naturais--Campus Trindade, 88010-970 Florianopolis, SC, Brazil

Tel.: ++5-04 83 319 219; Fax: ++5-04 83 319 711

e-mail: moacir@qmc.ufsc.br

M. G. Pizzolatti (1), A. H. Koga (2), E. C. Grisard (2), and M. Steindel (2)

(1) Departamento de Quimica, Laboratorio de Quimica de Produtos Naturais

(2) Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
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Author:Pizzolatti, M.G.; Koga, A.H.; Grisard, E.C.; Steindel, M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Jun 1, 2003
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