Effect of extracts and isolated compounds from Chresta scapigera on viability of Leishmania amazonensis and Trypanosoma cruzi

Efeito dos extratos e compostos isolados de Chresta scapigera sobre a viabilidade de Leishmania amazonensis e Trypanosoma cruzi

Abstracts

Fractionation of bioactive crude extracts of Chresta scapigera led to the isolation of four triterpenes and five flavonoids, among them beta-amyrin acetate (1), 11alpha,12alpha-oxidetaraxeryl acetate (2) and lupeol (3), as well as the flavonoids apigenin (6), kaempferol (7), crysoeriol (8) and luteolin (9) were active against Leishmania amazonensis amastigotes-like stages, while only the flavonoids (6), (7) and (9) showed toxicity towards bloods trypomastigote forms of Trypanosoma cruzi.

Chresta scapigera; Flavonoids; Triterpenes; Chresta scapigera

O fracionamento dos extratos bioativos de Chresta scapigera proporcionou o isolamento de triterpenos e flavonóides, dentre os quais acetato de beta-amirina (1), acetate de 11alfa,12-oxidotaraxeril (2) e lupeol (3), assim como os flavonóides apigenina (6), caenferol (7), crisoeriol (8) e luteolina (9) mostraram-se ativos contra formas amastigotas de Leishmania amazonensis, enquanto, apenas os flavonóides (6), (7) e (9) apresentaram toxicidade contra as formas tripomastigotas de Trypanosoma cruzi.

Chresta scapigera; Flavonóides; Triterpenos; Chresta scapigera

ORIGINAL PAPERS

Effect of extracts and isolated compounds from Chresta scapigera on viability of Leishmania amazonensis and Trypanosoma cruzi

Efeito dos extratos e compostos isolados de Chresta scapigera sobre a viabilidade de Leishmania amazonensis e Trypanosoma cruzi

Elisandra Cristina Schinor^I,*; Marcos José Salvador^{II, V}; Elisabeth Mieko Furusho Pral^III; Silvia Celina Alfieri^III; Sérgio Albuquerque^IV; Diones Aparecida Dias^{I, V}

^IDepartamento de Química da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo

IIUniversidade do Vale do Paraíba, Instituto de Pesquisa e Desenvolvimento

^IIIDepartamento de Parasitologia do Instituto de Ciências Biomédicas, Universidade de São Paulo

IVDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas da Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo

^VDepartamento de Física e Química da Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo

ABSTRACT

Fractionation of bioactive crude extracts of Chresta scapigera led to the isolation of four triterpenes and five flavonoids, among them b-amyrin acetate (1), 11a,12a-oxidetaraxeryl acetate (2) and lupeol (3), as well as the flavonoids apigenin (6), kaempferol (7), crysoeriol (8) and luteolin (9) were active against Leishmania amazonensis amastigotes-like stages, while only the flavonoids (6), (7) and (9) showed toxicity towards bloods trypomastigote forms of Trypanosoma cruzi.

Uniterms: Chresta scapigera/trypanocidal activity. Flavonoids. Triterpenes. Chresta scapigera/leishmanicidal activity

RESUMO

O fracionamento dos extratos bioativos de Chresta scapigera proporcionou o isolamento de triterpenos e flavonóides, dentre os quais acetato de b-amirina (1), acetate de 11a,12-oxidotaraxeril (2) e lupeol (3), assim como os flavonóides apigenina (6), caenferol (7), crisoeriol (8) e luteolina (9) mostraram-se ativos contra formas amastigotas de Leishmania amazonensis, enquanto, apenas os flavonóides (6), (7) e (9) apresentaram toxicidade contra as formas tripomastigotas de Trypanosoma cruzi.

Unitermos:Chresta scapigera/atividade tripanocida. Flavonóides. Triterpenos. Chresta scapigera/atividade leishmanicida

INTRODUCTION

Plants have been traditionally used for the treatment of diseases of different aethiology. Especially in the last decade, phytotherapy has received considerable attention in the search for alternatives to chemotherapy in parasitic diseases control (Muelas-Serrano et al., 2000). Many of these studies involve species of the Asteraceae family, among which can be mentioned Lychnophora granmongolense (Grael et al., 2000), Munnozia maronii (Fournet et al., 1993), Vernonia brachycalyx (Oketch-Rabah et al., 1998), Neurolaena lobata (Berger et al., 2001), whose crude extracts and isolated compounds presented antiprotozoal activity.

Leishmaniasis is a protozoan disease and occurs widely in tropical areas in South America (Sauvain et al., 1996). According to the WHO there are 2 million new cases of leishmaniasis per year worldwide (Barata et al., 2000). This disease is caused by haemoflagellate protozoan parasites that survive and multiply in magrophages in the mammalian host and are transmitted by phlebotomine sandflies (Salvador et al., 2002). The incidence of the disease has increased in the last years, and the therapy available mainly relies on antimonials, pentamidine and amphotericin B, which are toxic, difficult to apply in the field and not always effective (Mafezoli et al., 2000).

Another protozoan disease is South America trypanosomiasis caused by Trypanosoma cruzi and widespread though out the subcontinent with an estimated 18 million people infected (Barata et al., 2000). In Brazil about 5-6 million people are infected, and of which 300,000 are located in São Paulo State. The population has been infected mainly by contaminated blood transfusion for the control of the insect vector (Ribeiro, Veloso, 1997). Gentian violet is the only effective chemoprophylatic agent available in blood banks. However, it is not completely accepted by clinicians or patients because of its undesirable effects such as coloring the skin and possible mutagenic effects (Grael et al., 2000; Coura, Castro, 2002). For the acute and chronic diseases, the treatment is still a challenge today, since the available drugs possess severe side effects and are not completely efficient (Coura, Castro, 2002).

Thus, there is an urgent need for new drugs active against Leishmania spp and Trypanosoma cruzi.

Chresta, belonging to Asteraceae family, is a small genus that comprises 12 endemic species of the "Cerrado brasileiro" (Robinson, 1999). There are no prior studies about biological activity of this genus. The present studies, crude extracts (roots, stem, leaves and inflorescences) and some compounds isolated from Chresta scapigera were evaluated for their ability to reduce viability of axenic Leishmania amazonensis amastigotes and T. cruzi trypomastigotes forms, in vitro.

MATERIAL AND METHODS

Plant material

Chresta scapigera was collected in Furnas (MG-Brazil) in July, 1998, and identified by Dr. João Semir (Instituto de Biologia, Unicamp, Campinas, SP-Brazil). A voucher of each specimen is deposited in the Herbarium of FFCLRP/USP (SPFR6874).

Extraction and isolation process

Roots (188.0 g), stem (252.0 g), leaves (313.0 g) and inflorescences (99.0 g) were separated, dried, pulverized and stored in dark bags to protect them from humidity and light. The powdered material was extracted by maceration with n-hexane, dichloromethane and ethanol, respectively, at room temperature. The n-hexanic extract of roots (9.521 g) was chromatographed on a column of silica gel 60 (0.063-0.200 mm, Merck), eluted with hexane, ethyl acetate, methanol and mixtures of these solvents of increasing polarity. This extract furnishes 18 fractions and the fractions 3 and 4 were purified by preparative TLC yielding b-amyrin acetate (1) (16 mg) and 11a,12a-oxidetaraxeryl acetate (2) (10 mg), respectively. The n-hexanic extract of inflorescences (2.594 g) was chromatographed in the same conditions and furnishes 14 fractions. Preparative TLC was used to purify the fractions 4 and 7, which yield lupeol (3) (30 mg) and its acetate (4) (6 mg), respectively. The ethanolic crude extract of inflorescence (2.6 g) was partitioned between dichloromethane and methanol. The hidroalcoholic phase (1.3 g) was submitted to filtration on sephadex LH-20 and the fractions 6 and 7 were purified by preparative HPLC in reverse phase (column ODS Shimadzu 5 µm, 20 x 250 mm, eluent: methanol:water in gradient, flow 9mL/min, UV detection: 280 nm) affording tiliroside (5) (22 mg), apigenin (6) (12 mg) and kaempferol (7) (10 mg), respectively. While the dichloromethanic phase (391 mg) was chromatographed on a silica gel column eluted with hexane, ethyl acetate, methanol and mixtures of these solvents of increasing polarity. The fraction 3 was purified by preparative TLC affording crysoeriol (8) (3 mg). The ethanolic crude extract of stem was chromatographed on PVP by vacuum liquid chromatography eluted with CHCl₃, methanol and mixtures of these solvents of increasing polarity. Filtration on sephadex LH-20 was used to purify the fraction 1, which yield crysoeriol (8) (3 mg), apigenin (6) (2 mg) and luteolin (9) (3 mg).

The structures of all compounds were determined by spectroscopic methods (IR, ¹H- and ¹³C-NMR). All extracts and isolated compounds were evaluated against axenic L. amazonensis amastigotes and T. cruzi trypomastigotes forms, in vitro, according to a previous report (Mafezoli et al., 2000; Bastos et al., 1999).

Trypanocidal activity

For the trypanocidal activity, blood of Swiss albino mice infected with Trypanosoma cruzi (Y strain) was used, which was collected by cardiac puncture at the peak of parasitemia and was diluted to contain 10⁶ trypomastigotes/mL. Stock solutions (crude extracts) were prepared in 2.5% of dimethyl sulfoxide (DMSO) and were added to blood samples to provide a final concentration of 4000 µg/mL. After incubation for 24 hours at 4 ºC, the number of parasites was determined according to Brener (1962). In the tests, gentian violet (250 µg/mL) was used as positive control and DMSO 2.5% as negative control. All experiments were performed in triplicate.

Leishmanicidal activity

Axenic Leishmania (L.) amazonensis (strain designation MPRO/BR/72/M 1841) amastigotes were serially cultivated at 33 ºC in modified UM-54 mediun (Pral et al., 2003) and were used at the beginning of the stationary phase. Washed parasites were resuspended in RPMI-1640 medium supplemented with 4% fetal calf serum, pH 5.0 and incubated at 33 ºC for 24 hours with crude extracts (1000 µg/mL) dissolved previously in RPMI-1640. As controls, parasite suspensions were incubated in RPMI alone or RPMI containing 0.1% DMSO. Amastigote viability was assessed colorimetrically by reduction of a tetrazolium salt (MTT) as described by Mosmann (1983). Amphotericin B (20 µg/mL) was used as the positive control and DMSO/RPMI-1640 (1:99) negative control. The experiments were carried out in triplicate.

Figure 1

RESULTS AND DISCUSSION

The results are showed in Tables I and ^II. All the crude extracts evaluated markedly reduce the viability of L. amazonensis amastigotes (at 1000 µg/mL), while only the extracts hexanic of inflorescence, dichloromethanic of root and ethanolic (root and stem) did not interfere appreciably with lysis percentages of T. cruzi trypomastigotes forms (at 4000 µg/mL).

Data in Tables I and ^II show that effect of the evaluated compounds varies depending on the different concentrations used in the bioassay.

Compound (5) was not active against L. amazonensis and T. cruzi at the concentrations evaluated, while the compound (1) reduced the viability of L. amazonensis at the lower concentration used (at 14 µg/mL), but not interfere with lysis percentages of T. cruzi trypomastigote forms. Flavonoids (6) and (9) were the most active ones when assayed at 84 µg/mL against L. amazonensis, but a slight activity was observed against T. cruzi.

Compound (6) presents a 5,7,4'-hydroxylation pattern and showed active since 100 µg/mL with 54% of lysis. Ribeiro et al. (1997) and Grael et al. (2000) have demonstrated that hydroxylated flavonoids were more active than methoxylated flavonoids when evaluated against trypomastigote forms of T. cruzi. The authors believed that the probable mechanism of action for this activity might be explained from drugs that are able to generate reactive oxygen species in which T. cruzi is susceptible.

The other compounds interfered with viability of L. amazonensis and T. cruzi at concentration evaluated.

The results presented can be considered promising and these extracts may represent a source of active substances against mainly amastigote forms of L. amazonensis.

However, further studies (in vitro and in vivo) should be accomplished in order to understand the mechanisms of action, as well as to evaluate the toxicity, looking for a clinical employment of those bioactive compounds.

ACKNOWLEDGEMENTS

We wish to thank Fapesp for the financial support and Prof. Dr. Walter Vichnewski for the collection of plant material.

Recebido para publicação em 16 de março de 2006.

Aceito para publicação em 07 de março de 2007.

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Publication Dates

• Publication in this collection
  03 Sept 2007
• Date of issue
  June 2007

History

• Accepted
  07 Mar 2007
• Received
  16 Mar 2006