Trypanocidal activity of Meliaceae and Rutaceae plant extracts

ANTI-PARASITE DRUGS ACTIVITIES

Trypanocidal activity of meliaceae and rutaceae plant extracts

Alessandra Regina Pepe Ambrozin^I; Paulo Cezar Vieira^I,1; João Batista Fernandes^I; Maria Fátima das Graças Fernandes da Silva^I; Sérgio de Albuquerque^II

^IDepartamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz km 235, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil

^IIDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

ABSTRACT

The in vitro trypanocidal activity of 22 extracts and 43 fractions of plants belonging to the families Meliaceae and Rutaceae was evaluated. The extracts from leaves of Conchocarphus heterophyllus and branches of Trichilia ramalhoi were the most active. The trypanocidal activity seems to be increased by fractionation of the extracts. Fractions from C. heterophyllus and Galipea carinata were the most active and a 100% lysis of the parasites was observed for five fractions. From one of them were isolated two flavonoids: flavone and 7-methoxyflavone, which showed weak trypanocidal activity. The results obtained from the extracts and fractions revealed that the order Rutales is a promising source for the search of new drugs for Chagas disease. Phytochemical studies with the other active fractions are underway in order to isolate compounds, which could be associated with observed activities.

Key words: Meliaceae - Rutaceae - Chagas disease - trypanocidal activity

Chagas disease (American trypanosomiasis) is caused by the flagellate protozoan Trypanosoma cruzi (Kine-toplastida, Trypanosomatina), and affects more than 18 million people in Latin America, leading to approximately 400,000 deaths per year (WHO 1997). Its treatment is still a challenge, since the only drug commercially available (benznidazole) possesses severe side effects and its activity is limited to the acute phase of the disease (De Castro 1993, Fairlamb 1999). Coura and De Castro (2002) mention that an effective chemotherapy is needed for the people who are already infected. The demonstration of parasites in chronic patients reinforces the need of finding more efficient and less toxic drugs.

In the context of efforts to improve the therapy of Chagas disease, higher plants are a potential source of new drugs, with high activity and low toxicity (Phillipson & Wright 1991). Sepúlveda-Boza and Cassels (1996) mentioned a broad spectrum of chemical classes of substances showing activity against the parasite. Other promising compounds are the 2-aryl and 2-alkylquinoline alkaloids isolated from the extracts of the stem bark, root bark and leaves of Galipea longiflora (Rutaceae)(Fournet et al. 1994) and lignans from Zanthoxylum naranjillo(Rutaceae) (Bastos et al. 1999). In addition, we have been studying species of the order Rutales (Rutaceae, Meliaceae, Simaroubaceae, Burseraceae, and Cneoraceae) and several active substances have been isolated, mainly trypanocidal compounds (Mafezoli et al. 2000, Tomazela et al. 2000, Vieira et al. 2001).

In this paper, we present the results of the trypanocidal activity of some extracts and fractions of Almeidea coerulea, Almeidea rubra, Conchocarpus heterophyllus, and Galipea carinata (from Rutaceae family), as well as Trichilia ramalhoi (from the family Meliaceae). Also, the results of the in vitro trypomastigote bioassay with flavone (1) and 7-methoxyflavone (2), the major compounds of one active fraction, are described.

MATERIALS AND METHODS

Plant material - All screened plants were collected in Southeastern Brazil, and identified by Dr José R Pirani from the Department of Botany, University of São Paulo, Brazil. The voucher herbarium specimens were deposited at the Herbarium of that Department (Table I).

Preparation of crude extracts - Selected parts of the plants (leaves, stems, and/or branches) were dried carefully by forced air at 40°C and reduced to powder, followed by extraction three times with hexane by maceration at room temperature for 72 h. After the evaporation of the solvent under reduced pressure, crude hexane extracts were obtained. This process was repeated with methanol. The hexane and methanol extracts so obtained were assayed against T. cruzi.

Fractionation of crude extracts - Methanol extracts of T. ramalhoi were fractionated through liquid-liquid partition with hexane-methanol-water, dichloromethane-methanol-water, ethyl acetate-methanol-water, and butanol-methanol-water. The crude extracts of A. coerulea, A. rubra, C. heterophyllus, G. carinata, and the hexane extract of the leaves of T. ramalhoi were submitted to vacuum liquid chromatography over silica gel using a gradient hexane, dichloromethane, ethyl acetate, and methanol, to yield the corresponding fractions, which were subsequently tested for their tryponocidal activity.

Isolation of flavone and 7-methoxyflavone - The ethyl acetate fraction of the hexane extract from the leaves of C. heterophyllus (AHFHA) (7.3 g) was chromatographed on a Florisil column (8 x 5.9 cm) and eluted with solvents of increasing polarity (hexane ® methanol) to afford 6 fractions. The third fraction (1.6 g) was rechromatographed on a Silica gel column (230-400 mesh, 3.9 x 25.2 cm) using hexane-EtOAc mixtures. Eight fractions were obtained. The seventh one (1.27 g) was identificated as flavone (1) by comparison of the ¹³C NMR data with the literature(Kingsburry & Looker 1975). Further purification of fraction 4 (3.12 g) performed on a Silica gel column (230-400 mesh, 3.9 x 25.2 cm; CH₂Cl₂ ® MeOH) followed by gel filtration on Sephadex LH-20 (3.2 x 49.2 cm; MeOH) led to the isolation of 7-methoxyflavone (2) (22.7 mg)(King-sburry & Looker 1975). These compounds were assayed against T. cruzi.

Trypanocidal activityin vitro - The bioassays were carried out using blood of infected Swiss albino mice, which was collected by cardiac puncture at the peak of parasitemic infection (7th day of infection for Y strain of T. cruzi). The infected blood was diluted with healthy mice blood to achieve a concentration of 2.10⁶ forms/ml. Stock solutions of the extracts/fractions/compounds were prepared by dissolving in dimethylsulfoxide (DMSO). The activity of crude extracts was evaluated at 4 mg/ml, fractions at 2 mg/ml, pure substances at 500, 250, and 100 µg/ml. The bioassays were performed in triplicate on titration microplates (96 wells) which contained 400 µl of mixture/well. The plates were incubated at 4ºC, and the number of parasites counted after 24 h, following the method described by Brener (1962). Infected blood with the same volume of DMSO was used as control, and gentian violet to a concentration of 250 µg/ml was used as positive control. The activity is expressed as percent reduction of the parasite number (lysis) and IC₅₀ (mg/ml) for flavone and 7-methoxyflavone were calculated using the program GraphPad Prims v.3.0.

RESULTS AND DISCUSSION

In the present study, the trypanocidal activity of 22 extracts and 43 fractions of plants of Meliaceae and Rutaceae family was evaluated. Table II summarizes the results obtained from the crude extracts. Sixteen extracts showed significant activity (lysis % > 50). The extracts from the leaves of C. heterophyllus (AHFH) and from the branches of T. ramalhoi (TRGH, TRGM) were the most active ones. Also the results obtained from the extracts (Table II) revealed that these species are rich sources of trypanocidal compounds, therefore the order Rutales is a promising source of new drugs for Chagas disease. The species A. coerulea and C. heterophyllus had already been tested by Mafezoli et al. (2000), however different parts of the plants were investigated.

Table III shows the results obtained from the in vitro assay of fractions against the trypomastigote form of T. cruzi. Trypanocidal activity seems to be enriched by fractionation of the extracts. Only fractions of T. ramalhoi showed a lower percentage of lysis than those of the extracts. Ten fractions exhibited lysis above 80%, among them: the dichloromethane fraction from the methanolic extract of A. coerulea, AGMD (100%); the ethyl acetate fraction from the methanolic extract of the stem of A. rubra, ALCMA (80%); the ethyl acetate fraction from the hexane extract and the hexane and methanol fractions from the methanolic extract of the leaves and the ethyl acetate fraction from the methanolic stem extract of C. heterophyllus - AHFHA (100%), AHFMH (97.1%), AHFMM (98.6%), and AHCMA (100%); the dichloromethane and ethyl acetate fractions from the methanolic extract and the hexane fraction from the hexane extract of leaves of G. carinata GFMD (82.6%), GFMA (96.4%), GCFHH (100%) as well as the dichloromethane fraction from the methanolic extract of the stem of the same plant - GCCMD (100%). It will be noted that the best results were obtained from Conchocarpus and Galipea fractions. Phytochemical studies of all active fractions are underway in order to isolate the compounds which could be associated with observed activities.

One active fraction from C. heterophyllus (AHFHA) was investigated leading to the isolation of flavone (1) and 7-methoxyflavone (2), which were assayed against T. cruzi (Table IV). The results showed that these compounds have weak trypanocidal activity, particularly when compared to other flavonoids isolated from Trixis vauthieri(Ribeiro et al. 1997). The trypanocidal effect of the fraction AHFHA may be due to a combination effect between flavone and 7-methoxyflavone, which are the major compounds in this fraction. This possibility will be assessed by new bioassays with mixtures of these substances, which exist in the same proportion in the plant.

It is possible that the activity of the Rutaceae may be associated with coumarins and alkaloids (derived from anthranilic acid), which are characteristic of this family(Gray 1983, Mester 1983). C-glucosyl flavones(Jay et al. 1979, Wirasutisna et al. 1986), 2-quinolone and other alkaloids (Moulis et al. 1983), cycloartane triterpenoids and further alkaloids(Santos et al. 1998) have already been isolated from Almeidea. Galipea has afforded a hydro-xychalcone(López et al. 1998), a chromone(López et al. 1997), a coumarin(Wirasutisna et al. 1987), O- and C-glycosylflavones(Bakhtiar et al. 1990, 1994), and several 2-quinoline alkaloids(Fournet et al. 1989, 1993, Vieira & Kubo 1990, Rakotoson et al. 1998, Jacquemond-Collet et al. 1999). The structural diversity of metabolites from Rutaceae and the trypanocidal activity of 2-quinoline alkaloids and the lignan methylpluviatolide isolated from G. longiflora and Z. naranjillo (Fournet et al. 1994, Bastos et al. 1999) stimulated us to choose these plants. The trypanocidal activities observed confirm the previously noted potential of plants of the Rutaceae.

T. ramalhoi was the only Meliaceae species tested and no previous work on this plant was found. Meliaceae are a rich source of limonoids, however it seems that this class of compounds has never been evaluated for trypanocidal activity before (Champagne et al. 1992).

Finally, the results obtained in this study confirm the order Rutales as a fruitful source of new antichagasic compounds, since in the present work 16 active extracts and several fractions have been obtained, 5 of them showing 100% reduction of the parasite number. The more promising species seem to be C. heterophyllus and G. carinata although the weakly trypanocidal flavone (1) and 7-methoxyflavone (2) in their isolated form do not reproduce the activity of the crude extract. The full explanation of the observed activity of these fractions must await the results of the ongoing phytochemical studies.

ACKNOWLEDGEMENTS

To Dr José Rubens Pirani for collecting and identificating all the mentioned plants.

Received 5 September 2003

Accepted 12 January 2004

Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo

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Publication Dates

• Publication in this collection
  24 June 2004
• Date of issue
  Mar 2004

History

• Accepted
  12 Jan 2004
• Received
  05 Sept 2003