In Vitro Trypanocidal Activity of Triterpenes from Miconia Species Wilson Roberto Cunha1, Camila Martins1, Daniele da Silva Ferreira1, Antonio Eduardo Miller Crotti2, Norberto Peporine Lopes2, SØrgio Albuquerque3
Abstract
Letter
The bioassay-guided fractionation of methylene chloride extracts of Miconia fallax DC. and Miconia stenostachya DC. led to the isolation of five triterpene acids. The triterpenes ursolic acid, oleanolic acid and gypsogenic acid were active against blood trypomastigote forms of Trypanosoma cruzi. In contrast, the acetyl and methyl ester derivatives were not found to potentiate the trypanocidal activity. These results suggest the importance of the polar groups for activity.
Chagas' disease is endemic in Latin America, affecting 16 ± 18 million people, with more than 100 million exposed to the risk of infection [1]. Trypanosoma cruzi, the etiologic agent of the disease, causes a pathology of which the features depend upon both the inherent characteristics of the host and in the virulence of the parasite [2]. Blood transfusion has been recognized as having an important role in the transmission of Chagas' disease [3]. Gentian violet is the only effective compound available which eliminates the parasites in the blood prior to its transfusion. Despite its effectiveness, there are several restrictions for its use [4].
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In the last decade there has been an intensification in the search of trypanocidal compounds from natural sources, resulting in several classes of active plant metabolites [5], [6], [7]. Miconia, a genus with approximately 1000 species [8], belongs to Melastomataceae family. Miconia extracts and their isolated compounds have demonstrated biological activities such as antibiotic, antitumoral, analgesic and antimalarial effects [9], [10], [11]. In our laboratory, several plant extracts from Miconia were screened in vitro against trypomastigote blood forms of T. cruzi. The methylene chloride extracts of M. fallax [12] and M. stenostachya showed trypanocidal activity (M. fallax: 100 %, M. stenostachya: 94 %, 4 mg/mL) and some active compounds have been isolated and identified. The isolation strategy of the com-
Affiliation: 1 Ncleo de Pesquisas em Cincias Exatas e Tecnológicas da Universidade de Franca, Franca, SP, Brasil ´ 2 Departamento de Física e Química da Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo, Ribeiro Preto, SP, Brasil ´ 3 Departamento de Cincias da Sade da Faculdade de Cincias Farmacuticas de Ribeiro Preto, Universidade de So Paulo, Ribeiro Preto, SP, Brasil Correspondence: Prof. Dr. Wilson Roberto Cunha ´ Ncleo de Cincias Exatas e Tecnológicas da Universidade de Franca ´ Av. Dr. Armando Salles de Oliveira 201 ´ Parque Universitµrio ´ 14404-600 Franca- SP ´ Brazil ´ Fax: +55-16-37118886 ´ E-mail:
[email protected] Received: September 2, 2002 ´ Accepted: January 25, 2003 Bibliography: Planta Med 2003; 69: 470±472 ´ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943
pounds was the same for both species. Fig. 1 shows the chemical structures evaluated in this work. The structural elucidation of the isolated compounds and synthetic derivatives were performed using spectroscopic methods (MS, 1H- and 13C-NMR) in comparison with published data [13], [14], [15], [16]. Copies of the original spectra are obtainable from the author of correspondence. The trypanocidal activity was tested according to a previous report [7]. Table 1 shows lysis percentages of T. cruzi tripomastigotes forms induced by addition to the infected blood of the isolated compounds and their synthetic derivatives. The highest
IC50 values were obtained for natural compounds 1, 2 and 5 that contain OH and COOH groups. The IC50 values for synthetic derivatives were lower. Statistical analysis was performed using nonlinear regression to obtain the IC50 values followed by one-way analysis of variance (ANOVA). The results obtained are in accordance with those obtained for diterpene acids where the presence of polar groups was shown to be important for trypanocidal activity [17]. This is the first report of in vitro trypanocidal activity of triterpene acids.
Material and Methods
Table 1
In vitro trypanocidal activity of triterpenes isolated from two species of Miconia and their synthetic derivatives against the Y strain of Trypanosoma cruzi
Compounds
% lysis S.D./concentration (mg/mL)b 100
b
IC50 (mM)
500
1
91.1 1.4
95.5 0.7
99.6 0.7
2
69.2 7.4
90.7 2.5
99.2 0.7
80.4
mixture 1 + 2
60.3 3.7
91.1 1.4
95.1 1.2
109.4
mixture 1a + 2a
2.8 4.8
15.8 11.0
16.9 6.7
4,238.9
mixture 1b + 2b
42.9 4.4
44.1 0
47.4 5.1
610.5
3
19.8 8.3
76.4 11.5
97.2 1.4
294.9
4
46.6 4.8
47.4 5.1
70.4 1.8
402.3
5
73.1 8.4
97.4 2.9 100.0 0
gentian violeta a
250
100.0 0
100.0 0
100.0 0
21.3
56.6 76.0
Positive control. Mice infected blood containing the same DMSO concentration used in the stocks solution (negative control) did not present reduction of the parasite number.
A previous study on M. stenostachya has revealed the presence of sumaresinolic acid [15]. The reinvestigation of the methylene chloride extract of Miconia stenostachya produced, besides sumaresinolic acid (30 mg), 200 mg of gypsogenic acid (5) as a white amorphous solid, [a]D26: + 77.48 (c 0.19, MeOH) [16] and ursolic acid (200 mg). The mixture of 1 and 2 (100 mg) was treated with an excess of acetic anhydride in pyridine to give the C-3 acetoxy derivatives 1a and 2a. About 150 mg of the mixture of 1 and 2 was treated with CH2N2 in Et2O yielding a mixture of their respective C-28 methyl esters derivatives 1b and 2b. The isolated compounds and synthetic derivatives were tested against blood trypomastigote forms of T. cruzi according to a previous report [7]. Letter ¼ Planta Med 2003; 69: 470 ± 472
Letter
Fig. 1 Chemical structures of the triterpene acids isolated from Miconia species and synthetic derivatives tested for trypanocidal activity.
M. fallax and M. stenostachya were collected along Franca-Claraval highway, So Paulo, Brazil and identified by Dr. Angela Borges Martins, Instituto de Biologia, UNICAMP, Brazil. Voucher specimens (UEC 65132/UEC 21058) have been deposited in the Herbarium of the same Institute. The aerial parts of the plants were dried at 40 8C and the dried material was powdered (M. fallax: 1.3 kg, M. stenostachya: 1.0 kg) and extracted by maceration with methylene chloride (5 L, 3 days 3) and ethanol (5 L, 3 days 3) at room temperature. The methylene chloride extract of M. fallax (13.0 g) was chromatographed over 300 g silica gel 60 (0.063 ± 0.200 mm, Merck) by vacuum liquid chromatography [18], eluted with hexane, mixtures of hexane:ethyl acetate and ethyl acetate : ethanol of increasing polarity, resulting in six fractions of 1000 mL each [F1:hexane, F2:hexane : AcOEt (3 : 1), F3:hexane : AcOEt (1 : 1), F4:AcOEt , F5:AcOEt: EtOH (3 : 1), F6:AcOEt : EtOH (1 : 1)]. Fractions F-4 (1.76 g) and F-5 (1.23 g) exhibited a significant trypanocidal effect and were combined. The 1H- and 13C-NMR data of this fraction revealed the presence of a mixture of ursolic acid and oleanoic acid. An aliquot of this combined fraction (500 mg) was filtered over 60 g of mixture of celite:norit (3 : 1) eluted with ethyl acetate and the product was analyzed by HPLC (column silica Shimadzu, 5 mm, 20 250 mm, eluent: hexane:isopropyl alcohol, 98 : 2, flow 9 mL/min, UV detection: 225 nm) affording 15 mg of ursolic acid (1) as a white amorphous solid (tR: 5.7 min), [a]26 D : + 63.48 (c 0.18, MeOH) [13] and 20 mg of oleanolic acid (2) as a white amorphous solid (tR: 5.0 min), [a]26 D : + 68.28 (c 0.18, MeOH) [13]. Fraction F-3 (1.46 g) was washed with cold ethyl ether and afforded after recrystallization from MeOH, about 30 mg of oleanonic acid (3) was obtained as white amorphous solid, [a]26 D : ±58.38 (c 0.02, MeOH) [14]. A portion (500 mg) of Fraction F-6 (1.15 g) was treated like fraction F-3 yielding 40 mg of sumaresinolic acid (4) as a white amorphous solid, [a]26 D : + 22.48 (c 0.18, MeOH) [15].
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Acknowledgements This study was supported by FAPESP. We wish to thank Alba Regina Barbosa and Maria Ins Junqueira Garcia Teixeira for helping with plant collection. We are grateful to Dr. Paul J. Gates (University of Cambridge) for English revision.
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Letter ¼ Planta Med 2003; 69: 470 ± 472
