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- Medicinal Plants of Chile: Evaluation of their Anti- Trypanosoma cruzi Activity
- Results and Discussion
| © 2013 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com Introduction Chagas’ disease, caused by Trypanosoma cruzi, is among the most important endemic parasitic diseases. Approximately 16 to 18 million peo- ple are infected in large areas of Latin America (Fournet and Munoz, 2002). Over 1 million of them will die of the disease unless considerable advances are made (Maguire, 2006). Further- more, there is increasing evidence that it may be an emerging problem in the developed world, with more than 100,000 infected persons living in the United States of America alone (Maguire, 2006).
The drugs used for the treatment of this dis- ease are nifurtimox, a nitrofuran derivative, and benznidazole, a nitroimidazole derivative. Both drugs generate severe side effects, and nifurtimox is no longer used in several countries because of its toxicity (Castro et al., 2006; Croft et al., 2005). Consequently, the need for effective anti- Trypa- nosoma compounds with less toxicity stimulates the search for natural products as novel drug can- didates with a potential clinical use (Hoffmann
The aim of the present work was to assess the in vitro activity of some Chilean plant extracts against the trypomastigote forms of Trypanosoma cruzi. Thirty-one species of plants belonging to 28 genera in 21 families were investigated. The most active extracts were those obtained from
nomic and social importance with medicinal properties in Chile. This plant contains several sesquiterpenes of the drimane type. Leaves of Drimys winteri are used in Chilean folk medicine as analgesic and in anti-infl ammatory medications (Houghton and Manby, 1998; San Martin, 1983; Cotoras
et al., 2001; Muñoz and Fajardo, 2005; Muñoz
et al., 2001; Ruiz et al., 2010). Experimental General Column chromatography (CC) was carried out using silica gel 60G (Merck Darmstadt, Ger- many). Thin-layer chromatography (TLC) was performed on silica gel GF254 (Merck) with (i)
ane/acetone (8:2) as eluents. Spots were detected under UV light or by spraying with Liebermann- Burchard reagent and heating to 110 °C for 2 min. Medicinal Plants of Chile: Evaluation of their Anti-Trypanosoma cruzi Activity Orlando M. Muñoz a, *, Juan D. Maya b , Jorge Ferreira b , Philippe Christen c , José San Martin d , Rodrigo López-Muñoz b , Antonio Morello b , and Ulrike Kemmerling b a Department of Chemistry, Faculty of Science, University of Chile, Santiago, Chile. Fax: 56-2 2713888. E-mail: omunoz@uchile.cl b Institute of Biological Sciences, Faculty of Medicine, University of Chile, Santiago, Chile c School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland d Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Talca, Chile * Author for correspondence and reprint requests Z. Naturforsch. 68 c, 198 – 202 (2013); received December 15, 2011/March 7, 2013 The extracts of several plants of Central Chile exhibited anti- Trypanosoma cruzi try- pomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC 50 51.2 µg/mL) was purifi ed and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of
50 values of 27.9 and 25.1 µ M , respectively. Key words: Anti-Trypanosoma cruzi, Drimenol, Isodrimenin O. M. Muñoz et al. · Antichagasic Plant Extracts 199
Preparative TLC was performed on 2 mm thick silica gel F254 plates (Merck) and on a chroma- totron (Harrison-Research Model 7924 T; Palo Alto, CA, USA) with 1-mm and 2-mm discs, using silica gel 60 PF 254 (Merck). Flash chromatogra- phy was performed on silica gel 60 H (Merck) with an
10, 50, 100% ethyl acetate). Melting points are un- corrected. Optical rotations were measured with a Perkin Elmer 241 MC polarimeter (Waltham, MA, USA). 1 H and 13 C NMR spectra were recorded in CDCl 3
1 H NMR and 100 and 125 MHz for 13 C NMR, on a Bruker Avance AM-400 spectrometer (Karlsruhe, Ger- many).
The 1 H NMR spectra used for the measure- ment of the coupling constants and the HMBC spectra used for the determination of the con- nectivity of substituents were recorded in CDCl 3
on a Bruker-DRX 500 MHz instrument. The 1 H Larmor frequency was determined using a 5-mm QPN direct detection probe. Chemical shifts (δ in ppm) are relative to the internal standard tetra- methylsilane (TMS). 1D ( 1 H,
13 C) and 2D (COSY, HMQC, HMBC) experiments were performed using standard Bruker microprograms. Plant materials Ethnopharmacological and ethnobotanical lite- rature was obtained from Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Talca, Chile, and from the Departamento de Botanica, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. Thirty-one plant species were included in the study and collected in Cen- tral Chile (Coastal Range, Maule Region) during the fl owering season in January 2009 and 2010, and identifi ed by one of us (J. S. M.). Voucher specimens are deposited and kept in the Instituto de Biología Vege tal y Biotecnología, Universidad de Talca. Plants used in this study are listed in Table I.
The collected plants were washed with distilled water and dried on absorbing paper at an ambi- ent temperature of 25 – 30 °C in open air in the shade for 5 – 10 d. The dried plant samples were powdered and stored at ambient temperature in amber glass bottles until use. The powdered plant materials (10.0 – 80.0 g) were extracted with dichloromethane (4.0 mL/g plant) or methanol/ water (4:1, v/v; 4.0 mL/g plant) for 4 h at room temperature. The extracts were fi ltered and evap- orated to dryness under vacuum. The residues Table I. Chilean plant species used in this study. No. Species, family 1 Podanthus ovatifolius Lag., Asteraceae 2 Cryptocarya alba (Mol.) Looser, Lauraceae 3 Escallonia illinita K. Presl., Saxifragaceae 4 Blepharocalyx cruckshanksii (H. et A.) Nied., Myrtaceae 5 Satureja gilliesii (Graham) Briq., Lamiaceae 6 Drimys winteri J. R. Forst. et G. Forst., Winteraceae 7 Luma chequen (Mol.) A. Gray, Myrtaceae 8 Luma apiculata (DC.) Burret, Myrtaceae 9 Fuchsia magellanica Lam., Onagraceae 10 Colliguaja odorifera Mol., Euphorbiaceae 11 Ugni molinae Turcz., Myrtaceae 12 Alstroemeria revoluta Ruiz et Pav., Amaryllidaceae 13 Pitavia punctata Mol., Rutaceae 14 Podocarpus saligna D. Don, Podocarpaceae 15 Lapageria rosea Ruiz et Pav., Philesiaceae 16 Schinus latifolius (Gill. ex Lindl.) Engler, Anacar- diaceae
17 Kageneckia oblonga Ruiz et Pav., Rosaceae
No. Species, family 18 Margyricarpus pinnatus (Lam.) Kuntze, Rosaceae 19 Pseudognaphalium vira vira (Mol.) A. Anderb., Asteraceae 20 Eupatorium salvia Colla, Asteraceae 21 Baccharis concava (Ruiz et Pav.) Pers., Asteraceae 22 Viviania crenata (Hook.) G. Don ex H. et A., Ru- biaceae 23 Fabiana imbricata Ruiz et Pav., Solanaceae 24 Myoschilos oblonga Ruiz et Pav., Santalaceae 25 Berberis darwinii Hook., Berberidaceae 26 Maytenus chubutensis (Speg.) Lourt., O’Donell et Sleum., Celastraceae 27 Myrceugenia chrysocarpa (O. Berg) Kausel, Myrta- ceae
28 Elytropus chilensis (A. DC.) Muell. Arg., Apocyn- aceae
29 Berberis serrato-dentata Lechler, Berberidaceae 30 Berberis microphylla G. Forst., Berberidaceae 31 Pseudopanax laetevirens (Gay) Franch., Araliaceae
200 O. M. Muñoz et al. · Antichagasic Plant Extracts were weighed and solubilized in dimethylsulfox- ide (DMSO) for biological assays.
African green monkey Vero cells were infected by co-incubation of a culture of epimastigotes in the late stationary phase, which contains about 5% of the infective trypomastigote form (Con- treras
et al., 1985). Subsequently, the trypomas- tigotes harvested from this culture were used to further reinfect cultures of Vero cells at a density of 1 · 10 6 cells/25 cm 2 , in a proportion of parasites to cells of 2:1. Vero cell cultures infected with trypomastigotes were incubated at 37 °C in hu- midifi ed air and 5% CO 2 for 5 – 7 d. After that time, the culture medium was collected and cen- trifuged at 3,000 x g for 5 min, and the resulting trypomastigote-containing pellet was resuspend- ed at a density of 1 · 10 7 parasites/mL in RPMI 1640 culture medium (without phenol red). Trypomastigote viability assay Viability assays were performed using the formazan formation method, called 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide (MTT) assay, as previously described (Faun- dez
et al., 2005; Mosmann, 1986). Briefl y, 1 · 10 7
trypomastigotes were incubated in RPMI 1640 culture medium at 37 °C for 24 h with and with- out addition of the extracts at fi nal concentra- tions of 10 – 500 µg/mL. Formazan formation was measured at 570 nm in a multi-well reader (Lab systems Multiskan MS, Vantaa, Finland).
Barks of D. winteri were dried in an air-forced oven at 40 °C for 48 h. Four hundred fi fty g of chopped and powdered bark material were ex- tracted with n-hexane (2 x 2.0 L) for 24 h in a Soxhlet extractor. Filtration followed by evapora- tion of the solvent under reduced pressure (0.21 atm) at 25 – 30 °C gave a yellowish oily residue (30.0 g). The crude n-hexane extract was fi rst subjected to fl ash CC (silica gel, 230 – 400 mesh, 650 g), then fractionated by gradient elution (100%
to 100% ethyl acetate) to give individual frac- tions which were further purifi ed by two silica gel columns. Elution with n-hexane/ethyl acetate (98:2 – 90:10) yielded 2.84 g of a yellow mixture consisting of two compounds. Further separation on the chromatotron afforded 0.85 g of polygo- dial and 0.015 g of drimenol. The ethanol fraction (2.5 g) was subjected to CC on silica gel and sepa- rated by gradient elution (dichloromethane/meth- anol) to afford isodrimenin. The compounds were identifi ed by spectral data (IR, 1 H NMR, and 13 C NMR), which were in good agreement to those previously published (Cicció, 1984; Jansen and Groot,
2004; McCallion, 1982; Aasen et al., 1977; White and Burton, 1985), and by direct compari- son with authentic samples.
Table I shows the 31 plants used in this study from which dichloromethane and methanol/wa- ter extracts were prepared and tested against trypomastigotes in concentrations of up to 500
µg/mL. Table II shows the activities of ex- tracts of Podanthus ovatifolius, Berberis micro- phylla, Kageneckia oblonga, and Drimys winteri, the four plants that produced signifi cant inhibi- tion in the MTT test. These plant extracts ex- hibited activities between 7 and 5% of those of the standard compounds benznidazole or nifur- timox, respectively. The extracts from all other plants in Table I showed an IC 50 value higher than 500 µg/mL. Table III shows the activities of isodrimenin, drimenol, and polygodial, the three Table II. Activity of Chilean plant extracts on the
Plant
Extract MTT viability test IC 50
µg/mL] Mitique ( Podanthus ovatifolius) Methanol/water 40.1 3.0 Michay (
Berberis microphylla) Methanol/water 38.4 5.0 Bollén (
Kageneckia oblonga) Methanol/water 35.7 4.0 Canelo
(Drimys winteri) Dichloro- methane 51.2 2.0 Nifurtimox - 4.6 0.1 Benznidazole - 8.4 0.1 The MTT assay was carried out with the trypomastigote form of
T. cruzi. Extracts were used at fi nal concen- trations of 10, 15, 20, 50, 100, and 500 µg/mL. IC 50 val- ues were calculated from drug concentration-response curves (see Experimental). Results are shown as the average standard deviation of three independent ex- periments. O. M. Muñoz et al. · Antichagasic Plant Extracts 201
sesquiterpenes isolated from the stem bark of Drimys winteri (Fig. 1). The results show that polygodial, the most active antifungal (Kubo et al., 2001), antifeedant (Zapata et al., 2009), and antibacterial (Kubo et al., 2005) from D. winteri, has a weak anti- Trypanosoma activity with re- spect to the other sesquiterpenes. Isodrimenin and drimenol showed similar activities as nifur- timox or benznidazole (Table III). Some com- pounds of the other three active plant extracts are being isolated and their antiparasite activity will be tested later. Plant metabolites active against T. cruzi have been recently reviewed by our laboratories (Maya et al., 2007; Salas et al., 2011), and we concluded that the compounds isodrimenin and drimenol isolated from
lead structures to obtain new selective anticha- gasic drugs.
This research was funded by World Bank-CON- ICYT ACT-112 and Fondecyt-Chile No. 1090078 and 11110182. The authors thank Dr. Yedy Israel for his comments on the manuscript. Aasen A. J., Nishida J., Enzell C. D., and Appel H. H. (1977), The structure of (11ξ,12ξ)-11,12-di(7-drimen- 11-oxy)-11,12-epoxy-7-drimene. Acta Chem. Scand. 31B, 51 – 55. Castro J. A., Mecca M. M., and Bartel L. C. (2006), Toxic side effects of drugs used to treat Chagas’ disease (American trypanosomiasis). Hum. Exp. Toxicol. 25, 471 – 479. Cicció J. (1984), Poligodial, constituyente mayoritario de la corteza de Drimys granadensis L. F. (Winter- aceae). Ing. Cienc. Quím. 8, 45 – 46. Contreras V. T., Salles J. M., Thomas N., Morel C. M., and Goldenberg S. (1985),
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Compound IC 50 [ µ M ] Isodrimenin 27.9 0.3 Drimenol 25.1 0.5 Polygodial 120.4 1.5 Nifurtimox 16.1 0.2 Benznidazole 32.2 0.3 The activity was measured on
using the MTT method at the concentrations of 10, 15, 20, 50, 100, and 200
M . IC 50 values were calculated from drug concentration-response curves (see Experimental). Results are shown as the average standard deviation of three independent experiments. Fig. 1. Chemical structure of compounds isolated from Drimys winteri. R 1 R 2 O O Polygodial R 1 = R
2 = CHO
Drimenol R 1 = CH 2 OH; R
2 =CH
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