San Martin, J (San Martin, Jose). Univ Talca, Inst Biol Vegetal & Biotecnol, Talca, ChileThe extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 51.2 mu g/mL) was purified and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of T cruzi with IC50 values of 27.9 and 25.1 mu M, respectivel

Christen, P.

Ferreira, J.

Kemmerling, U.

Lopez-Munoz, R.

Maya, J.D.

Morello, A.

Munoz, O.M.

San Martin, J.

Zeitschrift für Naturforschung C

The extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 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 T. cruzi with IC50 values of 27.9 and 25.1 μM, respectively.</jats:p

Orlando M. Muñoz

Juan D. Maya

Jorge Ferreira

Philippe Christen

José San Martin

Rodrigo López-Muñoz

Antonio Morello

Ulrike Kemmerling

Crossref

Medicinal Plants of Chile: Evaluation of their Anti-Trypanosoma cruzi Activity

The extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 51.2 μg/mL) was purified and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of T. cruzi with IC50 values of 27.9 and 25.1 μM, respectively. © 2013 Verlag der Zeitschrift für Naturforschung, Tübingen

Muñoz, Orlando M.

Maya Arango, Juan

Ferreira, Jorge

Christen, Philippe

San Martín, José

López Muñoz, Rodrigo

Morello Casté, Antonio

Kemmerling Weis, Ulrike

Repositorio Académico de la Universidad de Chile

© 2013 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.comIntroductionChagas’ 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 et al., 1992; González et al., 1990).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 Podan-thus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri.Drimys winteri (Winteraceae) is a tree of eco-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).ExperimentalGeneralColumn 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) n-hexane/ethyl acetate (8:2, v/v) and (ii) n-hex-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 ActivityOrlando M. Muñoza,*, Juan D. Mayab, Jorge Ferreirab, Philippe Christenc, José San Martind, Rodrigo López-Muñozb, Antonio Morellob, and Ulrike Kemmerlingba Department of Chemistry, Faculty of Science, University of Chile, Santiago, Chile. Fax: 56-2 2713888. E-mail: omunoz@uchile.clb Institute of Biological Sciences, Faculty of Medicine, University of Chile, Santiago, Chilec School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerlandd Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Talca, Chile* Author for correspondence and reprint requestsZ. Naturforsch. 68 c, 198 – 202 (2013); received December 15, 2011/March 7, 2013The 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 (IC50 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 T. cruzi with IC50 values of 27.9 and 25.1 µM, respectively.Key words: Anti-Trypanosoma cruzi, Drimenol, IsodrimeninO. M. Muñoz et al. · Antichagasic Plant Extracts 199Preparative 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 n-hexane/ethyl acetate gradient (0, 1, 5, 10, 50, 100% ethyl acetate). Melting points are un-corrected. Optical rotations were measured with a Perkin Elmer 241 MC polarimeter (Waltham, MA, USA).1H and 13C NMR spectra were recorded in CDCl3, at 400 and 500 MHz for 1H NMR and 100 and 125 MHz for 13C NMR, on a Bruker Avance AM-400 spectrometer (Karlsruhe, Ger-many).The 1H 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 CDCl3 on a Bruker-DRX 500 MHz instrument. The 1H 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 (1H, 13C) and 2D (COSY, HMQC, HMBC) experiments were performed using standard Bruker microprograms.Plant materialsEthnopharmacological 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.ExtractionThe 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, family1 Podanthus ovatifolius Lag., Asteraceae2 Cryptocarya alba (Mol.) Looser, Lauraceae3 Escallonia illinita K. Presl., Saxifragaceae4 Blepharocalyx cruckshanksii (H. et A.) Nied., Myrtaceae5 Satureja gilliesii (Graham) Briq., Lamiaceae6 Drimys winteri J. R. Forst. et G. Forst., Winteraceae7 Luma chequen (Mol.) A. Gray, Myrtaceae8 Luma apiculata (DC.) Burret, Myrtaceae9 Fuchsia magellanica Lam., Onagraceae10 Colliguaja odorifera Mol., Euphorbiaceae11 Ugni molinae Turcz., Myrtaceae12 Alstroemeria revoluta Ruiz et Pav., Amaryllidaceae13 Pitavia punctata Mol., Rutaceae14 Podocarpus saligna D. Don, Podocarpaceae15 Lapageria rosea Ruiz et Pav., Philesiaceae16 Schinus latifolius (Gill. ex Lindl.) Engler, Anacar-diaceae17 Kageneckia oblonga Ruiz et Pav., Rosaceae No. Species, family18 Margyricarpus pinnatus (Lam.) Kuntze, Rosaceae 19 Pseudognaphalium vira vira (Mol.) A. Anderb., Asteraceae20 Eupatorium salvia Colla, Asteraceae21 Baccharis concava (Ruiz et Pav.) Pers., Asteraceae22 Viviania crenata (Hook.) G. Don ex H. et A., Ru-biaceae23 Fabiana imbricata Ruiz et Pav., Solanaceae24 Myoschilos oblonga Ruiz et Pav., Santalaceae25 Berberis darwinii Hook., Berberidaceae26 Maytenus chubutensis (Speg.) Lourt., O’Donell et Sleum., Celastraceae27 Myrceugenia chrysocarpa (O. Berg) Kausel, Myrta-ceae28 Elytropus chilensis (A. DC.) Muell. Arg., Apocyn-aceae29 Berberis serrato-dentata Lechler, Berberidaceae30 Berberis microphylla G. Forst., Berberidaceae 31 Pseudopanax laetevirens (Gay) Franch., Araliaceae200 O. M. Muñoz et al. · Antichagasic Plant Extractswere weighed and solubilized in dimethylsulfox-ide (DMSO) for biological assays.Culture of trypomastigotesAfrican 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 · 106 cells/25 cm2, 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% CO2 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 · 107 parasites/mL in RPMI 1640 culture medium (without phenol red).Trypomastigote viability assayViability 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 · 107 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).Characterization of compounds isolated from Drimys winteriBarks 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% n-hexane 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, 1H NMR, and 13C 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.Results and DiscussionTable 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 IC50 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 Tryp-anosoma cruzi trypomastigotes.Plant Extract MTT viability testIC50 [µg/mL]Mitique (Podanthus ovatifolius)Methanol/water 40.1  3.0Michay (Berberis microphylla)Methanol/water 38.4  5.0Bollén (Kageneckia oblonga)Methanol/water 35.7  4.0Canelo (Drimys winteri)Dichloro-methane51.2  2.0Nifurtimox - 4.6  0.1Benznidazole - 8.4  0.1The 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. IC50 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 201sesquiterpenes 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 Drimys winteri represent new basic lead structures to obtain new selective anticha-gasic drugs.AcknowledgementsThis 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), In vitro differentiation of Trypanosoma cruzi under chemically defi ned condi-tions. Mol. Biochem. Parasitol. 16, 315 – 327.Cotoras M., Garcia C., Lagos C., Folch C., and Mendoza L. (2001), Antifungal activity on Botrytis cinerea of fl avonoids and diterpenoids isolated from the surface of Pseudognaphalium spp. Bol. Soc. Chil. Quím. 46, 433 – 440.Croft S. L., Barrett M. P., and Urbina J. (2005), Chemo-therapy of trypanosomiases and leishmaniasis. Trends Parasitol. 21, 508 – 512.Faundez M., Pino P., Letelier P., Ortiz C., López R.,  Seguel C., Ferreira J., Pavani M., Morello A., and Maya J. D. (2005), Buthionine sulfoximine increases the toxicity of nifurtimox and benznidazole to Tryp-anosoma cruzi. Antimicrob. Agents Chemother. 49, 126 – 130.Fournet A. and Munoz V. (2002), Natural products as trypanocidal, antileishmanial and antimalarial drugs. Curr. Top. Med. Chem. 2, 1215 – 1237.González A. G., Bermejo J., Diaz J., Rodriguez E. M., Perez A., Yanes A., Rauter P., and Pozo J. (1990), Di-terpenes and other constituents of Eupatorium sal-via. Phytochemistry 29, 321 – 323.Hoffmann A., Lastra J., Veghazi E., and Farga C. (1992), Plantas medicinales de uso común en Chile, 2 ed. Fundación Cludio Gay, Santiago, Chile, p. 273.Table III. Activity of compounds isolated from Drimys winteri upon the Trypanosoma cruzi trypomastigote form.Compound IC50 [µM]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 T. cruzi trypomastigotes using the MTT method at the concentrations of 10, 15, 20, 50, 100, and 200 µM. IC50 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.R1R2OOPolygodial     R1= R2 = CHODrimenol       R1= CH2OH; R2 =CH3Isodrimenin202 O. M. Muñoz et al. · Antichagasic Plant ExtractsHoughton P. and Manby J. (1998), Medicinal plants of the Mapuche. J. Ethnopharmacol. 13, 89 – 103.Jansen B. M. and Groot A. (2004), Occurrence, bio-logical activity and synthesis of drimane sesquiterpe-noids. Nat. Prod. Rep. 21, 449 – 477.Kubo I., Fujita K., and Lee S. H. (2001), Antifungal mechanism of polygodial. J. Agric. Food Chem. 49, 1607 – 1611.Kubo I., Fujita K., Lee B., and Ha T. J. (2005), Anti-bacterial activity of polygodial. Phytother. Res. 19, 1013 – 1017.Maguire J. H. (2006), Chagas’ disease – Can we stop the deaths? N. Engl. J. Med. 355, 760 – 761.Maya J. D., Cassels B. K., Vasquez P., Ferreira J.,  Faúndez M., Galanti N., Ferreira A., and Morello A. (2007), Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host. Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol. 146, 601 – 620.McCallion R. F. (1982), Antibiotic substances from New Zealand plants. II. Polygodial, an anti-Candida agent from Pseudowintera colorata. Planta Med. 44, 134 – 138.Mosmann T. (1983), Rapid colorimetric assay for cel-lular growth and survival: application to prolifera-tion and cytotoxicity assays. J. Immunol. Methods 65, 55 – 63.Muñoz O. and Fajardo V. (eds.) (2005), Flora de Chile: Biologí a, farmacologí a y quí mica. Universidad de Playa Ancha, Valparaíso, Chile, p. 227.Muñoz O., Montes M., and Wilkomirsky T. (2001), Plantas medicinales de uso en Chile: Quí mica y Farmacologí a, 2 ed. Editorial Universitaria, Santiago, Chile.Ruiz A., Gutierrez I., Mardones C., Vergara C., Herlitz E., Vega M., Dorau C., Winterhalter P., and Von Baer D. (2010), Polyphenols and antioxidant activity of ca-lafate (Berberis microphylla ) fruits and other native berries from Southern Chile. J. Agric. Food Chem. 58, 6081 – 6089.Salas C., Faundez M., Morello A., Maya J. D., and Tapia R. (2011), Natural and synthetic naphthoquinones active against Trypanosoma cruzi: an inicial step towards new drugs for Chagas disease. Curr. Med. Chem. 18, 144 – 161.San Martin J. (1983), Medicinal plants in Central Chile. Econ. Bot. 37, 216 – 227.White J. D. and Burton P. J. (1985), Synthese of the insect antifeedant ()-cinnamodial and the drimane sesquiterpenoids ()-isodrimenin and ()-fragrolide. J. Org. Chem. 50, 357 – 364.Zapata N., Budia F., Viñuela E., and Medina P. (2009), Antifeedant and growth inhibitory effects of extracts and drimanes of Drimys winteri stem bark against Spodoptera littoralis (Lep., Noctuidae). Ind. Crops Prod. 30, 119 – 125.

Medicinal plants of Chile: Evaluation of their anti-trypanosoma cruzi activity

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Medicinal Plants of Chile: Evaluation of their Anti-Trypanosoma cruzi Activity

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