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Traditions &Transformationin Ethnobotany ICEB

2009

Tradiciones &transformacionesen Etnobotánica

MARÍA LELIA POCHETTINOANA H. LADIO

PATRICIA M. ARENAS(EDITORAS)

Editor responsable:

CYTED - Programa Iberoamericano Ciencia

y Tecnología para el Desarrollo

Coordinación de la edición:

Red Iberoamericana de Saberes y Prácticas Locales

sobre el Entorno Vegetal (RISAPRET)

Nilda Dora Vignale (Coordinadora)

Facultad de Ciencias Agrarias

Universidad Nacional de JujuyAlberdi 47 4600 San Salvador de Jujuy ArgentinaTel.: + 54 388 422-1548ndvignale@yahoo.com.arwww.fca.unju.edu.ar/risapret

© 2010

ISBN: 978-84-96023-95-6

Impreso en Argentina.

Todos los derechos reservados. Esta publicación no puede ser reproducida, en todo ni en parte, ni registrada en o transmitida por un sistema de recu-peración de información, en ninguna forma ni por ningún medio, sea me-cánico, fotoquímico, electrónico, magnético, electroóptico, por fotocopia, o cualquier otro, sin el permiso previo por escrito de los autores.

COMITÉ EDITORIAL

Emanuela Appetiti. The Institute for the Preservation of Medical Traditions hosted by the Smithsonian Institution Washington, D.C. USA.

Patricia M. Arenas. Laboratorio de Etnobotánica y Botánica Aplicada (LEBA), Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata. Calle 64 nº 3, 1900 La Plata, Argentina. CONICET.

Lidia S. Burry. Laboratorio de Palinología, Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata. Funes 3250, 7600 Mar del Plata, Argentina.

Aylen Capparelli. Departamento Científico de Arqueología, Museo de La Plata, Paseo del Bosque s/n, 1900 La Plata, Argentina. CONICET.

Laure Emperaire. Institut de Recherche pour le Développement. Unité Mixte de Recherche 208 (IRD-MNHN) PALOC (Patrimoines locaux). Francia.

Füsun Ertuğ. Consultant of ethnobotany at the Turkish Academy of Science (TUBA), Turkey.

Leonardo Galetto. Instituto Multidisciplinario de Biología Vegetal, Universidad Nacional de Córdoba. Argentina. CONICET.

Michael Heinrich. School of Pharmacy, London, UK.Norma Hilgert. Instituto de Biología Subtropical,

Facultad de Ciencias Forestales, Universidad Nacional de Misiones. CeIBA- CONICET. Puerto Iguazú, Misiones, Argentina.

Ana Ladio. INIBIOMA (CONICET - UNComahue). Quintral 1250. 8400 Bariloche, Río Negro, Argentina.

Verónica Lema. Laboratorio de Etnobotánica y Botánica Aplicada (LEBA), Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata. Calle 64 nº 3, 1900 La Plata, Argentina. CONICET.

María Rosa Martínez. Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata. Paseo del Bosque s/n, 1900 La Plata, Argentina. CONICET

Soledad Molares. INIBIOMA (CONICET - UNComahue). Quintral 1250. 8400 Bariloche. Río Negro, Argentina.

Ramón Morales. Real Jardín Botánico de Madrid, CSIC. Plaza de Murillo, 2. E-28014 Madrid, España.

Manuel Pardo de Santayana. Universidad Autónoma de Madrid, España.

Cecilia Perez de Micou. CONICET-INAPL, Universidad de Buenos Aires, Tres de Febrero 1378, 1426 Buenos Aires, Argentina.

María Lelia Pochettino. Laboratorio de Etnobotánica y Botánica Aplicada (LEBA), Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata. Calle 64 nº 3, 1900 La Plata, Argentina. CONICET.

Gustavo Scarpa. Centro de Estudios Farmacológicos y Botánicos-CONICET. Paraguay 2155, p.16º, Buenos Aires, Argentina.

Alain Touwaide. The Institute for the Preservation of Medical Traditions hosted by the Smithsonian Institution Washington, D.C. USA.

Matilde Trivi de Mandri. Laboratorio de Palinología, Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250. 7600 Mar del Plata, Argentina.

Soultana Valamoti. Department of Archaeology, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece.

Michèle Wollstonecroft. The Institute of Archaeology, University College London, UK.

REVISORES INVITADOS

José Luis Fernández Alonso. Instituto de Ciencias Naturales, Herbario Nacional Colombiano. Apart. 7495 Universidad Nacional de Colombia, Bogotá, D.C. Colombia.

Kevin N. Laland. School of Biology, St. Andrews University, St. Andrews, Scotland, UK.

Lía Montti. Instituto de Ecología Regional (IER), Universidad Nacional de Tucumán, Casilla de Correo 34, 4107 Yerba Buena, Tucumán, Argentina y Centro de Investigaciones del Bosque Atlántico (CeIBA), Andresito 21, Puerto Iguazú, Misiones, Argentina.

INDICE

1. INTRODuCCIóN ........................................................................................................................................... 1 2. IMpLICANCIAS TEóRICAS y EpISTEMOLógICAS DE LAS ESTRATEgIAS DE INVESTIgACIóN

ETNOBOTáNICA ............................................................................................................... 3 2.01 Crivos, M. Implicancias teóricas y epistemológicas de las estrategias de investigación

etnobotánica ........................................................................................................................................... 5

3. LAS pLANTAS y EL hOMBRE EN EL pASADO ....................................................................... 11 3.0.01 Capparelli, a. & l. prates. Identificación específica de frutos de algarrobo (Prosopis spp. L.,

Fabaceae) y mistol (Ziziphus mistol Griseb., Rhamnaceae) en un sitio arqueológico de Patagonia ........................................................................................................................................... 13

3.0.02 rodríguez, M. F. & z. e. rúgolo de agrasar. Gramíneas herbáceas y leñosas en espacios domésticos y rituales de la Puna meridional argentina durante el Holoceno ............................ 20

3.0.03 Colobig, M. M., a. F. zuCol & d. l. Mazzanti. Análisis de microrestos silíceos en secuencias sedimentarias del Sitio 1 de la localidad arqueológica Lobería I, Buenos Aires, Argentina .... 27

3.0.04 andreoni, d., a. gil & a. Capparelli. Efectos de la carbonización en especies leñosas de las provincias fitogeográficas Patagónica y del Monte (Mendoza, Argentina): una perspectiva arqueológica .............................................................................................................. 33

3.0.05 leMa, v. s., M. l. poChettino, M. pueblas, M. C. paleo & M. pérez Meroni. La etnobotánica como herramienta interpretativa en arqueología: prácticas de recolección en el Holoceno Tardío del Litoral Bonaerense (Argentina) ......................................................................................... 38

3.0.06 llano, C. & a. ugan. Rendimiento económico de plantas silvestres del sur de Mendoza: valores nutricionales, costos de manejo e interpretación del registro arqueológico ............... 44

3.0.07 leMa, v. s. Plant domestication and man-plant degrees of dependence in the pre-hispanic cultural development of Argentinean Northwest ............................................................................. 49

3.0.08 touwaide, a. History of Botany as Ethnobotany. Proposals toward a new approach to the ancient legacy ............................................................................................................................ 55

3.0.09 azar, p. F. El añil: una planta para teñir, un color para recordar ...................................................... 64 3.0.10 toMei, p. e., s. triMarChi s. & C. lenzi. An ancient ethnobotanic herbarium in the district

of Pisa (Tuscany, Italy) .......................................................................................................................... 69

3.1 HACIA UNA INTEGRACIÓN DE METODOLOGÍAS PARA OPTIMIZAR EL ESTUDIO DEL HOMBRE CON SU AMBIENTE EN EL PASADO

3.1.01 burry, l., M. trivi de Mandri & C. perez de MiCou. Hacia una integración de metodologías para optimizar el estudio del hombre con su ambiente en el pasado ........................................ 73

3.1.02 Korstanje, M. a. Lo lúdico, lo creativo y lo formalizado en la investigación del pasado: expandiendo saberes en arqueobotánica .......................................................................................... 76

3.1.03 Chaves, s. a. de Miranda. Paleopharmacology and palinological research in coprolites ................ 83 3.1.04 yagueddú, C. Identificación de restos vegetales en coprolitos ............................................................ 90 3.1.05 d’antoni, h. l. Arqueoecología: la humanidad en los ecosistemas del pasado .............................. 96

3.2 RECENT RESEARCH IN POST-HARVEST TRADITIONS IN HUMAN PREHISTORY: OLD AND NEW WORLD PALAEOETHNOBOTANICAL APPROACHES TO LINKING THE ARCHAEOLOGY AND ETHNOBOTANY OF PLANT PROCESSING.

3.2.01 Capparelli, a., t. valaMoti & M. wollstoneCroFt. Recent research in post-harvest traditions in human prehistory: old and new world palaeoethnobotanical approaches to linking the archaeology and ethnobotany of plant processing .................................................................. 104

3.2.02 Capparelli, a. Archaeobotanical recognition of food products derived from the algarrobo [Prosopis flexuosa DC and P. chilensis (Mol.) Stuntz] charred remains and its application to the case of El Shincal, Hualfin Valley, Argentina ....................................................................... 105

3.2.03 leMa, v. Cultivation, production and domestication: evaluating the archaeological visibility of interactions between human societies and plant populations in the past ........................... 110

3.2.04 lópez, M. l., a. Capparelli & a. nielsen. Modern practices of quinoa (Chenopodium quinoa W. Chenopodiaceae) processing and consumption in Lípez, Bolivia: an ethnoarchaeological approach .................................................................................................................................................. 117

3.2.05 ertuğ, F. The consumption of flax: postharvest activities linked to linseed oil production in Turkey .................................................................................................................................................. 123

3.2.06 valaMoti, s. M. The prehistory and modern survival of bulk processing cereals for piecemeal consumption: case studies from Greece ............................................................................................ 128

3.2.07 hosoya, l. a. Staple or famine food ? Ethnographic and archaeological approaches to nut processing in East Asian prehistory .................................................................................................... 133

3.2.08 wollstoneCroFt, M. Evolutionary trends in human eating behaviours: food processing and niche construction ............................................................................................................................................. 139

4. puEBLOS y pLANTAS quE ALIMENTAN ............................................................................................... 145 4.01 rapoport, e. ¿Cuán sabios son los sabios? .............................................................................................. 147 4.02 MCClatChey, w. & d. reedy. A mouthful of water: Do cider producers recognize the ancient

clean water potential of their products? ........................................................................................... 149 4.03 baser, K. h. C. Wild food plants of Turkey ............................................................................................. 161 4.04 CáMara hernández, j. Las tradiciones culinarias de los pueblos del noroeste argentino como

base de la conservación de la diversidad de los maíces nativos .................................................. 166

5. LA CONSTRuCCIóN DE LA DIVERSIDAD AgRÍCOLA: CONCEpTOS y pRáCTICAS ........................ 173 5.01 eMperaire, l. La construcción de la diversidad agrícola: conceptos y prácticas .............................. 175 5.02 oliveira, j. Cabral de. The role of social networks in agriculture biodiversirty: the Wajãpi case 176 5.03 eMperaire, l. & j. oliveira. Redes sociales y diversidad agrícola en la Amazonía brasileña:

un sistema multicéntrico ...................................................................................................................... 180 5.04 poChettino, M. l. Huertos peri-urbanos como aporte a la diversidad agrícola, Provincia de

Buenos Aires, Argentina ....................................................................................................................... 186 5.05 Freire, g. Ecología política del cambio agrícola: El dilema de la agroforestería indígena en un

petroestado ............................................................................................................................................. 193 5.06 deMeulenaere, e. Farmers’ seed systems in industrial agricultures: new research object, renewed

approaches in Ethnobotany ................................................................................................................. 197

6. FITOTERApIA y pLANTAS MEDICINALES ............................................................................................... 205 6.01 elvin-lewis, M. Ways in which integrated studies can identify meaningful remedies

for populations that use them ............................................................................................................ 207 6.02 Martínez, g. j. Los criterios terapéuticos en la farmacopea natural de los tobas bermejeños

del Chaco Central (Argentina) ............................................................................................................. 213 6.03 KutsChKer, a., C. ezCurra & v. balzaretti. Valeriana en los Andes Australes: biodiversidad y

compuestos activos ................................................................................................................................ 219 6.04 del vitto, l. a., e. M. petenatti & M. e. petenatti. Ethnomedical plants from Cuyo region,

Argentina: uses and conservational status ....................................................................................... 225 6.05 gheno heredia, y. a., a. r. Martínez CaMpos, e. sánChez vera & g. nava bernal. Plantas

medicinales de la organización de parteras y médicos indígenas tradicionales de Ixhuatlancillo, Veracruz, México .......................................................................................................... 232

6.06 pino benítez, n. & y. s. rentería. Plantas usadas en el tratamiento contra el estrés en una comunidad del Nor-occidente colombiano: un enfoque cuantitativo ......................................... 239

6.07 Mijares, g., C. Meneses & M. teixeira. Etnofarmacología de las helmintiasis en la comunidad de Mendoza, Municipio Acevedo. Estado Miranda, Venezuela ....................................................... 244

6.08 verde, a., d. rivera, j. Fajardo, C. obón, a. valdés, j. r. p. ruíz-gallardo, v. benlloCh, r. Ciudad, núñez & a. piera. Medicina popular y salud como materia curricular transversal en educación secundaria en Castilla La Mancha (España) ........................................................................................ 250

6.09 Madureira, M. do Céu. Antimalarial drug development research and the ancient knowledge of traditional medicines in S. Tomé e Principe Islands .................................................................. 256

6.10 Fajardo, j., a. verde, d. rivera, a. valdés, C. obón, M. heinriCh & F. Cebrián. Plantas medicinales de Castilla-La Mancha (España) merecedoras de investigación farmacológica y fitoquímica por la importancia de sus usos tradicionales en humanos .............................................................. 265

6.11 parra-sánChez, e., o. pérez-esCobar & l. sánChez. Exploración de saberes de plantas medicinales de Fusagasugá, Colombia ..................................................................................................................... 270

6.12 baCh, h. g. & r. h. Fortunato. Estudio de patrones de elección de plantas medicinales en una zona urbana del Oeste Bonaerense (Argentina) ................................................................. 277

6.13 Cristina, i. & p. M. arenas. Plantas de uso tradicional en Argentina de posible aplicación como adaptógenos ............................................................................................................................................ 282

6.14 Molares, s. & a. ladio. Criterios ambientales y organolépticos en los patrones de selección y uso de plantas medicinales en una comunidad Mapuche de la Patagonia semiárida Argentina. 286

6.15 Cuassolo, F., a. ladio & s. Calvelo. Los líquenes medicinales comercializados en S. C. de Bariloche: importancia local y control sanitario ........................................................ 291

6.16 riCheri, M., a. M. beesKow & a. ladio. Las plantas y la salud en la comunidad boliviana, Madryn (Argentina) .............................................................................................................................................. 297

6.17 gonzález, s. b., l. n. huaiquinao, a. gonzález, C. van baren, p. di leo lira & a. l. bandoni. Uso popular del paico y composición química de su aceite esencial en la zona de Esquel (Chubut, Argentina) .............................................................................................................................. 303

6.18 arenas, p. M. Consumo de algas en la provincia de Buenos Aires, Argentina: tradición o nuevo uso de los recursos? .............................................................................................................................. 308

6.19 lewis, w. h. Evaluating and protecting indigenous pharmacopeas and traditional knowledge .. 313 6.20 buCCiarelli, a. & M. sKliar. Evaluation of antiulcer activity of Solidago chilensis (Asteraceae)

in mice ..................................................................................................................................................... 317 6.21 buCCiarelli, a., C. bras, n. gandini, a. Minetti & M. sKliar. Estudio toxicológico de la fracción

acetato de etilo de capítulos de Solidago chilensis (Asteraceae) ................................................ 322 6.22 pérez Cuadra, v. & v. CaMbi. Foliar crystals in ornamental Araceae, their risk for health .............. 327 6.23 Flores, e. n. & n. d. vignale. Caracterización micrográfica de órganos vegetativos y

reproductivos de interés etnobotánico de Geoffroea decorticans (Gill. ex Hook. et Arn.) Burkart (Fabaceae) ................................................................................................................................ 330

7. RECuRSOS VEgETALES, MICOLógICOS O FICOLógICOS VALIOSOS pARA LA huMANIDAD .... 337 7.01 suárez, M. e. Recursos forestales no madereros (RFNM) entre wichís del Chaco semiárido salteño,

Argentina ................................................................................................................................................. 339 7.02 hurrell, j. a., e. ulibarri, g. deluCChi, M. l. pérez & n. anglese. Plantas condimenticias

utilizadas en la ciudad de Buenos Aires y en el Noreste Bonaerense (Argentina) .................... 344 7.03 seoane n. & j. oChoa. Uso del neneo en la región sur de la provincia de Río Negro (Argentina)

y su relación con la producción ovina ............................................................................................... 350 7.04 rodríguez M. r., M., l. galiCia s., w. sánChez, l. góMez M., a. e. zarCo a. & e. CeCCon.

Usos actuales, distribución potencial y etnolingüística del bambú leñoso (Bambuseae) en México ................................................................................................................................................. 355

7.05 MassoCa, p. e. & g. b. Frausin. Botánica funeraria en el cementerio de Araçá (São Paulo, Brasil) .. 364 7.06 de robert, p. & e. Katz. Usos alimentarios de palmeras un estudio comparativo

en Amazonía Brasileña .......................................................................................................................... 370 7.07 barriuso, M. a. & M. garCía-barriuso. Plantas tintóreas históricamente utilizadas en el centro

de la Península Ibérica ......................................................................................................................... 376 7.08 Caballero roque, a., j. perez, r. esCobar, j. ballinas & g. vela. Uso de nanche (Byrsonima

crassifolia, Malpighiaceae) como saborizante natural de una bebida refrescante ..................... 380 7.09 jabeen, a. Ethnobotany of fodder species of Ayubia National Park, Pakistan,

its conservation status and impacts on environment .................................................................... 384 7.10 Morales, r. Labiadas de España en América. Intercambio de Usos ................................................... 391 7.11 Marrero, a. Las labiadas en Canarias. Encrucijada en el Atlántico .................................................... 401

7.12 sChMidt-lebuhn, a. n. An update on the state of research on Minthostachys (Lamiaceae) .......... 408 7.13 albreCht, r., j. Montenegro, r. roldan, a. gurni, n. vignale & g. bassols. Análisis de las

composiciones nutricionales de cultivos andinos ........................................................................... 414 7.14 guiaMet, p., p. M. arenas, p. lavin, p. battistoni & s. góMez de saravia. Utilidad de extractos

obtenidos de plantas en el control de microorganismos que afectan al patrimonio cultural .... 419 7.15 staMpella, p., p. M. arenas, a. lópez, s. borrego, i. vivar & n. Cabrera. Plantas útiles en el

control de insectos bibliófagos ........................................................................................................... 423 7.16 vignale, n. d., M. a. rivas, e. jiMénez & a. a. gurni. Identificación micrográfica de semillas

de “chía” (Salvia hispanica L. - Lamiaceae) ..................................................................................... 428 7.17 CaMbi, v. & v. pérez Cuadra. Anatomía foliar de portulacas bonaerenses de importancia económica ... 433 7.18 guevara valenCia, M., M. t . gonzález-arnao, y. M. Martínez oCaMpo & j. Molina torres. Aplicación

de alcamidas presentes en extractos de Chrysanthemum morifoliun al cultivo in vitro de Vanilla planifolia ..................................................................................................................................... 437

8. ETNOBOTáNICA ECOLógICA .................................................................................................................... 443 8.01 villar, l. La toponimia de origen vegetal refleja el saber etnoecológico:

el caso del Pirineo Central (España) .................................................................................................. 445 8.02 Calvet-Mir, l., M. Calvet-Mir & v. reyes-garCía. Traditional ecological knowledge and landraces

in situ conservation in high mountain home gardens of Vall Fosca, Catalan Pyrenees, Iberian Peninsula ................................................................................................................................... 457

8.03 oChoa, j., a. ladio & M. lozada. Aspectos ecológicos y socioculturales asociados al uso de plantas silvestres en la población rural de Arroyo Las Minas (Río Negro, Argentina) .............. 465

8.04 ahuMada, a., M. l. CiaMpagna, j. vera bahiMa, j. j. garat & j. otero. Prácticas culturales en la selección y conservacion de hortalizas locales en el Cinturón Hortícola Platense ......... 472

8.05 buet Costantino, F., e. ulibarri & j. a. hurrell. Las huertas familiares en la isla Paulino (Buenos Aires, Argentina) .................................................................................................................... 479

8.06 eyssartier, C., a. ladio & M. lozada. Conocimiento tradicional sobre plantas en huertas, invernaderos y jardines en dos poblaciones rurales de la estepa patagónica ........................... 485

8.07 Cardoso, M. b., a. ladio & M. lozada. Utilización de especies combustibles en una comunidad rural de la estepa patagónica ........................................................................... 496

8.08 girsCht, a. M., a. e. rovere & s. Molares. El conocimiento etnobotánico en la generación de propuestas de restauración y uso sustentable de un área de reserva nacional de la Patagonia Argentina .................................................................................................................... 502

8.09 planChuelo, a. M. Therapeutic gardening survey in the State of Missouri, USA ............................. 509

9. TRADICIóN E INNOVACIóN EN LA RELACIóN ENTRE EL hOMBRE y LAS pLANTAS ............. 515 9.01 palaCio, M. & e. Carrizo. El conocimiento sobre plantas tintóreas en teleras de dos

departamentos de Santiago Del Estero .............................................................................................. 517 9.02 reMorini, C., M. Crivos, M. r. Martínez & a. aguilar Contreras. El “susto”: “síndrome culturalmente

específico” en contextos pluriculturales. Algunas consideraciones sobre su etiología y terapéutica en México y Argentina ...................................................................................................... 523

9.03 Katz, e. Cruzeiro do Sul Market (Acre, Brazilian Amazon): reflection of the regional culture and agricultural diversity .............................................................................................................................. 531

9.04 bonet, M. À., M. roldán, j. CaMprubí & j. vallès. Etnobotánica de Gallecs (Cataluña, Península Ibérica). Plantas y tradición en un entorno social y natural cambiante ..................................... 540

9.05 KujawsKa M. & Ł. ŁuCzaj. Studies of wild food plants in communist and post-communist Poland: changes in use and in research methodology .................................................................................. 545

9.06 pérez, M. l. & M. l. poChettino. Oficinal u oficial? Plantas de uso corriente denominadas a partir de fármacos (La Plata y alrededores, Buenos Aires, Argentina) ........... 552

9.07 gandolFo, e. s., t. M. ribeiro, & n. hanazaKi. Can the homegardens persist with the urbanization of the coastline? ................................................................................................................................... 557

pRóLOgO

La Red Iberoamericana de Saberes y Prácticas Locales sobre el Entorno vegetal (RISAPRET) del Programa Iberoamericano Ciencia y Tecnología para el Desarrollo (CYTED), fuertemente compro-metida con el desarrollo de la Etnobotánica en el ámbito geográfico en el que se desempeña (Ibero-américa) pero sin descuidar la importancia de las in-terrelaciones con otras áreas del planeta, ofrece hoy a la comunidad este interesante libro que constituye una muestra de los avances de las investigaciones en la actualidad.

En sus páginas, el lector encontrará valiosos apor-tes a este campo científico multidisciplinario, cuida-dosamente compilados en Capítulos, que permiten efectuar un recorrido progresivo por una parte impor-tante de los diferentes espacios que la Etnobotánica comprende, resultando por ello de utilidad tanto para quienes tienen interés en conocer de qué trata, para quienes ya están iniciando sus investigaciones en esta área como para quienes están en dicho camino.

Incluye los aportes de investigadores integran-tes de los Grupos Participantes de RISAPRET, a los que se suman voluntariamente quienes participaron del V CONGRESO INTERNACIONAL DE ETNOBOTÁNICA (ICEB), que tuvo lugar en S. C. de Bariloche (ARGEN-TINA) en octubre de 2009, otorgándole ello una en-riquecedora variedad de áreas geográficas diferentes de análisis, posibilitando que la Red amplíe, de esta manera, sus contactos y la posibilidad de ofrecer la presente obra.

Por un lado están representados los Grupos de las UNIVERSIDADES DE SALAMANCA y de ALICANTE (ESPAÑA), de la UNIVERSIDAD NACIONAL AUTÓNOMA

(MÉXICO) y por el otro la totalidad de los Grupos de ARGENTINA, país anfitrión de este Congreso V ICEB.

Cabe expresar un particular agradecimiento a to-dos los autores de los trabajos que orgullosamente presenta RISAPRET en este Libro.

Dicho agradecimiento se transforma en ESPECIAL cuando va dirigido a las tres Editoras del mismo. Se trata de las Dras. María Lelia POCHETTINO - Respon-sable del Grupo Participante del Laboratorio de Etno-botánica y Botánica Aplicada (LEBA) de la Facultad de Ciencias Naturales y Museo de la UNIVERSIDAD NACIONAL DE LA PLATA - Patricia M. ARENAS, inte-grante del mismo y de Ana H. LADIO, Responsable del Grupo Participante de la UNIVERSIDAD NACIONAL DEL COMAHUE, con sede en Bariloche (ARGENTINA), quienes gracias a su extensa y destacada trayectoria y experiencia en la especialidad así como a su respon-sabilidad, han logrado, después de una ardua tarea que insumió extensas horas de trabajo, el producto que con honor esta Coordinación de RISAPRET ofrece a la comunidad.

Si los lectores a quienes llegue esta obra logran ampliar su visión acerca de los diferentes aspectos que conforman la Etnobotánica; si es capaz de des-pertar pasión por el trabajo con las comunidades; si constituye nuevas oportunidades para incrementar las comunicaciones entre investigadores, teniendo en cuenta el elevado número de autores y las dife-rentes procedencias y áreas de estudio; si enriquece las bibliotecas… significará que la obra ha sido útil, que el esfuerzo de autores y editores tuvo sentido y que por lo tanto, RISAPRET continúa cumpliendo su cometido.

Dra. Nilda Dora VIGNALECoordinadora

Red Iberoamericana de Saberes y Prácticas Locales sobre el Entorno Vegetal

(RISAPRET)

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anTimalarial drug dEvElopmEnT rEsEarch and ThE anciEnT KnowlEdgE of TradiTional mEdicinEs in s. Tomé E principE islands

M. C. Madureira

Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, 2829-511 Monte de Caparica, Portugal, mcmadureira@oninet.pt.

6.09

AbstrACt

Madureira, M. C. 2010. Antimalarial Drug Development Research and the Ancient Knowledge of Traditional Medicines in S. Tomé e Principe Islands. Malaria is the most important parasitic disease in tropical areas, especially in many African countries due to resistance of Plasmodium falciparum to the clinical available drugs. In S. Tomé e Príncipe (STP), traditional medicine plays a crucial role, as population has few means of accessing medical treatment. Antimalarial activity and toxicity of 13/medicinal plants, used by traditional healers in STP for the treatment of malaria and fever, was studied. Tithonia diversifolia (Asteraceae) was selected for phytochemical sudies, which included bioguided fractionation, isolation and structural elucidation of its active compound. The sesquiterpenic lactone tagitinin C, was identified, and its antiplasmodial activity was verified (IC50 ≈ 0.24 µg/ml and IC50 ≈ 0.25 µg/ml against the chloroquine resistant P. falciparum strains FCB1 and W2), thus being a good candidate for the development of a new antimalarial product. All these results were reported to the STP government (Minister of Health.

Keywords: malaria, medicinal plants, São Tomé e Príncipe, Tithonia diversifolia; traditional medicine.

resuMen

Madureira, M. C. 2010. Investigación para el de-sarrollo de drogas antimaláricas y el conocimiento ancestral sobre medicinas tradicionales en S. Tomé e Islas Principe. La malaria es la enfermedad parasita-ria más importante en áreas tropicales, especialmente en muchos países africanos debido a la resistencia de Plasmodium falciparum a las drogas disponibles. En S. Tomé y Príncipe (STP), la medicina tradicional juega un rol crucial, dado que la población carece de medios para acceder a tratamientos médicos. Se estu-dió la actividad y toxicidad antimalárica en 13 plantas medicinales usadas por curadores tradicionales para el tratamiento de malaria y fiebre en STP. Se selec-cionó Tithonia diversifolia (Asteraceae) para realizar estudios fitoquímicos, incluyendo fraccionamiento bioguiado, aislamiento y elucidación estructural del principio activo. Se identificó la lactona sesquiter-pénica tagitinina C, cuya actividad se verificó (IC50 ≈ 0.24 µg/ml e IC50 ≈ 0.25 µg/ml contra P. falciparum cloroquino resistentes FCB1 y W2), resultando un buen candidato para el desarrollo de un nuevo pro-ducto antimalárico. Los resultados se comunicaron al Ministerio de Salud de STP.

Palabras clave: malaria, plantas medicinales, São Tomé e Príncipe, Tithonia diversifolia, medicina tra-dicional.

IntrODuCtIOnMalaria is the most important parasitic disease

in tropical areas, especially in many African coun-tries due to resistance of Plasmodium falciparum to the clinical available drugs. In S. Tomé e Prínci-pe (STP), traditional medicine plays a crucial role, as population has few means of accessing medical treatment.

Studies of plants used in traditional medicine for the treatment of malaria in various cultures have yielded important drugs that are critical to modern medicine (quinine from bark of the Pe-ruvian Cinchona tree, and artemisinin from the Chinese Artemisia annua). The continued reliance of contemporary indigenous populations on plant medicines warrants close scrutiny of those species

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for pharmacological action, adverse interactions (e.g., with pharmaceuticals), and long-range toxi-city. In view of the growing rate of drug- and pes-ticide-resistance among plasmodia and anophe-lines, respectively, these plants may eventually contribute as well to antimalarial drug discovery and the development of more effective mosquito repellents and antibreeding agents (Etkins et al., 2004). In fact, plants used in traditional medicine may hold keys to the secrets of many potent an-timalarial drugs, and over the shorter term, these plants can also continue to play a role in primary health care and insect management, especially where pharmaceuticals and other biomedical te-chnology is not available on a sustainable basis. We argue that the ethnopharmacological appro-ach is the best means to explore these plants, and present some of the results achieved during an ethnopharmacological study of medicinal plants from STP islands, in the Gulf of Guinea (Madureira et al., 2002a). Following this trend, and since ma-laria is one of the major health problems of the re-gion, and as there is a high prevalence of chloro-quine resistant Plasmodium falciparum (Loureiro et al., 1996), a study of 13 medicinal plants, used by the local traditional healers for the treatment of malaria / fevers, have been undertaken.

The “in vitro” inhibitory effects of the plant extracts on chloroquine resistant P. falciparum strains were evaluated, as well as their hepatic schizontocidal activity in P. berghei (Hep G2 cell line). Similarly, the “in vivo” antimalarial proper-

ties of these plant extracts have been studied in mice (Madureira et al., 2002b), and its “in vitro” cytotoxicity evaluated (hepatotoxiciy, mutage-nicity and acute toxicity). Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae) was selected for phytochemical sudies, which included bioguided fractionation, isolation and structural elucidation of its active compound (Goffin et al., 2002). Its antiplasmodial activity was confirmed, as well as its citotoxicity, thus being a good candidate for the development of a new antimalarial product.

MAterIAL AnD MetHODs1. Plant Preparation

The fresh leaves or bark of 13 medicinal plants were collected in S. Tomé island (S. Tomé e Prín-cipe, Gulf of Guinea), under the guidance of the healers. The identification and authentication was done by Prof. Jorge Paiva from Instituto Botânico da Universidade de Coimbra (COI), Portugal, where voucher specimens were deposited (Table 1).

Dried material was powdered and extracted with ethanol 70%, concentrated by rotary vacuum evaporation and lyophilized (CEE-crude ethanol extracts). Fresh material were initially homoge-nized, followed by same procedure. The CEE of each plant was dissolved in methanol and water 1:2 (MW) and subsequently partitioned between petroleum ether (PE), dichloromethane (DM) and ethyl acetate (EA); all the resulting fractions were lyophilized after concentration by rotary vacuum evaporation. Samples were frozen at -20 ºC.

Table 1. Medicinal plants traditionally used for malaria and fevers. Abbreviations: a, b, Different chemotypes of the same species, but locally distinguished by the traditional healers.

Number Botanical Name (Family) Local Name Plant part usedVoucher Nº (COI)

1 struchium sparganophora (asteraceae) Libô-d’áua Leaves mm 125

2 Vernonia amygdalinaa (asteraceae) Libô-mucambu Leaves mm 114

3 Vernonia amygdalinab (asteraceae) Libo-qué Leaves mm 21

4 ageratum conyzoides (asteraceae) fiá-malé aerial parts mm 28

5 cinchona succirubra (rubiaceae) Quina bark mm 25

6 aloe humilis (aloeaceae) áliba-babosa Leaf-sap mm 354

7 Tithonia diversifolia (asteraceae) Girassol aerial parts mm 625

8 cedrela odorata (meliaceae) cidlela bark mm 321

9 Premna angolensis (Verbenaceae) Pó-ama bark mm 619

10 Pycnanthus angolensis (myristicaceae) Pó-cassom bark mm 426

11a morinda lucida (rubiaceae) Gligô bark mm 26

11b morinda lucida (rubiaceae) Gligô Leaves mm 26

12 cestrum laevigatum (solanaceae) coedano Leaves mm 102

13 canna indica (cannaceae) salaconta roots mm 14

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Table 2. Antimalarial activity of plant extracts against P. falciparum (Dd2) “in vitro”. Abbreviations: CEE, crude ethanolic extracts; PE, petroleum ether fraction, DM, dichloromethane fraction; EA, ethyl acetate fraction; MW, remanescent methanol and water fraction; n.d., not determined. IC50 (chloroquine) = 0.094 mg/ml.

Number Botanical NameIC50, median values (mg/ml)

CEE PE DM EA MW1 struchium sparganophora 180 <10 100 100 240

2 Vernonia amygdalinaa 120 170 235 500 n.d.

3 Vernonia amygdalinab 340 200 80 10 n.d.

4 ageratum conyzoides 150 110 55 220 n.d.

5 cinchona succirubra <10 <10 <10 <10 <10

6 aloe humilis 260 150 150 25 500

7 Tithonia diversifolia 15 <10 <10 140 500

8 cedrela odorata 190 110 50 n.d. n.d.

9 Premna angolensis 180 250 250 250 n.d.

10 Pycnanthus angolensis <5 100 100 100 n.d.

11a morinda lúcida <10 50 50 100 500

11b morinda lúcida 10 130 60 500 125

12 cestrum laevigatum 100 100 50 150 135

13 canna indica 500 130 25 245 500

2. Determination of “in vitro” antiplasmodial activity in plant extracts2.1. Blood stage assays. To test the antiplasmodial activity of plant extracts we used the susceptibility microassay technique (Desjardins et al., 1979; Car-valho & Krettli, 1991). Two strains of Plasmodium falciparum: 3D7 - chloroquine sensitive clone of isolate NF54, and Dd2 - chloroquine resistant clone from the W2-MEF line (Wellems et al., 1990) were continuously maintained in culture, by the method of Trager & Jensen (1976) and used in these assays. Each CEE and fraction were dissolved in ethanol and diluted with RPMI 1640 culture medium, with 10% human serum, to prepare a series of concentrations of 500, 250, 100, 50, 25,10, 5 and 1 µg/ml. Chloro-quine was used as control and negative controls were prepared with the culture medium and 0.1% ethanol content. Three duplicate assays were carried out per parasite line.

The results are expressed as percentage of growth inhibition. The concentration, which inhi-bited growth by 50% (IC50) in comparison to con-trol wells, was estimated by linear interpolation between drug concentrations values (Hills et al., 1986) - Table 2.2.2. Hepatic stage assays. These assays evaluate the inhibition of development of the liver stage pa-rasite, as well as the toxicity of the plant extracts to the hepatic host cells. Viability assays (hepatotoxi-city test): an “in vitro” culture of HepG2 A16 hepatic

cells was used in this assay; plant extracts were dis-solved with ethanol and diluted with William’s E cul-ture medium to obtain six concentrations: 500, 250, 100, 50, 25 and 10 µg/ml, with a maximum of 0.1% in ethanol content. Primaquine was used as positi-ve control, and the negative controls were prepared with culture medium and 0.1% ethanol. These as-says were performed in triplicate, to determine the maximum non-toxical concentration to the hepatic cells (Sinden et al., 1990). Results were obtained by ELISA with a filter of 570 nm and a background of 630 nm and the minimum lethal dose (MLD) was de-termined for each extract (concentration that inhi-bits 30% of cell growth).

Antimalarial exoerythrocytic schizontocidal assay: female Balb/C mice were infected with Plasmodium berghei ANKA malaria parasites, and 5 - 8 days later, submitted to insectory maintai-ned Anopheles stephensi females for an infectious blood meal; 14 - 18 days later sporozoites were collected from dissected mosquito salivary glands and used to infect Hep G2 A16 cells (Trager & Jensen,1976; Hills et al., 1986) “in vitro” cultu-res (Calvo-Calle et al., 1994; Hollingdale et al., 1983; Sinden, 1991; Denizot & Lang, 1986). For each plant extract and fraction, 300 µl of the con-centration which exhibited 70% of cell growth in the viability assay (MLD), as well as its immediate lower concentration, were now tested in triplicate in the “in vitro” culture of the exoerythrocytic

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Table 3 - Cytotoxicity and exoerythrocytic antimalarial activity of plant extracts against Plasmodium berghei “in vitro” (Hep G2 cells). Abbreviations: MLD (minimal lethal dose)*/dose that inhibits 30% of cell growth; CEE, crude ethanolic extracts; PE, petroleum ether fraction; DM, dichloromethane fraction; EA, ethyl acetate fraction; MW, remanescent methanol and water fraction; n.a., not active; n.d., not determined. IC50 (primaquine) = 0.003 mg/ml. MLD (primaquine) = 0.1 mg/ml.

Extract Nº Plant speciesCitotoxicity MLD Tested Doses Schizontocidal activity

(µg/ml) (µg/ml) IC50 (µg/ml)

1 struchium sparganophorum 50 50 / 25 24

1eP struchium sparganophorum 100 100 / 50 n.d.

1dm struchium sparganophorum 25 25 / 10 35

1ae struchium sparganophorum 50 50 / 25 78

1ma struchium sparganophorum 500 500 / 250 482

2 Vernonia amygdalinaa 250 250 / 100 n.a.

3 Vernonia amygdalinab 250 250 / 100 305

4 ageratum conyzoides 100 100 / 50 135

5 cinchona succirubra 250 250 / 100 n.a.

6 aloe humilis 500 500 / 250 n.a.

6eP aloe humilis 100 100 / 50 n.a.

6dm aloe humilis 10 10 / 1 19

6ae aloe humilis 500 500 / 250 354

6ma aloe humilis 100 100 / 50 344

7 Tithonia diversifolia 100 100 / 50 287

7eP Tithonia diversifolia 50 50 / 25 18

7dm Tithonia diversifolia 10 10 / 1 n.a.

7ae Tithonia diversifolia 250 250 / 100 117

7ma Tithonia diversifolia 500 500 / 250 n.a.

8 cedrela odorata 250 250 / 100 158

9 Premna angolensis 500 500 / 250 n.a.

10 Pycnanthus angolensis 250 250 / 100 n.a.

10eP Pycnanthus angolensis 100 100 / 50 34

10dm Pycnanthus angolensis 500 500 / 250 n.a.

10ae Pycnanthus angolensis 25 25 /10 22

11a morinda lucida (bark) 500 500 / 250 415

11aeP morinda lucida (bark) 10 10 / 1 5

11adm morinda lucida (bark) 10 10 / 1 n.d.

11aae morinda lucida (bark) 50 50 / 25 137

11ama morinda lucida (bark) 500 500 / 250 n.a.

11b morinda lucida (folhas) 50 50 / 25 76

11beP morinda lucida (leaves) 250 250 / 100 103

11bdm morinda lucida (leaves) 100 100 / 50 167

11bae morinda lucida (leaves) 500 500 / 250 n.a.

12 cestrum laevigatum 250 250 / 100 n.a.

13 canna bidentata 500 500 / 250 n.a.

stage of P. berghei in HepG2 cells (Hollingdale et al., 1983; Millet et al., 1986). Results were obtai-ned through immunofluorescence assay (Danforth et al., 1978). Tests were repeated in two separate experiments. The concentration, which inhibited

the development of hepatic schizonts growth by 50% (IC50), in comparison to control wells, was estimated by linear interpolation between drug concentrations values and negative controls (Ta-ble 3).

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Table 4 – Antimalarial Activity and Toxicity of Medicinal Plant Extracts. Abbreviations: IC – Inhibitory concentra-Abbreviations: IC – Inhibitory concentra-: IC – Inhibitory concentra-tion; MLD – minimum letal dose; LD – letal dose; n.d.- not determinate; n.a – not active; M – mutagenic; NM – Not mutagenic for TA98; WM- weakly mutagenic; RI – relatively innocuous; RI* - relatively innocuous until tested dose. WT – weakly toxic.

Plant speciesExtract/Fraction

MutagenicityAcute Toxicity, “in vivo”

Schizonticidal Activity (Blood stage)

Schizonticidal Activity (Liver stage)

Hepatotoxicity

(TA98) IC50 (µg/ml) IC50 (µg/ml) MLD (µg/ml)

struchium sparganophora

eb n.d ri 180 24 50

eP n.d ri <10 n.d 100

dm n.d WT 100 35 25

ae n.d ri* 100 78 50

aloe humilis

eb n.d ri* 260 n.a 500

eP n.d n.d 150 n.a 100

dm n.d n.d 150 19 10

ae n.d ri * 25 354 500

Tithonia diversifolia

eb nm ri 15 287 100

eP nm ri* <10 18 50

dm nm WT <10 n.a. 10

ae m ri* 140 117 250

cedrela odorata

eb n.d ri 190 158 250

eP n.d ri* 110 n.d n.d

dm n.d ri* 50 n.d n.d

ae n.d ri* n.d n.d n.d

Pycnanthus angolensis

eb nm ri* <5 n.a. 250

eP nm ri 100 34 100

dm nm ri* 100 n.a. 500

ae Wm ri 100 22 25

morinda lucida (bark)

eb n.d ri <10 415 500

eP n.d ri* 50 5 10

dm n.d n.d 50 n.d 10

ae n.d n.d 100 137 50

3. Determination of “in vivo” antimalarial activity in plant extracts. For “in vivo” tests the ‘Four-day suppressive test’ (O’Neill et al., 1987) was used, with female Balb/C mice (6-week old; ± 20 g weigh) infec-ted by intraperitoneal innoculations of 107 erythro-cytes parasitized with P. berghei ANKA malaria para-site. Plant extracts and fractions were solubilized or suspended in ethanol at the maximum proportion of 1% (v/v) in distilled water, tested in two concentra-tions (500 and 1000 mg/kg body weight), adminis-tered daily, orally, during 4 days, to infected mice (in groups of five animals). Blood films were taken on the fourth day (96 h after the first dose), Giemsa stained and examined microscopically and the level of parasitaemia was calculated. Extract activity was determinate by percent reduction of parasitaemia in treated groups compared with untreated infected mice. The statistical analysis was based on Student’s t-test transformed in P-values (P > 0.05 is not sig-nificant; P < 0.05 is significant). Controls with local Cinchona extracts, at the same doses, were inclu-

ded. Survival studies were also carried out with the-se animals.4. Toxicity Assays. Two types of toxicity assays were performed: Genotoxicity (“in vitro”) – Ames Test, and Acute Toxicity “in vivo”. The first was perfor-med at Centro de Malária e Outras Doenças Tropicais (CMDT) and Centro de Investigação de Genética Hu-mana da Faculdade de Ciências Médicas da Universi-dade Nova de Lisboa, and the second, with the cola-boration of Biotério, Instituto de Higiene e Medicina Tropical (IHMT).4.1. Genotoxicity “in vitro” Assay – Ames Test. The lyophilized extracts/fractions (EB, EP, AE, DM) of Ti-thonia diversifolia and Pycnanthus angolensis, were dissolved in DMSO, to prepare a series of concentra-tions: 1, 5, 10, 50, 100, 150, 300, 600, 750, 1000, 1500, 2000 µg/ml, giving the final concentration/plaque of 0,04; 0,2; 0,4: 2; 4; 6; 12; 24; 30; 40; 60; 80 µg.

The bacteria used in the tests was Salmonella ti-phimurium (TA97), was obtained from Bruce Ames,

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through Laboratory of Genética Humana, Faculdade de Ciências Médicas, University Nova de Lisboa. In order to detect either the pro-mutagenic compounds (that needs a previously metabolization to induce a mutation), as the mutagenic compounds, we have performed two series of tests: with biotransforma-tion (using S9-mix) and without biotransformation (with phosphate buffer 0,1 M pH 7,4). All the tests were performed in triplicate, with a negative con-trol (25 µl of DMSO), and a positive control (5 µl of 2-nitroflurene / 2-NF,1µg/mL) for assays without biotransformation, and 10 µl of benzo-α-pirene / B-α-P, 0,5µg/mL) for assays with biotransforma-tion (Maron et al., 1983).

After the incubation period, the grown colo-nies (revertents) will be manually counted.4.2. Acute Toxicity (“in vivo”). Briefly, Mus muscu-lus (strain CD1) male and female mice, weighing 20-30g, with 8 weeks old were used, divided in groups of 5 males and 5 females for each dose of plant ex-tract/fraction.

The lyophilized extracts/fractions were dis-solved in ethanol and 5% of arabic-gum solution to prepare a series of concentrations: 2,5; 25; 50; 125 mg/ml (maximum 10% ethanol). Having in mind that we have to administer orally a single dose of 0,4 ml, the final doses will correspond to a concentration of 50, 500, 1000 and 2500 mg/Kg respectively (Cyted, 1995). A negative con-trol with ethanol and a 5% arabic-gum solution (maximum 10% ethanol) have been used.

After oral administration, mice were observed daily, during 7 days, and the number of deaths recorded. The lethal dose (LD50) was determined for each extract (concentration that determines 50% of population death).5. Phytochemical Studies. Tithonia diversifolia (Fig. 1) was the selected plant for phytochemical sudies, in order to identify the most active compounds. For the phytochemical study of the most active plant extract (PE), which included bioguided fractiona-tion, isolation and structural elucidation of its acti-ve compound, we used several classical and modern analytical techniques (TLC, CC, HPLC, UV, IV, MS, 1H-NMR and 13C-NMR (Goffin et al., 2002).

resuLts AnD DIsCussIOn1. Antimalarial Activity

The results of the “in vitro” tests with plant extracts against chloroquine resistant P. falcipa-rum strain (Dd2) are presented in Table 2. The extracts of A. humilis (EA), C. odorata (DM), C. laevigatum (DM) and C. bidentata (DM) showed moderate antiplasmodial activity with IC50 values 5 - 50 µg/ml. Six other medicinal plant extracts had evident antiplasmodial activity against chlo-roquine resistant P. falciparum, with IC50 values 5 -10 µg/ml: S. sparganophorum (PE), V. amygdali-na b (EA), Cinchona succirubra (CEE and all frac-tions), T. diversifolia (PE, DM), P. angolensis (CEE) and M. lucida (CEE). Comparing with the IC50 value obtained for Artemisia annua (3.9 µg/ml) and for Azadirachta indica (10 µg/ml) this group of plants has promising results (Phillipson et al., 1987).

In the cytotoxicity and exoerythrocytic anti-malarial activity of plant extracts against P. berg-hei “in vitro” (Hep G2 cells) (Table 3), eleven of

Figure 1. Tithonia diversifolia (Hemsl.) A. Gray

Figure 2. Tagitinin C.

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the tested extracts and fractions revealed hepatic schizontocidal activity, in concentrations below the respective MLD values. From these, four plants revealed evident schizontocidal activity with IC50 values from 5 to 34 mg/ml (S. sparganophorum, T. diversifolia, P. angolensis and M. lucida bark).

The plant extracts and fractions that showed significant activity “in vitro” tests have been se-lected for “in vivo” assays for antimalarial activi-ty (S. sparganophorum, C. succirubra, T. diversifo-lia, C. odorata and P. angolensis). In general, the tested plant extracts were partly active against the rodent malaria, except for Cinchona extracts that showed a total chemosuppression of parasi-taemia in mice infected with P. berghei. However, two of them, S. sparganophorum and C. odorata crude extracts, exhibited a significant inhibition (P < 0.05) of the parasite multiplication (85 and 73%, respectively), at oral doses of 1000 mg/kg per day. The parasitaemia (mean standard devia-tion) of the infected control group at the end of the 4-day test was 10.34 ± 2.11%; for the mice treated with S. sparganophorum the parasitaemia was 1.54 ± 1.38% and for the mice treated with C. odorata the parasitaemia was 2.84 ± 0.99%. The median time of mice survival of the infected trea-ted animals versus the control group (23 ± 3 days) was calculated and one plant extract increased this time by 35% (C. odorata).2. Toxicity Tests2.1. Mutagenicity Assay (Ames Test). The results of the genotoxicity assays are presented in Table 4. The number of revertents by plate is calculated as well as the RI (Reverse Index), taking into account the number of expontaneous revertents (Mortelmans et al., 2000). In order to consider a substance as a mutagenic it is necessary to have simultaneously a number of revertents higher than the number of ex-pontaneous revertents (20-50 Rev/Plate, for TA98) and a RI > 2,0. A substance can be classified as a week mutagenic when the number of revertents is higher than the number of expontaneous revertents for the strain, but it has a RI < 2,0.

The plant extracts/fractions CEE, PE and DM, from Tithonia diversifolia and Pycnathus angolen-sis do not present signs of genotoxicity, for the TA98 strain, in both assays with and without bio-transformação. However, the AE fraction of both

plants, presents a weak mutagenicity, for the TA98 strain, in the assay with biotransformation, and are not mutagenic in the assay without bio-transformation.2.2. Acute Toxicity Assay. The classification of plant extracts, as toxic agents, could be done according to their LD50 (Cyted, 1995). In table 4 are presented the calculated values of LD50 for the plant extracts/frac-tions studied. Thus, and according to the criteria of Cyted (1995), we can consider the extracts/fractions as following:• innocuous:Struchium sparganophora (CEE, EA,

PE), Tithonia diversifolia (CEE, EA, PE), Pycnan-thus angolensis (EA); Aloe humilis (EA); Cedrela odorata (EA); Morinda lucida (PE).

• weakly toxics: Struchium sparganophora (DM), Tithonia diversifolia (DM).For the extracts or fractions without deaths

within the maximum tested concentration (2.500 mg/kg PV), even if we can not calculated the LD50, we could confirm that they are not toxics until this concentration (Aloe humilis CEE; Cedrela odo-rata CEE, PE, DM; Pycnanthus angolensis CEE; DM, PE; Morinda lucida CEE).3. Phytochemical Studies

From the results obtained it was possible to identify the main active antimalarial compound in Tithonia diversifolia, the sesquiterpenic lactone ta-gitinin C – Figure 2. Its antiplasmodial activity was verified, showed a IC50 ≈ 0.24 µg/ml and IC50 ≈ 0.25 µg/ml against the chloroquine resistant P. falcipa-rum strains FCB1 and W2. However, tagitinin C also showed some cytotoxicity (IC50 = 0.706 µg/ml) to the cell line HTC-116 (Goffin et al., 2002).

COnCLusIOnsThe resistance of P. falciparum to chloroquine

is now a major health problem in STP, as in many parts of Africa, and these data suggest a potential role for medicinal plant extracts in the treatment of chloroquine-resistant falciparum malaria.

Our study indicates that traditional healers in STP use several medicinal plants against fever and/or malaria which have demonstrated varying activity against blood stage parasites of chloro-quine resistant P. falciparum, as well as against the hepatic development of P. berghei in Hep G2 liver cells. The effectiveness against liver stage de-

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velopment suggests that they could also be used as preventive medicines. The development of new drugs with these capacities could be of relevance for the protection of risk groups, in areas with such high levels of resistance.

Plants commonly used in traditional medicine are assumed to be safe due to their long usage in the treatment of diseases according to knowledge accumulated over centuries. However, recent scien-tific findings has shown that many plants used as food or in traditional medicine are potentially toxic, mutagenic and carcinogenic (Ajaiyeoba et al., 2006). Regarding the toxicity of the plant ex-tracts and fractions studied, some of them revealed some toxicity (Struchium sparganophora and Titho-nia diversifolia DM fractions), but their crude ex-tracts didn’t present any acute toxicity. This could be due to the presence in the EB of several other compounds that could inhibit the toxicity of the toxic substances presented in the DM fractions.

These results proved the relative innocuously of the studied plants crude extracts, thus being these ones the more approximate formula, in terms of composition, comparing to the tradicio-nal medicines, used by traditional healers of São Tomé e Príncipe.

Relating the results of the toxicity tests with the results of the antimalarial activity (Table 4), we verify that the more active extracts and frac-tions were in general the crude extracts (CEE) and the PE fractions with IC50 < 10 µg/ml (in blood stage), and with LD50 values lower than the toxic letal doses (in liver stage). Thus, is essential to notice that, in general, in the extracts/fractions that revealed an important antimalarial activity, the active concentrations are considerably lower than the concentrations that showed some toxic-ity. In fact, the selected plant for phytochemical studies, Tithonia diversifolia, presents a very in-teressant antimalarial activity either in the blood stage and in the liver stage for the PE fraction; this fraction was considered not mutagenic for the TA98 strain and innocuous in the acute toxicity test, thus being a good candidate for the develop-ment of a new antimalarial drug.

Phytochemical studies of the most active plant (T. diversifolia) have being performed, and the ses-quiterpenic lactone Tagitinin C was identified as

the most active compound. Bearing in mind that the optimal product may not always correspond to single compounds, and in order to develop more appropriate formulations for local production and administration of these products, further studies have been conducted, allowing the development of a HPLC method to achieve a rapid quantifica-tion of the active compound from several aerial plant extracts (Goffin et al., 2003). According to these results the water decoction seems to be the less effective way to prepare a traditional remedy, presenting the lowest concentration of tagitinina C and the lowest antimalarial activity, “in vitro”. On the other hand, leaves are the plant material with the highest level of tagitinin C (1,15%), thus should be preferentially used in the preparation of traditional remedies.

These informations have been transmitted to the Minister of Health and to the Traditional Healers Association, allowing a more safe and effective use of the traditional remedy. This will have an obvious economic advantage for the local populations, as the developed medicinal plant products will be the cheapest therapeutic alternative for malaria.

ACknOwLeDgeMentsWe would like to thank to traditional healers of S. Tomé e Príncipe, in particular to Sum Gino and Sum Pontes, with whom we learnt and worked, and to the Ministry of Health of RDSTP. We also express our gratitude to Virgílio do Rosário, Ana Paula Martins, Miléne Gomes and Filipe Santos (CMDT/IHMT), Luc Angenot and Eric Gof-fin for the phytochemical studies (University of Liége), Laurent Rénia for providing the biological material for the hepatic stage tests. A special thanks to Jorge Paiva for the identification of plant specimens. This study was financed by Fundação para a Ciência e Tecnologia (PRAXIS/PSAU/P/SAU/38/96).

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