Antimalarial Drug Development Research and the Ancient Knowledge of Traditional Medicines in S. Tomé E Principe Islands

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Antimalarial Drug Development Research and the Ancient Knowledge of Traditional Medicines in S. Tomé e Principe Islands

Conference Paper · October 2009

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Tradiciones & transformaciones en Etnobotánica MARÍA LELIA POCHETTINO ANA 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 Jujuy Alberdi 47 4600 San Salvador de Jujuy Argentina Tel.: + 54 388 422-1548 [email protected] www.fca.unju.edu.ar/risapret

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 Ramón Morales. Real Jardín Botánico de Madrid, Preservation of Medical Traditions hosted by the CSIC. Plaza de Murillo, 2. E-28014 Madrid, Smithsonian Institution Washington, D.C. USA. España. Patricia M. Arenas. Laboratorio de Etnobotánica Manuel Pardo de Santayana. Universidad y Botánica Aplicada (LEBA), Facultad de Autónoma de Madrid, España. Ciencias Naturales y Museo, Universidad Cecilia Perez de Micou. CONICET-INAPL, Nacional de La Plata. Calle 64 nº 3, Universidad de Buenos Aires, Tres de Febrero 1900 La Plata, Argentina. CONICET. 1378, 1426 Buenos Aires, Argentina. Lidia S. Burry. Laboratorio de Palinología, María Lelia Pochettino. Laboratorio de Departamento de Biología, Facultad de Etnobotánica y Botánica Aplicada (LEBA), Ciencias Exactas y Naturales, Universidad Facultad de Ciencias Naturales y Museo, Nacional de Mar del Plata. Funes 3250, 7600 Universidad Nacional de La Plata. Calle 64 Mar del Plata, Argentina. nº 3, 1900 La Plata, Argentina. CONICET. Aylen Capparelli. Departamento Científico Gustavo Scarpa. Centro de Estudios de Arqueología, Museo de La Plata, Paseo Farmacológicos y Botánicos-CONICET. Paraguay del Bosque s/n, 1900 La Plata, Argentina. 2155, p.16º, Buenos Aires, Argentina. CONICET. Alain Touwaide. The Institute for the Preservation Laure Emperaire. Institut de Recherche pour le of Medical Traditions hosted by the Développement. Unité Mixte de Recherche 208 Smithsonian Institution Washington, D.C. USA. (IRD-MNHN) PALOC (Patrimoines locaux). Matilde Trivi de Mandri. Laboratorio de Francia. Palinología, Departamento de Biología, Füsun Ertuğ. Consultant of ethnobotany at the Facultad de Ciencias Exactas y Naturales, Turkish Academy of Science (TUBA), Turkey. Universidad Nacional de Mar del Plata, Funes Leonardo Galetto. Instituto Multidisciplinario 3250. 7600 Mar del Plata, Argentina. de Biología Vegetal, Universidad Nacional de Soultana Valamoti. Department of Archaeology, Córdoba. Argentina. CONICET. Aristotle University of Thessaloniki, 54 124 Michael Heinrich. School of Pharmacy, London, UK. Thessaloniki, Greece. Norma Hilgert. Instituto de Biología Subtropical, Michèle Wollstonecroft. The Institute of Facultad de Ciencias Forestales, Universidad Archaeology, University College London, UK. Nacional de Misiones. CeIBA- CONICET. Puerto Iguazú, Misiones, Argentina. Ana Ladio. INIBIOMA (CONICET - UNComahue). Quintral 1250. 8400 Bariloche, Río Negro, REVISORES INVITADOS Argentina. Verónica Lema. Laboratorio de Etnobotánica José Luis Fernández Alonso. Instituto de Ciencias y Botánica Aplicada (LEBA), Facultad de Naturales, Herbario Nacional Colombiano. Apart. Ciencias Naturales y Museo, Universidad 7495 Universidad Nacional de Colombia, Bogotá, Nacional de La Plata. Calle 64 nº 3, 1900 La D.C. Colombia. Plata, Argentina. CONICET. Kevin N. Laland. School of Biology, St. Andrews María Rosa Martínez. Facultad de Ciencias University, St. Andrews, Scotland, UK. Naturales y Museo, Universidad Nacional de Lía Montti. Instituto de Ecología Regional (IER), La Plata. Paseo del Bosque s/n, 1900 La Plata, Universidad Nacional de Tucumán, Casilla Argentina. CONICET de Correo 34, 4107 Yerba Buena, Tucumán, Soledad Molares. INIBIOMA (CONICET - Argentina y Centro de Investigaciones del Bosque UNComahue). Quintral 1250. 8400 Bariloche. Atlántico (CeIBA), Andresito 21, Puerto Iguazú, Río Negro, Argentina. Misiones, Argentina.

Indice

1. Introducción ...... 1

2. Implicancias teóricas y epistemológicas de las estrategias de investigación etnobotánica...... 3 2.01 Cr i v o s , 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 Ca p p a r e l l i , A. & L. Pr a t e s . Identificación específica de frutos de algarroboProsopis ( spp. L., Fabaceae) y mistol (Ziziphus mistol Griseb., Rhamnaceae) en un sitio arqueológico de Patagonia ...... 13 3.0.02 Ro d r í g u e z , M. F. & Z. E. Rú g o l o d e Ag r a s a r . Gramíneas herbáceas y leñosas en espacios domésticos y rituales de la Puna meridional argentina durante el Holoceno ...... 20 3.0.03 Co l o b i g , M. M., A. F. Zu c o l & D. L. Ma z z a n t i . Análisis de microrestos silíceos en secuencias sedimentarias del Sitio 1 de la localidad arqueológica Lobería I, Buenos Aires, Argentina..... 27 3.0.04 An d r e o n i , D., A. Gi l & A. Ca p p a r e l l i . Efectos de la carbonización en especies leñosas de las provincias fitogeográficas Patagónica y del Monte (Mendoza, Argentina): una perspectiva arqueológica ...... 33 3.0.05 Le m a , V. S., M. L. Po c h e t t i n o , M. Pu e b l a s , M. C. Pa l e o & M. Pé r e z Me r o n i . La etnobotánica como herramienta interpretativa en arqueología: prácticas de recolección en el Holoceno Tardío del Litoral Bonaerense (Argentina)...... 38 3.0.06 Ll a n o , C. & A. Ug a n . Rendimiento económico de plantas silvestres del sur de Mendoza: valores nutricionales, costos de manejo e interpretación del registro arqueológico ...... 44 3.0.07 Le m a , V. S. Plant domestication and man-plant degrees of dependence in the pre-hispanic cultural development of Argentinean Northwest ...... 49 3.0.08 To u w a i d e , A. History of Botany as Ethnobotany. Proposals toward a new approach to the ancient legacy ...... 55 3.0.09 Az a r , P. F. El añil: una planta para teñir, un color para recordar ...... 64 3.0.10 To m e i , P. E., S. Tr i m a r c h i S. & C. Le n z i . 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 Bu r r y , L., M. Tr i v i d e Ma n d r i & C. Pe r e z d e Mi c o u . Hacia una integración de metodologías para optimizar el estudio del hombre con su ambiente en el pasado ...... 73 3.1.02 Ko r s t a n j e , M. A. Lo lúdico, lo creativo y lo formalizado en la investigación del pasado: expandiendo saberes en arqueobotánica ...... 76 3.1.03 Ch a v e s , S. A. d e Mi r a n d a . Paleopharmacology and palinological research in coprolites ...... 83 3.1.04 Ya g u e d d ú , C. Identificación de restos vegetales en coprolitos...... 90 3.1.05 D’An t o n i , 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 Ca p p a r e l l i , A., T. Va l a m o t i & M. Wo l l s t o n e c r o f t . 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 Ca p p a r e l l i , 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 Le m a , V. Cultivation, production and domestication: evaluating the archaeological visibility of interactions between human societies and plant populations in the past ...... 110 3.2.04 Ló p e z , M. L., A. Ca p p a r e l l i & A. Ni e l s e n . Modern practices of quinoa (Chenopodium quinoa W. Chenopodiaceae) processing and consumption in Lípez, Bolivia: an ethnoarchaeological approach ...... 117 3.2.05 Er t u ğ, F. The consumption of flax: postharvest activities linked to linseed oil production in Turkey ...... 123 3.2.06 Va l a m o t i , S. M. The prehistory and modern survival of bulk processing cereals for piecemeal consumption: case studies from Greece ...... 128 3.2.07 Ho s o y a , L. A. Staple or famine food ? Ethnographic and archaeological approaches to nut processing in East Asian prehistory...... 133 3.2.08 Wo l l s t o n e c r o f t , M. Evolutionary trends in human eating behaviours: food processing and niche construction...... 139

4. Pueblos y plantas que alimentan ...... 145 4.01 Ra p o p o r t , E. ¿Cuán sabios son los sabios? ...... 147 4.02 McCl a t c h e y, W. & D. Re e d y. A mouthful of water: Do cider producers recognize the ancient clean water potential of their products? ...... 149 4.03 Ba s e r, K. H. C. Wild food plants of Turkey ...... 161 4.04 Cá m a r a He r n á n d e z , 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 Em p e r a i r e , L. La construcción de la diversidad agrícola: conceptos y prácticas ...... 175 5.02 Ol i v e i r a , J. Ca b r a l d e . The role of social networks in agriculture biodiversirty: the Wajãpi case. 176 5.03 Em p e r a i r e , L. & J. Ol i v e i r a . Redes sociales y diversidad agrícola en la Amazonía brasileña: un sistema multicéntrico ...... 180 5.04 Po c h e t t i n o , M. L. Huertos peri-urbanos como aporte a la diversidad agrícola, Provincia de Buenos Aires, Argentina ...... 186 5.05 Fr e i r e , G. Ecología política del cambio agrícola: El dilema de la agroforestería indígena en un petroestado ...... 193 5.06 De m e u l e n a e r e , E. Farmers’ seed systems in industrial agricultures: new research object, renewed approaches in Ethnobotany ...... 197

6. FITOTERAPIA Y PLANTAS MEDICINALES ...... 205 6.01 El v i n -Le w i s , M. Ways in which integrated studies can identify meaningful remedies for populations that use them ...... 207 6.02 Ma r t í n e z , G. J. Los criterios terapéuticos en la farmacopea natural de los tobas bermejeños del Chaco Central (Argentina) ...... 213 6.03 Ku t s c h k e r , A., C. Ez c u r r a & V. Ba l z a r e t t i . Valeriana en los Andes Australes: biodiversidad y compuestos activos...... 219 6.04 De l Vi t t o , L. A., E. M. Pe t e n a t t i & M. E. Pe t e n a t t i . Ethnomedical plants from Cuyo region, Argentina: uses and conservational status ...... 225 6.05 Gh e n o He r e d i a , Y. A., A. R. Ma r t í n e z Ca m p o s , E. Sá n c h e z Ve r a & G. Na v a Be r n a l . Plantas medicinales de la organización de parteras y médicos indígenas tradicionales de Ixhuatlancillo, Veracruz, México...... 232 6.06 Pi n o Be n í t e z , N. & Y. S. Re n t e r í a . Plantas usadas en el tratamiento contra el estrés en una comunidad del Nor-occidente colombiano: un enfoque cuantitativo ...... 239 6.07 Mi j a r e s , G., C. Me n e s e s & M. Te i x e i r a . Etnofarmacología de las helmintiasis en la comunidad de Mendoza, Municipio Acevedo. Estado Miranda, Venezuela ...... 244 6.08 Ve r d e , A., D. Ri v e r a , J. Fa j a r d o , C. Ob ó n , A. Va l d é s , J. R. P. Ru í z -Ga l l a r d o , V. Be n l l o c h , R. Ci u d a d , Nú ñ e z & A. Pi e r a . Medicina popular y salud como materia curricular transversal en educación secundaria en Castilla La Mancha (España) ...... 250 6.09 Ma d u r e i r a , M. d o Cé u . Antimalarial drug development research and the ancient knowledge of traditional medicines in S. Tomé e Principe Islands ...... 256 6.10 Fa j a r d o , J., A. Ve r d e , D. Ri v e r a , A. Va l d é s , C. Ob ó n , M. He i n r i c h & F. Ce b r i á n . 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 Pa r r a -Sá n c h e z , E., O. Pé r e z -Es c o b a r & L. Sá n c h e z . Exploración de saberes de plantas medicinales de Fusagasugá, Colombia ...... 270 6.12 Ba c h , H. G. & R. H. Fo r t u n a t o . Estudio de patrones de elección de plantas medicinales en una zona urbana del Oeste Bonaerense (Argentina) ...... 277 6.13 Cr i s t i n a , I. & P. M. Ar e n a s . Plantas de uso tradicional en Argentina de posible aplicación como adaptógenos ...... 282 6.14 Mo l a r e s , S. & A. La d i o . 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 Cu a s s o l o , F., A. La d i o & S. Ca l v e l o . Los líquenes medicinales comercializados en S. C. de Bariloche: importancia local y control sanitario ...... 291 6.16 Ri c h e r i , M., A. M. Be e s k o w & A. La d i o . Las plantas y la salud en la comunidad boliviana, Madryn (Argentina) ...... 297 6.17 Go n z á l e z , S. B., L. N. Hu a i q u i n a o , A. Go n z á l e z , C. v a n Ba r e n , P. Di Le o Li r a & A. L. Ba n d o n i . Uso popular del paico y composición química de su aceite esencial en la zona de Esquel (Chubut, Argentina) ...... 303 6.18 Ar e n a s , P. M. Consumo de algas en la provincia de Buenos Aires, Argentina: tradición o nuevo uso de los recursos? ...... 308 6.19 Le w i s , W. H. Evaluating and protecting indigenous pharmacopeas and traditional knowledge ... 313 6.20 Bu cc i a r e l l i , A. & M. Sk l i a r . Evaluation of antiulcer activity of Solidago chilensis (Asteraceae) in mice ...... 317 6.21 Bu cc i a r e l l i , A., C. Br a s , N. Ga n d i n i , A. Mi n e t t i & M. Sk l i a r . Estudio toxicológico de la fracción acetato de etilo de capítulos de Solidago chilensis (Asteraceae) ...... 322 6.22 Pé r e z Cu a d r a , V. & V. Ca m b i . Foliar crystals in ornamental Araceae, their risk for health ...... 327 6.23 Fl o r e s , E. N. & N. D. Vi g n a l e . 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 Su á r e z , M. E. Recursos forestales no madereros (RFNM) entre wichís del Chaco semiárido salteño, Argentina ...... 339 7.02 Hu r r e l l , J. A., E. Ul i b a r r i , G. De l u cc h i , M. L. Pé r e z & N. An g l e s e . Plantas condimenticias utilizadas en la ciudad de Buenos Aires y en el Noreste Bonaerense (Argentina)...... 344 7.03 Se o a n e N. & J. Oc h o a . Uso del neneo en la región sur de la provincia de Río Negro (Argentina) y su relación con la producción ovina ...... 350 7.04 Ro d r í g u e z M. R., M., L. Ga l i c i a S., W. Sá n c h e z , L. Gó m e z M., A. E. Za r c o A. & E. Ce cc o n . Usos actuales, distribución potencial y etnolingüística del bambú leñoso (Bambuseae) en México ...... 355 7.05 Ma s s o c a , P. E. & G. B. Fr a u s i n . Botánica funeraria en el cementerio de Araçá (São Paulo, Brasil)... 364 7.06 De Ro b e r t , P. & E. Ka t z . Usos alimentarios de palmeras un estudio comparativo en Amazonía Brasileña ...... 370 7.07 Ba r r i u s o , M. A. & M. Ga r c í a -Ba r r i u s o . Plantas tintóreas históricamente utilizadas en el centro de la Península Ibérica ...... 376 7.08 Ca b a l l e r o Ro q u e , A., J. Pe r e z , R. Es c o b a r , J. Ba l l i n a s & G. Ve l a . Uso de nanche (Byrsonima crassifolia, Malpighiaceae) como saborizante natural de una bebida refrescante...... 380 7.09 Ja b e e n , A. Ethnobotany of fodder species of Ayubia National Park, Pakistan, its conservation status and impacts on environment ...... 384 7.10 Mo r a l e s , R. Labiadas de España en América. Intercambio de Usos ...... 391 7.11 Ma r r e r o , A. Las labiadas en Canarias. Encrucijada en el Atlántico ...... 401 7.12 Sc h m i d t -Le b u h n , A. N. An update on the state of research on Minthostachys (Lamiaceae) ...... 408 7.13 Al b r e c h t , R., J. Mo n t e n e g r o , R. Ro l d a n , A. Gu r n i , N. Vi g n a l e & G. Ba s s o l s . Análisis de las composiciones nutricionales de cultivos andinos ...... 414 7.14 Gu i a m e t , P., P. M. Ar e n a s , P. La v i n , P. Ba t t i s t o n i & S. Gó m e z d e Sa r a v i a . Utilidad de extractos obtenidos de plantas en el control de microorganismos que afectan al patrimonio cultural ..... 419 7.15 St a m p e l l a , P., P. M. Ar e n a s , A. Ló p e z , S. Bo r r e g o , I. Vi v a r & N. Ca b r e r a . Plantas útiles en el control de insectos bibliófagos ...... 423 7.16 Vi g n a l e , N. D., M. A. Ri v a s , E. Ji m é n e z & A. A. Gu r n i . Identificación micrográfica de semillas de “chía” (Salvia hispanica L. - Lamiaceae) ...... 428 7.17 Ca m b i , V. & V. Pé r e z Cu a d r a . Anatomía foliar de portulacas bonaerenses de importancia económica.... 433 7.18 Gu e v a r a Va l e n c i a , M., M. T . Go n z á l e z -Ar n a o , Y. M. Ma r t í n e z Oc a m p o & J. Mo l i n a To r r e s . Aplicación de alcamidas presentes en extractos de Chrysanthemum morifoliun al cultivo in vitro de Vanilla planifolia ...... 437

8. Etnobotánica Ecológica ...... 443 8.01 Vi l l a r, L. La toponimia de origen vegetal refleja el saber etnoecológico: el caso del Pirineo Central (España) ...... 445 8.02 Ca l v e t -Mi r, L., M. Ca l v e t -Mi r & V. Re y e s -Ga r c í a . Traditional ecological knowledge and landraces in situ conservation in high mountain home gardens of Vall Fosca, Catalan Pyrenees, Iberian Peninsula ...... 457 8.03 Oc h o a , J., A. La d i o & M. Lo z a d a . Aspectos ecológicos y socioculturales asociados al uso de plantas silvestres en la población rural de Arroyo Las Minas (Río Negro, Argentina)...... 465 8.04 Ah u m a d a , A., M. L. Ci a m p a g n a , J. Ve r a Ba h i m a , J. J. Ga r a t & J. Ot e r o . Prácticas culturales en la selección y conservacion de hortalizas locales en el Cinturón Hortícola Platense...... 472 8.05 Bu e t Co s t a n t i n o , F., E. Ul i b a r r i & J. A. Hu r r e l l . Las huertas familiares en la isla Paulino (Buenos Aires, Argentina) ...... 479 8.06 Ey s s a r t i e r, C., A. La d i o & M. Lo z a d a . Conocimiento tradicional sobre plantas en huertas, invernaderos y jardines en dos poblaciones rurales de la estepa patagónica ...... 485 8.07 Ca r d o s o , M. B., A. La d i o & M. Lo z a d a . Utilización de especies combustibles en una comunidad rural de la estepa patagónica ...... 496 8.08 Gi r s c h t , A. M., A. E. Ro v e r e & S. Mo l a r e s . 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 Pl a n c h u e l o , 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 Pa l a c i o , M. & E. Ca r r i z o . El conocimiento sobre plantas tintóreas en teleras de dos departamentos de Santiago Del Estero ...... 517 9.02 Re m o r i n i , C., M. Cr i v o s , M. R. Ma r t í n e z & A. Ag u i l a r Co n t r e r a s . El “susto”: “síndrome culturalmente específico” en contextos pluriculturales. Algunas consideraciones sobre su etiología y terapéutica en México y Argentina ...... 523 9.03 Ka t z , E. Cruzeiro do Sul Market (Acre, Brazilian Amazon): reflection of the regional culture and agricultural diversity ...... 531 9.04 Bo n e t , M. À., M. Ro l d á n , J. Ca m p r u b í & J. Va l l è s . Etnobotánica de Gallecs (Cataluña, Península Ibérica). Plantas y tradición en un entorno social y natural cambiante...... 540 9.05 Ku j a w s k a M. & Ł. Łu c z a j . Studies of wild food plants in communist and post-communist Poland: changes in use and in research methodology ...... 545 9.06 Pé r e z , M. L. & M. L. Po c h e t t i n o . Oficinal u oficial? Plantas de uso corriente denominadas a partir de fármacos (La Plata y alrededores, Buenos Aires, Argentina) ...... 552 9.07 Ga n d o l f o , E. S., T. M. Ri b e i r o , & N. Ha n a z a k i . Can the homegardens persist with the urbanization of the coastline? ...... 557 Prólogo

La Red Iberoamericana de Saberes y Prácticas (MÉXICO) y por el otro la totalidad de los Grupos de Locales sobre el Entorno vegetal (RISAPRET) del ARGENTINA, país anfitrión de este Congreso V ICEB. Programa Iberoamericano Ciencia y Tecnología Cabe expresar un particular agradecimiento a to- para el Desarrollo (CYTED), fuertemente compro- dos los autores de los trabajos que orgullosamente metida con el desarrollo de la Etnobotánica en el presenta RISAPRET en este Libro. ámbito geográfico en el que se desempeña (Ibero- Dicho agradecimiento se transforma en ESPECIAL américa) pero sin descuidar la importancia de las in- cuando va dirigido a las tres Editoras del mismo. Se terrelaciones con otras áreas del planeta, ofrece hoy trata de las Dras. María Lelia POCHETTINO - Respon- a la comunidad este interesante libro que constituye sable del Grupo Participante del Laboratorio de Etno- una muestra de los avances de las investigaciones en botánica y Botánica Aplicada (LEBA) de la Facultad la actualidad. de Ciencias Naturales y Museo de la UNIVERSIDAD En sus páginas, el lector encontrará valiosos apor- NACIONAL DE LA PLATA - Patricia M. ARENAS, inte- tes a este campo científico multidisciplinario, cuida- grante del mismo y de Ana H. LADIO, Responsable dosamente compilados en Capítulos, que permiten del Grupo Participante de la UNIVERSIDAD NACIONAL efectuar un recorrido progresivo por una parte impor- DEL COMAHUE, con sede en Bariloche (ARGENTINA), tante de los diferentes espacios que la Etnobotánica quienes gracias a su extensa y destacada trayectoria comprende, resultando por ello de utilidad tanto para y experiencia en la especialidad así como a su respon- quienes tienen interés en conocer de qué trata, para sabilidad, han logrado, después de una ardua tarea quienes ya están iniciando sus investigaciones en que insumió extensas horas de trabajo, el producto esta área como para quienes están en dicho camino. que con honor esta Coordinación de RISAPRET ofrece Incluye los aportes de investigadores integran- a la comunidad. tes de los Grupos Participantes de RISAPRET, a los Si los lectores a quienes llegue esta obra logran que se suman voluntariamente quienes participaron ampliar su visión acerca de los diferentes aspectos del V CONGRESO INTERNACIONAL DE ETNOBOTÁNICA que conforman la Etnobotánica; si es capaz de des- (ICEB), que tuvo lugar en S. C. de Bariloche (ARGEN- pertar pasión por el trabajo con las comunidades; si TINA) en octubre de 2009, otorgándole ello una en- constituye nuevas oportunidades para incrementar riquecedora variedad de áreas geográficas diferentes las comunicaciones entre investigadores, teniendo de análisis, posibilitando que la Red amplíe, de esta en cuenta el elevado número de autores y las dife- manera, sus contactos y la posibilidad de ofrecer la rentes procedencias y áreas de estudio; si enriquece presente obra. las bibliotecas… significará que la obra ha sido útil, Por un lado están representados los Grupos de que el esfuerzo de autores y editores tuvo sentido y las UNIVERSIDADES DE SALAMANCA y de ALICANTE que por lo tanto, RISAPRET continúa cumpliendo su (ESPAÑA), de la UNIVERSIDAD NACIONAL AUTÓNOMA cometido.

Dra. Nilda Dora VIGNALE Coordinadora Red Iberoamericana de Saberes y Prácticas Locales sobre el Entorno Vegetal (RISAPRET)

Tradiciones y transformaciones en Etnobotánica | Traditions and transformations in Ethnobotany

6.09 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, [email protected].

Abstract Resumen

Madureira, M. C. 2010. Antimalarial Drug Madureira, M. C. 2010. Investigación para el de- Development Research and the Ancient Knowledge of sarrollo de drogas antimaláricas y el conocimiento Traditional Medicines in S. Tomé e Principe Islands. ancestral sobre medicinas tradicionales en S. Tomé e Malaria is the most important parasitic disease in Islas Principe. La malaria es la enfermedad parasita- tropical areas, especially in many African countries ria más importante en áreas tropicales, especialmente due to resistance of Plasmodium falciparum to en muchos países africanos debido a la resistencia the clinical available drugs. In S. Tomé e Príncipe de Plasmodium falciparum a las drogas disponibles. (STP), traditional medicine plays a crucial role, En S. Tomé y Príncipe (STP), la medicina tradicional as population has few means of accessing medical juega un rol crucial, dado que la población carece de treatment. Antimalarial activity and toxicity of 13/ medios para acceder a tratamientos médicos. Se estu- medicinal plants, used by traditional healers in STP dió la actividad y toxicidad antimalárica en 13 plantas for the treatment of malaria and fever, was studied. medicinales usadas por curadores tradicionales para Tithonia diversifolia (Asteraceae) was selected for el tratamiento de malaria y fiebre en STP. Se selec- phytochemical sudies, which included bioguided cionó Tithonia diversifolia (Asteraceae) para realizar fractionation, isolation and structural elucidation estudios fitoquímicos, incluyendo fraccionamiento of its active compound. The sesquiterpenic lactone bioguiado, aislamiento y elucidación estructural del tagitinin C, was identified, and its antiplasmodial principio activo. Se identificó la lactona sesquiter- activity was verified (IC50 ≈ 0.24 µg/ml and IC50 ≈ pénica tagitinina C, cuya actividad se verificó (IC50 ≈

0.25 µg/ml against the chloroquine resistant P. 0.24 µg/ml e IC50 ≈ 0.25 µg/ml contra P. falciparum falciparum strains FCB1 and W2), thus being a good cloroquino resistentes FCB1 y W2), resultando un candidate for the development of a new antimalarial buen candidato para el desarrollo de un nuevo pro- product. All these results were reported to the STP ducto antimalárico. Los resultados se comunicaron al government (Minister of Health. Ministerio de Salud de STP.

Keywords: malaria, medicinal plants, São Tomé e Palabras clave: malaria, plantas medicinales, São Príncipe, Tithonia diversifolia; traditional medicine. Tomé e Príncipe, Tithonia diversifolia, medicina tradicional.

INTRODUCTION Studies of plants used in traditional medicine Malaria is the most important parasitic disease for the treatment of malaria in various cultures in tropical areas, especially in many African coun- have yielded important drugs that are critical to tries due to resistance of Plasmodium falciparum modern medicine (quinine from bark of the Pe- to the clinical available drugs. In S. Tomé e Prínci- ruvian Cinchona tree, and artemisinin from the pe (STP), traditional medicine plays a crucial role, Chinese Artemisia annua). The continued reliance as population has few means of accessing medical of contemporary indigenous populations on plant treatment. medicines warrants close scrutiny of those species

256 · Sección 6.09 Madureira, Antimalarial Drugs from Medicinal Plants of S. Tomé e Príncipe for pharmacological action, adverse interactions ties of these plant extracts have been studied in (e.g., with pharmaceuticals), and long-range toxi- mice (Madureira et al., 2002b), and its “in vitro” city. In view of the growing rate of drug- and pes- cytotoxicity evaluated (hepatotoxiciy, mutage- ticide-resistance among plasmodia and anophe- nicity and acute toxicity). Tithonia diversifolia lines, respectively, these plants may eventually (Hemsl.) A. Gray (Asteraceae) was selected for contribute as well to antimalarial drug discovery phytochemical sudies, which included bioguided and the development of more effective mosquito fractionation, isolation and structural elucidation repellents and antibreeding agents (Etkins et al., of its active compound (Goffin et al., 2002). Its 2004). In fact, plants used in traditional medicine antiplasmodial activity was confirmed, as well as may hold keys to the secrets of many potent an- its citotoxicity, thus being a good candidate for timalarial drugs, and over the shorter term, these the development of a new antimalarial product. plants can also continue to play a role in primary health care and insect management, especially MATERIAL AND METHODS where pharmaceuticals and other biomedical te- 1. Plant Preparation chnology is not available on a sustainable basis. The fresh leaves or bark of 13 medicinal plants We argue that the ethnopharmacological appro- were collected in S. Tomé island (S. Tomé e Prín- ach is the best means to explore these plants, and cipe, Gulf of Guinea), under the guidance of the present some of the results achieved during an healers. The identification and authentication was ethnopharmacological study of medicinal plants done by Prof. Jorge Paiva from Instituto Botânico from STP islands, in the Gulf of Guinea (Madureira da Universidade de Coimbra (COI), Portugal, where et al., 2002a). Following this trend, and since ma- voucher specimens were deposited (Table 1). laria is one of the major health problems of the re- Dried material was powdered and extracted gion, and as there is a high prevalence of chloro- with ethanol 70%, concentrated by rotary vacuum quine resistant Plasmodium falciparum (Loureiro evaporation and lyophilized (CEE-crude ethanol et al., 1996), a study of 13 medicinal plants, used extracts). Fresh material were initially homoge- by the local traditional healers for the treatment nized, followed by same procedure. The CEE of of malaria / fevers, have been undertaken. each plant was dissolved in methanol and water The “in vitro” inhibitory effects of the plant 1:2 (MW) and subsequently partitioned between extracts on chloroquine resistant P. falciparum petroleum ether (PE), dichloromethane (DM) and strains were evaluated, as well as their hepatic ethyl acetate (EA); all the resulting fractions were schizontocidal activity in P. berghei (Hep G2 cell lyophilized after concentration by rotary vacuum line). Similarly, the “in vivo” antimalarial proper- 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.

Voucher Nº Number Botanical Name (Family) Local Name Plant part used (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.

IC50, median values (mg/ml) Number Botanical Name CEE PE DM EA MW 1 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 cells was used in this assay; plant extracts were dis- activity in plant extracts solved with ethanol and diluted with William’s E cul- 2.1. Blood stage assays. To test the antiplasmodial ture medium to obtain six concentrations: 500, 250, activity of plant extracts we used the susceptibility 100, 50, 25 and 10 µg/ml, with a maximum of 0.1% microassay technique (Desjardins et al., 1979; Car- in ethanol content. Primaquine was used as positi- valho & Krettli, 1991). Two strains of Plasmodium ve control, and the negative controls were prepared falciparum: 3D7 - chloroquine sensitive clone of with culture medium and 0.1% ethanol. These as- isolate NF54, and Dd2 - chloroquine resistant clone says were performed in triplicate, to determine the from the W2-MEF line (Wellems et al., 1990) were maximum non-toxical concentration to the hepatic continuously maintained in culture, by the method cells (Sinden et al., 1990). Results were obtained by of Trager & Jensen (1976) and used in these assays. ELISA with a filter of 570 nm and a background of Each CEE and fraction were dissolved in ethanol and 630 nm and the minimum lethal dose (MLD) was de- diluted with RPMI 1640 culture medium, with 10% termined for each extract (concentration that inhi- human serum, to prepare a series of concentrations bits 30% of cell growth). of 500, 250, 100, 50, 25,10, 5 and 1 µg/ml. Chloro- Antimalarial exoerythrocytic schizontocidal quine was used as control and negative controls were assay: female Balb/C mice were infected with prepared with the culture medium and 0.1% ethanol Plasmodium berghei ANKA malaria parasites, and content. Three duplicate assays were carried out per 5 - 8 days later, submitted to insectory maintai- parasite line. ned Anopheles stephensi females for an infectious The results are expressed as percentage of blood meal; 14 - 18 days later sporozoites were growth inhibition. The concentration, which inhi- collected from dissected mosquito salivary glands and used to infect Hep G2 A16 cells (Trager & bited growth by 50% (IC50) in comparison to control wells, was estimated by linear interpolation Jensen,1976; Hills et al., 1986) “in vitro” cultu- between drug concentrations values (Hills et al., res (Calvo-Calle et al., 1994; Hollingdale et al., 1986) - Table 2. 1983; Sinden, 1991; Denizot & Lang, 1986). For 2.2. Hepatic stage assays. These assays evaluate each plant extract and fraction, 300 µl of the con- the inhibition of development of the liver stage pa- centration which exhibited 70% of cell growth in rasite, as well as the toxicity of the plant extracts to the viability assay (MLD), as well as its immediate the hepatic host cells. Viability assays (hepatotoxi- lower concentration, were now tested in triplicate city test): an “in vitro” culture of HepG2 A16 hepatic in the “in vitro” culture of the exoerythrocytic

258 · Sección 6.09 Madureira, Antimalarial Drugs from Medicinal Plants of S. Tomé e Príncipe

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.

Citotoxicity MLD Tested Doses Schizontocidal activity Extract Nº Plant species (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 the development of hepatic schizonts growth by al., 1983; Millet et al., 1986). Results were obtai- 50% (IC50), in comparison to control wells, was ned through immunofluorescence assay (Danforth estimated by linear interpolation between drug et al., 1978). Tests were repeated in two separate concentrations values and negative controls (Ta- experiments. The concentration, which inhibited ble 3).

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Table 4 – Antimalarial Activity and Toxicity of Medicinal Plant Extracts. Abbreviations��:�� IC – Inhibitory concentration; 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.

Schizonticidal Schizonticidal Acute Toxicity, Extract/ Mutagenicity Activity Activity Hepatotoxicity Plant species “in vivo” Fraction (Blood stage) (Liver stage) (TA98) IC50 (µg/ml) IC50 (µg/ml) MLD (µg/ml) EB n.d RI 180 24 50 Struchium EP n.d RI <10 n.d 100 sparganophora DM n.d WT 100 35 25 AE n.d RI* 100 78 50 EB n.d RI* 260 n.a 500 EP n.d n.d 150 n.a 100 Aloe humilis DM n.d n.d 150 19 10 AE n.d RI * 25 354 500 EB NM RI 15 287 100 EP NM RI* <10 18 50 Tithonia diversifolia DM NM WT <10 n.a. 10 AE M RI* 140 117 250 EB n.d RI 190 158 250 EP n.d RI* 110 n.d n.d Cedrela odorata DM n.d RI* 50 n.d n.d AE n.d RI* n.d n.d n.d EB NM RI* <5 n.a. 250 EP NM RI 100 34 100 Pycnanthus angolensis DM NM RI* 100 n.a. 500 AE WM RI 100 22 25 EB n.d RI <10 415 500 EP n.d RI* 50 5 10 Morinda lucida (bark) DM n.d n.d 50 n.d 10 AE n.d n.d 100 137 50

3. Determination of “in vivo” antimalarial activity ded. Survival studies were also carried out with the- in plant extracts. For “in vivo” tests the ‘Four-day se animals. suppressive test’ (O’Neill et al., 1987) was used, with 4. Toxicity Assays. Two types of toxicity assays were female Balb/C mice (6-week old; ± 20 g weigh) infec- performed: Genotoxicity (“in vitro”) – Ames Test, ted by intraperitoneal innoculations of 107 erythro- and Acute Toxicity “in vivo”. The first was perfor- cytes parasitized with P. berghei ANKA malaria para- med at Centro de Malária e Outras Doenças Tropicais site. Plant extracts and fractions were solubilized or (CMDT) and Centro de Investigação de Genética Hu- suspended in ethanol at the maximum proportion of mana da Faculdade de Ciências Médicas da Universi- 1% (v/v) in distilled water, tested in two concentra- dade Nova de Lisboa, and the second, with the cola- tions (500 and 1000 mg/kg body weight), adminis- boration of Biotério, Instituto de Higiene e Medicina tered daily, orally, during 4 days, to infected mice Tropical (IHMT). (in groups of five animals). Blood films were taken 4.1. Genotoxicity “in vitro” Assay – Ames Test. The on the fourth day (96 h after the first dose), Giemsa lyophilized extracts/fractions (EB, EP, AE, DM) of Ti- stained and examined microscopically and the level thonia diversifolia and Pycnanthus angolensis, were of parasitaemia was calculated. Extract activity was dissolved in DMSO, to prepare a series of concentra- determinate by percent reduction of parasitaemia tions: 1, 5, 10, 50, 100, 150, 300, 600, 750, 1000, in treated groups compared with untreated infected 1500, 2000 µg/ml, giving the final concentration/ mice. The statistical analysis was based on Student’s plaque of 0,04; 0,2; 0,4: 2; 4; 6; 12; 24; 30; 40; 60; t-test transformed in P-values (P > 0.05 is not sig- 80 µg. nificant; P < 0.05 is significant). Controls with local The bacteria used in the tests was Salmonella ti- Cinchona extracts, at the same doses, were inclu- phimurium (TA97), was obtained from Bruce Ames,

260 · Sección 6.09 Madureira, Antimalarial Drugs from Medicinal Plants of S. Tomé e Príncipe 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 biotransformation (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 control (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 / Figure 1. Tithonia diversifolia (Hemsl.) A. Gray B-α-P, 0,5µg/mL) for assays with biotransformation (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 extract/fraction. The lyophilized extracts/fractions were dissolved 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 Figure 2. Tagitinin C. mind that we have to administer orally a single dose of 0,4 ml, the final doses will correspond to RESULTS AND DISCUSSION a concentration of 50, 500, 1000 and 2500 mg/ 1. Antimalarial Activity Kg respectively (Cyted, 1995). A negative con- The results of the “in vitro” tests with plant trol with ethanol and a 5% arabic-gum solution extracts against chloroquine resistant P. falcipa- (maximum 10% ethanol) have been used. rum strain (Dd2) are presented in Table 2. The After oral administration, mice were observed extracts of A. humilis (EA), C. odorata (DM), C. daily, during 7 days, and the number of deaths laevigatum (DM) and C. bidentata (DM) showed recorded. The lethal dose (LD ) was determined 50 moderate antiplasmodial activity with IC values for each extract (concentration that determines 50 5 - 50 µg/ml. Six other medicinal plant extracts 50% of population death). had evident antiplasmodial activity against chlo- 5. Phytochemical Studies. Tithonia diversifolia (Fig. roquine resistant P. falciparum, with IC values 5 1) was the selected plant for phytochemical sudies, 50 -10 µg/ml: S. sparganophorum (PE), V. amygdali- in order to identify the most active compounds. For na b (EA), Cinchona succirubra (CEE and all frac- the phytochemical study of the most active plant tions), T. diversifolia (PE, DM), P. angolensis (CEE) extract (PE), which included bioguided fractiona- and M. lucida (CEE). Comparing with the IC value tion, isolation and structural elucidation of its acti- 50 obtained for Artemisia annua (3.9 µg/ml) and for ve compound, we used several classical and modern Azadirachta indica (10 µg/ml) this group of plants analytical techniques (TLC, CC, HPLC, UV, IV, MS, 1H- has promising results (Phillipson et al., 1987). NMR and 13C-NMR (Goffinet al., 2002). In the cytotoxicity and exoerythrocytic antimalarial activity of plant extracts against P. berghei “in vitro” (Hep G2 cells) (Table 3), eleven of

Sección 6.09 · 261 Tradiciones y transformaciones en Etnobotánica | Traditions and transformations in Ethnobotany the tested extracts and fractions revealed hepatic plants, presents a weak mutagenicity, for the schizontocidal activity, in concentrations below TA98 strain, in the assay with biotransformation, the respective MLD values. From these, four plants and are not mutagenic in the assay without bio- revealed evident schizontocidal activity with IC50 transformation. values from 5 to 34 mg/ml (S. sparganophorum, T. 2.2. Acute Toxicity Assay. The classification of plant diversifolia, P. angolensis and M. lucida bark). extracts, as toxic agents, could be done according to

The plant extracts and fractions that showed their LD50 (Cyted, 1995). In table 4 are presented the significant activity “in vitro” tests have been se- calculated values of LD50 for the plant extracts/frac- lected for “in vivo” assays for antimalarial activi- tions studied. Thus, and according to the criteria of ty (S. sparganophorum, C. succirubra, T. diversifo- Cyted (1995), we can consider the extracts/fractions lia, C. odorata and P. angolensis). In general, the as following: tested plant extracts were partly active against • innocuous: Struchium sparganophora (CEE, EA, the rodent malaria, except for Cinchona extracts PE), Tithonia diversifolia (CEE, EA, PE), Pycnan- that showed a total chemosuppression of parasi- thus angolensis (EA); Aloe humilis (EA); Cedrela taemia in mice infected with P. berghei. However, odorata (EA); Morinda lucida (PE). two of them, S. sparganophorum and C. odorata • weakly toxics: Struchium sparganophora (DM), crude extracts, exhibited a significant inhibition Tithonia diversifolia (DM). (P < 0.05) of the parasite multiplication (85 and For the extracts or fractions without deaths 73%, respectively), at oral doses of 1000 mg/kg within the maximum tested concentration (2.500 per day. The parasitaemia (mean standard devia- mg/kg PV), even if we can not calculated the LD50, tion) of the infected control group at the end of we could confirm that they are not toxics until the 4-day test was 10.34 ± 2.11%; for the mice this concentration (Aloe humilis CEE; Cedrela odo- treated with S. sparganophorum the parasitaemia rata CEE, PE, DM; Pycnanthus angolensis CEE; DM, was 1.54 ± 1.38% and for the mice treated with PE; Morinda lucida CEE). C. odorata the parasitaemia was 2.84 ± 0.99%. The 3. Phytochemical Studies median time of mice survival of the infected trea- From the results obtained it was possible to ted animals versus the control group (23 ± 3 days) identify the main active antimalarial compound in was calculated and one plant extract increased Tithonia diversifolia, the sesquiterpenic lactone ta- this time by 35% (C. odorata). gitinin C – Figure 2. Its antiplasmodial activity was

2. Toxicity Tests verified, showed a IC50 ≈ 0.24 µg/ml and IC50 ≈ 0.25 2.1. Mutagenicity Assay (Ames Test). The results µg/ml against the chloroquine resistant P. falcipa- of the genotoxicity assays are presented in Table 4. rum strains FCB1 and W2. However, tagitinin C also

The number of revertents by plate is calculated as showed some cytotoxicity (IC50 = 0.706 µg/ml) to well as the RI (Reverse Index), taking into account the cell line HTC-116 (Goffinet al., 2002). the number of expontaneous revertents (Mortelmans et al., 2000). In order to consider a substance as a Conclusions mutagenic it is necessary to have simultaneously a The resistance of P. falciparum to chloroquine number of revertents higher than the number of ex- is now a major health problem in STP, as in many pontaneous revertents (20-50 Rev/Plate, for TA98) parts of Africa, and these data suggest a potential and a RI > 2,0. A substance can be classified as a role for medicinal plant extracts in the treatment week mutagenic when the number of revertents is of chloroquine-resistant falciparum malaria. higher than the number of expontaneous revertents Our study indicates that traditional healers for the strain, but it has a RI < 2,0. in STP use several medicinal plants against fever The plant extracts/fractions CEE, PE and DM, and/or malaria which have demonstrated varying from Tithonia diversifolia and Pycnathus angolen- activity against blood stage parasites of chloro- sis do not present signs of genotoxicity, for the quine resistant P. falciparum, as well as against TA98 strain, in both assays with and without bio- the hepatic development of P. berghei in Hep G2 transformação. However, the AE fraction of both liver cells. The effectiveness against liver stage de-

262 · Sección 6.09 Madureira, Antimalarial Drugs from Medicinal Plants of S. Tomé e Príncipe velopment suggests that they could also be used the most active compound. Bearing in mind that as preventive medicines. The development of new the optimal product may not always correspond to drugs with these capacities could be of relevance single compounds, and in order to develop more for the protection of risk groups, in areas with appropriate formulations for local production and such high levels of resistance. administration of these products, further studies Plants commonly used in traditional medicine have been conducted, allowing the development are assumed to be safe due to their long usage in of a HPLC method to achieve a rapid quantifica- the treatment of diseases according to knowledge tion of the active compound from several aerial accumulated over centuries. However, recent scien- plant extracts (Goffin et al., 2003). According to tific findings has shown that many plants used these results the water decoction seems to be the as food or in traditional medicine are potentially less effective way to prepare a traditional remedy, toxic, mutagenic and carcinogenic (Ajaiyeoba et presenting the lowest concentration of tagitinina al., 2006). Regarding the toxicity of the plant ex- C and the lowest antimalarial activity, “in vitro”. tracts and fractions studied, some of them revealed On the other hand, leaves are the plant material some toxicity (Struchium sparganophora and Titho- with the highest level of tagitinin C (1,15%), thus nia diversifolia DM fractions), but their crude ex- should be preferentially used in the preparation tracts didn’t present any acute toxicity. This could of traditional remedies. be due to the presence in the EB of several other These informations have been transmitted to compounds that could inhibit the toxicity of the the Minister of Health and to the Traditional Healers toxic substances presented in the DM fractions. Association, allowing a more safe and effective use These results proved the relative innocuously of the traditional remedy. This will have an obvious of the studied plants crude extracts, thus being economic advantage for the local populations, as these ones the more approximate formula, in the developed medicinal plant products will be the terms of composition, comparing to the tradicio- cheapest therapeutic alternative for malaria. nal medicines, used by traditional healers of São Tomé e Príncipe. Acknowledgements Relating the results of the toxicity tests with We would like to thank to traditional healers of S. Tomé the results of the antimalarial activity (Table 4), e Príncipe, in particular to Sum Gino and Sum Pontes, with whom we learnt and worked, and to the Ministry of we verify that the more active extracts and frac- Health of RDSTP. We also express our gratitude to Virgílio tions were in general the crude extracts (CEE) and do Rosário, Ana Paula Martins, Miléne Gomes and the PE fractions with IC50 < 10 µg/ml (in blood Filipe Santos (CMDT/IHMT), Luc Angenot and Eric Gof- fin for the phytochemical studies (University of Liége), stage), and with LD50 values lower than the toxic letal doses (in liver stage). Thus, is essential to Laurent Rénia for providing the biological material for the hepatic stage tests. A special thanks to Jorge Paiva notice that, in general, in the extracts/fractions for the identification of plant specimens.This study that revealed an important antimalarial activity, was financed by Fundação para a Ciência e Tecnologia the active concentrations are considerably lower (PRAXIS/PSAU/P/SAU/38/96). than the concentrations that showed some toxicity. In fact, the selected plant for phytochemical studies, Tithonia diversifolia, presents a very in- BIBLIOGRAPHY Ajaiyeoba, E., M. Falade, O. Ogbole, L. Okpako, & D. teressant antimalarial activity either in the blood Akinboye. 2006. In vivo antimalarial and cytotoxic stage and in the liver stage for the PE fraction; properties of Annona senegalensis extract. Afr. J. Trad. this fraction was considered not mutagenic for the CAM 3 (1): 137 – 141. Calvo-Calle, J. M., A. Moreno, W. M. C. Eling & E. H. Nardin. TA98 strain and innocuous in the acute toxicity 1994. In vitro development of infectious liver stages of test, thus being a good candidate for the develop- Plasmodium yoelli and Plasmodium berghei malaria in ment of a new antimalarial drug. human cell lines. Experimental Parasitology 79, 362-373. Carvalho, L. H., A. U. Krettli. 1991. Antimalarial Phytochemical studies of the most active plant chemotherapy with natural products and chemically (T. diversifolia) have being performed, and the ses- defined molecules.Memórias do Instituto Oswaldo Cruz quiterpenic lactone Tagitinin C was identified as 86 (suppl. II), 181 -184.

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Cyted. 1995. Búsquedas de princípios bioactivos en plantas Madureira M. C., A. P. Martins, L. Salgueiro, J. Paiva, de la región, Manual de tecnicas de investigation. A. Proença da Cunha. 2002a. Medicinal Plants and Programa Iberoamericano de Ciencia y Tecnología para Traditional Medicine in the Gulf of Guinea – S. Tomé el Desarrollo, Proyecto X-1, 1995. and Príncipe Islands, in: Recent Progress in Medicinal Danforth, H. D., A. U. Orjih, R. S. Nussenzweig. 1978. Plants, pp. 361-381. New Delhi, India Research Book Immunofluorescent staining of exoerythrocytic Centre, Vol.7. schizonts of Plasmodium berghei in fixed liver Madureira, M. C., A. P. Martins, M. Gomes, J. Paiva, A. tissue with stage-specific immune serum.Journal of Proença da Cunha & V. E. Rosário. 2002b. Antimalarial Parasitology 64, 1123-1125. activity of medicinal plants used in traditional medicine Denizot, F., Lang, R. 1986. Rapid colorimetric assay in S. Tomé and Príncipe islands. J. Ethnopharmacol. for cell growth and survival. Modifications to the 81(1):23-9. tetrazolium dye procedure giving improved sensitivity Maron, D. M.& B. N. Ames. 1983. Revised methods for and reliability. Journal of Immunological Methods 89, Salmonella mutagenicity test, Mutation Research, 113: 271-277. 173-215. Desjardins, R., C. Canfield, J. Haynes & J. Chulay. 1979. Millet, P., I. Landau & W. Peters. 1986. In vitro testing of Quantitative assessment of antimalarial activity in antimalarial exo-erythrocytic schizontocides in primary vitro by a semiautomated microdilution technique. cultures of hepatocytes. Memórias do Instituto Oswaldo Antimicrobial Agents and Chemotherapy 16, 710-718. Cruz 81 (suppl. II), 135-141. Etkins, N., Madureira, M. C., et al. 2004. Guidelines for O’Neill, M. J., D. H. Bray, P. Boardmann, K. L. Chan, J. D. Ethnobotanical studies on Traditional Antimalarials, Phillipson, D. C. Warhurst & W. Peters. 1987. Plants in Tradicional Medicines for Malaria – Traditional as sources of antimalarial drugs, Part 4: activity of Medicines for Modern Times (Cap. 14), CRC Press, 2004. Brucea javanica fruits against chloroquine-resistant Goffin, E., E. Ziemons, P. De Mol, M. C. Madureira, A. P. Plasmodium falciparum in vitro and against Martins, A. P., A. P. Da Cunha, G. Phillips, M. Tits, L. Plasmodium berghei in vivo. Journal of Natural Products Angenot, & M. Frederich. 2002. In vitro antiplasmodial 51, 41-48. activity of Tithonia diversifolia and identification of its Phillipson, J. D., M. J. O’Neill, C. W. Wright, D. H. Bray & D. main active constituent: Tagitinin C. Planta Med. 68 C. Warhurst. 1987. Plants as sources of antimalarial and (6): 543-545. amoebicidal compounds. In: Medicinal and Poisonous Goffin E, A. P. da Cunha, E. Ziemons, M. Tits, L. Angenot Plants of the Tropics, Proceedings of Symposium of the & M. Frederich. 2003. Quantification of tagitinin 14th International Botanic Congress, Berlin, Pudoc, C in Tithonia diversifolia by reversed-phase high- Wageningen, pp. 5-35. performance liquid chromatography. Phytochem Anal. Sinden, R. E. 1991. Assexual blood stages of malaria 14(6): 378-80. modulate gametocyte infectivity to the mosquito Hills, M., C. Hudson & P. G. Smith. 1986. Global monitoring vector*/possible implications for control strategies. of the resistance of malarial parasites to drugs: Parasitology 103, 191-196. statistical treatment of the micro-test data. Working Sinden, R. E., A. Suhrbier, C. S. Davies, S. L. Fleck, K. paper No. 2.8.5. for the informal consultation on the Hodivala & J. C. Nicholas. 1990. The development and epidemiology of malaria parasites, WHO, Geneva. routine application of high-density exoerythrocytic- Hollingdale, M. R., P. Leland, A. L. Schwartz. 1983. In vitro stage cultures of Plasmodium berghei. Bulletin of the cultivation of the exoerythrocytic stage of Plasmodium World Health Organization 68 (Suppl.), 115-125. berghei in an hepatoma cell line. American Journal of Trager, W. & J. B. Jensen. 1976. Human malaria parasites in Tropical Medicine and Hygiene 32, 682-684. continues cultures. Science 193: 673-675. Loureiro, L.F., A. M. Cesário, A. S. Franco, V. E. Rosário, et Wellems, T. E., L. J. Panton, I. Y. Gluzman, V. E. Rosário, al. 1996. Malaria in São Tomé and Príncipe: prevalence R. W. Gwadz, A. Walker-Jonah & D. J. Krogstad. 1990. and drug susceptibility. Annals of Tropical Medicine Chloroquine resistance not linked to mdr-like genes in and Parasitology 90, 223-224. Plasmodium falciparum cross. Nature 345: 253-255.

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