07 04

ISSN 0717 7917 www.blacpma.
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Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas

Volumen 7, Nmero 4, 2008
Martha TILAAR et al. Review of Lansium domesticum Corra and its use in cosmetics BLACPMA, 7(4):183 - 189. Susana Gattuso et al. Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora Palau) from Argentina. BLACPMA, 7(4):190 - 198. Janne ROJAS et al. Evaluation of antibacterial activity on different solvent extracts of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae) collected in Venezuela BLACPMA, 7(4):199 - 202. Tzasna HERNNDEZ et al. Antifungal Activity of the Essential Oils of Two Verbenaceae: Lantana achyranthifolia and Lippia graveolens of Zapotitln de las Salinas, Puebla (Mxico) BLACPMA, 7(4):203 - 207. Paloma M. SALES et al. The use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia, Brazil BLACPMA, 7(4):208 217. Ana Laura FAZIO, et al. An ethanolic extract of Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth in C57BL/6 mice. BLACPMA, 7(4):218 - 225. Guillermo SCHINELLA et al. On the anti-trypanosomal, anti-inflammatory and toxicological activities of Haplophyllum linifolium and its diphyllin derivatives. BLACPMA, 7(4):226 - 228.
Comit Editorial
Fundadores Jos L. Martnez (Chile) - Jorge Rodrguez (Cuba) Editor Jefe Jos L. Martnez, Facultad de Medicina Veterinaria y Ciencias Pecuarias, UNICIT, Santiago, Chile. Editor Jefe Cientfico Jos M. Prieto, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, Reino Unido. Editores Ejecutivo Gabino Garrido, Centro de Qumica Farmacutica, La Habana, Cuba. Damaris Silveira, Facultade de Cincias da Sade, Universidade de Braslia, Brasil. Editores de Calidad Carla Delporte, Facultad de Ciencias Qumicas y Farmacuticas, Universidad de Chile. Peter Taylor, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Cientficas, Caracas, Venezuela. Editores de eventos Mara Ins Isla, Facultad de Farmacia y Bioqumica, Universidad Nacional de Tucumn, Argentina. Marcelo Wagner, Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Argentina. Editores Asociados Patricia Arenas, Facultad de Ciencias Naturales, Universidad Nacional de La Plata, Argentina. Marco Dehesa, Laboratorio RENASE, Quito, Ecuador. Jannette Gavillan, Instituto de Investigaciones Interdisciplinarias, Universidad de Puerto Rico Vicente Martnez, Escuela de Agricultura, Universidad de San Carlos, Guatemala. Leonora Mendoza, Facultad de Qumica y Biologa, Universidad de Santiago de Chile. Editores Asesores Arnaldo Bandoni; Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Argentina. Norman Farnsworth, College of Pharmacy, University of Illinois at Chicago, Estados Unidos. Michael Heinrich, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, Reino Unido. Francisco Morn, Laboratorio Central, Universidad de Ciencias Medicas de la Havana, Cuba. Patrick Moyna, Facultad de Qumica, Universidad La Repblica, Montevideo, Uruguay. Pulok K. Mukherjee, School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. Lionel Germosen Robineau, Facultad de Ciencias Exactas y Naturales, Universidad de las Antillas y Guyana (UAG), Pointe Pitre, Guadalupe Horacio Heinzein, Facultad de Qumica, Universidad La Repblica, Montevideo, Uruguay. Consejo Editorial
Christian Agyare, College of Health Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutics, KNUST, Kumasi, Ghana. Julio Alarcn, Facultad de Ciencias Bsicas, Universidad del Bio Bio, Chilln, Chile. Roco Alarcn, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, Reino Unido. Jorge Alonso, Asociacin de Fitoterapia de Argentina, Buenos Aires, Argentina. Giovanni Apendino, DISCAFF, Universidad del Piemonte Oriental, Novara, Italia. Elizabeth Barrera, Seccin Botnica, Museo Nacional de Historia Natural, Santiago, Chile. Geofrey Cordell, College of Pharmacy, Illinois University at Chicago, Estados Unidos. Rene Delgado, Centro de Qumica Farmacuticas, La Habana, Cuba. Eduardo Dellacasa, Facultad de Qumica, Universidad de La Repblica, Montevideo, Uruguay. Luis Doreste, Laboratorio Vitaplant, Mrida, Venezuela. Alina Freire-Fierro, Botany Department, Academy of Natural Sciences, Philadelphia, Estados Unidos. Mildred Garca, Escuela de Medicina, Universidad de Costa Rica Martha Gattusso. rea de Biologa Vegetal, Universidad Nacional de Rosario, Argentina. Harold Gmez, Facultad de Ciencias Qumicas y Farmacuticas, Cartagena de Indias, Colombia. Peter Houghton, Pharmaceutical Sciences Research Division, King's College London, Reino Unido. Ana Ladio, Departamento de Ecologa, Universidad Nacional del Comahue, San Carlos de Bariloche, Argentina. Patricia Landazuri, Facultad de Ciencias de la Salud, Universidad del Quindo, Armenia, Colombia. Claudio Laurido, Facultad de Qumica y Biologa, Universidad de Santiago de Chile. Abdul Manan Mat-Jais, Department of Biosciences, University of Putra, Putra, Malasia. Olga Lock de Ugaz, Departamento de Ciencias, Pontificia Universidad Catlica del Per. Pedro Melillo de Magalhaes, Centro Pluridisciplinar de Pesquisas Qumicas e Biolgicas, UNICAMP, Campinas, Brasil. John A.O. Ojewole, Faculty of Health Sciences, University of KwaZulu-Natal, Sudafrica. Edgar Pastene, Facultad de Farmacia, Universidad de Concepcin, Concepcin, Chile Mahendra Rai, Department of Biotechnology, Amravati University, Maharashtra, India. Luca Rastrelli, Dipartamento di Scienze Farmaceutiche, Universita de Salerno, Salerno, Italia. Elsa Rengifo, Instituto de Investigaciones de la Amazona Peruana, Iquitos, Per Jos Lus Ros, Facultad de Farmacia, Universidad de Valencia, Espaa Alicia Rodrguez, University of Havana, Havana, Cuba. Aurelio San Martn, Facultad de Ciencias, Universidad de Chile, Santiago, Chile Guillermo Schinella, Facultad de Ciencias Mdicas, Universidad Nacional de La Plata, Argentina. Yen-Jen Sung, National Yang-Ming University Taipei, Taiwn Nilka Torres, Centro Regional Universitario de Azuero, Universidad de Panam. Ren Torres, Facultad de Qumica y Biologa, Universidad de Santiago de Chile. Carlos Urza, Facultad de Qumica y Biologa, Universidad de Santiago de Chile. Beatriz Varela, Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Argentina. Carlos Vicente, Editor de la Revista Biodiversidad, sustento y culturas. Revista Biodiversidad, REDES-AT, Montevideo, Uruguay. Elisabeth Williamson, School of Pharmacy, University of Reading, Reino Unido. Talal Zari, Faculty of Science, King Abdulaziz University, Arabia Saudita.
Bol. Latinoam. Caribe Plant. Med. Aromat. Vol.7 (4) 2008
BLACPMA
OBJETIVOS DEL BOLETN Estimular a los grupos de trabajo existentes en Latinoamrica, sean investigadores, productores, funcionarios o simplemente interesados en las plantas medicinales y aromticas, poniendo a su disposicin este Boletn para la difusin y la divulgacin de sus investigaciones y de las actividades que en general desarrollen en torno a plantas. Ser una herramienta de difusin para la Sociedad Latinoamericana de Fitoqumica, principalmente, y de otras sociedades y agrupaciones que se sientan representadas por este Boletn. Constituir un nexo entre los profesionales de habla hispana, francesa, portuguesa e inglesa de la regin, relacionados con el tema central del Boletn OBJECTIVES OF THE BULLETIN To stimulate the existing work groups interested in the medicinal and aromatic plants in Latin America, investigators, producers, governmental agencies or general public interested in the subject, by publishing a Bulletin dedicated to the dissemination of their investigations and the activities that in general they develop around plants and natural products in general. To be a tool of diffusion for the Latin American Society of Fitoqumica, mainly, and of other societies and groupings that feel represented by this Bulletin. To constitute a nexus between the professionals of Hispanic, French, Portuguese and English speech of the region, related to the central subject of the Bulletin The LATIN AMERICAN BULLETIN AND OF the CARIBBEAN OF MEDICINAL AND AROMATIC PLANTS (BLACPMA), ISSN 0717 7917, is a bimonthly, electronic, scientific publication directed to any professional working in the field of the medicinal and aromatic plants. BLACPMA is a non profit organization. Although supported by Latin American Phytochemical Society (SLF), this bulletin is not property of Club or Association some. Neither BLACPMA nor the SLF are not responsible for the opinions published in this bulletin, that are unique responsibility of their respective authors. All the graphical material is original or published with the permission of its original authors. All the marks and logos referred in these pages are property of their respective authors or companies. In Chile, 1st January of 2008.
El BOLETN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES Y AROMTICAS (BLACPMA), ISSN 0717 7917, es una publicacin cientfica electrnica bimensual dirigida a diversos profesionales y tcnicos vinculados al campo de las plantas medicinales y aromticas. BLACPMA es una entidad sin nimo de lucro. Aunque auspiciada por la Sociedad Fitoqumica Latinoamericana (SLF), este boletn no es propiedad de Club o Asociacin alguna. Ni BLACPMA ni la SLF son responsables en ningn momento de las opiniones vertidas en sus pginas, que son responsabilidad nica de sus respectivos autores. Todo el material grfico ha sido creado de manera genuina o bien remitido por sus autores con el permiso de stos. Todas las marcas y logos referidos en estas pginas son propiedad de sus respectivos autores o empresas. En Chile, 1 de Enero de 2008.
BLACPMA WEB Site: www.blacpma.cl Envio de trabajos Online a nuestra editorial Authors Submission Package http://www.blacpma.cl/submissions.htm (Online Submission) Blacpma_editorial@hotmail.com
BLACPMA esta Indexada por: CHEMICAL ABSTRACTS CAB ABSTRACTS NAPRALERT INDEX COPERNICUS (Impacto 4.80) IMBIOMED LATINDEX QUALIS REDALYC
Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008
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Instrucciones para los autores

INSTRUCCIONES PARA LOS AUTORES El BOLETN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES (BLACPMA), ISSN 0717 7917, es una publicacin cientfica electrnica bimensual dirigida a profesionales y tcnicos que trabajen tanto en productos naturales de plantas medicinales o nutracuticos en general como en plantas medicinales y aromticas. Sern aceptados aqullos trabajos relacionados con alguna de las reas abarcadas por el Boletn tales como agronoma, antropologa y etnobotnica, aplicaciones industriales, botnica, calidad y normalizacin, ecologa y biodiversidad, economa y marketing, farmacologa, fotoqumica, farmacognosia, aspectos de regulacin y legislacin, informacin y difusin de eventos, cursos, premios, novedades, monografas, revisin de libros y cualquier otro tipo de material que se considere importante comunicar. tipo DE LA CONTRIBUCIN Los autores podrn presentar revisiones sobre un tema en particular as como un trabajo original de una investigacin cientfica, en la forma de trabajo completo o comunicacin corta. Cualquiera de estas contribuciones debern estar escritas en espaol, ingls, portugus o francs, sin lmite de extensin la cual deber estar razonablemente ajustada al objetivo del trabajo. Sin embargo, los anuncios, novedades y eventos no debern exceder una pgina. En todos los casos, las figuras estn includas. FORMATO DE LA CONTRIBUCIN La contribucin deber realizarse mediante el uso del Documento nico para Autores, tambin abreviado ASP (Authors Submission Package), que se puede descargar de la pagina Web www.blacpma.cl o en caso necesario se puede pedir a los editores por correo electrnico a blacpma_editorial@hotmail.com . El estilo de nuestra revista se detalla a continuacin: Los trabajos sern presentados en formato Microsof Word (versin 3.1 o superior usando Times New Roman nmero 11). Los trabajos constarn de Introduccin, Material y Mtodos, resultados, Discusin, Conclusiones y Bibliografa. En cualquiera de las modalidades en las que se presente el trabajo, en la primera pgina deber constar el Ttulo del trabajo (en espaol y en ingls), autores, institucin a la que pertenecen, la direccin y correo electrnico del autor principal. Tambin deber llevar un resumen en espaol y en ingls de no ms de 100 palabras, un ttulo corto y un mximo de 6 palabras clave. Los nmeros de las tablas y figuras deben ser arbigos. Resumen Deber llevar no ms de 150 palabras e incluir los mtodos usados, los resultados relevantes y las conclusiones. Texto Artculos originales: constarn de Introduccin, Materiales y Mtodos (descripcin extensa), Resultados (referidos a las tablas y figuras), Discusin (extensin libre), y Conclusiones (lo ms corta posible). Revisiones estarn estructuradas de acuerdo a las necesidades del autor. Comunicaciones breves o notas: debern llevar una breve Introduccin, Materiales y Mtodos (breve descripcin o slo referencia al protocolo publicado), Resultados y Conclusin. El nombre completo de la especie en latn y la familia (ej: Inula viscosa (L.) Aiton. Asteraceae) debern ser mencionados in extenso al menos en la seccin Materiales. A lo largo del trabajo slo se usar el nombre corto en latn (I. viscosa) Tablas Las tablas debern ser escritas usando un procesador Word y nunca seran figuras. Favor de no usar otras lneas distintas de las negras de 1 pt. El texto deber estar en Times New Roman 10 9 puntos. Incluir siempre Ttulo (numerado y citado en el trabajo) y la leyenda de las abreviaturas, en los casos en que corresponda. Figuras Incluir las referencias por separado (no incluir las leyendas en la figura). Necesitamos la imagen en cualquiera de los siguientes formatos (JPEG, JPG; GIF, BMP o TIFF). Sin embargo evitar TIFF si es demasiado grande y GIF si la imagen es de baja calidad. No hay restricciones en el nmero y color de las figuras, pero la inclusin de cualquier figura debe estar justificada. No es posible publicar una imagen que haya sido copiada de otra publicacin..Slo es posible publicar copias de imgenes libres de derecho de autor, de lo contrario debern ser rediseadas con un programa adecuado. Puede hallar versiones libres en Internet. Le sugerimos: MarvinSketch (para Windows y otros sistemas) (descargar gratis luego de registrarse http://www.chemaxon.com/product/msketch.html ) EasyChem for MacOS (http://sourceforge.net/project/showfiles.php?group_id=90102 ) Referencias Las citas en el texto debern incluir el apellido del autor y el ao, separado por coma y colocados entre parntesis (ej. Bruneton, 1995); si hay ms de un trabajo del mismo autor, se separarn por comas (ej. Bruneton, 1987, 1995, 2001). Si hay dos autores se citarn separados por y o su equivalente, respetando el idioma original de la fuente. Si hay ms de dos autores, slo se citar el primero seguido de la expresin et al. En tanto que en bibliografa debern figurar todos los autores. Si hay varios trabajos de un mismo autor y ao, se citar con una letra en secuencia adosada al ao (ejemplo: Mayer et al. 1987a, 1987b). Si un trabajo no tiene autor se lo citar como Annimo, seguido de la fecha de publicacin. Si hubiera ms de una cita de esta tipo en el mismo ao, se adosar una letra correlativamente (ejemplo: Annimo, 2002a, Annimo, 2002b). La bibliografa incluir SLO las referencias mencionadas en el texto, ordenadas alfabticamente por el apellido del primer autor, sin nmero que lo anteceda y sin sangra. Apellido/s del autor seguido de las iniciales del nombre sin puntos ni separacin entre ellas. El nombre de la revista se colocar abreviado segn normativas ISO de acuerdo con el Botanico Periodicum Huntianum (disponible solamente en edicin impresa) o Pubmed Journals Database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Journal, ISO abbreviation) que ofrece al posibilidad de confirmar on line el nombre y abreviatura de un enorme nmero de revistas. Por ltimo se citar el volumen de la publicacin, seguido del nmero entre parntesis, dos puntos y el nmero de pginas desde x hasta y, sin espacios entre medio. Las citas de libros deben explicitar las pginas consultadas as como el ao de edicin. No se admitirn citas incompletas y el incumplimiento de estas normas ser motivo de retraso del artculo hasta su correccin. Modelos Publicaciones peridicas Grove H, Rovirosa J, San Martin TO, Argandoa V. 1994. Secondary Metabolites of Dictyota crenulata. Bol. Soc. Chil. Qum. 39(3):173-178. Libros Durand AND, Miranda M, Cuellar A.1986. Manual of practical of laboratory of Pharmacognosy. Ed. I People and Education, Havana, Cuba, pp. 90, 120-121. Captulos de libros editados Lopes of Almeida JM. 2000. Pharmaceutical formulation of phytotherapeutic products, pp. 113-124. In Sharapin N: Foundations of technology of phytotherapeutic products. Ed. CAB and CYTED, Bogot, Colombia. Tesis (aceptable slo si no hay fuente alternativa) Gonzlez of Cid D. 2000. Cianobacteria study with noxious effects (deleterious and toxic) in aquatic atmospheres of the county of San Lus. Doctoral thesis, National University of San Lus, Argentina, pp. 234, 245-244. Comunicaciones a Congresos Si no hay libro oficial de Abstracts: Novak TO, Brown of Santayana M, Blackish JM. 2006. Antioxidant activity and fingerprinting of Spanish Bupleurum species used ace anti-inflammatory remedies. Communication to the British Pharmaceutical Conference 2006 (Royal Pharmaceutical Society of Great Britain, Manchester, UK, 4-6 September). Si hay libro official de Abstracts: Novak TO, Brown of Santayana M, Blackish JM. 2006. Antioxidant activity and fingerprinting of Spanish Bupleurum species used ace anti-inflammatory remedies. Summaries of the British Pharmaceutical Conference 2006 (Royal Pharmaceutical Society of Great Britain, Manchester, UK, 4-6 September) p.23. Si los resmenes fueron publicados en una revista, se menciona SLO la revista como si fuera un artculo ms. Novak TO, Brown of Santayana M, Blackish JM. 2006. Antioxidant activity and fingerprinting of Spanish Bupleurum species used ace anti-inflammatory remedies. J. Pharm. Pharmacol. 58(Suppl. 1): 82. Recursos electrnicos Nota: si es necesario cortar alguna direccin se recomienda hacerlo despus de una barra inclinada. ATENCIN: hoy existen muchos otros tipos de dominios que no son http. Por ejemplo los hay https o ftp. Igualmente existen muchos dominios que no son www, sino www2 u otros. Por tanto preste atencin a la direccin completa y no suma que por defecto van a ser http o www. Duncan R. 2000. Nano-sized particles ace "nanomedicines". http://www.mhra.gov.uk/home/idcplg?IdcService=GET_FILE&dDocName=con2022821&Revi sionSelectionMethod=Latest. [Consulted October 6, 2006]. En caso de no haber un autor, o cuando no hay un responsable principal, se toma la institucin responsable como equivalente al autor y en el texto se cita (CNN,200). CNN. Cuba's health care manages despite seizure. http://www.cnn.com/TRANSCRIPTS/0108/18/yh.00.html [Consulted 5 October, 2006]. Boletines o revistas on line con ISSN, la fuente debe ser citada como cualquier otra revista. Prieto JM. 2005. El Blsamo de Fierabrs. BLACPMA 4(3):48-51. Importante NOTA sobre la citacin de pginas Web En stos das se est comprobando el creciente ABUSO de la citacin de pginas Web para avalar afirmaciones cientficas hechas por los autores . resulta muy peligroso para su credibilidad como autor, y APRA la credibilidad de este Boletn, citar informacin obtenida en pginas Web que no tengan ninguna entidad cientficamente reconocida que se haga responsable de la susodicha informacin. Las pginas Web annimas slo deben ser usadas en casos muy justificados y ante la absoluta ausencia de ninguna otra fuente primaria cientficamente reconocida. El Comit editorial de esta revista realizar todo esfuerzo para eliminar el recurso fcil a pginas Web seudocientficas y desde luego los autores deben dar en todo caso una explicacin de por qu han recurrido a ste tipo de fuente. Todo abuso ser motivo de rechazo para su publicacin, incluso si este ya fue (errneamente) aceptado por los revisores. Si se trata de boletines o revistas on-line con ISSN, la fuente debe ser citada como cualquier otra revista. Envo de los trabajos y procedimiento de edicin Se podrn enviar tanto por correo electrnico a la direccin blacpma_editorial@hotmail.com o por correo areo en disco de 3.5 pulgadas a Lic. Jos Luis Martnez, Editor, Casilla de Correo 70036, Santiago 7, Chile. Los trabajos se acompaarn de una lista conteniendo el correo electrnico y la direccin de TODOS los autores. El autor principal ser el responsable de manifestar su conformidad en nombre de todos los autores, en relacin a la publicacin en BLACPMA as como de cualquier problema que se origine por la autoridad y/o originalidad del trabajo. Esto quedar claramente establecido en una nota formal que acompaar el trabajo enviado. Una vez recibido, el trabajo ser arbitrado por un par de revisores, que podrn ser miembros de nuestro comit editorial, acadmicos o profesionales reconocidos, quienes decidirn su aprobacin o rechazo. De todas maneras, el editor tiene la facultad para decidir si el trabajo cumple con el enfoque del boletn y tiene la libertad de modificar el manuscrito definitivo (ver el apartado siguiente). Autoridad final del Comit Editorial. Los editores se reservan el derecho de corregir o modificar el manuscrito aceptado para su publicacin en BLACPMA, previa consulta con el autor para que se adecue mejor al estilo y objetivos del boletn. Este procedimiento tendr lugar en aquellos casos en que los manuscritos no concuerdan con los modelos cientficos generalmente aceptados o si el contenido es innecesariamente largo, redundante o no suficientemente claro. Estas modificaciones pueden ser requeridas directamente a los autores y podrn retrasar la publicacin del manuscrito.
Gracias por su importante contribucin y por tener en cuenta estas normas. Comit Editorial BLACPMA
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Guidelines for Authors

The LATIN AMERICAN AND CARIBBEAN BULLETIN OF MEDICINAL AND AROMATIC PLANTS (BLACPMA), ISSN 0717 7917, it is a bimonthly electronic scientific publication directed to any professional or technician working both on natural products of medicinal or nutraceutical interest in general or with interest on medicinal and aromatic plants . Works related with any of the areas covered by the Bulletin will be accepted such as: agronomy, anthropology and ethnobotany, industrial applications, botany, quality and normalization, ecology and biodiversity, economy and marketing, pharmacology, phytochemistry, pharmacognosy, legislation and regulatory affaires, information and diffusion of events, courses, prizes, news, reports, book reviews, or any other material type which may be important to communicate. Type of contributions Authors will be able to present reviews on a particular subject as well as original scientific research, in the form of both full papers and short communications. Essays on hot topics for debate are also welcome. Any of these contributions may be written in Spanish, English, Portuguese or French , without limits on their extension which must be reasonably adjusted to the objective of the work. However Announcements, news and events reports should not exceed one page. In all the cases figures are included. Format of the contributions Authors must submit their contributions using the ASP (Authors Submission Package) downloadable from www.blacpma.cl . Otherwise they will not be considered. If you experience any problem in obtaining this document please ask our editorial office for a copy (blacpma_editorial@hotmail.com). What follows are the style accepted for publication in BLACPMA: The works will be presented in Microsoft Word format (version 3.1 or superior, using Times New Roman size 11 points). The works will be conformed by Introduction, Material and Methods, Results, Discussion, Conclusions and Bibliography. In anyone of the modalities in which the works be presented, in the first page it should appear: Title of the work (in Spanish and English), authors, the institution they belong to, the main author's address and e-mail. It should also appear a summary in Spanish and English with not more than 100 words, a short title and a maximum of 6 key words. The numbers of the tables and the figures should be Arabic. Abstract It must be not more than 200 words and contain the name of the methods used, all relevant results and conclusions, Text Original articles: divided it in Introduction, Materials and Methods (extended description), Results (refer to tables and figures), Discussion (free extension), and Conclusions (must be as short as possible). Reviews are structured according the authors needs. Short Communications or letters must have a brief intro, Materials and Methods (brief description or only reference to the protocol already published), Results and Conclusion. Speciess complete Latin name and family ( like Inula viscosa (L.) Aiton. Asteraceae- ) is required to be mentioned in extenso at least in Materials section. Throughout the paper only use the short latin name (I. viscose). Tables Tables must be written using a word processor. Images of tables ARE NOT ACCEPTED Please do not use other lines than black 1 pt. Text must be Times New Roman 10 or 9 points. Always Provide Title (numbered and cited in the paper). Provide abbreviations legend when necessary. Figures Provide the caption in a separate (do not include legends or captions in the figure) We need the images in any of the following formats (JPEG, JPG, GIF, BMP or TIFF). However, avoid TIFF as they are too big and GIF as they are low-quality. There are not restrictions on the number and colours of figures, but the inclusion of any figure must be justified. It is not possible to publish an image that has been copied from another journal wich owns the copyright. It is only possible to publish copies of copyright free images, otherwise you must redraw them in a suitable software. You can find free versions of such a software in the internet. We can suggest: MarvinSketch (for windows and other systems) (free download after registration http://www.chemaxon.com/product/msketch.html ) EasyChem for MacOS (http://sourceforge.net/project/showfiles.php?group_id=90102 ) References The citations in the text will include the author's last name and year, separated by coma and placed between parenthesis (example: Bruneton, 1995); if there is more than one work by the same author, they must be separated by comas (example: Bruneton, 1987, 1995, 2001). If there are two authors they must do a separated citation by "and" or its equivalent one, respecting the original source language. If there are more than two authors, it will be cited only the first one followed by the expression et al. highlighted in italic letter (example: Dixon et al., 1999), as long as in the bibliography all the authors will figure. If there are several works of the same author and year, it will make an appointment with a letter in sequence joined to the year (example: Mayer et al. 1987a, 1987b). If a work doesn't have author, it should be done a citation as Anonymous, followed by the publication date. If there were more than an anonymous citation in the same year, it will be correlatively joined to a letter (example Anonymous, 2002a, Anonymous, 2002b). The bibliography must include ONLY the references mentioned in the text, in alphabetical order for the first author's last name, without preceding by number or sangria. The author's last name followed by the first name initials without points neither separation among them. The journals name will be placed abbreviated according to ISO normative and in italic letter in agreement with the Botanical Periodicum Huntianum, (available only in printed edition) or with the more convenient Pubmed Journals Database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Journal, ISO abbreviation) that offers the possibility of checking online the name and abbreviation (in both senses) of an enormous number of magazines. At last it should be cited the volume of the publication, better if followed by the number between parenthesis, two points and the page number from x until y, without spaces between them. The citations of books must explain which pages were consulted and the year of edition (you must pay attention to not mistaking the year of the first edition with of the edition you are consulting). Incomplete citations won't be admitted and any other defect will be late reason of the article until its correction agreement to these norms. Models Periodic publications Grove H, Rovirosa J, San Martin TO, Argandoa V. 1994. Secondary Metabolites of Dictyota crenulata. Bol. Soc. Chil. Qum. 39(3):173-178. Books Durand AND, Miranda M, Cuellar A.1986. Manual of practical of laboratory of Pharmacognosy. Ed. I People and Education, Havana, Cuba, pp. 90, 120-121. Chapters of published books Lopes of Almeida JM. 2000. Pharmaceutical formulation of phytotherapeutic products, pp. 113-124. In Sharapin N: Foundations of technology of phytotherapeutic products. Ed. CAB and CYTED, Bogot, Colombia. Thesis (acceptable only if there is not alternative source) Gonzlez of Cid D. 2000. Cianobacteria study with noxious effects (deleterious and toxic) in aquatic atmospheres of the county of San Lus. Doctoral thesis, National University of San Lus, Argentina, pp. 234, 245-244. Communications to Congresses If there is not official book of abstracts: Novak TO, Brown of Santayana M, Blackish JM. 2006. 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EDITORIAL
BLACPMA DESDE EL 2009 SERA MAS GRANDE Si miramos hacia el ao 2002 en Buenos Aires, Argentina en donde gracias a una invitacin del Dr. Arnaldo Bandoni se realiz la presentacin de BLACPMA en el Congreso Latinoamericano de Fitoqumica nos damos cuento que en poco ms de 6 aos hemos alcanzado un sitial espectacular y que da a da vamos ascendiendo cada vez mas. El titulo de esta editorial obedece que a contar de 2009 cuando entremos al volumen 8 nuestra pgina Web oficial dejara de ser www.blacpma.cl y pasar a ser www.blacpma.org y en donde todos los miembros del Comit Editorial podrn tener e-mail del estilo xxxx@blacpma.org . La nueva pagina esta dotada de un sistema automatico de gestion en linea. Comentemos algo del Congreso a realizarse en enero de 2009 en Chilln (Chile) y que corresponder a la Tercera Reunin de BLACPMA (Varadero 2006, La Plata 2007). El sbado 26 de Julio se realiz la ltima reunin del Comit Organizador en el Centro de Biologa Marina en Dichato, hermoso lugar al lado del mar. Entre las decisiones se analizaron las mesas redondas que se realizarn una de las cuales estar a cargo de BLACPMA y que coordinar el Dr. Julio Alarcn las otras estarn a cargo de especialistas chilenos, si bien la nica que esta ya confirmada en su totalidad es la que coordina el Dr. Edgar Pastene (Chile) y tambin miembro de BLACPMA y en donde participara nuestro Editor Jefe Cientfico Dr. Jos Mara Prieto sobre antioxidantes. Las otras mesas redondas sern sobre Fitoqumica, Biotecnologa, Agronoma y Etnobotnica. Del mismo modo la Conferencia de Clausura ser dictada por otro miembro de BLACPMA el Dr. Gabino Garrido de Cuba. Adems de nuestra familia BLACPMAea participaran varios con Conferencias, entre ellos estarn Rene Torres (Chile), Leonora Mendoza (Chile), Damaris Silveira (Brasil), Vicente Martnez (Guatemala), Marcelo Wagner (Argentina), Mara Ins Isla (Argentina), Patricia Arenas (Argentina), Geofrey Cordell (USA), entre otros. Debo agradecer a travs de estas lneas la amistad que me une desde hace ya varios aos con la Dra. Marlene Porto, Directora del Centro de Investigacin y Desarrollo de Medicamentos de Cuba, quien ha permitido que varios cientficos de su centro hayan venido a diversos eventos en los cuales he estado ligado, es as como en esta oportunidad tendremos la visita de Janet Piloto Ferrer. Como Editor Jefe de BLACPMA deseo agradecer la confianza que ha tenido la Dra. Porto para facilitar la salida de miembros de su equipo. Tambin y volviendo hacia nuestra publicacin, BLACPMA ha sido aceptada en BVS (Biblioteca Virtual em Sade Brasil). Esta ha sido otra gestin de nuestra activsima compaera Damaris Silveira. Finalmente deseo agradecer a travs de estas lneas la participacin en los ltimos aos de la Dra. Patricia Arenas, de la Universidad Nacional de La Plata (Argentina), quien por motivos laborales y familiares se alejar de nuestra lnea editorial pero que seguir ligada como evaluadora, ser reemplazada a partir de 2009 por la Dra. Mara Ins Isla (Universidad Nacional de Tucumn, Argentina) quin hasta ahora se desempae como Editora encargada de resmenes de Congresos. En su cargo asumir el Dr. Marcelo Wagner (Universidad de Buenos Aires, Argentina), quien ser acompaado en dicha labor por el Dr. Harold Gmez (Universidad de Cartagena de Indias, Colombia). Esperando comunicarnos en el prximo nmero de BLACPMA, les saluda a nombre del Comit Editorial Jos Luis Martnez, Editor Jefe Jos Mara Prieto, Editor Jefe Cientfico Gabino Garrido, Editor Asociado Damaris Silveira, Editor Asociado
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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2008 Los Autores Derechos de Publicacin 2007 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 7 (4), 183 - 189 BLACPMA ISSN 0717 7917 Revisin | Review
Review of Lansium domesticum Corra and its use in cosmetics

[Revisin de Lansium domesticum Corra y sus usos en cosmtica]
Martha TILAAR1,Wong Lip WIH1*, Anna S. RANTI1, S. M. WASITAATMADJA2, SURYANINGSIH1, F.D.JUNARDY1, MAILY1 1Martha Tilaar Innovation Center, Jakarta Indonesia; 2 University of Indonesia, Yakarta, Indonesia..
Contacts: lwwong@martinaberto.co.id Received 7 October 2007; Accepted 30 November 2007; Minor corrections 5 April 2008; Online 5 April 2008
Abstract This article focuses on the indonesian tropical plant, Lansium domesticum. Its botany and phytochemistry as well as its medicinal, nutritional and cosmeceutical value, which include antioxidant, moisturizing, whitening and lightening effects, are reviewed. This plant could be of interest for the Latinamerican community as it can grow in several parts of America.
Keywords: Lansium domesticum, anti oxidant, moisturizing, cosmetic use.
Resumen Este articulo trata de la planta tropical Indonesia Lansium domesticum. Su botnica y fitoqumica as como sus usos medicinales, nutricionales y cosmticos, que incluyen efectos de antioxidantes, hidratantes, despigmentantes e iluminadores del cutis son revisados. Esta planta puede ser de inters para la comunidad latinoamericana ya que puede adaptarse y crecer en algunas partes de Amrica.
Palabras clave: Lansium domesticum, antioxidante, hidratante, uso cosmetico.
INTRODUCTION Indonesia, a tropical country, is one of the biggest archipelages in the world, full of plant diversity (Sutarjadi, 1992). Almost 11 percent of 30,000 species of plants have used in health and beauty care. (Sutarjadi, 1992). As a country rich in natural resources, including medicinal plants and marine resources, the botanists use to say that Indonesia is a mega biodiversity country. While the Dutch complimented Indonesia as de Smaragd Gordel which means the green (like emerald) and full of riches islands (Heyne, 1987). Indonesia, with its more than 200 million population, is known for its heritage in the utilization of plant for medicinal as well as cosmetic use. This article reviews the Indonesian tropical plant, Lansium domesticum, that has been used as the source of an extract for natural whitening in cosmetics. BOTANICAL DATA Family Lansium domesticum Corra belongs to the Meliaceae family (Heyne, 1987) Common names
(Heyne, 1987; Verheij, 1992; MMPND, 2007)

Langsat, Duku, Kokosan [Indonesia]; Langsat, Duku, Duku-Langsat Malaysia]; Lansones (Tagalog), Langsat, Duku, Longkong [Thailand]; Bon-bon [Vietnam]; Langsat [Brumese], Lan sa [Chinese]; Lan sa guo [Taiwan]; Langsat, Langsep [Danish]; Kokosan, Langsep [Dutch]; Langsat [English]; Langsep, Langsium [French]; Ecther Lansabaum, Langsat, Lansabaum, Lansibaum [German]; Lansio, Lanzone [Italian]; Ransa [Japanese]; Lang sat [Korean]; Arbol-do-lanza [Portuguese]; Arbol de lanza, Lanzon [Spanish].
Review of Lansium domesticum
Tilaar et al.
Synonyms (Heyne, 1987; Verheij, 1992; Nationalherbarium, 2007) Aglaia aquea (Jack) Kosterm., Aglaia domestica (Correa) Pellegr., Aglaia dookoo Griff., Aglaia intricatoreticulata Kosterm., Aglaia merrillii Elmer, Aglaia sepalina (Kosterm.) Kosterm., Aglaia steenisii Kosterm., Lansium aqueum (Jack) M.Roem., Lansium domesticum var. aqueum Jack, Lansium domesticum var. pubescens Koord. & Valet., Lansium domesticum var. typicum Backer, Lansium javanicum Koord. & Valet. ex Moll & Janss., Lansium javanicum M. Roem., Lansium parasiticum Sahni & Bennet, Lansium parasiticum var. aqueum (Jack) Sahni & Bennet, Lansium pedicellatum Kosterm., Lansium sepalinum Kosterm, Taeniochlaena polyneura Schellenb. Description
Seeds:
Seeds 1-3, enveloped by a closely adhering, thick, fleshy, translucent white aril; cells without developed seed are also filled with aril tissue (Heyne, 1987; Morton, 1987).
Figure 1. Lansium domesticum, tree and fruit
Bark:
Tree up to 30 m tall and trunk 75 cm in diameter, in cultivation usually only 5-10 m tall; bole up to 25 m, irregularly fluted, with steep buttresses; bark mottled grey and orange, furrowed, containing milky, sticky resinous sap; twigs glabrous to pilose. Origin and Geographic Distribution Langsat originates in western South-East Asia, from Peninsular Thailand in the west to Borneo in the east (Indonesia). It still occurs wild or naturalized in this area and is one of the major cultivated fruits. In Borneo it is found throughout the island. On a small scale, langsat is also cultivated in Vietnam, Burma, India, Sri Lanka, Hawaii, Australia, Surinam and Puerto Rico. In Indonesia, langsat can also be found in Banyuwangi, Palembang, Bangka, West of Kalimantan, and in some areas of Sulawesi (Celebes) (Heyne, 1987; Verheij, 1992). Langsat was introduced into Hawaii before 1930, and is frequently grown at low elevations. An occasional tree may be found on other Pacific islands. The species is little known in the American tropics, except in Surinam. Seeds were sent from Java to the Lancetilla Experimental Garden at Tela, Honduras, in 1926 and plants arrived from the same source in 1927. The trees have grown well and there are bearing trees in Trinidad, where the langsat was established in 1938, and a few around Mayaguez, Puerto Rico, that have been bearing well for about 60 years. There were young specimens growing on St. Croix in 1930. Southern Florida does not have the climatic and soil conditions favorable to the langsat. There have been attempts to maintain langsats at the University of Floridas Agricultural Research and Education Center in Homestead, but the trees have
Leaves:
Leaves alternate, odd-pinnate, 30-50 cm long, glabrous to densely pilose, petiole up to 7 cm long; leaflets alternate, 6-9, elliptical to oblong, 9-21 cm x 5-10 cm, glossy, chartaceous-corieceous, base what some asymmetric, apex short acuminate, lateral veins 10-14 pairs, petiolules 5-12mm long, thickened at base.
Flowers:
Inflorescence many flowered, solitary or in fascicles of 2-10 on the trunk or largest branches; recemes simple or branched at the base, 10-30 cm long; flowers bisexual, sessile to pedicelled, solitary, small; calyx fleshy, cup-shaped, 5-lobed, greenishyellow; petals fleshy, erect, ovate, 2-3 mm x 4-5 mm, white to pale yellow; staminal tube subglobose, up to 2 mm high, anthers in one whorl; ovary globose, appressed pilose, 4-5-celled; style short, thick, stigma broad.
Fruits:
Fruit an ellipsoid or globose berry, up to 2-4(-7) cm x 1.5-5 cm, yellowish pubescent, calyx persistent with reflexed lobes; fruit-wall thin (1-1.5 mm) or thick (up to 6mm).
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succumbed either to the limestone terrain or low temperatures. Varieties There are two distinct botanical varieties: Lansium domesticum var. pubescens, the typical wild langsat, which is a rather slender, open tree with hairy branchlets and nearly round, thick-skinned fruits having much milky latex; Lansium domesticum var domesticum , called the duku, doekoe, or dookoo, which is a more robust tree, broad-topped and densely foliaged with conspicuously-veined leaflets; the fruits, borne few to a cluster, are oblong-ovoid or ellipsoid, with thin, brownish skin, only faintly aromatic and containing little or no milky latex. The former is often referred to as the wild type, but both varieties are cultivated and show considerable range of form, size and quality. (Heyne, 1987; Morton, 1987; Verheij, 1992; MSC, 2002) Climate The langsat grows in ultra-tropical climate. Even in its native territory it cannot be grown at an altitude over 2,100 to 2,500 ft (650-750 m). It needs a humid atmosphere, plenty of moisture, and will not tolerate long, dry seasons. Some shade is beneficial, especially during the early years (Morton, 1987). CHEMICAL COMPOSITION The edible portion is 68% of the fruit weight. Per 100 g it contains: water 84 g, a little protein and fat, carbohydrates 14.2 g, mainly reducing sugars, predominantly glucose, fibre 0.8 g, ash 0.6 g, Ca 19 mg, K 275 mg, some vitamin B1 and B2 but little vitamin C. The energy value is 238 kJ/100g. The fresh peel contains 0.2% of a light-yellow volatile oil, a brown resin and reducing acids. From the dried peel, there is obtained a dark, semi-liquid oleoresin composed of 0.17% volatile oil and 22% resin. (Heyne, 1987; Verheij, 1992). Five tetranorterpenoid, domesticulide A-E (1-5), were isolated from seed of Lansium domesticum Corr. together with 11 known triterpenoids (6-16). It is worth nothing that the seed extract of L. domesticum are a rich source of limonoids. Six classes of the limonids have been isolated , including andirobin derivates (1-2), methyl angolensates (3, 4, 8, 9 and 10), mexicanolides (5-7), an azadiradione (11), onoceranoids (12-13) and dukunolides (14-16). Compounds 2, 3, 4, 7, 8, 10, 11, and 15 showed
antimalarial activity against Plasmodium falciparum with IC50's of 2.4-9.7 g/ml (Saewan, 2006).
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USAGE OF LANSIUM Food Uses The peel of the langsat can be easily removed and the flesh is practically eaten fresh out of hand, or served as a dessert, and may be cooked in various ways. Seedless fruit may be bottled in syrup or sometimes candied (Morton, 1987).
Table 1. Food value of L. domesticum Food value per 100 g of edible portion* 86.5 g 0.8 g 9.5 g 2.3 g 20.0 mg 30.0 mg 13.0 I.U. 89 mcg 124 mcg 1.0 mg mg (dry weight) *According to analysis made in India. The edible flesh may constitute 60% of the fruit. (Morton, 1987) Moisture Protein Carbohydrates Fiber Calcium Phosphorus Carotene (Vit.A) Thiamine Riboflavin Ascorbic Acid Phytin
Three new onoceranoid triterpenes, lansionic acid (17), 3 -hydroxyonocera-8(26),14-dien-21-one, and 21-hydroxyonocera-8(26), and 14-dien-3-one, were isolated from the fruit peel of Lansium domesticum var domesticum. These triterpenoids exhibited mild toxicity against brine shrimp (Artemia salina) (Tanaka, 2002).
Medicinal Uses The peel, rich in oleoresin, is used against diarrhea. This resin is non-toxic and is administered to halt diarrhea and intestinal spasms; However, Morton (1987) shown that it can contracts rabbit intestine in vitro. Other parts of the plant employed medicinally include the crushed seeds used to treat fevers and the astringent bark which is administered (p.o) against dysentery and malaria. The powdered bark is used in poultices against scorpion stings. (Verheij, 1992) Other Uses The dried peel is burned in Java, the aromatic smoke serving as a mosquito repellent and as incense in the rooms of sick people. The light-brown wood is tough and durable and used for house pots, tools, handles, etc (Heyne, 1987). Cosmetic Uses The dried Hydroethanol extract of Lansium domesticum fruit can be used as cosmetic. The dry extract is re-dissolved in propylene glycol to obtain the final product. It is used as a skin care product for skin depigmentation and moisturizing. The
17. Lansionic acid (1-16 from the authors, 17 from PubChem Substance Database)
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recommended dose of the liquid extract is 2 5% (Tilaar, 2007b). STUDIES ON BIOLOGICAL ACTIVITIES In Vitro Studies on Biological Activities From an in vitro studies it was shown that the extract of Lansium domesticum has antioxidant activity against DPPH free radical and anti tyrosinase activity (Vanni, 1990; Shimada, 1992; Tilaar , 2007a; TIlaar, 2007b). Clinical Study on Skin moisturizing and lightening effect This was performed according to Good Clinical Practice on a panel of 30 female volunteers aged 32 52 years old during 4 weeks. Skin moisture content was measured using Corneometer CM 820. Lightening effect was measured using a Mexameter MX 16 and data was statistically evaluated. The result showed that Lansium extract can significantly increase skin moisture content and decrease the skin melanin index (Serup, 1995; Anonymous, 1998a; Anonymous, 1998b; Tilaar, 2007a). Dosage and Safety Dermatological safety evaluation was performed using Repeated Opened Patch Test (ROPT) and SCPT. ROPT showed that L. domesticum extract did not cause any irritation or allergic skin reaction. Single Closed Patch Test (SCPT), showed that concentration of 1% and 3% of extracts did not cause any irritation or allergic skin reaction in all volunteers, while concentration of 5% caused irritation in 1.9% of all subjects. According to the method of HET-CAM (Hens Egg Testing of Chorioallantoic Membrane). Fifty milligrams (50mg) of Lansium extract in a lotion base was applied onto the chorioallantoic membrane and left in contact for 20 seconds. The membrane was then evaluated for 5 minutes for any appearance of hyperemia, hemorrhage, and opacity (Curry, 1991; Serup, 1995; SCCNFP, 2000; Ranti, 2007; Luepke,1985). CONCLUSION Lansium domesticum is an Indonesian plant species that has been successfully grown in certains parts of Latin-America. Several parts of the plant are used in popular medicine but it is more important as an economic crop as the edible fruit is widely eaten fresh as dessert. However it can also be used in cosmetics as we found that its extract has antioxidant
property as well as moisturizing and lightening effects with a good safety profile. REFERENCES
Anonymous. 1998a. Manual Instruction for Corneometer CM 820. Courage + Khazaka electronic GmbH. Germany. Anonymous. 1998b. Manual Instruction for Mexameter MX 16. Courage + Khazaka electronic GmbH. Germany. Curry AS, Getting SD, McEwen GN. 1991. CTFAs Safety Testing Guidelines. The Cosmetic Toiletry, and Fragrance Association. Washington. pp 1-5. Heyne K. 1987. Tumbuhan Berguna Indonesia II. Badan Litbang Departement Kehutanan. Jakarta. pp 11261128. Serup J, Jemec GBE. 1995. Handbook of Non-Invasive Methods and The Skin. CRC Press. London, UK. pp 168-169, 587-589, 598-600. Luepke NP. 1985. Hens Egg Chorioallantoic Membrane Test For Irritation Potential. Food Chem. Toxic. 23 (2):287-291. Morton J. 1987. Langsat. In: Fruits of Warm Climates. Julia F. Marton. Miami. Florida. pp. 201-203. Tilaar M, Wong LW, Ranti AS, Setiawati, E., Wasitaatmadja, SW, Maily, Maily, 2007a, Penelitian Bahan Pencerah dan Pelembab Kulit dari Tanaman Indonesia. Kongres ISFI XV. Jakarta. SCCNFP. 2000. Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation, Brussel. Shimada K, Fujikawa K, Yahara K, and Nakamura T. 1992. Antioxidative Properties of Xanthan on the Autoxidation of Soybean Oil in Cyclodextrin Emulsion. J. Agric. Food Chem. 40. pp 945-948. Sutarjadi, 1992. Tumbuhan Indonesia Sebagai Sumber Obat, Kosmetik dan Jamu. In: Positioning Seminar dan Lokakarya Nasional Etnobotani, Perpustakaan Nasional, Jakarta. Tilaar M, Wong LW, Ranti AS, Wasitaatmadja, SW, Suryaningsih, Junardy FD., Maily, 2007b. In Search of Naturally Derived Whitening agent-Pragmatic approach. Asian Societies of Cosmetic Scientists 8th Conference ASCS 2007. Delivering Science to the Depths of Asian Skin. Singapore. pp 116-117. Vanni A, Gastaldi D, and Giunata G. 1990. Kinetic Investigation on the Double Enzyme Activity of the Tyrosinase Mushroom. Annalli di Chimica. 80. pp 3560. Verheij EWM, and Coronel RE (Ed). 1992. Plant Resources of South-East Asia No.2. Edible Fruits and Nuts. Prosea Foundation. Bogor. Indonesia. pp. 186190. Waggoner WC. 1990. Clinical Safety and Efficacy Testing of Cosmetic. Cosmetic Science and Technology Series, vol.5. Marcel Dekker. Inc. New York. pp.2342. 188
Review of Lansium domesticum Tanaka T, Ishibashi M, Fujimoto H, Okuyama E, Koyano T, Kowithayakorn T, Hoyashi M, Komiyama K, 2002. New Onoceranoid Triterpene Constituents From Lansium domesticum. J. Natural Products. 65 (11). pp 1709-11. MMPND: Multilingual Multiscript Plant Name Database. Sorting Lansium Names. (http://www.plantnames.unimelb.edu.au/Sorting/Lansi um.html) (consulted 29 November 2007). MSC Lanzones. http://msc.edu.ph/cyberfair_entry_03/fruits/lanzones.h tm) (consulted 7 September 2007) Nationalherbarium. Lansium domesticum Correa. (http://www.nationalherbarium.nl/sungaiwain/meliace ae/Lansium_domesticum.htm) (consulted 7 September 2007).
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2008 Los Autores Derechos de Publicacin 2008 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 7 (4), 190 - 198 BLACPMA ISSN 0717 7917 Artculo Original | Original Article
Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora palau) from Argentina.
[Parmetros morfo-histolgicos cuantitativos en la caracterizacin de lemon verbena (Aloysia citriodora palau) de Argentina]
Susana GATTUSO1*, Catalina M. van BAREN2, Alejandra GIL3, Arnaldo BANDONI2; Graciela FERRARO2 y Martha GATTUSO1 1. 2. Ctedra de Botnica, Facultad de Ciencias Bioqumicas y Farmacuticas, Universidad Nacional de Rosario Suipacha 531, S2002LRK, Rosario, Argentina. Ctedra de Farmacognosia-IQUIMEFA. Facultad de Farmacia y Bioqumica.UBA-CONICET. Junn 956, 2 piso. C1113AAD Buenos Aires Argentina. 3. Facultad de Agronoma, Universidad de Buenos Aires, Av. San Martn 4453, (C 1417 DSE) Buenos Aires - Argentina.
*Contacto: sgattuso@fbioyf.unr.edu.ar . Received | Recibido 20/02/2008; Corregido | Corrected 24/04/2008; Aceptado | Accepted: 14/05/2008.
Abstract Lemon verbena, Aloysia citriodora Palau (Verbenaceae) is worldwide used due to the sensorial and medicinal properties of its leaves and essential oil. Micrographic identification of several samples from the most representative growing areas in our country was made in order to define the quality of a typical Argentine lemon verbena. Plant materials were fixed, freehand sectioned and stained with Safranin and Fast Green. Leaves are simples, their blades exhibit anomocytic stomata on the abaxial side, non-glandular and glandular trichomes and dorsiventral mesophyll. The stem, in incipient secondary growth, presents epidermis, angular collenchyma and the vascular bundles which are open collaterally. The morphoanatomical characterization, stomatic index, pallisade ratio and length of stem elements to be used as quantitative parameters of the leaves and stems were determined, in order to contribute to the identification of the plant material. Key words: Aloysia citriodora, lemon verbena, morpho-anatomical, standardization. Resumen Lemon verbena, Aloysia citriodora Palau (Verbenaceae) es usada mundialmente por las propiedades medicinales y sensoriales de sus hojas y aceite esencial. Se realiz la identificacin microgrfica de numerosos especmenes provenientes de reas de crecimiento ms representativas de nuestro pas, con el propsito de definir la calidad de un lemon verbena, tpicamente argentino. El material vegetal fue fijado, seccionado a mano alzada y teido con Safranina-Fast green. Posee hojas simples, sus lminas presentan estomas anomocticos en su hipofilo, pelos glandulares, eglandulares y un mesfilo dorsiventral. El tallo, de escaso crecimiento secundario, posee epidermis uniestrata, colnquima angular y haces colaterales abiertos. La caracterizacin morfoanatmica, el ndice estomtico, la proporcin de empalizada y las magnitudes de los elementos foliares y caulinares que se determinaron, contribuyen a la correcta identificacin del material vegetal. Palabras clave: Aloysia citriodora Palau, estandarizacin, lemon verbena, morfo-anatmico.
Characterization of lemon verbena
Gattuso et al. climatic conditions of their habitat (Molina et al., 2003) our research group has undertaken this study in order to define analytical parameters for the morphohistological characterization of lemon verbena growing in Argentina. These specifications could be used for the pharmacopoeial standardization of this plant. MATERIALS AND METHODS Plant material Fresh and dried plant materials were used for the macroscopic, micro-morphologic and quantitative studies. Samples were obtained from the Herbarium collection of the University of Rosario (UNR), Argentina. ARGENTINA: Santa Fe Province: San Jernimo Dept, Loc. Pto Gaboto, 24/I/2006, Gattuso, S 781 (UNR); 19/II/2006, Gattuso, S 785 (UNR). Rosario Dept, Loc. Rosario, 5/X/1996, Gattuso, S 676 (UNR). Buenos Aires Province, General Madariaga Dept, Loc. Pinamar, 13/I/1995, Gattuso, M 137 (UNR). Further plant materials used in this study, were collected by our group from stake accessions, originally collected from Buenos Aires, Mendoza, Salta and San Luis, and grown in the Experimental Field of the Facultad de Agronoma (Universidad de Buenos Aires). Methods Fresh material consisting of leaves and stems were fixed in F.A.A. (formaldehyde, ethanol, acetic acid, water, 2:10:1:3,5). Dried herbarium material was hydrated by boiling it in water with a few drops of detergent. Transverse sections of the stems measuring 10 m were prepared with a manual microtome and stained with Safranin and Fast Green (Strittmatter, 1979). The leaves were imbedded in paraffin and serial transverse sections, 10 to 12 m thick, were performed and stained with Fast Green Safranin (Ruzin, 1999). Epidermis was obtained by scraping fixed material and then stained with diluted Fast Green and Cresyl Violet (Strittmatter, 1980). Sections were mounted in synthetic balsam. The stems were macerated and leaves were cleared by conventional methods (Boodle, 1916, Strittmatter, 1973). The terminology proposed by Hickey (1979), was used for the description of leaf architecture. The distribution of calcium oxalate crystals was observed with a polarizing microscope (Johansen, 1940, OBrian and McCully, 1981). Original drawings were made with a drawing device. Symbols proposed by 191
INTRODUCTION The genus Aloysia Ortega ex Jussieu belongs to the Verbenaceae. This genus comprises about 30 species, distributed in the American continent from the south of the United States down to Chile and Argentina. Species belonging to this genus are shrubs, rarely trees, with three leaves per node and small flowers usually assembled in racemose inflorescences (Botta, 1978). Aloysia citriodora Palau (Aloysia triphylla (L'Hr.) Britt., Verbena triphylla L'Hrit., Zapania citriodora Lam., Lippia citriodora (Ort.) H.B.K., Lippia triphylla (L'Hrit) Kuntze, Aloysia sleumeri Mold., Verbenaceae) (Botta, 1978) is native to the geographical region that includes territories belonging to Argentina, Uruguay, Paraguay and Chile. It is commonly known as lemon verbena, cedrn, cidron, hierba Luisa or verveine odorante according to the country. The importance of lemon verbena can be inferred from the number of commercial crops present in different European, African and South American countries. Its significance is due to the sensorial and medicinal properties of its leaves and essential oil. The dried plant and its extracts are included in different food and medicinal preparations. Its processed leaves are used to manufacture tea and as an ingredient of alcoholic beverages or other non-alcoholic herbal drinks. It is also often included in phytomedicines as a result of its accepted traditional uses based on reported pharmacological activities such as digestive and diuretic (Arias and Costas, 1948, Duke, 1985, Torrent Mart, 1985), antispasmodic (Arias and Costas, 1948, Torrent Mart, 1976, 1985), carminative and sedative, (Soraru and Bandoni, 1978, Ratera and Ratera, 1980; Wannmacher et al., 1990a, 1990b, Alonso Paz et al., 1992, Mors et al., 2000), antimicrobial and local analgesic (Hieronymus, 1882, Dellacasa and Bandoni, 2003). It is codified by the Argentine Pharmacopoeia (1978), Francaise Pharmacopeia (1996) and Argentine Alimentary Codex. (1969-2007). Furthermore, it is included in the FDA's GRAS list, i.e. the list of food additives which are Generally Regarded As Safe (Newall et al., 1996). Recently a monograph under the name: lemon verbena leaf was included in the European Pharmacopoeia (2007). In view of the commercial value of lemon verbena, and considering that plants can display significant genetic biodiversity and variations in they morpho type due to differences in the edaphic or
Characterization of lemon verbena Metcalfe and Chalk (1972) were used in the diagrams. Statistics ANOVA was performed to determine differences of stomata index and palisade ratio of Aloysia citriodora from different regional origin. Post hoc Scheffes test was applied for multiple comparisons whenever differences were significant. Statistic significance admitted p <0.05. Mean Standard Deviation (SD) were from 5 replications of each species. Data were processed by commercial software STATGRAPHICS Plus-5.0 (2000). RESULTS Macroscopical characteristics Aloysia citriodora Palau is an erect, deciduous perennial shrub which grows to a height of 1 to 3 meters and exudes a powerful lemony scent. The leaves are simple with short petioles. They are narrow, lanceolate and 3 form at each node of the stems. The lamina is pale green, 5 to 10 cm long, entire with a rough margin and slight teeth, having parallel veins at 90 to the mid-rib and flat bristles along the edges. The inflorescence is a slim terminal panicle, the flowers consisting of a small, tubular calyx with 4 long narrow teeth and a pale, greenishmauve corolla divided into 4 acuminate lobes (Fig. 1 A) Plant material of interest Dried leaves. They are thin and fragile, deepgreen in color. Shortly petiolate, ovate-lanceolate, acuminated, measuring 5-10 cm long, with glandular hairs, shortly pilose on the lower surface. Microscopical characteristics Leaf anatomy Foliar architecture The analysis of the foliar architecture shows pinnate, camptodromous, brochidodromous venations (Fig.1, A, B) The primary vein is stout and shows a straight course. Secondary veins are thick and show a straight course. The marginal ultimate venation is looped. Limited, simple and curved venules. Welldeveloped areola randomly arranged, quadrangular, polygonal (Fig 1, C).
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Surface view of epidermis The epidermal cells of the adaxial face show straight, anticlinal walls of 4 to 5 sides (Fig. 1, D). Epidermic abaxial cells present anticlinal walls slightly undulating with anomocytic stomata and striate cuticule around of the stomata (Fig. 1, E; Fig. 2 E). Two types of trichomes are present on both sides of the leaf surfaces. Non glandular trichomes: athe upper epidermis shows numerous conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a rosette of cells at the base, with cystoliths, (Fig.1, D; Fig. 2 F); b- the lower epidermis shows simple, short, unicellular, thickened, cystolithic trichomes, (Fig. 1, E, F). Glandular trichomes: a- with foot simple or compound; stalk, 1 to 2 celled and head unicellular, cell mostly broader than long, outer walls convex, thin, smooth, content scanty and translucent, in both epidermis, more abundant in the lower one, (Fig. 1, E, Fig. 2 E). b- with foot simple, head small, relatively narrow, unicellular, contens dense, commonly persistent. Outer walls slightly convex, thin, smooth. Cuticular vesicle restricted to the apex, persistent or collapsing early, they occur widely and are aggregated in depressions on the lower surface (Fig. 1 E; Fig. 2 E). Cross-sections It is dorsiventraly compressed. Unstratified adaxial epidermis with cuticule thin and smooth, without stomata can be observed. Mesophyll is dorsiventral, with a 2-3 layer of pallisade parenchyma (Fig. 1, I, J; Fig. 2 A). The spongy parenchyma cells are located next to the abaxial epidermis, loosely arranged, with many intercellular spaces between them. The midrib consists of a collateral vascular bundle. The lower epidermis is unstratified and presents stomata in columns (Fig.1, K; Fig. 2 D). Stem anatomy Transverse sections show a circular margin with 6 ribs (Fig. 1 L). The epidermis is unstratified, with stomata which are composed of rectangular, rolled cells, trichomes are scarce. At the level of the ribs and in a subepidermal position, 3-4 layers of angular collenchyma can be observed (Fig. 1 L). Cork usually appears near the phloem (Fig. 1 M). Vascular bundles are open collaterally and are arranged forming a eustele, reinforced by conspicuous sclerenchymatic fibers. . 192
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Figure 1. Aloysia citriodora Palau. A-K leaf. A, vegetative branch. B-F, superficial view of the lamina. B, foliar architecture. C, last marginal venation and areolae. D, adaxial epidermis with conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a resette of cells at the base, with cystoliths. E, abaxial epidermis. F, simple trichomes, slightly verrucose, with cystoliths of calcium carbonate. G-M, cross section. G, glandular trichomes with unicellular head, in depressions on the leaf surface. H, glandular trichomes with unicellular foot and head 1-2 cells. I, schematic representation of the limb. J, detail of the limb indicated in I. K, stomata on columns. L-M, stem in cross section. L, schematic representation of the stem. M, detail of the stem indicated in L. N-R, dissociated cellular elements. N, fibre. O, vessel. P, tracheids. Q, parenchyma cells. R, xilematic parenchyma. S, collenchimatic cells. Scale bars 1 to B. 2 to I, L. 3 to D-H, JK, M-S. 4 to C.
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Figure 2. A-E: Aloysia citriodora Palau. Photomicrographs by light microscope. Transverse sections of leaf. A, main nerve, B, C, D mesophyll, in B, glandular trichomes in depressions on the leaf surface; C, glandular trichomes with unicellular foot and head 1-2 cells, D, stomata on columns. E-F, superficial view of the lamina. E, abaxial epidermis with glandular trichomes. F, adaxial epidermis whith conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a resette of cells at the base, with cystoliths. a, glandular trichomes with unicellular head, in depressions on the leaf surface. b, glandular trichomes with unicellular foot and head 1-2 cells. c, cystoliths. e, epidermis. pp, pallisade parenchyma. s, stomata.
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Tabla 1: Stomatic index and pallisade ratio data of reference plant material, experimental samples and the different materials studied.
Origin of the material Buenos Aires Santa Fe San Luis Mendoza Salta Jujuy Interval for Argentine Material Rancagua (Chile) Talca (Chile)
Stomatic index 5,45 (7,05) 10,20 5,16 (9,23) 13,43 8,33 (10,38) 12,69 8,64 (10,35) 12,09 8,93 (10,73) 13,73 7,27 (9,67) 11,11 5,16 - 13,73 7,14 (8,97) 11,67 5,77 (7,57) 9,26
Pallisade ratio 8,00 (9,31) 10,50 5,50 (6,70) 8,25 9,25 (10,60) 12,25 11,50 (11,81) 12,50 7,50 (8,20) 8,75 10,75 (14,30) 17,25 5,50 - 17,24 8,25 (9,65) 11,75 7,00 (8,40) 10,25
Figure 3: Bar graph of stomatic index and pallisade ratio of Aloysia citriodora samples of different origin. Mean SD. p < 0,05 ().
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Figure 4: Pallisade ratio Aloysia citriodora samples of different origin. Test multiple comparisons. Scheffe (95%). BA, Buenos Aires; JU, Jujuy; ME, Mendoza; RA, Rancagua (Chile); SA, Salta; SF, Santa Fe; SL, San Luis; TA, Talca (Chile).
Gattuso et al. addition, according to this last author, the leaves have a striking aromatic characteristic. The leaf venation, is brochidodromous type and quantitative parameters of vein islet index is 24, 50 - 29,10 - 33,75 (Albrecht et al. 2005). Metcalfe and Chalk (1972) mentioned for the genus Aloysia the occurrence of anomocytic stomata, we could ascertain this feature in this species. Trichomes are epidermal outgrowths of considerable value for taxonomic purposes. In A. citriodora there are unicellular, thick-walled cystolithic trichomes, trichomes arising from rosette of cell at the base, and they are by far the most prevalent and numerous subsessile glandular trichomes with a globular head according to Metcalfe and Chalk (1972) and Barbosa et al., (2001). According to Metcalfe and Chalk (1972) members of the family usually have dorsiventral mesophyll and include several (2-3) layers of pallisade parenchyma, according with the two strata found in this study. The stem organization of A. citriodora observed in this study corresponds to a typical herbaceous pattern (Barbosa et al., 2001, Bonzani et al., 2003). These results together with the quantitative parameters accomplish the morpho-histological characters stated in European Pharmacopeia. CONCLUSION The following diacritical macroscopic and micro morphological characteristics and quantitative parameters were established after the analysis of the studied plant material. The presence of the below described elements is considered to be useful for the botanical identification of the A. citriodora. Morphological characteristics Three leaves per node, simple, entire, lanceolate, petiolate. Stem terete, with ribs. Flower white, small, arranged in a cymose inflorescence. Anatomical characteristic Leaves: Venation pinnate, camptodromous, brochidodromous. Adaxial epidermis with anticlinal thin walls and strains. Abaxial epidermis with anticlinal thin and sinuous walls. Anomocytic stomata, slightly prominent or in column. Non glandular trichomes: a- the upper epidermis there are numerous conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a rosette of cells at the base, with cystoliths, b- the lower epidermis shows simple, short, unicellular, thickened, cystolithic trichomes, Glandular trichomes: a- with foot simple or compound; stalk, 1 196
Dissociated stems The following cellular elements can be observed in this preparation: 632 m long fibers (Fig.1 N); long vessels with appendage and simple terminal plates measuring 360 x 56 m (Fig. 1 O); tracheids measuring 552 m (Fig.1 P); thin walled parenchyma cells (Fig.1 Q); xylem axial parenchyma (Fig.1 R); collenchyma cells (Fig. 1 S). DISCUSSION The correct taxonomical nomenclature of the species was the first feature to harmonize in the search for data that could contribute to the normalization of lemon verbena. Palau (1784) used the binomial Aloysia citrodora in the original identification of the species. However, in most bibliographical references it is recurrently and almost universally named as Aloysia citriodora. We consulted several taxonomists about this situation, among them, Dr. Charlie Jarvis and Dr. Norman Robson (Kew Royal Botanic Gardens), as authorities in taxonomical nomenclature. They both agreed on the use of Aloysia citriodora because it appears to be the result of the correction of the former name to conform the current nomenclatural botanical rules, i.e., if a name is the result of the conjunction of two or more words, in this case, citro and odora, it is necessary to add a link vowel i or o between the Latin or Greek words, respectively. For this reason we assume that Aloysia citriodora Palau is the correct taxonomical nomenclature for this species. The leaf morphology and phyllotaxy of A. citriodora are similar to the description made by Botta (1979). In
Characterization of lemon verbena to 2 celled and head unicellular, cell mostly broader than long. b- with foot simple, head small, relatively narrow, unicellular, content dense, commonly persistent, occur in depressions on the lower surface. Mesophyll dorsiventral, hypostomatic. Quantitative assessments The following values were established for the cellular elements of macerated stems: vessel bordered pits, with a 360 x 56 m appendix, 552 m tracheids and 632 m fibres. The data resulting from the analysis of the stomatic index and pallisade ratio of reference plant material, experimental samples and the different chemotypes studied are summarized in Table 1 grouped according to their origin. Statistical analysis. Stomata index sample data yielded non significant differences among them (Fig.3). Statistical process of palisade ratio in the same samples (Fig.3, Fig. 4), showed that the sample values from Jujuy (JU) was higher, a fact that could be explained by the arid environment, demonstrating that regarding the structure, leaves are the most important organ reflecting the environmental influence. Samples from Mendoza (ME) and San Luis (SL) showed a similar behaviour. The sample from Santa Fe (SF) presented significant differences with the former ones, expressing itself as a mesophyte, since the palisade ratio value correspond to environments with middle temperature and humidity. The remaining samples are the reflection of the edaphically characteristics of the communities in which they develop, therefore, determining a great variety of ecological niches evidenced by intermediate values for each analysed index. REFERENCES
Albrecht RA, Bassols GB, Gurni AA. 2005. Aplicacin de parmetros cuantitativos en el estudio de Aloysia citrodora Palau (Verbenaceae). Acta Farm. Bonaerense 24(2): 239-241. Alonso Paz E, Bassagoda MJ, Ferreira F. 1992. Uso Racional de las Plantas Medicinales. Ed. Fin de Siglo, Montevideo, Uruguay, pp. 42-43. Arias H, Costas F. 1948. Plantas Medicinales. Ed. Biblioteca Nueva, Buenos Aires, Argentina, pp. 3435. Barbosa G, Bonzani N, Filippa E, Lujn M, Morero R, Bugatti M, Decolatti N, Arizar Espinar L. 2001. Atlas Histo-morfolgico de Plantas de Inters Medicinal de Uso Corriente en Argentina. Museo Botnico Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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Crdoba, Serie Especial I, Ed. Graphyon, Crdoba, Argentina, pp. 8-11. Bonzani N, Filippa E, Barbosa G. 2003. Estudio anatmico comparativo de tallo en algunas especie de Verbenaceae. Anales del Instituto de Biologa Universidad Autnoma de Mxico, Serie Botnica 74(1): 31-45. Boodle LA. 1916. A method of macerating fibres. Roy. Bot. Gard. Kew Bull. Misc.Inform. 5: 108-110. Botta S. 1979. Las especies argentinas del gnero Aloysia (Verbenaceae). Darwiniana 22(1-3): 67-108. Cdigo Alimentario Argentino. (1969-2007) : Ed. De la Canal y Asoc, Buenos Aires, Argentina.. Dellacasa E, Bandoni AL. 2003. Hierbaluisa. Aloysia citriodora Palau. Revista de Fitoterapia. 3(1): 19-25. Duke JA. 1985. Handbook of Medicinal Herbs. Ed. CRC Press, Florida, Estados Unidos, p. 33. European Pharmacopoeia 2007. Supplement 5.8, Monograph: Lemon verbena leaf Strasbourg: Council of Europe. Farmacopea Nacional Argentina, VI edicin, 1978, Buenos Aires, Argentina. Francais Pharmacope X Ed. (1996), Maisonneuve, Pars, Francia. Hieronymus J. 1882. Plantas Diafricas de la Flora Argentina. Bo.l Acad. Nac. de Ciencias Crdoba, Tomo IV, pp. 404-405. Hickey J. 1973. Classification of the architecture of Dicotyledons leaves. Am. J. Bot. 60: 17-33. Johansen DA. 1940. Plant microtechnique. Mc Graw-Hill, New York, pp. 184-202. Metcalfe CR, Chalk L. 1972. Anatomy of Dicotyledons. Vol. I Clarendon Press, Oxford, Inglaterra, pp. i-iii. Metcalfe CR, Chalk L. 1972. Anatomy of Dicotyledons. Vol. II. Clarendon Press, Oxford, Inglaterra, pp. 10301041. Molina AC, Viturro CI and Heit C. 2003. I. Estudio qumico de aceites esenciales de Aloysia triphylla. Resmenes del XIV Congreso de la Sociedad Argentina de Investigacin en Qumica Orgnica (SAIQO), Santa Fe, Argentina. 07 al 10 de noviembre de 2003 Mors WB, Rizzini CT, Alvarez P. 2000. Medicinal Plants of Brazil. Reference Publications INC, Algonac, Michigan, Estados Unidos, p. 30. Newall CA, Anderson LA, Phillipson JD. 1996. Herbal Medicines. A Guide for Health-care Professionals. Ed. The Pharmaceutical Press, London OBrian TP, McCully ME. 1981. The study of plant structure, principles and selected methods. Termarcarphi Pty Ltd, Melbourne, Australia, pp. 5, 15, 14. Palau A. 1784. Parte Prctica de Botnica del Caballero Carlos Linneo I. Imprenta Real, Madrid, Espaa. Ratera EL, Ratera MO. 1980. Plantas de la Flora Argentina Empleadas en Medicina Popular. Ed. Hemisferio Sur, Buenos Aires, Argentina, pp. 62-64.
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Ruzin S. 1999. Plant Microtechnique and Microscopy. Ed. Oxford University Press New York, pp. 1-125. Soraru SB and Bandoni AL. 1978. Plantas de la Medicina Popular Argentina. Ed. Albatros, Buenos Aires, Argentina, pp. 107-109. Strittmatter C. 1973. Nueva tcnica de diafanizacin. Bol. Soc. Argent. Bot. 15(1): 126-129. Strittmatter C. 1979. Modificacin de una tcnica de coloracin safranina-fast-green. Bol. Soc. Argent. Bot. 18(3-4): 121-122. Strittmatter C. 1980. Coloracin con Violeta de Cresyl. Bol. Soc. Argent. Bot. 19(1-2): 273-276. Torrent Mart MT. 1976. Algunos aspectos farmacognsticos y farmacodinmicos de Lippia citriodora HBK. Rev. Real Acad. Farm. 14: 39-55. Torrent Mart MT. 1985. Accin farmacolgica de algunas esencias de origen biolgico. Rev. Real Acad. Farm. (3 poca) (1): 43-56. Wannmacher L, Fuchs FD, Paoli CL, Fillman HS, Gianlupi A, Lubiancaneto JF, Hassegawa CY, Guimares FS. 1990a. Plant employed in the treatment of anxiety and Insomnia. I. An Ethnopharmacological Survey in Porto Alegre, Brazil. Fitoterapia 61(5): 445448. Wannmacher L, Fuchs FD, Paol CL, Fillman HS, Gianlupi A, Lubiancaneto JF, Hassegawa, CY, Guimares FS. 1990b. Plant employed in the treatment of anxiety and insomnia. Effect of infusions of Aloysia triphylla on experimental anxiety in normal volunteers. Fitoterapia 61(5): 449-453.
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2008 Los Autores Derechos de Publicacin 2008 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 7 (4), 199 - 201 BLACPMA ISSN 0717 7917 ArtculoOriginal | Original Article
Evaluation of antibacterial activity on different solvent extracts of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae) collected in Venezuela
[Evaluacin de la actividad antibacteriana en extractos de diferentes solventes de Euphorbia caracasana Boiss y Euphorbia cotinifolia L. (Euphorbiaceae) recolectadas en Venezuela]
Janne ROJAS1*, Judith VELASCO2, Antonio MORALES1, Tulia DAZ2, Gina MECCIA 1 1.
2.
Organic Biomolecular Research Group, Research Institute, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mrida, Venezuela. Microbiology and Parasitology Department, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mrida, Venezuela.
*Contacto: janner@ula.ve Recibido | Received 13/02/2008; Aceptado | Accepted 03/04/2008; Online 12/07/2008
Abstract In the present investigation the evaluation of the antibacterial activity of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. has been evaluated against Gram positive and Gram negative bacteria using the disc diffusion agar method. E. caracasana and E. cotinifolia leaves were extracted with isopropyl alcohol. These extracts, after concentration, were fractionated by column chromatography and selected fractions, based on their TLC profiles, tested for antibacterial activity. Fractions eluted with dichloromethane/methanol 1:1 from the column chromatography of E. caracasana extract showed antibacterial activity against S. aureus ATCC 25923 at concentrations of 336 g/mL and 33,6 g/mL (dilution 1:10 of the same extract). Fractions eluted with n-hexane-dichloromethane 1:1 and dichloromethane from the same column, lacked of antibacterial activity. None of the fractions eluted from the column separation of E. cotinifolia extract showed antibacterial activity in any of bacteria tested.
Keywords: Antibacterial activity, Euphorbia caracasana, Euphorbia cotinifolia, Euphorbiaceae, Staphylococcus aureus.
Resumen En la presente investigacin se evalu la actividad antibacteriana de Euphorbia caracasana Boiss y Euphorbia cotinifolia L. contra bacterias Gram positivas y Gram negativas usando el mtodo de difusin en agar con discos de papel. Las hojas de E. caracasana y E. cotinifolia fueron extradas con alcohol isoproplico. Los extractos, luego de concentrados, fueron separados por cromatografa en columna y las fracciones eludas de sta, fueron seleccionadas basandose en el perfil cromatogrfico TLC, y analizadas para determinar su actividad antibacteriana. Solo las fracciones eludas con diclorometano 1:1 de la cromatografa en columna del extracto de E. caracasana, mostraron actividad antibacteriana contra S. aureus ATCC 25923 a las concentraciones de 336 g/mL y 33,6 g/mL (dilucin 1:10 del mismo extracto). Las fracciones eludas con n-hexano-diclorometano 1:1 y diclorometano de la misma columna, no mostraron actividad antibacteriana. Ninguna de las fracciones eludas de la separacin cromatogrfica de E. cotinifolia mostraron actividad antibacteriana en las bacterias ensayadas.
Palabras clave: Actividad Antibacteriana, Euphorbia caracasana, Euphorbia cotinifolia, Euphorbiaceae, Staphylococcus aureus.
Antibacterial activity of E. caracasana Boiss and E. cotinifolia .
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INTRODUCTION Euphorbia genus belongs to the family Euphorbiaceae. This family comprises about 300 genus and 5000 species distributed mainly in America and tropical Africa. (Webster, 1994) The plants of the family Euphorbiaceae contain skin irritating and tumour-promoting diterpenoids, which have tigliane, ingenane, and daphnane skeletons (Evans & Taylor, 1983). Some species are used in folk medicine to treat skin diseases, gonorrhoea, migraines, intestinal parasites, and warts (Singla & Pathak, 1990), In addition, several macrocyclic diterpenoids with antibacterial, anticancer, PGE2inhibitory, anti-multidrug-resistant, prolyl endopeptidase inhibitory, antifeedant, anti-HIV, and analgesic activity have recently been isolated from different Euphorbia species. (Hohmann et al, 2002; Hohmann et al, 2003). They include jatrophane, ingol, myrsinane diterpenoids, leucocyanidol, quercitol, camphol, quercetrin, dihydroellagitannins and dimeric hydrolysable tannins-euphorbins. (Haba et al, 2007; Jasbi, 2006). The antibacterial activity of several Euphorbia species has been evaluated in different occasions. The aqueous extract of Euphorbia hirta, used in traditional medicine for the treatment of dysentery, colic, ulcers, asthma and chronic bronchial infections showed activity against Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus (Hore et al, 2006; Sudhakar et al, 2006). The methanolic and acetone leaf extracts of Euphorbia fusiformis showed growth inhibition of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhii A and Salmonella typhii B. (Natarajan et al, 2005). Ethanolic extract of Euphorbia australis showed activity against Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium (Palambo & Semple, 2001). Similarly the ethanol, acetone and water extracts of Euphorbia fruticosa (Alasbahi et al, 1999) and methanol extracts of Euphorbia macroclada (Darwish et al, 2002) showed inhibitory effects against Staphylococcus aureus. These results may support the popular use of these plants in traditional medicine for the treatment of fever, wound infections, and intestinal disorders. In the present investigation the evaluation of the antibacterial activity of Euphorbia caracasana and Euphorbia cotinifolia has been evaluated against
Gram positive and Gram negative bacteria using the disc diffusion agar method. To the best of our knowledge there are no reports regarding the antibacterial activity of these two species. MATERIALS AND METHODS Plant material The leaves of E. caracasana were collected in March 2005 in sector La Conquista, Pueblo Llano, at 1800 m above sea level and leaves of E. cotinifolia were collected in April 2005 in Sector La Pedregosa, Mrida State at 1520 m. Voucher specimens (JR 19 and JR 20, respectively) were deposited in the Dr Luis E Ruiz T. Herbarium, Faculty of Pharmacy and Biomedical Sciences, University of Los Andes, Venezuela. Extraction and column chromatographic separation The plant material (2.8 kg, E. caracasana and 2.0 kg, E. cotinifolia), dried and powdered, was extracted by soaking in isopropyl alcohol (8 L each) for 3 days at room temperature. After filtration, the extracts were concentrated to dryness (190 g E. caracasana and 171 g E. cotinifolia, respectively) and further fractionated on a column (30 cm x 6 cm) containing silica gel (230-400 mesh). Elution was initially with n-hexane (4 L), followed by mixtures of n-hexanedichloromethane (CH2Cl2) 1:1 (4 L), CH2Cl2 (4 L), CH2Cl2-methanol (MeOH) 1:1 (3 L), and MeOH (3 L) of increasing polarity. Fractions (90), each of 200 mL, were collected and examined by TLC. Selected fractions, based on their TLC profiles, were used for the antibacterial assay. Thin layer chromatography (TLC) TLC analysis was carried out on all the fractions eluted from the column. Oven dried (100oC for 30 min) silica gel 60 F254 (Merck, UK) layers, 250 m thick were utilized and different mixtures of nhexane, CH2Cl2, and MeOH were used, depending on the polarity of the eluted fractions to be analyzed. The plates were sprayed with sulphuric acid diluted to 10 % v/v with water and heating in an oven at 80oC for 10 min. Bacterial strains The microorganisms used were Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25992),
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Klebsiella pneumoniae (ATCC 23357) Pseudomonas aeruginosa (ATCC 27853).
and
Antibacterial method Antibacterial activity was determined using the disc diffusion agar method described by Velasco et al, (2005). The strains were maintained in agar conservation at room temperature. Each bacterial inoculum was incubated in 2.5 mL Meller-Hinton broth at 37 C for 18 hours. The bacterial inoculum was diluted in sterile 0.85 % saline to obtain turbidity visually comparable to a McFarland N 0.5 standard (106-8 CFU/mL). Every inoculum was spread over plates containing Meller-Hinton agar. Paper filter discs (6 mm) saturated with 20 L of every selected fraction, eluted from the column chromatography with the following solvent mixtures (nhexane/CH2Cl2 1:1, CH2Cl2 and CH2Cl2/MeOH 1:1) as well as dilutions 1:10, using the same solvents, were placed over the plates. These were preincubated at 4 C for 18 h and finally incubated at 37 C for 24 h. The inhibitory zone around the disc was measured and expressed in mm. A positive control was also assayed to check the sensitivity of the tested organisms using the following antibiotics: Ampicillin-sulbactam (10 g/10 g) for S. aureus (inhibition zone 50 mm), Vancomycin (30 g) for E. faecalis (inhibition zone 30 mm), Gentamicin (10 g) for E. coli (inhibition zone 42 mm), Cefotaxime (30 g) for K. pneumoniae (inhibition zone 40 mm) and Cefoperazone (75 g) for P. aeruginosa (inhibition zone 36 mm). A negative control was also included in the test using a filter paper disc saturated with hexane, CH2Cl2 and MeOH to check the possible activity of these solvents against the bacteria assayed. The experiments were repeated at least twice.
column, lacked of antibacterial activity. None of the fractions eluted from the column separation of E. cotinifolia extract showed antibacterial activity in any of the bacteria tested. Antibacterial activity has been reported for different Euphorbia species (Annapurna et al, 2004; Gonalves et al, 2006; Sudhakar et al, 2006). The ethanol extract of E. fusiformis showed antibacterial activity against S. aureus (Natarajan et al, 2005). Similar studies carried out with methanol extract of E. hirta and E. tirucalli revealed antibacterial activity against Staphylococcus epidermidis (Parekh et al, 2005). In recent years, multi-resistant bacterial strains have increased dramatically, and thus the treatment of several infections has become very difficult, reducing the therapeutic options. (Velazco et al, 2002; Velsquez et al, 2004; Hsueh et al, 2004; Alavarez et al, 2006). However, the antibacterial activity of E. caracasana semipurified extract against S. aureus, offers an option to the pharmaceutical industry of new natural medicine sources with activity against these bacterial strains that represent an important public health problem. Acknowledgments The authors would like to acknowledge Consejo de Desarrollo Cientifico, Humanistico y Tecnolgico (CDCHT) Mrida, Venezuela for the financial support (FA-304-03-08-A) of this investigation. REFERENCES
Alasbahi R, Safiyeva S, Craker L. 1999. Antimicrobial activity of some Yemeni medicinal plants. J Herbs Spices Med Plants. 6:75-83. Annapurna J, Chowdary I, Lalitha G, Ramakrishna S, Iyengar D. 2004. Antimicrobial Activity of Euphorbia nivulia leaf extract. Pharm Biol. 42: 91-93. Alvarez C, Cortes J, Arango A, Correa C, Leal A, Grebo. 2006. Resistencia antimicrobiana en unidades de cuidado intensivo de Bogot, Colombia, 2001-2003. Salud Pblica. 8: 86-101. Darwish R, Aburjai T, Al-Khalil S, Mahafzah A. 2002. Screening of antibiotic resistant inhibitors form local plant material against two strains of Staphylococcus aureus. J Ethnopharmacol. 79: 359-364. Evans F, Taylor S. 1983. Pro-inflammatory, tumor promoting and antitumor diterpene of the plant families Euphorbiaceae and Thymelaeaceae. In: Herz W, Grisebach H, Kirby G. (Eds.), Progress in the Chemistry of Organic Natural Products, 44. SpringerVerlag, New York, pp. 1-99.
Results and discussion

E. caracasana and E. cotinifolia leaves were extracted with isopropyl alcohol. These extracts, after concentration, were fractionated by column chromatography and selected fractions, based on their TLC profiles, tested for antibacterial activity. Fractions eluted with CH2Cl2/MeOH 1:1 from the column chromatography of E. caracasana extract showed antibacterial activity against S. aureus ATCC 25923 at concentrations of 336 g/mL (inhibition zone 10 mm) and 33,6 g/mL (dilution 1:10 of the same extract, inhibition zone 7 mm). Fractions eluted with n-hexane-CH2Cl2 1:1 and CH2Cl2 from the same
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Antibacterial activity of E. caracasana Boiss and E.cotinifolia . Gonalves B, Ramos V, Soares D, Mashrouah C, Silveira D. 2006. Antimicrobial and cytotoxic activities screening of some Brazilian medicinal plants used in Governador Valadares district. Rev Bras Cienc Farm. 42: 195-202. Haba H, Lavaud C, Harkat H, Alabdul A, Marcourt L, Benkhaled M. 2007. Diterpenoids and triterpenoids from Euphorbia guyoniana. Phytochemistry. 68: 1255-1260 Hohmann J, Molnar J, Redei D, Evanics F, Forgo P, Kalman A, Argay G, Szabo P. 2002. Discovery and biological evaluation of a new family of potent modulators of multidrug resistance: reversal of MDR of mouse lymphoma cells by new natural jatrophane diterpenoids isolated from Euphorbia species. J Med Chem, 45: 2425-2431. Hohmann J, Redei D, Forgo P, Molnar J, Dombi G, Zorig T. 2003. Jatrophane diterpenoids from Euphorbia mongolica as modulators of the multidrug resistance of L5128 mouse lymphoma cells. J Nat Prod. 66: 976-979. Hore S, Ahuja V, Mehta G, Kumar P, Pandey S, Ahmad A. 2006. Effect of aqueous Euphorbia hirta leaf extract on gastrointestinal motility. Fitoterapia. 77: 35-38. Hsueh P, Teng L, Chen W, Pan H, Chen M, Chang S. 2004. Increasing prevalence of methicillin-resistant Staphylococcus aureus causing nosocomial infections at a University Hospital in Taiwan from 1986 to 2001. Antimicrob Agents Chemother. 48: 1361-1364. Jassbi A. 2006. Chemistry and biological activity of secondary metabolites in Euphorbia from Iran. Phytochemistry. 67: 1977-1984 Natarajan D, Britto S, Srinivasan K, Nagamurugan N, Mohanasundari C, Perumal G. 2005. Anti-bacterial activity of Euphorbia fusiformis. A rare medicinal herb. J Ethnopharmacol. 102: 123-126 Singla A, Pathak K. 1990. Phytoconstituents of Euphorbia species. Fitoterapia. 61: 483516. Sudhakar M, Rao Ch, Rao P, Raju D, Venkateswarlu Y. 2006. Antimicrobial activity of Caesalpinia pulcherrima, Euphorbia hirta and Asystasia gangeticum. Fitoterapia. 77: 378-380. Palambo E, Semple S. 2001. Antibacterial activity of traditional medicinal plants. J Ethnopharmacol. 77: 151-157 Parekh J, Jadeja D, Chanda S. 2005. Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turk J Biol. 29: 203210. Velasco J, Contreras E, Buitrago D, Velazco E. 2005. Efecto antibacteriano de Virola sebifera sobre Staphylococcus aureus resistente a Meticilina. Ciencia. 13: 411-415. Velazco E, Nieves B, Araque M, Calderas Z. 2002. Epidemiologa de infecciones nosocomiales por Staphylococcus aureus en una unidad de alto riesgo neonatal. Enfer Infecc Microbiol Clin. 20: 321-325. Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (x) 2008
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Velazquez-Meza M, Aires de Sousa M, Echaniz-Aviles G, Solrzano-Santos F, Miranda-Novales G, SilvaSanchez J, de Lencastre H. 2004. Surveillance of Methicillin-Resistant Staphylococcus aureus in a Pediatric Hospital in Mexico City during a 7-Year Period (1997 to 2003): Clonal evolution and impact of infection control. J Clin Microbiol. 42: 3877-3880. Webster G. 1994. Systematic of the Euphorbiaceae: Introduction. Annal Miss Bot Gard. 81: 33-144.
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Antifungal Activity of the Essential Oils of Two Verbenaceae: Lantana achyranthifolia and Lippia graveolens of Zapotitln de las Salinas, Puebla (Mxico)
[Actividad antifngica del aceite esencial de dos Verbenaceae: Lantana achyranthifolia and Lippia graveolens de Zapotitln de las Salinas, Puebla (Mxico)]
Tzasna HERNNDEZ *, Margarita CANALES, Ana Maria GARCA , ngel DURAN, Samuel MERZ, Patricia DVILA, J. Guillermo VILA
1.
Laboratorio de Fitoqumica, Unidad de Biologa, Tecnologa y Prototipos (UBIPRO), Facultad de Estudios Superiores Iztacala (FES Iztacala) Universidad Nacional Autnoma de Mxico (UNAM).
*Contact: tzasna@servidor.unam.mx Submitted on 19/04/08 Revised on 18/05/2008; Corrected on 20/05/2008; Accepted on 21/05/2008; Online 06/06/2008
Abstract The present work documents the antifungal activity of the essentials oils of Lantana achyranthifolia Desf. and Lippia graveolens H.B.K. The essential oils of aerial parts of L. achyranthifolia and L. graveolens were obtained by steam distillation and examined by GC and GC-MS. Antifungal activity of the essentials oils was evaluated towards five fungal strains by the disk-diffusion method. The estimation of the Medium Inhibitory Concentration (IC50) was carried out by the inhibition of fungal growth method. The oils presented antifungal activity against five fungal strains; the essential oil of L. graveolens presented higher antifungal activity (IC50= 1090 g/mL) than L. achyranthifolia (IC50= 100180 g/mL). The present study validates the use in the folk medicine of L. achyranthifolia and L. graveolens for treating dermatological diseases and could be an option for the biological control of phytopathogen strains.
Keywords: Antifungal activity, Essential oil, Lantana achyranthifolia, Lippia graveolens.
Resumen Se documenta la actividad antifngica de los aceites esenciales de Lantana achyranthifolia y Lippia graveolens. Los aceites esenciales de la parte area de L. achyranthifolia y L. graveolens fueron obtenidos mediante arrastre de vapor y examinados por GC y GC-MS. La actividad antifngica fue evaluada frente a cinco cepas de hongos con el mtodo de difusin en agar. La concentracin inhibitoria media (IC50) se determin mediante el mtodo de inhibicin del crecimiento radial. Los aceites exhibieron actividad antifngica contra las cepas desafiadas. El aceite esencial de L. graveolens present mayor actividad (IC50= 1090 g/mL) que L. achyranthifolia (IC50= 100180 g/mL). Con el presente estudio se valida el uso de L. achyranthifolia y L. graveolens en la medicina tradicional para el tratamiento de enfermedades dermatolgicas y podra ser una buena opcin para el control biolgico de cepas fitopatgenas.
Palabras clave: Actividad antifngica, Aceite esencial, Lantana achyranthifolia, Lippia graveolens.
INTRODUCTION Fungi are ubiquitous in the environment, and infections due to fungal pathogens have become more frequent. With the rise of HIV cases and with the introduction of immunosuppresive agents, opportunistic fungal pathogens have become common. As a result, antifungal therapy is playing a greater role in health care and the screening of traditional plants in search of novel antifungals is now more frequently performed (Webster et al., 2008).
Among plant pathogens, fungi are the main pathogens and cause many diseases of plants. Mexico produces a great quantity of grains that are an important part of the Mexican diet. Several fungal species are responsible for important plant diseases associated with marked economic losses, wheat and corn are the cereals more susceptible (grains and seeds) to the attack of these organisms that degrade their quality in diverse forms (Matos et al., 1999; Tequida-Meneses et al., 2002). Traditional Mexican medicine has used a wide variety of plants to treat many ailments, particularly
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infectious diseases which are particularly common in rural areas of the country (Viseca, 1976). The information directly obtained from the people is a good option to preserve and improve the human health in geographically and culturally isolated communities (Argueta and Cano, 1994; McGaw et al., 2000). Lantana achyranthifolia and Lippia graveolens are shrubs distributed in the Americas (Argueta & Cano, 1994). Several species of the genus Lantana and Lippia are used in folk medicine in gastrointestinal, dermatological and respiratory affections (Barre et al., 1997; Argueta and Cano, 1994; Ghisalberti, 2000; Pascual et al., 2001; Hernandez et al., 2003). Several reports on the composition and antibacterial activity of the essentials oils from L. achyranthifolia (Deena and Thoppil, 2000; Hernandez et al., 2005) and L. graveolens (Compadre et al., 1987; Dominguez et al., 1989; Vernin et al., 2001; Salgueiro et al., 2003), can be found in the literature; therefore, there are not report about the antifungal activity of these species. Thus, the aim of this work was to document the antifungal activity of the essentials oils of L. achyranthifolia and L. graveolens to validate their use in the folk medicine and in the biological control of phytopathogens strains. MATERIALS AND METHODS Plant material Aerial parts of L. achyranthifolia and L. graveolens were collected in Zapotitln de las Salinas, Puebla (July 2001) and were identified by Dra. Edith Lpez Villafranco. Voucher specimens were deposited in the herbarium IZTA of the Facultad de Estudios Superiores Iztacala, Universidad Nacional Autnoma de Mxico (Vouchers n 26472 and 26474). Isolation of the essential oil The essentials oils were obtained by steam distillation (1 Kg of fresh plant) during 4 h in a Cleavenger-type apparatus (yield 0.34% v/w, d25 = 0.90 g/mL for L. achyranthifolia and 1.15% v/w, d25 = 0.93 g/mL for L. graveolens), and stored at 4C until tested and analyzed. GC-MS Analysis Conditions The analysis of the essentials oils was performed using a Hewlett Packard 5890-II gas chromatograph
equipped with a DB WAX column (30 m x 0.32 mm). The temperature of the column was programmed from 80 C to 220 C at 8C/min. The injector and detector temperatures were 225 C. The gas carrier was He, at a flow rate of 1 mL/min. Peak areas were measured by electronic integration. The relative amount of the individual components was based on the peak areas. GC-MS analysis was performed on a Jeol AX50HA using a DB Wax (30 m x 0.32 mm) capillary column. The temperature of both, column and injector was the same used at GC. Mass spectra were recorded at 70 eV. The oil components were identified by comparison of their retention indices and mass spectra with the NIST/EPA/NIH Mass Spectral Library (the results were compared with previous reports) (Salgueiro et al., 2003; Hernandez et al., 2005). Fungal Strains Five fungal pathogens were used: Fusarium sporotrichum ATCC NRLL 3299, the strains of Aspergillus niger, Trichophyton mentagrophytes, Fusarium moniliforme were donated by Dr. Cesar Flores (Laboratory of Plant Physiology of UBIPRO, FES Iztacala). In addition, the strain of Rhyzoctonia solani was donated by Dr. Raul Rodriguez (INIFAPTexcoco). The stock culture was maintained on Czapek Dox Agar (Sigma). Antifungal Activity The assay of antifungal activity was carried out in Petri dishes containing Czapek Dox agar (20 mL). After the mycelial colony had developed, sterile blank paper disks (5 mm diameter) were placed at 0.5 cm away from the rim of the mycelial colony. An aliquot of the essential oil (1 L containing 0.90 mg from L. achyranthifolia and 0.93 mg from L. graveolens) was added to the disks. The Petri dishes were incubated at 23 C for 72 h, until mycelial growth had enveloped. Disks containing samples had formed crescents of inhibition were consided with antifungal activity (Ye et al., 1999). For the quantitative assays, ten doses of each essential oil (2.0, 1.5, 1.0, 0.75, 0.5, 0.25, 0.12, 0.06, 0.03, 0.01 mg/mL) were added to Czapek Dox Agar (5 mL) at 45 C, rapidly mixed and poured into 6 cm Petri dishes. After the agar had cooled down to room temperature, a small amount (1 x 1 mm) of mycelia was inoculated. After incubation at 23 C for 72 h, the area of the mycelial colony was measured and the
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inhibition of fungal growth and hence, the Medium Inhibitory Concentration (IC50) was determined. Ketoconazole was used as reference and appropriate controls with no essential oil were used. Each experiment was repeated three times. Statistical analysis All experiments were performed in triplicate. The mean and standard deviation of the three experiments were determined. Statistical analysis of the differences between mean values obtained for experimental groups was done by an analysis of variance (ANOVA multifactorial model), where pvalues of 0.001 or less were considered statistically significant. The IC50 values were calculated by logarithmic model using the Microsoft Excel program. RESULTS As shown in Table 1, seventeen compounds of the essential oil of L. achyranthifolia were identified by GC/MS analysis representing 82.02%. The main compounds showing concentrations higher than 5% as percentage peak area were the monoterpenes: carvacrol (30.64%), 1,8-cineole (5.03%), and the sesquiterpenes: isocaryophyllene (10.73%), bisabolol (11.23%) and -bisabolene (5.68%). For L. graveolens nine compounds from the essential oil were identified by GC/MS analysis representing 94.58%. The main compounds, with concentrations higher than 5% as percentage peak area, were the monoterpenes: carvacrol (37.84%), -terpinyl acetate (22.35%), m-cymene (20.42%), and thymol (6.72%). DISCUSSION The essential oil of L. achyranthifolia (Table 1) is constituted mainly by sesquiterpenes. The major components were carvacrol (30.64%), -bisabolol (11.23%) and isocaryophyllene (10.73%) (Hernandez et al, 2005). On the other hand, the essential oil of L. graveolens (Table 1) is constituted mainly by monoterpenes and the main components were carvacrol (37.84%), -terpinyl acetate (22.35%), mcymene (20.42%) and thymol (6.72%).
Table 1. Composition of essentials oils of L. achyranthifolia and L. graveolens. L. achyranthifolia L. graveolens RT (min) % RT (min) % Thujene 4.51 1.03 6.13 2.54 -pinene m-cymene 7.30 20.42 1,8-Cineole 7.86 5.03 8.52 0.74 8.16 22.35 -terpinyl acetate Linalool 9.69 1.26 8.79 0.26 Camphor 10.96 0.49 Terpinen-4-ol 11.83 0.72 Bornyl acetate 14.50 0.56 Carvacrol 15.38 30.64 13.98 37.84 Thymol 15.05 6.72 Cadina-4(5),10(14)-dieno 16.79 3.38 Guaia-1(10),11(12)-dieno 17.15 2.76 Isocaryophyllene 18.01 10.73 15.96 2.18 Humulene 18.71 3.78 16.60 1.24 Aristol-1(10)-eno 18.83 0.65 19.68 5.68 -Bisabolene 19.88 11.23 -Bisabolol 20.11 2.77 -Cadinene Himachalene 20.38 1.17 Cedrene 25.67 0.43 Total 82.02 94.58 Compounds are listed considering the order of elution from a DB WAX column. RT, retention time. Compounds
The biological activity results are presented in Table 2. All the tested strains were sensitive to the essentials oils. There are significant differences between the fungal strains (F = 249.8, P < 0.001), the concentration (F= 7928.8, P < 0.001), and plant specie (F= 4565.0, P < 0.001). The more sensitive strains were F. moniliforme, to L. achyranthifolia and R. solani, to L. graveolens.
Table 2. Antifungal activity (IC50) of Lantana achyranthifolia and Lippia graveolens. Strain Controls (g/mL)
Ketoconazole Aspergillus niger Fusarium moniliforme Fusarium sporotrichum Trichophyton mentagrophytes Rhyzoctonia solani
Essential oil (g/mL)

L. achyranthifolia L. graveolens
672.0 0.2 0.1 0.1 0.2
180 100 130 110 140
50 50 90 20 10
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The essentials oils of L. achyranthifolia and L. graveolens presented antifungal activity against all the tested strains (Table 2). Lippia graveolens presented higher antifungal activity (IC50 = 10-90 g/mL), than L. achyranthifolia (IC50 = 100-180 g/mL). This result probably is due the existent differences of components and concentrations of the active compounds presenting in the essentials oils of both species. In fact, the observed antifungal activity can be attributed to the presence of some components such as carvacrol, -terpinyl acetate, cymene, thymol, pinene, linalool, etc., which are already known to exhibit antimicrobial activity (Knobloch et al, 1985; Juven et al, 1994; Harborne and Williams, 1995; Cimanga et al, 2002). It was observed that F. moniliforme for L. achyranthifolia and R. solani for L. graveolens were the more sensitive strains to the essentials oils and extract (IC50= 100 g/mL, 10 g/mL respectively). Aspergillus niger turned out to be more sensitive to the essential oil of L. achyranthifolia (IC50 = 180 g/mL) and L. graveolens (IC50 = 50 g/mL), than the positive control (ketoconazole IC50 = 612 g/mL). The observed differences (yield, composition and concentrations of each compound presented by the essential oil samples) among our L. graveolens sample and other L. graveolens samples previously reported (from different geographic origin), should maybe due to factors such as climate, time of collection, mode of extraction, etc. (Compadre et al, 1987; Dominguez et al, 1989; Vernin et al, 2001; Cimanga et al, 2002). CONCLUSION The present study confirms the use of L. achyranthifolia and L. graveolens in the folk medicine to treat dermatological diseases. In addition, could be an alternative way for the biological control of phytopathogen strains like Aspergillus, Fusarium and Rhyzoctonia strains. Acknowledgments The authors are grateful to Roco Serrano and Edith Lpez Villafranco for their technical assistance and to Javier Prez and Luis Velasco for their support on gas-mass measurement. This research has been supported by UNAM-PAPCA (2007-2008).
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Argueta VA, Cano AJ. 1994. Atlas de las Plantas de la Medicina Tradicional Mexicana. Mxico, Instituto Nacional Indigenista pp. 15-34, 804, 1073-1074. Barre JT, Borden BF, Coll JC, De Jess J, De la Fuente VE, Janairo GC, Ragasa CY. 1997. A bioactive triterpene from Lantana camara. Phytochem. 45:321324. Cimanga K, Kambu K, Tona L, Apers S, De Bruyne T, Hermans N, Tott J, Pieters L, Vlietinck AJ. 2002. Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J. Ethnopharmacol. 79:213-220. Compadre CM, Hussain RA, Leon I, Enriquez RG. 1987. Volatile constituents of Montanoa tomentosa and Lippia graveolens. Planta Med. 53:495-496. Deena MJ, Thoppil JE. 2000. Antimicrobial activity of the essential oil of Lantana. Fitoterapia. 71:453-455. Dominguez XA, Sanchez VH, Surez M, Baldas JH, Gonzlez MR. 1989. Chemical constituents of Lippia graveolens. Planta Med. 55:208. Ghisalberti EL. 2000. Lantana camara L. (Verbenaceae). Fitoterapia. 71:467-486. Harborne JB, Williams CA. 1995. Anthocyanins and other flavonoids. Nat. Prod. Rep. 7:639-657. Hernandez T, Canales M, Avila JG, Duran A, Caballero J, Romo de Vivar A, Lira R. 2003. Ethnobotany and antibacterial activity of some plants used in tradicional medicine of Zapotitln de las Salinas, Puebla (Mxico). J. Ethnopharmacol. 88:181-188. Hernandez T, Canales M, Avila JG, Garca AM, Martnez A, Caballero J, Romo de Vivar A, Lira R. 2005. Composition and antibacterial activity of essential oil of Lantana achyranthifolia Desf. (Verbenaceae). J. Ethnopharmacol. 96:551-554. Juven BJ, Kanner J, Schved F, Weisslowicz H. 1994. Factors that interact with the antibacterial action of thyme essential oil and its active constituents. J. Applied Bacteriol. 76:626-631. Knobloch L, Weigand H, Weis N, Schwarn HM, Vigenschow H. 1985. Action of terpenoids on energy metabolism. In: Progress in Essential Oil Research. USA, Eds.: Ernst-Joachim Brunke Editor, Walter de Gruyter. pp. 429-448. Matos OC, Baeta J, Silva MJ, Pinto-Ricardo CP. 1999. Sensitivity of Fusarium strains to Chelidonium majus L. extracts. J. Ethnopharmacol. 66:151-158. McGaw LJ, Jager AC, van Staden J. 2000. Antibacterial, anthelmintic and antiamoebic activity in South African medicinal plants. J. Ethnopharmacol. 72:247263. Pascual ME, Slowing K, Carretero E, Snchez MD, Villar A. 2001. Lippia: Traditional uses, chemistry and pharmacology: A review. J. Ethnopharmacol. 76:201214. 205
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The use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia, Brazil
[La utilizacin de hierbas medicinales por pacientes con SIDA del Hospital Universitario de Brasilia, Brasil]
Paloma M. SALES*, Patricia M de SOUSA, Celeste A. da SILVEIRA, Dmaris SILVEIRA*
1.
Faculdade de Cincias da Sade Universidade de Braslia. Asa Norte, Braslia, Brazil. CEP: 70910-900.
*Contacto: palomasalles@yahoo.com.br; damaris@unb.br Recibido | Received18/03/08; Aceptado | Accepted 02/06/2008; Online 12/07/2008
Abstract An epidemiological study was carried out to verify the use of medicinal plants and herbal medicines by HIV-infected patients attended at Hospital Universitrio de Braslia (HUB), and to evaluate the potential pharmacological interactions between prescribed antiretroviral medicines and such herbal preparations. A questionnaire was elaborated containing questions on the socio-demographic characteristics, time of diagnosis, how the patient discovered his disease, time of treatment with antiretroviral medicines, and the most frequent adverse effects, according to the patient. From the 128 interviewed patients, 52.3% claimed to be medicinal plants and/or herbal medicines users. Among patients using medicinal plant and/or herbal medicines, 77% did not inform the medical staff about this consumption practice. The results were higher than others similar reports. And several of the mentioned medicinal plants/ herbal medicine can potentially present important pharmacological interactions with anti-retroviral pharmacotherapy usually prescribed to the interviewed patients.
Keywords: AIDS, medicinal plants, herbal medicine, drug-herb interaction, ethnomedicine, anti-retroviral therapy.
Resumen Un estudio epidemiolgico fue realizado con el objetivo de evaluar la utilizacin y potencial interaccin farmacolgica entre hierbas/plantas medicinales y frmacos antiretrovirales prescritos a pacientes infectados con el VIH en el Hospital Universitario de Brasilia (HUB). Un cuestionario fue elaborado abordando preguntas sobre las caractersticas sociodemogrficas, el tiempo de diagnstico, como los pacientes se enteraron de sus enfermedades, el tiempo de tratamiento con los frmacos antiretrovirales, y los efectos adversos ms frecuentes, de acuerdo a que informaran los pacientes. De un total de 128 pacientes entrevistados, 52.3 % informaron utilizar plantas e/o hierbas medicinales. Entre los pacientes utilizando plantas medicinales o hierbas, 77 % no informaron al equipo medico acerca de sus prcticas del uso de plantas/hierbas medicinales, porcentual este elevado se comparado a otros estudios. Muchas de las plantas/hierbas medicinales pueden potencialmente presentar importantes interacciones farmacolgicas con la farmacoterapia antiretroviral usualmente prescrita a los pacientes entrevistados.
Palabras clave: SIDA, plantas medicinales, hierbas medicinales, interaccin frmaco-planta, etnomedicina, terapia antiretroviral.
INTRODUCTION A significant part of the traditional therapies used by communities, mainly from developing countries involves the use of plant extracts and their active principles(Farnsworth et al., 1985; Kong et al., 2003). A research carried out in the United States (2002), detected a prevalence of 18.6% in the utilization of medicinal plants in alternative medical treatments, and it is 6.8% higher than 1997 (Tindle et al., 2005). In fact, the Organic Trade Association (OTA), in conjunction with Nutrition Business Journal, recorded United States sales of organic products at $14.7 billion and growing at 17% annually (Starling, 2006). A study performed in Europe involving cancer patients showed that the frequent use of medicinal
plants was the most mentioned alternative treatment (Molassiotis et al., 2005). Similar results were found in Buenos Aires, with a percentage of 37.4% of utilization by the group researched (Franco and Pecci, 2003). According to the Brazilian Phytotherapy Industry Association (ABIFISA), 82% of the Brazilian people use medicinal plant-based products, in agreement with the World Health Organization (WHO) data; and this consumption is based on little or absent scientific corroboration in relation to efficiency, safety and toxicity of the herbs (ABIFISA, 2002; Veiga et al., 2005). However, the defenders of the free and uncontrolled use of medicinal plants claim that they have already been tested and ratified by the long history of the human, and these facts make them
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safe, effective and without significant side effects, common to synthesized products (De Smet, 2002, 2004; Ernst, 2004, 2006; De Smet, 2007). Thus, the apparent inoffensive image of herbal remedies encourages self-medication. The phytomedicine market is responsible for a yearly turnover of R$ 1 billion in all its productive chain and employs over than 100 thousand people in Brazil (ABIFISA, 2002). Studies have indicated that patients with chronic diseases including those infected with the human immunodeficiency virus (HIV) make use of medicinal plants or herbal remedies to improve their quality of life and increasing their life expectancy (Ness et al., 1999; Duggan et al., 2001; Colebunders et al., 2003; Sugimoto et al., 2005; Taylor et al., 2006; Sharma et al., 2006). However, considering the large variety of medicines used in the anti-retroviral therapy, such combination would increase the possibility for the occurrence of drug-medicinal plant interactions that could promote reduction of the therapeutic effects and/or increase the anti-retroviral toxicity, as well as, the reduction on therapeutic effects and/or increase on the medicinal plants or herbal medicine toxicity. The aim of this study was verify the prevalence of medicinal plants and herbal medicines used by HIV-infected patients attended at Hospital Universitrio de Braslia (HUB) and evaluate the potential pharmacological interactions between prescribed antiretroviral medicines and medicinal plants/herbal medicine. MATERIALS AND METHODS From a total of 199 patients with AIDS (Acquired Immunodeficiency Syndrome) attended at the Ambulatrio do Hospital Universitrio de Braslia (HUB) making regular use of anti-retroviral medicines from December 2002 to March 2004, a sample composed of 128 patients was calculated with confidence interval (CI) of 95%. A transversal-type analytical epidemiological study was conducted to obtain data on the sociodemographic and clinical-epidemiological characteristics as well as information about the use of medicinal plants and/or herbal medicine by the participants of this survey. The statistical strategy was based on the analysis of variance (ANOVA) and in the chi-squared test. The results were considered as significant when p<0.05. The analysis of data was performed by using the Statistical Program for Social Sciences (SPSS) version 10.0.
The inclusion criteria were: age (not younger than 18 years of age); HIV infected; use of anti-retroviral drugs; ambulatory treatment. This research was previously submitted to approval from the Ethics Research Committee from the Faculdade de Cincias da Sade, Universidade de Braslia. The following background characteristics were used in the analysis: age, gender, marital status, educational level, employment situation, social interaction, monthly familiar income and address, time of diagnosis, how the patient discovered his disease, time of treatment with antiretroviral medicines, the pharmacotherapy used and the most frequent adverse effects, according to the patient. About medicinal plants and/or herbal medicine, the following aspects were investigated: herbal medicine or medicinal plant used, form and time of utilization, purchase site, reasons and outcomes of the utilization and information to the medical staff in relation of this utilization.
Table 1. Socio-demographic characteristics of the 67 HIVinfected patients that reported the use of medicinal plants and/or phytomedicine Parameters Average age ( SD)* Male/ Female Number (%) 38.8 (9.49) 44 (65.67)/ 23 (34.33)
Marital status Singles 38 (56.71) Married or live matrimonially 22 (32.83) Divorced/widowered 7 (10.5) Educational level Illiterate 2 (3.0) From 1 to 4 years of school 11 (16.24) From 5 to 8 years of school 26 (38.8) Graduated from High-School 23 (34.33) Graduated from College 5 (7.5) Monthly familiar income** < 1 minimum wage 1 (1.52) Between 1 and 5 minimum wages 52 (78.79) Between 6 and 10 minimum wages 7 (10.61) > 10 minimum wages 6 (9.1) Social interaction Family 53 (79.09) Alone 10 (14.93) Other (s) or in philanthropic 4 (6.0) institution Residence area Urban 65 (97) Rural 2 (3) * SD Standard Deviation ** Minimum wage in 03/01/2004 = US$100.00; one interviewed refused to inform the monthly familiar income.
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Table 2 Reasons for the use of medicinal plants and/or phytomedicine* Reasons for the use of the Medicinal Plant/Herbal remedy Gastrointestinal disturbances
Pleasure or personal satisfaction
N (%) 24 (38.1) 18 (28.6) 8 (12.7) 6 (9.5) 3 (4.8) 2 (3.2) 1 (1.6) 1 (1.6)
Pain and inflammation control or colds Increase on the immunological response Sedative and/or sleep inducer Gastrointestinal disturbances and personal satisfaction Weight reduction Sedative and/or sleep inducer and due to gastrointestinal disturbances *Some patients mentioned more than one reason.
Some interviewed reported the use of more than one medicinal plant or herbal medicine. In addition, the medical staff was inquired with reference to their knowledge about properties of the herbal medicine, the frequency that their patients were using these ones, and the orientations provided to them. The physicians were also asked about their knowledge on possible interactions of medicinal plants and/or herbal medicine with the usual pharmacoterapy and the possible alterations on biological/pharmacological activities of these pharmaceuticals. For the analysis of the pharmacokinetic and pharmacodynamic potential drug interactions between medicinal plants and herbal medicines and the prescribed pharmacotherapy, a research was performed using the following database: MEDLINE (1966-2007) and LILACS, pharmacology and medicinal plants textbooks. RESULTS From the 128 interviewed patients, 52.3% (95% CI; 40.3%-64.4%) claimed to be medicinal plants and/or herbal medicines users. The use in the last 4 years represented 25% (95% CI; 14.6%-35.4%) of consumption affirmatives and the use in the last year represented 18% (95% CI; 8.8%-27.2%). The sociodemographic characteristics of these patients are presented in Table 1. Asked about why they initiate using medicinal plants, most of them reported that such habit is a familiar traditional practice (56, 6%; 95% CI 44.7%68.5%). The recommendations received from relatives and friends represented 83.6% (95% CI; 74.7%-92.5%). The interviewed also accepted medical indication (3%), induced by TV programs,
outdoor advertisements, radio, magazines and newspaper (4.5%). The use of medicinal plants and/or herbal medicines, were associated to the reduction on the acute symptoms from adjacent pathologies [91.1% (95% CI; 84.3%-97.9%)]. Other reasons were: reduction on the disease symptoms (4.5%), cure for AIDS (2.9%) and to reduce of side and adverse effects caused by medications (1.5%) (Table 2). When queried about the results obtained with the use of medicinal plants and/or herbal remedies, 86% (95% CI; 78.4%-94.8%) of them reported to have obtained positive results; 14% reported adverse effects or the lack of any result at all. Patients using medicinal plant and/or herbal medicines usually did not inform the medical staff about this consumption practice [77% (95% CI; 67%87%)]. Both patients who informed the medical staff about the use of medicinal plants and/or herbal medicines and those who did not inform the medical staff said had obtained positive results with this utilization (93% and 84%, respectively). The most mentioned herbal medicine pharmaceutical forms were: decoction with water, decoction with milk, maceration, infusion, garrafada (a mixture of several medicinal plants on ethanol or cachaa), syrup and capsule. In relation to the acquisition of the medicinal plant and/or herbal medicines, 61% (95% CI; 49.5%75.9%) of users reported to have obtained the plant through own garden, 30% purchased the plant in free markets or known root shops and only 9% bought the plant in specialized pharmacies and drugstores. Tables from 3 to 6 show the possible effects of cited medicinal plants and/or herbal medicines on the antiretroviral drugs action. DISCUSSION The obtained results were higher than others reports about HIV-infected patients using medicinal plants and/or herbal medicines (ranged from 20 to 40 %) (Kassler et al., 1991; Duggan et al., 2001; Colebunders et al., 2003; Sugimoto et al., 2005; Dhalla et al., 2006).
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Table 3 Plants and herbal remedies that can interact with antiretroviral drugs. Latin name (vernacular) Ilex paraguariensis (erva mate) Chenopodium ambrosioides Bert. Ex Stend. (erva de santa maria); Paullinia cupana Kunth (guaran); Smilax spp. (salsaparrilha); Camellia sinensis L. (ch preto) Symphytum officinale L. (confrei); Sambucus nigra L. (sabugueiro); Hymenaea courbaril L. (jatob); Gossypium barbadense L. (algodo) Possible action Can promote unbalance of the acid-gastric buffer. Interaction outcome Cause increase of gastrointestinal discomfort and alteration in the ARV absorption Can reduce ARV absorption, either by the revetment of the gastric wall or by the adsorption of the medicine. References (Fetrow and Avila, 2000; Cowl, 2003; Philp, 2004; Simes et al., 2004; Taketa et al., 2004) (Fetrow and Avila, 2000; Abdel-Kader et al., 2002; Cowl, 2003; LimaNishimura et al., 2003; Philp, 2004; Santos et al., 2004; Simes et al., 2004) (Fetrow and Avila, 2000; Butterweck et al., 2004; delCastillo et al., 2004; Unger and Frank, 2004) (Fetrow and Avila, 2000; Simes et al., 2004). (Havey, 1999; Fetrow and Avila, 2000; Philp, 2004; Simes et al., 2004)
Can form a viscous gel of the mucilage and gastric solution.
Matricaria chamomilla L. (camomila); Mentha spp. (hortel)
Antispasmodic
Can reduce the motility of the gastrointestinal tract causing reduction of ARV absorption. Can promote changes in the ARV absorption due the increasing on the gastric emptying. Can promote competition with ARV for the bond to the plasmatic protein.
Aloe spp. (babosa) Allium cepa L.( cebola); Eucalyptus globulus Labill. (eucalipto) Ilex paraguariensis (erva mate); Serenoa repens (saw palmetto); Sambucus nigra L. (sabugueiro); Maytenus ilicifolia Mart. ex Reiss (espinheira santa); Paullinia cupana Kunth (guaran); Camellia sinensis L. (ch preto) Eucalyptus globulus Labill. (eucalipto) Matricaria chamomilla L. (camomila) Mentha spp. (hortel) Uncaria tomentosa (Willd. Ex Roem. & Schult.) DC. (unha de gato)
Can increase the gastrointestinal peristaltic movement Quercetin associates to the plasmatic protein in around 98%.
(Fetrow and Avila, 2000; Izzo and Ernst, 2001; Cowl, 2003; Ohsaki et al., 2004; Philp, 2004; Simes et al., 2004; Taketa et al., 2004) (Fetrow and Avila, 2000; Williams, Inhibition of CYP450, in vitro. Can increase the ARV bioavailability 2001; Cowl, 2003; and/or toxicity. Butterweck et al., 2004; Philp, 2004; Simes et al., 2004) Induction of CYP450. Cmax SQV: reduction of 54% (Santos et al., 2004; Santos and Boullata, Allium sativum L. (alho) AUC SQV: reduction of 51% Cmax PI: reduction 2005) Allium cepa L. (cebola) Induction of CYP450. Cmax and AUC of PI: reduction (Havey, 1999) ARV=antiretroviral; PI=Protease inhibitor; CYP450 = cytochrome P450; PI=protease inhibitors; Cmax = maximum concentration; AUC=area under curve; SQV=saquinavir Formation of tannin insoluble complexes with proteins. Can increase the bioavailability of PI promoting higher bond to the plasmatic protein.
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Table 4 Plants and phytomedicines that can cause side effects synergism Latin name (vernacular) Peumus boldus Molina (boldo do chile) Symphytum officinale L. (confrei) Possible action Anticholinergic action Hepatotoxicity Interaction outcome Can increase neuropsychiatric EFV side effects. Can increase NRTI, NNRTI and PI-induced hepatic damage. Can increase NRTI, NNRTI and PI-induced hepatic damage. References (Fetrow and Avila, 2000). (Fetrow and Avila, 2000; Philp, 2004; Simes et al., 2004) (Fetrow and Avila, 2000; Lorenzo et al., 2002) (Fierro et al., 1999)
Mentha pulegium L. (poejo) Mikania glomerata Spreng.(guaco);
Hepatotoxicity
Formation of chelate by cumarin and Can promote pancytopenia with iron ions NRTI, NVP and PI. EFV=efavirenz; PI=protease inhibitors; NRTI= nucleoside analogous reverse transcriptase inhibitors; NNRTI= nucleoside non-analogous reverse transcriptase inhibitors; = increase. Table 5 Plants and their phytochemicals: possible interactions mechanisms Latin name (vernacular) Ilex paraguariensis (erva mate); Paullinia cupana Kunth (guaran); Camellia sinensis L. (ch preto). Operculina alata (Ham) Urban. (Batata de Ti) Myristica fragrans Houtt (nutmeg) EFV=efavirenz; Phytochemicals Psychoactive substances Interaction outcome Can increase neuropsychiatric EFV side effects. References
Psychoactive substances Psychoactive substances
Can increase neuropsychiatric EFV side effects. Can increase neuropsychiatric EFV side effects.
(Fetrow and Avila, 2000; Cowl, 2003; Philp, 2004; Simes et al., 2004; Taketa et al., 2004) (Simes et al., 2004) (Fetrow and Avila, 2000; Cowl, 2003; Simes et al., 2004)
Our research revealed that the recommendations for the use of medicinal plants and/or herbal remedies were usually obtained from relatives (greatgrandparents, grandparents, parents, brothers, etc) and friends; only 3% of them from medical prescription. In another study, realized in Thailand, that evaluated the use of medicinal plants by HIVinfected patients, the recommendations obtained from relatives and friends represented 14% and under medical prescription represented 36% of cases (Sugimoto et al., 2005). In a quest performed with Brazilian cancer patients the phytotherapy, when used alone, represented 71.64% of the non-conventional therapeutic modalities reported by patients, being also recommended by relatives and friends in 80.6% of cases (Elias and Alves, 2002). The same data obtained in relation to the prevalence and recommendation for the use of medicinal plants and herbal remedies were also observed in pre-surgical patients from Lenox Hill Hospital, New York, being the prevalence of 57% for the use and 63% for recommendations received (Adusumilli et al., 2004).
Overall, 53 types of preparations involving medicinal plants were identified. From these, 6 (11.32%) could not be identified due to the lack of access to the medicinal plant or herbal medicine because the interviewed could not make a clear description of the plant or herbal medicine, or because the patient used popular nomenclature for different species employed at distinct pathological conditions. Also, it was not possible identifying components present in preparations as garrafadas and others herbal complex mixtures (usually powdered) presenting no label containing the product description. These results are in agreement with a study performed with HIV/AIDS patients from University of California, San Francisco: from 22% of patients reporting the use of medicinal plants during three months preceding the interview, 24% could not identify which medicinal plant (s) they were making use of (Kassler et al., 1991). The lack of quality, unknown origin and misidentification of in natura medicinal plants, in addition to the adulteration of herbal medicines with heavy metals, hormones, stimulants, antiinflammatory agents and other drugs, are well
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reported problems involved with herbal medicine (Camargo, 1998; Winslow and Kroll, 1998; Corns, 2003; Betti and Thomsen, 2005; Grollman, 2005; Gomez et al., 2007; Latif and Rahman, 2007). These aspects must be carefully observed, once in the present study, 91% of the in natura medicinal plants mentioned by the interviewed were obtained through own plantation garden, free markets and root shops. Such practice may result in the use of potentially toxic species by a mix up due to similarities in relation to the macroscopic characteristics, or popular name of the medicinal species. Another recurrent problem is the contamination of herbs or herbal medicines by fungi and pathogenic bacteria that may promote toxic reactions, being also responsible for alterations in the expected effects of these medicines (Themboa et al., 2007). HIV-infected patients under drug therapy use antiretroviral triple scheme and, no rare, additional medicines for the treatment of opportunistic infections. Medicinal plants and/or herbal medicines are composed of several chemical components (Taylor et al., 2006; Tirona and Bailey, 2006; De Smet, 2007; Langlois-Klassen et al., 2007), thus, the association anti-retroviral therapy/medicinal plants may increase the risk of developing adverse drug reactions (Zhou et al., 2007). The physicians and the patients must perform a careful evaluation in relation to this potential risk, before starting the phytotherapy (Foster et al., 2003; Foti et al., 2007). Pharmacokinetic interactions may reduce the drug bioavailability and hence its efficiency or increase its bioavailability resulting in possible increased toxicity. The pharmacokinetic interactions presented in this work were mainly related to the medicine absorption, bonding to the plasmatic protein and induction or inhibition of the drug in the cytochrome P450 (CYP) (Foti et al., 2007). Among plants and herbal medicines used by the interviewed, the phytochemicals are worth of emphasis because they may change the antiretroviral drugs absorption, reducing the area under plasma concentration vs time curve. Absorption alterations that promote the reduction of the drug efficiency, antiretroviral included, may be caused by plants and/or herbal medicines containing saponins (Castilla et al., 2006), such as Ilex paraguariensis, Chenopodium ambrosioides, Paullinia cupana, Smilax spp and Camellia sinensis. The mucilage present in Symphytum officinale, Sambucus nigra, Hymenaea courbaril and
Gossypium barbadense, can form a viscous thin layer on the gastrointestinal mucosa causing reduction on the antiretroviral drug absorption, or through the revetment of the gastric wall or through the absorption of the medicine molecule (Haq, 2004) . Medicinal plants with antispasmodic action as Matricaria chamomilla and Mentha spp. reduce the motility of the gastrointestinal tract, thus reducing the antiretroviral medicines absorption. In fact, Mentha spp reduces total gastrointestinal transit or gastric emptying, decreases the basal tone in the gastrointestinal tract, and inhibits potassium depolarization-induced responses in the intestine (Rodriguez-Fragoso et al., 2008). The anthraquinones present in aloe can increase the peristaltic movements; therefore, can reduce the bioavailability of antiretroviral medicines. (Laitinen et al., 2007). Quercetin, widely found in nature and present in Allium cepa and Eucalyptus globulus, binds to the plasmatic proteins in around 98% and when administered with other medicine with high binding to the plasmatic protein, may lead to a pharmacological competition, and this interaction may form both a dislocated drug and a dislocating drug (Havey, 1999; Philp, 2004; Farkas et al., 2007). Thus, herbal medicines or large amounts of food containing onion or eucalyptus when used concomitantly with efavirenz, which present binding percentage to the plasmatic protein higher than 99% (deMaat et al., 2003), may promote a competition for this binding. It is known that, in this case, in other words, if the efavirenz is dislocated, its excitability symptoms in the central nervous system would become exacerbated. Considering the results obtained, it was verified that 28.1% of patients used zidovudine, lamivudine and efavirenz as therapeutic scheme and that A. cepa is a seasoning with large presence in the Brazilian cookery, it becomes crucial to inform patients on their concomitant use with the pharmacotherapy. The tannins present in I. paraguariensis, Serenoa repens, S. nigra, Maytenus ilicifolia, E. globulus, P. cupana and C. sinensis can form insoluble complexes with plasmatic proteins (Rivera et al., 2006), leading to an increase on the bioavailability of protease inhibitors, once this antiretroviral class presents high binding to the plasmatic protein. The increase on the bioavailability of this medicine class may lead to exacerbation on its side effects, among them, the blood stasis (Izzo and Ernst, 2001; Ohsaki et al., 2004; Philp, 2004; Taketa et al., 2004; Izzo, 2005).. The increase on the efavirenz side effects may also
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occur as a result of its increased bioavailability (deMaat et al., 2003). Other clinical relevant pharmacokinetic interactions can occur in the cytochrome P450 (CYP450). Alterations on the medicine bioavailability or efficiency may occur with the inhibition or induction of the enzymatic metabolism, respectively. The CYP450 is the isoform involved in the metabolism of at least 50% of drugs including the protease inhibitors (antiretroviral drugs). The CYP3A4 is more quantitatively expressed in the liver and on the intestine wall (Harris et al., 2003). Among medicinal plants/phytomedicine that can interact with CYP450, increasing the bioavailability or toxicity of antiretroviral drugs, Uncaria tomentosa Matricaria chamomilla and Mentha spp. stand out (Foster et al., 2005). An in vitro study showed the U. tomentosa inhibitory activity in the CYP3A4 (57%), CYP2D6 (13%) and CYP2C9 (11%). Matricaria chamomilla, in turn, also presented inhibitory activity in CYP3A4 (57%), CYP2D6 (54%) and CYP2C9 (61%) (Foster et al., 2003). The essential oil from Matricaria recutita L and the constituents cis-spiroether, trans-spiroether, alpha-bisabolol, and chamazulene can promote inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2D6, and CYP3A4) in vitro (Ganzera et al., 2006). Another in vitro study showed the Mentha spp. inhibitory activity in the CYP450 (Unger and Frank, 2004). The concern about action of medicinal plants or herbal medicines on CYP is increasing, meanly at countries when the use of medicinal plants sometimes is the one therapeutical resource (Mills et al., 2005). Antiretroviral drugs belonging to the nucleoside non-analogous reverse transcriptase inhibitors class and protease inhibitors present hepatic metabolism, especially by isoforms CYP3A4, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 (deMaat et al., 2003; Foster et al., 2005) and the concomitant use of U. tomentosa, M. chamomilla and Mentha spp with the class mentioned above may lead to an increase on their bioavailability/toxicity. In the same way medicinal plants and/or herbal medicines may also induce their metabolism in this enzymatic system, reducing the antiretroviral medicine efficiency. Among the mentioned plants/herbal remedies, garlic and onion are included. In a study conducted in humans aliin, present in Allium spp. promoted inductive activity of the CYP450 enzymatic system, causing reduction on the saquinavir maximum concentration in 54% and its
area under curve in 51% (Piscitelli et al., 2002). This compound is also present in the A. cepa (Havey, 1999), suggesting the same inductive activity on CYP450 enzymatic system. In relation to pharmacodynamic interactions, the present study identified some medicinal plants/herbal medicines can promote side effects synergism if administered concomitantly with some antiretroviral medicines. Psychoactive substances present in I. paraguariensis, Operculina alata, Myristica fragrans and C. sinensis may enhance the efavirenz-induced neuropsychiatric side effects. The Peumus boldus, due to its anticholinergic action, also can enhance the efavirenz-induced stimulation of the central nervous system. Antiretroviral drugs belonging to the nucleoside analogous reverse transcriptase inhibitors and nucleoside non-analogous reverse transcriptase inhibitors classes and the protease inhibitors cause increases on the alanine transaminase (ALT) and aspartate transaminase (AST) hepatic expression, indicating a chronic or acute hepatotoxicity. Symphytum officinale and Mentha (Fetrow and Avila, 2000; Lorenzo et al., 2002; Philp, 2004) are hepatotoxic and when administered with antiretroviral drugs may enhance hepatic injuries. Zidovudine, didanosine, estavudine, lamivudine, tenofovir, nevirapine, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir produce hematological substances causing pancytopenia. This side effect is exacerbated with the concomitant administration of Mikania glomerata, due to its iron ions chelating action. Interactions involving phytochemicals and conventional drugs give the wrong impression of being less frequent and severe than drug-drug interactions. This induces the false idea that plants and/or phytomedicine present irrelevant pharmacological profile and that their actual pharmacological potential should not be taken into account. Such fact leads to the occurrence of misidentification and lack of notification about the adverse effects of plants and herbal medicines that, in addition to a small number of scientific researches about them, contribute to the maintenance of the condition of being inappropriately used by the population, with increased risk to the users health. In fact, the market of largely unregulated herbal medicines presents significant risks to public health. In May 2006, the Brazilian Ministry of Health published a Government announcement that regulates
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integrative and complementary practices in the Brazilian Public Health System (Sistema nico de Sade - SUS), including phytotherapy. Thus, the capability of researches on the safety in relation to the use of this therapeutic arsenal should be encouraged, and information in relation to cautions of their use should be given to patients. However, it was observed that the parcel of patients (77.6%) who reported not to inform the medical staff on the use of medicinal plant/herbal remedy is relevant, corroborating previous works (Eisenberg et al., 1998; Adusumilli et al., 2004; Sharma et al., 2006), which reported that patients seldom inform voluntarily to the medical team on this practice. Although not included in the questionnaire applied, patients explained the reasons why they did not disclosure to the medical staff on his habit of using plants or herbal medicines. The fear of a negative reaction by the physician was relevant reason presented by them. The lack of scientific corroboration and the misconceptions about herbal medicine such if it does not do any good, it will not do any bad either, among other explanations, were the most heard responses from the medical staff in relation to patients who reported this practice. On the other hand, according to results from survey performed with the medical staff responsible for the care of these patients, it was verified that among the physicians inquired, 66.67% reported that routinely ask the patient about the use of medicinal plants/herbal remedies, but only half reported to provide information to the patient on this habit. However, the opinion in relation to the high interference level that medicinal plants and/or herbal remedies may cause to the pharmacotherapy was unanimous. When inquired in relation to some information on medicinal plants and/or herbal medicines, only 33% of the physicians provided the right answer for more than half of the questions about differences between medicinal plant and herbal medicines and their biological/pharmacological activity. CONCLUSION Considering the wide and growing use of medicinal plants and/or herbal medicines, researches focuses on phytochemical identification, with emphasis on their pharmacological effects, as well as their action mechanisms, efficiency and safety should be stimulated. The physicians and patients must
perform a careful evaluation about the potential risks, before starting the phytotherapy (Foster et al., 2003) REFERENCES
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An ethanolic extract of Uncaria tomentosa reduces inflammation and B16BL6 melanoma growth in C57BL/6 mice.
[Un extracto etanlico de Uncaria tomentosa reduce la inflamacin y el crecimiento del melanoma B16/BL6 en ratones C57BL/6]
Ana Laura FAZIO1, Diana BALLN2, Italo M. CESARI2, Mara Jess ABAD1, Miriam ARSENAK1, Peter TAYLOR1 1. 2. Centro de Medicina Experimental , Instituto Venezolano de Investigaciones Cientficas, Apartado 20632, Caracas 1020-A, Venezuela. Centro de Microbiologa y Biologa Celular, Instituto Venezolano de Investigaciones Cientficas, Apartado 20632, Caracas 1020A, Venezuela.
Contact : Tel +58 212 504 1097; Fax: +58 212 504 1086. E-Mail: ptaylor@ivic.ve Recibido | Received 4/06/2008; Aceptado | Accepted 30/06/2008; Online 12/07/2008
Abstract Extracts of the bark of Uncaria tomentosa (Cats Claw Ua de Gato) have been used traditionally for their anti-inflammatory and anticancer properties. We investigated the effect of a hydroethanolic extract (UT) of U. tomentosa on a) the viability of primary and tumor cells, b) the inflammatory response (tumor necrosis factor alpha [TNF-], interleukin-6 [IL-6] and nitric oxide [NO]) both in vitro and in vivo, c) B16/BL6 melanoma cell growth and metastasis in the C57BL/6 mouse, and d) nuclear factor B (NF-B) activity in LPS-stimulated HeLa cells. UT did not show an important cytotoxic effect in vitro at the doses up to 300 g/ml, but did inhibit tumor growth and metastasis in vivo. UT inhibited TNF-, IL-6 and NO production in vitro. NF-B activity was also inhibited. Our studies show that UT merits further study for its effects on processes common to inflammation and cancer.
Keywords: Uncaria tomentosa, cancer, NF-B, inflammation, B16/BL6 mouse
Resumen Los extractos de la corteza de Uncaria tomentosa (Ua de Gato) han sido usados tradicionalmente por sus propiedades anti-inflamatorias y antitumoral. Investigamos el efecto de un extracto hidroetanlico (UT) de U. tomentosa sobre: a) la viabilidad de clulas normales y tumorales, b) la respuesta inflamatoria (factor de necrosis tumoral alfa (TNF-a), interleuquina-6 (IL-6) y xido ntrico (ON) in vitro e in vivo, c) crecimiento y metstasis de clulas de melanoma B16/BL6 en ratones C57BL/6, y d) actividad del factor nuclear B (NF-B) en clulas HeLa estimuladas con LPS. UT no mostr un efecto citotxico importante in vitro hasta 300 g/ml, pero si inhibi el crecimiento de tumor primario y de metstasis in vivo. UT inhibi la produccin de TNF-, IL-6 y ON in vitro. La actividad de NF-B tambin result inhibida. Nuestros estudios muestran que UT contiene compuestos que ameritan ser ms estudiados por sus efectos sobre procesos comunes a inflamacin y cncer.
Palabras clave: Uncaria tomentosa, cncer, NF-B, inflammacin, B16/BL6
INTRODUCTION Uncaria tomentosa is a species of vine from the Rubiaceae family, widely distributed throughout South and Central America, which has a long tradition of use as a folk medicine for the treatment of a variety of conditions including inflammation, cancer and gastrointestinal disorders (Reinhard, 1999). Previous studies have shown antiinflammatory activity both in vitro (Sandoval-Chacn et al., 1998) and in mouse models of inflammation (Aquino et al., 1991; Caballero et al., 2005), and growth inhibitory effects in both tumor (Sheng et al., 1998) and normal cells (Akesson et al., 2003) in vitro. The role of chronic inflammation in tumor initiation and growth is well established (Coussens
and Werb, 2002) so a dual action against both inflammation and cancer is not surprising. Studies have shown that anti-inflammatory drugs may be effective in cancer therapy and/or prevention (Thun et al., 2002), and the possible mechanisms of action of anti-inflammatory phytochemicals have been reviewed (Surh et al., 2001). U. tomentosa extracts have been shown to inhibit NF-B (Sandoval-Chacn et al., 1998; Akesson et al., 2003), a transcription factor which represents an important link between chronic inflammation and cancer (Li et al., 2005) and which has been suggested as a possible target for the therapy of both (Bremner and Heinrich, 2002). We have previously shown that intraperitoneal (i.p.) injection of an aqueous extract of U. tomentosa
Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
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inhibited the TNF- and IL-6 response to lipopolysaccharide (LPS) challenge, and the growth of both primary tumors and metastasis in mice (Caballero et al., 2005). In the present study, we report the in vivo anti-inflammatory and antitumor activities of a hydroethanolic extract, as well as an inhibitory effect on NF-B. MATERIALS AND METHODS Plant material. The bark of Uncaria tomentosa (Cats claw, Ua de gato), was obtained from the Peruvian Amazon region and identified by us. Aliquots were ground then macerated in a 70% ethanol in water solution for 21 days in the dark at room temperature. The suspension was then filtered under sterile conditions using Whatman No. 1 filter paper then adjusted to a stock concentration of 5 mg/ml, which was calculated from the dry weight of a lyophilized sample. This extract shall be termed UT. Cells and animals. The cell lines, B16/BL6 (murine melanoma), K1735 (amelanotic murine melanoma), HT29 (human colon carcinoma), A549 (human lung carcinoma), WEHI 164 (mouse fibrosarcoma), LEC (mouse liver endothelial cell line) were cultured in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS - Gibco, BRL, USA), penicillin (100 Units/ml), streptomycin (100 g/ml) and containing in addition glucose 0.45% (HT29 cells), and Lglutamine 2 mM (A549 cells). Human peripheral blood monocytes (huPBMC) were obtained from healthy donors by standard Ficoll/Hypaque gradient centrifugation and cultured in RPMI-1640 10% FBS. Chopped spleens from C57BL/6 mice were ground through a wire mesh screen. After removal of detritus and lysis of red blood cells with 0.085% sodium citrate, adherent cells were removed by overnight incubation in plastic culture flasks. The non-adherent cells (muSplen) were harvested, counted and cultured in RPMI-1640 10% FBS. Murine peritoneal macrophages (muPM) were collected from C57BL/6 mice 4 days after a peritoneal injection of 2 ml of 4% thioglycollate. The cells were washed, seeded into culture flasks in RPMI-1640 10% FBS, and nonadherent cells discarded after 3 h. The adherent cells were then used immediately.
Female C57BL/6 mice (79 weeks old) were obtained from the Animal Facility, IVIC and fed with standard pellet diet and water ad libitum. All animal experiments were performed according to internationally accepted guidelines for the treatment of animals in research. Cytotoxicity. Cells were plated at 2.5 - 5 x 104 cells / well in 100 l of culture medium in flat-bottomed 96 well plates and allowed to attach for 24 h. Different concentrations of UT in 100 l culture medium were then added. Control wells were set up containing equivalent quantities of ethanol, which in no case exceeded 1%. No effect was observed due to the ethanol. After a further 24 h, the number of viable cells was assessed using the MTS/PMS chromogenic assay (Promega Corp., USA) according to the manufacturers instructions. Inflammatory response in vitro. Peritoneal macrophages were activated with 10 g/ml lipopolysaccharide (LPS - E. coli serotype 055:B5, Sigma, USA) for 24 h in the presence of UT, and then the concentrations of TNF- , IL-6 and nitric oxide (NO) were measured in the supernatants. TNF- was quantified using the WEHI 164 cell bioassay (Espevik and Nissen-Meyer, 1986), IL-6 with a commercial ELISA assay (R & D Systems Inc., MN, USA) and NO using the Griess reaction (Sandoval-Chacn et al., 1998). Inflammatory response in vivo. Mice were injected intraperitoneally (i.p.) with different doses of LPS in 100 l of PBS. After 1 h, blood was collected by heart puncture under ether anesthesia. Serum was separated and assayed for the two cytokines and NO as described above. In order to evaluate the effect of UT on the inflammatory response, mice were injected i.p. with 50 g UT on 3 consecutive days prior to LPS challenge. Lung metastasis. At day 0, mice were inoculated in the lateral tail vein (i.v.) with 105 B16/BL6 cells in 100 l PBS. Two treatment protocols with UT were performed a) intraperitoneal (i.p.) injection of 50 g of UT in 100 l PBS / 25% ethanol on days -2, -1 and 0, and b) i.p. injection of the same dose of extract 5 times per week starting from on day 0 up to day 21. Control
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animals received 100 l PBS / 25% ethanol. On day 23, the animals were sacrificed with ether; the lungs were removed, placed for 5 min in 3% H2O2 in H2O and fixed in Bouin's solution. The purpose of the H2O2 was twofold: to bleach hemorrhages which could be mistaken for metastases, and to inflate the lungs, facilitating the evaluation of metastases under the dissecting microscope. Animals were challenged with LPS prior to sacrifice, in order to measure serum TNF- and IL-6 levels as described above. Primary tumors. Primary tumors were induced by the subcutaneous (s.c.) injection of 5 x 104 B16/BL6 cells in 100 l PBS into the hind limb. The mice were injected i.p. with 50 g of UT 5 times per week starting from on day 0 up to day 21. Tumor size was measured in two dimensions with a vernier gauge. Animals were challenged with LPS prior to sacrifice, in order to measure serum TNF- and IL-6 levels as described above. NF-B luciferase assay. HeLa cells were transiently transfected with the NF-B luciferase reporter system (Stratagene, La Jolla, CA, USA) according the manufacturers instructions. Transfected cells were seeded into 96well plates, allowed to adhere overnight then treated for 1 h with different concentrations of UT. The cells were then stimulated for a further 4 h with 25 ng/ml huTNF- (BD Biosciences, Palo Alto, CA, USA). Luciferase activity was measured using the SteadyGlo assay kit (Promega, Madison, WI, USA), in a 96well luminometer. Dexamethasone and paclitaxel, which are known to inhibit and stimulate NF-B activity respectively, were included as controls. Statistical analysis. Each experiment was performed at least three times and results are expressed as the mean standard deviation. The unpaired Students t test with the Welch correction was used to assess the statistical significance of the differences. A confidence level of P < 0.05 was considered significant. RESULTS Cytotoxicity. Initial in vitro experiments (Fig. 1) showed an inhibitory effect of UT on 24 h cell growth only at
the higher concentrations of 100 and 300 g/ml. Inhibition was less than 50% in all cases. Three tumor cell lines (HT-29, K1735 and WEHI) and the three primary cell preparations (huPBMC, muSplen and muPM) were more sensitive to the extract than the B16/BL6, A549 and LEC cells.
Figure 1. Effect of the plant extracts on the viability of tumor cell lines in vitro. Cell viability was measured by the MTS chromogenic assay after a 24 h incubation in the presence of UT at the concentrations indicated. huPBMC - human peripheral blood monocytes, muSplen non-adherent mouse splenocytes, muPM - murine peritoneal macrophages. Results are expressed as the percentage of the absorbance in the absence of the plant extract (mean S.E.M., n = 3).
Effect of UT on the inflammatory response in vitro and in vivo. The TNF- response of mouse peritoneal macrophages to LPS was reduced by 48% in the presence of 100 g/ml UT (Fig. 2), although this reduction was not significant due to variability in the results. The IL-6 and NO responses were significantly reduced by 35% and 62% respectively (P < 0.001). This result was not due to a direct cytotoxic effect, as no change in the viability of these activated cells was observed at this concentration of UT (results not shown). A similar reduction in the inflammatory response was observed in vivo, when mice were injected i.p. with 50 g UT on 3 consecutive days prior to challenge with different doses of LPS (Fig. 3). The TNF, IL-6 and NO responses were reduced by 63, 59 and 57% respectively when the animals treated with UT were challenged with the highest dose of LPS. Again, only the results for IL-6 and NO were significant due to variability in the TNF response to LPS.
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Figure 2. Inhibition by UT of the inflammatory response of mouse peritoneal macrophages to LPS. Cells were activated with 10 g/ml LPS for 24 h in the presence of 100 g/ml UT extract. TNF- , IL-6 and NO levels were then measured in the supernatants. (mean S.E.M., n = 3). ** P < 0.001
Figure 3. Inhibition by UT of the inflammatory response to LPS in mice. Animals were pretreated with 50 g/ml UT i.p. on 3 days, then challenged with different doses of LPS. After 1 h, blood was extracted and the serum assayed for TNF- , IL-6 and NO. (mean S.E.M., n = 3). * P < 0.05.
Inhibition by UT of primary tumor growth and metastasis in mice. Mice were inoculated s.c. with B16/BL6 cells and the effect of i.p. UT on primary tumor growth was measured. At all time points after the appearance of the tumor, there was a very significant inhibition of tumor growth in the animals treated with UT (Fig. 4A). This effect was most notable at earlier times with the treated tumors measuring approximately 75% less than the controls up to day 16, but only 50% less at day 22. Effect of UT on the inflammatory response to LPS in tumor-bearing animals. As basal levels of serum TNF- and IL-6 are very low in animals with either primary tumors or metastases, we evaluated the effect of UT on the inflammatory response to a low dose of LPS (3 g / animal) in tumor-bearing animals, prior to sacrifice.
Figure 5 shows that both the TNF- and IL-6 responses to low dose LPS were much greater in animals with primary tumors than in animals without tumors (compare with Fig. 3). In contrast, very little priming of the inflammatory response to low dose LPS was observed in the animals with metastasis. The TNF- and IL-6 responses in animals with primary tumors were greatly inhibited by UT treatment, (85% and 81% respectively). Although TNF- levels were lower in the untreated animals with metastasis, an important inhibitory effect (81%) was observed after UT treatment. The already almost basal levels of IL-6 in these animals were unaffected by UT.
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Figure 5. Effect of UT on the inflammatory response to LPS in tumor-bearing mice. C57Bl/6 mice were inoculated s.c. or i.v. with B16/BL6 tumor cells to produce primary tumors or metastases, respectively, then injected i.p. 5 times a week up to day 21 with 50 g UT (corresponding to Fig 4A and 4C). One h before sacrifice, the animals were challenged with 3 g LPS. Blood was extracted and the serum assayed for TNF- and IL-6. (mean S.E.M., n = 3). * P < 0.05.
Figure 4. Effect of UT treatment on primary tumor growth and metastasis in mice. A. C57Bl/6 mice were inoculated s.c. with B16/BL6 tumor cells to initiate a primary tumor and injected i.p. 5 times a week up to day 21 with 50 g UT. Tumor growth was assessed with a vernier gauge. B. Mice were inoculated i.v. with B16/BL6 cells. Treatment with UT consisted of 50 g i.p. and the 3 days prior to inoculation. Lung metastases were counted on day 22. C. Mice were inoculated i.v. with tumor cells, as in B., but treated with UT post-inoculation as in A. (mean S.E.M., n = 10). * P < 0.005, ** P < 0.0005.
Effect of UT on the NF-B response to activation by TNF- in HeLa cells. The effect of UT on the NF-B response of HeLa cells to TNF- was determined in a luciferase reporter assay. UT at 10 g/ml slightly stimulated NF-B activity, but was inhibitory (47%) at 100 g/ml (Fig. 6). The highest dose of dexamethasone used (30 M), a known NF-B inhibitor, inhibited the response by 38%. A change of medium after a preincubation with UT only slightly reduced the degree of inhibition, suggesting that the effect was not due to a direct interaction between the crude extract and LPS or TNF- (results not shown). Preliminary experiments also confirmed that UT was not cytotoxic for these cells at the doses used in these short term assays (results not shown).
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Figure 6. Effect of UT treatment on NF-B activity. HeLa cells transfected with the NF-B/luciferase reporter plasmid were treated for 1 h with different concentrations of UT, then stimulated for a further 4 h with 25 ng/ml huTNF-. DEX dexamethasone (30 M), PAC paclitaxel (1 M) see Materials and Methods.. (mean S.E.M., n = 3).
DISCUSSION Our previous studies indicated that an aqueous extract of UT was not cytotoxic for a range of cells at concentrations up to 3 mg/ml (Caballero et al., 2005). Here we show that the hydroethanolic extract of UT, was more cytotoxic for some of the tumor cell lines and the primary cells, but not for the B16/BL6 melanoma cells. We were unable to test concentrations higher than 300 g/ml, as a precipitate began to appear in the cultures at 1 mg/ml. A review of several reports suggests that extracts of U. tomentosa may show some antiproliferative activity (Sandoval-Chacn et al., 1998; Sheng et al., 1998; Riva et al., 2001; Akesson et al., 2003; De Martino et al., 2006) although the effects were observed in the mg/ml range. Santa Mara showed that aqueous extracts of U. tomentosa were not toxic for Chinese hamster ovary cells (CHO) in 3 different bioassay systems (Santa Maria et al., 1997). In contrast, U. tomentosa was reported to attenuate peroxynitriteinduced apoptosis in HT29 and RAW 264.7 cells (Sandoval-Chacn et al., 1998). Considering these results and the dose of UT we used in the in vivo experiments (50 g UT/day), it is difficult to conclude that the inhibitory effect seen with the primary tumors and metastasis was due to a direct effect on tumor cell proliferation or viability. Our results confirm previous reports of the antiinflammatory action of extracts of Cats Claw (Aquino et al., 1991). Although it has been reported
that an aqueous extract of this plant inhibited TNF- and nitrite production by the RAW 264.7 mouse macrophage cell line in vitro (Sandoval et al., 2000; Sandoval et al., 2002), as far as we are aware this is the first report of its inhibitory effect on inflammatory cytokine production in vivo while there have been two reports of a stimulatory effect of Uncaria extracts on IL-6 production (Lemaire et al., 1999; Eberlin et al., 2005). Evidently such results must be interpreted taking into account the nature of the preparation used, and its route of administration. Aguilar reported that a hydroethanolic extract was more active than a water extract in the mouse paw edema assay (Aguilar et al., 2002). Such extracts may be expected to contain compounds with a wider range of polarities, including bioactive alkaloids, than a water extract but also less desirable compounds such as tannins (Sheng et al., 2005; Pilarski et al., 2006). Our finding that UT inhibits the inflammatory response as well as tumor growth and metastasis, in the same mouse model is interesting considering the proven relationship between inflammation and cancer. UT may contain compounds which separately exert anti-inflammatory or anticancer effects through different mechanisms, or the results may be due to a compound, or group of compounds, acting through a common mechanism. Plant components with both activities including flavonoids and terpenes have been reported (Evans and Taylor, 1983; Middleton et al., 2000). In a previous study, we showed that blocking TNF- with a TNF receptor construct decreased serum TNF- and IL-6 levels after LPS challenge in tumor-inoculated mice (Cubillos et al., 1997) as well as reducing the number of lung metastases in the same animals. Previous studies have shown that aqueous extracts of U. tomentosa inhibit NF-B activity (SandovalChacn et al., 1998; Akesson et al., 2003), whereas Aguilar reported greater inhibitory activity in a hydroethanolic extract (Aguilar et al., 2002). Here, we confirm that our hydroethanolic extract was also inhibitory for NF-B, at concentrations that were not cytotoxic. However, NF-B is an anti-apoptotic factor in cells and its constitutive expression may be important in the survival of tumor cells (Aggarwal, 2004). Thus, the anticancer effects of anti- NF-B compounds derived from plants may derive from an inhibition of inflammatory cells, as well as a direct effect, at higher concentrations, on the tumor cells themselves.
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The biomedical applications of plants with antiinflammatory activity are becoming increasingly important given the links which have been established over the last decade or so between cancer and inflammation. This field becomes increasingly interestingly since it has been found that many antitumor drugs, such as paclitaxel activate NF-B, thus counteracting their own cytotoxicity (Karin et al., 2002). Anti-inflammatory drugs that act through NF-B, whether derived from plants or not, may be potential sensitizers to enhance the effectiveness of conventional cancer chemotherapy. CONCLUSION The results suggest that the anticancer activity of this hydroethanolic extract of U. tomentosa in this model may, to a large extent, be due to its antiinflammatory properties rather than to a direct cytotoxic effect. REFERENCES
Aggarwal BB. 2004. Nuclear factor-kappaB: the enemy within. Cancer Cell 6(3): 203-8. Aguilar JL, Rojas P, Marcelo A, Plaza A, Bauer R, Reininger E, Klaas CA, Merfort I. 2002. Antiinflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae). J Ethnopharmacol 81(2): 271-6. Akesson C, Lindgren H, Pero RW, Leanderson T, Ivars F. 2003. An extract of Uncaria tomentosa inhibiting cell division and NF-kappa B activity without inducing cell death. Int Immunopharmacol 3(13-14): 18891900. Aquino R, De Feo V, De Simone F, Pizza C, Cirino G. 1991. Plant metabolites. New compounds and antiinflammatory activity of Uncaria tomentosa. J Nat Prod 54(2): 453-459. Bremner P, Heinrich M. 2002. Natural products as targeted modulators of the nuclear factor-kappaB pathway. J Pharm Pharmacol 54(4): 453-72. Caballero M, Arsenak M, Abad MJ, I.M. C, Taylor P. 2005. Effect of plant extracts on B16-BL6 melanoma cell growth and metastasis in C57BL/6 mice. Acta Cient Venez 55(7-8): 21-27. Coussens LM, Werb Z. 2002. Inflammation and cancer. Nature 420(6917): 860-7. Cubillos S, Scallon B, Feldmann M, Taylor P. 1997. Effect of blocking TNF on IL-6 levels and metastasis in a B16-BL6 melanoma/mouse model. Anticancer Res 17(3C): 2207-11. De Martino L, Martinot JLS, Franceschelli S, Leone A, Pizza C, De Feo V. 2006. Proapoptotic effect of
Uncaria tomentosa extracts. J Ethnopharmacol 107(1): 91-94. Eberlin S, dos Santos LM, Queiroz ML. 2005. Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes. Int Immunopharmacol 5(7-8): 1235-46. Espevik T, Nissen-Meyer J. 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods 95(1): 99-105. Evans FJ, Taylor SE. 1983. Pro-inflammatory, tumourpromoting and anti-tumour diterpenes of the plant families Euphorbiaceae and Thymelaeaceae. Fortschr Chem Org Naturst 44: 1-99. Karin M, Cao Y, Greten FR, Li ZW. 2002. NF-KB in cancer: From innocent bystander to major culprit. Nature Rev Cancer 2(4): 301-310. Lemaire I, Assinewe V, Cano P, Awang DV, Arnason JT. 1999. Stimulation of interleukin-1 and -6 production in alveolar macrophages by the neotropical liana, Uncaria tomentosa (una de gato). J Ethnopharmacol 64(2): 109-15. Li Q, Withoff S, Verma IM. 2005. Inflammationassociated cancer: NF-KB is the lynchpin. Trends Immunol 26(6): 318-325. Middleton E, Jr., Kandaswami C, Theoharides TC. 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52(4): 673-751. Pilarski R, Zielinski H, Ciesiolka D, Gulewicz K. 2006. Antioxidant activity of ethanolic and aqueous extracts of Uncaria tomentosa (Willd.) DC. J Ethnopharmacol 104(1-2): 18-23. Reinhard KH. 1999. Uncaria tomentosa (Willd.) D.C.: cat's claw, una de gato, or saventaro. J Altern Complement Med 5(2): 143-51. Riva L, Coradini D, Di Fronzo G, De Feo V, De Tommasi N, De Simone F, Pizza C. 2001. The antiproliferative effects of Uncaria tomentosa extracts and fractions on the growth of breast cancer cell line. Anticancer Res 21(4A): 2457-61. Sandoval-Chacn M, Thompson JH, Zhang XJ, Liu X, Mannick EE, Sadowska-Krowicka H, Charbonnet RM, Clark DA, Miller MJ. 1998. Antiinflammatory actions of cat's claw: the role of NF-B. Aliment Pharmacol Ther 12(12): 1279-1289. Sandoval M, Charbonnet RM, Okuhama NN, Roberts J, Krenova Z, Trentacosti AM, Miller MJ. 2000. Cat's claw inhibits TNFalpha production and scavenges free radicals: role in cytoprotection. Free Radic Biol Med 29(1): 71-78. Sandoval M, Okuhama NN, Zhang XJ, Condezo LA, Lao J, Angeles FM, Musah RA, Bobrowski P, Miller MJ. 2002. Anti-inflammatory and antioxidant activities of cat's claw (Uncaria tomentosa and Uncaria guianensis)
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are independent of their alkaloid content. Phytomedicine 9(4): 325-37. Santa Maria A, Lopez A, Diaz MM, Albn J, Galn de Mera A, Vicente Orellana JA, Pozuelo JM. 1997. Evaluation of the toxicity of Uncaria tomentosa by bioassays in vitro. J Ethnopharmacol 57(3): 183-187. Sheng Y, Akesson C, Holmgren K, Bryngelsson C, Giamapa V, Pero RW. 2005. An active ingredient of Cat's Claw water extracts identification and efficacy of quinic acid. J Ethnopharmacol 96(3): 577-84. Sheng Y, Pero RW, Amiri A, Bryngelsson C. 1998. Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa. Anticancer Res 18(5A): 3363-3368. Surh YJ, Chun KS, Cha HH, Seong Su H, Keum YS, Park KK, Sang Sup L. 2001. Molecular mechanisms underlying chemopreventive activities of antiinflammatory phytochemicals: Down-regulation of COX-2 and iNOS through suppression of NF-B activation. Mutat Res 480-481: 243-268. Thun MJ, Henley SJ, Patrono C. 2002. Nonsteroidal antiinflammatory drugs as anticancer agents: Mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 94(4): 252-266.
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On the preclinical anti-trypanosomal, anti-inflammatory and toxicological activities of H. linifolium (L.) G. Don and its diphyllin derivatives.
[Sobre las actividades pre-clnicas antitripanosmica, antiinflamatoria y toxicolgica de H. linifolium (L.) G. Don y sus derivados difilnicos]
Guillermo SCHINELLA1,2,*, Horacio TOURNIER1,2, Anibal ZAIDENBERG1 and Jos M. PRIETO3 1. Catedra de Farmacologa, Facultad de Ciencias Mdicas, Universidad Nacional de La Plata, Argentina 2. Comisin de Investigaciones Cientficas de la Provincia de Buenos Aires, La Plata, Argentina 3. Departament de Farmacologa, Facultat de Farmcia, Universitat de Valencia, Valencia, Spain
*Contact: E-mail: Guillermo.Schinella@uv.es T +54 221 421 6932 F +54 221 423 6710 Recibido | Received 15/02/2008; Aceptado | Accepted 9/07/2008; Online 12/07/2008
Abstract
Haplophyllum linifolium (L.) G. Don -synonym H. hispanicum Spach - is an endemic species of the East of Spain, with topical antiinflammatory and antitrypanosome activities. In this work we aim to further gain insight into these activities by A) evaluating the efficacy of the methanolic plant extract in a TPA-induced chronic model of topical inflammation in mouse ears and B) test the effect of two arylnaphthalene lignans, diphyllin apioside (1) and diphyllin acetylapioside (2) against epimastigotes of T. cruzi in axenic cultures. Our results, show that the diphyllin derivatives (1) and (2) present therein are only endowed with a mild in vitro antitrypanosome activity, with IC50 values of 62.9 and 60.1 M, respectively, being toxic to normal mammal cells at the same concentration. On the other hand the plant methanolic extract exhibits a potent topical toxicity, causing necrosis of the skin, when applied chronically. The benefits and risks of therapies involving H. linifolium (L.) G. Don extracts or derivate products must be carefully balanced and always done under tight medical control.
Keywords: Haplophyllum linifolium(L.) G. Don , Trypanosoma cruzi, inflammation, toxicity, lignans.
Resumen
Haplophyllum linifolium (L.) G. Don sinonimo: H. hispanicum Spach- es una especie endmica de la regin oriental de Espaa con actividades antiinflamatoria tpica y antitripanosmica. En este trabajo nuestro objetivo fue obtener informacin adicional sobre estas actividades mediante la evaluacin de A) la eficacia del extracto metanlico de la planta en un modelo de inflamacin tpica crnica inducida por TPA en oreja de ratn y B) el efecto de dos lignanos arilnaftalnicos, apisido (1) y acetil apisido (2) de difilina contra epimastigotes de T. cruzi en cultivos axnicos. Nuestros resultados demuestran que los derivados (1) y (2) presentes en el extracto, estn dotados de una leve actividad antitripanosmica in vitro con valores de CI50 de 62,9 y 60,1 M, respectivamente, concentraciones que son txicas para clulas normales de mamferos. Por otra parte, el extracto metanlico aplicado en forma tpica y utilizado crnicamente, muestra una potente actividad txica manifestada por necrosis de la piel. Los beneficios y riesgos de terapias que involucren extractos de H.linifolium (L.) G. Don o productos derivados de los mismos deben ser cuidadosamente balanceados y su utilizacin debe realizarse siempre bajo estricto control mdico.
Palabras clave: Haplophyllum linifolium (L.) G. Don, Trypanosoma cruzi, inflamacin, toxicidad, lignanos.
INTRODUCTION Haplophyllum linifolium (L.) G. Don (Rutaceae) or H. hispanicum Spach- which is a synonym- is an endemic species of Spain, where it grows in semiarid lands, and is known for its high skin reactivity being employed clinically against vitiligo with some success (Massmanian and Prieto, 1996). In a previous paper we reported the topical anti-inflammatory activity of the methanolic extract from aerial parts of H. linifolium in acute models identifying two arylnaphthalene lignans, diphyllin apioside (1) and diphyllin acetylapioside (2) (Figure 1) as bioactive principles (Prieto et al., 1996). We further
characterized the 5-LOX inhibitory activity of these compounds as one of the mechanisms underlying this pharmacological effect (Prieto et al., 2002). This plant species was also one of the hits during a screening program for potential antiprotozoal drugs. Among 18 plant extracts and two fungi used in Chinese and Mediterranean traditional medicine, the methanolic extract of flowering aerial parts of H. linifolium turned out to be a promising source for anti-trypanosome drugs (Schinella et al, 2002). Other reported activities of these arylnaphtalene lignans include cytotoxic, and antiviral ones (Gonzlez et al., 1979).
Preclinical bioactivities of H. linifolium and its diphyllin derivatives
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We aim to further gain insight into these activities and so we decided (A) to evaluate the efficacy of the plant extract in a chronic model of topical inflammation and (B) to test the effect of the arylnaphtalene compounds against epimastigotes of T. cruzi in axenic cultures. MATERIAL AND METHODS Chemicals The extraction was performed with methanol PA (Merck, Germany). The culture medium base used was Medium 199 (Gibco BRL, Life Technologies, USA). The chemicals for the assays were of analytical grade (Sigma Co., St. Louis, USA). Compounds 1 and 2 were obtained as described by Prieto et al. (1996). Flowering aerial parts of Haplophyllum linifolium (L.) G. Don - also referred in literature by its synonym Haplophyllum hispanicum L. Spach (Tutin et al., 1968)- and commonly known in the province of Valencia as ruda or ruda mascle- were collected at El Vedat de Torrent (Valencia, Spain). The samples were identified by Dr. J. Peris (Department of Botany, University of Valencia) and a voucher specimen (VAL3368) was deposited in the herbarium of the Botanical Garden of the University of Valencia. The extract (yield 8 % w/w fresh plant) was obtained by maceration in methanol, solvent removed under reduced pressure at 40C and then freeze-dried. Antiprotozoal assay It was performed as previously described by Schinella et al. (2002). Briefly, an inoculum of 5105 cells mL1 T. cruzi epimastigotes (clone Bra C15C2) were cultured in F29 media supplemented with 10% (v/v) heat-inactivated fetal calf serum at 27 C. Compounds were added at different concentrations. All assays were carried out in triplicate. Final dimethylsulphoxide (DMSO) concentration was less than 0.5%. Parasites were counted after 72 h of contact with the samples in a Neubauer chamber. The activity of the compounds was assessed by comparison with a negative control (DMSO). Benznidazole was used as a reference drug. HPLC quantitative analyses HPLC-DAD analysis was performed using a Merck-Hitachi system (Intelligent Pump L-6200) equipped with an L-7455 Diode Array Detector, an L-7200 Autosampler and a Lichrospher C18 (250
4 mm, 5 m, Merck) column. Samples (40-60 L) were injected using a loop (20 L) trough a Rheodyne injection valve. Elution conditions: isocratic with MeOH/H2O (65:35), 1 mL/min, Pressure 60-80 bar. Data were processed using DAD Manager software (Hitachi-Merck). Cytotoxicity studies The cytotoxicities of both plant extracts and compounds were assessed on elicited peritoneal leukocytes obtained from rats with viability greater than 95% as determined by the trypan blue exclusion test. The cell viability was assessed by the capacity of PMN mitochondrial dehydrogenase enzymes to convert the 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) into a dark blue formazan. The reaction was followed spectrophotometrically at 490 nm (Mossman, 1983). Chlorpromazine (200 M) was used as a reference compound (positive control). Multiple TPA application The methanol extract of H. linifolium was applied twice daily (15 L, 1 mg.mL1) during four days onto the left ears of Swiss mice (25-30 g, n=6) as follows: in the morning immediately after TPA 2.5 g/ear application and 6 h later. Dexamethasone, 0.05 mg/ear, was used as the reference drug. The vehicle (ethanol) was applied onto the right ears as control. The swelling was expressed as an increase in the ear thickness due to TPA application, and swelling inhibition was expressed as a reduction in thickness with respect to the control group (Cullar et al., 2001) Statistical analysis Data were expressed as mean S.D. Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Dunnetts t-test for multiple comparisons. Differences were considered significant at p < 0.05. The inhibitory concentration 50% (IC50) was calculated from the concentration/effect regression line. In each case, an appropriate range of 45 concentrations was used. RESULTS AND DISCUSSION The lignans (1) (12 mg) and (2) (9 mg) (Figure 1) were isolated following the same chromatographic procedure reported by Prieto et al. (1996) and identified and tested for purity by HPLC-UV. The spectra of pure standards of diphyllin apioside and
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Figure 2. HPLC-UV Chromatogram (260 nm) and UV-spectra of diphyllin apioside and diphyllin acetylapioside standards injected altogether (elution conditions: MeOH/H2O (65:35) isocratic, 1 mL/min).
diphyllin acetylapioside were identical with a maximum at 260 nm. Their retentions times under our conditions were 2.58 min and 3.86 min respectively (Figure 2). The spectra and retention times of the isolated compounds (1) and (2) were identical to those of the standards allowing their identification as diphyllin apioside and diphyllin acetylapioside, respectively. The compounds were >95% pure (Data not shown). In the antitrypanosomal assay, The results were as shown in Table 1. In a previous work (Schinella et al., 2002), the methanol extract of flowering aerial parts of Haplophyllum linifolium (H. hispanicum) resulted very active against epimastigotes of T. cruzi, with a 65% of growth inhibition at 250 g ml-1. The results obtained indicate that the arylnaphtalene lignans contribute to this anti-protozoal activity of the extract. However, an important criterion in the search of active compounds against T. cruzi with therapeutic perspectives implies to determine any toxic effect on mammalian host cells at the active concentrations. For that purpose, the cytotoxicity of the compounds against rat peritoneal polimorphonuclear leukocytes was assessed. In our case, we found that the arylnaphtalene lignans were cytotoxic just over 40 M. Considering all these data as a whole we can not discard any unspecific cytotoxity towards the parasite.
Figure 1. Structures of diphyllin apioside (1) and diphyllin acetylapioside (2)
Table 1. Inhibitory Concentrations 50% (IC50, M) and Confidence Interval 95% (CI95) of the growth of epimastigotes of T. cruzi in axenic cultures shown by the assayed compounds.
Compound Diphyllin apioside Diphyllin acetylapioside Benznidazole
IC50 CI95 62.9 (46.0 79.8) 60.1 (50.1 70.1) 50.0 (41.2 58.9)
(1) R=H; (2) R=CH3CO
It is known that H. linifolium is endowed with a potent topical photodynamic activity (Massmanian and Prieto, 1996). Skin contact with the plant, followed by exposure to the sunlight or UV radiation, induces burns and hyperpigmentation. This phenomena is a consequence of the arylnaphatlene lignans presence, an effect used both ethnopharmacologically for the treatment of warts (Gonzlez et al., 1979) and under medical supervision in the treatment of vitiligo and leukodermia by treating patients topically with the tincture (Massmanian and Prieto, 1996). When used in single doses in murine models of TPA induced ear oedema the extract has a potent topical antiinflammatory activity with no apparent toxicity (Prieto et al., 1996). This effect is due, at least in part, to the inhibition of 5-LOX activity (Prieto et al., 2002). However, the plant extract was very toxic in a murine model of chronic topical application (1 mg/ear/day), resulting on the ulceration and loss of tissue of the treated ears. The general toxicity of
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Schinella et al. Prieto JM, Recio MC, Giner RM, Mez S, Massmanian A, Waterman PG, Ros JL. 1996. Topical antiinflammatory lignans from Haplophyllum hispanicum. Z. Naturforsch. 51c: 618-22. Schinella GR, Tournier HA, Prieto JM, Ros JL, Buschiazzo H, Zaidenberg A. 2002. Inhibition of Trypanosoma cruzi growth by medical plant extracts. Fitoterapia 73:569-575. Tselin AL, Vermel EM, Kuznetsova GA, Kuzmina LV, Markova LN, Medvedeva LI, Pimenov MG, Shagova LI. 1972. [Biological activity of phenolic and other natural compounds contained in different species of the genus Haplophyllum (Rutaceae family)]. Tr. Bot. Inst. Akad. Nauk SSSR, Ser.5, 16:3-6. Tutin, TG, Heywood, VH, Burges, NA, Moore, DM, Valentina, DH, Walters, SM, Webb, DA. 1968. Flora Europaea. Vol. II: Rosaceae-Umbelliferae. University Press, Cambridge
Haplophyllum extracts was reported by Tselin et al. (1972) and it may be due to the important cytotoxic activity of diphyllin and diphyllinin derivatives, first described in this genus by (Gonzlez et al., 1974) which can block the DNA synthesis in both normal and malignant cells in a similar fashion to justicidine A (Gonzlez et al., 1979). CONCLUSIONS Our results, and previous works, show that the crude methanolic extract of Haplophyllum linifolium (L.) G. Don and the diphyllin derivatives present therein are endowed with a strong and mild in vitro unspecific anti-trypanosome activity. The extract containing diphyllin derivatives has a potent topical anti-inflammatory effect when used at a single dose but causes necrosis of the skin, when applied chronically. People must avoid using this plant remedy both externally and internally without tight medical control. Acknowledges We are grateful to Dr. Jose Luis Rios, Dr. Rosa Giner, and Dr. Maria del Carmen Recio for their comments on the manuscript. REFERENCES
Cullar MJ, Giner RM, Recio MC, Mez S, Ros JL. 2001. Topical anti-inflammatory activity of some Asian medicinal plants used in dermatological disorders. Fitoterapia 72:221-229. Gonzlez AG, Darias V, Alonso G. 1979. Cytostatic lignans isolated from Haplophyllum hispanicum. Planta Med 36:200-203. Gonzlez Gonzlez A, Moreno Ordez R, Rodrguez LF. 1974. Lignans from Haplophyllum hispanicum. An. Quim. 70: 234-238. Prieto JM, Giner RM, Recio MC, Schinella G, Mez S, Ros JL. 2002. Diphyllin acetylapioside: a 5lipoxygenase inhibitor from Haplophyllum hispanicum. Planta Med. 68:359-360. Massmanian A, Prieto JM. 1996. Dermatitis de Contacto por Haplophyllum hispanicum. 2 parte. Perfil de una planta agresiva y curativa a la vez. Ponencia Cientfica, 36 Reunin del GEIDC, Grupo Espaol de Dermatitis de Contacto, Sevilla, Espaa, 4 Mayo. Mosmann TJ. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. Prieto JM, Giner RM, Recio MC, Schinella G, Mez S, Ros JL. 2002. Diphyllin acetylapioside, a 5lipoxygenase inhibitor from Haplophyllum hispanicum. Planta Med 68:359-360. Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (5) 2008
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TERCERA REUNION ANUAL DE BLACPMA

PRIMER CONGRESO INTERNACIONAL DE FARMACOBOTANICA DE CHILE TERCERA REUNION ANUAL DE BLACPMA CHILLN CHILE 6 AL 9 DE ENERO DE 2009 SEDE FACULTAD DE AGRONOMA UNIVERSIDAD DE CONCEPCIN SEDE CHILLN www.farmacobotanica.cl CONFERENCISTAS CONFIRMADOS INTERNACIONALES PETER TAYLOR (VENEZUELA) GEOFREY CORDELL (USA) JOS MARA PRIETO (INGLATERRA) GABINO GARRIDO (CUBA) VICENTE MARTINEZ (GUATEMALA) DAMARIS SILVEIRA (BRASIL) ALBERTO ANGEL GURNI (ARGENTINA) DORITA VIGNALE (ARGENTINA) GRACIELA PONESSA (ARGENTINA) ANA LADIO (ARGENTINA) MARA INS ISLA (ARGENTINA) MARCELO WAGNER (ARGENTINA) SANDRA PATRICIA GUZMAN (COLOMBIA) GABINO GARRIDO (CUBA) JANET PILOTO FERRER (CUBA) PATRICIA ARENAS (ARGENTINA) NACIONALES BRUCE CASSELS MARIO SILVA CARLOS CESPEDES EDUARDO SARU RENE TORRES HUGO MORALES CARLOS CESPEDES MARISOL BERTI HERMINE VOGEL SUSANA FISHER EDGAR PASTENE XIMENA POLANCO HERNAN SPEISKY - JUAN IZQUIERDO MANUEL CORTES MARA TERESA AEDO JULIO ALARCN ROBERTO RODRIGUEZ SIMPOSIOS TEMAS LIBRES EXPOSICIN COMIT ORGANIZADOR Anglica Urbina Presidente Magalis Bittner Vicepresidente Fidelina Gonzlez e Iris Pereira Secretarias Guisella Reyes Jos Luis Martnez Gloria Bernal Coordinadores Ral Cerda Carlos Calvo Alejandro Chanda Tesoreros Julio Alarcn Presidente BLACPMA 3 Nelsn Zapata Presidente Comisin Cientfica Nacional Mara Ins Isla Presidente Comisin Cientfica Internacional
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Eventos
II CONGRESO DE MEDICINA ALTERNATIVA 14 16 de Julio de 2008 Ciudad de Panam - Panam http://www.otramedicina.com/2007/12/11/ii-congreso-demedicina-alternativa-en-panama/ IX WORLD CONFERENCE ON CLINICAL PHARMACOLOGY AND THERAPEUTICS - CPT 2008 27 de Julio 1 de Agosto de 2008 Quebec Canad www.cpt2008.org ; cpt2008@ncr-cnrc.gc.ca XXVIII CONGRESO LATINOAMERICANO DE QUMICA 27 de julio 1 de agosto de 2008 San Juan, Puerto Rico progflaq2008@gmail.com VII JOINT MEETING OF AFERP, ASP, GA, PSE AND SIF ON NATURAL PRODUCTS WITH PHARMACEUTICAL, NUTRACEUTICAL, COSMETIC AND AGROCHEMICAL INTEREST. 3 8 de Agosto de 2008 Atenas Grecia www.jointmeeting.2008athens.gr afea.jointmeeting@2008athens.gr facabz@unicauca.edu.co IV CONGRESO LATINOAMERICANO Y DEL CARIBE DE CACTCEAS Y OTRAS SUCULENTAS XXX CONGRESO DE LA ORGANIZACIN INTERNACIONAL PARA EL ESTUDIO DE LAS PLANTAS SUCULENTAS LIX CONGRESO NACIONAL DE BOTNICA DE BRASIL 4 8 de Agosto de 2008 Natal Rio Grande do Norte Brasil aalbesiano@yahoo.com. VI SEMINARIO ESTUDIANTIL DE CIENCIAS BIOLOGICAS & III ENCUENTRO NACIONAL DE INVESTIGADORES 11- 15 de Agosto de 2008 Tunja Colombia semillerobiofilia@hotmail.com XII INTERNATIONAL SYMPOSIUM ON MICROBIAL ECOLOGY Cairns Australia 17 al 22 de Agosto de 2008 www.kenes.com/isme12 CURSO Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 MTODOS DE MODELAMIENTO DE DISTRIBUCIN DE ESPECIES Y SUS APLICACIONES II VERSIN 25 29 de Agosto de 2008 La Paz Bolivia http://www.geocities.com/cae.biologia VIII CONGRESO DE FICOLOGA DE AMRICA LATINA Y EL CARIBE 25 29 de Agosto de 2008 Lima Per. http://biologia.unmsm.edu.pe/sfalyc/prog.htm I CONGRESO DE ATENCIN PRIMARIA DE SALUD Talca Chile 28 al 30 de Agosto de 2008 www.congresoaps.ucm.cl 12TH INTERNATIONAL PALYNOLOGICAL CONGRESS 30 de Agosto 5 de septiembre de 2008 Bonn, Alemania http://www.paleontology.unibonn.de/kongress08/index.htm JORNADAS IBEROAMERICANAS SOBRE ETNOBOTANICA Y DESARROLLO SOCIAL 25 al 29 de Agosto de 2008 Antigua - Guatemala ndvignale@yahoo.com.ar; normahilgert@yahoo.com.ar SIMPOSIO INTERNACIONAL DE TOMATE EN EL TRPICO. Villa de Leyva, 9-13 de septiembre de 2008. Sociedad Colombiana de Ciencias Hortcolas http://www.soccolhort.com/tomato soccolhort@gmail.com Telfonos: (57-1) 3165000, Ext. 19041 y 19043 y (57-1) 8650218/19 V CONGRESO COLOMBIANO DE CROMATOGRAFA, SIMPOSIO IBEROAMERICANO DE EVALUACIN SENSORIAL (SENSIBER V); X CONGRESO COLOMBIANO DE FITOQUMICA 4 6 de septiembre de 2008. Bogot Colombia socolquim@gmail.com
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Eventos
SIMPSIO DE PLANTAS MEDICINAIS DO BRASIL & INTERNATIONAL CONGRESS OF ETHNOPHARMACOLOGY 16 19 de septiembre de 2008 Sao Paulo Brasil www.plantasmedicinais.unifesp.br plantasmedicinais@eventus.com.br CURSO "CONSERVACIN DE REAS IMPORTANTES PARA PLANTAS EN AMRICA LATINA" Santo Domingo Repblica Dominicana 28 de Septiembre 4 de Octubre cursoconservaciondeareas@gmail.com CURSO CONSERVACION DE LA BIODIVERSIDAD DE ANFIBIOS Copacabana Bolivia 29 de Septiembre 5 de Octubre www.durrellwildlife.org CONGRESO MUNDIAL PARA LA NATURALEZA 5 14 de octubre de 2008 Barcelona Espaa sonsoles.sanroman@iucn.org XLIII CONGRESO NACIONAL DE CIENCIAS BIOLGICAS 7 10 de octubre de 2008 Yopal Colombia www.asociacioncolombianadecienciasbiologicas.org VIII JORNADAS NACIONALES III CONGRESO INTERNACIONAL DE ENSEANZA DE LA BIOLOGA La Educacin en Biologa como Respuesta a la Demanda Social Mar del Plata, 9, 10 y 11 de Octubre de 2008 informacin: jneb8@yahoo.com.ar XVIII CONGRESO ASOCIACIN LATINOAMERICANA DE FARMACOLOGA III CONGRESO IBEROAMERICANO DE FARMACOLOGA XXX CONGRESO ANUAL DE LA SOCIEDAD DE FARMACOLOGA DE CHILE 12 15 de Octubre de 2008 Coquimbo, Chile. http://www.biologiachile.cl/socfarmch/. CONGRESO IBEROAMERICANO DE QUIMICA & XXIV CONGRESO PERUANO DE QUIMICA 13 17 de Octubre de 2008 El Cusco Per sqpcongreso@yahoo.com Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
1st INTERNATIONAL SYMPOSIUM ON YERBA MAT AND HEALTH School of Medicine (Facultad de Medicina) & School of Chemistry (Facultad de Quimica) October 17-18, 2008 Montevideo, Uruguay http://209.209.34.25/webdocs/ISYM/Home.html III CONGRESO INTERNACIONAL DE PLANTAS MEDICINALES 30 de octubre 1 de noviembre Palmira Colombia mssanchezo@palmira.unal.edu.co IV ENCUENTRO INTERREGIONAL DE INVESTIGACION EN EDUCACION Concepcin Chile 5 al 7 de Noviembre de 2008 www.enin.cl VI CONGRESO INTERNACIONAL DE FITOTERAPIA 7 9 de Noviembre De 2008 Oviedo Espaa www.astumed.org/congreso XII CONGRESO SOCIEDAD MESOAMERICANA PARA LA BIOLOGA Y LA CONSERVACIN 10 14 de Noviembre de 2008 San Salvador El Salvador www.smbcelsalvador2008.com WOCMAP IV 9 14 de Noviembre de 2008 Cape Town Sudafrica VIII CONGRESO LATINOAMERICANO DE HERPETOLOGA 24 29 de Noviembre de 2008 Topes de Collantes Sancti Spiritus Cuba http://fbio.uh.cu/herpetologia/index.htm 8voclah@fbio.uh.cu VIII CONGRESO NACIONAL DE FARMACOLOGA Y TERAPUTICA (CENTROFARMACOL 2008) Y II TALLER NACIONAL DE SERVICIOS FARMACUTICOS CLNICOS (SERVIFARMA 2008) 26 29 de Noviembre de 2008 Santa Clara Cuba servifarma@iscm.vcl.sld.cu V SIMPOSIO UNIVERSITARIO IBEROAMERICANO SOBRE MEDIO AMBIENTE 1 5 de Diciembre de 2008 La Habana, Cuba Informaciones: suima@quimica.cujae.edu.cu
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I CONGRESO INTERNACIONAL DE FARMACOBOTANICA DE CHILE TERCERA REUNION DE BLACPMA 6 9 DE ENERO DE 2009 CHILLAN CHILE www.farmacobotanica.cl FAPRONATURA 2009 17 20 de abril de 2009 Varadero - Cuba http://www.scf.sld.cu/fapronatura2009/fapronatura09.htm V INTERNATIONAL CONGRESS OF ETHNOBOTANY (ICEB 2009) TRADITIONS AND TRANSFORMATIONS IN ETHNOBOTANY 21 24 se Septiembre de 2009 San Carlos de Bariloche, Argentina pochett@fcnym.unlp.edu.ar aladio@crub.uncoma.edu.ar
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BLACPMA es la primera publicacin cientfica Latinoamericana sobre Productos Naturales con vocacin Internacional totalmente Independiente y de Libre Acceso. The Latin American Bulletin of Medicinal and Aromatic Plants is an independent, peer reviewed, open access, scientific journal for the advancement of natural products research in Latin America and beyond.
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ISSN 0717 7917 www.blacpma.

Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas

Bol. Latinoam. Caribe Plant. Med. Aromat. Vol.7 (4) 2008

Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008

Instrucciones para los autores

Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008

Guidelines for Authors

Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Review of Lansium domesticum Corra and its use in cosmetics

(Heyne, 1987; Verheij, 1992; MMPND, 2007)

Review of Lansium domesticum

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Review of Lansium domesticum

Review of Lansium domesticum

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Review of Lansium domesticum

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Review of Lansium domesticum

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Characterization of lemon verbena

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Characterization of lemon verbena

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Characterization of lemon verbena

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Characterization of lemon verbena

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Characterization of lemon verbena

Characterization of lemon verbena

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Antibacterial activity of E. caracasana Boiss and E. cotinifolia .

Antibacterial activity of E. caracasana Boiss and E.cotinifolia .

Klebsiella pneumoniae (ATCC 23357) Pseudomonas aeruginosa (ATCC 27853).

Results and discussion

Antifungal Activity of the Essential Oils of Two Verbenaceae

Antifungal Activity of the Essential Oils of Two Verbenaceae

Essential oil (g/mL)

672.0 0.2 0.1 0.1 0.2

180 100 130 110 140

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Antifungal Activity of the Essential Oils of Two Verbenaceae

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Antifungal Activity of the Essential Oils of Two Verbenaceae

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

N (%) 24 (38.1) 18 (28.6) 8 (12.7) 6 (9.5) 3 (4.8) 2 (3.2) 1 (1.6) 1 (1.6)

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Can form a viscous gel of the mucilage and gastric solution.

Matricaria chamomilla L. (camomila); Mentha spp. (hortel)

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Mentha pulegium L. (poejo) Mikania glomerata Spreng.(guaco);

Psychoactive substances Psychoactive substances

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Use of herbal medicine by AIDS patients from Hospital Universitrio de Braslia

Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008