Boletín Latinoamericanoy del Caribe de Plantas

Medicinales y Aromáticas

Volumen 7, Número 4, 2008

Martha TILAAR et al.Review of Lansium domesticum Corrêa and its use in cosmeticsBLACPMA, 7(4):183 - 189.

Susana Gattuso et al.Morpho-histological and quantitative parameters in thecharacterization of lemon verbena (Aloysia citriodora Palau) fromArgentina. BLACPMA, 7(4):190 - 198.

Janne ROJAS et al. Evaluation of antibacterial activity on different solvent extracts ofEuphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae) collected in VenezuelaBLACPMA, 7(4):199 - 202.

Tzasna HERNÁNDEZ et al. Antifungal Activity of the Essential Oils of Two Verbenaceae: Lantana achyranthifolia and Lippia graveolens of Zapotitlán de las Salinas, Puebla (México)BLACPMA, 7(4):203 - 207.

Paloma M. SALES et al. The use of herbal medicine by AIDS patients from Hospital Universitário de Brasília, BrazilBLACPMA, 7(4):208 – 217.

Ana Laura FAZIO, et al. An ethanolic extract of Uncaria tomentosa reduces inflammationand B16-BL6 melanoma growth in C57BL/6 mice. BLACPMA, 7(4):218 - 225.

Guillermo SCHINELLA et al.On the anti-trypanosomal, anti-inflammatory and toxicologicalactivities of Haplophyllum linifolium and its diphyllin derivatives.BLACPMA, 7(4):226 - 228.

ISSN 0717 7917www.blacpma.cl

Comité Editorial

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

Fundadores José L. Martínez (Chile) - Jorge Rodríguez (Cuba)

Editor Jefe José L. Martínez, Facultad de Medicina Veterinaria y Ciencias Pecuarias, UNICIT, Santiago, Chile.

Editor Jefe Científico José M. Prieto, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, Reino Unido.

Editores Ejecutivo Gabino Garrido, Centro de Química Farmacéutica, La Habana, Cuba. Damaris Silveira, Facultade de Ciências da Saúde, Universidade de Brasília, Brasil. Editores de Calidad Carla Delporte, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. Peter Taylor, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Editores de eventos María Inés Isla, Facultad de Farmacia y Bioquímica, Universidad Nacional de Tucumán, Argentina. Marcelo Wagner, Facultad de Farmacia y Bioquímica, 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 Martínez, Escuela de Agricultura, Universidad de San Carlos, Guatemala. Leonora Mendoza, Facultad de Química y Biología, Universidad de Santiago de Chile.

Editores Asesores Arnaldo Bandoni; Facultad de Farmacia y Bioquímica, 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 Morón, Laboratorio Central, Universidad de Ciencias Medicas de la Havana, Cuba. Patrick Moyna, Facultad de Química, Universidad La República, 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 à Pitre, Guadalupe Horacio Heinzein, Facultad de Química, Universidad La República, Montevideo, Uruguay.

Consejo Editorial Christian Agyare, College of Health Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutics, KNUST, Kumasi, Ghana. Julio Alarcón, Facultad de Ciencias Básicas, Universidad del Bio Bio, Chillán, Chile. Rocío Alarcón, Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, Reino Unido. Jorge Alonso, Asociación de Fitoterapia de Argentina, Buenos Aires, Argentina. Giovanni Apendino, DISCAFF, Universidad del Piemonte Oriental, Novara, Italia. Elizabeth Barrera, Sección Botánica, Museo Nacional de Historia Natural, Santiago, Chile. Geofrey Cordell, College of Pharmacy, Illinois University at Chicago, Estados Unidos. Rene Delgado, Centro de Química Farmacéuticas, La Habana, Cuba. Eduardo Dellacasa, Facultad de Química, Universidad de La República, Montevideo, Uruguay. Luis Doreste, Laboratorio Vitaplant, Mérida, Venezuela. Alina Freire-Fierro, Botany Department, Academy of Natural Sciences, Philadelphia, Estados Unidos. Mildred García, Escuela de Medicina, Universidad de Costa Rica Martha Gattusso. Área de Biología Vegetal, Universidad Nacional de Rosario, Argentina. Harold Gómez, Facultad de Ciencias Químicas y Farmacéuticas, Cartagena de Indias, Colombia. Peter Houghton, Pharmaceutical Sciences Research Division, King's College London, Reino Unido. Ana Ladio, Departamento de Ecología, Universidad Nacional del Comahue, San Carlos de Bariloche, Argentina. Patricia Landazuri, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia. Claudio Laurido, Facultad de Química y Biología, 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 Católica del Perú. Pedro Melillo de Magalhaes, Centro Pluridisciplinar de Pesquisas Químicas e Biológicas, UNICAMP, Campinas, Brasil. John A.O. Ojewole, Faculty of Health Sciences, University of KwaZulu-Natal, Sudafrica. Edgar Pastene, Facultad de Farmacia, Universidad de Concepción, Concepción, 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 Amazonía Peruana, Iquitos, Perú José Luís Ríos, Facultad de Farmacia, Universidad de Valencia, España Alicia Rodríguez, University of Havana, Havana, Cuba. Aurelio San Martín, Facultad de Ciencias, Universidad de Chile, Santiago, Chile Guillermo Schinella, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Argentina. Yen-Jen Sung, National Yang-Ming University Taipei, Taiwán Nilka Torres, Centro Regional Universitario de Azuero, Universidad de Panamá. René Torres, Facultad de Química y Biología, Universidad de Santiago de Chile. Carlos Urzúa, Facultad de Química y Biología, Universidad de Santiago de Chile. Beatriz Varela, Facultad de Farmacia y Bioquímica, 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 nido. UTalal Zari, Faculty of Science, King Abdulaziz University, Arabia Saudita.

BLACPMA

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

OBJETIVOS DEL BOLETÍN • Estimular a los grupos de trabajo existentes en

Latinoamérica, sean investigadores, productores, funcionarios o simplemente interesados en las plantas medicinales y aromáticas, poniendo a su disposición este Boletín para la difusión y la divulgación de sus investigaciones y de las actividades que en general desarrollen en torno a plantas.

• Ser una herramienta de difusión para la Sociedad Latinoamericana de Fitoquímica, principalmente, y de otras sociedades y agrupaciones que se sientan representadas por este Boletín.

• Constituir un nexo entre los profesionales de habla hispana, francesa, portuguesa e inglesa de la región, relacionados con el tema central del Boletín

El BOLETÍN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES Y AROMÁTICAS (BLACPMA), ISSN 0717 7917, es una publicación científica electrónica bimensual dirigida a diversos profesionales y técnicos vinculados al campo de las plantas medicinales y aromáticas. BLACPMA es una entidad sin ánimo de lucro. Aunque auspiciada por la Sociedad Fitoquímica Latinoamericana (SLF), este boletín no es propiedad de Club o Asociación alguna. Ni BLACPMA ni la SLF son responsables en ningún momento de las opiniones vertidas en sus páginas, que son responsabilidad única de sus respectivos autores. Todo el material gráfico ha sido creado de manera genuina o bien remitido por sus autores con el permiso de éstos. Todas las marcas y logos referidos en estas páginas son propiedad de sus respectivos autores o empresas. En Chile, 1 de Enero de 2008.

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 Fitoquímica, 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.

BLACPMA WEB Site: www.blacpma.clEnvio de trabajos Online a nuestra editorial Author’s 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™

Instrucciones para los autores

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

INSTRUCCIONES PARA LOS AUTORES El BOLETÍN LATINOAMERICANO Y DEL CARIBE DE PLANTAS MEDICINALES (BLACPMA), ISSN 0717 7917, es una publicación científica electrónica bimensual dirigida a profesionales y técnicos que trabajen tanto en productos naturales de plantas medicinales o nutracéuticos en general como en plantas medicinales y aromáticas. Serán aceptados aquéllos trabajos relacionados con alguna de las áreas abarcadas por el Boletín tales como agronomía, antropología y etnobotánica, aplicaciones industriales, botánica, calidad y normalización, ecología y biodiversidad, economía y marketing, farmacología, fotoquímica, farmacognosia, aspectos de regulación y legislación, información y difusión de eventos, cursos, premios, novedades, monografías, revisión de libros y cualquier otro tipo de material que se considere importante comunicar. tipo DE LA CONTRIBUCIÓN Los autores podrán presentar revisiones sobre un tema en particular así como un trabajo original de una investigación científica, en la forma de trabajo completo o comunicación corta. Cualquiera de estas contribuciones deberán estar escritas en español, inglés, portugués o francés, sin límite de extensión la cual deberá estar razonablemente ajustada al objetivo del trabajo. Sin embargo, los anuncios, novedades y eventos no deberán exceder una página. En todos los casos, las figuras están incluídas. FORMATO DE LA CONTRIBUCIÓN La contribución deberá realizarse mediante el uso del Documento Único para Autores, también abreviado ASP (Author’s 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 electrónico a blacpma_editorial@hotmail.com . El estilo de nuestra revista se detalla a continuación: Los trabajos serán presentados en formato Microsof Word (versión 3.1 o superior usando Times New Roman número 11). Los trabajos constarán de Introducción, Material y Métodos, resultados, Discusión, Conclusiones y Bibliografía. En cualquiera de las modalidades en las que se presente el trabajo, en la primera página deberá constar el Título del trabajo (en español y en inglés), autores, institución a la que pertenecen, la dirección y correo electrónico del autor principal. También deberá llevar un resumen en español y en inglés de no más de 100 palabras, un título corto y un máximo de 6 palabras clave. Los números de las tablas y figuras deben ser arábigos. Resumen Deberá llevar no más de 150 palabras e incluir los métodos usados, los resultados relevantes y las conclusiones. Texto Artículos originales: constarán de Introducción, Materiales y Métodos (descripción extensa), Resultados (referidos a las tablas y figuras), Discusión (extensión libre), y Conclusiones (lo más corta posible). Revisiones estarán estructuradas de acuerdo a las necesidades del autor. Comunicaciones breves o notas: deberán llevar una breve Introducción, Materiales y Métodos (breve descripción o sólo referencia al protocolo publicado), Resultados y Conclusión. El nombre completo de la especie en latín y la familia (ej: Inula viscosa (L.) Aiton. – Asteraceae) deberán ser mencionados in extenso al menos en la sección Materiales. A lo largo del trabajo sólo se usará el nombre corto en latín (I. viscosa) Tablas Las tablas deberán ser escritas usando un procesador Word y nunca seran figuras. Favor de no usar otras líneas distintas de las negras de 1 pt. El texto deberá estar en Times New Roman 10 ó 9 puntos. Incluir siempre Título (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 número y color de las figuras, pero la inclusión de cualquier figura debe estar justificada. No es posible publicar una imagen que haya sido copiada de otra publicación..Sólo es posible publicar copias de imágenes libres de derecho de autor, de lo contrario deberán ser rediseñadas 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 deberán incluir el apellido del autor y el año, separado por coma y colocados entre paréntesis (ej. Bruneton, 1995); si hay más de un trabajo del mismo autor, se separarán por comas (ej. Bruneton, 1987, 1995, 2001). Si hay dos autores se citarán separados por “y” o su equivalente, respetando el idioma original de la fuente. Si hay más de dos autores, sólo se citará el primero seguido de la expresión et al. En tanto que en bibliografía deberán figurar todos los autores. Si hay varios trabajos de un mismo autor y año, se citará con una letra en secuencia adosada al año (ejemplo: Mayer et al. 1987a, 1987b). Si un trabajo no tiene autor se lo citará como Anónimo, seguido de la fecha de publicación. Si hubiera más de una cita de esta tipo en el mismo año, se adosará una letra correlativamente (ejemplo: Anónimo, 2002a, Anónimo, 2002b). La bibliografía incluirá SÓLO las referencias mencionadas en el texto, ordenadas alfabéticamente por el apellido del primer autor, sin número que lo anteceda y sin sangría. Apellido/s del autor seguido de las iniciales del nombre sin puntos ni separación entre ellas. El nombre de la revista se colocará abreviado según normativas ISO de acuerdo con el Botanico Periodicum Huntianum (disponible solamente en edición 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 número de revistas. Por último se citará el volumen de la publicación, seguido del número entre paréntesis, dos puntos y el número de páginas desde x hasta y, sin espacios entre medio. Las citas de libros deben explicitar las páginas consultadas así como el año de edición. No se admitirán citas incompletas y el incumplimiento de estas normas será motivo de retraso del artículo hasta su corrección.

Modelos Publicaciones periódicas Grove H, Rovirosa J, San Martin TO, Argandoña V. 1994. Secondary Metabolites of Dictyota crenulata. Bol. Soc. Chil. Quím. 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. Capítulos 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, Bogotá, Colombia. Tesis (aceptable sólo si no hay fuente alternativa) González of Cid D. 2000. Cianobacteria study with noxious effects (deleterious and toxic) in aquatic atmospheres of the county of San Luís. Doctoral thesis, National University of San Luís, 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 resúmenes fueron publicados en una revista, se menciona SÓLO la revista como si fuera un artículo más.

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 electrónicos Nota: si es necesario cortar alguna dirección se recomienda hacerlo después de una barra inclinada. ATENCIÓN: 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 atención a la dirección 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&RevisionSelectionMethod=Latest. [Consulted October 6, 2006].

En caso de no haber un autor, o cuando no hay un responsable principal, se toma la institución 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 Bálsamo de Fierabrás. BLACPMA 4(3):48-51.

Importante NOTA sobre la citación de páginas Web En éstos días se está comprobando el creciente ABUSO de la citación de páginas Web para avalar afirmaciones científicas hechas por los autores . resulta muy peligroso para su credibilidad como autor, y APRA la credibilidad de este Boletín, citar información obtenida en páginas Web que no tengan ninguna entidad científicamente reconocida que se haga responsable de la susodicha información. Las páginas Web “anónimas” sólo deben ser usadas en casos muy justificados y ante la absoluta ausencia de ninguna otra fuente primaria científicamente reconocida. El Comité editorial de esta revista realizará todo esfuerzo para eliminar el recurso fácil a páginas Web seudocientíficas y desde luego los autores deben dar en todo caso una explicación de por qué han recurrido a éste tipo de fuente. Todo abuso será motivo de rechazo para su publicación, incluso si este ya fue (erróneamente) aceptado por los revisores. Si se trata de boletines o revistas on-line con ISSN, la fuente debe ser citada como cualquier otra revista. Envío de los trabajos y procedimiento de edición Se podrán enviar tanto por correo electrónico a la dirección blacpma_editorial@hotmail.com o por correo aéreo en disco de 3.5 pulgadas a Lic. José Luis Martínez, Editor, Casilla de Correo 70036, Santiago 7, Chile. Los trabajos se acompañarán de una lista conteniendo el correo electrónico y la dirección de TODOS los autores. El autor principal será el responsable de manifestar su conformidad en nombre de todos los autores, en relación a la publicación en BLACPMA así como de cualquier problema que se origine por la autoridad y/o originalidad del trabajo. Esto quedará claramente establecido en una nota formal que acompañará el trabajo enviado. Una vez recibido, el trabajo será arbitrado por un par de revisores, que podrán ser miembros de nuestro comité editorial, académicos o profesionales reconocidos, quienes decidirán su aprobación o rechazo. De todas maneras, el editor tiene la facultad para decidir si el trabajo cumple con el enfoque del boletín y tiene la libertad de modificar el manuscrito definitivo (ver el apartado siguiente). Autoridad final del Comité Editorial. Los editores se reservan el derecho de corregir o modificar el manuscrito aceptado para su publicación en BLACPMA, previa consulta con el autor para que se adecue mejor al estilo y objetivos del boletín. Este procedimiento tendrá lugar en aquellos casos en que los manuscritos no concuerdan con los modelos científicos generalmente aceptados o si el contenido es innecesariamente largo, redundante o no suficientemente claro. Estas modificaciones pueden ser requeridas directamente a los autores y podrán retrasar la publicación del manuscrito.

Gracias por su importante contribución y por tener en cuenta estas normas.

Comité Editorial BLACPMA

Guidelines for Authors

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

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 (Author’s 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 author’s 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. Species’s 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 abbreviation’s 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 re-draw 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 journal’s 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.

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EDITORIAL

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 182

BLACPMA DESDE EL 2009 SERA MAS GRANDE Si miramos hacia el año 2002 en Buenos Aires, Argentina en donde gracias a una invitación del Dr. Arnaldo Bandoni se realizó la presentación de BLACPMA en el Congreso Latinoamericano de Fitoquímica nos damos cuento que en poco más de 6 años hemos alcanzado un sitial espectacular y que día a día vamos ascendiendo cada vez mas. El titulo de esta editorial obedece que a contar de 2009 cuando entremos al volumen 8 nuestra página Web oficial dejara de ser www.blacpma.cl y pasará a ser www.blacpma.org y en donde todos los miembros del Comité Editorial podrán 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 Chillán (Chile) y que corresponderá a la Tercera Reunión de BLACPMA (Varadero 2006, La Plata 2007). El sábado 26 de Julio se realizó la última reunión del Comité Organizador en el Centro de Biología Marina en Dichato, hermoso lugar al lado del mar. Entre las decisiones se analizaron las mesas redondas que se realizarán una de las cuales estará a cargo de BLACPMA y que coordinará el Dr. Julio Alarcón las otras estarán a cargo de especialistas chilenos, si bien la única que esta ya confirmada en su totalidad es la que coordina el Dr. Edgar Pastene (Chile) y también miembro de BLACPMA y en donde participara nuestro Editor Jefe Científico Dr. José María Prieto sobre antioxidantes. Las otras mesas redondas serán sobre Fitoquímica, Biotecnología, Agronomía y Etnobotánica. Del mismo modo la Conferencia de Clausura será dictada por otro miembro de BLACPMA el Dr. Gabino Garrido de Cuba. Además de nuestra familia BLACPMAeña participaran varios con Conferencias, entre ellos estarán Rene Torres (Chile), Leonora Mendoza (Chile), Damaris Silveira (Brasil), Vicente Martínez (Guatemala), Marcelo Wagner (Argentina), María Inés Isla (Argentina), Patricia Arenas (Argentina), Geofrey Cordell (USA), entre otros. Debo agradecer a través de estas líneas la amistad que me une desde hace ya varios años con la Dra. Marlene Porto, Directora del Centro de Investigación y Desarrollo de Medicamentos de Cuba, quien ha permitido que varios científicos de su centro hayan venido a diversos eventos en los cuales he estado ligado, es así 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. También y volviendo hacia nuestra publicación, BLACPMA ha sido aceptada en BVS (Biblioteca Virtual em Saúde – Brasil). Esta ha sido otra gestión de nuestra activísima compañera Damaris Silveira. Finalmente deseo agradecer a través de estas líneas la participación en los últimos años de la Dra. Patricia Arenas, de la Universidad Nacional de La Plata (Argentina), quien por motivos laborales y familiares se alejará de nuestra línea editorial pero que seguirá ligada como evaluadora, será reemplazada a partir de 2009 por la Dra. María Inés Isla (Universidad Nacional de Tucumán, Argentina) quién hasta ahora se desempañe como Editora encargada de resúmenes de Congresos. En su cargo asumirá el Dr. Marcelo Wagner (Universidad de Buenos Aires, Argentina), quien será acompañado en dicha labor por el Dr. Harold Gómez (Universidad de Cartagena de Indias, Colombia). Esperando comunicarnos en el próximo número de BLACPMA, les saluda a nombre del Comité Editorial

José Luis Martínez, Editor Jefe José María Prieto, Editor Jefe Científico

Gabino Garrido, Editor Asociado Damaris Silveira, Editor Asociado

© 2008 Los Autores Derechos de Publicación © 2007 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (4), 183 - 189

BLACPMA ISSN 0717 7917

Revisión | Review

Review of Lansium domesticum Corrêa and its use in cosmetics [Revisión de Lansium domesticum Corrêa y sus usos en cosmética]

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.idReceived 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 botánica y fitoquímica así como sus usos medicinales, nutricionales y cosméticos, que incluyen efectos de antioxidantes, hidratantes, despigmentantes e iluminadores del cutis son revisados. Esta planta puede ser de interés para la comunidad latinoamericana ya que puede adaptarse y crecer en algunas partes de América.

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 Corrêa 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

B a r k : 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. L e a v e s :

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. F l o w e r s :

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, greenish-yellow; 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).

S e e d s : 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

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 Florida’s Agricultural Research and Education Center in Homestead, but the trees have

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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 μg/ml (Saewan, 2006).

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Three new onoceranoid triterpenes, lansionic acid (17), 3 β -hydroxyonocera-8(26),14-dien-21-one, and 21α-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).

17. Lansionic acid

(1-16 from the authors, 17 from PubChem Substance Database)

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*

Moisture 86.5 g Protein 0.8 g Carbohydrates 9.5 g Fiber 2.3 g Calcium 20.0 mg Phosphorus 30.0 mg Carotene (Vit.A) 13.0 I.U. Thiamine 89 mcg Riboflavin 124 mcg Ascorbic Acid 1.0 mg Phytin mg (dry

weight) *According to analysis made in India. The edible flesh may constitute 60% of the fruit. (Morton, 1987)

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

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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 (Hen’s 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. CTFA’s 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 1126-1128.

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. Hen’s 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.

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© 2008 Los Autores

Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (4), 190 - 198 BLACPMA ISSN 0717 7917

Artículo Original | Original Article

Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora palau) from Argentina.

[Parámetros morfo-histológicos cuantitativos en la caracterización de lemon verbena (Aloysia citriodora palau) de Argentina]

Susana GATTUSO1*, Catalina M. van BAREN2, Alejandra GIL3, Arnaldo BANDONI2; Graciela FERRARO2 y Martha GATTUSO1

1. Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario Suipacha 531, S2002LRK, Rosario, Argentina.

2. Cátedra de Farmacognosia-IQUIMEFA. Facultad de Farmacia y Bioquímica.UBA-CONICET. Junín 956, 2º piso. C1113AAD Buenos Aires Argentina.

3. Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 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 realizó la identificación micrográfica de numerosos especímenes provenientes de áreas de crecimiento más representativas de nuestro país, con el propósito de definir la calidad de un lemon verbena, típicamente argentino. El material vegetal fue fijado, seccionado a mano alzada y teñido con Safranina-Fast green. Posee hojas simples, sus láminas presentan estomas anomocíticos en su hipofilo, pelos glandulares, eglandulares y un mesófilo dorsiventral. El tallo, de escaso crecimiento secundario, posee epidermis uniestrata, colénquima angular y haces colaterales abiertos. La caracterización morfoanatómica, el índice estomático, la proporción de empalizada y las magnitudes de los elementos foliares y caulinares que se determinaron, contribuyen a la correcta identificación del material vegetal.

Palabras clave: Aloysia citriodora Palau, estandarización, lemon verbena, morfo-anatómico.

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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'Hér.) Britt., Verbena triphylla L'Hérit., Zapania citriodora Lam., Lippia citriodora (Ort.) H.B.K., Lippia triphylla (L'Hérit) 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”, “cedrón”, “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 Martí, 1985), antispasmodic (Arias and Costas, 1948, Torrent Martí, 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

climatic conditions of their habitat (Molina et al., 2003) our research group has undertaken this study in order to define analytical parameters for the morpho-histological 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 Jerónimo 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 Agronomía (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 μm 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 μm 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, O´Brian and McCully, 1981). Original drawings were made with a drawing device. Symbols proposed by

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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 Scheffe’s 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, greenish-mauve corolla divided into 4 acuminate lobes (Fig. 1 A)

Plant material of interest Dried leaves. They are thin and fragile, deep-

green 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. Well-developed areola randomly arranged, quadrangular, polygonal (Fig 1, C).

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: a- the 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. .

Characterization of lemon verbena Gattuso et al.

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, J-K, M-S. 4 to C.

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Characterization of lemon verbena Gattuso et al.

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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Characterization of lemon verbena Gattuso et al.

Tabla 1: Stomatic index and pallisade ratio data of reference plant material, experimental samples and the different materials studied.

Origin of the material Stomatic index Pallisade ratio Buenos Aires 5,45 (7,05) 10,20 8,00 (9,31) 10,50 Santa Fe 5,16 (9,23) 13,43 5,50 (6,70) 8,25 San Luis 8,33 (10,38) 12,69 9,25 (10,60) 12,25 Mendoza 8,64 (10,35) 12,09 11,50 (11,81) 12,50 Salta 8,93 (10,73) 13,73 7,50 (8,20) 8,75 Jujuy 7,27 (9,67) 11,11 10,75 (14,30) 17,25

Interval for Argentine Material 5,16 - 13,73 5,50 - 17,24 Rancagua (Chile) 7,14 (8,97) 11,67 8,25 (9,65) 11,75 Talca (Chile) 5,77 (7,57) 9,26 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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Characterization of lemon verbena Gattuso et al.

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).

Dissociated stems The following cellular elements can be observed

in this preparation: 632 µm long fibers (Fig.1 N); long vessels with appendage and simple terminal plates measuring 360 x 56 µm (Fig. 1 O); tracheids measuring 552 µm (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

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

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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 µm appendix, 552 µm tracheids and 632 µm 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.

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ArtículoOriginal | Original Article

Evaluation of antibacterial activity on different solvent extracts of Euphorbia caracasana Boiss and Euphorbia cotinifolia L. (Euphorbiaceae)

collected in Venezuela

[Evaluación 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 DÍAZ2, Gina MECCIA 1

1. Organic Biomolecular Research Group, Research Institute, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mérida, Venezuela.

2. Microbiology and Parasitology Department, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mérida, 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 μg/mL and 33,6 μg/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 investigación se evaluó la actividad antibacteriana de Euphorbia caracasana Boiss y Euphorbia cotinifolia L. contra bacterias Gram positivas y Gram negativas usando el método de difusión en agar con discos de papel. Las hojas de E. caracasana y E. cotinifolia fueron extraídas con alcohol isopropílico. Los extractos, luego de concentrados, fueron separados por cromatografía en columna y las fracciones eluídas de ésta, fueron seleccionadas basandose en el perfil cromatográfico TLC, y analizadas para determinar su actividad antibacteriana. Solo las fracciones eluídas con diclorometano 1:1 de la cromatografía en columna del extracto de E. caracasana, mostraron actividad antibacteriana contra S. aureus ATCC 25923 a las concentraciones de 336 μg/mL y 33,6 μg/mL (dilución 1:10 del mismo extracto). Las fracciones eluídas con n-hexano-diclorometano 1:1 y diclorometano de la misma columna, no mostraron actividad antibacteriana. Ninguna de las fracciones eluídas de la separación cromatográfica de E. cotinifolia mostraron actividad antibacteriana en las bacterias ensayadas.

Palabras clave: Actividad Antibacteriana, Euphorbia caracasana, Euphorbia cotinifolia, Euphorbiaceae, Staphylococcus aureus.

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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, PGE2-inhibitory, 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, Mérida 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-hexane-dichloromethane (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 μm thick were utilized and different mixtures of n-hexane, 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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Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (x) 2008 200

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

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 Müeller-Hinton broth at 37 ºC for 18 hours. The bacterial inoculum was diluted in sterile 0.85 % saline to obtain turbidity visually comparable to a McFarland Nº 0.5 standard (106-8 CFU/mL). Every inoculum was spread over plates containing Müeller-Hinton agar. Paper filter discs (6 mm) saturated with 20 μL of every selected fraction, eluted from the column chromatography with the following solvent mixtures (n-hexane/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 ºC for 18 h and finally incubated at 37 ºC 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 μg/10 μg) for S. aureus (inhibition zone 50 mm), Vancomycin® (30 μg) for E. faecalis (inhibition zone 30 mm), Gentamicin® (10 μg) for E. coli (inhibition zone 42 mm), Cefotaxime® (30 μg) for K. pneumoniae (inhibition zone 40 mm) and Cefoperazone® (75 μg) 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.

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 μg/mL (inhibition zone 10 mm) and 33,6 μg/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

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; Gonçalves 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; Velásquez 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 Tecnológico (CDCHT) Mérida, Venezuela for the financial support (FA-304-03-08-A) of this investigation.

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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. Springer-Verlag, New York, pp. 1-99.

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Hohmann J, Re´dei D, Forgo P, Molna´r 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: 483–516.

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

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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. Epidemiología de infecciones nosocomiales por Staphylococcus aureus en una unidad de alto riesgo neonatal. Enfer Infecc Microbiol Clin. 20: 321-325.

Velazquez-Meza M, Aires de Sousa M, Echaniz-Aviles G, Solórzano-Santos F, Miranda-Novales G, Silva-Sanchez 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.

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Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (4), 202 - 206 BLACPMA ISSN 0717 7917

ArtículoOriginal | Original Article

Antifungal Activity of the Essential Oils of Two Verbenaceae: Lantana achyranthifolia and Lippia graveolens of Zapotitlán de las Salinas, Puebla

(México) [Actividad antifúngica del aceite esencial de dos Verbenaceae: Lantana achyranthifolia and Lippia graveolens de

Zapotitlán de las Salinas, Puebla (México)] Tzasna HERNÁNDEZ *, Margarita CANALES, Ana Maria GARCÍA , Ángel DURAN, Samuel MERÁZ, Patricia DÁVILA,

J. Guillermo ÁVILA

1. Laboratorio de Fitoquímica, Unidad de Biología, Tecnología y Prototipos (UBIPRO), Facultad de Estudios Superiores Iztacala (FES Iztacala) Universidad Nacional Autónoma de México (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= 10–90 μg/mL) than L. achyranthifolia (IC50= 100–180 μg/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 antifúngica de los aceites esenciales de Lantana achyranthifolia y Lippia graveolens. Los aceites esenciales de la parte aérea de L. achyranthifolia y L. graveolens fueron obtenidos mediante arrastre de vapor y examinados por GC y GC-MS. La actividad antifúngica fue evaluada frente a cinco cepas de hongos con el método de difusión en agar. La concentración inhibitoria media (IC50) se determinó mediante el método de inhibición del crecimiento radial. Los aceites exhibieron actividad antifúngica contra las cepas desafiadas. El aceite esencial de L. graveolens presentó mayor actividad (IC50= 10–90 μg/mL) que L. achyranthifolia (IC50= 100–180 μg/mL). Con el presente estudio se valida el uso de L. achyranthifolia y L. graveolens en la medicina tradicional para el tratamiento de enfermedades dermatológicas y podría ser una buena opción para el control biológico de cepas fitopatógenas.

Palabras clave: Actividad antifúngica, 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 Zapotitlán de las Salinas, Puebla (July 2001) and were identified by Dra. Edith López Villafranco. Voucher specimens were deposited in the herbarium IZTA of the Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (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 4°C 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 8°C/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 (INIFAP-Texcoco). 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 μL 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 p-values 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%), α-bisabolol (11.23%) and β-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%), α-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%), α-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%), α-terpinyl acetate (22.35%), m-cymene (20.42%) and thymol (6.72%).

Table 1. Composition of essentials oils of L. achyranthifolia and L. graveolens.

Compounds L. achyranthifolia L. graveolens RT (min) % RT (min) % Thujene - - 4.51 1.03 β-pinene - - 6.13 2.54 m-cymene - - 7.30 20.421,8-Cineole 7.86 5.03 - - α-terpinyl acetate 8.52 0.74 8.16 22.35Linalool 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 37.84Thymol - - 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 - - β-Bisabolene 19.68 5.68 - - α-Bisabolol 19.88 11.23 - - β-Cadinene 20.11 2.77 - - Himachalene 20.38 1.17 - - Cedrene 25.67 0.43 - - Total 82.02 94.58Compounds are listed considering the order of elution from a DB WAX column. RT, retention time.

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.

Controls (μg/mL)

Essential oil (μg/mL) Strain

Ketoconazole L. achyranthifolia L. graveolens Aspergillus niger 672.0 180 50 Fusarium moniliforme 0.2 100 50 Fusarium sporotrichum 0.1 130 90 Trichophyton mentagrophytes

0.1 110 20

Rhyzoctonia solani 0.2 140 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 μg/mL), than L. achyranthifolia (IC50 = 100-180 μg/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, α-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 μg/mL, 10 μg/mL respectively). Aspergillus niger turned out to be more sensitive to the essential oil of L. achyranthifolia (IC50 = 180 μg/mL) and L. graveolens (IC50 = 50 μg/mL), than the positive control (ketoconazole IC50 = 612 μg/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 Rocío Serrano and

Edith López Villafranco for their technical assistance and to Javier Pérez and Luis Velasco for their support on gas-mass measurement. This research has been supported by UNAM-PAPCA (2007-2008).

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Salgueiro LR, Cavaleiro C, Goncalves MJ, Proenca da Cunha A. 2003. Antimicrobial activity and chemical composition of the essential oil of Lippia graveolens from Guatemala. Planta Med. 69:80-83.

Tequida-Meneses M, Cortez-Rocha M, Rosas-Burgos C, Lopez-Sandoval S, Corrales-Maldonado C. 2002. Efecto de extractos alcoholicos de plantas silvestres sobre la inhibición de crecimiento de Aspergillus flavans, Aspergillus níger, Penicilliun expansum, Fusarium moniliforme y Fusarium poae. Rev. Iberoamericana de Micología 19:84-88.

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ArtículoOriginal | Original Article

The use of herbal medicine by AIDS patients from Hospital Universitário de Brasília, Brazil

[La utilización de hierbas medicinales por pacientes con SIDA del Hospital Universitario de Brasilia, Brasil] Paloma M. SALES*, Patricia M de SOUSA, Celeste A. da SILVEIRA, Dâmaris SILVEIRA*

1. Faculdade de Ciências da Saúde Universidade de Brasília. Asa Norte, Brasília, 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 Universitário de Brasília (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 epidemiológico fue realizado con el objetivo de evaluar la utilización y potencial interacción farmacológica entre hierbas/plantas medicinales y fármacos antiretrovirales prescritos a pacientes infectados con el VIH en el Hospital Universitario de Brasilia (HUB). Un cuestionario fue elaborado abordando preguntas sobre las características sociodemográficas, el tiempo de diagnóstico, como los pacientes se enteraron de sus enfermedades, el tiempo de tratamiento con los fármacos antiretrovirales, y los efectos adversos más 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 prácticas 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 farmacológicas con la farmacoterapia antiretroviral usualmente prescrita a los pacientes entrevistados.

Palabras clave: SIDA, plantas medicinales, hierbas medicinales, interacción fármaco-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 Universitário de Brasília (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 Ambulatório do Hospital Universitário de Brasília (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 socio-demographic 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 Ciências da Saúde, Universidade de Brasília.

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 HIV-infected patients that reported the use of medicinal plants and/or phytomedicine

Parameters Number (%) Average age (± SD)* 38.8 (±9.49) Male/ Female 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 institution

4 (6.0)

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

N (%)

Gastrointestinal disturbances 24 (38.1)Pleasure or personal satisfaction 18 (28.6)Pain and inflammation control or colds 8 (12.7)Increase on the immunological response 6 (9.5)Sedative and/or sleep inducer 3 (4.8)Gastrointestinal disturbances and personal satisfaction 2 (3.2)Weight reduction 1 (1.6)Sedative and/or sleep inducer and due to gastrointestinal disturbances 1 (1.6)

*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 socio-demographic 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 cachaça), 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) Possible action Interaction outcome References Ilex paraguariensis (erva mate) Chenopodium ambrosioides Bert. Ex Stend. (erva de santa maria); Paullinia cupana Kunth (guaraná); Smilax spp. (salsaparrilha); Camellia sinensis L. (chá preto)

Can promote unbalance of the acid-gastric buffer.

Cause increase of gastrointestinal discomfort and alteration in the ARV absorption

(Fetrow and Avila, 2000; Cowl, 2003; Philp, 2004; Simões et al., 2004; Taketa et al., 2004)

Symphytum officinale L. (confrei); Sambucus nigra L. (sabugueiro); Hymenaea courbaril L. (jatobá); Gossypium barbadense L. (algodão)

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

Can reduce ARV absorption, either by the revetment of the gastric wall or by the adsorption of the medicine.

(Fetrow and Avila, 2000; Abdel-Kader et al., 2002; Cowl, 2003; Lima-Nishimura et al., 2003; Philp, 2004; Santos et al., 2004; Simões et al., 2004)

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

Antispasmodic Can reduce the motility of the gastrointestinal tract causing reduction of ARV absorption.

(Fetrow and Avila, 2000; Butterweck et al., 2004; delCastillo et al., 2004; Unger and Frank, 2004)

Aloe spp. (babosa) Can increase the gastrointestinal peristaltic movement

Can promote changes in the ARV absorption due the increasing on the gastric emptying.

(Fetrow and Avila, 2000; Simões et al., 2004).

Allium cepa L.( cebola); Eucalyptus globulus Labill. (eucalipto)

Quercetin associates to the plasmatic protein in around 98%.

Can promote competition with ARV for the bond to the plasmatic protein.

(Havey, 1999; Fetrow and Avila, 2000; Philp, 2004; Simões et al., 2004)

Ilex paraguariensis (erva mate); Serenoa repens (saw palmetto); Sambucus nigra L. (sabugueiro); Maytenus ilicifolia Mart. ex Reiss (espinheira santa); Paullinia cupana Kunth (guaraná); Camellia sinensis L. (chá preto) Eucalyptus globulus Labill. (eucalipto)

Formation of tannin insoluble complexes with proteins.

Can increase the bioavailability of PI promoting higher bond to the plasmatic protein.

(Fetrow and Avila, 2000; Izzo and Ernst, 2001; Cowl, 2003; Ohsaki et al., 2004; Philp, 2004; Simões et al., 2004; Taketa et al., 2004)

Matricaria chamomilla L. (camomila) Mentha spp. (hortelã) Uncaria tomentosa (Willd. Ex Roem. & Schult.) DC. (unha de gato)

Inhibition of CYP450, in vitro.

Can increase the ARV bioavailability and/or toxicity.

(Fetrow and Avila, 2000; Williams, 2001; Cowl, 2003; Butterweck et al., 2004; Philp, 2004; Simões et al., 2004)

Allium sativum L. (alho)

Induction of CYP450. Cmax SQV: reduction of 54% AUC SQV: reduction of 51% Cmax PI: reduction

(Santos et al., 2004; Santos and Boullata, 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

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Table 4 – Plants and phytomedicines that can cause side effects synergism

Latin name (vernacular) Possible action Interaction outcome References Peumus boldus Molina (boldo do chile) Anticholinergic action Can increase neuropsychiatric EFV

side effects. (Fetrow and Avila, 2000).

Symphytum officinale L. (confrei) Hepatotoxicity Can increase NRTI, NNRTI and PI-induced hepatic damage.

(Fetrow and Avila, 2000; Philp, 2004; Simões et al., 2004)

Mentha pulegium L. (poejo) Hepatotoxicity Can increase NRTI, NNRTI and PI-induced hepatic damage.

(Fetrow and Avila, 2000; Lorenzo et al., 2002)

Mikania glomerata Spreng.(guaco);

Formation of chelate by cumarin and iron ions

Can promote pancytopenia with NRTI, NVP and PI.

(Fierro et al., 1999)

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) Phytochemicals Interaction outcome References

Ilex paraguariensis (erva mate); Paullinia cupana Kunth (guaraná); Camellia sinensis L. (chá preto).

Psychoactive substances

Can increase neuropsychiatric EFV side effects.

(Fetrow and Avila, 2000; Cowl, 2003; Philp, 2004; Simões et al., 2004; Taketa et al., 2004)

Operculina alata (Ham) Urban. (“Batata de Tiú”)

Psychoactive substances Can increase neuropsychiatric EFV side effects.

(Simões et al., 2004)

Myristica fragrans Houtt (nutmeg) Psychoactive substances Can increase neuropsychiatric EFV side effects.

(Fetrow and Avila, 2000; Cowl, 2003; Simões et al., 2004)

EFV=efavirenz;

Our research revealed that the recommendations for the use of medicinal plants and/or herbal remedies were usually obtained from relatives (great-grandparents, 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 HIV-infected 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, anti-inflammatory 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 anti-retroviral 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 Único de Saúde - 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)

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BLACPMA ISSN 0717 7917

ArtículoOriginal | Original Article

An ethanolic extract of Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth in C57BL/6 mice.

[Un extracto etanólico de Uncaria tomentosa reduce la inflamación y el crecimiento del melanoma B16/BL6 en ratones C57BL/6]

Ana Laura FAZIO1, Diana BALLÉN2, Italo M. CESARI2, María Jesús ABAD1, Miriam ARSENAK1, Peter TAYLOR1

1. Centro de Medicina Experimental , Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A, Venezuela.

2. Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A, 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 (Cat’s Claw – Uña 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-α], 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 κB (NF-κB) activity in LPS-stimulated HeLa cells. UT did not show an important cytotoxic effect in vitro at the doses up to 300 μg/ml, but did inhibit tumor growth and metastasis in vivo. UT inhibited TNF-α, IL-6 and NO production in vitro. NF-κB 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-κB, inflammation, B16/BL6 mouse

Resumen Los extractos de la corteza de Uncaria tomentosa (Uña de Gato) han sido usados tradicionalmente por sus propiedades anti-inflamatorias y antitumoral. Investigamos el efecto de un extracto hidroetanólico (UT) de U. tomentosa sobre: a) la viabilidad de células normales y tumorales, b) la respuesta inflamatoria (factor de necrosis tumoral alfa (TNF-a), interleuquina-6 (IL-6) y óxido nítrico (ON) in vitro e in vivo, c) crecimiento y metástasis de células de melanoma B16/BL6 en ratones C57BL/6, y d) actividad del factor nuclear κB (NF-κB) en células HeLa estimuladas con LPS. UT no mostró un efecto citotóxico importante in vitro hasta 300 µg/ml, pero si inhibió el crecimiento de tumor primario y de metástasis in vivo. UT inhibió la producción de TNF-α, IL-6 y ON in vitro. La actividad de NF-κB también resultó inhibida. Nuestros estudios muestran que UT contiene compuestos que ameritan ser más estudiados por sus efectos sobre procesos comunes a inflamación y cáncer.

Palabras clave: Uncaria tomentosa, cáncer, NF-κB, inflammación, 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 anti-inflammatory activity both in vitro (Sandoval-Chacón 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-κB (Sandoval-Chacón 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 Fazio et al.

inhibited the TNF-α 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-κB.

MATERIALS AND METHODS

Plant material. The bark of Uncaria tomentosa (Cat’s claw, Uña

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 Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS - Gibco, BRL, USA), penicillin (100 Units/ml), streptomycin (100 μg/ml) and containing in addition glucose 0.45% (HT29 cells), and L- glutamine 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 non-adherent cells discarded after 3 h. The adherent cells were then used immediately.

Female C57BL/6 mice (7–9 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 µl of culture medium in flat-bottomed 96 well plates and allowed to attach for 24 h. Different concentrations of UT in 100 µl 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 manufacturer’s instructions.

Inflammatory response in vitro. Peritoneal macrophages were activated with 10

µg/ml lipopolysaccharide (LPS - E. coli serotype 055:B5, Sigma, USA) for 24 h in the presence of UT, and then the concentrations of TNF- α, IL-6 and nitric oxide (NO) were measured in the supernatants. TNF- α 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-Chacón et al., 1998).

Inflammatory response in vivo. Mice were injected intraperitoneally (i.p.) with

different doses of LPS in 100 μl 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 μg 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 µl PBS. Two treatment protocols with UT were performed a) intraperitoneal (i.p.) injection of 50 μg of UT in 100 µl 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 μl 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-α 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 µl PBS into the hind limb. The mice were injected i.p. with 50 μg 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-α and IL-6 levels as described above.

NF-κB luciferase assay. HeLa cells were transiently transfected with the

NF-κB luciferase reporter system (Stratagene, La Jolla, CA, USA) according the manufacturer’s instructions. Transfected cells were seeded into 96-well 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-α (BD Biosciences, Palo Alto, CA, USA). Luciferase activity was measured using the Steady-Glo assay kit (Promega, Madison, WI, USA), in a 96-well luminometer. Dexamethasone and paclitaxel, which are known to inhibit and stimulate NF-κB 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 Student’s 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 μg/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-α response of mouse peritoneal macrophages to LPS was reduced by 48% in the presence of 100 μg/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 μg 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 µg/ml LPS for 24 h in the presence of 100 μg/ml UT extract. TNF- α, IL-6 and NO levels were then measured in the supernatants. (mean ± S.E.M., n = 3). ** P < 0.001

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-α 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 μg / animal) in tumor-bearing animals, prior to sacrifice.

Figure 3. Inhibition by UT of the inflammatory response to LPS in mice. Animals were pretreated with 50 μg/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- α, IL-6 and NO. (mean ± S.E.M., n = 3). * P < 0.05.

Figure 5 shows that both the TNF-α 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-α and IL-6 responses in animals with primary tumors were greatly inhibited by UT treatment, (85% and 81% respectively). Although TNF-α 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 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 μg 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 μg 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.

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 μg UT (corresponding to Fig 4A and 4C). One h before sacrifice, the animals were challenged with 3 μg LPS. Blood was extracted and the serum assayed for TNF- α and IL-6. (mean ± S.E.M., n = 3). * P < 0.05.

Effect of UT on the NF-κB response to activation by TNF-α in HeLa cells.

The effect of UT on the NF-κB response of HeLa cells to TNF-α was determined in a luciferase reporter assay. UT at 10 μg/ml slightly stimulated NF-κB activity, but was inhibitory (47%) at 100 μg/ml (Fig. 6). The highest dose of dexamethasone used (30 μM), a known NF-κB 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-α (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-κB activity. HeLa cells transfected with the NF-κB/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-α. DEX – dexamethasone (30 μM), PAC – paclitaxel (1 μM) – 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 μg/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-Chacón 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 María 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 peroxynitrite-induced apoptosis in HT29 and RAW 264.7 cells (Sandoval-Chacón et al., 1998). Considering these results and the dose of UT we used in the in vivo experiments (50 μg 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 anti-inflammatory action of extracts of Cat’s Claw (Aquino et al., 1991). Although it has been reported

that an aqueous extract of this plant inhibited TNF-α 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-α with a TNF receptor construct decreased serum TNF-α 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-κB activity (Sandoval-Chacón 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-κB, at concentrations that were not cytotoxic. However, NF-κB 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-κB 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 anti-inflammatory 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-κB, thus counteracting their own cytotoxicity (Karin et al., 2002). Anti-inflammatory drugs that act through NF-κB, 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 anti-inflammatory properties rather than to a direct cytotoxic effect.

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BLACPMA ISSN 0717 7917

ArtículoOriginal | Original Article

On the preclinical anti-trypanosomal, anti-inflammatory and toxicological activities of H. linifolium (L.) G. Don and its diphyllin derivatives.

[Sobre las actividades pre-clínicas antitripanosómica, antiinflamatoria y toxicológica de H. linifolium (L.) G. Don y sus derivados difilínicos]

Guillermo SCHINELLA1,2,*, Horacio TOURNIER1,2, Anibal ZAIDENBERG1 and José M. PRIETO3

1. Catedra de Farmacología, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Argentina 2. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, La Plata, Argentina

3. Departament de Farmacología, Facultat de Farmácia, 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 anti-inflammatory 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 ear’s 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 µM, 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 endémica de la región oriental de España con actividades antiinflamatoria tópica y antitripanosómica. En este trabajo nuestro objetivo fue obtener información adicional sobre estas actividades mediante la evaluación de A) la eficacia del extracto metanólico de la planta en un modelo de inflamación tópica crónica inducida por TPA en oreja de ratón y B) el efecto de dos lignanos arilnaftalénicos, apiósido (1) y acetil apiósido (2) de difilina contra epimastigotes de T. cruzi en cultivos axénicos. Nuestros resultados demuestran que los derivados (1) y (2) presentes en el extracto, están dotados de una leve actividad antitripanosómica in vitro con valores de CI50 de 62,9 y 60,1 µM, respectivamente, concentraciones que son tóxicas para células normales de mamíferos. Por otra parte, el extracto metanólico aplicado en forma tópica y utilizado crónicamente, muestra una potente actividad tóxica 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 utilización debe realizarse siempre bajo estricto control médico.

Palabras clave: Haplophyllum linifolium (L.) G. Don, Trypanosoma cruzi, inflamación, 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 (González et al., 1979).

Preclinical bioactivities of H. linifolium and its diphyllin derivatives Schinella et al.

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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 40°C and then freeze-dried.

Antiprotozoal assay It was performed as previously described by

Schinella et al. (2002). Briefly, an inoculum of 5×105 cells mL–1 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 μm, Merck) column. Samples (40-60 μL) were injected using a loop (20 μL) 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, 5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan. The reaction was followed spectrophotometrically at 490 nm (Mossman, 1983). Chlorpromazine (200 µM) was used as a reference compound (positive control).

Multiple TPA application The methanol extract of H. linifolium was applied

twice daily (15 μL, 1 mg.mL–1) 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 μg/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 (Cuéllar 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 Dunnett’s 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 4–5 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

Preclinical bioactivities of H. linifolium and its diphyllin derivatives Schinella et al.

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 anti-trypanosomal 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 µg 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 µM. Considering all these data as a whole we can not discard any unspecific cytotoxity towards the parasite.

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Figure 1. Structures of diphyllin apioside (1) and diphyllin acetylapioside (2)

(1) R=H; (2) R=CH3CO

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).

Table 1. Inhibitory Concentrations 50% (IC50, µM) and Confidence Interval 95% (CI95) of the growth of epimastigotes of T. cruzi in axenic cultures shown by the assayed compounds.

Compound IC50 CI95Diphyllin apioside 62.9 (46.0 – 79.8)

Diphyllin acetylapioside 60.1 (50.1 – 70.1) Benznidazole 50.0 (41.2 – 58.9)

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 (González 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 anti-inflammatory 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

Preclinical bioactivities of H. linifolium and its diphyllin derivatives Schinella et al.

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (5) 2008 228

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 (González et al., 1974) which can block the DNA synthesis in both normal and malignant cells in a similar fashion to justicidine A (González 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 Cuéllar MJ, Giner RM, Recio MC, Máñez S, Ríos JL.

2001. Topical anti-inflammatory activity of some Asian medicinal plants used in dermatological disorders. Fitoterapia 72:221-229.

González AG, Darias V, Alonso G. 1979. Cytostatic lignans isolated from Haplophyllum hispanicum. Planta Med 36:200-203.

González González A, Moreno Ordóñez R, Rodríguez LF. 1974. Lignans from Haplophyllum hispanicum. An. Quim. 70: 234-238.

Prieto JM, Giner RM, Recio MC, Schinella G, Máñez S, Ríos JL. 2002. Diphyllin acetylapioside: a 5-lipoxygenase inhibitor from Haplophyllum hispanicum. Planta Med. 68:359-360.

Massmanian A, Prieto JM. 1996. Dermatitis de Contacto por Haplophyllum hispanicum. 2ª parte. Perfil de una planta agresiva y curativa a la vez. Ponencia Científica, 36 Reunión del GEIDC, Grupo Español de Dermatitis de Contacto, Sevilla, España, 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, Máñez S, Ríos JL. 2002. Diphyllin acetylapioside, a 5-lipoxygenase inhibitor from Haplophyllum hispanicum. Planta Med 68:359-360.

Prieto JM, Recio MC, Giner RM, Máñez S, Massmanian A, Waterman PG, Ríos JL. 1996. Topical anti-inflammatory lignans from Haplophyllum hispanicum. Z. Naturforsch. 51c: 618-22.

Schinella GR, Tournier HA, Prieto JM, Ríos 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

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