from Argentina. - blacpma - Universidad de Santiago de Chile

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from Argentina. - blacpma - Universidad de Santiago de Chile
ISSN 0717 7917
www.blacpma.cl
Boletín Latinoamericano
y 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 cosmetics
BLACPMA, 7(4):183 - 189.
Susana Gattuso et al.
Morpho-histological and quantitative parameters in the
characterization of lemon verbena (Aloysia citriodora Palau) from
Argentina.
BLACPMA, 7(4):190 - 198.
Janne ROJAS et al.
Evaluation of antibacterial activity on different solvent extracts of
Euphorbia caracasana Boiss and Euphorbia cotinifolia L.
(Euphorbiaceae) collected in Venezuela
BLACPMA, 7(4):199 - 202.
Tzasna 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, Brazil
BLACPMA, 7(4):208 – 217.
Ana Laura FAZIO, et al.
An ethanolic extract of Uncaria tomentosa reduces inflammation
and B16-BL6 melanoma growth in C57BL/6 mice.
BLACPMA, 7(4):218 - 225.
Guillermo SCHINELLA et al.
On the anti-trypanosomal, anti-inflammatory and toxicological
activities of Haplophyllum linifolium and its diphyllin derivatives.
BLACPMA, 7(4):226 - 228.
Comité Editorial
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.
Bol. Latinoam. Caribe Plant. Med. Aromat. Vol.7 (4) 2008
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 Unido.
Talal Zari, Faculty of Science, King Abdulaziz University, Arabia Saudita.
i
BLACPMA
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
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
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.
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.cl
Envio de trabajos Online a nuestra editorial
Author’s Submission Package
http://www.blacpma.cl/submissions.htm
(Online Submission)
[email protected]
BLACPMA esta Indexada por:
CHEMICAL ABSTRACTS™
CAB ABSTRACTS™
NAPRALERT™
INDEX COPERNICUS™ (Impacto 4.80)
IMBIOMED™
LATINDEX™
QUALIS™
REDALYC™
Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008
ii
Instrucciones para los autores
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 [email protected] .
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.
Bol. Latinoam. Caribe Plant. Med. Aromat. Vol. 7 (4) 2008
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&Revi
sionSelectionMethod=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 [email protected] 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
iii
Guidelines for Authors
The LATIN AMERICAN AND CARIBBEAN BULLETIN OF MEDICINAL AND
AROMATIC PLANTS (BLACPMA), ISSN 0717 7917, it is a bimonthly electronic scientific
publication directed to any professional or technician working both on natural products of
medicinal or nutraceutical interest in general or with interest on medicinal and aromatic plants .
Works related with any of the areas covered by the Bulletin will be accepted such as: agronomy,
anthropology and ethnobotany, industrial applications, botany, quality and normalization, ecology
and biodiversity, economy and marketing, pharmacology, phytochemistry, pharmacognosy,
legislation and regulatory affaires, information and diffusion of events, courses, prizes, news,
reports, book reviews, or any other material type which may be important to communicate.
Type of contributions
Authors will be able to present reviews on a particular subject as well as original scientific
research, in the form of both full papers and short communications. Essays on hot topics for
debate are also welcome. Any of these contributions may be written in Spanish, English,
Portuguese or French , without limits on their extension which must be reasonably adjusted to the
objective of the work. However Announcements, news and events reports should not exceed one
page. In all the cases figures are included.
Format of the contributions
Authors must submit their contributions using the ASP (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
([email protected]). 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
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EDITORIAL
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
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Esperando comunicarnos en el próximo número de BLACPMA, les saluda a nombre del Comité Editorial
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José María Prieto, Editor Jefe Científico
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Damaris Silveira, Editor Asociado
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
182
© 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: [email protected]
Received 7 October 2007; Accepted 30 November 2007; Minor corrections 5 April 2008; Online 5 April 2008
Abstract
This article focuses on the indonesian tropical plant, Lansium domesticum. Its botany and phytochemistry as well as its medicinal, nutritional
and cosmeceutical value, which include antioxidant, moisturizing, whitening and lightening effects, are reviewed. This plant could be of
interest for the Latinamerican community as it can grow in several parts of America.
Keywords: Lansium domesticum, anti oxidant, moisturizing, cosmetic use.
Resumen
Este articulo trata de la planta tropical Indonesia Lansium domesticum. Su 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
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.
Tilaar et al.
Seeds:
Seeds 1-3, enveloped by a closely adhering, thick,
fleshy, translucent white aril; cells without developed
seed are also filled with aril tissue (Heyne, 1987;
Morton, 1987).
Figure 1. Lansium domesticum, tree and fruit
Description
Bark:
Tree up to 30 m tall and trunk 75 cm in diameter,
in cultivation usually only 5-10 m tall; bole up to 25
m, irregularly fluted, with steep buttresses; bark
mottled grey and orange, furrowed, containing milky,
sticky resinous sap; twigs glabrous to pilose.
Leaves:
Leaves alternate, odd-pinnate, 30-50 cm long,
glabrous to densely pilose, petiole up to 7 cm long;
leaflets alternate, 6-9, elliptical to oblong, 9-21 cm x
5-10 cm, glossy, chartaceous-corieceous, base what
some asymmetric, apex short acuminate, lateral veins
10-14 pairs, petiolules 5-12mm long, thickened at
base.
Flowers:
Inflorescence many flowered, solitary or in
fascicles of 2-10 on the trunk or largest branches;
recemes simple or branched at the base, 10-30 cm
long; flowers bisexual, sessile to pedicelled, solitary,
small; calyx fleshy, cup-shaped, 5-lobed, greenishyellow; petals fleshy, erect, ovate, 2-3 mm x 4-5 mm,
white to pale yellow; staminal tube subglobose, up to
2 mm high, anthers in one whorl; ovary globose,
appressed pilose, 4-5-celled; style short, thick, stigma
broad.
Fruits:
Fruit an ellipsoid or globose berry, up to 2-4(-7)
cm x 1.5-5 cm, yellowish pubescent, calyx persistent
with reflexed lobes; fruit-wall thin (1-1.5 mm) or
thick (up to 6mm).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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
184
Review of Lansium domesticum
succumbed either to the limestone terrain or low
temperatures.
Tilaar et al.
antimalarial activity against Plasmodium falciparum
with IC50's of 2.4-9.7 μg/ml (Saewan, 2006).
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
185
Review of Lansium domesticum
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Tilaar et al.
186
Review of Lansium domesticum
Tilaar et al.
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
Protein
Carbohydrates
Fiber
Calcium
Phosphorus
Carotene (Vit.A)
Thiamine
Riboflavin
Ascorbic Acid
Phytin
86.5 g
0.8 g
9.5 g
2.3 g
20.0 mg
30.0 mg
13.0 I.U.
89 mcg
124 mcg
1.0 mg
mg (dry
weight)
*According to analysis made in India. The edible flesh may
constitute 60% of the fruit. (Morton, 1987)
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).
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).
17. Lansionic acid
(1-16 from the authors, 17 from PubChem Substance Database)
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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
187
Review of Lansium domesticum
Tilaar et al.
recommended dose of the liquid extract is 2 – 5%
(Tilaar, 2007b).
property as well as moisturizing and lightening
effects with a good safety profile.
STUDIES ON BIOLOGICAL ACTIVITIES
REFERENCES
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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
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189
© 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.
2.
Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario Suipacha 531,
S2002LRK, Rosario, Argentina.
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: [email protected] .
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.
Characterization of lemon verbena
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Gattuso et al.
climatic conditions of their habitat (Molina et al.,
2003) our research group has undertaken this study in
order to define analytical parameters for the morphohistological characterization of lemon verbena
growing in Argentina. These specifications could be
used for the pharmacopoeial standardization of this
plant.
MATERIALS AND METHODS
Plant material
Fresh and dried plant materials were used for the
macroscopic, micro-morphologic and quantitative
studies. Samples were obtained from the Herbarium
collection of the University of Rosario (UNR),
Argentina.
ARGENTINA: Santa Fe Province: San 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
191
Characterization of lemon verbena
Metcalfe and Chalk (1972) were used in the
diagrams.
Statistics
ANOVA was performed to determine differences
of stomata index and palisade ratio of Aloysia
citriodora from different regional origin. Post hoc
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, greenishmauve corolla divided into 4 acuminate lobes (Fig. 1
A)
Plant material of interest
Dried leaves. They are thin and fragile, deepgreen in color. Shortly petiolate, ovate-lanceolate,
acuminated, measuring 5-10 cm long, with glandular
hairs, shortly pilose on the lower surface.
Microscopical characteristics
Leaf anatomy
Foliar architecture
The analysis of the foliar architecture shows
pinnate, camptodromous, brochidodromous venations
(Fig.1, A, B) The primary vein is stout and shows a
straight course. Secondary veins are thick and show a
straight course. The marginal ultimate venation is
looped. Limited, simple and curved venules. Welldeveloped areola randomly arranged, quadrangular,
polygonal (Fig 1, C).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Gattuso et al.
Surface view of epidermis
The epidermal cells of the adaxial face show
straight, anticlinal walls of 4 to 5 sides (Fig. 1, D).
Epidermic abaxial cells present anticlinal walls
slightly undulating with anomocytic stomata and
striate cuticule around of the stomata (Fig. 1, E; Fig.
2 E). Two types of trichomes are present on both
sides of the leaf surfaces. Non glandular trichomes: athe upper epidermis shows numerous conical,
unicellular, thick-walled, verrucose, cystolithic
trichomes, each arising from a rosette of cells at the
base, with cystoliths, (Fig.1, D; Fig. 2 F); b- the
lower epidermis shows simple, short, unicellular,
thickened, cystolithic trichomes, (Fig. 1, E, F).
Glandular trichomes: a- with foot simple or
compound; stalk, 1 to 2 celled and head unicellular,
cell mostly broader than long, outer walls convex,
thin, smooth, content scanty and translucent, in both
epidermis, more abundant in the lower one, (Fig. 1,
E, Fig. 2 E). b- with foot simple, head small,
relatively narrow, unicellular, contens dense,
commonly persistent. Outer walls slightly convex,
thin, smooth. Cuticular vesicle restricted to the apex,
persistent or collapsing early, they occur widely and
are aggregated in depressions on the lower surface
(Fig. 1 E; Fig. 2 E).
Cross-sections
It is dorsiventraly compressed. Unstratified
adaxial epidermis with cuticule thin and smooth,
without stomata can be observed. Mesophyll is
dorsiventral, with a 2-3 layer of pallisade
parenchyma (Fig. 1, I, J; Fig. 2 A). The spongy
parenchyma cells are located next to the abaxial
epidermis, loosely arranged, with many intercellular
spaces between them. The midrib consists of a
collateral vascular bundle. The lower epidermis is
unstratified and presents stomata in columns (Fig.1,
K; Fig. 2 D).
Stem anatomy
Transverse sections show a circular margin with 6
ribs (Fig. 1 L). The epidermis is unstratified, with
stomata which are composed of rectangular, rolled
cells, trichomes are scarce. At the level of the ribs
and in a subepidermal position, 3-4 layers of angular
collenchyma can be observed (Fig. 1 L). Cork usually
appears near the phloem (Fig. 1 M). Vascular bundles
are open collaterally and are arranged forming a
eustele, reinforced by conspicuous sclerenchymatic
fibers.
.
192
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, JK, M-S. 4 to C.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
193
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.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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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
Buenos Aires
Santa Fe
San Luis
Mendoza
Salta
Jujuy
Interval for Argentine Material
Rancagua (Chile)
Talca (Chile)
Stomatic index
5,45 (7,05) 10,20
5,16 (9,23) 13,43
8,33 (10,38) 12,69
8,64 (10,35) 12,09
8,93 (10,73) 13,73
7,27 (9,67) 11,11
5,16 - 13,73
7,14 (8,97) 11,67
5,77 (7,57) 9,26
Pallisade ratio
8,00 (9,31) 10,50
5,50 (6,70) 8,25
9,25 (10,60) 12,25
11,50 (11,81) 12,50
7,50 (8,20) 8,75
10,75 (14,30) 17,25
5,50 - 17,24
8,25 (9,65) 11,75
7,00 (8,40) 10,25
Figure 3: Bar graph of stomatic index and pallisade ratio of Aloysia citriodora samples of different origin. Mean ± SD. p < 0,05 (∗).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
195
Characterization of lemon verbena
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Gattuso et al.
addition, according to this last author, the leaves have
a striking aromatic characteristic. The leaf venation,
is brochidodromous type and quantitative parameters
of vein islet index is 24, 50 - 29,10 - 33,75 (Albrecht
et al. 2005). Metcalfe and Chalk (1972) mentioned
for the genus Aloysia the occurrence of anomocytic
stomata, we could ascertain this feature in this
species. Trichomes are epidermal outgrowths of
considerable value for taxonomic purposes. In A.
citriodora there are unicellular, thick-walled
cystolithic trichomes, trichomes arising from rosette
of cell at the base, and they are by far the most
prevalent and numerous subsessile glandular
trichomes with a globular head according to Metcalfe
and Chalk (1972) and Barbosa et al., (2001).
According to Metcalfe and Chalk (1972) members of
the family usually have dorsiventral mesophyll and
include several (2-3) layers of pallisade parenchyma,
according with the two strata found in this study. The
stem organization of A. citriodora observed in this
study corresponds to a typical herbaceous pattern
(Barbosa et al., 2001, Bonzani et al., 2003).
These results together with the quantitative
parameters accomplish the morpho-histological
characters stated in European Pharmacopeia.
CONCLUSION
The following diacritical macroscopic and micro
morphological characteristics and quantitative
parameters were established after the analysis of the
studied plant material. The presence of the below
described elements is considered to be useful for the
botanical identification of the A. citriodora.
Morphological characteristics
Three leaves per node, simple, entire, lanceolate,
petiolate. Stem terete, with ribs. Flower white, small,
arranged in a cymose inflorescence.
Anatomical characteristic
Leaves: Venation pinnate, camptodromous,
brochidodromous. Adaxial epidermis with anticlinal
thin walls and strains. Abaxial epidermis with
anticlinal thin and sinuous walls. Anomocytic
stomata, slightly prominent or in column. Non
glandular trichomes: a- the upper epidermis there are
numerous
conical,
unicellular,
thick-walled,
verrucose, cystolithic trichomes, each arising from a
rosette of cells at the base, with cystoliths, b- the
lower epidermis shows simple, short, unicellular,
thickened,
cystolithic
trichomes,
Glandular
trichomes: a- with foot simple or compound; stalk, 1
196
Characterization of lemon verbena
to 2 celled and head unicellular, cell mostly broader
than long. b- with foot simple, head small, relatively
narrow, unicellular, content dense, commonly
persistent, occur in depressions on the lower surface.
Mesophyll dorsiventral, hypostomatic.
Quantitative assessments
The following values were established for the
cellular elements of macerated stems: vessel bordered
pits, with a 360 x 56 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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198
© 2008 Los Autores
Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (4), 199 - 201
BLACPMA ISSN 0717 7917
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.
2.
Organic Biomolecular Research Group, Research Institute, Faculty of Pharmacy and Biomedical Sciences. University of Los
Andes. Mérida, Venezuela.
Microbiology and Parasitology Department, Faculty of Pharmacy and Biomedical Sciences. University of Los Andes. Mérida,
Venezuela.
*Contacto: [email protected]
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.
Antibacterial activity of E. caracasana Boiss and E. cotinifolia .
INTRODUCTION
Euphorbia genus belongs to the family
Euphorbiaceae. This family comprises about 300
genus and 5000 species distributed mainly in
America and tropical Africa. (Webster, 1994) The
plants of the family Euphorbiaceae contain skin
irritating and tumour-promoting diterpenoids, which
have tigliane, ingenane, and daphnane skeletons
(Evans & Taylor, 1983). Some species are used in
folk medicine to treat skin diseases, gonorrhoea,
migraines, intestinal parasites, and warts (Singla &
Pathak, 1990), In addition, several macrocyclic
diterpenoids with antibacterial, anticancer, PGE2inhibitory,
anti-multidrug-resistant,
prolyl
endopeptidase inhibitory, antifeedant, anti-HIV, and
analgesic activity have recently been isolated from
different Euphorbia species. (Hohmann et al, 2002;
Hohmann et al, 2003). They include jatrophane,
ingol, myrsinane diterpenoids, leucocyanidol,
quercitol, camphol, quercetrin, dihydroellagitannins
and dimeric hydrolysable tannins-euphorbins. (Haba
et al, 2007; Jasbi, 2006).
The antibacterial activity of several Euphorbia
species has been evaluated in different occasions.
The aqueous extract of Euphorbia hirta, used in
traditional medicine for the treatment of dysentery,
colic, ulcers, asthma and chronic bronchial infections
showed activity against Escherichia coli, Proteus
vulgaris,
Pseudomonas
aeruginosa
and
Staphylococcus aureus (Hore et al, 2006; Sudhakar et
al, 2006). The methanolic and acetone leaf extracts of
Euphorbia fusiformis showed growth inhibition of
Staphylococcus
aureus,
Escherichia
coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae,
Proteus vulgaris, Salmonella typhii A and Salmonella
typhii B. (Natarajan et al, 2005). Ethanolic extract of
Euphorbia australis showed activity against Bacillus
cereus, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas
aeruginosa
and
Salmonella
typhimurium (Palambo & Semple, 2001). Similarly
the ethanol, acetone and water extracts of Euphorbia
fruticosa (Alasbahi et al, 1999) and methanol extracts
of Euphorbia macroclada (Darwish et al, 2002)
showed inhibitory effects against Staphylococcus
aureus. These results may support the popular use of
these plants in traditional medicine for the treatment
of fever, wound infections, and intestinal disorders.
In the present investigation the evaluation of the
antibacterial activity of Euphorbia caracasana and
Euphorbia cotinifolia has been evaluated against
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Rojas et al.
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-hexanedichloromethane (CH2Cl2) 1:1 (4 L), CH2Cl2 (4 L),
CH2Cl2-methanol (MeOH) 1:1 (3 L), and MeOH (3
L) of increasing polarity. Fractions (90), each of 200
mL, were collected and examined by TLC. Selected
fractions, based on their TLC profiles, were used for
the antibacterial assay.
Thin layer chromatography (TLC)
TLC analysis was carried out on all the fractions
eluted from the column. Oven dried (100oC for 30
min) silica gel 60 F254 (Merck, UK) layers, 250 μm
thick were utilized and different mixtures of nhexane, CH2Cl2, and MeOH were used, depending on
the polarity of the eluted fractions to be analyzed.
The plates were sprayed with sulphuric acid diluted
to 10 % v/v with water and heating in an oven at
80oC for 10 min.
Bacterial strains
The microorganisms used were Staphylococcus
aureus (ATCC 25923), Enterococcus faecalis
(ATCC 29212), Escherichia coli (ATCC 25992),
199
Antibacterial activity of E. caracasana Boiss and E.cotinifolia .
Klebsiella pneumoniae (ATCC 23357)
Pseudomonas aeruginosa (ATCC 27853).
and
Antibacterial method
Antibacterial activity was determined using the
disc diffusion agar method described by Velasco et
al, (2005). The strains were maintained in agar
conservation at room temperature. Each bacterial
inoculum was incubated in 2.5 mL 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 (nhexane/CH2Cl2 1:1, CH2Cl2 and CH2Cl2/MeOH 1:1)
as well as dilutions 1:10, using the same solvents,
were placed over the plates. These were preincubated
at 4 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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (x) 2008
Rojas et al.
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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© 2008 Los Autores
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: [email protected]
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
Antifungal Activity of the Essential Oils of Two Verbenaceae
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Hernández et al.
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 (INIFAPTexcoco). The stock culture was maintained on
Czapek Dox Agar (Sigma).
Antifungal Activity
The assay of antifungal activity was carried out in
Petri dishes containing Czapek Dox agar (20 mL).
After the mycelial colony had developed, sterile
blank paper disks (5 mm diameter) were placed at 0.5
cm away from the rim of the mycelial colony. An
aliquot of the essential oil (1 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
203
Antifungal Activity of the Essential Oils of Two Verbenaceae
inhibition of fungal growth and hence, the Medium
Inhibitory Concentration (IC50) was determined.
Ketoconazole was used as reference and appropriate
controls with no essential oil were used. Each
experiment was repeated three times.
Statistical analysis
All experiments were performed in triplicate. The
mean and standard deviation of the three experiments
were determined. Statistical analysis of the
differences between mean values obtained for
experimental groups was done by an analysis of
variance (ANOVA multifactorial model), where pvalues of 0.001 or less were considered statistically
significant. The IC50 values were calculated by
logarithmic model using the Microsoft Excel
program.
RESULTS
As shown in Table 1, seventeen compounds of the
essential oil of L. achyranthifolia were identified by
GC/MS analysis representing 82.02%. The main
compounds showing concentrations higher than 5%
as percentage peak area were the monoterpenes:
carvacrol (30.64%), 1,8-cineole (5.03%), and the
sesquiterpenes: isocaryophyllene (10.73%), αbisabolol (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%), mcymene (20.42%) and thymol (6.72%).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Hernández et al.
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
6.13
2.54
β-pinene
m-cymene
7.30
20.42
1,8-Cineole
7.86
5.03
8.52
0.74
8.16
22.35
α-terpinyl acetate
Linalool
9.69
1.26
8.79
0.26
Camphor
10.96
0.49
Terpinen-4-ol
11.83
0.72
Bornyl acetate
14.50
0.56
Carvacrol
15.38
30.64 13.98
37.84
Thymol
15.05
6.72
Cadina-4(5),10(14)-dieno 16.79
3.38
Guaia-1(10),11(12)-dieno 17.15
2.76
Isocaryophyllene
18.01
10.73 15.96
2.18
Humulene
18.71
3.78
16.60
1.24
Aristol-1(10)-eno
18.83
0.65
19.68
5.68
β-Bisabolene
19.88
11.23 α-Bisabolol
20.11
2.77
β-Cadinene
Himachalene
20.38
1.17
Cedrene
25.67
0.43
Total
82.02
94.58
Compounds are listed considering the order of elution from a DB
WAX column. RT, retention time.
The biological activity results are presented in
Table 2. All the tested strains were sensitive to the
essentials oils. There are significant differences
between the fungal strains (F = 249.8, P < 0.001), the
concentration (F= 7928.8, P < 0.001), and plant
specie (F= 4565.0, P < 0.001). The more sensitive
strains were F. moniliforme, to L. achyranthifolia and
R. solani, to L. graveolens.
Table 2. Antifungal activity (IC50) of Lantana achyranthifolia
and Lippia graveolens.
Strain
Aspergillus niger
Fusarium moniliforme
Fusarium sporotrichum
Trichophyton
mentagrophytes
Rhyzoctonia solani
Controls
(μg/mL)
Essential oil (μg/mL)
Ketoconazole
L. achyranthifolia
L. graveolens
672.0
0.2
0.1
0.1
180
100
130
110
50
50
90
20
0.2
140
10
204
Antifungal Activity of the Essential Oils of Two Verbenaceae
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).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Hernández et al.
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© 2008 Los Autores
Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (4), 207 - 216
BLACPMA ISSN 0717 7917
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: [email protected]; [email protected]
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
$14.7 billion and growing at 17% annually (Starling,
2006). A study performed in Europe involving cancer
patients showed that the frequent use of medicinal
plants was the most mentioned alternative treatment
(Molassiotis et al., 2005). Similar results were found
in Buenos Aires, with a percentage of 37.4% of
utilization by the group researched (Franco and
Pecci, 2003).
According to the Brazilian Phytotherapy Industry
Association (ABIFISA), 82% of the Brazilian people
use medicinal plant-based products, in agreement
with the World Health Organization (WHO) data;
and this consumption is based on little or absent
scientific corroboration in relation to efficiency,
safety and toxicity of the herbs (ABIFISA, 2002;
Veiga et al., 2005). However, the defenders of the
free and uncontrolled use of medicinal plants claim
that they have already been tested and ratified by the
long history of the human, and these facts make them
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
safe, effective and without significant side effects,
common to synthesized products (De Smet, 2002,
2004; Ernst, 2004, 2006; De Smet, 2007). Thus, the
apparent inoffensive image of herbal remedies
encourages self-medication. The phytomedicine
market is responsible for a yearly turnover of R$ 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 sociodemographic
and
clinical-epidemiological
characteristics as well as information about the use of
medicinal plants and/or herbal medicine by the
participants of this survey. The statistical strategy
was based on the analysis of variance (ANOVA) and
in the chi-squared test. The results were considered as
significant when p<0.05. The analysis of data was
performed by using the Statistical Program for Social
Sciences (SPSS) version 10.0.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Sales et al.
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 HIVinfected patients that reported the use of medicinal plants and/or
phytomedicine
Parameters
Average age (± SD)*
Male/ Female
Number (%)
38.8 (±9.49)
44 (65.67)/ 23
(34.33)
Marital status
Singles
38 (56.71)
Married or live matrimonially
22 (32.83)
Divorced/widowered
7 (10.5)
Educational level
Illiterate
2 (3.0)
From 1 to 4 years of school
11 (16.24)
From 5 to 8 years of school
26 (38.8)
Graduated from High-School
23 (34.33)
Graduated from College
5 (7.5)
Monthly familiar income**
< 1 minimum wage
1 (1.52)
Between 1 and 5 minimum wages
52 (78.79)
Between 6 and 10 minimum wages
7 (10.61)
> 10 minimum wages
6 (9.1)
Social interaction
Family
53 (79.09)
Alone
10 (14.93)
Other (s) or in philanthropic
4 (6.0)
institution
Residence area
Urban
65 (97)
Rural
2 (3)
* SD – Standard Deviation
** Minimum wage in 03/01/2004 = US$100.00; one
interviewed refused to inform the monthly familiar income.
208
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
Table 2 – Reasons for the use of medicinal plants and/or
phytomedicine*
Reasons for the use of the Medicinal Plant/Herbal
remedy
Gastrointestinal disturbances
Pleasure or personal satisfaction
Pain and inflammation control or colds
Increase on the immunological response
Sedative and/or sleep inducer
Gastrointestinal disturbances and personal satisfaction
Weight reduction
Sedative and/or sleep inducer and due to
gastrointestinal disturbances
*Some patients mentioned more than one reason.
N (%)
24 (38.1)
18 (28.6)
8 (12.7)
6 (9.5)
3 (4.8)
2 (3.2)
1 (1.6)
1 (1.6)
Some interviewed reported the use of more than
one medicinal plant or herbal medicine.
In addition, the medical staff was inquired with
reference to their knowledge about properties of the
herbal medicine, the frequency that their patients
were using these ones, and the orientations provided
to them. The physicians were also asked about their
knowledge on possible interactions of medicinal
plants and/or herbal medicine with the usual
pharmacoterapy and the possible alterations on
biological/pharmacological activities of these
pharmaceuticals.
For the analysis of the pharmacokinetic and
pharmacodynamic potential drug interactions
between medicinal plants and herbal medicines and
the prescribed pharmacotherapy, a research was
performed using the following database: MEDLINE
(1966-2007) and LILACS, pharmacology and
medicinal plants textbooks.
RESULTS
From the 128 interviewed patients, 52.3% (95%
CI; 40.3%-64.4%) claimed to be medicinal plants
and/or herbal medicines users. The use in the last 4
years represented 25% (95% CI; 14.6%-35.4%) of
consumption affirmatives and the use in the last year
represented 18% (95% CI; 8.8%-27.2%). The sociodemographic characteristics of these patients are
presented in Table 1.
Asked about why they initiate using medicinal
plants, most of them reported that such habit is a
familiar traditional practice (56, 6%; 95% CI 44.7%68.5%). The recommendations received from
relatives and friends represented 83.6% (95% CI;
74.7%-92.5%). The interviewed also accepted
medical indication (3%), induced by TV programs,
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Sales et al.
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).
209
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
Sales et al.
Table 3 – Plants and herbal remedies that can interact with antiretroviral drugs.
Latin name (vernacular)
Ilex paraguariensis (erva mate)
Chenopodium ambrosioides Bert. Ex
Stend. (erva de santa maria);
Paullinia cupana Kunth (guaraná);
Smilax spp. (salsaparrilha);
Camellia sinensis L. (chá preto)
Symphytum officinale L. (confrei);
Sambucus nigra L. (sabugueiro);
Hymenaea courbaril L. (jatobá);
Gossypium barbadense L. (algodão)
Possible action
Can promote unbalance of the
acid-gastric buffer.
Can form a viscous gel of the
mucilage and gastric solution.
Interaction outcome
Cause increase of gastrointestinal
discomfort and alteration in the ARV
absorption
Can reduce ARV absorption, either by
the revetment of the gastric wall or by
the adsorption of the medicine.
Matricaria chamomilla L. (camomila);
Mentha spp. (hortelã)
Antispasmodic
Can reduce the motility of the
gastrointestinal tract causing reduction
of ARV absorption.
Aloe spp. (babosa)
Can increase the
gastrointestinal peristaltic
movement
Quercetin associates to the
plasmatic protein in around
98%.
Can promote changes in the ARV
absorption due the increasing on the
gastric emptying.
Can promote competition with ARV
for the bond to the plasmatic protein.
Allium cepa L.( cebola);
Eucalyptus globulus Labill. (eucalipto)
Ilex paraguariensis (erva mate);
Serenoa repens (saw palmetto);
Sambucus nigra L. (sabugueiro);
Maytenus ilicifolia Mart. ex Reiss
(espinheira santa);
Paullinia cupana Kunth (guaraná);
Camellia sinensis L. (chá preto)
Eucalyptus globulus Labill. (eucalipto)
Matricaria chamomilla L. (camomila)
Mentha spp. (hortelã)
Uncaria tomentosa (Willd. Ex Roem.
& Schult.) DC. (unha de gato)
References
(Fetrow and Avila,
2000; Cowl, 2003;
Philp, 2004; Simões
et al., 2004; Taketa et
al., 2004)
(Fetrow and Avila,
2000; Abdel-Kader et
al., 2002; Cowl,
2003; LimaNishimura et al.,
2003; Philp, 2004;
Santos et al., 2004;
Simões et al., 2004)
(Fetrow and Avila,
2000; Butterweck et
al., 2004; delCastillo
et al., 2004; Unger
and Frank, 2004)
(Fetrow and Avila,
2000; Simões et al.,
2004).
(Havey, 1999; Fetrow
and Avila, 2000;
Philp, 2004; Simões
et al., 2004)
(Fetrow and Avila,
2000; Izzo and Ernst,
2001; Cowl, 2003;
Ohsaki et al., 2004;
Philp, 2004; Simões
et al., 2004; Taketa et
al., 2004)
(Fetrow and Avila,
2000; Williams,
Inhibition of CYP450, in vitro.
Can increase the ARV bioavailability
2001; Cowl, 2003;
and/or toxicity.
Butterweck et al.,
2004; Philp, 2004;
Simões et al., 2004)
Induction of CYP450.
Cmax SQV: reduction of 54%
(Santos et al., 2004;
Santos and Boullata,
Allium sativum L. (alho)
AUC SQV: reduction of 51%
Cmax PI: reduction
2005)
Allium cepa L. (cebola)
Induction of CYP450.
Cmax and AUC of PI: reduction
(Havey, 1999)
ARV=antiretroviral; PI=Protease inhibitor; CYP450 = cytochrome P450; PI=protease inhibitors; Cmax = maximum concentration; AUC=area
under curve; SQV=saquinavir
Formation of tannin insoluble
complexes with proteins.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Can increase the bioavailability of PI
promoting higher bond to the
plasmatic protein.
210
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
Sales et al.
Table 4 – Plants and phytomedicines that can cause side effects synergism
Latin name (vernacular)
Peumus boldus Molina (boldo do chile)
Possible action
Anticholinergic action
Symphytum officinale L. (confrei)
Hepatotoxicity
Mentha pulegium L. (poejo)
Hepatotoxicity
Interaction outcome
Can increase neuropsychiatric EFV
side effects.
Can increase NRTI, NNRTI and
PI-induced hepatic damage.
Can increase NRTI, NNRTI and
PI-induced hepatic damage.
References
(Fetrow and Avila,
2000).
(Fetrow and Avila,
2000; Philp, 2004;
Simões et al.,
2004)
(Fetrow and Avila,
2000; Lorenzo et
al., 2002)
(Fierro et al., 1999)
Formation of chelate by cumarin and Can promote pancytopenia with
iron ions
NRTI, NVP and PI.
EFV=efavirenz; PI=protease inhibitors; NRTI= nucleoside analogous reverse transcriptase inhibitors; NNRTI= nucleoside non-analogous
reverse transcriptase inhibitors; ↑ = increase.
Mikania glomerata Spreng.(guaco);
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)
(Simões et al., 2004)
Operculina alata (Ham) Urban.
(“Batata de Tiú”)
Myristica fragrans Houtt (nutmeg)
Psychoactive substances
Psychoactive substances
Can increase neuropsychiatric EFV
side effects.
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 (greatgrandparents, grandparents, parents, brothers, etc)
and friends; only 3% of them from medical
prescription. In another study, realized in Thailand,
that evaluated the use of medicinal plants by HIVinfected patients, the recommendations obtained from
relatives and friends represented 14% and under
medical prescription represented 36% of cases
(Sugimoto et al., 2005).
In a quest performed with Brazilian cancer
patients the phytotherapy, when used alone,
represented 71.64% of the non-conventional
therapeutic modalities reported by patients, being
also recommended by relatives and friends in 80.6%
of cases (Elias and Alves, 2002). The same data
obtained in relation to the prevalence and
recommendation for the use of medicinal plants and
herbal remedies were also observed in pre-surgical
patients from Lenox Hill Hospital, New York, being
the prevalence of 57% for the use and 63% for
recommendations received (Adusumilli et al., 2004).
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Overall, 53 types of preparations involving
medicinal plants were identified. From these, 6
(11.32%) could not be identified due to the lack of
access to the medicinal plant or herbal medicine
because the interviewed could not make a clear
description of the plant or herbal medicine, or
because the patient used popular nomenclature for
different species employed at distinct pathological
conditions. Also, it was not possible identifying
components present in preparations as “garrafadas”
and others herbal complex mixtures (usually
powdered) presenting no label containing the product
description. These results are in agreement with a
study performed with HIV/AIDS patients from
University of California, San Francisco: from 22% of
patients reporting the use of medicinal plants during
three months preceding the interview, 24% could not
identify which medicinal plant (s) they were making
use of (Kassler et al., 1991).
The lack of quality, unknown origin and
misidentification of in natura medicinal plants, in
addition to the adulteration of herbal medicines with
heavy metals, hormones, stimulants, antiinflammatory agents and other drugs, are well
211
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
reported problems involved with herbal medicine
(Camargo, 1998; Winslow and Kroll, 1998; Corns,
2003; Betti and Thomsen, 2005; Grollman, 2005;
Gomez et al., 2007; Latif and Rahman, 2007).
These aspects must be carefully observed, once in
the present study, 91% of the in natura medicinal
plants mentioned by the interviewed were obtained
through own plantation garden, free markets and root
shops. Such practice may result in the use of
potentially toxic species by a mix up due to
similarities in relation to the macroscopic
characteristics, or popular name of the medicinal
species. Another recurrent problem is the
contamination of herbs or herbal medicines by fungi
and pathogenic bacteria that may promote toxic
reactions, being also responsible for alterations in the
expected effects of these medicines (Themboa et al.,
2007).
HIV-infected patients under drug therapy use antiretroviral triple scheme and, no rare, additional
medicines for the treatment of opportunistic
infections. Medicinal plants and/or herbal medicines
are composed of several chemical components
(Taylor et al., 2006; Tirona and Bailey, 2006; De
Smet, 2007; Langlois-Klassen et al., 2007), thus, the
association anti-retroviral therapy/medicinal plants
may increase the risk of developing adverse drug
reactions (Zhou et al., 2007). The physicians and the
patients must perform a careful evaluation in relation
to this potential risk, before starting the phytotherapy
(Foster et al., 2003; Foti et al., 2007).
Pharmacokinetic interactions may reduce the drug
bioavailability and hence its efficiency or increase its
bioavailability resulting in possible increased
toxicity. The pharmacokinetic interactions presented
in this work were mainly related to the medicine
absorption, bonding to the plasmatic protein and
induction or inhibition of the drug in the cytochrome
P450 (CYP) (Foti et al., 2007).
Among plants and herbal medicines used by the
interviewed, the phytochemicals are worth of
emphasis because they may change the antiretroviral
drugs absorption, reducing the area under plasma
concentration vs time curve.
Absorption alterations that promote the reduction
of the drug efficiency, antiretroviral included, may be
caused by plants and/or herbal medicines containing
saponins (Castilla et al., 2006), such as Ilex
paraguariensis,
Chenopodium
ambrosioides,
Paullinia cupana, Smilax spp and Camellia sinensis.
The mucilage present in Symphytum officinale,
Sambucus nigra, Hymenaea courbaril and
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Sales et al.
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
212
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Sales et al.
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
213
Use of herbal medicine by AIDS patients from Hospital Universitário de Brasília
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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Sales et al.
perform a careful evaluation about the potential risks,
before starting the phytotherapy (Foster et al., 2003)
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Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (5), 217 - 224
BLACPMA ISSN 0717 7917
ArtículoOriginal | Original Article
An ethanolic extract of Uncaria tomentosa reduces inflammation and B16BL6 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.
2.
Centro de Medicina Experimental , Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A,
Venezuela.
Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020A, Venezuela.
Contact : Tel +58 212 504 1097; Fax: +58 212 504 1086. E-Mail: [email protected]
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 antiinflammatory 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
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 Lglutamine 2 mM (A549 cells). Human peripheral
blood monocytes (huPBMC) were obtained from
healthy donors by standard Ficoll/Hypaque gradient
centrifugation and cultured in RPMI-1640 10% FBS.
Chopped spleens from C57BL/6 mice were ground
through a wire mesh screen. After removal of detritus
and lysis of red blood cells with 0.085% sodium
citrate, adherent cells were removed by overnight
incubation in plastic culture flasks. The non-adherent
cells (muSplen) were harvested, counted and cultured
in RPMI-1640 10% FBS. Murine peritoneal
macrophages (muPM) were collected from C57BL/6
mice 4 days after a peritoneal injection of 2 ml of 4%
thioglycollate. The cells were washed, seeded into
culture flasks in RPMI-1640 10% FBS, and nonadherent cells discarded after 3 h. The adherent cells
were then used immediately.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Fazio et al.
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
218
Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
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 96well plates, allowed to adhere overnight then treated
for 1 h with different concentrations of UT. The cells
were then stimulated for a further 4 h with 25 ng/ml
huTNF-α (BD Biosciences, Palo Alto, CA, USA).
Luciferase activity was measured using the SteadyGlo assay kit (Promega, Madison, WI, USA), in a 96well luminometer. Dexamethasone and paclitaxel,
which are known to inhibit and stimulate NF-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
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Fazio et al.
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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Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
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.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Fazio et al.
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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Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
Fazio et al.
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.
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.
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
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).
221
Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
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 peroxynitriteinduced 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 antiinflammatory action of extracts of Cat’s Claw
(Aquino et al., 1991). Although it has been reported
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
Fazio et al.
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 (SandovalChacón 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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Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth
The biomedical applications of plants with antiinflammatory activity are becoming increasingly
important given the links which have been
established over the last decade or so between cancer
and inflammation. This field becomes increasingly
interestingly since it has been found that many
antitumor drugs, such as paclitaxel activate NF-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 antiinflammatory properties rather than to a direct
cytotoxic effect.
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Derechos de Publicación © 2008 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 7 (5), 225 - 228
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: [email protected] T +54 221 421 6932 F +54 221 423 6710
Recibido | Received 15/02/2008; Aceptado | Accepted 9/07/2008; Online 12/07/2008
Abstract
Haplophyllum linifolium (L.) G. Don -synonym H. hispanicum Spach - is an endemic species of the East of Spain, with topical antiinflammatory and antitrypanosome activities. In this work we aim to further gain insight into these activities by A) evaluating the efficacy of
the methanolic plant extract in a TPA-induced chronic model of topical inflammation in mouse 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.
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.
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).
MATERIAL AND METHODS
Cytotoxicity studies
The cytotoxicities of both plant extracts and
compounds were assessed on elicited peritoneal
leukocytes obtained from rats with viability greater
than 95% as determined by the trypan blue exclusion
test. The cell viability was assessed by the capacity of
PMN mitochondrial dehydrogenase enzymes to
convert the 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) into a dark blue
formazan.
The
reaction
was
followed
spectrophotometrically at 490 nm (Mossman, 1983).
Chlorpromazine (200 µM) was used as a reference
compound (positive control).
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 ×
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (5) 2008
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
226
Preclinical bioactivities of H. linifolium and its diphyllin derivatives
diphyllin acetylapioside were identical with a
maximum at 260 nm. Their retentions times under
our conditions were 2.58 min and 3.86 min
respectively (Figure 2). The spectra and retention
times of the isolated compounds (1) and (2) were
identical to those of the standards allowing their
identification as diphyllin apioside and diphyllin
acetylapioside, respectively. The compounds were
>95% pure (Data not shown). In the antitrypanosomal assay, The results were as shown in
Table 1.
In a previous work (Schinella et al., 2002), the
methanol extract of flowering aerial parts of
Haplophyllum linifolium (H. hispanicum) resulted
very active against epimastigotes of T. cruzi, with a
65% of growth inhibition at 250 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.
Figure 1. Structures of diphyllin apioside (1) and diphyllin
acetylapioside (2)
(1) R=H; (2) R=CH3CO
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (5) 2008
Schinella et al.
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
Diphyllin apioside
Diphyllin acetylapioside
Benznidazole
IC50
CI95
62.9 (46.0 – 79.8)
60.1 (50.1 – 70.1)
50.0 (41.2 – 58.9)
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 antiinflammatory activity with no apparent toxicity
(Prieto et al., 1996). This effect is due, at least in part,
to the inhibition of 5-LOX activity (Prieto et al.,
2002). However, the plant extract was very toxic in a
murine model of chronic topical application (1
mg/ear/day), resulting on the ulceration and loss of
tissue of the treated ears. The general toxicity of
227
Preclinical bioactivities of H. linifolium and its diphyllin derivatives
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.
Schinella et al.
Prieto JM, Recio MC, Giner RM, Máñez S, Massmanian
A, Waterman PG, Ríos JL. 1996. Topical antiinflammatory 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
Acknowledges
We are grateful to Dr. Jose Luis Rios, Dr. Rosa
Giner, and Dr. Maria del Carmen Recio for their
comments on the manuscript.
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Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (5) 2008
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[email protected]
XII INTERNATIONAL SYMPOSIUM ON
MICROBIAL ECOLOGY
Cairns – Australia
17 al 22 de Agosto de 2008
www.kenes.com/isme12
CURSO
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
JORNADAS IBEROAMERICANAS SOBRE
ETNOBOTANICA Y DESARROLLO SOCIAL
25 al 29 de Agosto de 2008
Antigua - Guatemala
[email protected]; [email protected]
SIMPOSIO INTERNACIONAL DE TOMATE EN EL
TRÓPICO.
Villa de Leyva, 9-13 de septiembre de 2008.
Sociedad Colombiana de Ciencias Hortícolas
http://www.soccolhort.com/tomato
[email protected]
Teléfonos: (57-1) 3165000, Ext. 19041 y 19043 y (57-1)
8650218/19
V CONGRESO COLOMBIANO DE
CROMATOGRAFÍA, SIMPOSIO
IBEROAMERICANO DE EVALUACIÓN
SENSORIAL (SENSIBER V); X CONGRESO
COLOMBIANO DE FITOQUÍMICA
4 – 6 de septiembre de 2008.
Bogotá – Colombia
[email protected]
230
Eventos
SIMPÓSIO DE PLANTAS MEDICINAIS DO BRASIL
&
INTERNATIONAL CONGRESS OF
ETHNOPHARMACOLOGY
16 – 19 de septiembre de 2008
Sao Paulo – Brasil
www.plantasmedicinais.unifesp.br
[email protected]
CURSO
"CONSERVACIÓN DE ÁREAS IMPORTANTES
PARA PLANTAS EN AMÉRICA LATINA"
Santo Domingo – República Dominicana
28 de Septiembre – 4 de Octubre
[email protected]
CURSO
CONSERVACION DE LA BIODIVERSIDAD DE
ANFIBIOS
Copacabana – Bolivia
29 de Septiembre – 5 de Octubre
www.durrellwildlife.org
CONGRESO MUNDIAL PARA LA NATURALEZA
5 – 14 de octubre de 2008
Barcelona – España
[email protected]
XLIII CONGRESO NACIONAL DE CIENCIAS
BIOLÓGICAS
7 – 10 de octubre de 2008
Yopal – Colombia
www.asociacioncolombianadecienciasbiologicas.org
VIII JORNADAS NACIONALES
III CONGRESO INTERNACIONAL
DE ENSEÑANZA DE LA BIOLOGÍA
La Educación en Biología como Respuesta a la Demanda
Social
Mar del Plata, 9, 10 y 11 de Octubre de 2008
información: [email protected]
XVIII CONGRESO ASOCIACIÓN
LATINOAMERICANA DE FARMACOLOGÍA
III CONGRESO IBEROAMERICANO DE
FARMACOLOGÍA
XXX CONGRESO ANUAL DE LA SOCIEDAD DE
FARMACOLOGÍA DE CHILE
12 – 15 de Octubre de 2008
Coquimbo, Chile.
http://www.biologiachile.cl/socfarmch/.
CONGRESO IBEROAMERICANO DE QUIMICA
& XXIV CONGRESO PERUANO DE QUIMICA
13 – 17 de Octubre de 2008
El Cusco – Perú
[email protected]
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
1st INTERNATIONAL SYMPOSIUM ON YERBA
MATÉ AND HEALTH
School of Medicine (Facultad de Medicina) & School of
Chemistry (Facultad de Quimica)
October 17-18, 2008
Montevideo, Uruguay
http://209.209.34.25/webdocs/ISYM/Home.html
III CONGRESO INTERNACIONAL DE PLANTAS
MEDICINALES
30 de octubre – 1 de noviembre
Palmira – Colombia
[email protected]
IV ENCUENTRO INTERREGIONAL DE
INVESTIGACION EN EDUCACION
Concepción – Chile
5 al 7 de Noviembre de 2008
www.enin.cl
VI CONGRESO INTERNACIONAL DE
FITOTERAPIA
7 – 9 de Noviembre De 2008
Oviedo – España
www.astumed.org/congreso
XII CONGRESO SOCIEDAD MESOAMERICANA
PARA LA BIOLOGÍA Y LA CONSERVACIÓN
10 – 14 de Noviembre de 2008
San Salvador – El Salvador
www.smbcelsalvador2008.com
WOCMAP IV
9 – 14 de Noviembre de 2008
Cape Town – Sudafrica
VIII CONGRESO LATINOAMERICANO DE
HERPETOLOGÍA
24 – 29 de Noviembre de 2008
Topes de Collantes – Sancti Spiritus – Cuba
http://fbio.uh.cu/herpetologia/index.htm
[email protected]
VIII CONGRESO NACIONAL DE FARMACOLOGÍA
Y TERAPÉUTICA (CENTROFARMACOL 2008) Y II
TALLER NACIONAL DE SERVICIOS
FARMACÉUTICOS CLÍNICOS (SERVIFARMA 2008)
26 – 29 de Noviembre de 2008
Santa Clara – Cuba
[email protected]
V SIMPOSIO UNIVERSITARIO
IBEROAMERICANO SOBRE MEDIO AMBIENTE
1 – 5 de Diciembre de 2008
La Habana, Cuba
Informaciones: [email protected]
231
Eventos
I CONGRESO INTERNACIONAL DE
FARMACOBOTANICA DE CHILE
TERCERA REUNION DE BLACPMA
6 – 9 DE ENERO DE 2009
CHILLAN – CHILE
www.farmacobotanica.cl
FAPRONATURA 2009
17 – 20 de abril de 2009
Varadero - Cuba
http://www.scf.sld.cu/fapronatura2009/fapronatura09.htm
V INTERNATIONAL CONGRESS OF
ETHNOBOTANY (ICEB 2009)
“TRADITIONS AND TRANSFORMATIONS IN
ETHNOBOTANY”
21 – 24 se Septiembre de 2009
San Carlos de Bariloche, Argentina
[email protected] [email protected]
Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008
232
BLACPMA es la primera
publicación científica
Latinoamericana sobre
Productos Naturales con
vocación Internacional
totalmente Independiente
y de Libre Acceso.
The Latin American
Bulletin of Medicinal and
Aromatic Plants is an
independent, peer
reviewed, open
access, scientific journal
for the advancement of
natural products research
in Latin America and
beyond.
Indexada por:
CHEMICAL ABSTRACTS® (CAS)
CAB Abstracta®
NAPRALERT®
IMBIOMED®
INDEX COPERNICUS®
LATINDEX®
REDALYC®
QUALIS®

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