Plantas Iberoamericanas y Del Caribe

Boletín Latinoamericano y del Caribe
de Plantas Medicinales y Aromáticas

ISSN 0717 7917
Aloysia triphylla
Volumen 9, Número 1, Enero de 2010
Revisiones | Reviews
− De Souza et al. (Brasil) Produtos Naturais com atividade inibitória da Translocase I, uma promissora classe de compostos contra tuberculose.
Artículos | Articles
− Domínguez-Ortiz et al. (Mexico) Antioxidant and anti-inflammatory activity of Moussonia deppeana.
− Ascari et al. (Brasil) Phytochemical and biological investigations of Caryocar brasiliense Camb.
− Oliva et al. (Argentina) Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.) Britton from different regions of Argentina.
− Cortadi et al. (Argentina) Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae sensu lato de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia
parvifolia Engl., Picramnia sellowii Planch. y Castela coccinea Griseb.
− Rojas et al. (Argentina) Composición química y efecto antibacteriano del aceite esencial de Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios.
− Kader et al. (Bangladesh-Reino Unido) Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal activity.
− Letelier et al. (Chile) A protocol for evaluating the safety of herbal preparations in a rat model: the case of a supercritical fluid extract of Saw Palmetto.
Comunicaciones | Communications
− Pérez Colmenares et al. (Venezuela) Volatile components from the leaves of Solanum hypomalacophyllum Bitter.
Publicada por | Published by: Cooperación Latinoamericana y Caribeña en Plantas Medicinales y Aromáticas
Indexada por | Indexed by: SCOPUS, Science Citation Index Expanded (SCISEARCH), Journal Citation Reports/Science Edition, Biological
Abstracts y BIOThomson Reuters Master Journal List , Chemical Abstracts (CAS), NAPRALERT, CAB International (CAB Abstracts),
GlobalHEALTH, Index Copernicus, IMBIOMED, LATINDEX, QUALIS, REDALYC, Biblioteca Virtual da Saude (BVS).
© 2010 The Authors
© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), i
BLACPMA ISSN 0717 7917
Comité Editorial | Editorial Board
EDITOR JEFE | EDITOR IN CHIEF CONSEJO EDITORIAL | EDITORIAL

ADVISORY BOARD
José L. Martínez (Santiago, Chile)
Julio Alarcón (Chillán, Chile)
EDITORES CIENTIFICOS | SCIENTIFIC Talal Aburjai (Amman, Jordan)
EDITORS Arnaldo Bandoni (Buenos Aires, Argentina)
José María Prieto (London, UK) Elizabeth Barrera (Santiago, Chile)
Peter Taylor (Caracas, Venezuela) Armando Cáceres (Guatemala, Guatemala)
Salvador Cañigueral (Barcelona, España)
EDITOR EJECUTIVO | MANAGING EDITOR Bruce Cassels (Santiago, Chile)
Geoffrey Cordell (Illinois, USA)
Damaris Silveira (Brasilia, Brasil) Rosa Degen (Asunción, Paraguay)
Marco Dehesa (Quito, Ecuador)
EDITORES | EDITORS Olga Lock (Lima, Perú)
Carla Delporte (Santiago, Chile) Rodolfo Juliani (New Jersey, USA)
Gabino Garrido (Antofagasta, Chile) Patricia Landázuri (Armenia, Colombia)
Martha Gattuso (Rosário, Argentina) Norman Farnsworth (Illinois, USA)
Jeannette Gavillán (San Juan, Pto Rico) Elisabeth Williamson (London, UK)
Leonora Mendoza (Santiago, Chile) Michael Heinrich (London, UK)
Horacio Olivo (Iowa, USA) Peter Houghton (London, UK)
Edgar Pastene (Concepción, Chile) Luis Kanzaki (Brasilia, Brasil)
Verónica Rivas (Monterrey, México) Ana Ladio (Bariloche, Argentina)
Gabriela Ricciardi (Chaco, Argentina) Francisco Morón (La Habana, Cuba)
Luis A. Simeoni (Brasília, Brasil) Patrick Moyna (Montevideo, Uruguay)
Beatriz Varela (Buenos Aires, Argentina) Pulok Mukkerjee (Jadavpur, India)
Luca Rastrelli (Salerno, Italia)
EDITOR HONORARIO | HONORARY EDITOR Vicente Martínez (Guatemala, Guatemala)
John A. O. Ojewole (Natal, Sudafrica)
Jorge Rodríguez Chanfreau (La Habana, Cuba) Edison Osorio (Medellín, Colombia)
Mahendra Rai (Maharashtra, India)
Elsa Rengifo (Iquitos, Perú)
José Luis Ríos (Valencia, España)
Lionel Robineau (Pointe à Pitre, Guadalupe)
Gloria Saavedra (Cochabamba, Bolivia)
Marcelo Wagner (Buenos Aires, Argentina)
Talal Zari (Arabia Saudita)
BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas
This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. () which permits to copy, distribute and transmit the
work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get
permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.
Este es un articulo de Acceso Libre bajo los términos de una licencia Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar
públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su
apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra.
Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada
en esta licencia menoscaba o restringe los derechos morales del autor.
© 2010 The Authors
© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), ii-iii
Nota Editorial | Editorial Notes
Una nueva década

José L MARTINEZ1, Damaris SILVEIRA2, José M PRIETO3, Gabino GARRIDO4 & Peter TAYLOR5
1
Universidad Santo Tomás, Talca, Chile; 2Universidad de Brasilia, Brasil; 3Universidad de Londres, Inglaterra; 4Universidad de
Antofagasta, Chile; 5IVIC, Caracas, Venezuela
Estamos comenzando una nueva década del s. misma han sido dos proyectos presentados antes que
XXI y un nuevo año, el noveno, del Boletín desafortunadamente no resultaron agraciados, pero
Latinoamericano y del Caribe de Plantas Medicinales no por ello nos daremos por vencidos. Próximamente
y Aromáticas (BLACPMA) con muchas novedades. su Presidente José María Prieto tratará de quitarse
Durante el año anterior nos sometimos a evaluación horas de sueño y editar un boletín de noticias
en SCIELO y recibimos muchas recomendaciones CLACPMA para dinamizar esta asociación y darla a
que nos han permitido mejorar. Sin embargo una conocer allende Latinoamérica.
entre ellas nos ha causado especial dolor de corazón y Peter Taylor (Venezuela) ha tomado para si la
cabeza: SCIELO nos llamo a re-configurar el Comité ingrata tarea de reconsiderar todo el flujo editorial
Editorial ya que en su opinión estaba sobrecargado y elaborando un cronograma, un manual de funciones y
no permitía el crecimiento sostenible de la revista. un texto con las instrucciones a autores y revisores
BLACPMA ha sido siempre un foro de profesionales que se dará a conocer en próximos números. Con esto
unidos en la pasión de divulgar ciencia, y siempre se pretendemos agilizar aun más los procesos
hemos considerado plasmar a todos ellos en nuestro evaluativos ya que la entrada en ISI de BLACPMA
comité editorial lo cual era consustancial a esta ha supuesto una carga extraordinaria para el equipo
filosofía. Sin embargo la profesionalización de este editorial y educar a autores y revisores en cuanto a
boletín parece incompatible con ello y hemos debido los mínimos de calidad exigibles por nuestra revista.
aceptar cambios profundos. Empezando por los roles Por otro lado, nuevamente hemos recibido el
principales, asumiendo Peter Taylor (Venezuela) y interés de SILAE para establecer una Asociación
Damaris Silveira un rol mas importante dentro de la entre nuestra publicación y su Sociedad. Desde ya
jerarquía, aliviando un poco a Gabino Garrido estamos invitados a su reunión en Cagliary (Italia)
(Chile).y a José María Prieto (Inglaterra) que habían para Septiembre de 2010 pero esperamos poder
llevado un gran peso hasta ahora. En los roles de reunirnos en forma previa en el primer semestre de
Editores hemos decidido convocar a aquellos colegas este año.
que se mostraron mas proactivos durante el 2009: Una de nuestras principales colaboradoras la Dra.
desde Argentina Martha Gattuso (Rosario), Gabriela Gabriela Ricciardi y amiga de muchos años, este mes
Ricciardi (Resistencia) y Beatriz Varela (Buenos de febrero de 2010 contrae matrimonio, como Comité
Aires), además de Verónica Rivas (México) y Ejecutivo de BLACPMA deseamos para ella un
Horacio Olivo (USA) y Edgar Pastene (Chile). mundo lleno de felicidades en esta nueva vida que
Así que ¿qué paso con todos nuestros grandes inicia.
amigos que no podemos mantener nominalmente en Todas estas buenas nuevas se han empañado.
el comité editorial? Simplemente siguen vinculados Hace poco más de un mes, el pueblo Haitiano ha
igual que antes al BLACPMA a través de la naciente sufrido una de sus peores tragedias y como Boletín
CLACPMA (ver boletín de XXX 2009). CLACPMA Latinoamericano y del Caribe no nos hemos querido
ya se está mostrando como un catalizador del trabajo mantener al margen. No tenemos las herramientas
en red de sus integrantes y como resultados de la para poder estar más cerca pero de todos modos
This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. () which permits to copy, distribute and transmit the
work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work. Any of these conditions can be waived if you get
permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.
Este es un articulo de Acceso Libre bajo los términos de una licencia Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar
públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su
apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede alterar, transformar o generar una obra derivada a partir de esta obra.
Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada
en esta licencia menoscaba o restringe los derechos morales del autor.
Prieto et al. Guia de Autores
queremos enviar a través de estas líneas un noble Queremos terminar agradeciendo a aquellos que
saludo de apoyo y solidaridad. El Editor Jefe ha de una u otra manera hacen que este Boletín ocupe el
invitado al Sr. Benito Baranda, Presidente de la sitial que tiene en este momento todos los autores y
Organización de Voluntariado “Fundación América revisores que durante el 2009 han contribuido a esta
Solidaria” para que en un número próximo nos ilustre revista y especialmente por su importante apoyo a
un poco sobre la situación de ese país caribeño y Arnaldo Bandoni y Mariela Marinoff (Argentina),
quizás nos invité de alguna forma a solidarizar ya sea Guillermo Padrón (México), Carlos Céspedes (Chile)
a través de la Fundación que él dirige o de otras y Carolina Baquero (Colombia).
instancias. Lamentablemente para este número su Les dejamos ya con este su Boletín, y como
tiempo ha sido escaso debido a sus innumerables siempre les rogamos nos hagan llegar sus sugerencias
labores para apoyar a Haití. y contribuciones que siempre serán bienvenidas y
acreditadas.
www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 9 (1) 2010 | iii
© 2010 The Authors
© 2010 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 9 (1), 1 - 12
Revisión | Review
Produtos Naturais com atividade inibitória da Translocase I, uma

promissora classe de compostos contra tuberculose
[Natural Products with Translocase I inhibitory activity, as new lead compounds against tuberculosis]
Marcus Vinícius Nora DE SOUZA* , Victor FACCHINETTI, Danielle CARDINOT, Claudia Regina Brandão GOMES
Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far Manguinhos, R. Sizenando Nabuco 100, Manguinhos, 21041-
250, Rio de Janeiro, RJ, Brazil.
Abstract
Nowadays, Tuberculosis (TB), a contagious infectious disease caused by Mycobacterium tuberculosis, is becoming again a worldwide health problem.
The major causes that increase TB cases in the twenty one century are the rapid spread of multi-drug resistant strains, and TB association with the human
immunodeficiency virus (HIV) infection, which started in the mid-1980s. Considering that, the development of new drugs is urgently needed or a human
tragedy could happen. In this context, Translocase I, an enzyme involved in the biosynthesis of peptidoglycan, can be an important target for the development
of new drugs against this disease. Considering that, the aim of the present review is to highlight a series of new promising anti-TB agents, which have been
reported as Translocase I inhibitors namely Liposidomicines, Caprazamicines, Capuramicines, Pacidamicines, Mureidomicines e Napsamicines,
Muraimicines, and Tunicamicines all of these structural templates isolated from Streptomyces species.
Keywords: tuberculosis; translocase I; streptomyces
Resumo
Atualmente, a tuberculose (TB), doença infecto-contagiosa cujo agente etiológico é o Mycobacterium tuberculosis é um grave problema de saúde
mundial. Os principais fatores responsáveis pelo ressurgimento dessa doença no século vinte um foram o rápido desenvolvimento de cepas multiresistentes e
a associação do Mycobacterium tuberculosis com o vírus da imunodeficiência humana (HIV) no início da década de 1980. Nesse contexto, torna-se
necessário o desenvolvimento de novos fármacos ou uma tragédia humana pode ocorrer. Considerando esses fatores, a translocase I, enzima envolvida na
biossíntese de peptideoglicanas, pode ser um importante alvo no desenvolvimento de novos fármacos no combate a essa doença. Assim sendo, o objetivo
dessa revisão é destacar uma série de novas substâncias promissoras para o tratamento da TB, isoladas de diferentes espécies de Streptomyces, que vem sendo
relatadas como inibidores da translocase I como Liposidomicinas, Caprazamicinas, Capuramicinas, Pacidamicinas, Mureidomicinas e Napsamicinas,
Muraimicinas, and Tunicamicinas.
Palavras Chave: tuberculose; translocase I; Streptomyces
List of Abbreviations
AIDS (Acquired Immune Deficiency Syndrome); BCG (Bacilo de Calmette-Guérin); CDC (Center for Disease Control and Prevention); CPZs
(caprazamicinas); LPMs (liposidomicinas); LPS (lipopolissacarídeo); MDR (Multidrug resistant); MIC (Minimum inhibitory concentration); MRYs
(muraimicinas); OMS (Organização Mundial de Saúde); Human Immunodeficiency Virus (HIV); Tuberculosis (TB); TCM (tunicamicinas); UPAs (uridil
peptídeos); XDR (Extensively Drug Resistant)
Recibido | Received: July, 10, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: September 8, 2009.
Publicado en Línea | Published Online 15 December 2010
Declaración de intereses | Declaration of interests: authors have no competing interests.
Financiación | Funding: This work has not received funds.
This article must be cited as: Marcus Vinícius Nora de Souza*, Victor Facchinetti, Danielle Cardinot, Claudia Regina Brandão Gomes. 2009. Produtos Naturais com atividade
inibitória da Translocase I, uma promissora classe de compostos contra tuberculose. Bol Latinoam Caribe Plant Med Aromat 9(1):1 – 12. {EPub 15 DEcember 2009 }.
*Contactos | Contacts: marcos_souza@far.fiocruz.br
This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ )
which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work.
Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.
Este es un articulo de Acceso Libre bajo los términos de una licencia “Atribución Creativa Común-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-
nd/3.0/deed.es) Usted es libre de copiar, distribuir y comunicar públicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los créditos de la obra de la manera especificada por el autor
o el licenciador (pero no de una manera que sugiera que tiene su apoyo o apoyan el uso que hace de su obra). No comercial. No puede utilizar esta obra para fines comerciales. Sin obras derivadas. No se puede
alterar, transformar o generar una obra derivada a partir de esta obra. Al reutilizar o distribuir la obra, tiene que dejar bien claro los términos de la licencia de esta obra. Alguna de estas condiciones puede no
aplicarse si se obtiene el permiso del titular de los derechos de autor. Nada en esta licencia menoscaba o restringe los derechos morales del autor.
De Souza et al. Translocase I, um importante alvo terapêutico no combate à tuberculose
INTRODUÇÃO MATERIAIS E MÉTODOS

A tuberculose (TB) é uma doença crônica
O presente review foi organizado
endêmica na maioria dos países em desenvolvimento,
basicamente em três secções: A primeira delas
transmitida pelo ar e existente há milhares de anos,
apresenta a tuberculose e o seu ressurgimento, devido
causada pelo agente etiológico Mycobacterium
ao aparecimento de super-bactérias resistentes aos
tuberculosis, que, por ser uma bactéria aeróbia,
fármacos utilizados. A segunda seção destaca a
desenvolve-se principalmente nos pulmões, mas
importância dos produtos naturais no tratamento da
também pode atacar outras áreas do corpo humano.
tuberculose, bem como uma importante enzima
Atualmente, estima-se que um terço da população
conhecida como translocase presente na parede
mundial é portadora assintomática do Bacilo de
cellular do Mycobacterium tuberculosis, agente
Koch, dos quais 5% a 10% irão manifestar a doença
etiológico da TB. Finalmente, a última seção destaca
que tem como principais sintomas tosse crônica
produtos naturais e sua relação estrutura-atividade
persistente, suor noturno, dor no tórax e perda de
capazes de inibir essa enzima.
peso devido à falta de apetite. ( De Souza and
Vasconcelos, 2005a) MULTIDRUG RESISTANT (MDR) E
A prevenção da TB é feita por meio da vacinação EXTENSIVELY DRUG RESISTANT (XDR)
de recém-nascidos em seus primeiros 30 dias de vida TUBERCULOSE
com a vacina BCG (Bacilo de Calmette-Guérin), e o
tratamento da doença é realizado, preferencialmente, O abandono do tratamento tem provocado o
através da combinação de quatro fármacos: aparecimento de cepas resistentes aos fármacos de 1ª
Isoniazida, Rifampicina, Pirazinamida e Etambutol, escolha. Multidrug resistant (MDR) TB é definido,
utilizados durante um período de 6 meses que pode segundo a OMS (Organização Mundial da Saúde),
ser estendido para 9 meses em casos especiais. como doenças causadas pelo M. tuberculosis
Apesar de ser um tratamento eficaz e barato, a taxa resistente a Isoniazida e Rifampicina que são os
de abandono é extremamente elevada em alguns fármacos mais eficazes no tratamento da tuberculose.
países por diversos motivos como a longa duração do Essas cepas resistentes foram identificadas no final
tratamento, falta de informação e de dos anos 80 e início dos anos 90, representando uma
acompanhamento médico e a grande quantidade de ameaça ao controle da doença.
efeitos colaterais associados ao uso desses fármacos. De acordo com a OMS, as infecções por cepas
(De Souza, 2006a, 2006b) MDR-TB devem ser tratadas com pelo menos quatro
A questão da TB torna-se ainda mais delicada medicamentos de segunda escolha nunca usados
quando inserida no contexto da pandemia de anteriormente pelos pacientes, incluindo
HIV/AIDS, já que a co-infecção por M. tuberculosis Capreomicina, Amicacina ou Canamicina injetáveis e
e HIV tem se mostrado uma combinação letal. Dados um derivado fluorquinolônico como a Ciprofloxacina
da Organização Mundial de Saúde (OMS) indicam a e a Ofloxacina. O uso dos fármacos de segunda
ocorrência de mais de 300 casos anuais de TB a cada escolha apresenta como desvantagens a maior
100.000 habitantes em áreas da África subsaariana, quantidade de efeitos colaterais, alto custo e maior
local em que há maior incidência do vírus HIV. tempo de tratamento, que pode chegar a até 24 meses
Nessas áreas, mais de dois terços das pessoas de duração.
infectadas por TB estão co-infectadas por esse vírus. No tratamento de casos de infecções por cepas
No Brasil, dados da OMS mostram que no período MDR-TB, também é comum o uso de Etionamida e
entre 2000 e 2006 foram notificados quase 700.000 Ácido p-aminossalicílico e dos fármacos de primeira
casos de TB, sendo o Rio de Janeiro o estado com escolha Pirazinamida e Etambutol. (De Souza, 2006c)
maior número de casos registrados por ano, e pouco Uma nova ameaça ao controle da TB é a
mais de 60.000 óbitos causados por essa doença. identificação recente de cepas do tipo Extensively
Aproximadamente 20% dessas mortes estão Drug Resistant (XDR) TB, que é definida, de acordo
associadas a pacientes co-infectados pelo vírus HIV. com a OMS, como cepas do tipo MDR-TB
(WHO, 2009) resistentes a fluorquinolonas e a pelo menos um dos
três medicamentos injetáveis de segunda escolha
anteriormente citados. Como essas bactérias
apresentam resistência aos fármacos de primeira e de
www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 9 (1) 2010 | 2
segunda escolha mais eficazes, a doença se torna Após a descoberta da estreptomicina, outros
virtualmente incurável através da utilização dos aminoglicosídeos foram descobertos e utilizados até
medicamentos atualmente existentes no mercado para os dias de hoje no tratamento e controle da
tratamento da TB. (CDC, 2009) tuberculose, podendo-se destacar os
Uma pesquisa conduzida pela OMS em conjunto aminoglicosídeos canamicida, obtida a partir da
com o CDC (Center for Disease Control and cultura de fungos Streptomyces capreolus, amicacina,
Prevention) entre 2000 e 2004 identificou cepas do derivado semisintético obtido partir da canamicina A
tipo XDR-TB em todo o mundo, principalmente nos e capreomicina 1A, obtida a partir da cultura de
países da antiga União Soviética e da Ásia. Outra fungos Streptomyces Kanamyceticus. Além dos
pesquisa realizada pela OMS, dessa vez na África do aminoglicosídeos, pode-se mencionar também a D-
Sul, mostrou resultados alarmantes. De 544 pacientes cicloserina, obtida a partir da fermentação de
estudados, 221 estavam infectados com cepas do tipo Streptomyces sps.
MDR-TB e desses, 53 foram enquadrados na Com o crescente surgimento de super bactérias
definição de XDR-TB, sendo 44 HIV-positivos. Dos resistentes a praticamente todos os fármacos
53 pacientes XDR-TB, 52 morreram em até 25 dias, utilizados no tratamento da tuberculose, diversos
incluindo os beneficiados pelo uso de antirretrovirais, grupos de pesquisa tem demonstrado mais uma vez, o
fato esse que desperta a preocupação e demonstra a papel fundamental da mãe natureza na descoberta de
urgência de novos esforços com relação à novos fármacos no combate à tuberculose. Nesse
identificação de novos alvos terapêuticos e ao contexto, pode-se destacar o crescente número de
desenvolvimento de novos fármacos no combate à publicações científicas na área, identificando diversos
TB. (WHO, 2009) produtos naturais isolados de diversas fontes com
promissoras perspectivas no combate a tuberculose
IMPORTÂNCIA DOS PRODUTOS NATURAIS multiresitente. Na figura abaixo se encontra alguns
NO TRATAMENTO DA TUBERCULOSE exemplos de produtos naturais obtidos de plantas
De maneira similar ao que aconteceu com outras com atividade tuberculostática. (De Souza, 2005b)
doenças, tais como câncer, a malária, e certas (De Souza, 2006d).
doenças inflamatórias, o sucesso dos produtos Figura 2: Produtos naturais com atividade tuberculostática.
naturais merece destaque também na descoberta do H3CHN
tratamento e cura da tuberculose. Vale ressaltar que a 7 O O

B
descoberta da estreptomicina (Figura 1), isolada a 8
O
partir de culturas de Streptomyces griséus, pela A H3 C

OCOCH3
CH3
H
equipe liderada pelo bioquímico norte-americano, O
C
O O H OH HO
CH3
Selman Waksman, em 1943, foi uma das mais 12 OH
H
H
relevantes da história da medicina moderna e da O H

(+) - Calanolide A
humanidade. Esse medicamento foi responsável pela MIC = 3.13 μ g mL-1
H CH2 OH
AcO O
OAc
MIC = 3.13 μ g mL-1 MIC = 12.5 μ g mL-1

cura e pelo controle da tuberculose, doença que na (XU et al., 2004)
(CHUMKAEW et. al, 2003)
(KANOKMEDHAKUL et al., 2005)
época de seu surgimento causava a morte de um
número incontável de indivíduos. (De Souza, 2009) OH
CHO
O
Figura 1. Estrutura da Estreptomicina. N
N
HO O
NH
NH
Solsodomina A
MIC = 10 μ g mL-1
OH O (SAYED et al., 1998)
MIC = 6.3 μ g mL-1

(KANOKMEDHAKUL et al., 2005)
MYCOBACTERIUM E TRANSLOCASE I
Em geral, o citoplasma bacteriano é separado do
meio extracelular por uma membrana citoplasmática
formada por uma bicamada lipídica com boa fluidez,
que age como uma barreira seletivamente permeável,
e por uma parede celular constituída por nos animais e na maioria das plantas). A biossíntese
peptideoglicanas que confere a resistência necessária do peptideoglicano consiste é constituida de três
para suportar a alta pressão osmótica interna (Nikaido estágios: síntese do lipídeo I e do lipídeo II, seguido
and Saier JR, 1994). A biossíntese da parede celular da polimerização do lipídeo II por transpeptidação e
vem sendo explorada como alvo farmacológico para transglicosilação. Como exemplo de fármacos em uso
a pesquisa de novos antibióticos desde a descoberta clínico que inibem a polimerização do lipídeo II na
da penicilina por Fleming em 1929. superfície da bactéria, pode-se mencionar as β-
Bactérias Gram-positivas como o Staphylococcus lactamas e as vancomicinas. Esses fármacos possuem
aureus possuem parede celular formada por uma um mecanismo de ação diferente dos utilizados no
espessa camada de peptideoglicanas que confere tratamento para tuberculose, tornando-se possível
pouca resistência à difusão de pequenas moléculas. Já combater as infecções causadas por micobactérias
as bactérias Gram-negativas, como a Escherichia multirresistentes. (Boyle and Donachie, 1998),
coli, contêm em sua parede celular, além da camada (Hirano et al, 2008a)
de peptideoglicanas, uma outra membrana bilipídica Estudos têm sido realizados visando à descoberta
externa. A superfície externa dessa membrana é de substâncias capazes de inibir a síntese do lipídeo I.
recoberta por um lipídeo não usual, o Nessa etapa, a enzima fosfo-MurNAc-pentapeptideo
lipopolissacarídeo (LPS), que possui estrutura de translocase, também chamada de translocase I
pouca fluidez. Foi demonstrado que até mesmo (MraY), catalisa a reação entre o UDP-MurNAc-
moléculas lipofílicas apresentam dificuldades para pentapeptideo e o undecaprenil-fosfato formando
atravessar a parte hidrofóbica desse lipídeo, o qual se UMP e o Lipídeo I, que é o primeiro intermediário da
torna uma barreira eficiente contra a rápida difusão síntese de peptideoglicanas. Essa catálise requer a
de antibióticos lipofílicos. Em contraste, a parede presença de Mg+2 como cofator para a ativação da
celular das micobactérias é constituída por três enzima (Figura 4).(Struve et al, 1966), (Heydanek et
subestruturas covalentemente interligadas: as al, 1969)
peptideoglicanas, arabinogalactanas e os ácidos Figura 4: Biossíntese do Lipídeo I
micólicos, sendo os últimos formados por longas
cadeias de ácidos graxos contendo diferentes grupos
funcionais, como ligações duplas, cetona, éster,
epóxido, metóxi e ciclopropano. Esses compostos são
de extrema importância para a sobrevivência das
micobactérias, pois dificultam a penetração de drogas
hidrofóbicas, evitam a desidratação e permitem que a Várias substâncias têm sido relatadas como
bactéria se desenvolva no sistema imune do inibidoras da enzima translocase I, dentre elas as
hospedeiro (Figura 3). (Bugg and Walsh, 1992), liposidomicinas, as caprazamicinas, as capuramicinas
(Heijenoort, 2001), (Kimura and Bugg, 2003) (De e as pacidamicinas, que serão abordadas a seguir.
Souza, 2008)
Figura 3. Representação da parede celular de bactérias.
INIBIDORES DA TRANSLOCASE I
Liposidomicina
As liposidomicinas (LPMs) são produtos naturais
da classe dos antibióticos 6’-N-alquil-5’-β-O-
aminoribosilgliciluridina. Em 1998, foram reportadas
liposidomicinas do tipo I, II, III e IV, isoladas da
cepa mutante de Streptomyces griseosporeus SN-
1061M. As LPMs originais do tipo I possuem as
porções sulfato e ácido 3-metilglutárico. Os
A biossíntese de peptideoglicanas é essencial para compostos do tipo II não contêm a porção ácido 3-
a sobrevivência da bactéria (seres que possuem metilglutárico, os do tipo III não contêm a porção
células procarióticas) e se torna um alvo interessante sulfato e os do tipo 4 não possuem nenhuma das duas
no desenvolvimento de antibióticos, já que não existe porções (Figura 5). (Kimura et al, 2003)
correspondência em células eucarióticas (presente
A capacidade das liposidomicinas (LPMs) em estruturais das LPMs, que foram testadas como
inibir seletivamente a síntese de peptideoglicanas foi inibidoras da translocase I. Algumas dessas
descoberta em 1985. (Isono et al, 1985). Testes in moléculas estão representadas na figura 6.
vitro indicam que as LPMs do tipo I e III são os Figura 6. Simplificações estruturais das liposidomicinas
melhores inibidores da enzima translocase I, quando O O
comparadas as LPMs do tipo II e IV, indicando que a R
NH
O N O NH
O O 5" N
porção ácido 3-metilglutárico exerce papel R1
O
H2N O O
O
fundamental na inibição da translocase I. Entretanto, HO OH HO OH
R3" R2" R
3' R2'
as LPMs do tipo I, assim como a LPMs do tipo II,

Substância R R1 MraY Substância R2' R3' R2'' R3'' MraY
apresentam baixa atividade antimicrobiana in vivo IC (μΜ) IC (μΜ)
devido à presença da porção sulfato, hidrofílica, na 1 50 H OH OH OH 80

H NH2 9
posição 2” da 5-aminopentose, fato que confere às (R) - CH2OH 425
2 NH2 OH OH OH 10
10 H
LPMs baixa permeabilidade através da membrana
3 (S) - CH2OH NH2 5
celular. As LPMs do tipo III e IV são mais 11 OH OH H OH 115
4 CH(NH)NH
lipofílicas devido à ausência da porção sulfato, H 30
12 OH OH OH H
>1000
consequentemente, possuem maior atividade 5 H H2NC(NH)NH 25

OH OH 120
13 H H
antimicobacteriana in vivo. 6 H MeNH 45
A presença do ácido graxo nessa classe de 7 H EtNH 150
substâncias também é extremamente importante para 8 H n-PrNH 140
a atividade biológica, provavelmente porque confere

um aumento da lipofilicidade. (Kimura et al, 1998) A substância 1 apresentou moderada atividade
Figura5: Liposidomicinas dos tipos I, II, III e IV. inibitória (IC50: 50µM). A introdução do grupamento
H2N H2N
CH2OH na posição 5”, indicou que o isômero R (2) é
HO HO inativo, enquanto o isômero S (3) possui maior
O
HO3SO HO3SO
O
atividade inibitória do que a substância 1 (IC50:
CH3
R O 3''' N
6'
O O
H
N O
R O 3''' N
CH3
O O
H
N O 5µM). (Dini et al, 2000)
6' 5'
HO O O
HO2C
2'''
N
5' O N
O
HO2C
2'''
N
O N
Um grupamento básico, tal como o grupamento
O
O CH3 O
H3C
HO OH H3C
O
HO OH amino, aminas secundárias, amidina e guanidina são
Liposidomicina I Liposidomicina II
H2N
importantes para a atividade inibitória da MraY.
H2N
HO HO
Sendo que, no caso das aminas secundárias, o
HO
O
HO
O
tamanho da cadeia lateral influência diretamente na
R O 3''' N
CH3
O O
H
N O
O 3''' N
CH3
O O
H
N O
atividade inibitória. A metilamina (6) é ativa (IC50:
R
HO O O
2'''
N
6' 5' O N
O
2'''
6'
5' O N 45µM), enquanto que aminas com grupamento
HO2C HO2C N
O CH3 O
H3C
O
HO OH
H3C
O
HO OH
alquílico (7 e 8) maior são inativas. (Dini et al,
Liposidomicina III Liposidomicina IV 2001a)
Liposidomicinas R
A comparação da atividade inibitória dos
I-A; II-A; III-A; IV-A H3C
compostos 9-13 indicou que presença da hidroxila na
I-B; III-B (H3C)2HC
posição 3” é essencial para a atividade. Entretanto, o
H3C
I-C; II-C; III-C; IV-C
composto 3’-dexoxi é 5 vezes mais ativo do que o
I-G; III-G H3C
correspondente derivado 3’-hidroxilado, sugerindo
I-H; III-H H3C
H3C
que apenas a hidroxila na posição 3” é essencial para
I-K; III-K
(H3C)2HC
a inibição da translocase I. (Dini et al, 2001b)
I-L; III-L
I-M; III-M H3C
I-N; III-N H3C

Caprazamicinas
III-X H3C As caprazamicinas (CPZs) (Figura 7) são
III-Y H3C
produtos naturais que, assim como as LPMs,
I-Z; III-Z H3C
pertencem a classe dos antibióticos 6’-N-alquil-5’-β-
O-aminoribosilgliciluridina. As CPZs são isoladas a
partir de culturas de Streptomyces sp. e mostram uma
Dini e colaboradores em 2000 e 2001 sintetizaram excelente atividade contra bactérias Gram-positivas.
várias substâncias baseadas em simplificações (Igarashi et al, 2005)
NH2 NH2
Figura 7. Estrutura das Caprazamicinas. HO HO
HO O O HO O
O O
Me
NH2 HO N O O H2N O
HO NH NH
5'' O N O N
HO2C N O O HO2C N O O
HO O Me Me
HO OH HO OH
1'' 5 O 15
Me Caprazol
R O 3''' N O (MIC = 2,50 µg/mL)
6' (inativa)
O NH
O O 2''' 5' N NH2
Me O O
HO2C N O 1'
O
HO
O Me O Me
MeO OMe HO OH O O HO O
O
OMe Me
Caprazamicinas O N OH Me
O
O N
O NH
N
HO2C N O O HO2C N O
Me
Caprazamicinas Me
R OH
Me
HO
16 17
(inativa) (inativa)
A H3C
B (H3C)2HC
O palmitoilcaprazol, quando testado contra
C H3C Mycobacterium smegmatis ATCC607, apresentou
D
(H3C)2HC
MIC (Minimum inhibitory concentration –
E
concentração inibitória mínima) de 6,25 µg/mL,
H3C
similar ao das CPZs, e quatro vezes maior do que o
F (H3C)2HC do composto 14 (25 µg/mL), mostrando que a
G H3C ausência do grupamento metila na porção da
CH3
diazepanona influencia negativamente a atividade
antimicobacteriana. (Hirano et al, 2008a). Outros
As CPZs apresentam atividade in vitro contra
análogos foram testados contra Mycobacterium
Mycobacterium tuberculosis, tanto em cepas
tuberculosis H37Rv, sendo que o composto 15
sensíveis como em cepas multirresistente e não
apresentou atividade ligeiramente reduzida quando
demonstraram toxicidade significativa em ratos.
comparado ao palmitoilcaprazol (MIC = 2,50 e 6,25
Devido às excelentes propriedades biológicas, as
µg/mL, respectivamente), sugerindo que a abertura
CPZs são substâncias promissoras para a síntese de
do anel diazepanona diminui a atividade inibitória
novos agentes anti-TB, com um novo mecanismo de
sem, contudo, tornar a substância inativa. A
ação. Nesse contexto, quando comparadas às LPMs,
fragmentação da molécula nas porções aminoribose
as CPZs possuem importantes semelhanças
ou uridina tornou as moléculas 16 e 17 inativas e,
estruturais e biológicas sugerindo que essas classes
portanto, são cruciais para a atividade antibacteriana.
de compostos possuem o mesmo mecanismo das
(Hirano et al, 2008b) O caprazol, análogo das CPZs
LPMs. (Ichikawa, 2008)
sem a porção alquílica no anel diazepanona, também
Hirano e colaboradores sintetizaram diversos
não apresentou atividade antimicobacteriana. Essas
análogos da caprazamicina (Figura 8), incluindo o
alterações realizadas na parte lipofílica da cadeia
caprazol e o palmitoilcaprazol, que foram testados
lateral são de fundamental importância para a
contra o Mycobacterium sp.
permeabilidade na célula bacteriana. (Hirano et al,
Figura 8. Análogos das Caprazamicinas. 2008a)
NH2 NH2
Capuramicina
HO HO
A capuramicina (figura 9) foi originalmente
O HO O O HO O
Me
O H
O isolada a partir de culturas de Streptomyces griseous
O O
O N
O NH
O N
O N
NH 466-S3 e possui a capacidade de inibir a enzima
N
HO2C N O
Me
O HO2C N O
Me
O translocase I, no entanto seu espectro de ação é baixo.
HO OH HO OH
Palmitoilcaprazol 14
(MIC = 6,25 µg/mL) (MIC = 25 µg/mL)
Figura 9. Análogos da Capuramicina e sua attividade As substâncias obtidas foram testadas frente ao M.
antimicrobiana (Hotoda et al, 2003a) smegmatis e a enzima Translocase I, ficando evidente
OH
OH O
OH
OH O
a importância do grupamento NH da amida para a
CONH2 CONH2
O H
N NH NH
atividade dessas substâncias. Os melhores resultados
O N R1 O N
HN O O
O
O O
O
foram observados com os compostos 18-24 que
R O O
RO OH HO OH foram, então, testados frente às micobactérias de
Capuramicina (R = H)
Análogos da Capuramicina 18-24 maior relevância clínica: Mycobacterium avium
A-500359A (R = Me) NIHJ1605, Mycobacterium intracellulare ATCC1954
E-3 e Mycobacterium kansasii ATCC12478. Suas
Compostos R1 1a 2b 3c 4d 5e atividades foram comparadas com a da rifampicina e
Capuramicina - 10 12,5 8 8 8 isoniazida sendo que o análogo 18 apresentou MIC
A-500359A - 10 6,25 8 4 16
semelhante ao da capuramicina, variando entre 4 e 16
μg/mL. (Hotoda et al, 2003a)
A-500359E MeO- 27 >100 - - -
Hotoda e Colaboradores também obtiveram
18 PhNH- 6,5 6,25 16 4 8 derivados acilados a partir das moléculas de
19 3-Me-PhNH- 7,6 12,5 4 1 8 capuramicina e de seu derivado metilado A500359A
20 3-F-PhNH- 10 6,25 2 2 8 (Figura 10)
Observou-se que o aumento no tamanho da cadeia
21 4-F-PhNH- 37 6,25 4 2 2
lateral provoca diminuição na atividade das
22 3,4-di-F- 9 6,25 2 0,5 1 moléculas frente à enzima Translocase I, porém,
PhNH-
cadeias de tamanho ideal como a duodecanoíla e a
23 4-Cl-PhNH- 18 6,25 4 2 16 decanoíla presentes, respectivamente, nos derivados
24 4-Br-PhNH- 20 6,25 8 0,5 8 da capuramicina (25, MIC variando entre 0,063 e
Rifampicina - - - 0,125 0,125 0,125 3,13 μg/mL) e do A-500359A (26, MIC variando
entre 0,063 e 6,25 μg/mL), apresentaram excelente
Isoniazida - - -
a
atividade frente às micobactérias, provavelmente
Translocase I, IC50 (ng/mL)
b
M. smegmatis SANK75075, MIC (µg/mL)
devido à lipofilicidade conferida a esses compostos
c
M. avium NIHJ1605, MIC (µg/mL) que permite uma melhor penetração através da
d
M. intracellulareATCC1954 E-3, MIC (µg/mL) membrana celular desses microorganismos. Os
e
M. kansasii ATCC12478, MIC (µg/mL) valores de MIC observados para estes compostos
Em geral, a capuramicina apresenta maior foram iguais ou melhores do que os observados para
atividade frente à micobactérias e pouca atividade a isoniazida (MIC entre 0,125 e 0,25 μg/mL) e a
frente a bactérias Gram-positivas. É interessante rifampicina (MIC entre 1 e 2 μg/mL). (Hotoda et al,
notar que as micobactérias apresentam resistência 2003b)
contra muitos derivados β-lactâmicos, medicamentos Figura 10. Análogos acilados da Capuramicina e sua attividade
antimicrobiana (Hotoda et al, 2003b)
que pertencem à mesma classe das capuramicinas, OH
O
devido à presença da enzima β-lactamase. A
OH
CONH2
O H
N NH
atividade dessas substâncias poderia ser então HN O O O N
explicada pela estabilidade do seu anel β-lactâmico R O O

MeO O (CH2)nH
frente a essas enzimas. O mecanismo de 25 e 26
O
permeabilidade das capuramicinas através da Compostos R n 1a 2b 3c 4d 5e
membrana das micobactérias ainda não está
completamente elucidado, sendo atualmente, muitos Capuramicina H - 10 12,5 8 8 8
análogos de capuramicinas vêm sendo isolados, A-500359A Me - 10 6,25 8 4 16
sintetizados e testados como inibidores da enzima 25 H 11 n.d. 3,13 <0,063 0,125 0,125
translocase I. Como exemplo, pode-se citar análogos 26 Me 9 50 6,25 <0,063 <0,063 <0,063
da capuramicina que não contém a porção azepan-2-
ona (Figura 7) obtidos por Hotoda e Colaboradores, a Rifampicina - - n.d 0,125 0,125 0,125 0,25
partir da reação entre diversas aminas e o produto Isoniazida - - n.d - 1 8 2
a
natural A500359E, também isolado de cepas S. Translocase I, IC50 (ng/mL); bM.smegmatis SANK75075, MIC
griseous. (µg/mL);c M. Avium NIHJ1605, MIC (µg/mL); dM.
e
intracellulareATCC1954 E-3, MIC (µg/mL); M. Kansasii 2003), (Chatterjee et al, 1994), (Chen et al, 1989),
ATCC12478, MIC (µg/mL) (Fernandes et al, 1989)
Os compostos (RS-124922) e (RS-118641) Figura 12. Estrutura das napsamicinas, mureidomicinas e
pacidamicinas.
(Figura 11), sintetizados pela empresa japonesa
SCH3 SCH3
Sankyo, foram avaliados frente ao Mycobacterium O CH3 O OH
O CH3 O OH H H
tuberculosis, apresentando melhor atividade contra HO
N
H
N
N N CO2H R
N N
N N CO2H
M. tuberculosis (Cepa H37Rv) (MIC = 8 μg/mL e

H H H H
NH CH3 O CH3 O
O NH O NH
O R1
MIC = 1 μg/mL, respectivamente), do que o
R O R1
OH
composto A500359A (MIC = 16 μg/mL). A atividade OH OH
Napsamicina R R1 Mureidomicina R R1
dessas substâncias frente à cepas MDR-TB não A H uracil A H uracil
sofreu variações estatisticamente significativas, B

C
CH3
H
uracil
diidrouracil
B
C
H diidrouracil
glicina uracil
quando comparadas às observadas nas cepas H37Rv. D CH3 diidrouracil D glicina diidrouracil
Não foram realizados testes in vivo para o composto O CH3 CH3 O

R1
H H
(RS-124922) devido a sua baixa solubilidade no R
N
N
N
N N CO2H
H H
modelo de tratamento utilizado. Na avaliação in vivo CH3
O NH
O O
O N NH
dos outros, observou-se uma considerável redução no OH O
número de organismos viáveis nos pulmões em OH
Pacidamicina R R1
comparação com o grupo de controle. Os estudos 1 alanil 3-indol
desenvolvidos mostram que os análogos de 2 alanil fenil

alanil 3-hidroxi-fenil
capuramicinas possuem grande potencial 3
3-indol
4 H
antimicobacteriano e são bons candidatos a posterior 5 H fenil
avaliação no tratamento de infecções causadas por M. 6 glicil 3-indol
7 glicil fenil
tuberculosis. (Koga et al, 2004)
Figura 11. Análogos da capuramicina sintetizados pela empresa Compostos da classe das UPAs apresentam
japonesa Sankyo e sua attividade antimicrobiana (Koga et al,
2004). excelente atividade contra Pseudomonas aeruginosa
OH OH
devido as suas propriedades toxicológicas e
O H
OH
CONH2
O
H
OH
CONH2
O
farmacológicas favoráveis. Cepas bacterianas
HN
N
O O O N
NH F N
O O O N
NH
resistentes, a ofloxacin e β-lactamas, permanecem
O O O O
Me
MeO OH
F
MeO OH sensíveis à substâncias dessa classe de antibióticos.
A500359-A (RS-124922)
Entretanto, os UPAs possuem espectro de ação muito
estreito, sendo ativo especificamente contra cepas de
OH
OH
CONH2
O Pseudomonas, e muito pouco ativo contra bactérias
O
HN
H
N
O O O N
NH Gram-negativas e Gram-positivas, o que pode ser
Me O O explicado pelo fato desses compostos possuírem
MeO O
(RS-118641) O
acesso restrito à célula bacteriana, devido a sua
polaridade e ao seu grande potencial para fazer
Substâncias Cepa H37Rv Cepa MDR-TB ligações de hidrogênio com a água. (Isono et al,
MIC ( μg/mL) MIC ( μg/mL) 1992), (Boojamra et al, 2001), (Boojamra et al, 2003)
A500359-A 16 16 Boojamra e colaboradores, sintetizaram várias
(RS-124922) 8 4 diidropacidamicinas D, a partir da hidrogenação do
(RS-118641) 1 0,5
Rifampicina < 0,03 > 32
produto natural (pacidamicina 4), ou de síntese total,
Isoniazida 0,06 4 introduzindo diferentes aminoácidos na cadeia
acíclica (Figura 13). (Boojamra et al, 2003) As
Pacidamicinas, mureidomicinas e napsamicinas substâncias sintetizadas foram testadas contra quatro
cepas de Mycobacterium tuberculosis, das quais duas,
As pacidamicinas são antibióticos da classe uridil
a W e a P, são resistentes a todo o tipo de tratamento
peptídeos (UPAs), assim como as mureidomicinas e
anti-tuberculose, observando-se que as moléculas em
napsamicinas (Figura 12), capazes de inibir a enzima
que NHCH(R3)COOH é um resíduo de um
translocase I (MraY), isoladas de cultura de
aminoácido aromático, e as porções H2NCH(R1)C(O)
Streptomices sp. (Isono et al, 1992), (Boojamra et al,
e (CO)CH(R2)NH possuem resíduos hidrofóbicos
(substâncias 27 e 32-35) apresentaram melhor culturas de Streptomices sp. (Figura 14). (McDonald
atividade antimicobacteriana (MIC variando entre 4 e et al, 2002) Dentre estas, as muraimicinas A e B, que
10 µg/mL), inclusive quando comparadas a possuem um grupamento éster com uma cadeia
pacidamicina 4 (MIC maior que 30 µg/mL), sendo alquilica longa, geralmente possuem melhor atividade
ativo inclusive frente às cepas multirresistentes. antibacteriana do que as muraimicinas que não
Figura 13. Derivados da diidropacidamicina 4 e sua attividade apresentam o grupamento éster, demonstrando a
antimicrobiana (Boojamra et al, 2003) necessidade da porção lipofílica para a penetração na
R2
parede celular das bactérias.
O CH3 O R3
H McDonald e colaboradores relataram que algumas
H2N N
N N N CO2H
R1 CH3 O
H H
O muraimicinas (muraimicinas A1, A5, B6, C2 e C3)
O NH
O N NH
inibem a síntese do lipídeo II e a biossíntese da
O
camada peptideoglicana em concentrações de 0,027
OH µg/mL, indicando que a atividade antibacteriana das
H2N
O R2 R3 MRYs é comparável a da LPM C e a da
Substâncias N
R1
O
N
H H
CO2H mureidomicina A, que inibem a translocase de E.
Pacidamicina 4 meta-tirosina alanina triptofano colii, in vitro, com valores de IC50 de 0,05 e 0,03 µg
27 alanina 4-flúor-fenilalanina tirosina /mL, respectivamente.(Ichikawa and Matsuda, 2007)
28 glicina leucina triptofano Figura 14. Estrutura das muraimicinas.
29 alanina metionina tirosina
R1
30 alanina fenilalanina (2-naftil)alanina O
H H H H H H O
HO2C N N N N CO2H
31 leucina fenilalanina triptofano N
H NH
O O O N
HN H2N O O
32 alanina (4-bifenil)alanina triptofano H H
O
O N
33 alanina fenilalanina triptofano H HO R2 HO OH
34 alanina 4-flúor-fenilalanina triptofano
35 alanina 4-trifluormetilfenilalanina triptofano Muraimicina R1 R2 Muraimicina R1 R2

A1 O2C(CH2)12N(OH)C(NH)NH2 OCH3 C1 OH OCH3
A2 O2C(CH2)10N(OH)C(NH)NH2 OCH3 C2 OH OH
Substâncias 1a 2b 3c 4d A3 O2C(CH2)12NHC(NH)NH2 OCH3 C3 OH H
A4 O2C(CH2)12N(OH)C(NH)NH2 OH D1 H OCH3
Pacidamicina 4 >30 >30 >30 >30 B1 O2C(CH2)6CH(CH3)CH2CH3 OCH3 D2 H OH
27 10 8 8 10 B2 O2C(CH2)6CH(CH3)2 OCH3 D3 H H
B3 O2C(CH2)4CH(CH3)CH2CH3 OCH3
28 >30 30 10 10
B4 O2C(CH2)5CH(CH3)2 OCH3
29 >30 >30 >30 >30 B5 O2C(CH2)5CH(CH3)2 OH
B6 O2C(CH2)4CH(CH3)2 OCH3
30 30 30 30 10
B7 O2C(CH2)4CH(CH3)2 OH
31 30 30 30 10
R1
32 8 4 4 10 H H H
O
H H H
HO2C N N N N CO2H O
N
33 8 8 4 8 H NH
O O O N
HN HO
H H
34 8 8 4 8 O
O N
H HO OH
35 8 4 4 8
a Muraimicina R1
Cepa H37Rv, MIC (µg/mL)
b A5 O2C(CH2)12N(OH)C(NH)NH2
Cepa TN913, MIC (µg/mL)
c C4 OH
Cepa TN565 (W), MIC (µg/mL)
d
Cepa TN1618(P), MIC (µg/mL)
Lin e colaboradores sintetizaram derivados da
Muraimicinas muraimicina C1, através de reações seletivas nos
grupamentos amino primário e secundário (Figura
As muraimicinas (MRYs) são substâncias
15).
estruturalmente semelhantes aos antibióticos uridil
peptídeo (UPAs), como as mureimicinas,
napsamicinas, pacidamicinas e liposidomicina.
Dezenove muraimicinas foram isoladas a partir de
Figura 15. Derivados da muraimicina C1. DISCUSSÃO

R
O
OH O
N
Tendo em vista o grande número de efeitos
H H H H
HO2C N N
N
N N O O colaterais associados aos fármacos mais utilizados no
H
O
HN
O
O O O N
NH tratamento da TB, a duração de seu tratamento e os
H H2N H
O N
O adventos MDR e XDR-TB, faz-se necessário o
OH
H HO OMeHO desenvolvimento de novos medicamentos ou uma
36: R = (CH2)4CH3 (MIC = 6,25 µg/mL) grande tragédia poderá ocorrer, voltando-se ao tempo
37: R = CH2Ph (MIC = 6,25 µg/mL) em que não se existia a cura para essa doença. Nesse
Os derivados onde ocorreu substituição tanto na contexto, a translocase I (Mra Y), uma das enzimas
amina primária como na secundária foram inativos envolvidas na fase inicial da biossíntese de
como inibidores da MraY, em concentrações < 100 peptideoglicanas, tem se mostrado um alvo promissor
µg/mL. Entretanto, os compostos onde houve na busca de novos fármacos, sendo representada por
substituição apenas na amina secundária diversos produtos naturais e seus derivados sintéticos,
apresentaram boa atividade inibitória, que representando uma nova classe no combate às
demonstrou estar correlacionada com a lipofilicidade infecções bacterianas modernas. Nesse contexto,
dos substituintes introduzidos. Dentre estes, podemos destacar as liposidomicinas (LPMs) e as
destacou-se as hidantóinas 36 e 37, que inibiram a caprazamicinas (CPZs), produtos naturais da classe
MraY na mesma concentração que a muraimicina C1 dos antibióticos 6’-N-alquil-5’-β-O-
(6,25 µg/mL). (Lin et al, 2002) aminoribosilgliciluridina, que apresentam excelentes
atividades frente a enzima translocase. Dentre os
Tunicamicinas compostos avaliados dessas classes, merece destaque,
A classe de produtos naturais conhecidas como a capuramicina, substância pertencente a família das
tunicamicinas (TCM) (Figura 16) foi isolada da CPZs, que apresentou excelentes perspectivas sendo
fermentação de Streptomyces lysosuperficius sendo o estudo de sua estrutura atividade importante para
capazes de inibir a replicação de vírus, bactérias e identificação de novos derivados mais potentes,
fungos baseada na glicosidação de proteínas. simplificados, com melhores propriedade
farmacocinéticas, bem como capazes de ajudar na
O
melhor compreenção desse mecanismo de ação.
HO OH O
R N OH
H
O
O
O N NH
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© 2009 The Authors
Artículo Original | Original Article
Antioxidant and anti-inflammatory activity of Moussonia deppeana

[Actividad antioxidante y anti-inflamatoria de Moussonia deppeana]
Miguel A. DOMÍNGUEZ-ORTIZ,1* Omar MUÑOZ-MUÑIZ,2* Rosa Virginia GARCÍA-RODRÍGUEZ,2

Maribel VÁZQUEZ-HERNÁNDEZ,2 Janeth GALLEGOS-ESTUDILLO,2 Jesús Samuel CRUZ-SÁNCHEZ.1
1
Instituto de Ciencias Básicas, Universidad Veracruzana; 2 Unidad de Servicios de Apoyo en Resolución Analítica, Universidad
Veracruzana, Luis Castelazo Ayala S/N, Col. Industrial Ánimas, CP. 91190 Xalapa, Ver. México.
Abstract
Moussonia deppeana (Schldl. & Cham) Hanst is a species of Mexican Medicinal Flora used in Veracruz state, to treat sufferings related to stomach
pain, renal diseases, cough, tumors and inflammation. Obtained results showed that EtOAc extract was the most active in free radical scavenging test DPPH
(CI50 18.3±3.4 µg/mL) with 41% of reducing power respect to ascorbic acid and total content of polyphenols was smaller (328.9±7.6 mg GAE/g) than the
found in the ethanol extract (388.6±6.2 mg GAE/g). Anti-inflammatory activity was evaluated by topical application of the extracts (doses 2 mg/ear) giving a
greater inhibition in hexane and EtOAc extracts (39 and 28%, respectively). The model of paw edema was evaluated in EtOAc extract, observing a similar
inhibition to indomethacin (43% with 100 mg of dose) at the first hour. These results support the biological effect attributed in their traditional use.
Keywords: Anti-inflammatory, Antioxidant, Moussiana deppeana, Ethnopharmacology, Medicinal Plants.
Resumen
Moussonia deppeana (Schldl. & Cham) Hanst, es una especie de la Flora Medicinal Mexicana usada en el estado de Veracruz, para tratar padecimientos
relacionados con dolor estomacal, enfermedades renales, tos, tumores e inflamación. Los resultados obtenidos mostraron que el extracto de EtOAc fue el más
activo en la prueba de DPPH (CI50 18.3±3.4 µg/mL), con un poder reductor de 41% respecto al ácido ascórbico y el contenido total de polifenoles fue menor
(328.9±7.6 mg GAE/g) al encontrado en el extracto etanólico (388.6±6.2 mg GAE/g). La actividad anti-inflamatoria evaluada mediante aplicación tópica de
los extractos (dosis de 2 mg/oreja) dio mayor inhibición con el extracto hexánico, seguida del EtOAc (39 y 28%, respectivamente). El modelo del edema
plantar fue evaluado únicamente en el extracto de EtOAc observándose una inhibición similar a indometacina (43% a dosis de 100 mg de extracto) en la
primera hora. Los resultados apoyan el efecto biológico atribuido en su uso tradicional.
Palabras Clave: Anti-inflamatorio, Antioxidante, Moussiana deppeana, Etnofarmacología, Plantas Medicinales.
Recibido | Received: May, 29, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: October 15, 2009.
Declaración de intereses | Declaration of interests: authors have no competing interests. Todos los autores contribuyeron de igual manera en el manuscrito.
Financiación | Funding: This work was financed by Universidad Veracruzana.
This article must be cited as: Miguel A. Domínguez-Ortiz, Omar Muñoz-Muñiz, Rosa Virginia García-Rodríguez, Maribel Vázquez-Hernández, Janeth Gallegos-Estudillo, Jesús
Samuel Cruz-Sánchez. 2010. Antioxidant and anti-inflammatory activity of Moussonia deppeana. Bol Latinoam Caribe Plant Med Aromat 9(1):13 – 19. {EPub 15 December
2009}.
*Contactos | Contacts: omunoz@uv.mx; Phone: +52-228-841-8900 ext. 13554; Fax: +52-228-841-8917.
Este es un articulo de Acceso Libre bajo los terminos de una licencia “Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-
aplicarse si se obtiene el permiso del titular de los derechos de autor Nada en esta licencia menoscaba o restringe los derechos morales del autor.
Domínguez-Ortíz et al. Antioxidant and Anti-inflammatory Activity of Moussonia deppeana
with FeCl3 test (Oyaizu, 1986); total polyphenols by

INTRODUCTION Folin-Ciocalteau assay (Spanos and Wrosltad, 1990)
Moussonia deppeana (Schldl. & Cham) as well as 12-O-tetradecanoylphorbol 13-acetate
Hanst, belongs to Gesneriaceae family (synonyms: (TPA) induced mouse ear edema model (Young and
Kolheria deppeana, Gesneria deppeana, Moussonia De Young, 1989) and carrageenan induced mouse
elongate), this species is broad distributed since paw edema model (Levy, 1969) for determination of
Mexico to Panama. In folklore medicinal is anti-inflammatory properties.
commonly known as clachichinole, tlachichinole,
MATERIALS AND METHODS
tochomitillo or valletina (Escalante, 1988). This plant
is frequently used by Mexican people in traditional All chemical used were analytical grade. 1, 1-
medicine because of their curative properties Diphenyl-2-picryl hydrazyl (DPPH), Folin-
(stomach inflammation, diarrhea, ulcer, kidney Ciocalteu´s reagent, potassium ferricyanide
disease, vaginal infection and some tumors). In this (K3Fe[CN]6), ferric chloride (FeCl3), gallic acid
sense, several extracts of these plants have been monohydrate, ascorbic acid, carrageenan lambda, 12-
studied as antiprotozoal (Calzada et al., 1998) and O-tetradecanoylphorbol 13-acetate and solvents were
some reports revealed the presence of β-sitosterol, β- obtained from Sigma-Aldrich (México).
D-glucosyl-sitosterol, ursolic acid, oleanolic acid, Absorbance in colorimetric determinations
2β,3β-dihydroxy-olean-12-en-28-oic acid, 2α,3α- was measured in a UV-Vis Varian spectrophotometer
dihydroxy-olean-12-en-28-oic acid (Noguera et al., Cary100 model.
1994); 2-methyl-anthraquinone, chromanone and
stigmasterol (Reyes-Blas, 1995). Vegetal material
Gesneriaceae family is very extensive and M. deppeana was collected in Rancho Viejo-
includes tropical herbs and shrubs; many of them are Cinco Palos Municipality of Coatepec, Veracruz
growth as ornamentals (Martínez, 1969; Alcántara State, Mexico in October 2007. The taxonomic
and Luna, 2001). In some members of this family identification of plants was confirmed by Luis
have been isolated anthocyanins, flavonoids and Hermann Bojorquez Galván, a taxonomist. A
flavones (Díaz, 1976; Gould and Lister, 2006). Of voucher specimen (CIB8987) has been deposited in
particular interest was the isolation of some the Instituto de Investigaciones Biológicas Herbarium
glycosylated rutine derivatives by Robinson and of Universidad Veracruzana.
Tood (Robinson et al., 1934), which have a well-
defined anti-inflammatory activity. Preparation of crude plant extracts
By the other hand, is well known that oxygen About 800 g of aerial part of plant material
and nitrogen reactive species play important roles in were cut into small pieces, dried at room temperature
normal physiological processes, protection from and extracted by exhaustive maceration in darkness
pathogens, cellular signaling pathways, and with different solvents. Three extracts were obtained
regulation of vascular tone (Valko et al., 2007); also, successively and solvent was removed using rotary
they are related to development of tissue damage in evaporator (Hexane, 1.68 g; EtOAc, 2.01 g and
various human diseases such as cancer, aging, EtOH, 7.3 g). Crude extracts were kept in amber
neurodegenerative disease, malaria and pathological colored glass vials at room temperature for further
events in living organism (Gutteridge, 1994). use.
In this sense, antioxidant capacity of
medicinal plants and herbs has been linked to in vivo Phytochemical analysis
protection from oxidative stress in numerous studies
Phytochemical analysis of the plant extracts
(Prior et al., 2005) but rarely has been associated with
was undertaken using standard qualitative methods
anti-inflammatory capacity (Jensen et al., 2008). For
(color test and/or Thin Layer Chromatography, TLC).
this reason, the present study is aimed to the
Two milligrams of each extract were dissolved in
evaluation of antioxidant and acute anti-inflammatory
chloroform (5 mL) before application to TLC plates
effect of several extracts of M. deppeana growing in
Mexico by using 1,1-diphenyl-2-picrylhydrazyl (2×6 cm). The elution systems were benzene/acetone
(9:1) for hexane and EtOAc extracts meanwhile for
(DPPH) radical scavenging assay (Brand-Williams et
the ethanol extract was used a mixture butanol/water/
al., 1995; Miliauskas et al., 2004); reducing power
www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (1) 2010 | 14
acetic acid (6:3:1). The revealing agents were: µL of each sample (1 mg/mL, three replicates), 2.5
Dragendorff solution (for alkaloids), AlCl3 1% in mL 1/10 dilution of Folin-Ciocalteu´s reagent and 2
ethanol (for flavonoids), ZnCl2 (for sapogenins), mL of Na2CO3 (7.5 %, w/v) were added and
KOH 10% in ethanol (for coumarins), perchloric acid incubated at 45 °C for 15 min. The absorbance of all
(for sterols) and NaOH (for quinones) (Domínguez, samples was measured at 765 nm using a UV-Vis
1973; Kaufman et al., 2006). spectrophotometer. Results were expressed as gallic
acid equivalent (µg/mL) by using the following
Animals equation, which was obtained from standard gallic
All animals employed in the experiments acid graph (range 20 to1000 µg/mL).
were CD1 male mice (20-25 g) obtained from
Facultad de Medicina of the Universidad Absorbance  0.001 [GAE(g / mL)]  0.075
Veracruzana, Xalapa. The animals were acclimated
for one week in photoperiods adjusted to 12 hours of Determination of reducing power
light and 12 hours darkness daily and 50-55% The reducing power was determined
relative humidity with standard pellet diet (Rodent according to the method described by Oyaizu, 1986.
chow) and drink ad libitum. This work was A 0.125 mL aliquot of extract (1 mg/mL) was mixed
performed according to the Guide for the Care and with 1.25 mL of 200 mM sodium phosphate buffer
Use of Laboratory Animals (National Academy of (pH 6.6) and 1.25 mL of 1% of potassium
Sciences, 1996) and the Official Mexican Norm ferricyanide. The mixture was then incubated at 50
(NOM-062-ZOO-1999). °C for 20 min. After 1.25 mL of 10% trichloroacetic
acid (w/v) were added, the mixture was centrifugated
Determination of Antioxidant Capacity at 650 g for 10 min. A 2.5 mL aliquot of the upper
layer was mixed with 5 mL of destilled water and 1
Free radical scavenging by the use of DPPH mL of 0.1% ferric chloride, and the absorbance at
radical 700 nm was measured. The obtained value was
The DPPH radical scavenging capacity of compared with the ascorbic acid value as standard.
each extract was determined according to Brand-
Williams method modified by Miliauskas, 2004. Anti-inflammatory evaluation
DPPH radicals have and absorption maximum at 517
nm, which disappears with reduction by an 12-O-tetradecanoylphorbol 13-acetate (TPA)-
antioxidant compound. The DPPH radical solution in induced mouse ear edema.
methanol (9 x 10-5 M) was freshly prepared, and 2.9 Irritant dermatitis was induced on the right
mL of this solution was mixed with 100 µL of ear by topical application of 2.5 g TPA in 25 L of
methanolic solutions of plant extracts at several acetone according to the methodology reported by
concentrations (33, 16.5 and 8.25 µg/mL). The Young and De Young, 1989. TPA was applied on
samples were incubated for 30 min at 37 °C in a both the inner and outer surfaces of the ears. The
water bath, and decrease in absorbance at 517 nm extracts (doses 2 mg/ear) and the indomethacin
was measured (AE). A blank sample containing 100 (doses 2 mg/ear) were applied topically 30 minutes
µL of methanol in the DPPH radical solution was after TPA to the right ear (E´t), the left ear received
prepared daily, and its absorbance was measured vehicle (E´0). In the control group the right ear
(AB). Radical scavenging activity was calculated received only TPA (Et) and the other ear acetone (E0).
using the following formula: In all groups the edema was allowed to develop for 6
hours; afterwards the animals were sacrificed by
 A  AE  dislocation cervical and plugs (diameter of 6 mm) of
% Inhibition   B  x 100 the central portion were taken from both ears and
 AB  weighted. The inhibition of auricular edema was
calculated with the difference between weight ears of
Determination of total phenolic content the animal treatment with the extract or indomethacin
The total phenolic concentration was and the control group.
determined using the Folin-Ciocalteu´s reagent
according to the Spanos and Wrosltad, 1990. To 50
 ( E  E0 )control  ( E´t  E´0 )treated  quinones, which supports the presence of some
% Inhibition   t  x 100
 ( Et  E0 )control 
metabolites previously reported (Noguera et al., 1994
and Reyes-Blas, 1995).
Carrageenan-induced mouse paw edema Table 1. Phytochemical screening.
20 µL/paw of a 1% carrageenan solution was Test

Extracts
injected into the sub-plantar region of the left hind Hexane EtOAc EtOH
Alkaloids - - -
paw of the mouse. The increment in the paw thick Flavonoids - + +
was determined with a digital micrometer at 1, 3, 5 Sapogenins - - +
and 7 h after carrageenan administration. A reference Coumarins - + +
group was administered intraperitoneally with Quinones + + -
indomethacin (doses 5 mg/kg). The extract was Sterols + + -
administered at doses of 100 and 300 mg/kg by the
same via before carrageenan administration. The In many cases, the presence of flavonoids,
control group received vehicle only. The percent quinones and coumarins are associated with
edema inhibition was calculated for each animal antioxidant properties due to their role in several
group in comparison to group treated with vehicle human diseases where ROS could be involved. On
(10% Tween 80) according to the Olajide et al, 2000. the other hand, these components could be presents
actively in inflammatory processes, in fact,
Statistical analysis of data antioxidant/anti-inflammatory activity have been
related intrinsically to these chemical substances
Data are presented as means ± S. D. of at
(Takahashi and Shibamoto, 2008; Jensen et al.,
least triplicate experiments. For in vivo experiments,
2008).
data is reported as the means ± S.E.M. Significant
In order to evaluate the antioxidant efficiency
differences between groups were determined by
of plant extracts, the radical scavenging capacity
analysis of variance (ANOVA) complemented with
based in DPPH assay was determined and the results
Dunnett´s test using the software Statistica version 7
are shown in Table 2. In this sense, the percentage of
from StatSoft, Inc. (2004), p<0.05 was considered
inhibition of the DPPH radical varied from 41.1% for
significant.
the hexane extract to 92.4% for the EtOAc extract,
which represents a variation of approximately 2-fold
RESULTS AND DISCUSSION
respect to the hexane extract. The EtOAc extract
Reactive oxygen species (ROS) may be showed the highest antioxidant activity index (AAI)
involved in the etiologies of several human diseases (Scherer and Teixeira-Godoy, 2009) following EtOH
as atherosclerosis, ischemic injury, cancer and extract and Hexane extract (1.9, 1.6 and 0.9,
neurodegenerative diseases, as well as in processes respectively).
like inflammation and ageing (Edmonds, 2000). Also, Table 2. Antioxidant capacity of the extracts of M. deppeana
there is evidence that antioxidants may be useful in based on DPPH test.
preventing the deleterious consequences of oxidative
Extract DPPH DPPH AAI*
stress and there is increasing interest in the protective Inhibition IC50
biochemical functions of natural antioxidants (%) (µg/mL)
containing in medicinal plants. In this sense, our Hexane 41.1 ± 1.2 40.5 ± 1.3 0.9
attention has been focused in the antioxidant and EtOAc 92.4 ± 2.1 18.3 ± 3.4 1.9
anti-inflammatory properties of M. deppeana, a EtOH 70.1 ± 1.8 22.0 ± 0.4 1.6
Phenylbutazone 47.8 ± 4.4 34.4 ± 3.2 1.0
medicinal plant for the treatment of diabetes, stomach Rutin 93.5 ± 1.1 4.8 ± 0.1 7.4
inflammation, kidney diseases, cough, and tumoral Ascorbic acid** 100 0.6 ± 0.1 61.2
disease (Cano-Asseleih, 1997). Data expressed as mean ± SD (n=3). DPPH radical solution in
The phytochemical screening (Table 1) methanol (9 x 10-5 M, 35.48 µg/mL) using (33µg/mL) of plant
extract and ascorbic acid and rutin (5 µg/mL). * AAI = Final
revealed the presence of phenolic compounds
concentration of DPPH (μg.ml-1) / IC50 (μg.ml-1). ** No
(flavonoids and coumarins) in EtOAc and EtOH significance differences in IC50 were observed between this study
extracts. In the hexane and EtOAc extracts were and results previously reported (Sharma and Bhat, 2009).
possible to observe a positive result for sterols and
The DPPH free radical scavenger capacity of between hexane extract and esculetin (39 and 38%
the extracts was compared with well known respectively, Table 4) but lower than indomethacin
antioxidant (ascorbic acid and rutin) and anti- (69%). In this test, ethanol extract did not show a
inflammatory compound phenylbutazone. Despite the significant inhibition.
fact that extracts had been a significant antioxidant Table 4. Anti-inflammatory activity of M. deppeana on ear
activity index (ca. 2, see Table 2), the obtained values edema induced with TPA.
were lower than rutine and ascorbic acid (7.4, 61.2
Treatment Extract Dose Edema Inhibition
respectively) but slightly higher than phenylbutazone (mg/ear) inhibition (%)
in the case of EtOAc extract (1.0 vs 1.9). (mg)
Taking into account the results in radical- Control 14.4 ± 0.6 0
scavenging assay, it was expected that total phenol M. deppeana
Hexane 2 8.8 ± 0.6* 39
content and reducing power had presented the same
EtOAc 2 10.4 ± 0.7 28
behavior (Paśko et al., 2009); the EtOH extract had Ethanol 2 13.8 ± 0.5 4
the highest concentration of total phenol (388.6±6.2 Indomethacin 2 4.5 ± 0.5* 69
µg GA/mL, Table 3) close to the EtOAc extract β-sitosterol 1 9.9 ± 0.5* 31
(328.9±7.6 µgGA/mL, Table 3). In the reducing Esculetin 1 8.9 ± 0.8* 38
Values are expressed as mean ± S. E. M. The inhibition (%) was
power assay the EtOAc extract had shown the highest calculated respect to control group. * ANOVA-Dunnett´s, p <
value over the EtOH extract (41.3% vs 29.0% 0.05, n = 6.
respectively, Table 3). These observations indicated a
linkage between phenolics concentration, reducing In this regard, inflammation induced by TPA
power and antioxidant activity; on the other hand, in leads to protein kinase C activation, which is related
all assays hexane extract always showed the lowest to activation of phospholipase A2 with releases
values in each test (Table 2 and 3). arachidonic acid from membrane cells that is
metabolized to prostaglandins and leukotrienes
Table 3. Total phenolic content and power reducing of the (Recio et al., 2000). The inhibition observed in
extracts of M. deppeana. hexane extract could be related to this mechanism.
Extract Total phenols Reducing power According to the obtained results in
(%) preliminary anti-inflammatory test (TPA), the hexane
Hexane 20.6 ± 1.5 5.3 ± 0.4 and EtOAc extract have the major inhibition;
EtOAc 328.9 ± 7.6 41.3 ± 0.5 however, we chosen the EtOAc, due to the low
EtOH 388.6 ± 6.2 29.0 ± 1.1
solubility presents in hexane extract, in order to
Data expressed as mean ± SD (n=3). Total phenols expressed in
mgs. of Gallic acid equivalent per g of sample (mgGAE/g). confirm the biological effect with the paw edema
Reducing power is expressed with respect to ascorbic acid induced with the carrageenan model in mouse applied
(33µg/mL). by systemic route.
The EtOAc extract of M. deppeana (100 and
The propagation of free radical can bring 300 mg/kg dose) applied 30 min before of
many adverse reactions leading to extensive tissue carrageenan did not reduce the edema formation
damage. Lipids, proteins and DNA are very significantly respect to the control group. In contrast,
susceptible to attack by free radical (Yu et al, 1992). the indomethacin to 5 mg/kg dose reduced the edema
In this way, all antioxidant test evaluated in this work formation in all time registered.
showed that M. deppeana has a great amount of Surprisingly, paw edema decrease 43% with
antioxidant compounds that may offer resistance lower doses of the extract (100 mg/Kg) only at the
against oxidative stress by scavenging the free first hour; this effect was similar to indomethacin
radicals and inhibiting lipid peroxidation. (Table 5).
Given the results in the antioxidant assays, In the acute inflammation induced by
our attention now was focused in the evaluation of carrageenan, levels of substance P in inflamed paw
the anti-inflammatory activity. In the ear edema increase within 15 minutes after induction, these
induced with topic application of TPA, the hexane levels remained elevated during the first 2 h of
and EtOAc extracts produced the mayor inhibition of inflammation (Gilligan et al., 1994). Other mediators
the edema, 39 and 28%, respectively (Table 4). At 2 of inflammation as serotonin, histamine and
mg/ear dose, the inhibition edema was similar bradykinin play important roles during the early
phase of carrageenan edema (Di Rosa et al., 1971). Hidalgo, México: Eloxochitlán y Tlahuelompa. Acta
According that the observed effect in EtOAc extract Botánica Mexicana. 54: 51-87.
could be related to some of these inflammation Brand-Williams W, Cuvelier ME, Berset C. 1995. Use of a
mediators. free radical method to evaluate antioxidant capacity.
LWT. 28(1): 25-30.
Table 5. Anti-inflammatory activity of EtOAc extract of M. Calzada F, Meckes M, Cedillo-Rivera R, Tapia-Contreras
deppeana on paw edema produced with carrageenan test. A, Mata R. 1998. Screening of Mexican Medicinal
Time Paw edema (mm) and % inhibition Plants for Antiprotozoal Activity. Pharm. Biol. 36(5):
(h) 305-309.
Control Indomethacin M. deppeana M. deppeana
5 mg/kg 100 mg/kg 300 mg/kg
Cano-Asseleih LM. 1997. Flora medicinal de Veracruz,
1 0.7±0.1 0.4 ± 0.1* 0.4± 0.1* 0.6 ± 0.1 pp. 224. Ed. Universidad Veracruzana, Xalapa,
(43%) (43%) (14%) Veracruz-México.
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(36%) (9%) (9%) the mediatiors of the acute inflammatory response
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(42%) (17%) (8%) turpentine. J. Pathol. 104:15-29.
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(30%) (NE) (NE) medicinales de México. pp. 59, 259. Ed. Instituto
Values are expressed as mean ± S.E.M. The inhibition (%) was
Mexicano para el Estudio de las Plantes Medicinales,
calculated respect to control group. * ANOVA-Dunnett´s, p <
0.05, n = 8. México.
Domínguez XA. 1973. Métodos de Investigación
Fitoquímica, pp. 81-85. Ed. Limusa, México.
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hexanic extract was the best and the EtOAc extract licenciatura, Universidad Nacional Autónoma de
had the higher antioxidant activity. Further México, México, D. F., pp. 9-11.
investigations in the phytochemistry field and other Gilligan JP, Lovato SJ, Erion MD, Jeng AY. 1994.
Modulation of carrageenan-induced hind paw edema
biological activities are being developed in our group
by substance P. Inflammation. 18: 285-292.
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mechanism and chemical composition. Plants, pp. 397-442. In Andersen ØM and Markham
KR: Flavonoids: chemistry, biochemistry, and
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and mechanism of antioxidant protection. Chem. Biol.
the hexanic and EtOAc extracts of M. deppeana only
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low doses of EtOAc extract produced a similar Scherwits L, Beaman R, Endres JR, Schauss AG.
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a short period of time. In the case of the antioxidant inflammatory Capacities of an Antioxidant-Rich Fruit
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© 2010 The Authors
Articulo Original | Original Article
Phytochemical and biological investigations of Caryocar brasiliense Camb

[Estudios fitoquímicos y biológicos sobre Caryocar brasiliense Camb]
Jociani ASCARI , Jacqueline Aparecida TAKAHASHI, Maria Amélia Diamantino BOAVENTURA*
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP 31270-970. Belo
Horizonte – MG, Brasil.
Abstract
Caryocar brasiliense epicarp and external mesocarpe were chemically and biologically evaluated. From the phytochemical study, ethyl gallate, 5-
hydroxyfurfural, gallic acid, methyl shikimate, and mixtures of β-D-fructopyranose and β-D-fructofuranose, α-and β-D-glucose, lupeol and oleic acid and β-
sitosterol, stigmasterol and oleic acid were isolated and spectroscopically identified by NMR (1D and 2D). Tests on the antioxidant, allelopathic and
antimicrobial activities were carried out for the crude extract, fractions and pure compounds. Extract and pure compounds showed good activities in all
bioassays.
Keywords: Caryocar brasiliense Camb.; phenolic compounds; triterpenes; antimicrobial; antioxidant; allelopathic activity
Resumo
El epicarpo y el mesocarpo externo de Caryocar brasiliense Camb. fueron evaluados química y biologicamente. Del estudio fitoquímico, fueron
aislados e identificados por RMN (1D y 2D) galato de etilo, 5-hidroximetilfurfural, ácido gálico, chiquimato de metilo y mezclas de β-D-fructopiranosa y β-
D-fructofuranosa, α- y β-D-glucosa, lupeol y ácido oléico, β-sitosterol, estigmasterol y ácido oléico. Fueron realizados ensayos de evaluación del efecto anti-
oxidante, anti-microbiano y alelopático para el extracto etanólico crudo, fraciones y compuestos puros, los cuales presentaron buena actividad.
Palavras Chave: Caryocar brasiliense Camb.; compuestos fenólicos; triterpenos; actividad anti-oxidante; actividad antimicrobiana; actividad alelopática.
Recibido | Received: August, 9, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: October 26, 2009.
Publicado en Línea | Published Online December 15, 2009.
Financiación | Funding: CNPq, JAT and MADB for grants. FAPEMIG, for financial support.
This article must be cited as: . Jociani Ascari , Jacqueline Aparecida Takahashi, Maria Amélia, Diamantino Boaventura. 2010. Phytochemical and biological investigations of
Caryocar brasiliense Camb. Bol Latinoam Caribe Plant Med Aromat 9(1):20 – 28. {EPub 15 Dec 2009 }.
*Contactos | Contacts: dianadb@netuno.lcc.ufmg.br
Ascari et al. Phytochemical and biological investigations of Caryocar brasiliense Camb
INTRODUCTION spectrophotometric assay, as well as for their

allelopatic activity, evaluating their effect on the
Caryocaraceae is a small botanic family
growth of radicule and shoot of Lactuca sativa
widely distributed in Central and South America. It is
(lettuce).
constituted by two genera, Caryocar and Anthodicus,
which include 25 species. Caryocar genus presents the
higher number of species (16), being the most
economically important since their very nutritive fruits General Experimental Procedures
are used as source of edible oils and in the preparation
of juices and liqueurs (Prance, 1990). Melting points were determined with a Kofler
Caryocar brasiliense Camb., known in Brazil hot plate apparatus and are uncorrected. Infrared (IR)
as pequizeiro, deserves a distinguished position due to spectra were recorded with a Spectrum One with ATR-
the commercial, nutritional and gastronomic IR, from Perkin Elmer. Nuclear Magnetic Resonance
importance of its fruit, named pequi. Pequi is a (NMR) spectra were recorded in CD3OD, D2O, CDCl3
spherical green fruit, presenting 1-4 segments. Its and C5D5N, at room temperature, on Bruker Avance
structure is composed by an epicarp (very thin peel), DX-200 and DRX 400 MHz spectrometers.
an external pulpy mesocarp, internal mesocarp (light- Absorbance, in DPPH assay, was measured in a
yellow, pulpy, rich in oil, vitamins and proteins), that Hitashi 2010 spectrophotometer. Silica gel Merck
involve a layer of thin and rigid endocarp spines (Darmstadt, Germany) 100-200 and 200-425 mesh
(approximately 2-5 mm large) and white nut (seed). were used for column chromatography and silica gel
Together, internal mesocarp, needle endocarp and seed Merck 60G was used for thin layer chromatography.
constitute one segment (Damiani, 2006). The fruit is Polyamide was purchased from Macherey Nagel
used in Central Brazil culinary; fruits and leaves of C. (Düren, Germany). Solvents PA and HPLC grade were
brasiliense are used in the folk medicine to treat cold, purchased from Vetec (Brazil) and Sigma Chemicals
edema, bronchitis, cough, burns and is also used as a Co (St. Louis, USA), respectively. BHT (2,6-di-tert-
scaring agent (Vieira and Martins, 2000; Magalhães et butyl-4-methylphenol) and DPPH (1,1-diphenyl-2-
al., 1988). picrylhydrazyl), were also purchased from Sigma.
From C. brasiliense leaves β-amyrin, oleanoic
Plant Material
and ellagic acids and a mixture of β-sitosterol and
stigmasterol were isolated. Edible pulp showed to be Fruits of Caryocar brasiliense were collected
rich in vitamins A, C, riboflavin, thiamin and in January 2008, in Curvelo region, Minas Gerais,
carotenoids (Azevedo and Rodriguez, 2004). The oil Brazil, and identified by Dr. João Renato Stehmann. A
extracted from C. brasiliense fruits presented several voucher specimen (No. 120826) was deposited at the
biological properties such as anti-fungal (Passos et al., Herbarium of the Natural History Museum of UFMG,
2002), against Trypanosoma cruzi (Herzog-Soares et Belo Horizonte, Minas Gerais, Brazil.
al., 2002) and Biomphalaria glabrata (Bezerra et al.,
2002) activities and also showed to be an effective Extraction and Isolation
antioxidant agent (Paula-Junior et al., 2006). The fruit was open and the internal mesocarp
The external mesocarp is the part that presents was discharged. The external mesocarp, together with
the biggest dimension in the pequi fruit but it is thrown the epicarp was grinded in ethanol, using a liquefier,
away since this is a non edible part of the fruit. The and after 14 days at room temperature, the mixture
disposal of the external mesocarp generates a huge was filtered through a cotton plug followed by
amount of solid residues that could be used, adding Whatman filter paper. The extract was concentrated
value to the plant. In this work, the phytochemical with a rotatory evaporator and 256.0 g of ethanol
study of this part of the fruit, together with the epicarp, extract were obtained. A portion (130.0 g) of it was
is presented, since no systematic phytochemical study chromatographed over polyamide column (106.0 g),
addressing these parts were found in the literature. eluted with water, methanol and ethyl acetate pure and
Twelve compounds were isolated, pure or as mixtures of decreasing polarities. Twenty-eight
constituents of mixtures. Pequi fruit ethanol extract, fractions of 100 mL each were collected, and reunited,
fractions and some of the isolated compounds were by silica gel TLC, in five groups of fractions (G-1 to
tested for their antioxidant activity using the radical G-5). G-1 (72.6 g) was dissolved in water and
DPPH (1,1-diphenyl-2-picrylhydrazyl) extracted with diethyl ether; subsequent evaporation of
the solvents afforded 6.0 g of ethereal fraction (G-1E) oleic acid (10) mixture were isolated. From fractions
and 66.6 g of insoluble residue (T1). 7-18, 1.15 g of ethyl gallate (1) were obtained by
G-1E (6.0 g) was submitted to a silica gel filtration.
column chromatography, with CH2Cl2, CH3OH and
H2O, as eluent, either pure or in mixtures of increasing Spectrometric data
polarity: 48 fractions of 50 mL each were collect, and Compound 1 (Ethyl gallate): 3.58 g, 14.0 % yield.
pooled in 11 groups of fractions. Group of fractions 2 white solid, m.p.: 170.2-172.1 oC. IR (KBr, cm-1):
(635.8 mg) was rechromatographed on silica gel 3446, 3290, 2973, 2933, 1704, 1615, 1533, 1469, 1309
column with hexane, CH2Cl2 and ethyl acetate as and 1250. 1H NMR (400 MHz, C5D5N) δH (H, m, J in
eluent. A white solid precipitated from several Hz): 7.9 (H-2), 7.9 (H-6), 4.3 (H-8, q, 7.2); 1.2 (H-9, t,
fractions and, by washing with CH2Cl2 and filtration, 7.2). 13C NMR (100 MHz, C5D5N) δC: 121.8 (C-1);
1.4 g of a pure compound were obtained. This 110.6 (C-2); 148.0 (C-3); 141.3 (C-4); 148.0 (C-5);
compound, pure by TLC, was identified as ethyl 110.6 (C-6); 167.5 (C-7); 60.8 (C-8); 14.8 (C-9).
gallate (1). The solvents from the filtration were Compound 2 (5-Hydroxymethylfurfural): 41.0 mg,
evaporated and the residue (150.6 mg) was 0.02 % yield, viscous oil. IR (KBr, cm-1): 3369, 1658,
rechromatographed on silica gel column: group of 1582, 1519 and 1018. 1H NMR (200 MHz, CDCl3) δH
fractions 31-36 (41.0 mg) was found to be pure by (H, m, J in Hz): 7.2 (H-3, d, 3.4); 6.5 (H-4, d, 3.4); 9.6
TLC and was identified as 5-hydroxy-methylfurfural (H-6, s); 4.7 (H-7, s); 2.7 (s, OH). 13C NMR (50 MHz,
(2)
CDCl3) δC: 152.3 (C-2); 123.0 (C-3); 110.0 (C-4);
Group of fractions 6 (618.1 mg), from G-1E,
160.8 (C-5); 177.8 (C-6); 57.6 (C-7).
was submitted to silica gel column chromatography
Compound 3 (Gallic acid). 201.0 mg, 0.08 % yield.
(eluents: hexane, dichloromethane and ethyl acetate,
white solid, m.p.: 245-248 oC. IR (KBr, cm-1): 3500,
either pure or in mixtures of increasing polarity). From
3450-2600, 1650, 1600, 1540 and 1350. 1H NMR (400
group of fractions 2 (fractions 20-24), gallic acid (3)
MHz, CD3OD) δH (H, m): 7.1 (H-2, H-6, s); 9.0 (OH,
was isolated (201.0 mg). From group of fractions 8,
from G-1E (594.6 mg), after silica gel column s). 13C NMR (100 MHz, CD3OD) δC: 122.1 (C-1);
chromatography, 8.0 mg of pure methyl shikimate (4) 110.5 (C-2 and C-6); 146.5 (C-3 and C-5); 139.7 (C-
were isolated. 4); 170.6 (C-7);
Part of G-2 (9.5 g), from polyamide column, Compound 4: Methyl shikimate (8.0 mg, 0.003 %
was chromatographed over silica gel column, with yield): white solid, m.p.: 108-109 oC. IR (KBr, cm-1):
dichloromethane, ethyl acetate and methanol, as 3303, 1715, 1657 and 1233. 1H NMR (400 MHz,
eluent. Eight group of fractions were obtained and CD3OD) δH (H, m, J in Hz): 6.8 (H-2, m); 4.4 (H-3, br
group of fractions 2 (fractions 16-19) furnished s); 3.7 (H-4, dd, 6.8 and 3.6); 4.0 (H-5, m); 2.2 and 2.7
additional 1.03 g of ethyl gallate (1), by precipitation. (H-6, m); 4.8 (H-8, s); 4.5 (OH, br s). 13C NMR (100
Group of fractions 4 (fractions 25-28, 2.1 g), from G- MHz, CD3OD) δC: 130.0 (C-1); 139.3 (C-2); 67.4 (C-
2, after rechromatography over silica gel column, gave 3); 72.7 (C-4); 68.6 (C-5); 31.6 (C-6); 168.9 (C-7);
146.0 mg of a mixture of β-D-fructopyranose (5) and 52.5 (C-8).
β-D-fructofuranose (6). Group of fractions 5 (fractions Mixture 1 [β-D-fructopyranose (5) and β-D-
29-30, 2.3 g), from G-2, was also submitted to fructofuranose (6)]: 146 mg, 0.06 % yield. β-D-
chromatography on silica gel and lead to the isolation Frutopyranose (5). 1H NMR (400 MHz, CD3OD)
of a white solid (8.5 mg), that was identified as a δH (H, m): 3.5 and 3.7 (H-1, m); 3.8 (H-3, br s); 3.9
mixture of α-D-glucose (7) and β-D-glucose (8). Part (H-4, br s); 4.0 (H-5, d); 3.7 and 4.0 (H-6, m). 13C
of G-3 (8.0 g), from polyamide column, after NMR (100 MHz, CD3OD) δC: 64.7 (C-1); 99.3 (C-2);
submitted to silica gel column chromatography, using 69.6 (C-3); 72.0 (C-4); 71.4 (C-5); 64.7 (C-6). β-D-
the same eluent described, gave 9 groups of fractions. fructofuranose (6). 1H NMR (400 MHz, CD3OD)
Group of fractions 1 (0.3 g) was rechromatographed δH (H, m): 3.6 and 3.7 (H-1, m); 4.0-4.1 (H-3, H-4 and
on silica gel column (eluent hexane/acetone, either H-5, m); 3.5 and 3.8 (H-6, m). 13C NMR (100 MHz,
pure or in mixtures of increasing polarity), and 23 CD3OD) δC: 64.4 (C-1); 103.3 (C-2); 77.7 (C-3); 77.0
fractions were obtained. From fractions 1-7 (0.27 g), (C-4); 83.5 (C-5); 64.4 (C-6).
8.0 mg of lupeol (9) and oleic acid (10) mixture and Mixture 2 [α-D-glucose (7) and β-D-glucose (8)]: 8.5
20.0 mg of β-sitosterol (11), stigmasterol (12) and mg, 0.0033 % yield. α-D-Glucose (7). 1H NMR (400
MHz, D2O) δH (H, m, J in Hz): 5.2 (H-1, d, 3.6); 3,4 reference compound. Samples and BHT (750.0 μL)
(H-2, m); 3.7 (H-3, t, 9.6); 3.4 (H-4, t, 9.6); 3.7–3.8 were prepared in triplicate for each concentration used
(H-5, m); 3.7 (H-6, dd, 5.6 and 12.4). 13C NMR (100 (1.0, 10.0 and 100.0 μg/mL) and, to each flask, the
MHz, D2O) δC: 92.1 (C-1); 71.5 (C-2); 72.8 (C-3); volume was adjusted to 2.0 mL by adding 1.5 mL of a
69.6 (C-4); 71.3 (C-5); 60.6 (C-6). β-D-Glucose (8). 0.002 % p/v solution of DPPH in methanol. The
1
H NMR (400 MHz, D2O) δH (H, m, J in Hz): 4.6 (H- solutions were shaken vigorously and kept in the dark
1, d, 8.2); 3.2 (H-2, t, 8.8); 3.4-3.5 (H-3, H-4, H-5, m); for 30 min. The control was prepared as above without
3.7–3.8 (H-6, m). 13C NMR (100 MHz, D2O) δC: 95.9 any extract or substance. Absorbance (measured on a
(C-1); 74.1 (C-2); 75.7 (C-3); 69.6 (C-4); 75.9 (C-5); Hitashi 2010 spectrophotometer) was measured at 517
60.7 (C-6). nm and methanol was used for the baseline correction.
Mixture 3 [lupeol (9) and oleic acid (10)]: 8.0 mg, Radical scavenging activity was expressed as
0.003 % yield: Lupeol (9). 13C NMR (100 MHz, the inhibition percentage and was calculated:
CDCl3) δC: 38.4 (C-1); 27.5 (C-2); 79.4 (C-3); 39.0 (C- {(Abscontrol - Abssample)/Abscontrol} x 100
4); 55.6 (C-5); 18.7 (C-6); 34.6 (C-7); 41.2 (C-8); where Abscontrol = absorbance of DPPH radical
50.8 (C-9); 37.5 (C-10); 21.3 (C-11); 25.0 (C-12); 38.4 in methanol and Abssample = absorbance of the extracts
(C-13); 43.2 (C-14); 27.5 (C-15); 35.9 (C-16); 43.3 and pure substances in methanol + DPPH. Scavenging
(C-17); 48.3 (C-18, C-19); 151.3 (C-20); 29.4 (C-21); activities were expressed in μg/mL. IC50 values
40.3 (C-22); 28.3 (C-23); 15.7 (C-24); 16.4 (C-25); (μg/mL) was expressed the concentration of sample
16.3 (C-26); 14.9 (C-27); 18.2 (C-28); 109.6 (C-29); necessary to scavenge 50% of DPPH free radicals.
19.6 (C-30). Oleic acid (10) 13C NMR (100 MHz,
CDCl3) δ: 178.7 (C-1); 34.1 (C-2); 25.0 (C-3); 29.6 Allelopathic Bioassay
(C-4); 29.4 (C-5); 29.5 (C-6); 29.6 (C-7); 27.8 (C-8); Lactuca sativa (cv Grand Rapids) seeds were
130.3 (C-9); 130.1 (C-10); 27.7 (C-11); 29.7 (C-12); purchased from Isla Pak, RS, Brazil. All undersized
29.9 (C-13); 29.8 (C-14 and C-15); 32.2 (C-16); 23.0 and damaged seeds were discarded. According to
(C-17); 14.4 (C-18). metodology described by Vieira et al. (2005),
Mixture 4 [β-sitosterol (11), stigmasterol (12) and germination and growth were conducted in 100 mm
oleic acid (10)]: 20.0 mg, 0.0075 % yield. β-Sitosterol Petri dishes containing 9.0 cm sheet of Whatman no. 1
(11) and Stigmasterol (12). 13C NMR (100 MHz, filter paper as suport. Then, 25 lettuce seeds were
CDCl3) δC: 37.3 (C-1); 31.7 (C-2); 71.9 (C-3); 42.3 placed per dish with 10 mL of a test (10-4, 10-6 and 10-8
(C-4); 140.8 (C-5); 121.8 (C-6); 32.0 (C-7); 31.9 (C- M) or a control solution (without substance). All
8); 50.2 (C-9); 36.2 (C-10); 21.1 (C-11); 39.7 (C-12); solutions were prepared with deionized water and their
42.3 (C-13); 56.9 (C-14); 24.4 (C-15); 28.9 (C-16); pH values [buffered with 10 mM 2-(N-morpholino)
56.0 (C-17); 12.1 (C-18); 19.4 (C-19); 36.2 (C-20 for ethanesulfonic acid, MES] were adjusted to 6.0 - 6.5
β-sitosterol); 40.5 (C-20 for stigmasterol); 19.0 (C-21 with NaOH solution. Concentrations lower than 10-4
for β-sitosterol); 21.2 (C-21 for stigmasterol); 34.0 (C- M were obtained by dilution series. All tests were
22 for β-sitosterol); 138.3 (C-22 for stigmasterol); 26.1 triplicated. Dishes were covered with Parafilm to
(C-23 for β-sitosterol); 129.8 (C-23 for stigmasterol); reduce evaporation and incubated in the dark at 25 oC,
in a controlled-environment growth chamber, for 5
45.9 (C-24 for β-sitosterol);51.3 (C-24 for
days. After this time, the number of germinated seeds
stigmasterol); 29.2 (C-25 for β-sitosterol); 31.9 (C-25
were counted (a seed was considered to be germinated
for stigmasterol); 19.0 (C-26); 19.8 (C-27 for β- when the radicle was at least 0.2 mm long), the
sitosterol); 19.0 (C-27 for stigmasterol); 22.7 (C-28 for lengths of radicle and shoots were measured (using a
β-sitosterol); 25.4 (C-28 for stigmasterol); 12.0 (C-29 paquimeter). During the measurement process, the
for β-sitosterol); 12.3 (C-29 for stigmasterol). dishes were kept at 4 oC to avoid subsequent growth.
The osmotic pressure values were measured on a
DPPH Radical Scavenging Assay microsmometer and ranged between 30 and 38
Radical scavenging activities of extracts and mOsmolar.
flavonoids (Table 1) were determined according to the
method described by Burda and Oleszek (2001). BHT
(2,6-di-tert-butyl-4-methylphenol) was used as
7 8 9 7
COOCH2CH3 COOH

1 3 4 1
2 6 2
6
2
H 6 OH
HO 4 OH 5 HO 4 OH
O 7
O
OH OH
Ethyl galate (1) 5-Hydroxymethylfurfural (2) Galic acid (3)
7 8
2 6
HO 3
1 CO2CH3 CH2OH OH
OH
O OH
6
2
4 1 5 2
6
O CH2OH
HO 5 CH2OH
4 OH 4
HO 3 1
HO OH
3 OH
Methyl shikimate (4) β-D-Frutopyranose (5) β-D-Frutofuranose (6)
30
20
29
19
H OH H OH 17
H O H O 25 26
H OH 14 28
HO HO 1
HO 10 8
H HO H
3 27
HO OH HO
H
H H HO
24 23
α-D-Glucose (7) β-D-Glucose (8) Lupeol (9)

29
29
26
21 22 26
25 21 22
19 23 25
O 20 27 19 23
20 27
18
1 H 18
H
1
H H 1
3
9 H H
5 3
10 HO
6 HO 5
6
Oleic acid (10) β-Sitosterol (11) Stigmasterol (12)
Table 1 – Antioxidant activity of ethanol extract and fractions from epicarp and external mesocarp of C. brasiliense Camb. (pequi),
determined by DPPH method, in three different concentrations.
Extracts (1 µg/mL) Inhibition (%) IC50 (µg/mL)
/fractions (10 µg/mL) (100 µg/mL)
EE 0.0 63.7 94.4 28.3±6.6
G-1 5.4 51.3 96.7 9.3±6.3
G -1E 16.6 95.5 96.9 2,4±0.8
T1 3.6 16.9 91.7 22.2±2.5
BHT 40.4 45.6 84.2 16.4 ±3.6
Table 2 – Antimicrobial activity, in vitro, of G-1E fraction, obtained from epicarp and external mesocarp of C. brasiliense Camb. (pequi)
towards several bacterial strain and the yeast C. albicans
Inhibition zones (mm)a
Microrganisms G –1E Chloranfenicol Miconazol
S. aureus 13.0±2.8 20±2.8 nt
S. typhymurium 11.0±1.41 20±0.0 nt
E. coli 12.0±2.8 23±1.4 nt
C. freundi 11.0±0.0 22±0.7 nt
B. cereus 11.0±2.8 24±1.4 nt
L. monocytogenes 18.0±2.8 30±1.4 nt
P. aeruginosa 10.0±1.4 14±2.1 nt
C. albicans 0 nt 25±2.1
nt = not tested; aValues are mean ± SD of triplicate determinations.
Figure 2. Effect of ethanol extract (EE) and G-1 group from epicarp and external mesocarp of C. brasiliense Camb. (pequi) on radical and
shoot length of L. sativa, in three different concentrations. Values are presented as percentage differences from the control, zero representing
an observed value identical to the control (solution without substance), a positive value representing stimulation and a negative value
representing inhibition.
40
30
Radical
20
G-1
Length (% control)
EE
10
0
EE G-1
Shoot
-10
-20 1.0 mg/ mL

0.2 mg/ mL
0.04 mg/ mL
-30
Figure 3. Effect of ethereal fraction (G1-E), ethereal insoluble by using Student’s t-test and the differences between
fraction (T1) and G-3, from epicarp and external mesocarp of C. the experiment and control were significant at a value
brasiliense Camb. (pequi) on radical and shoot length of L. of P ≤0.05. The inhibitory and stimulatory activities,
sativa, in three different concentrations. Values are presented as compared to those of the control, are shown in
percentage differences from the control (solution without
substance), zero representing an observed value identical to the
Figures 2, 3 and 4.
control, a positive value representing stimulation and a negative
value representing inhibition. Antimicrobial Bioassay
30
Samples were tested in duplicate by disc diffusion
15
G1-E T1 G-3
method in agar with minor modifications (Lana et al.,
0
2003). Microorganisms used were Staphylococcus
Length (% control)
-15
Radical
-30 aureus ATCC 29212, Salmonella typhimurium

-45
-60
ATCC 14028, Escherichia coli ATCC 25922,
-75 Citrobacter freundi ATCC 8090, Bacillus cereus
30
G1-E T1 G-3 ATCC 11778, Listeria monocytogenes ATCC 15313,
15
Pseudomonas aeruginosa ATCC 27853 and Candida
albicans ATCC 18804. For minimum inhibitory
0
Shoot
1.0 mg/ mL
concentration test, carried out in duplicate, samples

-15 0.2 mg/ mL
0.04 mg/ mL
0.008 mg/ mL
were serially diluted starting from concentration of
-30
0.0016 mg/ mL
512 to 5.0 μg/ml for each test microorganism. Tubes

Figure 4. Effect of ethyl galate (1), hydroxymethylfurfural (2)
and galic acid (3), isolated from epicarp and external mesocarp of
were incubated for 18 hours at 35°c. The results are
C. brasiliense Camb. (pequi) on radical and shoot length of L. shown in Table 2
sativa, in three different concentrations. Values are presented as
percentage differences from the control (solution without RESULTS AND DISCUSSION
substance), zero representing an observed value identical to the
control, a positive value representing stimulation and a negative A total of twelve compounds were isolated
value representing inhibition. from the pequi fruit epicarp and external mesocarp
parts. Their structures were determined based on the
analysis of spectroscopic data, especially NMR, and
20 literature data comparison. The pure compounds were
1 2 3 identified as ethyl gallate (1) (Ceruks et al., 2007), 5-
10
hydroxymethylfurfural (2) (Kuo et al., 2002), gallic
Length (% control)
acid (3) (Souza Filho et al., 2006) and methyl

0
shikimate (4) (Liu et al., 2004; Adrio et al., 1997).
-10 Additionally, mixtures containing β-D-
fructopyranose (5) and β-D-fructofuranose (6)
1 2 3
-20
Radical (Sobolev et al., 2003; Breitmaier and Voelter, 1987);
Shoot
-30 -3
α- and β-D-glucose (7 and 8) (Collins and Ferrier,
10 M
-5
10 M 1995); lupeol (9) (Mahato and Kundun, 1994) and
-40
-7
10 M oleic acid (10) (Oliveira et al., 2006); β-sitosterol
(11), stigmasterol (12) (Goulart et al., 1993) and oleic
acid (10) were obtained. The structures of these
Data Analysis isolated compounds were assigned on the basis of
The effect on germination and growth are spectroscopic data, including two-dimensional NMR
given as percent differences from control, and consist methods and by comparison of their spectral data
of the differences (in cm) between mean values of with values described in the literature. Structures of
seeds with tested compounds and mean values for compounds 1-12 can be found in Figure 1.
control (seeds grown without addition of tested Ethyl gallate (1) was isolated on a very high
compounds)/ mean values for control x 100. Thus, yield (14% from the crude ethanol extract, EE),
zero represents the control, positive values represent whereas compounds 2, 3 and 4 were isolated on a
stimulation of the studied parameter and negative very low yield from EE.
values represent inhibition. The data were evaluated
Ethyl gallate and gallic acid were previously all tested microorganisms, except for S. typhimurium
isolated from leaves of Caryocar microcarpum and C. albicans was 512 μg/mL.
(Kawanishi and Raffaud, 1986). Oleic acid, β-
sitosterol and stigmasterol have already been isolated CONCLUSIONS
from C. microcarpum and C. villosum (Kawanishi This work pointed out for the possibility to
and Raffaud, 1986; Marx et al., 1997). use the external mesocarp of pequi fruit as a rich
Crude ethanol extract (EE), fractions G-1 source of ethyl gallate, since this compound was
obtained from polyamide column, G-1E (fraction isolated in an expressive yield (14 % from crude
soluble in ethyl ether, obtained from G-1) and T1 ethanol extract). The biological potential of this crude
(ethyl ether insoluble residue, obtained from G-1) extract and isolated compounds were also noticeable
were evaluated for their antioxidant activities (Burda (high IC50 in the antioxidant evaluation of extracts,
and Oleszek, 2001). Table 1 shows the average and both inhibitory and stimulatory effect on radical
values for DPPH radical scavenging activity in each and shoot growth of L. sativa, respectively from
tested concentration, and IC50 values. G1-E presented gallic acid), since plant material used is a residue
the higher IC50, that can be associated to the presence produced in large scale in Brazil.
of gallic acid and ethyl gallate in higher
concentrations than in EE and T1. At 100 μg/mL, all AKNOWLEDGEMENTS
extracts tested were more active than BHT, the
To CNPq, for JAT e MADB grants. To
reference compound.
FAPEMIG, for financial help.
The obtained results from allelopathic
evaluation, according to methodology described by
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Camb. Caryocaraceae, sob o aspecto farmacoquímico dados. Braz. J Med. Plant. 3:13-36.
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Mahato SB, Kundun AP. 1994. 13C NMR spectra of MADZ. 2005. Effects of kaurane diterpene derivatives
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© 2010 The Authors
Articulo Original | Original Article
Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.)

Britton from different regions of Argentina
[Actividad antimicrobiana de aceites esenciales de Aloysia triphylla (L`Her.) Britton procedentes de
diversas regiones de Argentina]
María de las M. OLIVA1, Emilia BELTRAMINO1, Nicolás GALLUCCI1, Carina CASERO1, Julio ZYGADLO2, Mirta
DEMO1
1
Universidad Nacional de Río Cuarto. Dpto de Microb e Inmunol. Ruta 36. Km 601. Rio Cuarto. Córdoba. Argentina. 2 Instituto
Multidisciplinario de Biología Vegetal (IMBIV). Cat. Qca. Org. UNC. Córdoba. Argentina.
Abstract
Essential oils are known to exert antimicrobial activity. Differences in the chemical composition of them influence this activity. This work intends
to study the variability in the chemical composition and the antimicrobial activity of essential oils obtained from plants of A. triphylla collected from different
regions of Argentina. Essential oils were obtained by hydrodistillation and analyzed with GC-MS. The antimicrobial studies were carried out by the paper
disc diffusion method. The essential oils shared common components but presented differences in the quantity and quality of the rest of them. The essential
oil from La Paz showed the highest citral/limonene relation and the best antimicrobial activity. Yeasts resulted to be the most sensitive microorganisms,
followed by the Gram positive bacteria. Statistical analysis showed significative differences in the antimicrobial activity. The differences in the biological
activity of each essential oil could be attributed to the quantity and quality of the terpenic composition.
Keywords: Aromatic plants; Aloysia triphylla; Antibacterial; antifungic; terpenes.
Resumen
Los aceites esenciales poseen conocida actividad antimicrobiana. Esta actividad puede estar influenciada por la composición química de los
aceites. El objetivo del presente trabajo fue estudiar la variabilidad en la composición química y la actividad antimicrobiana del aceite esencial obtenido a
partir de plantas de A. triphylla recolectada de diferentes regiones de Argentina. Los aceites esenciales fueron obtenidos por hidrodestilación y analizados por
GC-MS. Los estudios antimicrobianos se llevaron a cabo por la técnica de difusión en disco. Los aceites esenciales presentaron componentes mayoritarios
comunes y presentaron diferencias en la cantidad y calidad del resto de los componentes. La mayor relación citral/limoneno y la mejor actividad
antimicrobiana fue obtenida con el aceite esencial de La Paz. Las levaduras resultaron ser los microorganismos más sensibles, seguidos por las bacterias Gram
positivo. El análisis estadístico mostró diferencias significativas en la actividad antimicrobiana de las distintas muestras. Las diferencias en la actividad
biológica de cada aceite esencial podría ser atribuido a la cantidad y calidad de los terpenos lo constituyen.
Palabras Clave: Plantas Aromaticas; Aloysia triphylla; Antibacterianos; antifungicos; terpenos.
Recibido | Received: September 9, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: December 10, 2009.
Publicado en Línea | Published Online: December 15, 2009.
Declaración de intereses | Declaration of interests: Authors have no competing interests.
Financiación | Funding: We are grateful to SECyT of Universidad Nacional de Río Cuarto for financial support.
This article must be cited as: María de las M. Oliva, Emilia Beltramino, Nicolás Gallucci, Carina Casero, Julio Zygadlo, Mirta Demo. 2010. Antimicrobial activity of essential oils
of Aloysia triphylla (L`Her.) Britton from different regions of Argentina. Bol Latinoam Caribe Plant Med Aromat 9(1):29 – 37. {EPub December 15, 2009}.
*Contactos | Contacts:. E-mail: mdemo@exa.unrc.edu.ar. Tel: +54-0358-4676434, Fax: +54-0358-4676231.
Oliva et al. Antimicrobial activity of essential oils of A. triphylla from different regions of Argentina
industries. The pharmaceutical industry uses A.

INTRODUCTION
triphylla for its carminative, antispasmodic and
For centuries, indigenous plants have been sedative properties. There are several scientific
used in herbal medicine for curing various diseases. studies that support the use of products obtained from
The acceptance of traditional medicine as an A. triphylla. It has been found good antimicrobial
alternative form for health care and the development activity of the methanolic and ethanolic extracts, as
of microbial resistance to the available antibiotics well as it has been described antimicrobial activity in
have led scientific groups to investigate the the EOs (Akroum et al. 2009; Demo et al. 2005;
antimicrobial activity of medicinal plants (Ozturk and Oskay et al. 2005; Sartoratto et al. 2004). The
Ercisli, 2007). In addition, a major problem in the increasing interest in this specie has largely
food industry is that the microbial activity is a contributed to expanding A. triphylla crops in
primary mode of deterioration of many foods. Argentina, Chile, Paraguay, Europe and Africa
Currently there is a growing interest to use natural Mediterranean regions (Gil et al., 2007; Pascual et
antibacterial compounds for the preservation of al., 2001, Sartoratto et al. 2004).
foods, as these possess a characteristic flavor and Cedrón is included in the Código Alimentario
sometimes show antioxidant activity as well as Argentino (CAA) as a corrective and coadjutant, in
antimicrobial activity (Schelz et al., 2006; Teixeira- the section referred to vegetal condiments (Código
Duarte et al., 2007). Alimentario Argentino). “Cedrón” is recognized and
Plants which are rich in a wide variety of described in the Farmacopea Nacional Argentina, VI
secondary metabolites belonging to chemical classes Edición (FNA) as “the dried leaves with young
(tannins, terpenoids, alkaloids, polyphenols) are stems, flowers and fruits of Aloysia triphylla
generally superior in their biological activities (L´Hérit) Britt”. It is also described in the
suggesting that this strength is dependent on the Pharmacopoeias of France, Spain, Mexico and
diversity and quantity of such constituents (Geyid et Europe (Bandoni. 2000). It is included in the GRAS
al, 2005). Most of the antimicrobial activity in list (Generally Regarded as safe) and the Food and
essential oils (EOs) appears to derive from Drug Administration (FDA) has categorized it as a
oxygenated terpenoids as alcoholic and phenolic dietary supplement due to the wide use in America
terpenes, while other constituents are believed to and Occidental Europe (Barboza y col. 2001).
contribute little to the antimicrobial effect (Burt. Many EOs are known to exert antimicrobial
2004; Koroch et al. 2007.; Zygadlo and Juliani. activity (Schelz et al., 2006, Teixeira Duarte et al.,
2000). Therefore, the determination of the 2007). Differences in the chemical composition of
compounds responsible for any biological activity them related to variety, agronomic practice and
would facilitate the selection of the plants for future processing are also likely to influence antimicrobial
investigation properties, since these factors contribute to both the
Aloysia triphylla (L`Her.) Britton, (Aloysia profile and relative concentrations of active
citriodora Palau,) popularly known as “cedrón”, is a ingredients (Delaquis et al., 2002). The EOs content
member of the Verbenaceae Family. It is perennial is influenced by genetic material, culture conditions,
and grows widely in North and South America and environment, season, crop and post-crop processing
also in northeast, northwest and central regions of (Gil et al., 2007; Hussain et al., 2008).
Argentine. It is cultivated from Mexico till the South The components commonly found in A.
region of the continent. It is a bush with white triphylla EOs are: neral, geranial limonene, geranyl
flowers and fruits, with an intense scent lemon-like, acetate, betacaryophyllene, ar-curcumene, and
sweet, lightly floral, and herbaceous (Barboza y col. spathulenol. Other compounds that could be founds
2001; Gil et al. 2007). This specie is used in folk in specific chemotypes are carvone, cedrol, 1,8-
medicine to treat many digestive disorders, as cineol, thujone isomers and citronellal (Gil et al.,
antiinflamatory, analgesic, antipyretic, tonic and 2007).
stimulating. It shares an important place on the Due to the differences described in the
international herbal market due to the sensory and chemical composition of the EOs of a particular
medicinal properties of it EOs. These attributes vegetable specie, the aim of this work was to study
determine its use as a primary ingredient for the chemical composition of EOs obtained from
infusions and nonalcoholic beverages as well as samples of A. triphylla collected in different regions
aromatic ingredient for the flavor and fragrance
of Argentine and the relationship with the references mass spectra and retention indices of
antimicrobial activity. volatile compounds. GC-MS analysis was still
performed using a column Supelcowax 10 with the
MATERIALS AND METHODS same conditions as describe above (Adams. 1989).
Plant material Microorganisms

The plants of Aloysia triphylla were obtained The activity of the EOs was tested against the
in March, 2005, from plants growing in farms following microorganisms: Gram positive bacteria:
(plantations) located in different regions of Staphylococcus aureus ATCC 25923, Staphylococcus
Argentina: from Córdoba Province: Río Primero and epidermidis (milk), Micrococcus luteus ATCC 9341,
La Paz, from Salta Province: Las Viñas, from Enterococcus faecalis ATCC 29212, Bacillus cereus
Mendoza Province, from San Luis Province and one (rice). Gram negative bacteria: Escherichia coli
sample from Paraguay Republic. (water), Proteus mirabilis (urine), Klebsiella
pneumoniae (composting of poultry), and
Essential oils obtention Pseudomonas aeruginosa (water). The yeasts
The EOs were obtained from dried vegetable Candida albicans (mouth), Rhodotorula sp. (cereal)
material, which was hydrodistilled in a Clevenger- and Hansenula sp. (cereal) were used in order to
like apparatus. The oil obtained was dried with probe antifungal activity.
anhydrous sodium sulphate and stored in the freeze
until analysis (De Feo et al., 1998). Antimicrobial assays
Gas Chromatography-FID Analysis of the antibacterial activity

The EO were analyzed with a Shimadzu GC- The antibacterial studies were carried out
R1A gas chromatograph equipped with a fused silica according to De Pooter et al., (1995). The paper disc
column (30 m x 0.25 mm) coated with CBP-1. The diffusion method was used to test the antimicrobial
temperature of the column was programmed from activity. Tubes containing Triptein Soy Broth (TSB)
600C to 2400C at 40C/min. The injector and detector inoculated with the microorganisms were incubated
temperatures were at 2700C. The gas carrier was He, during 18 h, at 37 ºC. From these tubes ten-fold
at a flow rate of 1 ml/min. Peak areas were measured dilutions were made, until an OD ≅ 0.04 (106 cfu/ml)
by electronic integration. The relative amounts of the was reached. The antifungal activity was determined
individual components are based on the peak areas with the same methodology but using that dilution
obtained, without FID response factor correction. with an OD ≅ 0.4 (106 cfu/ml). The inoculumm
Programmed temperature retention index of the (200μl) was spread over plates containing Mueller-
compounds were determined relative to n-alkanes. Hinton Agar and a paper filter disc (6mm) was
GC analysis was still performed using a column impregnated with 10μl of the EO and placed on the
Supelcowax-10 with the same conditions as surface of the media. The plates were left 30 minutes
described above (Zunino et al., 1998). at room temperature to allow the diffusion of the oil
in the agar; then they were incubated at 37°C during
Gas Chromatography-Mass Spectrometry 24 hours. After this time the inhibition zone around
GC-MS analyses were performed on a Perkin the disc was measured with a caliper. Discs with
Elmer Q-910 using a 30 m x 0.25 mm capillary gentamicine (10 μg) were used as positive control.
column coated with CBP-1. The temperature of the
column and the injector were the same than those Analysis of the antifungal activity
from GC. The carrier gas was He, at a flow rate of Antifungal experiments were performed in
1ml/min. Mass spectra were recorded at 70 eV. The the same way as those with bacteria using Sabouraud
oil components were identified by comparison of Agar (SA) for the plates. Discs with anfotericine B (2
their retention indices, mass spectra with those of μg/ml) were used as positive controls.
authentic samples, by peak enrichment, with
published data, mass spectra library of National
Institute of Standards and Technology (NIST 3.0)
and our mass spectra library which contains
Minimum inhibitory concentration assay (MIC) caryophyllene oxide while San Luis had D
The minimum inhibitory concentration germacrene and biciclogermacrene (Table1).
(MIC) was performed according to the method The relation between major terpenic
previously described by De Feo et al., (1998). It was components of an EO has been proposed as a
determined by two-fold dilutions of EOs in dimethyl criterion to identify chemotypes (Muñoz-Collazos et.
sulfoxide (DMSO), placing 10 μl of each dilution on al., 1993). In this work the rate between citral (neral
a filter paper disc. The EOs concentration range was + geranial) and limonene has been calculated. The
from 900 mg/ml to 7.03 mg/ml. The discs were EOs from La Paz showed the highest citral/limonene
placed on the surface of a TSA plate, previously relation (16.9) and Las Viñas the least relation (0.73).
inoculated with 200 μl of each inoculumm, and left at The rest of the EOs relations were located between
room temperature to allow the diffusion of the oil. both of them (Table 1).
Then they were incubated at 37 oC during 24 h. After The antimicrobial activity of the EOs was
this time the inhibition zone around the disc was assayed against Gram positive bacteria, Gram
measured with a caliper. MIC was defined as the negative bacteria and yeasts. The yeasts resulted to be
lowest concentration that inhibited visible growth. the most sensitive microorganisms to the effect of the
The MIC with fungus was determined in the same EOs, followed by the Gram positive bacteria and
way as with bacteria using Sabouraud Agar (SA) in lastly the Gram negative ones. It is interesting to note
the plates. The negative control consisted in a paper that the three yeasts were inhibited by all the EOs,
disc impregnated with 10 μl of DMSO. The positive with average inhibition zones diameters of 22 mm.
(Table 2)
control was a disc impregnated with the antibiotic
The EOs from La Paz showed the best
gentamicine (10 μg) for bacteria. For yeasts,
antimicrobial activity, inhibiting the growth of all
anfotericine B (2 μg/ml) was used.
tested microorganisms, except P. aeruginosa. The
inhibition zones obtained with this oil for B. cereus
Statistical analysis
(38mm), M. luteus (33mm) and C. albicans are
All the experiments were performed in remarkable. Mendoza`s EOs presented good
duplicate and statistical analysis of the data were inhibition activity against microorganisms with
performed using GraphPad Prism 4.0 program. A average diameters of 29 mm against B. cereus. The
probability value of p<0.05 was considered EOs from Las Viñas, Paraguay and San Luis showed
statistically significant. varied antimicrobial activity, with inhibition zones of
20 mm, 21 mm and 15 mm for B. cereus,
RESULTS respectively. (Table 2)
The chemical composition of the EOs from Previously, it had been reported good
Aloysia triphylla collected from Rio Primero, La Paz, antimicrobial activity for the EOs obtained from Río
Las Viñas, Mendoza, San Luis and Paraguay has Primero (Demo, et al. 2005). Taking into
been investigated by means of gas chromatographic consideration these previous studies, La Paz and Rio
techniques. The EOs average yield in all the samples Primero, both located in Córdoba Province, showed
was 0.4% (w/v) and the components which were the best inhibition spectrum of all the oily samples.
commonly found in all the EOs samples were: However there were differences in their chemical
limonene, neral, geranial, spathulenol and composition.
caryophyllene oxide, with intrinsic variations in the B. cereus, S. aureus and M. luteus were the
quantity and quality of the resting terpenes in each most susceptible Gram positive bacteria to the EOs
sample (Table 1). The samples analyzed showed that action. The Gram negative bacteria E. coli and K.
the EOs from Mendoza had the biggest proportion of pneumoniae were inhibited by the EOs from La Paz
neral and geranial, followed by La Paz. Las Viñas and Las Viñas.
had the biggest proportion of limonene and carvone,
while neral and geranial were in the least proportion.
Rio Primero EOs was the only one presenting
camphor and borneol and the biggest proportion of α-
thujone. The EOs from Paraguay had spathulenol and
Table 1: Components identified in the essential oils of A. triphylla (%) colleced in 6 different places. In bold data accounting for the main
differences in composition.
Components Rio Primero La Paz Las Viñas Paraguay Mendoza San Luis
α thujene 0,8 tr 0,6 tr tr tr
α pinene 1,2 0,3 1,5 tr tr tr
camphene 0,6 - 0,9 - - -
6-metil-5-hepten-2-one - tr 1,7 tr tr tr
myrcene 1,7 tr 0,9 tr tr tr
p-cymene 0,4 tr tr tr tr tr
limonene 6,9 2,9 21,3 19,1 14,2 17,9
cis ocimene 0,5 tr 1,2 tr tr tr
γ terpinene 0,7 tr 0,9 tr tr tr
sabinene hydrate 0,5 - tr - - -
camphenilone 0,7 tr 2,2 tr tr tr
linalool 0,3 0,5 2,2 0,6 tr 0,3
α thujone 13,1 0,5 1,7 0,6 tr 0,2
2,2 dimetil-3,4 octadienal 0,4 1,3 - 0,5 tr 0,4
camphenol (6) 0,3 - tr - - -
dihydrolinalool 0,1 - tr - - -
cis verbenol 0,2 - tr - - -
citronellal 0,1 tr 1,1 tr tr tr
menthone 0,3 - - - - -
isoborneol - tr 0,8 tr tr tr
terpin-4-ol 0,3 - tr - - -
α terpineol 0,5 - 2,4 - - -
trans carveol 0,5 - 3,3 - - -
cis carveol 0,4 - 1,1 - - -
(E) ocimenone 0,4 0,5 tr 0,8 tr 0,4
neral 18,7 20 12,4 15,5 31,5 13
carvone 1,2 - 13,1 - - -
carvotanacetone 0,1 - - - - -
geranial 21,3 29,2 3,3 19,5 22,6 18,5
camphor 4,1 - - - - -
borneol 1,2 - - - - -
α copaene 0,8 0,5 tr 0,3 tr 0,8
β bourbonene 1 1 0,6 0,8 tr 0,9
β cubebene 0,2 tr 4,2 tr tr tr
α cedrene 3,2 0,9 tr 2,8 tr 3,2
(E) caryophyllene 0,4 0,6 tr 0,7 tr 0,4
α humulene 0,6 1,1 tr 0,5 tr 0,6
Cisdihydroαterpineol - - tr - - -
curcumene (ar) 0,1 tr 3,3 tr tr tr
germacrene D 4,3 2,3 tr 5,3 tr 6,9
α zingiberene 0,4 0,5 tr 0,3 tr 0,4
bicyclogermacrene 3,8 4,2 tr 6,8 3,9 7,2
cubebol 0,1 0,9 tr 0,7 tr 1,3
β curcumene 0,1 0,4 0,5 tr tr 0,8
δ cadinene 0,2 0,3 4,2 tr 3,2 0,2
(E)nerolidol 0,5 0,6 tr 1,6 9,6 0,9
spathulenol 0,9 8,9 6,6 11,1 4,4 10,1
cariophyllene oxide 1 7 6,9 10,5 4,5 10
globulol 0,8 0,3 tr tr tr 0,3
viridiflorol 0,5 2,5 tr 0,6 tr 1,1
guaiol 0,3 0,3 0,8 tr tr 0,2
Total 95,3 87,5 99,7 98,6 93,9 96
Markers
Citral (Neral + geranial) 40 49,2 15,7 35 54,1 31,5
Citral:Limoneno 5,8 16,9 0,73 1,83 3,8 1,76
Table 2: Antimicrobial activity of EOs of A. triphylla. Inhibition zones in (mm)

La Paz Las Viñas Paraguay Mendoza San Luis
MO P(<0.05)
δ δ δ δ δ
S. aureus 20 5.95 9 6.3 17 0.7 10 5.42 NI 0 0.0001
S. epidermidis 22 2.83 NI 0 13 3.53 11 2.82 9 2.12 0.0001
M. luteus 33 1.41 8 0.95 11 0.7 14 0.7 23 4.24 0.0001
E. faecalis 13.5 5.53 NI 0 NI 0 NI 0 NI 0 0.0001
B. cereus 38 11.2 20 11.54 21 5.65 29 6.38 15 0.7 0.0265
E. coli 8 4.33 7 7.68 NI 0 9 1.74 NI 0 0.0208
K. pneumoniae 10 0 4 4.92 NI 0 NI 0 NI 0 0.0169
P. mirabilis 10 0.7 NI 0 NI 0 4 4.94 NI 0 0.0001
P. aeruginosa NI - NI - NI - NI - NI - -
C. albicans 39 18.18 14 4.76 9 12.72 27 8.81 9 12.02 0.0101
Hansenula sp 16 3.53 20 0 22 0 20 0 NI 0 0.0001
Rhodotorula sp 34 7.07 10 0.7 11 0 22 2.12 17 1.41 0.0054
NI: No inhibition; (-): Not done;
Table 3: Minimun Inhibitory Concentration of the EOs of A. triphylla (mg/ml)
MO Place/EOs Concentration (900-7 mg/ml)
La Paz Las Viñas Paraguay Mendoza San Luis

S. aureus 28.1 NI 900 56.25 NI
S. epidermidis 28.1 NI 900 112.5 225
M. luteus 7 900 450 225 900
E. faecalis 56.25 NI NI NI NI
B. cereus 7 900 7.03 7.03 56.25
E. coli 900 NI NI NI NI
K. pneumoniae 900 900 NI NI NI
P. mirabilis 450 NI NI NI NI
P. aeruginosa NI NI NI NI NI
C. albicans 28.1 7 56.25 56.25 14
Hansenula sp 7 56.25 7 225 NI
Rhodotorula sp 7 900 7 28.1 112.5
The yeasts were the most sensitive for M. luteus and B. cereus (Table 3). Gram negative
microorganisms, being inhibited by al the EOs. La bacteria were inhibited by pure compounds with the
Paz EOs showed the biggest inhibition zones, with exception of P. mirabilis that presented a CIM of 450
diameters of 39 mm against C. albicans and 34 mm mg/ml with La Paz EOs.
against Rhodotorula sp, while San Luis showed the The best MIC values for C. albicans were
smallest one. The others EOs samples showed obtained with the EOs of Las Viñas (MIC: 7 mg/ml)
different degrees of inhibition activity against the and the best MIC values for Rhodotorula sp and
yeasts. Hansenula sp were obtained with La Paz and
In order to analize if the differences found in Paraguay EOs (MIC: 7 mg/ml) (Table 3).
the antimicrobial activity could be attributed to the
origin and composition of the EOs, the statistical DISCUSSION
analysis was performed. This analysis showed Differences in the content and composition of
significative differences in the antimicrobial activity the EOs of A. triphylla have been reported
of the EOs from all the places collected. These previously. In these reports, the EOs content ranged
variability was present in all the tested between 0.2 and 1% on dry weight (Gil et al., 2007,
microorganisms (Table 2). Sartoratto, et al. 2004). In this study, the average
For Gram positive bacteria the best values yield obtained with the EOs samples (0.4% (w/v))
were obtained with La Paz EOs with values of 28.1 was included between these values.
mg/ml for S. aureus and S. epidermidis and 7 mg/ml
There were chemical differences in the activity, while Las Viñas EO showed the least
quantity and quality of the EOs obtained from A. terpenic relation and antimicrobial activity (Table 1
triphylla collected from different places. However, and 2). These results are suggesting that higher rates
all the EOs samples shared the terpenic components between both compounds could be determining a
limonene, neral, geranial, espathulenol and better antimicrobial ability and a broader microbial
cariophyllene oxide, which were described by other spectrum. Some authors suggested that the
authors as the characteristically constituents of A. compounds present in the greatest proportions are not
triphylla EOs (Gil et al., 2007; Pascual et al., 2001; necessarily responsible for the greatest share of the
Stashenko y col., 2003). Each EO sample showed total activity. The data on the activity of the essential
particular features in the rest of the constituents with oils, in some cases are not compatible with those of
variability in the composition of each one. The EOs the pure constituents in higher percentages Thus, the
content in vegetable species is influenced by genetic involvement of the less abundant constituents should
material, culture conditions, environment, season, be considered (Cimanga et al., 2002; Tampieri et al.,
extraction methods, crop and post-crop processing 2005; Zygadlo and Juliani. 2000).
(Gil et al., 2007; Hussain et al., 2008; Tampieri et al.; Mono and sesquiterpenes and the mixture
2005). The culture conditions, kind of soil and between them in the oil, could constitute a barrier to
climate were particular for each one of the samples microbial infections (Cowan. 1999; Lambert et al.,
collected. Río Primero and La Paz are located in the 2001; Tan et al., 1999; Vataru Nakamura et al.,
central region of Argentina, Mendoza and San Luis 2004). The biotic and abiotic factors (environment,
are located in the central-west of Argentina while Las specie, chemotype) of the places where specie are
Viñas and Paraguay are in the north. Previous studies collected have influence on the quantity and quality
on A. triphylla have reported that the production and of the terpenic composition of the EOs.
composition of the EOs vary according to the part of Consequently, differences in the EOs yielding and in
the plant, the stage of development and the harvesting the biological activity are observed, being active
locations of the plant (Gil et al., 2007). Other against bacteria and fungi or only one of them (De
investigations support the fact that the differences in Pooter et al., 1995; Hess et al., 2007; Zygadlo and
the quantity of the terpenes identified in an EO Juliani. 2000). A study made with O. basilicum EOs
obtained from a vegetable collected in the same zone with regard to seasonal variations, showed changes in
are due to the environment (Karaman, 2006; Merle et the antimicrobial activity, attributing these variations
al., 2004). It was found variability in the terpenic to the different chemical composition of the oils.
composition of Ocimun basilicum EOs collected in Some earlier reports showed that the changes in
different seasons, concluding that the growing season chemical composition of an EOs directly affected
was affecting the chemical content (Hussain et al., their biological activities (Hussain et al., 2008). The
2008). It is worth to mention that it was analyzed an differences in the biological activity of each A.
EO sample from La Paz collected the following year triphylla EOs could be attributed to the quantity and
(2006) in order to analyze if variations in the quality of the terpenic composition and the possible
chemical composition related to the harvesting year associations between them. In addition, it could be
happened and it was found the same terpenic deduced that the antimicrobial activity is not only
composition as in 2005 with variations in the quantity dependent on the quality and quantity of the EOs but,
of them. (Data not shown) on the particular sensibility of each particular strain.
Citral and limonene were the major The inhibitory activity against
components identified in these EOs. It has been microorganisms responsible for human and plant
reported antimicrobial activity of both of them diseases of EOs from A. triphylla has been described
(Demo y col. 2001, Di Pasqua et al., 2006; Wolken et in other investigations (Demo et al., 2005; Pascual et
al., 2002). The individual activity of citral and al., 2001; Sartoratto, et al. 2004). What is more,
limonene could be suggesting that the relationship Sartoratto, et al. 2004, described MIC values of A.
between them could be determining the antimicrobial tryphilla (L´Hér.) Britton lower than
activity. This justifies the study of the rate between chloramphenicol, when it was used as the positive
them and the possible relation between this value and control antibiotic, showing the antimicrobial potential
the antimicrobial activity of the EOs. In this work the of this EO. (Sartoratto, et al. 2004) But, a comparison
EO of A. triphylla from La Paz showed the biggest of the chemical composition and the antimicrobial
rate citral/limonene and the best antimicrobial activity of the EOs obtained from A. triphylla
collected in different regions, and the relation Burt, S. 2004. Essential oils: antibacterial properties and
between this two variables, have not been previously potential aplications in food – a review. Int. J. Food
reported. Microbiol. 94: 223-253
It is difficult to compare results with others Cimanga K, Kambu K, Tona L, Apers S, de Bruyne T,
Hermans N, Totté J, Pieters L, Vlietinck AJ. 2002.
reported in the literature because of the naturally
Correlation between chemical composition and
varying composition of EOs even in the same species antibacterial activity of essential oils of some aromatic
due to the presence of chemotypes, different harvest medicinal plants growing in the Democratic Republic
times, different extraction methods, etc. Furthermore, of Congo. J. Ethnopharmacol. 79: 213-220.
it is important to consider the different Código Alimentario Argentino. Capitulo XVI Correctivos
microbiological tests utilized and the different y Coadyuvantes. Articulo 1215
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of the Essential Oil of Minthostachys verticillata
the genotypical composition of this specie should be
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taken into consideration, in order to obtain the ideal 65.
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The production of EOs and their utilization as fractions of dill, cilantro, coriander and eucalyptus
potential therapeutic agents and natural food essential oils. Int. J. Food Microbiol. 74: 101–109.
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María de las Mercedes Oliva, Mauro Nicolás speciosa. Flavor Frag. J. 10:63-67.
Gallucci, Carina Caseros and Julio Alberto Zygadlo Di Pascua R, Hoskins N, Betts G, Mauriello G. 2006.
are researchers from CONICET. We are grateful to Changes in membrane fatty acids composition of
microbial cells induced bu addiction of thymol,
SECyT of Universidad Nacional de Río Cuarto for
carvacrol, limonene, cinnamaldehyde, and eugenol in
financial support. We thanks to Plantadroga S. A. the growing media. J. Agr. Food Chem. 54: 2745-
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Alvarez Toledo (Salta sample) for the provission of Geyid A, Abebe D, Debella A, Makonnen Z, Aberra F,
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© 2010 The Authors
Articulo original | Original article
Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae

sensu lato de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia
parvifolia Engl., Picramnia sellowii Planch. y Castela coccinea Griseb.
[Morphoanatomy of leaves barks and wood of Argentinean Simaroubaceae sensu latu. Part I: Alvaradoa
subovata Cronquist, Picramnia parvifolia Engl., Picramnia sellowii Planch. and Castela coccinea Griseb]
Adriana CORTADI1, Luisina ANDRIOLO1, María Noel CAMPAGNA1, María Laura MARTÍNEZ1, Osvaldo Di SAPIO1,
Adriana BROUSSALIS2, Martha GATTUSO1, Susana GATTUSO1.
1
Cátedra de Farmacobotánica. Departamento de Ciencias Biológicas. Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR. Suipacha
531. S2002 LRK. Rosario. República Argentina. 2Cátedra de Farmacognosia. Facultad de Farmacia y Bioquímica, UBA. Junín 956, 2º piso.
CP 1113 CABA.
Abstract
The Simaroubaceae (sensu lato) family is represented in Argentina by six genera and eight species, seven of which are native and only one non-
autochthonous; some are used in folk medicine as tonic, insecticides and pesticides. Leaves were cut previous paraffin embedding and diaphanization.
Longitudinal and cross-sectional cuts were made on cortex and leños and were cut and macerated. They were stained with Safranine-Fast-green and Cresyl
Violet. Microscopic examination was performed by light microscopy and SEM. In the present work leaves, cortex and leños from Alvaradoa subovata,
Picramnia parvifolia, Picramnia sellowii and Castela coccinea were morpho-anatomically analyzed in order to determine diagnosis characters to identify
diagnostic characters to ensure the identity and quality of these resources. In leaves, namely, 1. presence or absence of gland hairs, mesophile type, presence
of mucilage; 2. cortex: periderm, radii types, crystals, sclerenchyma elements; 3. leño size, pores location, radii, and axial parenchyma, among others.
Complete the presentation with photomicrographs and keys in order to provide adequate differentiation between entities.
Keywords: Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii, Castela coccinea, Simaroubaceae, morphoanatomical characters
Resumen
Las Simaroubaceae (sensu lato) está representada en Argentina, por 6 géneros y 8 especies, de las cuales 7 son nativas y una introducida; algunas
son utilizadas en medicina popular como tónicas, insecticidas y antiparasitarias. Las hojas se cortaron previa inclusión en parafina y se diafanizaron; las
cortezas y leños se cortaron longitudinal y transversalmente y se maceraron. Se colorearon con Safranina-Fast-green y Violeta de Cresyl. Las observaciones
se realizaron con microscopio óptico y microscopio electrónico de barrido. En este trabajo se han estudiado morfoanatómicamente las hojas, cortezas y leños
de Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii y Castela coccinea a fin de determinar caracteres diagnósticos para garantizar la identidad y
calidad de estos recursos. En hojas: presencia o ausencia de pelos glandulares, tipo de mesófilo, presencia de mucílagos; en cortezas: la peridermis, tipos de
radios, cristales, elementos esclerenquimáticos; en leños: tamaño y disposición de los poros, radios, y parénquima axial, entre otros. Se completa la
presentación con fotomicrografías y claves con el objeto de brindar una adecuada diferenciación entre las entidades.
Palabras Clave: Alvaradoa subovata; Picramnia parvifolia; Picramnia sellowii; Castela coccinea; Simaroubaceae; caracteres morfoanatómicos.
Recibido | Received: Agosto 4, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: Noviembre 30, 2009.
Publicado en Línea | Published Online Diciembre 15 2009
Financiación | Funding: ANPCyT, proyecto PICT BID/2007-1494 y SECYT BIO/2008-200.
This article must be cited as: Adriana Cortadi, Luisina Andriolo, María Noel Campagna, María Laura Martínez, Osvaldo Di Sapio, Adriana Broussalis, Martha Gattuso, Susana
Gattuso. 2010. Estudio farmacobotánico de hojas, cortezas y leños de Simaroubaceae (sensu lato) de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia parvifolia Engl.,
Picramnia sellowii Planch. y Castela coccinea Griseb. Bol Latinoam Caribe Plant Med Aromat 9(1):38 – 55. {EPub 15 December 2009 }.
*Contactos | Contacts: sgattuso@fbioyf.unr.edu.ar
Cortadi et al. Caracteres morfoanatómicos de especies de Simaroubaceae Part I
actividades biológicas in vivo y/o in vitro. Los

INTRODUCCIÓN cuasinoides de C-20 han sido extensamente
La familia Simaroubaceae incluye 30 géneros y estudiados por sus propiedades biológicas, dado que
200 especies de climas tropicales y subtropicales. se han encontrado algunos compuestos con marcada
Los estudios taxonómicos referidos a las actividad antileucémica (Guo et al. 2005); actividad
Simaroubaceae de América fueron abordados por antitumoral (Cuendet y Pezzuto 2004, Fukamiya et
Cronquist (1944), Pirani (1987). Este mismo autor se al. 1992, Guy Balansard y Hajime Ohigashi 2002,
ocupo de las especies de Picramnia de Brasil en un Ogura et al. 1977, Toyota et al. 1990), el modo de
detallado trabajo (Pirani 1990). acción de los cuasinoides en ésta actividad fue
En la Argentina las Simaroubaceae (sensu lato) encarada por Grieco et al. (1997) y Morre et al.
están representadas por una especie introducida (1998) entre otros; actividad antimalarica, (Bourdy et
Ailanthus altissima (Mill) Swingle y siete especies al. 2004, Castro et al. 2006, O´Neill et al. 1986, 1987,
nativas, Castela coccinea Griseb, Castela tweedii 1988, Ajaiyeoba et al. 1999, Bertani et al. 2007);
Planch., Picrasma crenata. (Vell.) Engl., Simaba actividad antiviral, (Grieco et al. 1997); actividad
glabra, Alvaradoa subovata Cronquist, Picramnia antifeedant-insecticida (Lidert et al. 1987); actividad
parvifolia Engl. y Picramnia sellowii Planch., antiparasitaria-antiprotozoaria (Camacho et al. 2003,
(Xifreda 1999, Xifreda y Seo, 2006). 2004, Guy Balansard y Hajime Ohigashi 2002,
Existen datos anatómicos de la madera, (Webber, Moncayo 2003, Martínez et al. 2009); actividad
1936; O´Donell, 1937; Heimsch 1942; Metcalfe y herbistática-herbicida (Grieco et al. 1997, Siwajinda
Chalk 1950, 1972), Wheeler et al. (1986, 1989) et al 2001) y actividad citotóxica (Anderson et al
trabajaron con maderas duras, incluyendo al género 1991, Cordell et al. 1993, Tagahara y Kuo-Hsiung
Picramnia confeccionaron una base de datos con 1993).
caracteres anatómicos; morfología del polen Actualmente, el análisis micrográfico de aquellas
(Erdtman 1986; Moncada y Machado 1987, estudios especies biológicamente activas, es requisito
fitoquímicos (Stuhlfauth et al. 1985; Da Silva y indispensable para las Farmacopeas herbarias de todo
Gottlieb 1987; Simao et al. 1991) y de la estructura el mundo, las cortezas y maderas de la flora de
del pericarpio (Fernando y Quinn 1992). Argentina han sido poco estudiadas, en este contexto,
Las hojas, cortezas y leños de algunas de estas el objetivo del presente trabajo es estudiar caracteres
especies de Simaroubaceae (sensu lato) son utilizadas morfoanatómicos de hojas, cortezas y leños de
en la medicina popular para el tratamiento de Alvaradoa subovata, Picramnia parvifolia,
enfermedades de la piel: Picramnia sellowii Picramnia sellowii y Castela coccinea, de modo de
(Balderrama et al. 2001); vermicida: Picramnia tener no sólo una identificación morfológica sino
antidesma y Picrasma exelsa (Kelly y Dickinson también micrográfica de las mismas, permitiendo la
1985) y en trastornos del tracto gastrointestinal: identificación de cada especie cuando se encuentren
Ailanthus altissima (Martínez Crovetto, 1981; molturadas.
Grieve, 1996); Castela tweedii y Picrasma crenata
MATERIALES Y MÉTODOS
(Martínez Crovetto, 1981, Simao et al. 1991) y
Castela coccinea (UMSA-Fundación, 2002, Bourdy
Materiales estudiados
et al. 2004). El leño de Alvaradoa amorphoides se
usa como tónico estomacal y la corteza como Se emplearon materiales fresco y de los herbarios
antipruriginosa (Martinez, 1969; Toursarkissian, BAA, MCNS, SF, SI y UNR, los que son citados
1980). conforme a las siglas respectivas (Holmgren et al.,
Las Simaroubaceae se caracterizan por la 1990). El material fresco fue coleccionado por los
presencia en todos sus representantes de cuasinoides, autores, en las provincias de Misiones, Corrientes,
principios amargos, derivados de triterpenos Chaco, Santiago del Estero, Tucumán, Jujuy, Salta y
degradados y alcaloides derivados del triptofano, β- Santa Fe. Para los estudios anatómicos fueron
carbolines, canthinones, clasificados en los tipos utilizados siete ejemplares de cada especie.
estructurales C1-C5, (Simao et al 1991). Los Se utilizó material de herbario y fresco, a este se
cuasinoides de acuerdo a su estructura química se lo fijó en FAA (alcohol etílico 70º, ácido acético
dividen en 5 grupos C-18, C-19, C-20, C-22 y C-25. glacial, formaldehído y agua 50:5:30:15) y al
muchos de éstos tienen un amplio rango de material de herbario se lo hidrató. Para el estudio de
las hojas, las láminas, se cortaron transversalmente en Bioquímicas y Farmacéuticas (UNR). Las
la parte media de las mismas con micrótomo tipo preparaciones histológicas se hallan depositadas en la
Minot, previa inclusión en parafina (Gattuso y histoteca de dicha Cátedra.
Gattuso 2002). Para el análisis de las epidermis,
venación y micrografía cuantitativa, las láminas RESULTADOS
foliares se diafanizaron (Strittmatter, 1973) y se La identificación de estas cuatro especies se
determinaron los siguientes parámetros: índice de realiza por una combinación de caracteres
estomas (Salisbury, 1927), estomas por milímetro morfológicos y anatómicos de las partes utilizadas,
cuadrado (Timmerman, 1927), índice de empalizada los que se presentan en cuadros, figuras y láminas.
(Zorning y Weiss, 1925) y pelos simples por
milímetro cuadrado; para todas estas medidas se
trabajó con objetivo de 40x con un ocular de 10x. DESCRIPCIÓN MACROSCÓPICA DE LAS
Para la descripción de la arquitectura foliar se utilizó ESPECIES ESTUDIADAS
la terminología de Hickey (1973) y para los pelos
Üphof et al. (1962). Las cortezas y leños se cortaron Alvaradoa subovata Cronquist. Brittonia 5 (2):134.
con xilótomo en forma transversal y longitudinal 1944.
(radial y tangencial), previo ablandado con agua Sinónimos: Alvaradoa amorphoides var. puberulenta
hirviendo adicionada de una gotas de detergente Monach. Lilloa 8: 407. 1942.
comercial; y se maceraron aplicando la técnica de Nombres vulgares: “Pichi-blanco”, Sacha ruda” o
Boodle (1916) y se midió con ocular micrométrico la “Chuquisaca”.
longitud y el diámetro de los elementos vasales como Uso vernáculo: El leño se utiliza como tónico
así también longitud y latitud de fibras. estomacal, y la corteza como antipurriginosa.
Las coloraciones empleadas fueron Safranina (Toursarkissian, 1980).
alcohólica 80º, Safranina-Fast-green (Strittmatter, Hojas: compuestas, pecioladas, alternas,
1979) y Violeta de Cresyl (Strittmatter, 1980). La imparipinnadas con 16-24 folíolos, de 2-4 cm de
distribución de los cristales de oxalato de calcio fue long. x 0,4-1 cm lat., oblongos, alternos, borde
analizada utilizando luz polarizada. entero, ápice retuso, base aguda (Fig.1 A, a).
Para la descripción de los elementos del leño se Corteza: color castaño grisáceo, escasas grietas
usó IAWA Committee (1989). longitudinales, numerosas lenticelas. Fractura entera.
Las ilustraciones son originales y fueron Leño: color amarillo pálido, poros apenas visibles,
realizadas con microscopio óptico (MO) Nikon anillos pocos diferenciables, albura blanquecina.
Alphaphot, con tubo de dibujo. Para los esquemas se
siguió la simbología de Metcalfe y Chalk (1950). Las Picramnia parvifolia Engl. Fl. Bras. 12(2): 242, pl.
fotomicrografías fueron obtenidas con Microscopio 49. 1874.
Carl Zeiss Axiolab y equipo fotográfico MC 80. Los Nombres vulgares: No conocidos.
detalles de las epidermis en superficie, cortezas y Uso vernáculo: No se registra en la bibliografía
leños fueron observados con microscopio electrónico consultada ningún uso medicinal para esta especie.
de barrido (MEB) Leitz AMR 1000; en el caso de la Hojas: compuestas, pecioladas, alternas,
láminas foliares las muestras fueron fijadas en imparipinnadas de 7-15 folíolos, de 2-10 cm de long.
glutaraldehído al 4 % deshidratadas en alcoholes x 0,8-2 cm lat., alternos a subopuestos,
ascendentes, se aplicó punto crítico y finalmente se membranáceos, oblongo-elíptico a oblongo
metalizó con oro paladio (O´Brien y McCully, 1981). lanceolados, margen poco revoluto, ápice
Las observaciones morfológicas se efectuaron con subacuminado o más raramente obtuso, base aguda y
microscopio estereoscópico Nikon SMZ-U asimétrica, a veces oval obtusa (Fig.1 B, b).
ZOOM1:1 con tubo de dibujo. Corteza: color pardo rojizo superficie uniforme,
Para los caracteres anatómicos cuantitativos se escasas lenticelas. Fractura entera.
calcularon las medias aritméticas (x) con su Leño: color amarillo brillante, poros imperceptibles,
correspondiente desvío estándar sobre 10 campos. anillos apenas diferenciables, albura y duramen de
Se acondicionaron los ejemplares para la aspecto homogéneo
incorporación a los herbarios UNR y de la Cátedra de
Farmacobotánica de la Facultad de Ciencias
Fig.1. A-V: Caracteres morfo-anatómicos foliares diferenciales. A-D: tipos de hojas, observación con microscopio estereoscópico. A-C:
compuestas pinadas: A, de 16-24 folíolos en A. subovata. B, de 7-15 folíolos en P. parvifolia. C, de 9-15 folíolos en P. sellowii. D, hoja
simple en C. coccinea. E-V: observación con MO. E-M, V: vista superficial de las epidermis: E-F, adax. y abax respectivamente en A.
subovata. G-H, adax. y abax. respectivamente en P. parvifolia. I-J, adax. y abax. respectivamente en P. sellowii. K-L, adax. y abax.
respectivamente en C. coccinea. M, pelo glandular en P. sellowii. V, cavidad esquizógena en P. parvifolia. N-U, sección transversal de las
láminas foliares: N-S, mesófilo dorsiventral: N-O, en A. subovata. P-Q, en P. parvifolia. R-S, P. sellowii. T-U, mesófilo céntrico en C.
coccinea. En todos, esquemas y detalle de lo indicado. Caracter anatómico foliar común: a-c, D: arquitectura foliar camptódroma
broquidódroma, a-c folíolos, D hoja. Escalas: 1 corresponde a O, Q, S, U. 2 corresponde a M, V. 3 corresponde a N, P, R, T. 4 corresponde a
E-L.
Fig.2. A-G: Caracteres anatómicos foliares diferenciales. Observación con MO. A-C, sección transversal de la lámina foliar; A-B, mesófilo
dorsiventral: A, A. subovata; B, P. parvifolia. C, mesófilo céntrico en C. coccinea. D-G: vista superficial: D-E en P. sellowii: D, pelos
simples y glandulares (flechas), E, pelo glandular y estomas anomocíticos. F, C. coccinea, pelos simples (flechas) y estomas. G, A. subovata,
pelos simples y estomas hundidos (flechas). ce, cavidad esquizógena; e, epidermis; ep, epidermis papilosa; et, estomas; h, hipodermis; m,
mucílagos.
Fig.3. A-R: Caracteres anatómicos diferenciales de las cortezas. Observación con MO. A-H: Representación esquemática de la corteza y
detalle de la sección transversal de las células del súber: A y E: C. coccinea. B y F: A. subovata. C y G: P. parvifolia. D y H: P. sellowii.
Macerado de corteza: I: Células del súber en vista superficial. J: fibras libriformes. K: parénquima axial. L: células de radio. M y N: drusas,
cristales poliédricos y estiloides de oxalato de calcio. O: braquiesclereidas. P: fibroesclereidas. Q: macroesclereidas. R: macroesclereidas
con inclusión de cristales poliédricos. Escalas: 1 corresponde a J, O-R. 2 corresponde a L-M. 3 corresponde a E-I, K. 4 corresponde a A-D.
Fig.4. A-D: Sección transversal de los leños .a-d: detalle de los vasos. Observación con MO A y a: A. subovata. B y b: P. parvifolia. C y c:
P. sellowii. D y d: C. coccinea
Fig.5. A-F: Caracteres anatómicos diferenciales y comunes del leño. Observación con MEB. A-D: Sección transversal del leño: A, A.
subovata poros solitarios con distribución radial y oblicua. B, P. parvifolia poros solitarios. C, P. sellowii poros solitarios y múltiples
radiales. D, C. coccinea poros solitarios, geminados, racemiformes y múltiples radiales. E-F: sección longitudinal: E, vasos. F, puntuaciones
areoladas alternas. G-I: Cristales de oxalato de calcio en corteza. Observación con MEB. G, solitarios poliédricos. H, drusa. I, estiloides
(flechas).
Picramnia sellowii Planch. London J. Bot. 5: 578. transversales cortas, lenticelas prominentes y notable
1846. depósito de líquenes. Fractura fibrosa.
Sinónimos:Picramnia sellowii fo. glabrescens Leño: color castaño amarillento, brillante, anillos de
Chodat & Hassl. Bull. Herb. Boissier, sér.2, 3:800. crecimiento medianamente visibles, delimitados por
1903 una franja pardusca. Porosidad difusa, con tendencia
Picramnia sellowii fo. hirsuta. Chodat & Hassl. Bull. a semicircular. Duramen y albura no diferenciables.
Herb. Boissier, sér.2, 3:800. 1903
Picramnia sellowii fo. intermedia Chodat y Hassl. CARACTERES ANATÓMICOS COMUNES
Bull. Herb. Boissier, sér.2, 3:800. 1903
Picramnia sellowii var. latifolia Engl. Fl. Bras. Hojas
12(2):232. 1874. 1. Lámina en vista superficial
Picramnia sellowii subsp. spruceana (Engl.) Pirani. Arquitectura: camptódroma, broquidódroma
Bol. Bot. Univ. Sao Paulo 12: 132. 1990. (Fig.1 a, b, c, D) En todas las especies hay de 4 a 5
Nombres vulgares: “Cedrillo”, “Cedrillo-na” y órdenes de venas, las secundarias son pinnadas,
“Tarirí” mientras que las de orden superior son reticuladas.
Uso vernáculo: Se utiliza como alterante Las venas marginales forman ojales cerrados con
(Toursarkissian, 1980). terminaciones vasculares libres. Las areolas son
poligonales dispuestas al azar, coexistiendo
Hojas: compuestas, pecioladas, alternas, terminaciones vasculares simples y ramificadas y
imparipinnadas de 9-15 folíolos, de 4-8 cm de long. x rectas o curvas. La red vascular es de densidad
1-3 cm lat., alternos a subopuestos en la misma hoja, intermedia.
membranáceos a cartáceos, oval lanceolados, margen Epidermis: las células de la epidermis adaxial
poco revoluto, ápice obtuso, base asimétrica obtusa o (Fig.1 E, G, I, K) son ligeramente más grandes que
raramente aguda (Fig.1 C, c). las de la epidermis abaxial (Fig.1 F, H, J, L),
Corteza: color gris pardusco; estrías longitudinales y elongadas sobre los nervios. Los estomas están
transversales poco profundas que delimitan pequeñas confinados a la epidermis abaxial. En ambas
placas. Abundantes lenticelas con importante reborde epidermis se observan escasos pelos simples,
y apertura en cruz. Fractura entera. unicelulares, de paredes delgadas, que se ubican con
Leño: color amarillo pardusco, poros no visibles, mayor densidad sobre las nervaduras, siendo la
anillos apenas perceptibles, albura y duramen no longitud de los mismos diferente para cada especie
diferenciables. (Fig.1 F, H, J, K, L. Fig.2 D, F, G).
2. Lámina en corte transversal
Castela coccinea Griseb. Abh. Konigl. Ges. Wiss. Ambas epidermis son uniestratificadas. La hoja es
Gottingen 19: 107. 1874. hipostomática (Fig.1 N, P, R, T). En posición
Sinónimo:Ximenia americana var. purbens Griseb. subepidérrmica la vena media se halla reforzada por
Symb. Fl. Argent. 149. 1879. colénquima de tipo laminar del lado adaxial y
Nombres vulgares: “Espada”, “Granadillo”, abaxial. El nervio medio está constituido por 5 a 7
“Meloncillo”, “Mistol del zorro”, “Mistol del chivo”, haces vasculares colaterales abiertos dispuestos en
“Molle Colorado”, “Sacha melón” y “Sacha arco, acompañados por una vaina conspicua de fibras
meloncillo”- (Fig.1 N, P, R, T).
Uso vernáculo: La corteza, hojas y raíz se utilizan en
contra de la disentería, diarreas y fiebres Cortezas
intermitentes; también se reconoce como tónico Felodermis pluriestratificada.
gástrico (Xifreda y Seo, 2006)
Hojas: simples, cortamente pecioladas, alternas, de Leños
1,5-3 cm de long. x 0,5-1 cm lat., oblongas
De porosidad difusa (Fig.4 A-D), la
pinnatinervias, de margen liso, ápice redondeado,
disposición de los poros es variable según las
base cuneada (FIg.1 D, d).
especies. Los miembros de vasos son en su mayoría
Corteza: color pardo grisáceo a pardo amarillento,
de contorno circular y se observan escasos elípticos,
muy rugosa, ligeras estrías longitudinales y
presentan placa perforada simple y oblicua, con
apéndices (Fig.4 a-d), las puntuaciones Las fibras son de dos tipo: 1- libriforme,
intervasculares son areoladas, de disposición alterna, dispuestas de manera no estratificada, algunas de
con abertura interna elíptica e inclusa (Fig.5 E, F). ellas son septadas y 2- fibrotraqueidas de paredes
El parénquima axial se encuentra presente. moderadamente engrosadas.
Los radios son no estratificados, las células
no dejan espacios intercelulares y son de paredes
medianamente engrosadas.
CARACTERES ANATOMICOS DIFERENCIALES

Tabla 1: Caracteres anatómicos diferenciales de las hojas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Alvaradoa subovata Picramnia parvifolia Picramnia sellowii Castela coccinea
Células de paredes
Epidermis abaxial rectas (Fig.1 F. Fig.2 Células de paredes ligeramente sinuosas (Fig.1 H, J, L. Fig.2 E, F)
G)
Vista superficial
Tipo Anomocíticos (Fig.1 Anomo y paracíticos Anomocíticos (Fig.1 Anomocíticos (Fig.1

Estomas F) (Fig.1 H) J. Fig.2 E) L. Fig.2F)
Posición Hundidos (Fig.2 G) A nivel
Pie uniseriado de
Ausentes longitud variable y
Pelos glandulares Ausentes
cabeza pluricelular
(Fig.1 M; Fig.2 D, E)
Gruesa y lisa (Fig.2
Cutícula en ambas epidermis Delgada y lisa
C)
Uniestratificada
Con algunas células
Epidermis adaxial Con hipodermis
Papilosa (Fig.1 O. septadas;
Uniestratificada mucilaginosa (Fig.1
Fig.2 A) mucilaginosas (Fig.2
T, U. Fig.2 C)
B)
Céntrica (Fig.1 T.
Dorsiventral (Fig.1 O, U, S)
Fig.2 C)
2-3 hileras de 2-3 hileras de
Sección transversal
Mesófilo empalizada (Fig.1 O. 1-2 hileras de empalizada. (Fig.1 Q, S. Fig.2 B) empalizada (Fig.1 U.
Fig.2 A) Fig.2 C)
Parénquima esponjoso
Parénquima esponjoso
con mucílagos (Fig.2 Parénquima esponjoso sin mucílagos
sin mucílagos
B)
Cavidad esquizógena
Estructuras secretoras internas Ausentes con gomorresinas Ausentes
(Fig.1 V; Fig.2 B)
Drusas y escasos Drusas y abundantes Drusas y escasos
Cristales de oxalato de calcio Drusas
estiloides cristales solitarios cristales solitarios
La vaina La vaina
Haces de nervios menores parenquimática no parenquimática La vaina parenquimática no alcanza ambas
alcanza ambas alcanza ambas epidermis
epidermis epidermis (Fig.2 B)
Uniestratificada Uniestratificada
Epidermis abaxial Uniestratificada
papilosa mucilaginosa
Tabla 2: Caracteres anatómicos diferenciales de las cortezas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Dimensiones variables, Paredes con Dimensiones variables, Homogéneas, no
paredes con engrosamiento mediano paredes con aplanadas, de paredes
engrosamiento mediano y heterogéneo, las engrosamiento mediano levemente engrosadas
Células del súber
y homogéneo (Fig.3 F). basales lignificadas y heterogéneo, las (Fig.3 E).
(Fig.3 G). basales en forma de “U”
o lignificadas (Fig.3 H).
Parénquima cortical Abundante (Fig3 B). Escaso (Fig.3 C, D). Ausente (Fig.3 A).
1-3-seriados con 1-5-seriados con

Radios secundarios 1-3-seriados. 1-2-seriados, raro 3. ensanchamiento distal. ensanchamiento distal.
Braquiesclereidas Fibroesclereidas del Braquiesclereidas Macro, braqui y

aisladas en parénquima sistema axial dispuestas aisladas en parénquima fibroesclereidas en el
cortical; macro y en forma de estratos cortical. límite con el floema.
braquiesclereidas en el tangenciales Macro, braqui y Fibras libriformes en el
Elementos esclerenquimáticos límite con el floema discontinuos (Fig.3 C). fibroesclereidas en el floema funcional (Fig.3
(Fig.3 B). límite con el floema. A).
Fibroesclereidas
aisladas en el floema
funcional (Fig.3 D).
Drusas y escasos Poliédricos en serie de Drusas y poliédricos Drusas y escasos
poliédricos en parénquima septado abundantes en poliédricos en los radios
parénquima cortical axial (Fig.3 C). parénquima cortical y secundarios (Fig.3 A).
(Fig.5 G, H). Estiloides escasos en el axial.
Cristales de oxalato de calcio abundantes en el Poliédricos incluidos en
parénquima axial (Fig.3 algunas macro y
B. Fig.5 I). braquiesclereidas (Fig.3
D).
Tabla 3: Caracteres anatómicos diferenciales de los leños de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Crecimiento en anillos Poco marcados. De transición gradual Marcados
Solitarios, geminados,
Solitarios y múltiples
Solitarios (escasos) y racemiformes y
con distribución radial Solitarios (Fig.4 B,
Disposición de poros múltiples radiales de 4- múltiples radiales
y oblicua (Fig.4 A; Fig.5 B).
10 (Fig.4 C Fig.5 C). cortos de 3-5 (Fig.4 D;
Fig.5 A).
Fig.5 D).
Apéndice Apéndice muy
Apéndice pronunciado Apéndice apenas
Elemento de vaso con apéndice medianamente pronunciado
(Fig.4, a) pronunciado (Fig.4, d)
pronunciado (Fig.4, b) (Fig.4, c)
Abundante paratraqueal
Escaso, difuso, Escaso, metatraqueal, Casi ausente o apenas
Parénquima axial vasicéntrico, aliforme o
apotraqueal. de paredes engrosadas. metatraqueal.
en bandas diagonales.
1-7 seriados (Fig.4 D),
1-3-seriados, Uniseriados (Fig. 4 B, C), homocelulares, con
homocelulares, con
Radios heterocelulares (Fig.4 células erectas cuadrangulares y 2-3 seriados,
abundantes cristales de
A). heterocelulares.
oxalato de calcio.
CARACTERES CUANTITATIVOS
Los datos cuantitativos obtenidos del estudio anatómico de las partes utilizadas, de las especies analizadas, se
expresan en las tablas 4, 5 y 6
Tabla 4: Resultados cuantitativos del estudio de las hojas de A. subovata, P. parvifolia; P. sellowii y C. coccinea.

Índice de estomas 9,02±0,91 12,81±1,76 11,04±1,28 13,36±2,65
Estomas (mm2) 4,50±0,85 9,70±2,06 8,20±2,10 13,70±2,50
Índice de empalizada 6,63±1,00 7,33±1,54 4,05±0,66 11,45±2,03
2
Pelos simple (mm ) 3,50±0,85 2,90±0,88 3,30±0,67 3,00±0,82
543,40±57,66 137,60±20,80
Longitud pelos simple (µm) 153,60±26,61 160,00±23,45 a a
203,20±21,73 90,60±3,27
Tabla 5: Resultados cuantitativos del estudio de las cortezas de A. subovata, P. parvifolia; P. sellowii y C. coccinea.
359,60±45,19 338,40±50,74 421,60±47,71 439,30±64,29
Longitud fibras (µm) a a a a
656,00±69,64 720,00±61,51- 876,00±121,16- 1082,00±176,05
Latitud fibras (µm) 22,40±3,37 24,80±5,90 24,80±4,54 11,40±2,32
Tabla 6: Resultados cuantitativos del estudio de los leños de A. subovata; P. parvifolia; P. sellowii y C. coccinea.

Vasos (mm2) 26,50±6,13 11,60±1,51 89,60±10,71 104,20±12,26
32,00±6,53 32,00±6,53
Diámetro miembros de vasos (µm) 64,80±7,00 76,00±6,80 a a
56,00±6,53 70,4±14,51
194,40±43,42
Longitud miembros de vasos (µm) a 422,40±47,97 506,40±54,00 173,90±24,55
560,00±132,09
unicelulares 8,50±1,43 7,20±1,14 12,4±2,80 16,50±5,23
Altura radios (nº
de células pluricelulares 23,50±3,37 16,00±1,94 29,30±6,75 31,90±5,97
350,40±43,79 355,20±44,01 347,20±41,86 340,90±49,83
Longitud fibras xilares (µm) a a a a
627,20±61,30 780,80±49,77 736,00±57,44 864,00±61,18
Latitud fibras xilares (µm) 26,40±5,40 24,00±5,33 24,80±4,54 11,60±1,57
DISCUSIÓN Y CONCLUSIONES En la bibliografía consultada, las obras clásicas

sobre anatomía de los órganos vegetativos de las
Las especies de la familia Simaroubaceae (sensu
Dicotiledóneas (Solereder, 1908; Metcalfe y Chalk,
lato) aquí estudiadas, muestran caracteres morfo-
1972), no se hallaron las entidades estudiadas en el
anatómicos de valor diagnóstico que permiten su
presente trabajo, todas nativas de Argentina, si se
reconocimiento cuando se encuentran molturadas,
mencionan los géneros, Alvaradoa, Picramnia y
que es la forma en que se utilizan y/o comercializan.
Castela.
De las especies estudiadas, P. parvifolia no cuenta,
El estudio morfoanatómico de las hojas ha
en la bibliografía consultada, con uso vernáculo
aportado los siguientes caracteres diagnósticos: pelos
reconocido; sin embargo por comunicaciones orales
glandulares presentes solo en P. sellowii acordando
se sabe que se la comercializa indistintamente con las
con Solereder (1908), quien señala que no ocurren
otras especies analizadas.
pelos glandulares en todos los miembros de esta un anillo discontinuo constituido por tres tipos
familia. La estructura del mesófilo es en C. coccínea celulares: macro, braqui y fibroesclereidas. En P.
céntrica y dorsiventral en las tres restantes, sellowii, las macro y braquiesclereidas muestran
coincidente con lo indicado por Metcalfe y Chalk incrustaciones de cristales poliédricos de oxalato de
(1972) para el género Castela; en esta misma especie calcio.
se observa una hipodermis mucilaginosa, mientras En la corteza interna de C. coccinea y P. sellowii
que en P. parvifolia los mucílagos están presentes en se observó un ensanchamiento distal de los radios
las epidermis, carácter no mencionado por los autores secundarios. Roth (1981) en sus estudios acerca de la
consultados para estos géneros. Con respecto a las estructura anatómica de la corteza de árboles
cavidades secretoras, solo se encontraron en los tropicales, reconoce la disposición de las fibras como
parénquimas de la lámina de P. parvifolia; Metcalfe y el criterio diagnóstico de mayor valor, coincidiendo
Chalk (1972) lo mencionan acompañando a los haces con esta observación, se ha encontrado que en C.
vasculares en numerosos géneros, indicando que son coccinea, hay abundantes estratos formados por
poco frecuentes en Picramnia y Alvaradoa, por lo fibras libriformes, en P. parvifolia 3-5 estratos de
que coincidimos en esta apreciación con respecto a fibroesclereidas y en P. sellowii aislados estratos de
Alvaradoa, no así con Picramnia. Los cristales de fibroesclereidas.
oxalato de calcio constituyen un caracteres valioso Los tipos de cristales de oxalato de calcio
para diferenciar a estas cuatro especies, se presentan coinciden con los mencionados para los caracteres
como cristales poliédricos solitarios y drusas en C. histofoliares; en cuanto a la distribución es típica para
coccínea y P. parvifolia, como drusas y estiloides en cada especie, así se observan estiloides en el floema
A. subovata, acordando con lo mencionado por funcional de A. subovata, para las dos especies de
Solereder (1908) y Metcalfe y Chalk (1972), mientras Picramnia los cristales poliédricos se ubican en el
que solo se presentan drusas en P. sellowii. El tamaño floema funcional siendo más abundantes en P.
y cantidad de los cristales varía según las especies; parvifolia y en C. coccinea las drusas y los
así, en A. subovata los estiloides son escasos, en P. poliédricos, que son escasos, están confinados a los
parvifolia son abundantes los cristales poliédricos y radios secundarios.
en C. coccinea abundan las drusas; estas La caracterización de las cortezas encaradas en
observaciones son indicadas también por Solereder este trabajo es la primera contribución al respecto, ya
(1908) y Metcalfe y Chalk (1972). No es un dato que las mismas no han sido descriptas con
menor la extensión de la vaina parenquimática de los anterioridad.
haces vasculares menores las que se presentan solo Del análisis de los resultados obtenidos sobre las
en P. parvifolia. estructuras de los leños de las especies aquí
Para cortezas, Solereder (1908) y Metcalfe y estudiadas, se puede afirmar que existen escasas
Chalk (1972), sólo se circunscriben a la especie analogías entre ellas, lo que permite lograr una
Ailanthus altisssima, en base a las descripciones de adecuada diferenciación de los mismos. Asimismo, y
Müller (1908). Di Sapio et al. (1997) analizan a pesar de algunas pequeñas discrepancias
caracteres anatómicos de cortezas y leños de observadas, sobretodo en los caracteres referidos al
Ailanthus altisssima, Quassia amara y Castela tamaño y número de los elementos celulares,
tweedii a fin de contribuir al conocimiento y originadas por las distintas condiciones climáticas en
delimitación de las citadas especies. sus hábitat, coincidimos con las observaciones
Existe uniformidad respecto del número de realizadas por O’Donell (1937), Heimsch (1942) y
peridermis excepto para Castella coccinea. Las Metcalfe y Chalk (1972), relativo a las descripciones
células del súber son muy variables en la constitución del leño de las especies de los géneros Alvaradoa,
de sus paredes ya que alternan notablemente estratos Picramnia y Castela.
celulares con escaso o fuerte depósito de material
graso o lignina en sus paredes, en algunos caso en
forma de “U” como sucede en P. sellowii. La
felodermis, es pluriestratificada.
La sección transversal de la corteza muestra, en la
zona límite entre el parénquima cortical y el floema
funcional en A. subovata, P. sellowii y C. coccinea
Clave para delimitar las especies por los caracteres morfo-anatómicos de la hoja
A- Hojas compuestas imparipinnadas.

B- 16-24 folíolos.
C- Mesófilo dorsiventral, 2-3 hileras de células en empalizada, ambas epidermis papilosas,
parénquima esponjoso sin mucílagos y sin cavidades secretoras, con drusas y estiloides de
oxalato de calcio. A. subovata
BB- 7-15 folíolos.
C- Epidermis adx. y parénquima esponjoso con mucílagos, cavidades secretoras, drusas y abundantes
cristales poliédricos de oxalato de calcio. P. parvifolia
CC- Epidermis adx. y parénquima esponjoso sin mucílagos, sin cavidades secretoras y con drusas de
oxalato de calcio. P. sellowii
AA- Hojas simples.
B- Mesófilo céntrico con 2-3 hileras de células en empalizada, parénquima esponjoso sin mucílagos
y con drusas y escasos cristales solitarios de oxalato de calcio. Hipodermis mucilaginosa.
C. coccinea
Clave para delimitar las especies por los caracteres anatómicos de la corteza
A- Células del súber con engrosamiento homogéneo.

B- Paredes de las células del súber levemente engrosadas, sin parénquima cortical, drusas de oxalato
de calcio en parénquima de radios secundarios. C. coccinea
BB- Paredes de las células del súber medianamente engrosadas, con abundante parénquima cortical
y estiloides de oxalato de calcio en el parénquima axial. A. subovata
AA- Células del súber con engrosamiento heterogéneo.
B- Células basales del súber lignificadas, radios secundarios 1-2 seriados (raro 3), cristales
poliédricos en parénquima septado axial. P. parvifolia
BB- Células basales del súber engrosadas en U con lignina, radios secundarios 1-3 seriados
con ensanchamiento distal, cristales poliédricos de oxalato de calcio incluidos en
macro y braquiesclereidas. P. sellowii
Clave para delimitar las especies por los caracteres anatómicos del leño
A- Poros solitarios.
B- Parénquima axial metatraqueal escaso. P. parvifolia
AA- Poros solitarios y múltiples, radiales.
B- Poros geminados y racemiformes. Parénquima axial paratraqueal vasicéntrico, aliforme o
en bandas diagonales. C. coccinea
BB- Parénquima axial metatraqueal, escaso.
C- Radios 1-3 seriados heterocelulares. A. subovata
CC-Radios uniseriados homocelulares y 2-3 seriados heterocelulares. P. sellowii
Picramnia parvifolia Engl.

ANEXO 1:
ARGENTINA. Prov. Misiones: Dpto. San
Materiales estudiados Pedro, Arroyo San Pedro, 1-XI-1958, Gamerro y
Toursarkissian 69 (SI). Dpto. Oberá, Oberá, 5-V-
Alvaradoa subovata Cronquist 2007, Oakley et al. s/nº (UNR). Prov. Corrientes:
Dpto. Santo Tomé, Santo Tomé, 7-XII-1997, Múlgura
ARGENTINA. Prov. Jujuy: Dpto. Valle et al. 1589 (SI)
Grande, R Jordán. 28-XII-1977, Kiesling y Ulibarry
1634 (SI); Dpto. Ledesma, camino de Río A. Negra a Picramnia sellowii Planch.
Abra de las Cañas, 21-III-1972, Legname et al 9153y
9155 (MCNS), Ruta provincial nº 83 Parque ARGENTINA. Prov.Formosa: s/f. Jorgensen
Nacional Calilegua Km 8-10 margen norte del río 3260 (SI). Prov. Misiones: Dpto. Gral. Belgrano,
San Lorenzo 10-12 Km al NW del pueblo Ledesma Bernardo de Irigoyen 2 Km al S de B. de Irigoyen
700-900 msm, 20-VI-1998, Novara et al 11083 sobre naciente del río Pepirí Guazú, 26º16´S
(MCNS); Dpto. Santa Bárbara, Abra de los 53º38´W, 15-X-1996, Morrone et al 1426, (SI); Dpto.
Morteros. 26-I-1975, Zuloaga y Deginiani 278 (SI). San Ignacio, Peñón del Teyucaré, 27º16´ S 55º35´W,
Prov. Salta: Dpto. Orán, ruta Prov.18 a 3-4 Km. del 20-IX-2000, Múlgura et al 2157 (SI); Dpto. Posadas,
Puente Internacional Argentina-Bolivia. 22º 43´S Posadas, 17-I-1930, Rodríguez 27 (SI). Prov. Chaco:
64º43´W, 1-V-2003, Morrone et al 4536 (SI); Dpto. Dpto. Bermejo, arroyo Zapirán y ruta 11, 23-XI-
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© 2010 The Authors
Composición química y efecto antibacteriano del aceite esencial de

Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios
[Chemical composition and antibacterial effects of the essential oil of Aloysia triphylla against genito-
urinary pathogens]
Luis B. ROJAS1, Judith VELASCO2, Tulia DÍAZ3, Ricardo GIL OTAIZA4, Juan CARMONA5, Alfredo USUBILLAGA1.
1
Instituto de Investigaciones, 2Departamento de Microbiología y Parasitología, 3Departamento de Bioanálisis Clínico, 4Cátedra de
Farmacognosia, 5Jardín de Plantas Medicinales “Dr. Luís Ruiz Terán”, Facultad de Farmacia y Bioanálisis. Universidad de Los Andes,
Mérida - Venezuela.
Abstract
The essential oil of Aloysia triphylla was obtained by hydrodistillation of the aerial parts of the plant and was analyzed by GC and GC-MS Twenty two
components were identified. The main constituents were geranial (27.3%) neral (22.5%), geraniol (6.2%), biciclogermacreno (5.2%) and nerol (4.9%).
Evaluation of antibacterial activity by the agar diffusion method with disks against clinical isolates from urinary tract infections and bacterial vaginosis
revealed inhibition of development of all isolates (Escherichia coli, Klebsiella ozaenae, Enterobacter aerogenes, Proteus mirabilis, Staphylococcus aureus
and Enterococcus sp.), with MIC values of 10-50 mg/ml. This is the first report about the antibacterial activity of this essential oil against genito-urinary
pathogens. The low doses observed, suggested it may be used in pharmaceutical preparations for the treatment of infections caused by these microorganisms.
Keywords: Aloysia triphylla, Verbenaceae, essential oil, antibacterial activity.
Resumen
El aceite esencial de Aloysia triphylla fue obtenido por hidrodestilación de las partes aéreas de la planta y fue analizado por CG y CG-EM, se identificaron 22
componentes, siendo los mayoritarios geranial (27,3 %), neral (22,5 %), geraniol (6,2 %), biciclogermacreno (5,2 %) y nerol (4,9 %). La evaluación de la
actividad antibacteriana del aceite esencial por el método de difusión en agar con discos contra aislados clínicos de infecciones del tracto urinario y de
vaginosis bacteriana, reveló inhibición del desarrollo de todos los aislados (Escherichia coli, Klebsiella ozaenae, Enterobacter aerogenes, Proteus mirabilis,
Staphylococcus aureus and Enterococcus sp.), con valores de CIM de 10-50 µg/ml. Este es el primer reporte sobre el efecto antibacteriano de este aceite
esencial contra patógenos genito-urinarios y la baja dosis observada, sugiere que este aceite podría ser usado en preparaciones farmacéuticas para el
tratamiento de infecciones causadas por estos micro-organismos.
Palabras Clave: Aloysia triphylla, Verbenaceae, aceite esencial, actividad antibacteriana.
List of Abbreviations
GC: Gas Chromatography ; GC-MS: Gas Chromatography-Mass Spectrometry ; MIC: Minimal Inhibitory Concentration; CLSI: Clinical and Laboratory
Standars Institute; CG-EM: Cromatografía de Gases acoplada a Espectrometría de Masas ; CG: Cromatografía de Gases; CIM: Concentración
Inhibitoria Mínima; ITU: Infección del Tracto Urinario ; VB: Vaginosis Bacteriana ; DMSO: Dimetilsulfóxido ;BLEE: β-Lactamasa de espectro
extensor; SV: Secreción vaginal; URO: Urocultivo
Recibido | Received: September, 30, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: November 16, 2009.
Financiación | Funding: This work was financed by el Consejo de Desarrollo Científico Humanístico y Tecnológico (CDCHT Universidad de los Andes, Mérida-Venezuela)
This article must be cited as: Luis B. Rojas, Judith Velasco, Tulia Díaz, Ricardo Gil Otaiza, Juan Carmona, Alfredo Usubillaga. 2009. Composición química y efecto
antibacteriano del aceite esencial de Aloysia triphylla (L’Hér.) Britton contra patógenos genito-urinarios. Bol Latinoam Caribe Plant Med Aromat 9(1): 56 - 62. {EPub 15 Dec
2009}.
*Contactos | Contacts: Email: judithvelasco2005@yahoo.es ; Tel: 0058-2742403568; 00582742403410; Fax: 0058-2742403568.
Este es un articulo de Acceso Libre bajo los terminos de una licencia “Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional” (http://creativecommons.org/licenses/by-nc-
aplicarse si se obtiene el permiso del titular de los derechos de autor Nada en esta licencia menoscaba o restringe los derechos morales del autor.
Rojas et al. Composición química del aceite esencial de A. triphylla y efecto contra patógenos genito-urinarios
et al., 2007, Di Leo Lira et al., 2008), un trabajo

INTRODUCCIÓN realizado en Marruecos reporta 1,8-cineol y citral
La infección del tracto urinario (ITU) es la (neral + geranial) (Bellakhdar & ldrissi, 1994), otro en
invasión, colonización y multiplicación de Argentina presenta a la Myrcenona y alfa-tujona
microorganismos en el aparato urinario (Lizama et al., (Zygadlo et al., 1994) y por último un estudio de
2005, Ochoa et al., 2005). Es la segunda causa de Colombia muestra citral (neral + geranial), nerol y
infección más frecuente en humanos, constituye un geraniol como componentes más abundantes
importante problema de salud que afecta a millones de (Jaramillo et al., 2003).
personas cada año (Echeverría et al., 2006). Motivado al incremento de patógenos genito-
La vaginosis bacteriana (VB) es una infección urinarios multirresistentes, en el presente estudio se
cervicovaginal, que resulta de alteraciones en la flora evaluó la actividad antibacteriana del aceite esencial
bacteriana aerobia y anaerobia, con disminución del de A. triphylla contra 9 aislados asociados a ITU, 11
número de bacilos de Döderlein con aparición de un aislados asociados a VB y 5 cepas de referencia por el
flujo genital, lo cual se traduce en cambios método de difusión en agar con discos, además se
fisicoquímicos de las secreciones vaginales. Es una de describe su composición química.
las dos infecciones genitales más frecuentes en las
MATERIALES Y MÉTODOS
mujeres con vida sexual activa (Hillier & Holmes,
1999, Caballero et al., 2000, Mota et al., 2008).
Material vegetal y extracción
A pesar de la amplia disponibilidad de
antibióticos para el tratamiento de las ITU y VB, en Las partes aéreas frescas (1 kg) de A. triphylla,
ocasiones la sintomatología no desaparece, por un fueron recolectadas en junio de 2007 en la localidad de
fenómeno creciente y que preocupa a la comunidad Cacute, Parroquia perteneciente al Municipio Rangel
médica denominado resistencia bacteriana. Cada vez del Estado Mérida (Venezuela), una muestra (voucher
es más frecuente el aislamiento de bacterias N° Juan Carmona 788) se depositó en el Herbario
multirresistentes como las productoras de β-Lactamasa MERF “Luís Ruiz Terán” de la Facultad de Farmacia
de espectro extenso (BLEE) (Beigi et al., 2004, y Bioanálisis de la Universidad de Los Andes. La
Karlowsky et al., 2006, Gobernado et al., 2007, muestra fue sometida a hidrodestilada durante 4 horas,
Colodner et al., 2008, Gagliotti et al., 2008, Guneysel usando un aparato tipo Clevenger. El aceite esencial
et al., 2009). obtenido se secó sobre sulfato de sodio anhidro y se
Frente a esta problemática, se destaca el importante guardó bajo refrigeración (4ºC) en la oscuridad.
papel que han desempeñado las plantas como fuente
de sustancias con importante actividad Cromatografía de gases (CG)
farmacoterapéutica. En tal sentido, a la especie Un cromatógrafo de gases, modelo Autosystem,
Aloysia triphylla se le ha descrito actividad marca Perkin Elmer, equipado con un detector de
antibacteriana y antifúngica (López et al., 2004, llama, fue usado para la determinación de los índices
Teixeira et al., 2005, 2007, Sartoratto et al., 2004, de Kováts, mediante et comparación con n-alcanos
Demo et al., 2005, Duarte 2006, Duarte et al., 2007). (C7-C22). Los análisis fueron llevados a cabo usando
Aloysia triphylla (L´Hér.) Britton (Aloysia una columna capilar HP-5 (30 m x 0,25 mm id, con
citriodora [Lam.] H.B.K., es originaria de Sudamérica una película de 0,25 μm de espesor). La temperatura
y pertenece a la familia Verbenaceae. La especie es del bloque de inyección fue de 200 ºC. La temperatura
ampliamente utilizada por los pobladores de la zona del horno del GC fue programada como sigue:
para resolver diversos problemas de salud. En trabajos temperatura inicial 60 ºC, se aplicó luego un
anteriores se reporta la presencia de un flavonoide, la incremento de 4 ºC.min-1 hasta una temperatura final
luteolin-7-diglucuronida y el compuesto fenólico de 260 ºC. Se usó helio como gas portador con un flujo
verbascoside (Carnat et al., 1995; Skaltsa & Shammas, de 1.0 ml/min a volumen constante (Rojas et al., 1999).
1988). La composición del aceite esencial ha sido
estudiada en diversos países, la mayoría presentan Cromatografía de gases acoplada a espectrometría
como componentes mayoritarios citral (neral + de masas (CG-EM)
geranial) y limoneno (Montes et al., 1973, Özek et al., El análisis se realizó en un cromatógrafo de gases
1996, Carnat et al., 1999, Sartoratto et al., 2004, Hewlett Packard 6890 serie II acoplado a un detector
Gomes et al., 2006, Argyropoulou et al., 2007, ; Díaz de masas Hewlett Packard 5973, equipado con un
inyector automático HP y una columna capilar HP- De los veintidós componentes identificados, 2,7 %
5MS de 30 m de largo (0.25 mm id; con una película resultaron ser compuestos alifáticos insaturados (de
de 0,25 μm de espesor). Se usó una energía de menos de 10 átomos de carbono), el 72,4 %
ionización de 70 eV. Se inyectó una muestra de 1.0 μl monoterpenos y el 22,3 % sesquiterpenos.
de 2% de solución de aceite en n-heptano con un Los componentes mayoritarios identificados
reparto de 100:1. La identificación de los componentes fueron: geranial (27,3 %), neral (22,5 %), geraniol
de la esencia fue establecida utilizando la base de (6,2%), bicyclogermacrene (5,2 %) y nerol (4,9 %).
datos Wiley (6a Ed.) y comparación de los índices de Este aceite guarda estrecha relación con el aceite
kováts obtenidos con datos publicados en la literatura esencial de la misma especie estudiada en Colombia
(Adams, 1995). La temperatura del inyector y el [geranial (38,1 %), neral (19,3 %), geraniol (5,4%),
programa de temperatura fueron los mismos usados nerol (4,7 %) y biciclogermacreno (3,4 %)] (Jaramillo
para la medición de los índices de Kováts. et al., 2003) y los componentes geranial y neral forman
parte de los aceites esenciales de A. triphylla
Análisis Microbiológico estudiadas en el ámbito mundial (Montes et al., 1973;
Carnat et al., 1999; Sartoratto et al., 2004; Gomes et
Cepas bacterianas al., 2006; Argyropoulou et al., 2007; Díaz et al., 2007;
Se sometieron al estudio las cepas bacterianas Di Leo Lira et al., 2008). Siendo Colombia un país
descritas en la Tabla N° 3, que fueron proporcionadas vecino al nuestro, se puede pensar que ambas especies
por el Laboratorio Clínico “Lic. Ana Aparicio” y por están relacionadas botánicamente.
el Departamento de Microbiología y Parasitología,
Facultad de Farmacia y Bioanálisis de la Universidad Evaluación de la actividad antibacteriana
de los Andes. Estas cepas fueron aisladas de pacientes El aceite de A. triphylla mostró fuerte actividad
con ITU y VB en Mérida - Venezuela. Además, se antibacteriana contra las cepas de referencia,
incluyeron las cepas de referencia que se mencionan observándose zonas de inhibición con diámetros entre
en la Tabla N° 2. 7 y 19 mm y valores de CIM entre 10 a 60 μg/mL, sin
embargo, no presentó actividad contra P. aeruginosa
Método antimicrobiano ATCC 27853 (Tabla N° 2). También fue activo contra
La evaluación de la actividad antibacteriana se todos los patógenos genito-urinarios probados, con
realizó de acuerdo al método de difusión en agar con rangos de zona de inhibición de 9 a 30 mm y valores
discos descrita por Velasco et al., 2007 y la de CIM de 10 a 50 μg/mL (Tabla N° 3). En resumen,
concentración inhibitoria mínima (CIM) solo contra la actividad del aceite contra todas las bacterias
los microorganismos que mostraron zonas de ensayadas mostró valores de CIM de 10 a 60 μg/mL y
inhibición. La CIM se determinó por dilución del la dosis de 20 μg/mL predominó en el 52 % del total
aceite con dimetilsulfóxido (DMSO) con un rango de de microorganismos.
10-160 μg/ml, definida como la concentración más La actividad biológica observada en el presente
baja capaz de inhibir el desarrollo bacteriano (CLSI, estudio coincide con los resultados obtenidos por
2009). Los ensayos se realizaron por duplicado. Demo et al., 2005 (Argentina), quienes observaron
actividad antibacteriana del aceite esencial de A.
RESULTADOS Y DISCUSIÓN triphylla contra S. aureus, E. faecalis, E. coli,
Klebsiella sp. y Proteus mirabilis y ausencia de
Caracterización Fitoquímica actividad frente a P. aeruginosa. Sin embargo, difiere
Por hidrodestilación de las hojas frescas de la A. de los hallazgos de Sartoratto et al., 2004, en cuanto a
triphylla se obtuvo un aceite esencial de color la actividad contra E. coli.
ligeramente amarillo y olor penetrante, con un
rendimiento del 0,2%. En la Tabla N° 1 se muestran
los componentes identificados, que constituyen el 97
% del total del aceite, los cuales fueron identificados
mediante búsqueda computarizada en la Librería
Wiley y por comparación de los Índices de Kováts
obtenidos experimentalmente, con los reportados en la
literatura (Adams, 1995).
Tabla N°1. Componentes volátiles del aceite esencial deAloysia triphylla en columna capilar HP-5
N° Compuesto % IKcala
1 1 – octen–3–ol 0,9 977
2 6–metil–5–hepten–2–ona 1,8 985
3 Limoneno 3,0 1030
4 Eucaliptol 1,4 1033
5 β– cis–ocimeno 1,5 1049
6 linalool 0,5 1099
7 cis-crisantenol 0,9 1169
8 mentol 1,3 1187
9 α -terpineol 1,0 1195
10 nerol 4,9 1235
11 neral 22,5 1250
12 geraniol 6,2 1261
13 geranial 27,3 1279
14 geranil acetato 1,9 1387
15 β-cariofileno 2,1 1427
16 germacreno-D 4,6 1494
17 α-zingibereno 0,6 1506
18 biciclogermacreno 5,2 1509
19 cis-gamma-bisaboleno 0,9 1522
20 trans-sesquisabinene-hidrato 2,1 1569
21 spatulenol 4,5 1583
22 cariofileno-oxido 2,3 1588
TOTAL 97,4 -
a
La composición del aceite esencial fue determinada por comparación de los espectros de masas de cada componente con la base de datos
Willey e índice de Kováts calculado (IKcal).
Tabla 2: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra cepas de referencia
Zona de inhibición (mm)*
CIM
Microorganismos Aceite Control positivo
μg/ml
esencial SAM VA GM AZT CAZ
Staphylococcus aureus
18,75±0,25 40,75±0,25 20
(ATCC 6538)
Enterococcus faecalis
17,75±0,25 20,50±0,50 60
(ATCC 29212)
Escherichia coli
12,50±0,50 21,00±0,00 10
(ATCC 25922)
Klebsiella pneumoniae
7,00±0,00 29,50±0,50 30
(ATCC 23357)
Pseudomonas
aeruginosa (ATCC NA 28,00±0,00 NP
27853)
* Zona de inhibición en mm, discos 6 mm diámetro, media de dos ensayos, SAM: Sulbactam-Ampicilina® (10μg/10 μg), VA:
Vancomicina® (30 μg), GM: Gentamicina® (10 μg), AZT: Aztreonam® (30μg), CAZ: Ceftazidima® (30 μg), NA: No activo, NP: No
probado. CIM: Concentración inhibitoria mínima, rango de concentración 10-160 μg/ml.
Tabla 3: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra patógenos genito-urinarios
Aceite esencial
CIM
Microorganismos Zona de inhibición
μg/ml
(mm)*
Aislados clínicos de ITU:
Escherichia coli BLEE (URO 1) 9,75±0,25 20
Escherichia coli (URO 5) 11,00±0,00 20
Escherichia coli (URO 8) 9,50±0,50 20
Klebsiella ozaenae (URO 2) 9,00±0,00 30
Klebsiella ozaenae (URO 4) 10,75±0,25 10
Klebsiella ozaenae BLEE (URO 7) 9,00±0,00 50
Klebsiella ozaenae multirresistente (URO 11) 8,00±0,00 20
Enterobacter aerogenes (URO 6) 8,75±0,25 30
Proteus mirabilis (URO 9) 7,00±0,00 10
Aislados clínicos de SV:
Escherichia coli (SV 5) 12,75±0,25 20
Klebsiella ozaenae (SV 1) 9,75±0,25 20
Staphylococcus aureus (SV 4) 30,00±0,00 20
Enterococcus faecalis (SV 2) 14.50±0,50 20
Enterococcus faecalis (SV 3) 23,00±0,00 20
Enterococcus faecium (SV 7) 21,75±0,25 20

Enterococcus faecium (SV 9) 25,00±0,00 20
*Zona de inhibición en mm, discos 6 mm diámetro, media de dos ensayos, BLEE: β-Lactamasa de espectro extenso. ITU: Infecciones del
tracto urinario; SV: Secreción vaginal. URO: Urocultivo.; CIM: Concentración inhibitoria mínima, rango de concentración 10-160 μg/ml.
Recientemente, Duarte et al., 2007 en una Universidad de los Andes, Mérida, Venezuela,
investigación sobre efecto inhibitorio de aceites Programa ADG, Grupo de investigación:
esenciales obtenidos de varias plantas medicinales de Bacteriología Clínica) por el soporte financiero para
Brasil contra cepas de E. coli diarreogénicas, señalan el desarrollo de esta investigación.
actividad inhibitoria del aceite de A. triphylla contra
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Infección del tracto urinario en un servicio de urgencia
© 2010 The Authors
Zederone from the rhizomes of Zingiber zerumbet and its anti-

staphylococcal Activity
[Aislamiento de Zederona de los rizomas de Zingiber zerumbet y su actividad antiestafilocócica]
M. Golam KADER1, M. Rowshanul HABIB1, Farjana NIKKON1, Tanzima YEASMIN1, Mohammad A. RASHID2, M.
Mukhlesur RAHMAN3*, Simon GIBBONS3
1
Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh; 2Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh; 3Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK
Abstract
A sesquiterpene, zederone (1), was isolated from the crude ethanolic extract of the rhizomes of Zingiber zerumbet (L.) Smith. It is the first time
report of isolation of this compound from the genus Zingiber. Its structure was established by a series of spectral data including high-field NMR (both 1D and
2D) and MS. The antibacterial activity of this compound was determined against a number of multi-drug resistant and methicillin-resistant Staphylococcus
aureus strains (SA1199B, ATCC25923, XU212, RN4220 and EMRSA15) and minimum inhibitory concentration (MIC) values were found to be in the range
of 64-128 μg/ml.
Keywords: Zingiber zerumbet; Zingiberaceae; Zederone; Antibacterial; Staphylococcus aureus
Resumen
Un sesquiterpeno, zederona (1), fue aislado del extracto crudo metanólico de los rizomas de Zingiber zerumbet (L.) Smith. Esta es la primera vez
que se reporta este compuesto en el género Zingiber. Su estructura se estableció tras una serie de análisis espectrales incluyendo NMR de alto campo (1D y
2D) y espectrometría de masa. La actividad antibacteriana de este compuesto se determine frente a varias cepas multi-fármaco resistentes y meticilina-
resistentes Staphylococcus aureus (SA1199B, ATCC25923, XU212, RN4220 and EMRSA15) y las concentraciones inhibidoras mínimas se encontraron en el
rango de 64-128 μg/ml.
Palabras Clave: Zingiber zerumbet; Zingiberaceae; Zederona; Actividad antibacteriana; Staphylococcus aureus.
Recibido | Received: December 01, 2009

Aceptado en Versión Corregida | Accepted in Corrected Version: December 15, 2009
Publicado en Línea | Published Online: December 17, 2009
Financiación | Funding: none declared
This article must be cited as:. M. Golam Kader, M. Rowshanul Habib, Farjana Nikkon, Tanzima Yeasmin, Mohammad A. Rashid, M. Mukhlesur Rahman, Simon Gibbons.
2010. Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal Activity. Bol Latinoam Caribe Plant Med Aromat 9(1):63 – 68. {EPub December 17, 2009}.
*Contactos | Contacts:. E-mail: mukhlesur.rahman@pharmacy.ac.uk

Kader et al. Zederone from the rhizomes of Zingiber zerumbet and its anti-staphylococcal activity

INTRODUCTION
Zingiber zerumbet (L.) Smith (Fam. General experimental procedures
Zingiberaceae) locally known as ‘Bon Ada’ is a NMR spectra (both 1D and 2D) were
vigorous ginger with leafy stems growing to about acquired on a Bruker Avance 500 MHz NMR (500
1.2 m in height that is widely cultivated throughout MHz for 1H and 125 MHz for 13C) spectrometer
the tropics including Southeast Asia, Korea, India using the residual solvent peak as internal standard.
and Bangladesh for its medicinal properties (Kritikar The IR spectra were recorded with a Perkin-Elmer
and Basu, 1984; Fansworth and Bunyapraphatsara, Lambda spectrophotometer and the Mass spectra
1992; Saadiah and Halijah, 1995). The most common were recorded with HRTOF-MS in positive mode.
use of Z. zerumbet is as a shampoo and conditioner The melting point were determined using a Digital
for the hair (Burkill, 1966; Petard, 1986). Its Melting point Apparatus (model IA 8103,
rhizomes are used in traditional medicine for the Electrothermal Engineering LTD, Southend-on-Sea,
treatment of inflammation, swelling, loss of appetite, Essex, UK) and are uncorrected. All solvents used in
lumbago, diabetes, chest pain, rheumatic pains, this study were of analytical grade and purchased
bronchitis, dyspepsia and sore throat (Burkill, 1966; from BDH and Merck.
Kritikar and Basu, 1984; Farnsworth and
Bunyapraphatsara, 1992). The juice of the boiled Plant materials
rhizomes has also been used in indigenous medicine Fresh rhizomes of Z. zerumbet were collected
for worm infestation in children (Petard, 1986). from the hilly areas of Chittagong, Bangladesh in
Previous phytochemical investigations on this plant October 2007 and identified by a taxonomist, Dr.
have revealed the isolation of several sesquiterpenes, Mohammed Yusuf, BCSIR Laboratory, Chittagong,
flavonoids and aromatic compounds (Matthes et al., Bangladesh where a voucher specimen (No. 1061) of
1980; Masuda et al., 1991; Dai et al., 1997; Jang et this collection has been maintained.
al, 2004; Jang et al, 2005). The volatile oil of the
rhizomes has been shown to contain zerumbone, Extraction and isolation of compound (1)
humulene, camprene α-caryophyllene and camphene
(Hasnah, 1991; Srivastava et al., 2000; Bhuiyan et al., The powdered plant material (800 g) of Z.
2009). Zerumbone, a predominant sesquiterpene from zerumbet was extracted with ethanol (4 L) in an
this plant, has been studied intensively for its use as aspirator bottle for a week and then filtered and
anti-inflammatory, and in chemoprevention and concentrated by using a rotary evaporator at 45oC
chemotherapy strategies (Dai et al., 1997; Kitayama under reduced pressure. The crude ethanol extract
et al., 2001; Murakami et al., 1999; Murakami et al., (2.0 g) was subjected to column chromatography
2002; Tanaka et al., 2001). From the pharmacological over silica gel (Merck) eluting with petroleum ether
point of view, Z. zerumbet has been reported to and ethyl acetate of increasing polarity and finally
inhibit colon and lung carcinogenesis in mice (Kim et with ethanol which yielded a total of 105 fractions.
al., 2009) and CXCL12-induced invasion of breast Based on TLC analysis, fractions 15-18 were
and pancreatic tumor cells (Sung et al., 2008), combined together and then subjected to preparative
suppresses phorbol ester-induced expression of TLC using the solvent system petroleum ether and
multiple scavenger receptor genes in THP-1 human ethyl acetate in a ratio of 20:1 to yield compound 1
monocytic cells and inhibits Epstein-Barr Virus (8.0 mg; white powder; Rf 0.45 in 5% EtOAc in
activation (Vimala et al., 1999; Murakami et al., petroleum ether). The structure of the compound (1)
1999). As a part of our research focused on bioactive was confirmed by analysis of its IR, 1H-NMR, 13C-
compounds from indigenous medicinal plants, we NMR and TOF- Mass spectral data at The School of
here report the isolation and identification of a Pharmacy, University of London, UK.
o
compound (1) from the ethanolic extract of Zingiber Compound 1: White powder; mp. 58-60 C;
zerumbet (L.) Smith and its ant-staphylococcal IR (KBr): 1662, 1521, 1533, 1558, 914, 861 cm-1; 1H-
activity against a series of multi-drug resistant NMR (500 MHz, CDCl3): δ 5.49 (1H, dd, J = 12.0,
(MDR) and methicillin resistant Staphylococcus 4.0 Hz , H-1), 2.25 (1H, m, H-2a), 2.53 (1H, m, H-
aureus strains: SA1199B, ATCC25923, XU212, 2b), 1.29 (1H, m, H-3a), 2.31 (1H, dt, J= 13.0, 3.5
RN4220 and EMRSA15. Hz, H-3b), 3.81 (1H, s, H-5), 3.69 (1H, d, J= 16.0
Hz, H-9a), 3.77 (1H, d, J= 16.0 Hz, H-9b), 7.10 (1H,
s, H-12), 1.61 (3H, s, H-13), 1.35 (3H, s, H-14), 2.12 RESULTS AND DISCUSSION
(3H, s, H-15); 13C-NMR (125 MHz, CDCl3): 131.4
(C-1), 24.9 (C-2), 38.2 (C-3), 64.2 (C-4), 66.8 (C-5),
Identification of compound (1)
192.4 (C-6), 122.5 (C-7), 157.3 (C-8), 42.1 (C-9), Compound (1) was isolated as an amorphous
131.3 (C-10), 123.5 (C-11), 138.3 (C-12), 16.0 (C- off-white powder from the crude ethanol extract of
the rhizomes of Zingiber zerumbet (L.) Smith. The
13), 15.4 (C-14), 10.5 (C-15); HR-TOF-ESIMS
+ high-resolution TOFMS showed the pseudo
[M+H] m/z 247.0889. molecular ion, [M+H]+ at m/z 247.0889,
corresponding to the molecular formula as C15H18O3.
Bacterial strains The 1H-NMR spectrum of compound 1 showed a
The antibacterial assay was performed downfield one proton singlet at δ 7.10, an olefinic
against a panel of multi-drug and methicillin- proton signal at δ 5.49, another methine signal at
resistant strains of Staphylococcus aureus. S. aureus δ 3.81, three methyl proton resonances at δ 1.35,
standard strain ATCC 25923 and tetracycline- 1.61, 2.12, and methylene proton resonances between
resistant strain XU212 which possesses the TetK δ1.29-3.77 integrating for four protons. The 13C-
tetracycline efflux protein provided by Dr Edet Udo NMR spectrum displayed a total of 15 carbons while
(Gibbons and Udo, 2000). Strain SA-1199B which the DEPT-135 and HMQC experiments indicated that
overexpresses the norA gene encoding the NorA 9 out of 15 carbons had attached protons. Analysis of
MDR efflux pump was provided by Professor Glenn the 13C and DEPT135 spectra allowed discernment of
Kaatz (Kaatz et al., 1993). Strain RN4220 which the carbon resonances into three methyls (δC 10.5,
possesses the MsrA macrolide efflux protein was
15.4, 16.0), three methylenes (δC 24.9, 38.2 and
provided by Dr Jon Cove (Ross et al., 1989).
42.1), three methines (δC 66.8, 131.4, 138.3), and six
EMRSA-15 (Richardson and Reith, 1993) was the
generous gift of Dr Paul Stapleton. quaternary carbons, including a carbonyl group (δC
192.4). The assignment of all carbons and protons
Minimum inhibitory concentration (MIC) assay. and thereby the structure of the compound was
resolved by 2D experiments, notably COSY, HMQC
All five S. aureus strains were cultured on and HMBC experiments. In the COSY experiment,
nutrient agar (Oxoid) and incubated for 24 h at 37°C the olefinic proton at δ 5.49 (H-1; δC 131.4 from
prior to MIC determination. Norfloxacin was HMQC) showed strong interaction with H2-2 protons
purchased from the Sigma Chemical Co. Mueller-
at δ 2.25 and 2.53 along with a weak connectivity
Hinton broth (MHB; Oxoid) was adjusted to contain
with the methyl singlet at δ 1.61 (H3-15). The
20 and 10 mg/l of Ca2+ and Mg2+, respectively. An
methylene protons (H2-2) showed coupling with H2-3
inoculum density of 5 x 105 cfu of each of the test
organisms was prepared in normal saline (9 g/l) by protons at δ 1.29 and δ 2.31 in the COSY experiment.
comparison with a 0.5 MacFarland standard. MHB The presence of a methine (66.8; δH 3.81 from
(125 μl) was dispensed into 10 wells of a 96 well HMQC) and a quaternary (64.2) in the 13C
microtitre plate (Nunc, 0.3 ml volume per well). A experiment confirmed the presence of an epoxide in
stock solution of norfloxacin was prepared by the molecule. The C-5 methine proton at δ 3.81
dissolving the antibiotic in DMSO (Sigma) and showed HMBC connectivities over two bonds (2J) to
dilution in MHB to give a final concentration of a quaternary carbon at δ 64.2 (C-4) and the carbonyl
0.625%. A DMSO control was included in all assays. group at δ 192.4 (C-6). The methyl protons at 1.35 (δC
Compounds were serially diluted into each of 15.4 from HMQC) revealed 3J connectivities to δ
the wells followed by the addition of the bacterial 38.2 (C-3) and δ 66.8 (C-5) and thereby confirmed its
inoculum and the microtitre plate was incubated at linkage at C-4. The methylene protons at δ 3.70 and δ
37°C for 18 h. The MIC recorded as the lowest 3.77 (H2-9, δC 42.1 from HMQC) showed 2J
concentration at which no growth was observed. This correlations over two bonds to quaternary carbons at
was facilitated by the addition of 20 μl of a 5 mg/ml δ 131.3 (C-10) and δ 157.3 (C-8) and a 3J interactions
methanolic solution of 3-[4,5-dimethylthiazol-2-yl]- to methyl (δC 16.0), olefinic (δC 131.4, C-1) and
2,5-diphenyltetrazolium bromide (MTT; Sigma) to quaternary (δC 122.5, C-7) carbons. Furthermore, 3J
each of the wells and incubation for 20 minutes. A connectivities from the methyl protons at δ 1.61 to δ
blue colouration indicated bacterial growth (Shiu and 131.4 (C-1) and δ 42.1 (C-9) confirmed its placement
Gibbons, 2006).
at C-10. The remaining methyl resonance at δ 2.12 The compound was tested for the
(δC 10.5 from HMQC) showed connectivities to δC antibacterial activity against a panel of five strains of
122.5 (C-7) and a methine carbon at δ 138.3 (C-12) Staphylococcus aureus: SA1199B, ATCC25923,
over three bonds. This allowed the placement of this XU212, RN4220 and EMRSA15 and showed weak
methyl group at C-11. Accordingly, compound 1 was activity with minimum inhibitory concentration
identified as zederone. Its NMR data were in (MIC) values in the range of 64-128μg/ml (Table 2).
agreement with those reported previously (Hikino et Figure 1. Structure of compound 1
al., 1971). Although, it is a known natural product
1 9
reported before from a number species of the genus 10 8 O
Curcuma including C. zedoaria (Matthes et al., 2
1980), C. aromatic (Phan and Phan 2000), C. 15 12
comosa (Qu et al., 2009), C. kwangsiensis ( Zhu et 5 7
3
al., 2009), C. ochrorhiza. (Sirat et al. 2009), C. 4 6
11
xanthorrhiza (Sukari et al., 2008), this is the first O
isolation from the genus, Zingiber. 14 O 13
Table 1 1NMR (500 MHz), 13C NMR (125 MHz) and HMBC data of compound 1 in CDCl3.
Position δH δC HMBC
2 3
J J
1 5.49, dd, J= 12.0, 4.0 Hz 131.4 C-2, C-10 C-9, C-15
2 2.25, br d; 2.53, m 24.9 C-3 -
3 1.29, m; 2.31, dt, J= 13.0, 3.5 Hz 38.2 C-2, C-4 -
4 - 64.2 - -
5 3.81, s 66.8 C-4, C-6 C-14
6 - 192.4 - -
7 - 122.5 - -
8 - 157.3 - -
9 3.69, d, J= 16.0 Hz; 3.77, d, J= 16.0 Hz 42.1 C-8, C-10 C-7, C-15
10 - 131.3 - -
11 - 123.5 - -
12 7.10, s 138.3 C-11 C-7, C-8, C-13
13 1.61, s 16.0 - C-7, C-12
14 1.35, s 15.4 C-4 C-3, C-5
15 2.12, s 10.5 - C-1, C-9
Table 2. MICs of 1 and standard antibiotic in μg/ml

SA1199B Xu212 ATCC 25943 RN 4220 EMRSA 15
Compound 1 128 64 128 64 128
Norfloxacin 16 4 0.5 0.5 0.5
glycosides from Zingiber zerumbet. Phytochemistry

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© 2010 The Authors
A protocol for evaluating the safety of herbal preparations in a rat model:

the case of a supercritical fluid extract of Saw Palmetto
[Un protocolo para evaluar la seguridad de preparaciones herbales en un modelo de ratas: el caso de un extracto
fluido supercrítico de Saw Palmetto]
María Eugenia LETELIER*, Paula ARACENA-PARKS, Liliana PEREDO-SILVA
Laboratory of Pharmacology and Toxicology, Department of Pharmacological and Toxicological Chemistry, School of Chemical and
Pharmaceutical Sciences, Universidad de Chile, Sergio Livingstone Pohlhammer (ex-Olivos) 1007, Independencia, Chile.
Abstract
Herbal extracts must be evaluated for their efficacy and safety. In vivo acute toxicity studies must consider the different mechanisms by which active
compounds may elicit toxicological outcomes. Thus, a methodology to test general parameters related to acute toxicity responses in a murine model was
developed, using a Saw Palmetto extract (HiPower®): adult male Sprague-Dawley rats were treated orally with two doses of HiPower® (the recommended
dose for humans and a dose 10-fold higher) for 10 days, to examine general homeostatic parameters (hemogram and clinical chemistry) as well as
morphological features of tissues involved in the response to xenobiotics (liver, kidney, spleen, and lymphatic ganglia). None of the parameters analyzed
underwent significant changes during treatment, suggesting that HiPower® displays a good safety profile for the period tested. This method may be adopted
for testing the in vivo acute toxicity of herbal extracts.
Keywords: Saw Palmetto, safety profile, Sprague-Dawley rats, acute toxicity.
Resumen
Los extractos herbales deben ser evaluados en cuanto a eficacia y seguridad. Estudios de toxicidad aguda in vivo deben considerar los diferentes
mecanismos por los cuales los principios activos pueden producir toxicidad. Por consiguiente, se desarrolló una metodología para examinar parámetros
generales relacionados con las respuestas de toxicidad aguda en un modelo murino, utilizando un extracto de Saw Palmetto (HiPower®): ratas Sprague-
Dawley macho fueron tratadas con dos dosis de HiPower® (la dosis recomendada para humanos y una dosis 10 veces mayor) durante 10 días, para ensayar
parámetros generales homeostáticos (hemograma y perfil bioquímico), así como características morfológicas de tejidos involucrados en la respuesta a
xenobióticos (hígado, riñón, bazo y ganglios linfáticos). Ninguno de los parámetros analizados sufrió cambios significativos durante el tratamiento, sugiriendo
que HiPower® presenta un buen perfil de seguridad durante el periodo evaluado. Este método puede ser adoptado para ensayar la toxicidad aguda in vivo de
extractos herbales.
Palabras Clave: Saw Palmetto, perfil de seguridad, ratas Sprague-Dawley, toxicidad aguda.
Recibido | Received: September, 16, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: December 16, 2009.
Financiación | Funding: This work was supported by Madreselva Producción y Desarrollo Ltda. (Santiago, Chile).
This article must be cited as: Names. 2009. Title. Bol Latinoam Caribe Plant Med Aromat 9(1):69 – 79. {EPub XX Month 200X }.
*Contactos | Contacts: E-mail: mel@ciq.uchile.cl; Tel: 56-2-9782885; Fax: 56-2-9782996;
Letelier et al. In vivo safety profile of a supercritical extract of Saw Palmetto
INTRODUCTION Fagelman, 2004; Ulbricht et al., 2006; Vallancien and

Pariente, 2001; Wilt et al., 2000), there are few
Compounds occurring in plants have been
classical toxicological studies in animals. Only a small
used for millennia to treat diseases; this use has set the
amount of studies have shown significant toxic effects
basis for the isolation of such compounds for
of a particular Saw Palmetto extract (PC-SPES), which
therapeutic use. Only recently, however, scientific
has been removed from the market (de la Taille et al.,
research on herbal extracts has begun to fully validate
2000; de la Taille et al., 1999; Small et al., 2000;
their therapeutic use and safety. Most of this research
Sovak et al., 2002). More recent clinical studies in
is focused on the therapeutic application of compounds
humans have found no serious adverse effects of Saw
found in herbal extracts. For instance, extracts from
Palmetto extracts (Avins et al., 2008; Boyle et al.,
leaves of plants are rich in antioxidant compounds,
2004; Ernst, 2002; Hizli and Uygur, 2007; Willetts et
such as polyphenols, which can be useful for the
al., 2003). Some hepatotoxic effects have been
treatment of pathologies associated to oxidative stress
associated to a n-hexane-based Saw Palmetto extract
(e.g. neurodegenerative diseases). Nevertheless,
(Hamid et al., 1997); indeed, a classical toxicity study
assessing the safety of herbal extracts is a challenging
in rats has been reported, showing an increase in
endeavour, due to their complex nature. In contrast to
oxidative stress associated with the intake of 2X and
purified compounds, herbal extracts display a number
5X the maximum dose recommended for humans
of different molecules with potentially very different
(480µl/day) for this type of preparation (Singh et al.,
biological targets; interaction of herbal compounds
2007). Nonetheless, no toxicological studies have been
with their targets may lead to beneficial and/or toxic
reported using the supercritical fluid extract of Saw
consequences. Therefore, it becomes necessary to
Palmetto. Therefore, to perform the first acute
develop a methodology that evaluates the safety
toxicological study in rats of a supercritical fluid Saw
profile of herbal extracts containing more than one
Palmetto extract (commercial name “HiPower®”),
active compound.
Sprague-Dawley rats were fed with 1X and 10X the
The present work is aimed to establish a
recommended dose for humans of this product (doses
methodology for assessing the acute toxicity of a
adjusted for rat metabolism) for 10 days. Rat blood
particular herbal extract of Saw Palmetto (Serenoa
samples obtained at different time intervals were
repens W. Bartram). Saw Palmetto belongs to the
analyzed through hemogram and clinical chemistry
Arecaceae (Palmae or Palmaceae) family. It is also
parameters; biopsies from liver, spleen, thymus and
known as Serenoa serrulatum Schultes, Serenoa
lymphatic ganglia were also collected and analyzed for
serrulata (Michaux) Nichols, or Sabal serrulata
possible morphological changes indicative of tissue
(Michaux) Nutall ex Schultes (Wilt et al., 2000)).
damage. This study showed no statistically significant
The therapeutic benefits of Saw Palmetto
changes compared to normal ranges of any of the
appear to be related to its fruits, rich in natural oils.
parameters analyzed; in addition, no significant
Currently, Saw Palmetto extract is widely used for the
changes were found in the morphological profile of the
treatment of benign prostate hyperplasia (BPH,
studied tissues. This demonstrates the safety of this
prostate enlargement) (Bent et al., 2006; Gerber and
particular Saw Palmetto extract at the doses and
Fitzpatrick, 2004; Hizli and Uygur, 2007; Wilt et al.,
periods tested.
2000; Wilt et al., 1998). The type and relative
The protocol followed for this study evaluates
abundance of characteristic compounds (phenolic
general homeostasis and the function of liver and
compounds, phytosterols, flavonoids, polyprenoids,
kidney, organs responsible for xenobiotic
sugars, fatty acids, etc.) of the oily extract from Saw
biotransformation and excretion, respectively.
Palmetto depends on the extraction procedure.
Therefore, the type of study presented here may be
Currently, the most used procedures to obtain Saw
adopted as a method for assessing the acute toxicity of
Palmetto extracts are: 1) n-hexane (100%) extraction
herbal extracts.
that produces a liposterolic extract (LESP) (Carraro et
al., 1996); 2) ethanol (70-95% w/w) extraction
(Derakhshani et al., 1997); or 3) supercritical fluid
extraction with liquid CO2 (Cristoni et al., 1997). Saw Palmetto extract HiPower®
Although there are several reports regarding
the therapeutical applications of Saw Palmetto extracts HiPower® is a supercritical fluid extract from Saw
(Bent et al., 2006; Carraro et al., 1996; Lowe and Palmetto fruits and was provided by Madreselva
Desarrollo y Producción Ltda. (Santiago, Chile). This phosphorus, glucose, blood ureic nitrogen, cholesterol,
extract contains: 3.8% palmitic acid, 1.7% stearic acid, total protein, albumin, total bilirubin, acid phosphatase
14.8% oleic acid, 44.2% linoleic acid, and 34.3% (AP), lactate dehydrogenase (LDH), and glutamyl
linolenic acid. oxaloacetic transaminase (GOT).
Animals Histopathology studies

Male Sprague-Dawley rats (200-230g) were These studies were performed by the Cyto-
maintained with normal pellet diet (Kimber), access to Histopathology Laboratory BiopsCyt (Santiago,
water ad libitum, in a 12:12 light/dark cycle at 21°C. Chile). Histomorphology studies were performed with
Animals were maintained in the vivarium of the haematoxylin-eosin on the biopsies obtained at the
School of Chemical and Pharmaceutical Sciences different intervals during treatment.
(Universidad de Chile, Santiago, Chile). All
procedures were performed according to the protocols Statistical Analyses
approved by the Ethical Committee of the institution Data presented in this study correspond to mean ±
and to the “Guide for the Care and Use of Laboratory 95% confidence intervals (95% CI). Statistical
Animals” (NRC, USA). significances between means of hemogram and
clinical chemistry data were obtained through
Treatment of rats ANOVA. Statistical significances between medians of
Dosage of HiPower® was calculated from the 1X data and reference values were obtained through
recommended dose for humans (480µl/day), and Wilcoxon Signed Rank tests. Reference values for
considering 70kg for normal human weight and that adult Sprague-Dawley rats were obtained from
rats display a 4-fold higher metabolic rate than observations from the Institute of Public Health of
humans. Doses were diluted in sunflower oil for oral Chile (Uribe et al., 1995) or according to Lillie et al.,
delivery. Rats were distributed in 3 experimental 1996. All statistical analyses were performed using
groups of 40 rats each: two groups that were given 28 GraphPad Prism 5.0. Significances were set at 95%
(HiPower® 1X group) and 280µl (HiPower® 10X confidence
group) extract/Kg/day, in two oral (gavage) doses,
respectively, and a control group that received RESULTS
sunflower oil alone in equivalent volumes (Sunflower
oil group). Following 2, 4, 6, 8, and 10 days of daily 1. Effect of HiPower® on hemogram
treatment, 8 rats from each group were anaesthetized parameters of Sprague-Dawley rats.
with ether and sacrificed by exsanguination through Adult male Sprague-Dawley rats were treated
cardiac puncture. Blood samples were used for orally for up to 10 days with vehicle (Sunflower oil
hemogram and biochemical profile, and biopsies of group), 1X (HiPower® 1X group), or 10X (HiPower®
liver, thymus, spleen and lymphatic ganglia were 10X group) the recommended dose of HiPower® for
collected for histopathology analysis. humans, as detailed in Material and Methods.
Following 2, 4, 6, 8, and 10 days of each treatment,
Hemogram study rats were sacrificed and blood samples were collected
It was performed by the Central Laboratory of the for hemogram analysis.
Clinical Hospital of the Universidad de Chile As shown in Figure 1, HiPower® treatment did
(Santiago, Chile). Parameters analyzed were red blood not significantly alter the following parameters,
cell count (RBC), hematocrit, haemoglobin, mean compared to normal ranges: red blood cells count
corpuscular volume (MCV), mean corpuscular (RBC) or hematocrit (Figure 1); haemoglobin or mean
haemoglobin (MCH), mean corpuscular haemoglobin corpuscular haemoglobin (MCHC, Figure 2); white
concentration (MCHC), and white blood cell, blood cells count (WBC, Figure 3); and platelet count
lymphocyte and platelet counts. (Figure 4). We also found complete absence of
bacilliform white cells, basophiles, eosinophiles,
Clinical chemistry study metamyelocytes, or myelocytes (not shown).
It was performed by the Central Laboratory of the
Clinical Hospital of the Universidad de Chile
(Santiago, Chile). Parameters analyzed were: calcium,
Figure 1. Effect of HiPower® acute treatment on red blood cell count (RBC) and hematocrit of Sprague-Dawley rats. Animals were treated
with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and
Methods. RBC and hematocrit were analyzed at different time points of a 10-day treatment. Data points represent the mean of each
determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each
parameter.
Figure 2. Effect of HiPower® acute treatment on red blood cell mean corpuscular volume (MCV) and hemoglobin-related parameters
of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and
HiPower® 10X cohorts), as detailed in Material and Methods. Hemoglobin, MCV, mean corpuscular hemoglobin (MCH) and mean
corpuscular hemoglobin concentration (MCHC) were analyzed at different time points of a 10-day treatment. Data points represent the mean
of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for
each parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according
to Wilcoxon Signed Rank test.
Figure 3. Effect of HiPower® acute treatment on white blood cell count (WBC) and lymphocytes of Sprague-Dawley rats. Animals
were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in
Material and Methods. WBC and lymphocytes were analyzed at different time points of a 10-day treatment. Data points represent the mean
of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for
each parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according
to Wilcoxon Signed Rank test.
Figure 4. Effect of HiPower® acute treatment on platelets of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or
with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Platelets were
analyzed at different time points of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95%
CI. Horizontal dotted lines represent the upper and lower limits of reference values for each parameter.
Figure 5. Effect of HiPower® acute treatment on blood levels calcium and phosphorus of Sprague-Dawley rats. Animals were treated
with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and
Methods. Blood levels of calcium and phosphorus were analyzed at different time points of a 10-day treatment. Data points represent the
mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values
for each parameter.
Figure 6. Effect of HiPower® acute treatment on blood levels of albumin and total protein of Sprague-Dawley rats. Animals were
treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts), as detailed in
Material and Methods. Blood levels of albumin and total protein were analyzed at different time points of a 10-day treatment. Data points
represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of
reference values for each parameter.
Figure 7. Effect of HiPower® acute treatment on blood levels of glucose, cholesterol, bilirubin, and urea nitrogen of Sprague-Dawley
rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and HiPower® 10X cohorts),
as detailed in Material and Methods. Blood levels of glucose, cholesterol, bilirubin, and urea nitrogen were analyzed at different time points
of a 10-day treatment. Data points represent the mean of each determination and error bars depict the 95% CI. Horizontal dotted lines
represent the upper and lower limits of reference values for each parameter.
Figure 8. Effect of HiPower® acute treatment on blood levels of alkaline phosphatase, aspartate aminotransferase, and lactate
dehydrogenase of Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower®
(HiPower® 1X and HiPower® 10X cohorts), as detailed in Material and Methods. Blood levels of alkaline phosphatase, aspartate
aminotransferase, and lactate dehydrogenase were analyzed at different time points of a 10-day treatment. Data points represent the mean of
each determination and error bars depict the 95% CI. Horizontal dotted lines represent the upper and lower limits of reference values for each
parameter. Stars indicate parameters with a median significantly different (p<0.05) than their corresponding reference range, according to
Wilcoxon Signed Rank test.
Figure 9. Effect of HiPower® acute treatment on morphological features of liver, spleen, lymphatic ganglia, and thymus biopsies from
Sprague-Dawley rats. Animals were treated with vehicle (sunflower oil) or with two different doses of HiPower® (HiPower® 1X and
HiPower® 10X cohorts), as detailed in Material and Methods. Biopsies from liver, spleen, lymphatic ganglia, and thymus were collected at
different time points of a 10-day treatment. Representative images from haematoxylin-eosin staining of samples are shown. A. 4X
magnification showing a typical hepatic lobule (bracket) with a central vein (arrow). B. 10X magnification of a hepatic biopsy showing a
better view of the central vein (arrow); hepatic trabecules (solid arrowhead) and sinusoids (open arrowheads) are also distinguishable. C. 20X
magnification showing a portal triad, with the portal venule (a), the bile duct surrounded by a cuboid epithelium (b), and the hepatic arteriole
(c). D. 20X magnification of a spleen biopsy showing white and red pulps, with a germinative center in the latter. E. Germinative center of a
splenic red pulp at 40X magnification. F. Germinative center of a splenic red pulp depicting apoptotic bodies (arrows). All structures shown
are representative of all groups and treatment intervals. G. 10X magnification of a lymphatic ganglion biopsy, in which cortex and part of the
medulla can be distinguished. Two lymphoid follicles can be discriminated, with their respective germinative centers. H. 20X magnification
of a germinative center of a ganglion lymphoid follicle, with numerous apoptotic bodies (arrows). I. 40X magnification of a germinative
center of a ganglion lymphoid follicle, with a better view of apoptotic bodies (arrows). J. 4X magnification of thymus lobule structure, with
clear discrimination of cortex (1) and medulla (2) K. 40X magnification of a Hassall body (black arrow), with normal architecture. L. 40X
magnification of a medullar zone of a thymus lobule, displaying scarce apoptotic bodies (white arrows).
Wilcoxon Signed Rank tests showed that central vein (Figure 9A and 9B) and portal triads
particular cohorts displayed medians significantly constituted by blood vessels and bile ducts (Figure
different from reference ranges in mean corpuscular 9C). Hepatocytes displayed a conserved structure,
volume (MCV), mean corpuscular haemoglobin without noticeable degeneration or necrosis. Most
(MCH) and the % lymphocytes (stars in Figures 2 liver biopsies displayed small hematopoietic loci,
and 3). Two-way ANOVA tests, however, showed with the same occurrence regardless of the cohort
that these differences are not a reflection of analyzed or the collection time. Liver parenchyma
significant deviations compared to the vehicle displayed no mitosis count per mm2 in most samples,
(p>0.05). with a mitosis count of 1-4 per mm2 in a few
biopsies, regardless of the cohort or collection time.
2. Effect of HiPower® on clinical chemistry Spleen biopsies had also normal architecture
parameters of Sprague-Dawley rats. of red or white pulp, regardless of the cohort or
In addition to hemogram analysis, blood treatment interval analyzed (Figure 9D-F). All
samples were also analyzed for several clinical samples displayed hematopoietic activity and a
chemistry parameters. As shown in Figures 5-8, mitotic count in germinative centers of 0-4 per mm2,
HiPower® treatment did not change the following regardless of the treatment or collection interval
parameters: calcium or phosphorus (Figure 5); blood (Figures 9D and 9E). Vehicle cohort displayed
levels of albumin or total proteins (Figure 6); blood virtually no apoptotic bodies, while 1X HiPower®
glucose, cholesterol, bilirubin, or urea nitrogen and 10X HiPower® cohorts showed some apoptotic
(Figure 7); and the activity of alkaline phosphatase bodies in seldom cases (Figure 9F).
(AP, Figure 8). Lymphatic ganglia biopsies from all three
Wilcoxon Signed Rank tests showed that cohorts displayed conserved histological structures
some cohorts displayed medians significantly higher (Figure 9G-I). We found lymphocytic elements and
than the upper limit of the normal range for aspartate reticulo-histocytic cells in the lymphoid tissue. Most
aminotransferase activity (stars in Figure 8, middle biopsies, regardless of the cohort or collection time,
panel). Two-way ANOVA tests, however, showed displayed secondary follicles with germinative
that these differences are not a reflection of centers (Figure 9G). With low frequency, some
significant deviations compared to the vehicle sinusal edema was found regardless of the cohort or
(p>0.05). Also, most cohorts showed medians the collection time. At the level of lymphoid follicles,
significantly lower than the lower limit of the normal some apoptotic activity was evidenced by the
range for lactate dehydrogenase (LDH) activity (stars occurrence of apoptotic bodies in the cytoplasm of
in Figure 8, bottom panel). Two-way ANOVA reticulo-histocytic cells (Figures 9H and 9I). The
analysis demonstrated that most of these deviations abundance of apoptotic bodies, however, was not
were not significantly different from the vehicle associated to a cohort or collection time. On the other
cohort, except for the cohorts treated with 1X hand, at the level of germinative center, we observed
HiPower® (8 and 10 days of treatment, p<0.001) and a mitotic count of 2-25 per mm2. Variability of
10X HiPower® (6 and 8 days of treatment, p<0.001). mitotic count was unchanged among cohorts or
collection times.
3. Effect of HiPower® on morphological Histological structures of the thymus were
features of rat tissues. conserved in all biopsies tested (Figure 9J-L),
We also obtained biopsies (hepatic, splenic, regardless of the cohort or the collection time, with
thymic, and lymphatic) from each group at different normal architectures of cortex and medulla (Figure
time intervals of the treatments. These biopsies were 9J). Normal lymphoid and epithelial (Hassall bodies,
used to perform histopathology analysis of tissue Figure 9K) components were also visualized.
sections with haematoxylin-eosin, as detailed in Apoptotic scores did not reveal significant
Material and Methods. differences between cohorts or collection times
All liver biopsies, regardless of the group or (Figure 9L). Mitotic count in germinative centers was
the time of collection, showed a conserved 1-2 per mm2, displaying no significant differences
histological architecture (Figure 9A-C). We observed between cohorts or collection times.
classical structures, such as hepatic lobules with a
DISCUSSION It has been shown that some Saw Palmetto

extracts can be toxic in acute toxicological studies
Our study was aimed to develop a
(Hamid et al., 1997; Singh et al., 2007). On the other
methodology that allows the evaluation of the safety
hand, several studies have demonstrated the safety of
profile of herbal extracts, using a supercritical fluid
these extracts in humans (Avins et al., 2008; Boyle et
Saw Palmetto extract (HiPower®) as a start point. To
al., 2004; Ernst, 2002; Hizli and Uygur, 2007;
this end, we evaluated possible changes in classical
Willetts et al., 2003). It is very difficult to address the
hemogram and clinical chemistry parameters (as a
safety of a Saw Palmetto extract due to the diversity
measure of general homeostasis) from Sprague-
of extraction methods (Habib and Wyllie, 2004).
Dawley rats fed for 10 days with two different doses
Although this extract appears to be more
of HiPower®, compared with rats fed with vehicle
concentrated than other commercial Saw Palmetto
(sunflower oil). We also evaluated potential gross
extracts, we postulated that the absence of organic
morphological alterations in key organs involved in
solvents in the supercritical fluid extraction of Saw
xenobiotic metabolism (to address possible toxicity
Palmetto fruits, leading to the preparation of an
associated with xenobiotic biotransformation) and
extract with a good safety profile. Due to their oily
immune system (to address potential toxic
nature, it is possible that Saw Palmetto extracts used
immunological response). With this purpose, blood
for the treatment of benign prostate hyperplasia
samples and tissue biopsies were collected from rats
contain lipoperoxides; these substances must be
at different intervals during the 10-day treatment.
controlled due to their potential for promoting lipid
Essentially none of the parameters tested
peroxidation in tissues. Noteworthy, the particular
underwent any statistically significant changes
Saw Palmetto extract used in this study did not lead
following this treatment, as assessed by Wilcoxon
to an increase in basal lipoperoxidation of rat liver
Signed Rank test. Although these analyses showed
microsomes (data not shown), a system routinely
some deviations of medians from the normal ranges,
used by our lab to test oxidative damage (Letelier et
most of such deviations were not significantly
al., 2007). The absence of pro-oxidant activity
different from those of the vehicle cohort. This may
corroborates the good safety profile of this Saw
be the reflection of parameters with high inter-
Palmetto extract. In light of our data, we can
individual dispersion. Noteworthy, lactate
conclude that HiPower® displays a safety profile of at
dehydrogenase activity values were lower than the
least one order of magnitude in dosage.
lower limit of normal ranges for this parameter in all
The general strategy used for the present
cohorts, especially at the beginning of the treatment.
study indeed allowed the assessment of potential
Although this parameter reached the normal interval
alterations in general homeostasis and morphological
in the case of the vehicle cohort, it remained under
features of key organs in xenobiotic
the lower limit for the 1X HiPower® and 10X
biotransformation (liver) and excretion (kidney).
HiPower® cohorts. Since lactate dehydrogenase
Therefore, we conclude that this type of studies may
activity is a classical marker for liver damage, these
be adopted for future evaluations of safety profiles of
data suggest that HiPower® may display
herbal extracts.
hepatoprotective activity.
In summary, hemogram and clinical
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© 2010 The Authors
Comunicación | Communication
Volatile Components from the Leaves of Solanum hypomalacophyllum

Bitter.
[Componentes volátiles de las hojas de Solanum hypomalacophyllum Bitter.]
Alida PÉREZ COLMENARES1, Luis B. ROJAS1*, Alfredo USUBILLAGA1
1
Instituto de Investigaciones, Facultad de Farmacia y Bioanálisis. Universidad de Los Andes, Mérida - Venezuela.
Abstract
The essential oil from the leaves of Solanum hypomalacophyllum (Solanaceae) collected in May 2007 at Páramo La Culata (Mérida State, Venezuela)
was obtained by hydrodistillation and its composition was determined by GC and GC/MS. Eleven compounds, representing 92.4 % of the oil, were identified
5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0%) and hexadecanoic acid (5.2 %) were the most abundant components.
Keywords: Solanum hypomalacophyllum; Solanaceae; essential oil; 5-octen-1-ol; GC-MS analysis.
Resumen
El aceite esencial de las hojas de Solanum hypomalacophyllum (Solanaceae) recogidas en mayo 2007 en el Páramo La Culata (Estado de Mérida,
Venezuela) fue obtenido por hidrodestilacion y fue analizado por GC y GC/MS. Se identificaron 11 componentes que representan el 92.4% del total del
aceite. Los componentes mayoritarios fueron identificados como 5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-hexanol (12.0 %) y ácido hexadecanoico
(5.2 %).
Palabras Clave: Solanum hypomalacophylllum; Solanaceae; aceite esencial; 5-octen-1-ol; GC-MS análisis.
Recibido | Received: November, 18, 2009.

Aceptado en Versión Corregida | Accepted in Corrected Version: January, 10, 2010.
Publicado en Línea | Published Online January 21, 2010.
Financiación | Funding: This work was supported by: Consejo de Desarrollo Científico, Humanístico y Tecnológico (CDCHT–Mérida–Venezuela, project FA-380-06-03-A),
FONACIT-PCP (46.255), University of Los Andes-Plan II.
This article must be cited as: Pérez Colmenares A, Rojas L B., Usubillaga A. 2010. Volatile Components from the Leaves of Solanum hypomalacophyllum. Bol Latinoam Caribe
Plant Med Aromat 9(1):80 – 83. {EPub 21 Jan 2010 }.
*Contactos | Contacts: E-mail: rojasl@ula.ve; Tel: 0058-2742403958 ; Fax: 0058-2742403455.
Pérez Colmenares et al. Essential Oil of Solanum hypomalacophyllum
INTRODUCTION Extraction and analysis of the essential oil

The Solanaceae family comprises about 90 Fresh leaves (2000 g) were cut into small
genera and 3.000 species which are widely pieces and submitted to hydrodestillation for 3 h
distributed in the world. They are a rich source of using a Clevenger-type apparatus. 0.1 mL of essential
active secondary metabolites (Coletto da Silva et al., oil was obtained that corresponds to a yield of
2004). Within this family, the genus Solanum is the 0.005% v/w.
largest and most complex with more than 1500 The composition of the essential oil was
species (Chowdhury et al., 2007) which yield a great determined by comparison of the mass spectrum of
variety of steroidal saponins and glycoalkaloids of each component with Wiley GC/MS library data and
interest from ecological and human health viewpoints also from retention index (RI) data (Morteza et al.,
(Roddick et al., 2001). Numerous species of Solanum 2004).
are known to possess a variety of biological activities
including antimycotic (Gonzales et al., 2004; Singh et Gas chromatography
al., 2007), antiviral (Arthan et al., 2002), GC analyses were performed using a Perkin-
molluscicidal (Silva et al., 2006), teratogenic (Keeler Elmer Autosystem gas chromatograph equipped with
et al., 1990), and cytotoxic properties (Nakamura et a FID detector and data-handling system. A 5%
al., 1996; Lu et al., 2009). Previous studies reported phenylmethylpolysiloxane fused-silica capillary
the chemical composition of the essential oil of column was used (30 m x 0.25 mm i.d., film
several species of Solanum, like S. verbascifolium thickness 0.25 μm; HP-5, Hewlett-Packard, CA,
(Ma et al., 2006), S. lyratum (Xu et al., 2006), S. USA). The oven temperature was programmed from
aculeastrum (Koduru et al., 2006) and of S. 60 ºC to 260 ºC at 4 ºC/min. The injector and detector
pseudocapsicum (Aliero et al., 2006, 2007), but no (Flame ionization detector, FID) temperatures were
reports have been published on the composition of 200ºC and 280 ºC, respectively. The carrier gas was
the volatile constituents of S. hypomalacophyllum helium at 0.8 mL/min. The sample (1.0 μL) was
Bitter. This species is a small tree native to the injected using a split ratio of 10:1. Retention indices
Venezuelan Andes where it grows wild in humid were calculated by comparing the retention times of
places at altitudes between 2150 and 3200 meters. the eluting peaks with those of standard C8-C24 n-
Previous studies reported the isolation of 4-keto alkanes. The percentage composition of the oil was
steroidal alkaloids solaphyllidine, calculated by the normalization method from the GC
deacetoxysolaphyllidine, desacetylsolaphyllidine, peak areas.
spirosolaphyllidine and solamaladine from green
berries and leaves (Usubillaga, 1970, 1984; Gas chromatography – mass spectrometry
Usubillaga et al., 1982; Alarcon et al., 2006). GC-MS analyses were carried out on a Model
Therefore, in continuation of our study of the 5973 Hewlett-Packard GC-MS system fitted with a
Venezuelan Solanaceae, we report here the first study HP-5MS fused silica column (30 m x 0.25 mm i.d.,
on the essential oil of S. hypomalacophyllum in order film thickness 0.25 μm, Hewlett-Packard). The oven
to obtain knowledge on its chemical composition. temperature program was the same as that used for
the HP-5 column for GC analysis; the transfer line
MATERIALS AND METHODS temperature was programmed from 150ºC to 280ºC;
source temperature, 230ºC; quadrupole temperature,
Plant Material 150ºC; carrier gas, helium, adjusted to a linear
Leaves of Solanum hypomalacophyllum were velocity of 34 cm/s; ionization energy, 70 eV; scan
collected in May 2007, in the area of El Páramo de la range, 40:500 amu; 3.9 scans/s. Sample (1.0 μL) was
Culata, Mérida State, Venezuela at 2800 m above sea injected using a Hewlett-Packard ALS injector with a
level. A voucher specimen has been deposited at the split ratio of 50:1. The identity of the oil components
MERF Herbarium (Herbarium of the Faculty of was established from their GC retention indices, by
Pharmacy and Bioanalysis in Mérida, Venezuela). comparison of their MS spectra with those of
The botanical identification was made by Forest standard compounds available in the laboratory, and
Engineer Juan Carmona, MERF Herbarium. by a library search (NIST 05 and Wiley)(Adams,
1995).
RESULTS AND DISCUSSION REFERENCES

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Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas
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Descripción
BLACPMA es una revista cientifica dedicada a las plantas medicinales, aromáticas, y económicas y a los productos naturales bioactivos.
Publica contribuciones originales en ocho áreas importantes:
1. Caracterización de los ingredientes activos de las plantas medicinales
2. Desarrollo de métodos para la estandarización para los extractos bioactivos y los productos naturales de la planta.
3. Identificación de la bioactividad de productos naturales vegetales.
4. Identificación de blancos y mecanismo de la actividad de productos naturales.
5. Producción y caracterización genómica de la biomasa de especies medicinales.
6. Química y bioquímica de productos naturales bioactivos.
7. Revisiones críticas de la personalidad histórica, clínica y jurídica de plantas medicinales.
8. Aspectos agrícolas de plantas medicinales y aromáticas.
Descargue el MODELO DE ARTICULO con las instrucciones de autor
Detalles Bibliográficos
ISSN: 0717 7917
Publicado por CLACPMA
6 numeros / 1 Volumen año
Licencia “Creative Commons Atribucion-No Comercial-No trabajos derivados 3.0 Internacional”
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Los articulos publicados electronicamente son gratuitos. Visite nuestro sitio web www.blacpma.org
Separatas impresas pueden ser encargadas a editorial@blacpma.org (10 separatas: USD $25 + cargas postales)
Audiencia
Farmacólogos, Etnofarmacologos, farmacéuticos, fitoquímicos, químicos orgánicos, toxicólogos, botánicos y antropólogos, entre otros.
Impacto
2009: 9.00 © Index Copernicus; La evaluación del Journal Citation Reports (Thomson Reuters) ESTA EN CURSO.
Publicada por | Published by: Cooperación Latinoamericana y Caribeña en Plantas Medicinales y Aromáticas
Indexada por | Indexed by: SCOPUS, Science Citation Index Expanded (SCISEARCH), Journal Citation Reports/Science Edition, Biological
Abstracts y BIOThomson Reuters Master Journal List , Chemical Abstracts (CAS), NAPRALERT, CAB International (CAB Abstracts),
GlobalHEALTH, Index Copernicus, IMBIOMED, LATINDEX, QUALIS, REDALYC, Biblioteca Virtual da Saude (BVS).

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Boletín Latinoamericano y del Caribe

de Plantas Medicinales y Aromáticas

EDITOR JEFE | EDITOR IN CHIEF CONSEJO EDITORIAL | EDITORIAL

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

Una nueva década

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

Produtos Naturais com atividade inibitória da Translocase I, uma

Keywords: tuberculosis; translocase I; streptomyces

Palavras Chave: tuberculose; translocase I; Streptomyces

Recibido | Received: July, 10, 2009.

*Contactos | Contacts: marcos_souza@far.fiocruz.br

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

INTRODUÇÃO MATERIAIS E MÉTODOS

tratamento e cura da tuberculose. Vale ressaltar que a 7 O O

partir de culturas de Streptomyces griséus, pela A H3 C

relevantes da história da medicina moderna e da O H

MIC = 3.13 μ g mL-1 MIC = 12.5 μ g mL-1

MIC = 6.3 μ g mL-1

as LPMs do tipo I, assim como a LPMs do tipo II,

devido à presença da porção sulfato, hidrofílica, na 1 50 H OH OH OH 80

consequentemente, possuem maior atividade 5 H H2NC(NH)NH 25

A presença do ácido graxo nessa classe de 7 H EtNH 150

substâncias também é extremamente importante para 8 H n-PrNH 140

a atividade biológica, provavelmente porque confere

I-M; III-M H3C

I-N; III-N H3C

explicada pela estabilidade do seu anel β-lactâmico R O O

M. tuberculosis (Cepa H37Rv) (MIC = 8 μg/mL e

composto A500359A (MIC = 16 μg/mL). A atividade OH OH

sofreu variações estatisticamente significativas, B

Não foram realizados testes in vivo para o composto O CH3 CH3 O

desenvolvidos mostram que os análogos de 2 alanil fenil

avaliação no tratamento de infecções causadas por M. 6 glicil 3-indol

34 alanina 4-flúor-fenilalanina triptofano

35 alanina 4-trifluormetilfenilalanina triptofano Muraimicina R1 R2 Muraimicina R1 R2

Substâncias 1a 2b 3c 4d A3 O2C(CH2)12NHC(NH)NH2 OCH3 C3 OH H

Figura 15. Derivados da muraimicina C1. DISCUSSÃO

Artículo Original | Original Article

Antioxidant and anti-inflammatory activity of Moussonia deppeana

Miguel A. DOMÍNGUEZ-ORTIZ,1* Omar MUÑOZ-MUÑIZ,2* Rosa Virginia GARCÍA-RODRÍGUEZ,2

Keywords: Anti-inflammatory, Antioxidant, Moussiana deppeana, Ethnopharmacology, Medicinal Plants.

Palabras Clave: Anti-inflamatorio, Antioxidante, Moussiana deppeana, Etnofarmacología, Plantas Medicinales.

Recibido | Received: May, 29, 2009.

*Contactos | Contacts: omunoz@uv.mx; Phone: +52-228-841-8900 ext. 13554; Fax: +52-228-841-8917.

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

with FeCl3 test (Oyaizu, 1986); total polyphenols by

Carrageenan-induced mouse paw edema Table 1. Phytochemical screening.

20 µL/paw of a 1% carrageenan solution was Test

Phytochemical and biological investigations of Caryocar brasiliense Camb

Recibido | Received: August, 9, 2009.

*Contactos | Contacts: dianadb@netuno.lcc.ufmg.br

BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas

INTRODUCTION spectrophotometric assay, as well as for their

Ethyl galate (1) 5-Hydroxymethylfurfural (2) Galic acid (3)

Methyl shikimate (4) β-D-Frutopyranose (5) β-D-Frutofuranose (6)

α-D-Glucose (7) β-D-Glucose (8) Lupeol (9)

Oleic acid (10) β-Sitosterol (11) Stigmasterol (12)

-20 1.0 mg/ mL

-30 aureus ATCC 29212, Salmonella typhimurium

concentration test, carried out in duplicate, samples

512 to 5.0 μg/ml for each test microorganism. Tubes

acid (3) (Souza Filho et al., 2006) and methyl

Antimicrobial activity of essential oils of Aloysia triphylla (L`Her.)

Keywords: Aromatic plants; Aloysia triphylla; Antibacterial; antifungic; terpenes.