BLACPMA Vol. 9 #1 (2010)

Publicada por | Published by: Cooperacin Latinoamericana y Caribea en Plantas Medicinales y Aromticas
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).

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

Aloysia triphylla
Volumen 9, Nmero 1, Enero de 2010

Revisiones | Reviews
De Souza et al. (Brasil) Recent advances in the use of Panax ginseng as an analgesic: a systematic review.
Artculos | Articles
Domnguez-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 farmacobotnico de hojas, cortezas y leos 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) Composicin qumica y efecto antibacteriano del aceite esencial de Aloysia triphylla (LHr.) Britton contra patgenos 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
Prez Colmenares et al. (Venezuela) Volatile components from the leaves of Solanum hypomalacophyllum Bitter.

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (1), i
BLACPMA ISSN 0717 7917
Comit Editorial | Editorial Board

BLACPMA es una publicacin de la Cooperacin Latinoamericana y Caribea de Plantas Medicinales y Aromticas

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
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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 trminos de una licencia Atribucin Creativa Comn-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar
pblicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los crditos 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.
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en esta licencia menoscaba o restringe los derechos morales del autor.

EDITOR JEFE | EDITOR IN CHIEF
Jos L. Martnez (Santiago, Chile)
EDITORES CIENTIFICOS | SCIENTIFIC
EDITORS
Jos Mara Prieto (London, UK)
Peter Taylor (Caracas, Venezuela)
EDITOR EJECUTIVO | MANAGING EDITOR
Damaris Silveira (Brasilia, Brasil)
EDITORES | EDITORS
Carla Delporte (Santiago, Chile)
Gabino Garrido (Antofagasta, Chile)
Martha Gattuso (Rosrio, Argentina)
Jeannette Gavilln (San Juan, Pto Rico)
Leonora Mendoza (Santiago, Chile)
Horacio Olivo (Iowa, USA)
Edgar Pastene (Concepcin, Chile)
Vernica Rivas (Monterrey, Mxico)
Gabriela Ricciardi (Chaco, Argentina)
Luis A. Simeoni (Braslia, Brasil)
Beatriz Varela (Buenos Aires, Argentina)
EDITOR HONORARIO | HONORARY EDITOR
Jorge Rodrguez Chanfreau (La Habana, Cuba)

CONSEJO EDITORIAL | EDITORIAL
ADVISORY BOARD
Julio Alarcn (Chilln, Chile)
Talal Aburjai (Amman, Jordan)
Arnaldo Bandoni (Buenos Aires, Argentina)
Elizabeth Barrera (Santiago, Chile)
Armando Cceres (Guatemala, Guatemala)
Salvador Caigueral (Barcelona, Espaa)
Bruce Cassels (Santiago, Chile)
Geoffrey Cordell (Illinois, USA)
Rosa Degen (Asuncin, Paraguay)
Marco Dehesa (Quito, Ecuador)
Olga Lock (Lima, Per)
Rodolfo Juliani (New Jersey, USA)
Patricia Landzuri (Armenia, Colombia)
Norman Farnsworth (Illinois, USA)
Elisabeth Williamson (London, UK)
Michael Heinrich (London, UK)
Peter Houghton (London, UK)
Luis Kanzaki (Brasilia, Brasil)
Ana Ladio (Bariloche, Argentina)
Francisco Morn (La Habana, Cuba)
Patrick Moyna (Montevideo, Uruguay)
Pulok Mukkerjee (Jadavpur, India)
Luca Rastrelli (Salerno, Italia)
Vicente Martnez (Guatemala, Guatemala)
John A. O. Ojewole (Natal, Sudafrica)
Edison Osorio (Medelln, Colombia)
Mahendra Rai (Maharashtra, India)
Elsa Rengifo (Iquitos, Per)
Jos Luis Ros (Valencia, Espaa)
Lionel Robineau (Pointe Pitre, Guadalupe)
Gloria Saavedra (Cochabamba, Bolivia)
Marcelo Wagner (Buenos Aires, Argentina)
Talal Zari (Arabia Saudita)

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (1), ii-iii
Nota Editorial | Editorial Notes


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 trminos de una licencia Atribucin Creativa Comn-No Comercial-No trabajos derivados 3.0 Internacional. Usted es libre de copiar, distribuir y comunicar
pblicamente la obra bajo las condiciones siguientes: Reconocimiento. Debe reconocer los crditos 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 trminos 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.
Una nueva dcada
Jos L MARTINEZ
1
, Damaris SILVEIRA
2
, Jos M PRIETO
3
, Gabino GARRIDO
4
& Peter TAYLOR
5

1
Universidad Santo Toms, Talca, Chile;
2
Universidad de Brasilia, Brasil;
3
Universidad de Londres, Inglaterra;
4
Universidad de
Antofagasta, Chile;
5
IVIC, Caracas, Venezuela

Estamos comenzando una nueva dcada del s.
XXI y un nuevo ao, el noveno, del Boletn
Latinoamericano y del Caribe de Plantas Medicinales
y Aromticas (BLACPMA) con muchas novedades.
Durante el ao anterior nos sometimos a evaluacin
en SCIELO y recibimos muchas recomendaciones
que nos han permitido mejorar. Sin embargo una
entre ellas nos ha causado especial dolor de corazn y
cabeza: SCIELO nos llamo a re-configurar el Comit
Editorial ya que en su opinin estaba sobrecargado y
no permita el crecimiento sostenible de la revista.
BLACPMA ha sido siempre un foro de profesionales
unidos en la pasin de divulgar ciencia, y siempre
hemos considerado plasmar a todos ellos en nuestro
comit editorial lo cual era consustancial a esta
filosofa. Sin embargo la profesionalizacin de este
boletn parece incompatible con ello y hemos debido
aceptar cambios profundos. Empezando por los roles
principales, asumiendo Peter Taylor (Venezuela) y
Damaris Silveira un rol mas importante dentro de la
jerarqua, aliviando un poco a Gabino Garrido
(Chile).y a Jos Mara Prieto (Inglaterra) que haban
llevado un gran peso hasta ahora. En los roles de
Editores hemos decidido convocar a aquellos colegas
que se mostraron mas proactivos durante el 2009:
desde Argentina Martha Gattuso (Rosario), Gabriela
Ricciardi (Resistencia) y Beatriz Varela (Buenos
Aires), adems de Vernica Rivas (Mxico) y
Horacio Olivo (USA) y Edgar Pastene (Chile).
As que qu paso con todos nuestros grandes
amigos que no podemos mantener nominalmente en
el comit editorial? Simplemente siguen vinculados
igual que antes al BLACPMA a travs de la naciente
CLACPMA (ver boletn de XXX 2009). CLACPMA
ya se est mostrando como un catalizador del trabajo
en red de sus integrantes y como resultados de la
misma han sido dos proyectos presentados antes que
desafortunadamente no resultaron agraciados, pero
no por ello nos daremos por vencidos. Prximamente
su Presidente Jos Mara Prieto tratar de quitarse
horas de sueo y editar un boletn de noticias
CLACPMA para dinamizar esta asociacin y darla a
conocer allende Latinoamrica.
Peter Taylor (Venezuela) ha tomado para si la
ingrata tarea de reconsiderar todo el flujo editorial
elaborando un cronograma, un manual de funciones y
un texto con las instrucciones a autores y revisores
que se dar a conocer en prximos nmeros. Con esto
se pretendemos agilizar aun ms los procesos
evaluativos ya que la entrada en ISI de BLACPMA
ha supuesto una carga extraordinaria para el equipo
editorial y educar a autores y revisores en cuanto a
los mnimos de calidad exigibles por nuestra revista.
Por otro lado, nuevamente hemos recibido el
inters de SILAE para establecer una Asociacin
entre nuestra publicacin y su Sociedad. Desde ya
estamos invitados a su reunin en Cagliary (Italia)
para Septiembre de 2010 pero esperamos poder
reunirnos en forma previa en el primer semestre de
este ao.
Una de nuestras principales colaboradoras la Dra.
Gabriela Ricciardi y amiga de muchos aos, este mes
de febrero de 2010 contrae matrimonio, como Comit
Ejecutivo de BLACPMA deseamos para ella un
mundo lleno de felicidades en esta nueva vida que
inicia.
Todas estas buenas nuevas se han empaado.
Hace poco ms de un mes, el pueblo Haitiano ha
sufrido una de sus peores tragedias y como Boletn
Latinoamericano y del Caribe no nos hemos querido
mantener al margen. No tenemos las herramientas
para poder estar ms cerca pero de todos modos
Prieto et al. Guia de Autores

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 9 (1) 2010 | iii

queremos enviar a travs de estas lneas un noble
saludo de apoyo y solidaridad. El Editor Jefe ha
invitado al Sr. Benito Baranda, Presidente de la
Organizacin de Voluntariado Fundacin Amrica
Solidaria para que en un nmero prximo nos ilustre
un poco sobre la situacin de ese pas caribeo y
quizs nos invit de alguna forma a solidarizar ya sea
a travs de la Fundacin que l dirige o de otras
instancias. Lamentablemente para este nmero su
tiempo ha sido escaso debido a sus innumerables
labores para apoyar a Hait.
Queremos terminar agradeciendo a aquellos que
de una u otra manera hacen que este Boletn ocupe el
sitial que tiene en este momento todos los autores y
revisores que durante el 2009 han contribuido a esta
revista y especialmente por su importante apoyo a
Arnaldo Bandoni y Mariela Marinoff (Argentina),
Guillermo Padrn (Mxico), Carlos Cspedes (Chile)
y Carolina Baquero (Colombia).
Les dejamos ya con este su Boletn, y como
siempre les rogamos nos hagan llegar sus sugerencias
y contribuciones que siempre sern bienvenidas y
acreditadas.

2010 The Authors
2010 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 9 (1), 1 - 12
Revisin | Review

BLACPMA es unapublicacin delaCooperacin Latinoamericanay CaribeadePlantas Medicinales y Aromticas

This is an open access articledistributed under theterms of aCreativeCommons Attribution-Non-Commercial-No DerivativeWorks 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ )
which permits to copy, distributeand transmit thework, provided theoriginal work is properly cited. You may not usethis work for commercial purposes. You may not alter, transform, or build upon this work.
Any of theseconditions can bewaived if you get permission fromthecopyright holder. Nothing in this licenseimpairs or restricts theauthor's moral rights.

Este es un articulo de Acceso Libre bajo los trminos de una licencia Atribucin Creativa Comn-No Comercial-No trabajos derivados 3.0 Internacional (http://creativecommons.org/licenses/by-nc-
nd/3.0/deed.es) Usted es libredecopiar, distribuir y comunicar pblicamentelaobrabajo las condiciones siguientes: Reconocimiento. Debereconocer los crditos delaobradelamaneraespecificadapor el autor
o el licenciador (pero no deunamaneraquesugieraquetienesu apoyo o apoyan el uso quehacedesu obra). No comercial. No puedeutilizar estaobraparafines comerciales. Sin obras derivadas. No sepuede
alterar, transformar o generar unaobraderivadaapartir deestaobra. Al reutilizar o distribuir laobra, tienequedejar bien claro los trminos delalicenciadeestaobra. Algunadeestas condiciones puedeno
aplicarsesi seobtieneel permiso del titular delos derechos deautor. Nadaen estalicenciamenoscabao restringelos derechos morales del autor.
Produtos Naturais com atividade inibitria da Translocase I, uma
promissora classe de compostos contra tuberculose
[Natural Products with Translocase I inhibitory activity, as new lead compounds against tuberculosis]
Marcus Vincius Nora DE SOUZA
*
,

Victor FACCHINETTI, Danielle CARDINOT,

Claudia Regina Brando GOMES

Fundao Oswaldo Cruz, Instituto de Tecnologia em Frmacos-Far Manguinhos, R. Sizenando Nabuco 100, Manguinhos, 21041-
250, Rio de Janeiro, RJ, Brazil.
Abstract
Nowadays, Tuberculosis (TB), a contagious infectious disease caused by Mycobacteriumtuberculosis, 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 aimof 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 fromStreptomyces species.
Keywords: tuberculosis; translocase I; streptomyces
Resumo
Atualmente, a tuberculose (TB), doena infecto-contagiosa cujo agente etiolgico o Mycobacterium tuberculosis umgrave problema de sade
mundial. Os principais fatores responsveis pelo ressurgimento dessa doena no sculo vinte umforamo rpido desenvolvimento de cepas multiresistentes e
a associao do Mycobacteriumtuberculosis como vrus da imunodeficincia humana (HIV) no incio da dcada de 1980. Nesse contexto, torna-se
necessrio o desenvolvimento de novos frmacos ou uma tragdia humana pode ocorrer. Considerando esses fatores, a translocase I, enzima envolvida na
biossntese de peptideoglicanas, pode ser umimportante alvo no desenvolvimento de novos frmacos no combate a essa doena. Assimsendo, o objetivo
dessa reviso destacar uma srie de novas substncias promissoras para o tratamento da TB, isoladas de diferentes espcies de Streptomyces, que vemsendo
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-Gurin); CDC (Center for Disease Control and Prevention); CPZs
(caprazamicinas); LPMs (liposidomicinas); LPS (lipopolissacardeo); MDR (Multidrug resistant); MIC (Minimum inhibitory concentration); MRYs
(muraimicinas); OMS (Organizao Mundial de Sade); Human Immunodeficiency Virus (HIV); Tuberculosis (TB); TCM (tunicamicinas); UPAs (uridil
peptdeos); XDR (Extensively Drug Resistant)

Recibido | Received: J uly, 10, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: September 8, 2009.
Publicado en Lnea | Published Online 15 December 2010
Declaracin de intereses | Declaration of interests: authors have no competing interests.
Financiacin | Funding: This work has not received funds.
This article must be cited as: Marcus Vincius Nora de Souza*, Victor Facchinetti, Danielle Cardinot, Claudia Regina Brando Gomes. 2009. Produtos Naturais comatividade
inibitria da Translocase I, uma promissora classe de compostos contra tuberculose. Bol LatinoamCaribe Plant Med Aromat 9(1):1 12. {EPub 15 DEcember 2009 }.
*Contactos | Contacts: marcos_souza@far.fiocruz.br
De Souza et al. Translocase I, um importante alvo teraputico no combate tuberculose

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 9 (1) 2010 | 2

INTRODUO
A tuberculose (TB) uma doena crnica
endmica na maioria dos pases em desenvolvimento,
transmitida pelo ar e existente h milhares de anos,
causada pelo agente etiolgico Mycobacterium
tuberculosis, que, por ser uma bactria aerbia,
desenvolve-se principalmente nos pulmes, mas
tambm pode atacar outras reas do corpo humano.
Atualmente, estima-se que um tero da populao
mundial portadora assintomtica do Bacilo de
Koch, dos quais 5% a 10% iro manifestar a doena
que tem como principais sintomas tosse crnica
persistente, suor noturno, dor no trax e perda de
peso devido falta de apetite. ( De Souza and
Vasconcelos, 2005a)
A preveno da TB feita por meio da vacinao
de recm-nascidos em seus primeiros 30 dias de vida
com a vacina BCG (Bacilo de Calmette-Gurin), e o
tratamento da doena realizado, preferencialmente,
atravs da combinao de quatro frmacos:
Isoniazida, Rifampicina, Pirazinamida e Etambutol,
utilizados durante um perodo de 6 meses que pode
ser estendido para 9 meses em casos especiais.
Apesar de ser um tratamento eficaz e barato, a taxa
de abandono extremamente elevada em alguns
pases por diversos motivos como a longa durao do
tratamento, falta de informao e de
acompanhamento mdico e a grande quantidade de
efeitos colaterais associados ao uso desses frmacos.
(De Souza, 2006a, 2006b)
A questo da TB torna-se ainda mais delicada
quando inserida no contexto da pandemia de
HIV/AIDS, j que a co-infeco por M. tuberculosis
e HIV tem se mostrado uma combinao letal. Dados
da Organizao Mundial de Sade (OMS) indicam a
ocorrncia de mais de 300 casos anuais de TB a cada
100.000 habitantes em reas da frica subsaariana,
local em que h maior incidncia do vrus HIV.
Nessas reas, mais de dois teros das pessoas
infectadas por TB esto co-infectadas por esse vrus.
No Brasil, dados da OMS mostram que no perodo
entre 2000 e 2006 foram notificados quase 700.000
casos de TB, sendo o Rio de J aneiro o estado com
maior nmero de casos registrados por ano, e pouco
mais de 60.000 bitos causados por essa doena.
Aproximadamente 20% dessas mortes esto
associadas a pacientes co-infectados pelo vrus HIV.
(WHO, 2009)
MATERIAIS E MTODOS
O presente review foi organizado
basicamente em trs seces: A primeira delas
apresenta a tuberculose e o seu ressurgimento, devido
ao aparecimento de super-bactrias resistentes aos
frmacos utilizados. A segunda seo destaca a
importncia dos produtos naturais no tratamento da
tuberculose, bem como uma importante enzima
conhecida como translocase presente na parede
cellular do Mycobacterium tuberculosis, agente
etiolgico da TB. Finalmente, a ltima seo destaca
produtos naturais e sua relao estrutura-atividade
capazes de inibir essa enzima.
MULTIDRUG RESISTANT (MDR) E
EXTENSIVELY DRUG RESISTANT (XDR)
TUBERCULOSE
O abandono do tratamento tem provocado o
aparecimento de cepas resistentes aos frmacos de 1
escolha. Multidrug resistant (MDR) TB definido,
segundo a OMS (Organizao Mundial da Sade),
como doenas causadas pelo M. tuberculosis
resistente a Isoniazida e Rifampicina que so os
frmacos mais eficazes no tratamento da tuberculose.
Essas cepas resistentes foram identificadas no final
dos anos 80 e incio dos anos 90, representando uma
ameaa ao controle da doena.
De acordo com a OMS, as infeces por cepas
MDR-TB devem ser tratadas com pelo menos quatro
medicamentos de segunda escolha nunca usados
anteriormente pelos pacientes, incluindo
Capreomicina, Amicacina ou Canamicina injetveis e
um derivado fluorquinolnico como a Ciprofloxacina
e a Ofloxacina. O uso dos frmacos de segunda
escolha apresenta como desvantagens a maior
quantidade de efeitos colaterais, alto custo e maior
tempo de tratamento, que pode chegar a at 24 meses
de durao.
No tratamento de casos de infeces por cepas
MDR-TB, tambm comum o uso de Etionamida e
cido p-aminossaliclico e dos frmacos de primeira
escolha Pirazinamida e Etambutol. (De Souza, 2006c)
Uma nova ameaa ao controle da TB a
identificao recente de cepas do tipo Extensively
Drug Resistant (XDR) TB, que definida, de acordo
com a OMS, como cepas do tipo MDR-TB
resistentes a fluorquinolonas e a pelo menos um dos
trs medicamentos injetveis de segunda escolha
anteriormente citados. Como essas bactrias
apresentam resistncia aos frmacos de primeira e de

segunda escolha mais eficazes, a doena se torna
virtualmente incurvel atravs da utilizao dos
medicamentos atualmente existentes no mercado para
tratamento da TB. (CDC, 2009)
Uma pesquisa conduzida pela OMS em conjunto
com o CDC (Center for Disease Control and
Prevention) entre 2000 e 2004 identificou cepas do
tipo XDR-TB em todo o mundo, principalmente nos
pases da antiga Unio Sovitica e da sia. Outra
pesquisa realizada pela OMS, dessa vez na frica do
Sul, mostrou resultados alarmantes. De 544 pacientes
estudados, 221 estavam infectados com cepas do tipo
MDR-TB e desses, 53 foram enquadrados na
definio de XDR-TB, sendo 44 HIV-positivos. Dos
53 pacientes XDR-TB, 52 morreram em at 25 dias,
incluindo os beneficiados pelo uso de antirretrovirais,
fato esse que desperta a preocupao e demonstra a
urgncia de novos esforos com relao
identificao de novos alvos teraputicos e ao
desenvolvimento de novos frmacos no combate
TB. (WHO, 2009)
IMPORTNCIA DOS PRODUTOS NATURAIS
NO TRATAMENTO DA TUBERCULOSE
De maneira similar ao que aconteceu com outras
doenas, tais como cncer, a malria, e certas
doenas inflamatrias, o sucesso dos produtos
naturais merece destaque tambm na descoberta do
tratamento e cura da tuberculose. Vale ressaltar que a
descoberta da estreptomicina (Figura 1), isolada a
partir de culturas de Streptomyces grisus, pela
equipe liderada pelo bioqumico norte-americano,
Selman Waksman, em 1943, foi uma das mais
relevantes da histria da medicina moderna e da
humanidade. Esse medicamento foi responsvel pela
cura e pelo controle da tuberculose, doena que na
poca de seu surgimento causava a morte de um
nmero incontvel de indivduos. (De Souza, 2009)
Figura 1. Estrutura da Estreptomicina.

Aps a descoberta da estreptomicina, outros
aminoglicosdeos foram descobertos e utilizados at
os dias de hoje no tratamento e controle da
tuberculose, podendo-se destacar os
aminoglicosdeos canamicida, obtida a partir da
cultura de fungos Streptomyces capreolus, amicacina,
derivado semisinttico obtido partir da canamicina A
e capreomicina 1A, obtida a partir da cultura de
fungos Streptomyces Kanamyceticus. Alm dos
aminoglicosdeos, pode-se mencionar tambm a D-
cicloserina, obtida a partir da fermentao de
Streptomyces sps.
Com o crescente surgimento de super bactrias
resistentes a praticamente todos os frmacos
utilizados no tratamento da tuberculose, diversos
grupos de pesquisa tem demonstrado mais uma vez, o
papel fundamental da me natureza na descoberta de
novos frmacos no combate tuberculose. Nesse
contexto, pode-se destacar o crescente nmero de
publicaes cientficas na rea, identificando diversos
produtos naturais isolados de diversas fontes com
promissoras perspectivas no combate a tuberculose
multiresitente. Na figura abaixo se encontra alguns
exemplos de produtos naturais obtidos de plantas
com atividade tuberculosttica. (De Souza, 2005b)
(De Souza, 2006d).
Figura 2: Produtos naturais com atividade tuberculosttica.
O
O O O
OH
(+) - Calanolide A
7
8
12
A
B
C
MIC=3.13 gmL
-1
O
H3C
OCOCH3
CH3
CH3
H
O
H3CHN
O
H
H
OH
H
H
CH2OH
MIC=3.13 gmL
-1
O
O OH
HO
O
OH
MIC =6.3
H
O AcO
OAc
HO
MIC=12.5 gmL
-1
N
NH
N
CHO
NH
SolsodominaA
MIC =10 gmL
-1
gmL
-1
(XU et al., 2004)
(CHUMKAEWet. al, 2003)
(KANOKMEDHAKUL et al., 2005)
(KANOKMEDHAKUL et al., 2005)
(SAYED et al., 1998)

MYCOBACTERIUM E TRANSLOCASE I
Em geral, o citoplasma bacteriano separado do
meio extracelular por uma membrana citoplasmtica
formada por uma bicamada lipdica com boa fluidez,
que age como uma barreira seletivamente permevel,


e por uma parede celular constituda por
peptideoglicanas que confere a resistncia necessria
para suportar a alta presso osmtica interna (Nikaido
and Saier J R, 1994). A biossntese da parede celular
vem sendo explorada como alvo farmacolgico para
a pesquisa de novos antibiticos desde a descoberta
da penicilina por Fleming em 1929.
Bactrias Gram-positivas como o Staphylococcus
aureus possuem parede celular formada por uma
espessa camada de peptideoglicanas que confere
pouca resistncia difuso de pequenas molculas. J
as bactrias Gram-negativas, como a Escherichia
coli, contm em sua parede celular, alm da camada
de peptideoglicanas, uma outra membrana bilipdica
externa. A superfcie externa dessa membrana
recoberta por um lipdeo no usual, o
lipopolissacardeo (LPS), que possui estrutura de
pouca fluidez. Foi demonstrado que at mesmo
molculas lipoflicas apresentam dificuldades para
atravessar a parte hidrofbica desse lipdeo, o qual se
torna uma barreira eficiente contra a rpida difuso
de antibiticos lipoflicos. Em contraste, a parede
celular das micobactrias constituda por trs
subestruturas covalentemente interligadas: as
peptideoglicanas, arabinogalactanas e os cidos
miclicos, sendo os ltimos formados por longas
cadeias de cidos graxos contendo diferentes grupos
funcionais, como ligaes duplas, cetona, ster,
epxido, metxi e ciclopropano. Esses compostos so
de extrema importncia para a sobrevivncia das
micobactrias, pois dificultam a penetrao de drogas
hidrofbicas, evitam a desidratao e permitem que a
bactria se desenvolva no sistema imune do
hospedeiro (Figura 3). (Bugg and Walsh, 1992),
(Heijenoort, 2001), (Kimura and Bugg, 2003) (De
Souza, 2008)
Figura 3. Representao da parede celular de bactrias.

A biossntese de peptideoglicanas essencial para
a sobrevivncia da bactria (seres que possuem
clulas procariticas) e se torna um alvo interessante
no desenvolvimento de antibiticos, j que no existe
correspondncia em clulas eucariticas (presente
nos animais e na maioria das plantas). A biossntese
do peptideoglicano consiste constituida de trs
estgios: sntese do lipdeo I e do lipdeo II, seguido
da polimerizao do lipdeo II por transpeptidao e
transglicosilao. Como exemplo de frmacos em uso
clnico que inibem a polimerizao do lipdeo II na
superfcie da bactria, pode-se mencionar as -
lactamas e as vancomicinas. Esses frmacos possuem
um mecanismo de ao diferente dos utilizados no
tratamento para tuberculose, tornando-se possvel
combater as infeces causadas por micobactrias
multirresistentes. (Boyle and Donachie, 1998),
(Hirano et al, 2008a)
Estudos tm sido realizados visando descoberta
de substncias capazes de inibir a sntese do lipdeo I.
Nessa etapa, a enzima fosfo-MurNAc-pentapeptideo
translocase, tambm chamada de translocase I
(MraY), catalisa a reao entre o UDP-MurNAc-
pentapeptideo e o undecaprenil-fosfato formando
UMP e o Lipdeo I, que o primeiro intermedirio da
sntese de peptideoglicanas. Essa catlise requer a
presena de Mg
+2
como cofator para a ativao da
enzima (Figura 4).(Struve et al, 1966), (Heydanek et
al, 1969)
Figura 4: Biossntese do Lipdeo I

Vrias substncias tm sido relatadas como
inibidoras da enzima translocase I, dentre elas as
liposidomicinas, as caprazamicinas, as capuramicinas
e as pacidamicinas, que sero abordadas a seguir.
INIBIDORES DA TRANSLOCASE I
Liposidomicina
As liposidomicinas (LPMs) so produtos naturais
da classe dos antibiticos 6-N-alquil-5--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
pores sulfato e cido 3-metilglutrico. Os
compostos do tipo II no contm a poro cido 3-
metilglutrico, os do tipo III no contm a poro
sulfato e os do tipo 4 no possuem nenhuma das duas
pores (Figura 5). (Kimura et al, 2003)


A capacidade das liposidomicinas (LPMs) em
inibir seletivamente a sntese de peptideoglicanas foi
descoberta em 1985. (Isono et al, 1985). Testes in
vitro indicam que as LPMs do tipo I e III so os
melhores inibidores da enzima translocase I, quando
comparadas as LPMs do tipo II e IV, indicando que a
poro cido 3-metilglutrico exerce papel
fundamental na inibio da translocase I. Entretanto,
as LPMs do tipo I, assim como a LPMs do tipo II,
apresentam baixa atividade antimicrobiana in vivo
devido presena da poro sulfato, hidroflica, na
posio 2 da 5-aminopentose, fato que confere s
LPMs baixa permeabilidade atravs da membrana
celular. As LPMs do tipo III e IV so mais
lipoflicas devido ausncia da poro sulfato,
consequentemente, possuem maior atividade
antimicobacteriana in vivo.
A presena do cido graxo nessa classe de
substncias tambm extremamente importante para
a atividade biolgica, provavelmente porque confere
um aumento da lipofilicidade. (Kimura et al, 1998)
Figura5: Liposidomicinas dos tipos I, II, III e IV.
O
HO OH
N
H
N O O
N
N
O
O
R
O
O CH3
O
HO
O
CH3
H3C
HO2C
Liposidomicina I
Liposidomicina III
2'''
3'''
6'
5'
Liposidomicina II
Liposidomicina IV
Liposidomicinas R
I-A; II-A; III-A; IV-A H3C
(H3C)2HC I-B; III-B
H3C I-C; II-C; III-C; IV-C
H3C I-G; III-G
H3C I-H; III-H
H3C
I-K; III-K
I-L; III-L
(H3C)2HC
I-M; III-M
I-N; III-N
I-Z; III-Z
III-X
III-Y
H3C
H3C
H3C
H3C
H3C
O
O
H2N
HO
HO3SO
O
HO OH
N
H
N O O
5'
O
O
H2N
HO
HO3SO
N
N
O
O
R
O
CH3
H3C
HO2C
2'''
3'''
6'
N
N
O
O
R
O
O CH3
O
HO
O
CH3
H3C
HO2C
6'
3'''
2''' O
HO OH
N
H
N O O
5'
O
O
H2N
HO
HO
N
N
O
O
R
O
CH3
H3C
HO2C
2'''
3'''
6'
O
HO OH
N
H
N O O
5'
O
O
H2N
HO
HO

Dini e colaboradores em 2000 e 2001 sintetizaram
vrias substncias baseadas em simplificaes
estruturais das LPMs, que foram testadas como
inibidoras da translocase I. Algumas dessas
molculas esto representadas na figura 6.
Figura 6. Simplificaes estruturais das liposidomicinas
Substncia R R
1
MraY
IC ()
1 H NH
2
50
2
(R) - CH
2
OH
NH
2
425
3
(S) - CH
2
OH
NH
2
5
4
H
CH(NH)NH 30
5 H H
2
NC(NH)NH 25
6
H MeNH 45
7 H
EtNH 150
8
H
n-PrNH 140
O
HO OH
N
NH
O
O
O
R
O
HO OH
R
1 O
R
3'
R
2'
N
NH
O
O
O O
R
3"
R
2"
H
2
N
5"
Substnci a R
2'
R
3'
R
2''
R
3''
9
H OH OH OH
10
OH
H OH OH
11 OH OH H OH
12 OH OH OH H
13 H H OH OH
MraY
IC ()
80
10
115
>1000
120

A substncia 1 apresentou moderada atividade
inibitria (IC
50
: 50M). A introduo do grupamento
CH
2
OH na posio 5, indicou que o ismero R (2)
inativo, enquanto o ismero S (3) possui maior
atividade inibitria do que a substncia 1 (IC
50
:
5M). (Dini et al, 2000)
Um grupamento bsico, tal como o grupamento
amino, aminas secundrias, amidina e guanidina so
importantes para a atividade inibitria da MraY.
Sendo que, no caso das aminas secundrias, o
tamanho da cadeia lateral influncia diretamente na
atividade inibitria. A metilamina (6) ativa (IC
50
:
45M), enquanto que aminas com grupamento
alqulico (7 e 8) maior so inativas. (Dini et al,
2001a)
A comparao da atividade inibitria dos
compostos 9-13 indicou que presena da hidroxila na
posio 3 essencial para a atividade. Entretanto, o
composto 3-dexoxi 5 vezes mais ativo do que o
correspondente derivado 3-hidroxilado, sugerindo
que apenas a hidroxila na posio 3 essencial para
a inibio da translocase I. (Dini et al, 2001b)
Caprazamicinas
As caprazamicinas (CPZs) (Figura 7) so
produtos naturais que, assim como as LPMs,
pertencem a classe dos antibiticos 6-N-alquil-5--
O-aminoribosilgliciluridina. As CPZs so isoladas a
partir de culturas de Streptomyces sp. e mostram uma
excelente atividade contra bactrias Gram-positivas.
(Igarashi et al, 2005)


Figura 7. Estrutura das Caprazamicinas.

Caprazamicinas
Caprazamicinas R
H
3
C
H
3
C
G
(H
3
C)
2
HC
O
HO OH
N
NH
O
O
O
O
HO
HO
NH
2
N
N
O
O
HO
2
C
Me
Me
O
R
O
O Me O
O O
MeO
Me
OMe
OMe
1'
5'
6'
2'''
3'''
1''
5''
5
A
B
C
D
F
E
(H
3
C)
2
HC
H
3
C
(H
3
C)
2
HC
H
3
C
CH
3

As CPZs apresentam atividade in vitro contra
Mycobacterium tuberculosis, tanto em cepas
sensveis como em cepas multirresistente e no
demonstraram toxicidade significativa em ratos.
Devido s excelentes propriedades biolgicas, as
CPZs so substncias promissoras para a sntese de
novos agentes anti-TB, com um novo mecanismo de
ao. Nesse contexto, quando comparadas s LPMs,
as CPZs possuem importantes semelhanas
estruturais e biolgicas sugerindo que essas classes
de compostos possuem o mesmo mecanismo das
LPMs. (Ichikawa, 2008)
Hirano e colaboradores sintetizaram diversos
anlogos da caprazamicina (Figura 8), incluindo o
caprazol e o palmitoilcaprazol, que foram testados
contra o Mycobacterium sp.
Figura 8. Anlogos das Caprazamicinas.

O
HO OH
N
NH
O
O
O
O
HO
HO
NH
2
N
N
O
O
HO
2
C
Me
H
O O
O
HO OH
N
NH
O
O
O
O
HO
HO
NH
2
N
N
O
O
HO
2
C
Me
Me
14
(MIC =25 g/mL)
Palmitoi lcaprazol
(MIC =6,25 g/mL)

O
HO OH
N
NH
O
O
O
O
HO
HO
NH
2
N
N
O
HO
HO
2
C
Me
Me
O
HO OH
N
NH
O
O
O
O
HO
HO
NH
2
H
2
N
N
O
O
HO
2
C
Me
O
O
HO OH
N
NH
O
O
OH
N
N
O
O
HO
2
C
Me
Me
O
Me
O
O
HO
HO
NH
2
N
N
O
O
HO
2
C
Me
Me
O
Caprazol
15
16 17
(MIC =2,50 g/mL)
(inativa)
(inativa) (inativa)

O palmitoilcaprazol, quando testado contra
Mycobacterium smegmatis ATCC607, apresentou
MIC (Minimum inhibitory concentration
concentrao inibitria mnima) de 6,25 g/mL,
similar ao das CPZs, e quatro vezes maior do que o
do composto 14 (25 g/mL), mostrando que a
ausncia do grupamento metila na poro da
diazepanona influencia negativamente a atividade
antimicobacteriana. (Hirano et al, 2008a). Outros
anlogos foram testados contra Mycobacterium
tuberculosis H37Rv, sendo que o composto 15
apresentou atividade ligeiramente reduzida quando
comparado ao palmitoilcaprazol (MIC =2,50 e 6,25
g/mL, respectivamente), sugerindo que a abertura
do anel diazepanona diminui a atividade inibitria
sem, contudo, tornar a substncia inativa. A
fragmentao da molcula nas pores aminoribose
ou uridina tornou as molculas 16 e 17 inativas e,
portanto, so cruciais para a atividade antibacteriana.
(Hirano et al, 2008b) O caprazol, anlogo das CPZs
sem a poro alqulica no anel diazepanona, tambm
no apresentou atividade antimicobacteriana. Essas
alteraes realizadas na parte lipoflica da cadeia
lateral so de fundamental importncia para a
permeabilidade na clula bacteriana. (Hirano et al,
2008a)
Capuramicina
A capuramicina (figura 9) foi originalmente
isolada a partir de culturas de Streptomyces griseous
466-S3 e possui a capacidade de inibir a enzima
translocase I, no entanto seu espectro de ao baixo.



Figura 9. Anlogos da Capuramicina e sua attividade
antimicrobiana (Hotoda et al, 2003a)
O
RO OH
N
NH
O
O
CONH
2
O O
H
N
O
OH
OH
HN
O
Capuramicina (R =H)
A-500359A (R =Me)
O
HO OH
N
NH
O
O
CONH
2
O O
R
1
O
OH
OH
R
Anlogos da Capuramicina 18-24

Compostos R
1
1
a
2
b
3
c
4
d
5
e
Capuramicina - 10 12,5 8 8 8
A-500359A - 10 6,25 8 4 16
A-500359E MeO- 27 >100 - - -
18 PhNH- 6,5 6,25 16 4 8
19 3-Me-PhNH- 7,6 12,5 4 1 8
20 3-F-PhNH- 10 6,25 2 2 8
21 4-F-PhNH- 37 6,25 4 2 2
22 3,4-di-F-
PhNH-
9 6,25 2 0,5 1
23 4-Cl-PhNH- 18 6,25 4 2 16
24 4-Br-PhNH- 20 6,25 8 0,5 8
Rifampicina - - - 0,125 0,125 0,125
Isoniazida - - -

a
Translocase I, IC
50
(ng/mL)
b
M. smegmatis SANK75075, MIC (g/mL)
c
M. avium NIHJ 1605, MIC (g/mL)
d
M. intracellulareATCC1954 E-3, MIC (g/mL)
e
M. kansasii ATCC12478, MIC (g/mL)
Em geral, a capuramicina apresenta maior
atividade frente micobactrias e pouca atividade
frente a bactrias Gram-positivas. interessante
notar que as micobactrias apresentam resistncia
contra muitos derivados -lactmicos, medicamentos
que pertencem mesma classe das capuramicinas,
devido presena da enzima -lactamase. A
atividade dessas substncias poderia ser ento
explicada pela estabilidade do seu anel -lactmico
frente a essas enzimas. O mecanismo de
permeabilidade das capuramicinas atravs da
membrana das micobactrias ainda no est
completamente elucidado, sendo atualmente, muitos
anlogos de capuramicinas vm sendo isolados,
sintetizados e testados como inibidores da enzima
translocase I. Como exemplo, pode-se citar anlogos
da capuramicina que no contm a poro azepan-2-
ona (Figura 7) obtidos por Hotoda e Colaboradores, a
partir da reao entre diversas aminas e o produto
natural A500359E, tambm isolado de cepas S.
griseous.
As substncias obtidas foram testadas frente ao M.
smegmatis e a enzima Translocase I, ficando evidente
a importncia do grupamento NH da amida para a
atividade dessas substncias. Os melhores resultados
foram observados com os compostos 18-24 que
foram, ento, testados frente s micobactrias de
maior relevncia clnica: Mycobacterium avium
NIHJ 1605, Mycobacterium intracellulare ATCC1954
E-3 e Mycobacterium kansasii ATCC12478. Suas
atividades foram comparadas com a da rifampicina e
isoniazida sendo que o anlogo 18 apresentou MIC
semelhante ao da capuramicina, variando entre 4 e 16
g/mL. (Hotoda et al, 2003a)
Hotoda e Colaboradores tambm obtiveram
derivados acilados a partir das molculas de
capuramicina e de seu derivado metilado A500359A
(Figura 10)
Observou-se que o aumento no tamanho da cadeia
lateral provoca diminuio na atividade das
molculas frente enzima Translocase I, porm,
cadeias de tamanho ideal como a duodecanola e a
decanola presentes, respectivamente, nos derivados
da capuramicina (25, MIC variando entre 0,063 e
3,13 g/mL) e do A-500359A (26, MIC variando
entre 0,063 e 6,25 g/mL), apresentaram excelente
atividade frente s micobactrias, provavelmente
devido lipofilicidade conferida a esses compostos
que permite uma melhor penetrao atravs da
membrana celular desses microorganismos. Os
valores de MIC observados para estes compostos
foram iguais ou melhores do que os observados para
a isoniazida (MIC entre 0,125 e 0,25 g/mL) e a
rifampicina (MIC entre 1 e 2 g/mL). (Hotoda et al,
2003b)
Figura 10. Anlogos acilados da Capuramicina e sua attividade
antimicrobiana (Hotoda et al, 2003b)
O
MeO O
N
NH
O
O
CONH
2
O O
H
N
O
OH
HN
O
OH
R
O
(CH
2
)nH
25 e 26

Compostos R n 1
a
2
b
3
c
4
d
5
e
Capuramicina H - 10 12,5 8 8 8
A-500359A Me - 10 6,25 8 4 16
25 H 11 n.d. 3,13 <0,063 0,125 0,125
26 Me 9 50 6,25 <0,063 <0,063 <0,063
Rifampicina - - n.d 0,125 0,125 0,125 0,25
Isoniazida - - n.d - 1 8 2
a
Translocase I, IC
50
(ng/mL);
b
M.smegmatis SANK75075, MIC
(g/mL);
c
M. Avium NIHJ 1605, MIC (g/mL);
d
M.

intracellulareATCC1954 E-3, MIC (g/mL);
e
M. Kansasii
ATCC12478, MIC (g/mL)

Os compostos (RS-124922) e (RS-118641)
(Figura 11), sintetizados pela empresa japonesa
Sankyo, foram avaliados frente ao Mycobacterium
tuberculosis, apresentando melhor atividade contra
M. tuberculosis (Cepa H37Rv) (MIC =8 g/mL e
MIC = 1 g/mL, respectivamente), do que o
composto A500359A (MIC =16 g/mL). A atividade
dessas substncias frente cepas MDR-TB no
sofreu variaes estatisticamente significativas,
quando comparadas s observadas nas cepas H37Rv.
No foram realizados testes in vivo para o composto
(RS-124922) devido a sua baixa solubilidade no
modelo de tratamento utilizado. Na avaliao in vivo
dos outros, observou-se uma considervel reduo no
nmero de organismos viveis nos pulmes em
comparao com o grupo de controle. Os estudos
desenvolvidos mostram que os anlogos de
capuramicinas possuem grande potencial
antimicobacteriano e so bons candidatos a posterior
avaliao no tratamento de infeces causadas por M.
tuberculosis. (Koga et al, 2004)
Figura 11. Anlogos da capuramicina sintetizados pela empresa
japonesa Sankyo e sua attividade antimicrobiana (Koga et al,
2004).
O
MeO O
O
CONH
2
O O
H
N
O
OH
OH
HN
O
Me
O
N
NH
O
O
MeO OH
N
NH
O
O
CONH
2
O O
H
N
O
OH
OH
F
F
A500359-A
O
MeO OH
N
NH
O
O
CONH
2
O O
H
N
O
OH
OH
HN
O
Me
(RS-118641)
(RS-124922)

Substncias Cepa H37Rv
MIC ( g/mL)
Cepa MDR-TB
MIC ( g/mL)
A500359-A 16 16
(RS-124922) 8 4
(RS-118641) 1 0,5
Rifampicina <0,03 >32
Isoniazida 0,06 4

Pacidamicinas, mureidomicinas e napsamicinas
As pacidamicinas so antibiticos da classe uridil
peptdeos (UPAs), assim como as mureidomicinas e
napsamicinas (Figura 12), capazes de inibir a enzima
translocase I (MraY), isoladas de cultura de
Streptomices sp. (Isono et al, 1992), (Boojamra et al,
2003), (Chatterjee et al, 1994), (Chen et al, 1989),
(Fernandes et al, 1989)
Figura 12. Estrutura das napsamicinas, mureidomicinas e
pacidamicinas.
Napsami cina
R R
1
A H uracil
B CH3 uracil
C H diidrouracil
D CH3
diidrouracil
O
OH
NH
O
H
N
O
N
H
O
N
H
CO
2
H
SCH
3
OH CH
3
N
CH
3
O
NH
HO
R R
1 O
OH
NH
O
H
N
O
N
H
O
N
H
CO
2
H
SCH
3
OH CH
3
CH
3
O
R
1
H
N
R
OH
O
OH
NH
O
H
N
O
N
H
O
N
H
R
1
CO
2
H
CH
3 CH
3
N
CH3
O
H
N
R
OH
N NH
O
O
Mureidomicina R R
1
A H uracil
B H diidrouracil
C
glicina uracil
D
glicina diidrouracil
Pacidamicina R R
1
1 alanil 3-indol
2 alanil fenil
3
alanil 3-hidroxi-fenil
4 H 3-indol
5
H
fenil
6 glicil 3-indol
7 glicil fenil

Compostos da classe das UPAs apresentam
excelente atividade contra Pseudomonas aeruginosa
devido as suas propriedades toxicolgicas e
farmacolgicas favorveis. Cepas bacterianas
resistentes, a ofloxacin e -lactamas, permanecem
sensveis substncias dessa classe de antibiticos.
Entretanto, os UPAs possuem espectro de ao muito
estreito, sendo ativo especificamente contra cepas de
Pseudomonas, e muito pouco ativo contra bactrias
Gram-negativas e Gram-positivas, o que pode ser
explicado pelo fato desses compostos possurem
acesso restrito clula bacteriana, devido a sua
polaridade e ao seu grande potencial para fazer
ligaes de hidrognio com a gua. (Isono et al,
1992), (Boojamra et al, 2001), (Boojamra et al, 2003)
Boojamra e colaboradores, sintetizaram vrias
diidropacidamicinas D, a partir da hidrogenao do
produto natural (pacidamicina 4), ou de sntese total,
introduzindo diferentes aminocidos na cadeia
acclica (Figura 13). (Boojamra et al, 2003) As
substncias sintetizadas foram testadas contra quatro
cepas de Mycobacterium tuberculosis, das quais duas,
a W e a P, so resistentes a todo o tipo de tratamento
anti-tuberculose, observando-se que as molculas em
que NHCH(R
3
)COOH um resduo de um
aminocido aromtico, e as pores H
2
NCH(R
1
)C(O)
e (CO)CH(R
2
)NH possuem resduos hidrofbicos

(substncias 27 e 32-35) apresentaram melhor
atividade antimicobacteriana (MIC variando entre 4 e
10 g/mL), inclusive quando comparadas a
pacidamicina 4 (MIC maior que 30 g/mL), sendo
ativo inclusive frente s cepas multirresistentes.
Figura 13. Derivados da diidropacidamicina 4 e sua attividade
antimicrobiana (Boojamra et al, 2003)
Substncias
R
1
H
2
N
O
R
2
N
H O
R
3
N
H
CO
2
H
Pacidamicina 4
meta-tirosina alanina
triptofano
27 alanina 4-flor-fenilalanina tirosina
28 glicina leucina triptofano
29
alanina metionina
tirosina
30
alanina fenilalanina (2-naftil)alanina
31
leucina fenilalanina triptofano
32 alanina (4-bifenil)alanina triptofano
33 alanina fenilalanina triptofano
34 alanina
4-flor-fenilalanina triptofano
35 alanina 4-trifluormetilfenilalanina triptofano
O
NH
O
H
N
O
N
H
O
N
H
R
3
CO
2
H
R
2
N
CH
3
O
H
2
N
N NH
O
O
OH
R
1
CH
3

Substncias 1
a
2
b
3
c
4
d
Pacidamicina 4 >30 >30 >30 >30
27 10 8 8 10
28 >30 30 10 10
29 >30 >30 >30 >30
30 30 30 30 10
31 30 30 30 10
32 8 4 4 10
33 8 8 4 8
34 8 8 4 8
35 8 4 4 8

a
Cepa H37Rv, MIC (g/mL)
b
Cepa TN913, MIC (g/mL)
c
Cepa TN565 (W), MIC (g/mL)
d
Cepa TN1618(P), MIC (g/mL)
Muraimicinas
As muraimicinas (MRYs) so substncias
estruturalmente semelhantes aos antibiticos uridil
peptdeo (UPAs), como as mureimicinas,
napsamicinas, pacidamicinas e liposidomicina.
Dezenove muraimicinas foram isoladas a partir de
culturas de Streptomices sp. (Figura 14). (McDonald
et al, 2002) Dentre estas, as muraimicinas A e B, que
possuem um grupamento ster com uma cadeia
alquilica longa, geralmente possuem melhor atividade
antibacteriana do que as muraimicinas que no
apresentam o grupamento ster, demonstrando a
necessidade da poro lipoflica para a penetrao na
parede celular das bactrias.
McDonald e colaboradores relataram que algumas
muraimicinas (muraimicinas A1, A5, B6, C2 e C3)
inibem a sntese do lipdeo II e a biossntese da
camada peptideoglicana em concentraes de 0,027
g/mL, indicando que a atividade antibacteriana das
MRYs comparvel a da LPM C e a da
mureidomicina A, que inibem a translocase de E.
colii, in vitro, com valores de IC
50
de 0,05 e 0,03 g
/mL, respectivamente.(Ichikawa and Matsuda, 2007)
Figura 14. Estrutura das muraimicinas.
Murai mici na R
1
R
2
A1 O
2
C(CH
2
)
12
N(OH)C(NH)NH
2 OCH
3
A2
O
2
C(CH
2
)
10
N(OH)C(NH)NH
2
OCH
3
A3 O
2
C(CH
2
)
12
NHC(NH)NH
2
OCH
3
A4 O
2
C(CH
2
)
12
N(OH)C(NH)NH
2 OH
B1
O
2
C(CH
2
)
6
CH(CH
3
)CH
2
CH
3 OCH
3
B2 O
2
C(CH
2
)
6
CH(CH
3
)
2
OCH
3
B3
O
2
C(CH
2
)
4
CH(CH
3
)CH
2
CH
3
OCH
3
B4 O
2
C(CH
2
)
5
CH(CH
3
)
2 OCH
3
B5
O
2
C(CH
2
)
5
CH(CH
3
)
2 OH
B6
O
2
C(CH
2
)
4
CH(CH
3
)
2 OCH
3
B7 O
2
C(CH
2
)
4
CH(CH
3
)
2 OH
O
HO OH
N
NH
O
O
O
CO
2
H
H
N
H
H
H
N
O
R
1
N
H
O
H
N
HN
N
H
H
H
O
O
H
N HO
2
C
O
HO R
2
H
2
N
O
HO OH
N
NH
O
O
CO
2
H
H
N
H H
N
O
R
1
N
H
O
H
N
HN
N
H
H
H
O
O
H
N HO
2
C
HO
H
Muraimici na
R
1
R
2
C1 OH OCH
3
C2 OH OH
C3 OH H
D1 H
OCH
3
D2 H OH
D3 H H
Muraimicina
R
1
A5 O
2
C(CH
2
)
12
N(OH)C(NH)NH
2
C4 OH

Lin e colaboradores sintetizaram derivados da
muraimicina C1, atravs de reaes seletivas nos
grupamentos amino primrio e secundrio (Figura
15).


Figura 15. Derivados da muraimicina C1.
O
HO OH
N
NH
O
O
O
N
H
H
N
O
OH
N
H
O
H
N
HN
N
H
H
H
O
O
H
N HO
2
C
O
HO OMe
H
2
N
N
O
R
O
36: R =(CH
2
)
4
CH
3
(MIC =6,25 g/mL)
37: R =CH
2
Ph (MIC =6,25 g/mL)

Os derivados onde ocorreu substituio tanto na
amina primria como na secundria foram inativos
como inibidores da MraY, em concentraes <100
g/mL. Entretanto, os compostos onde houve
substituio apenas na amina secundria
apresentaram boa atividade inibitria, que
demonstrou estar correlacionada com a lipofilicidade
dos substituintes introduzidos. Dentre estes,
destacou-se as hidantinas 36 e 37, que inibiram a
MraY na mesma concentrao que a muraimicina C1
(6,25 g/mL). (Lin et al, 2002)
Tunicamicinas
A classe de produtos naturais conhecidas como
tunicamicinas (TCM) (Figura 16) foi isolada da
fermentao de Streptomyces lysosuperficius sendo
capazes de inibir a replicao de vrus, bactrias e
fungos baseada na glicosidao de protenas.

Tunicamicina R
I (CH
2
)
7
CH(CH
3
)
2
II (CH
2
)
8
CH(CH
3
)
2
III
(CH
2
)
10
CH
3
IV (CH
2
)
11
CH
3
V (CH
2
)
9
CH(CH
3
)
2
VI
(CH
2
)
11
CH(CH
3
)
2
VII (CH
2
)
10
CH(CH
3
)
2
VIII (CH
2
)
12
CH
3
O
OH
N NH
O
O
HO
OH
O
O
HO OH
N
H
O
R
O
OH
HO
HO
H
3
C(OC)HN

Figura 16. Estrutura das Tunicamicinas.

Alm de sua ampla atividade, as Tunicamicinas
servem como ferramentas na elucidao de
mecanismos bacterianos. (Inuka et al, 1993),
(Ichikawa, 2008)
DISCUSSO
Tendo em vista o grande nmero de efeitos
colaterais associados aos frmacos mais utilizados no
tratamento da TB, a durao de seu tratamento e os
adventos MDR e XDR-TB, faz-se necessrio o
desenvolvimento de novos medicamentos ou uma
grande tragdia poder ocorrer, voltando-se ao tempo
em que no se existia a cura para essa doena. Nesse
contexto, a translocase I (Mra Y), uma das enzimas
envolvidas na fase inicial da biossntese de
peptideoglicanas, tem se mostrado um alvo promissor
na busca de novos frmacos, sendo representada por
diversos produtos naturais e seus derivados sintticos,
representando uma nova classe no combate s
infeces bacterianas modernas. Nesse contexto,
podemos destacar as liposidomicinas (LPMs) e as
caprazamicinas (CPZs), produtos naturais da classe
dos antibiticos 6-N-alquil-5--O-
aminoribosilgliciluridina, que apresentam excelentes
atividades frente a enzima translocase. Dentre os
compostos avaliados dessas classes, merece destaque,
a capuramicina, substncia pertencente a famlia das
CPZs, que apresentou excelentes perspectivas sendo
o estudo de sua estrutura atividade importante para
identificao de novos derivados mais potentes,
simplificados, com melhores propriedade
farmacocinticas, bem como capazes de ajudar na
melhor compreeno desse mecanismo de ao.
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2009 The Authors

Artculo Original | Original Article


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Antioxidant and anti-inflammatory activity of Moussonia deppeana
[Actividad antioxidante y anti-inflamatoria de Moussonia deppeana]
Miguel A. DOMNGUEZ-ORTIZ,
1*
Omar MUOZ-MUIZ,
2*
Rosa Virginia GARCA-RODRGUEZ,
2

Maribel VZQUEZ-HERNNDEZ,
2
Janeth GALLEGOS-ESTUDILLO,
2
Jess Samuel CRUZ-SNCHEZ.
1

1
Instituto de Ciencias Bsicas, Universidad Veracruzana;
2
Unidad de Servicios de Apoyo en Resolucin Analtica, Universidad
Veracruzana, Luis Castelazo Ayala S/N, Col. Industrial nimas, CP. 91190 Xalapa, Ver. Mxico.

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 18.33.4 g/mL) with 41% of reducing power respect to ascorbic acid and total content of polyphenols was smaller (328.97.6 mg GAE/g) than the
found in the ethanol extract (388.66.2 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 inflamacin. Los resultados obtenidos mostraron que el extracto de EtOAc fue el ms
activo en la prueba de DPPH (CI50 18.33.4 g/mL), con un poder reductor de 41% respecto al cido ascrbico y el contenido total de polifenoles fue menor
(328.97.6 mg GAE/g) al encontrado en el extracto etanlico (388.66.2 mg GAE/g). La actividad anti-inflamatoria evaluada mediante aplicacin tpica de
los extractos (dosis de 2 mg/oreja) dio mayor inhibicin con el extracto hexnico, seguida del EtOAc (39 y 28%, respectivamente). El modelo del edema
plantar fue evaluado nicamente en el extracto de EtOAc observndose una inhibicin similar a indometacina (43% a dosis de 100 mg de extracto) en la
primera hora. Los resultados apoyan el efecto biolgico atribuido en su uso tradicional.
Palabras Clave: Anti-inflamatorio, Antioxidante, Moussiana deppeana, Etnofarmacologa, Plantas Medicinales.

Recibido | Received: May, 29, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: October 15, 2009.
Declaracin de intereses | Declaration of interests: authors have no competing interests. Todos los autores contribuyeron de igual manera en el manuscrito.
Financiacin | Funding: This work was financed by Universidad Veracruzana.
This article must be cited as: Miguel A. Domnguez-Ortiz, Omar Muoz-Muiz, Rosa Virginia Garca-Rodrguez, Maribel Vzquez-Hernndez, Janeth Gallegos-Estudillo, Jess
Samuel Cruz-Snchez. 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.

Domnguez-Ortz et al. Antioxidant and Anti-inflammatory Activity of Moussonia deppeana

www.blacpma.org Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol.9 (1) 2010 | 14

INTRODUCTION
Moussonia deppeana (Schldl. & Cham)
Hanst, belongs to Gesneriaceae family (synonyms:
Kolheria deppeana, Gesneria deppeana, Moussonia
elongate), this species is broad distributed since
Mexico to Panama. In folklore medicinal is
commonly known as clachichinole, tlachichinole,
tochomitillo or valletina (Escalante, 1988). This plant
is frequently used by Mexican people in traditional
medicine because of their curative properties
(stomach inflammation, diarrhea, ulcer, kidney
disease, vaginal infection and some tumors). In this
sense, several extracts of these plants have been
studied as antiprotozoal (Calzada et al., 1998) and
some reports revealed the presence of -sitosterol, -
D-glucosyl-sitosterol, ursolic acid, oleanolic acid,
2,3-dihydroxy-olean-12-en-28-oic acid, 2,3-
dihydroxy-olean-12-en-28-oic acid (Noguera et al.,
1994); 2-methyl-anthraquinone, chromanone and
stigmasterol (Reyes-Blas, 1995).
Gesneriaceae family is very extensive and
includes tropical herbs and shrubs; many of them are
growth as ornamentals (Martnez, 1969; Alcntara
and Luna, 2001). In some members of this family
have been isolated anthocyanins, flavonoids and
flavones (Daz, 1976; Gould and Lister, 2006). Of
particular interest was the isolation of some
glycosylated rutine derivatives by Robinson and
Tood (Robinson et al., 1934), which have a well-
defined anti-inflammatory activity.
By the other hand, is well known that oxygen
and nitrogen reactive species play important roles in
normal physiological processes, protection from
pathogens, cellular signaling pathways, and
regulation of vascular tone (Valko et al., 2007); also,
they are related to development of tissue damage in
various human diseases such as cancer, aging,
neurodegenerative disease, malaria and pathological
events in living organism (Gutteridge, 1994).
In this sense, antioxidant capacity of
medicinal plants and herbs has been linked to in vivo
protection from oxidative stress in numerous studies
(Prior et al., 2005) but rarely has been associated with
anti-inflammatory capacity (Jensen et al., 2008). For
this reason, the present study is aimed to the
evaluation of antioxidant and acute anti-inflammatory
effect of several extracts of M. deppeana growing in
Mexico by using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging assay (Brand-Williams et
al., 1995; Miliauskas et al., 2004); reducing power
with FeCl
3
test (Oyaizu, 1986); total polyphenols by
Folin-Ciocalteau assay (Spanos and Wrosltad, 1990)
as well as 12-O-tetradecanoylphorbol 13-acetate
(TPA) induced mouse ear edema model (Young and
De Young, 1989) and carrageenan induced mouse
paw edema model (Levy, 1969) for determination of
anti-inflammatory properties.
MATERIALS AND METHODS
All chemical used were analytical grade. 1, 1-
Diphenyl-2-picryl hydrazyl (DPPH), Folin-
Ciocalteus reagent, potassium ferricyanide
(K
3
Fe[CN]
6
), ferric chloride (FeCl
3
), gallic acid
monohydrate, ascorbic acid, carrageenan lambda, 12-
O-tetradecanoylphorbol 13-acetate and solvents were
obtained from Sigma-Aldrich (Mxico).
Absorbance in colorimetric determinations
was measured in a UV-Vis Varian spectrophotometer
Cary100 model.
Vegetal material
M. deppeana was collected in Rancho Viejo-
Cinco Palos Municipality of Coatepec, Veracruz
State, Mexico in October 2007. The taxonomic
identification of plants was confirmed by Luis
Hermann Bojorquez Galvn, a taxonomist. A
voucher specimen (CIB8987) has been deposited in
the Instituto de Investigaciones Biolgicas Herbarium
of Universidad Veracruzana.
Preparation of crude plant extracts
About 800 g of aerial part of plant material
were cut into small pieces, dried at room temperature
and extracted by exhaustive maceration in darkness
with different solvents. Three extracts were obtained
successively and solvent was removed using rotary
evaporator (Hexane, 1.68 g; EtOAc, 2.01 g and
EtOH, 7.3 g). Crude extracts were kept in amber
colored glass vials at room temperature for further
use.
Phytochemical analysis
Phytochemical analysis of the plant extracts
was undertaken using standard qualitative methods
(color test and/or Thin Layer Chromatography, TLC).
Two milligrams of each extract were dissolved in
chloroform (5 mL) before application to TLC plates
(26 cm). The elution systems were benzene/acetone
(9:1) for hexane and EtOAc extracts meanwhile for
the ethanol extract was used a mixture butanol/water/


acetic acid (6:3:1). The revealing agents were:
Dragendorff solution (for alkaloids), AlCl
3
1% in
ethanol (for flavonoids), ZnCl
2
(for sapogenins),
KOH 10% in ethanol (for coumarins), perchloric acid
(for sterols) and NaOH (for quinones) (Domnguez,
1973; Kaufman et al., 2006).
Animals
All animals employed in the experiments
were CD1 male mice (20-25 g) obtained from
Facultad de Medicina of the Universidad
Veracruzana, Xalapa. The animals were acclimated
for one week in photoperiods adjusted to 12 hours of
light and 12 hours darkness daily and 50-55%
relative humidity with standard pellet diet (Rodent
chow) and drink ad libitum. This work was
performed according to the Guide for the Care and
Use of Laboratory Animals (National Academy of
Sciences, 1996) and the Official Mexican Norm
(NOM-062-ZOO-1999).
Determination of Antioxidant Capacity
Free radical scavenging by the use of DPPH
radical
The DPPH radical scavenging capacity of
each extract was determined according to Brand-
Williams method modified by Miliauskas, 2004.
DPPH radicals have and absorption maximum at 517
nm, which disappears with reduction by an
antioxidant compound. The DPPH radical solution in
methanol (9 x 10
-5
M) was freshly prepared, and 2.9
mL of this solution was mixed with 100 L of
methanolic solutions of plant extracts at several
concentrations (33, 16.5 and 8.25 g/mL). The
samples were incubated for 30 min at 37 C in a
water bath, and decrease in absorbance at 517 nm
was measured (A
E
). A blank sample containing 100
L of methanol in the DPPH radical solution was
prepared daily, and its absorbance was measured
(A
B
). Radical scavenging activity was calculated
using the following formula:

100 % x
A
A A
Inhibition
B
E B
(

=
Determination of total phenolic content
The total phenolic concentration was
determined using the Folin-Ciocalteus reagent
according to the Spanos and Wrosltad, 1990. To 50
L of each sample (1 mg/mL, three replicates), 2.5
mL 1/10 dilution of Folin-Ciocalteus reagent and 2
mL of Na
2
CO
3
(7.5 %, w/v) were added and
incubated at 45 C for 15 min. The absorbance of all
samples was measured at 765 nm using a UV-Vis
spectrophotometer. Results were expressed as gallic
acid equivalent (g/mL) by using the following
equation, which was obtained from standard gallic
acid graph (range 20 to1000 g/mL).

075 . 0 )] / ( [ 001 . 0 + = mL g GAE Absorbance
Determination of reducing power
The reducing power was determined
according to the method described by Oyaizu, 1986.
A 0.125 mL aliquot of extract (1 mg/mL) was mixed
with 1.25 mL of 200 mM sodium phosphate buffer
(pH 6.6) and 1.25 mL of 1% of potassium
ferricyanide. The mixture was then incubated at 50
C for 20 min. After 1.25 mL of 10% trichloroacetic
acid (w/v) were added, the mixture was centrifugated
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
mL of 0.1% ferric chloride, and the absorbance at
700 nm was measured. The obtained value was
compared with the ascorbic acid value as standard.
Anti-inflammatory evaluation
12-O-tetradecanoylphorbol 13-acetate (TPA)-
induced mouse ear edema.
Irritant dermatitis was induced on the right
ear by topical application of 2.5 g TPA in 25 L of
acetone according to the methodology reported by
Young and De Young, 1989. TPA was applied on
both the inner and outer surfaces of the ears. The
extracts (doses 2 mg/ear) and the indomethacin
(doses 2 mg/ear) were applied topically 30 minutes
after TPA to the right ear (E
t
), the left ear received
vehicle (E
0
). In the control group the right ear
received only TPA (E
t
) and the other ear acetone (E
0
).
In all groups the edema was allowed to develop for 6
hours; afterwards the animals were sacrificed by
dislocation cervical and plugs (diameter of 6 mm) of
the central portion were taken from both ears and
weighted. The inhibition of auricular edema was
calculated with the difference between weight ears of
the animal treatment with the extract or indomethacin
and the control group.



100
) (
) ( ) (
%
0
0 0
x
control E E
treated E E control E E
Inhibition
t
t t
(

=

Carrageenan-induced mouse paw edema
20 L/paw of a 1% carrageenan solution was
injected into the sub-plantar region of the left hind
paw of the mouse. The increment in the paw thick
was determined with a digital micrometer at 1, 3, 5
and 7 h after carrageenan administration. A reference
group was administered intraperitoneally with
indomethacin (doses 5 mg/kg). The extract was
administered at doses of 100 and 300 mg/kg by the
same via before carrageenan administration. The
control group received vehicle only. The percent
edema inhibition was calculated for each animal
group in comparison to group treated with vehicle
(10% Tween 80) according to the Olajide et al, 2000.
Statistical analysis of data
Data are presented as means S. D. of at
least triplicate experiments. For in vivo experiments,
data is reported as the means S.E.M. Significant
differences between groups were determined by
analysis of variance (ANOVA) complemented with
Dunnetts test using the software Statistica version 7
from StatSoft, Inc. (2004), p<0.05 was considered
significant.
RESULTS AND DISCUSSION
Reactive oxygen species (ROS) may be
involved in the etiologies of several human diseases
as atherosclerosis, ischemic injury, cancer and
neurodegenerative diseases, as well as in processes
like inflammation and ageing (Edmonds, 2000). Also,
there is evidence that antioxidants may be useful in
preventing the deleterious consequences of oxidative
stress and there is increasing interest in the protective
biochemical functions of natural antioxidants
containing in medicinal plants. In this sense, our
attention has been focused in the antioxidant and
anti-inflammatory properties of M. deppeana, a
medicinal plant for the treatment of diabetes, stomach
inflammation, kidney diseases, cough, and tumoral
disease (Cano-Asseleih, 1997).
The phytochemical screening (Table 1)
revealed the presence of phenolic compounds
(flavonoids and coumarins) in EtOAc and EtOH
extracts. In the hexane and EtOAc extracts were
possible to observe a positive result for sterols and
quinones, which supports the presence of some
metabolites previously reported (Noguera et al., 1994
and Reyes-Blas, 1995).

Table 1. Phytochemical screening.
Test
Extracts
Hexane EtOAc EtOH
Alkaloids - - -
Flavonoids - + +
Sapogenins - - +
Coumarins - + +
Quinones + + -
Sterols + + -

In many cases, the presence of flavonoids,
quinones and coumarins are associated with
antioxidant properties due to their role in several
human diseases where ROS could be involved. On
the other hand, these components could be presents
actively in inflammatory processes, in fact,
antioxidant/anti-inflammatory activity have been
related intrinsically to these chemical substances
(Takahashi and Shibamoto, 2008; Jensen et al.,
2008).
In order to evaluate the antioxidant efficiency
of plant extracts, the radical scavenging capacity
based in DPPH assay was determined and the results
are shown in Table 2. In this sense, the percentage of
inhibition of the DPPH radical varied from 41.1% for
the hexane extract to 92.4% for the EtOAc extract,
which represents a variation of approximately 2-fold
respect to the hexane extract. The EtOAc extract
showed the highest antioxidant activity index (AAI)
(Scherer and Teixeira-Godoy, 2009) following EtOH
extract and Hexane extract (1.9, 1.6 and 0.9,
respectively).
Table 2. Antioxidant capacity of the extracts of M. deppeana
based on DPPH test.
Extract DPPH
Inhibition
(%)
DPPH
IC
50
(g/mL)

AAI
*
Hexane 41.1 1.2 40.5 1.3 0.9
EtOAc 92.4 2.1 18.3 3.4 1.9
EtOH 70.1 1.8 22.0 0.4 1.6
Phenylbutazone 47.8 4.4 34.4 3.2 1.0
Rutin 93.5 1.1 4.8 0.1 7.4
Ascorbic acid** 100 0.6 0.1 61.2
Data expressed as mean SD (n=3). DPPH radical solution in
methanol (9 x 10
-5
M, 35.48 g/mL) using (33g/mL) of plant
extract and ascorbic acid and rutin (5 g/mL). * AAI = Final
concentration of DPPH (g.ml
-1
) / IC
50
(g.ml
-1
). ** No
significance differences in IC
50
were observed between this study
and results previously reported (Sharma and Bhat, 2009).



The DPPH free radical scavenger capacity of
the extracts was compared with well known
antioxidant (ascorbic acid and rutin) and anti-
inflammatory compound phenylbutazone. Despite the
fact that extracts had been a significant antioxidant
activity index (ca. 2, see Table 2), the obtained values
were lower than rutine and ascorbic acid (7.4, 61.2
respectively) but slightly higher than phenylbutazone
in the case of EtOAc extract (1.0 vs 1.9).
Taking into account the results in radical-
scavenging assay, it was expected that total phenol
content and reducing power had presented the same
behavior (Pako et al., 2009); the EtOH extract had
the highest concentration of total phenol (388.66.2
g GA/mL, Table 3) close to the EtOAc extract
(328.97.6 gGA/mL, Table 3). In the reducing
power assay the EtOAc extract had shown the highest
value over the EtOH extract (41.3% vs 29.0%
respectively, Table 3). These observations indicated a
linkage between phenolics concentration, reducing
power and antioxidant activity; on the other hand, in
all assays hexane extract always showed the lowest
values in each test (Table 2 and 3).

Table 3. Total phenolic content and power reducing of the
extracts of M. deppeana.
Extract Total phenols Reducing power
(%)
Hexane 20.6 1.5 5.3 0.4
EtOAc 328.9 7.6 41.3 0.5
EtOH 388.6 6.2 29.0 1.1
Data expressed as mean SD (n=3). Total phenols expressed in
mgs. of Gallic acid equivalent per g of sample (mgGAE/g).
Reducing power is expressed with respect to ascorbic acid
(33g/mL).

The propagation of free radical can bring
many adverse reactions leading to extensive tissue
damage. Lipids, proteins and DNA are very
susceptible to attack by free radical (Yu et al, 1992).
In this way, all antioxidant test evaluated in this work
showed that M. deppeana has a great amount of
antioxidant compounds that may offer resistance
against oxidative stress by scavenging the free
radicals and inhibiting lipid peroxidation.
Given the results in the antioxidant assays,
our attention now was focused in the evaluation of
the anti-inflammatory activity. In the ear edema
induced with topic application of TPA, the hexane
and EtOAc extracts produced the mayor inhibition of
the edema, 39 and 28%, respectively (Table 4). At 2
mg/ear dose, the inhibition edema was similar
between hexane extract and esculetin (39 and 38%
respectively, Table 4) but lower than indomethacin
(69%). In this test, ethanol extract did not show a
significant inhibition.
Table 4. Anti-inflammatory activity of M. deppeana on ear
edema induced with TPA.
Treatment Extract Dose
(mg/ear)
Edema
inhibition
(mg)
Inhibition
(%)
Control 14.4 0.6 0
M. deppeana
Hexane

2

8.8 0.6*

39
EtOAc
Ethanol
2
2
10.4 0.7
13.8 0.5
28
4
Indomethacin 2 4.5 0.5* 69
-sitosterol 1 9.9 0.5* 31
Esculetin 1 8.9 0.8* 38
Values are expressed as mean S. E. M. The inhibition (%) was
calculated respect to control group. * ANOVA-Dunnetts, p <
0.05, n = 6.

In this regard, inflammation induced by TPA
leads to protein kinase C activation, which is related
to activation of phospholipase A
2
with releases
arachidonic acid from membrane cells that is
metabolized to prostaglandins and leukotrienes
(Recio et al., 2000). The inhibition observed in
hexane extract could be related to this mechanism.
According to the obtained results in
preliminary anti-inflammatory test (TPA), the hexane
and EtOAc extract have the major inhibition;
however, we chosen the EtOAc, due to the low
solubility presents in hexane extract, in order to
confirm the biological effect with the paw edema
induced with the carrageenan model in mouse applied
by systemic route.
The EtOAc extract of M. deppeana (100 and
300 mg/kg dose) applied 30 min before of
carrageenan did not reduce the edema formation
significantly respect to the control group. In contrast,
the indomethacin to 5 mg/kg dose reduced the edema
formation in all time registered.
Surprisingly, paw edema decrease 43% with
lower doses of the extract (100 mg/Kg) only at the
first hour; this effect was similar to indomethacin
(Table 5).
In the acute inflammation induced by
carrageenan, levels of substance P in inflamed paw
increase within 15 minutes after induction, these
levels remained elevated during the first 2 h of
inflammation (Gilligan et al., 1994). Other mediators
of inflammation as serotonin, histamine and
bradykinin play important roles during the early


phase of carrageenan edema (Di Rosa et al., 1971).
According that the observed effect in EtOAc extract
could be related to some of these inflammation
mediators.
Table 5. Anti-inflammatory activity of EtOAc extract of M.
deppeana on paw edema produced with carrageenan test.
Time
(h)
Paw edema (mm) and % inhibition
Control Indomethacin
5 mg/kg
M. deppeana
100 mg/kg
M. deppeana
300 mg/kg
1 0.70.1 0.4 0.1*
(43%)
0.4 0.1*
(43%)
0.6 0.1
(14%)
3 1.10.1 0.7 0.1*
(36%)
1.0 0.1
(9%)
1.0 0.1
(9%)
5 1.20.1 0.7 0.1*
(42%)
1.0 0.1
(17%)
1.1 0.1
(8%)
7 1.00.1 0.7 0.1*
(30%)
1.1 0.2
(NE)
1.0 0.1
(NE)
Values are expressed as mean S.E.M. The inhibition (%) was
calculated respect to control group. * ANOVA-Dunnetts, p <
0.05, n = 8.

With these results, it was not possible to find
a correlation between antioxidant and anti-
inflammatory effects on the models used. In fact,
those observations indicate that responsible
compounds of both activities are not the same;
because in the anti-inflammatory topic test (TPA) the
hexanic extract was the best and the EtOAc extract
had the higher antioxidant activity. Further
investigations in the phytochemistry field and other
biological activities are being developed in our group
in order to get more information respect to action
mechanism and chemical composition.
CONCLUSION
Anti-inflammatory activity was observed in
the hexanic and EtOAc extracts of M. deppeana only
by topical application. The systemic administration of
low doses of EtOAc extract produced a similar
inhibition percent observed with indomethacine only
a short period of time. In the case of the antioxidant
activity, the EtOAc was the best extract evaluated
following by EtOH and Hexane extract; however, it
was not possible to find a correlation between the
anti-inflammatory and antioxidant activities with the
used assays. Finally, the biological activity of M.
deppeana observed in the polar extracts supports its
traditional use.
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2010 The Authors
Articulo Original | Original Article



Phytochemical and biological investigations of Caryocar brasiliense Camb
[Estudios fitoqumicos y biolgicos sobre Caryocar brasiliense Camb]
Jociani ASCARI

, Jacqueline Aparecida TAKAHASHI, Maria Amlia Diamantino BOAVENTURA*

Departamento de Qumica, Instituto de Cincias Exatas, Universidade Federal de Minas Gerais, Av. Antnio Carlos, 6627, CEP 31270-970. Belo
Horizonte MG, Brasil.

Abstract
Caryocar brasiliense epicarp and external mesocarpe were chemically and biologically evaluated. Fromthe phytochemical study, ethyl gallate, 5-
hydroxyfurfural, gallic acid, methyl shikimate, and mixtures of -D-fructopyranose and -D-fructofuranose, -and -D-glucose, lupeol and oleic acid and -
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 qumica y biologicamente. Del estudio fitoqumico, fueron
aislados e identificados por RMN (1D y 2D) galato de etilo, 5-hidroximetilfurfural, cido glico, chiquimato de metilo y mezclas de -D-fructopiranosa y -
D-fructofuranosa, - y -D-glucosa, lupeol y cido olico, -sitosterol, estigmasterol y cido olico. Fueron realizados ensayos de evaluacin del efecto anti-
oxidante, anti-microbiano y aleloptico para el extracto etanlico crudo, fraciones y compuestos puros, los cuales presentaron buena actividad.
Palavras Chave: Caryocar brasiliense Camb.; compuestos fenlicos; triterpenos; actividad anti-oxidante; actividad antimicrobiana; actividad aleloptica.

Recibido | Received: August, 9, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: October 26, 2009.
Publicado en Lnea | Published Online December 15, 2009.
Financiacin | Funding: CNPq, J AT and MADB for grants. FAPEMIG, for financial support.
This article must be cited as: . J ociani Ascari

, J acqueline Aparecida Takahashi, Maria Amlia, Diamantino Boaventura. 2010. Phytochemical and biological investigations of
Caryocar brasiliense Camb. Bol LatinoamCaribe 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
Caryocaraceae is a small botanic family
widely distributed in Central and South America. It is
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
are used as source of edible oils and in the preparation
of juices and liqueurs (Prance, 1990).
Caryocar brasiliense Camb., known in Brazil
as pequizeiro, deserves a distinguished position due to
the commercial, nutritional and gastronomic
importance of its fruit, named pequi. Pequi is a
spherical green fruit, presenting 1-4 segments. Its
structure is composed by an epicarp (very thin peel),
an external pulpy mesocarp, internal mesocarp (light-
yellow, pulpy, rich in oil, vitamins and proteins), that
involve a layer of thin and rigid endocarp spines
(approximately 2-5 mm large) and white nut (seed).
Together, internal mesocarp, needle endocarp and seed
constitute one segment (Damiani, 2006). The fruit is
used in Central Brazil culinary; fruits and leaves of C.
brasiliense are used in the folk medicine to treat cold,
edema, bronchitis, cough, burns and is also used as a
scaring agent (Vieira and Martins, 2000; Magalhes et
al., 1988).
From C. brasiliense leaves -amyrin, oleanoic
and ellagic acids and a mixture of -sitosterol and
stigmasterol were isolated. Edible pulp showed to be
rich in vitamins A, C, riboflavin, thiamin and
carotenoids (Azevedo and Rodriguez, 2004). The oil
extracted from C. brasiliense fruits presented several
biological properties such as anti-fungal (Passos et al.,
2002), against Trypanosoma cruzi (Herzog-Soares et
al., 2002) and Biomphalaria glabrata (Bezerra et al.,
2002) activities and also showed to be an effective
antioxidant agent (Paula-J unior et al., 2006).
The external mesocarp is the part that presents
the biggest dimension in the pequi fruit but it is thrown
away since this is a non edible part of the fruit. The
disposal of the external mesocarp generates a huge
amount of solid residues that could be used, adding
value to the plant. In this work, the phytochemical
study of this part of the fruit, together with the epicarp,
is presented, since no systematic phytochemical study
addressing these parts were found in the literature.
Twelve compounds were isolated, pure or as
constituents of mixtures. Pequi fruit ethanol extract,
fractions and some of the isolated compounds were
tested for their antioxidant activity using the radical
DPPH (1,1-diphenyl-2-picrylhydrazyl)
spectrophotometric assay, as well as for their
allelopatic activity, evaluating their effect on the
growth of radicule and shoot of Lactuca sativa
(lettuce).
General Experimental Procedures
Melting points were determined with a Kofler
hot plate apparatus and are uncorrected. Infrared (IR)
spectra were recorded with a Spectrum One with ATR-
IR, from Perkin Elmer. Nuclear Magnetic Resonance
(NMR) spectra were recorded in CD
3
OD, D
2
O, CDCl
3

and C
5
D
5
N, at room temperature, on Bruker Avance
DX-200 and DRX 400 MHz spectrometers.
Absorbance, in DPPH assay, was measured in a
Hitashi 2010 spectrophotometer. Silica gel Merck
(Darmstadt, Germany) 100-200 and 200-425 mesh
were used for column chromatography and silica gel
Merck 60G was used for thin layer chromatography.
Polyamide was purchased from Macherey Nagel
(Dren, Germany). Solvents PA and HPLC grade were
purchased from Vetec (Brazil) and Sigma Chemicals
Co (St. Louis, USA), respectively. BHT (2,6-di-tert-
butyl-4-methylphenol) and DPPH (1,1-diphenyl-2-
picrylhydrazyl), were also purchased from Sigma.
Plant Material
Fruits of Caryocar brasiliense were collected
in J anuary 2008, in Curvelo region, Minas Gerais,
Brazil, and identified by Dr. J oo Renato Stehmann. A
voucher specimen (No. 120826) was deposited at the
Herbarium of the Natural History Museum of UFMG,
Belo Horizonte, Minas Gerais, Brazil.
Extraction and Isolation
The fruit was open and the internal mesocarp
was discharged. The external mesocarp, together with
the epicarp was grinded in ethanol, using a liquefier,
and after 14 days at room temperature, the mixture
was filtered through a cotton plug followed by
Whatman filter paper. The extract was concentrated
with a rotatory evaporator and 256.0 g of ethanol
extract were obtained. A portion (130.0 g) of it was
chromatographed over polyamide column (106.0 g),
eluted with water, methanol and ethyl acetate pure and
mixtures of decreasing polarities. Twenty-eight
fractions of 100 mL each were collected, and reunited,
by silica gel TLC, in five groups of fractions (G-1 to
G-5). G-1 (72.6 g) was dissolved in water and
extracted with diethyl ether; subsequent evaporation of


the solvents afforded 6.0 g of ethereal fraction (G-1E)
and 66.6 g of insoluble residue (T1).
G-1E (6.0 g) was submitted to a silica gel
column chromatography, with CH
2
Cl
2
, CH
3
OH and
H
2
O, as eluent, either pure or in mixtures of increasing
polarity: 48 fractions of 50 mL each were collect, and
pooled in 11 groups of fractions. Group of fractions 2
(635.8 mg) was rechromatographed on silica gel
column with hexane, CH
2
Cl
2
and ethyl acetate as
eluent. A white solid precipitated from several
fractions and, by washing with CH
2
Cl
2
and filtration,
1.4 g of a pure compound were obtained. This
compound, pure by TLC, was identified as ethyl
gallate (1). The solvents from the filtration were
evaporated and the residue (150.6 mg) was
rechromatographed on silica gel column: group of
fractions 31-36 (41.0 mg) was found to be pure by
TLC and was identified as 5-hydroxy-methylfurfural
(2)
Group of fractions 6 (618.1 mg), from G-1E,
was submitted to silica gel column chromatography
(eluents: hexane, dichloromethane and ethyl acetate,
either pure or in mixtures of increasing polarity). From
group of fractions 2 (fractions 20-24), gallic acid (3)
was isolated (201.0 mg). From group of fractions 8,
from G-1E (594.6 mg), after silica gel column
chromatography, 8.0 mg of pure methyl shikimate (4)
were isolated.
Part of G-2 (9.5 g), from polyamide column,
was chromatographed over silica gel column, with
dichloromethane, ethyl acetate and methanol, as
eluent. Eight group of fractions were obtained and
group of fractions 2 (fractions 16-19) furnished
additional 1.03 g of ethyl gallate (1), by precipitation.
Group of fractions 4 (fractions 25-28, 2.1 g), from G-
2, after rechromatography over silica gel column, gave
146.0 mg of a mixture of -D-fructopyranose (5) and
-D-fructofuranose (6). Group of fractions 5 (fractions
29-30, 2.3 g), from G-2, was also submitted to
chromatography on silica gel and lead to the isolation
of a white solid (8.5 mg), that was identified as a
mixture of -D-glucose(7) and -D-glucose(8). Part
of G-3 (8.0 g), from polyamide column, after
submitted to silica gel column chromatography, using
the same eluent described, gave 9 groups of fractions.
Group of fractions 1 (0.3 g) was rechromatographed
on silica gel column (eluent hexane/acetone, either
pure or in mixtures of increasing polarity), and 23
fractions were obtained. From fractions 1-7 (0.27 g),
8.0 mg of lupeol (9) and oleic acid (10) mixture and
20.0 mg of -sitosterol (11), stigmasterol (12) and
oleic acid (10) mixture were isolated. From fractions
7-18, 1.15 g of ethyl gallate (1) were obtained by
filtration.
Spectrometric data
Compound 1 (Ethyl gallate): 3.58 g, 14.0 % yield.
white solid, m.p.: 170.2-172.1
o
C. IR (KBr, cm
-1
):
3446, 3290, 2973, 2933, 1704, 1615, 1533, 1469,

1309
and 1250.
1
H NMR (400 MHz, C
5
D
5
N)
H
(H, m, J in
Hz): 7.9 (H-2), 7.9 (H-6), 4.3 (H-8, q, 7.2); 1.2 (H-9, t,
7.2).
13
C NMR (100 MHz, C
5
D
5
N)
C
: 121.8 (C-1);
110.6 (C-2); 148.0 (C-3); 141.3 (C-4); 148.0 (C-5);
110.6 (C-6); 167.5 (C-7); 60.8 (C-8); 14.8 (C-9).
Compound 2 (5-Hydroxymethylfurfural): 41.0 mg,
0.02 % yield, viscous oil. IR (KBr, cm
-1
): 3369, 1658,
1582, 1519 and 1018.
1
H NMR (200 MHz, CDCl
3
)
H

(H, m, J in Hz): 7.2 (H-3, d, 3.4); 6.5 (H-4, d, 3.4); 9.6
(H-6, s); 4.7 (H-7, s); 2.7 (s, OH).
13
C NMR (50 MHz,
CDCl
3
)
C
: 152.3 (C-2); 123.0 (C-3); 110.0 (C-4);
160.8 (C-5); 177.8 (C-6); 57.6 (C-7).
Compound 3 (Gallic acid). 201.0 mg, 0.08 % yield.
white solid, m.p.: 245-248
o
C. IR (KBr, cm
-1
): 3500,
3450-2600, 1650, 1600, 1540 and 1350.
1
H NMR (400
MHz, CD
3
OD)
H
(H, m): 7.1 (H-2, H-6, s); 9.0 (OH,
s).
13
C NMR (100 MHz, CD
3
OD)
C
: 122.1 (C-1);
110.5 (C-2 and C-6); 146.5 (C-3 and C-5); 139.7 (C-
4); 170.6 (C-7);
Compound 4: Methyl shikimate (8.0 mg, 0.003 %
yield): white solid, m.p.: 108-109
o
C. IR (KBr, cm
-1
):
3303, 1715, 1657 and 1233.
1
H NMR (400 MHz,
CD
3
OD)
H
(H, m, J in Hz): 6.8 (H-2, m); 4.4 (H-3, br
s); 3.7 (H-4, dd, 6.8 and 3.6); 4.0 (H-5, m); 2.2 and 2.7
(H-6, m); 4.8 (H-8, s); 4.5 (OH, br s).
13
C NMR (100
MHz, CD
3
OD)
C
: 130.0 (C-1); 139.3 (C-2); 67.4 (C-
3); 72.7 (C-4); 68.6 (C-5); 31.6 (C-6); 168.9 (C-7);
52.5 (C-8).
Mixture 1 [-D-fructopyranose (5) and -D-
fructofuranose (6)]: 146 mg, 0.06 % yield. -D-
Frutopyranose (5).
1
H NMR (400 MHz, CD
3
OD)
H
(H, m): 3.5 and 3.7 (H-1, m); 3.8 (H-3, br s); 3.9
(H-4, br s); 4.0 (H-5, d); 3.7 and 4.0 (H-6, m).
13
C
NMR (100 MHz, CD
3
OD)
C
: 64.7 (C-1); 99.3 (C-2);
69.6 (C-3); 72.0 (C-4); 71.4 (C-5); 64.7 (C-6). -D-
fructofuranose (6).
1
H NMR (400 MHz, CD
3
OD)
H
(H, m): 3.6 and 3.7 (H-1, m); 4.0-4.1 (H-3, H-4 and
H-5, m); 3.5 and 3.8 (H-6, m).
13
C NMR (100 MHz,
CD
3
OD)
C
: 64.4 (C-1); 103.3 (C-2); 77.7 (C-3); 77.0
(C-4); 83.5 (C-5); 64.4 (C-6).
Mixture 2 [-D-glucose(7) and -D-glucose(8)]: 8.5
mg, 0.0033 % yield. -D-Glucose (7).
1
H NMR (400


MHz, D
2
O)
H
(H, m, J in Hz): 5.2 (H-1, d, 3.6); 3,4
(H-2, m); 3.7 (H-3, t, 9.6); 3.4 (H-4, t, 9.6); 3.73.8
(H-5, m); 3.7 (H-6, dd, 5.6 and 12.4).
13
C NMR (100
MHz, D
2
O)
C
: 92.1 (C-1); 71.5 (C-2); 72.8 (C-3);
69.6 (C-4); 71.3 (C-5); 60.6 (C-6). -D-Glucose (8).
1
H NMR (400 MHz, D
2
O)
H
(H, m, J in Hz): 4.6 (H-
1, d, 8.2); 3.2 (H-2, t, 8.8); 3.4-3.5 (H-3, H-4, H-5, m);
3.73.8 (H-6, m).
13
C NMR (100 MHz, D
2
O)
C
: 95.9
(C-1); 74.1 (C-2); 75.7 (C-3); 69.6 (C-4); 75.9 (C-5);
60.7 (C-6).
Mixture 3 [lupeol (9) and oleic acid (10)]: 8.0 mg,
0.003 % yield: Lupeol (9).
13
C NMR (100 MHz,
CDCl
3
)
C
: 38.4 (C-1); 27.5 (C-2); 79.4 (C-3); 39.0 (C-
4); 55.6 (C-5); 18.7 (C-6); 34.6 (C-7); 41.2 (C-8);
50.8 (C-9); 37.5 (C-10); 21.3 (C-11); 25.0 (C-12); 38.4
(C-13); 43.2 (C-14); 27.5 (C-15); 35.9 (C-16); 43.3
(C-17); 48.3 (C-18, C-19); 151.3 (C-20); 29.4 (C-21);
40.3 (C-22); 28.3 (C-23); 15.7 (C-24); 16.4 (C-25);
16.3 (C-26); 14.9 (C-27); 18.2 (C-28); 109.6 (C-29);
19.6 (C-30). Oleic acid (10)
13
C NMR (100 MHz,
CDCl
3
) : 178.7 (C-1); 34.1 (C-2); 25.0 (C-3); 29.6
(C-4); 29.4 (C-5); 29.5 (C-6); 29.6 (C-7); 27.8 (C-8);
130.3 (C-9); 130.1 (C-10); 27.7 (C-11); 29.7 (C-12);
29.9 (C-13); 29.8 (C-14 and C-15); 32.2 (C-16); 23.0
(C-17); 14.4 (C-18).
Mixture 4 [-sitosterol (11), stigmasterol (12) and
oleic acid (10)]: 20.0 mg, 0.0075 % yield. -Sitosterol
(11) and Stigmasterol (12).
13
C NMR (100 MHz,
CDCl
3
)
C
: 37.3 (C-1); 31.7 (C-2); 71.9 (C-3); 42.3
(C-4); 140.8 (C-5); 121.8 (C-6); 32.0 (C-7); 31.9 (C-
8); 50.2 (C-9); 36.2 (C-10); 21.1 (C-11); 39.7 (C-12);
42.3 (C-13); 56.9 (C-14); 24.4 (C-15); 28.9 (C-16);
56.0 (C-17); 12.1 (C-18); 19.4 (C-19); 36.2 (C-20 for
-sitosterol); 40.5 (C-20 for stigmasterol); 19.0 (C-21
for -sitosterol); 21.2 (C-21 for stigmasterol); 34.0 (C-
22 for -sitosterol); 138.3 (C-22 for stigmasterol); 26.1
(C-23 for -sitosterol); 129.8 (C-23 for stigmasterol);
45.9 (C-24 for -sitosterol);51.3 (C-24 for
stigmasterol); 29.2 (C-25 for -sitosterol); 31.9 (C-25
for stigmasterol); 19.0 (C-26); 19.8 (C-27 for -
sitosterol); 19.0 (C-27 for stigmasterol); 22.7 (C-28 for
-sitosterol); 25.4 (C-28 for stigmasterol); 12.0 (C-29
for -sitosterol); 12.3 (C-29 for stigmasterol).
DPPH Radical Scavenging Assay
Radical scavenging activities of extracts and
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
reference compound. Samples and BHT (750.0 L)
were prepared in triplicate for each concentration used
(1.0, 10.0 and 100.0 g/mL) and, to each flask, the
volume was adjusted to 2.0 mL by adding 1.5 mL of a
0.002 % p/v solution of DPPH in methanol. The
solutions were shaken vigorously and kept in the dark
for 30 min. The control was prepared as above without
any extract or substance. Absorbance (measured on a
Hitashi 2010 spectrophotometer) was measured at 517
nm and methanol was used for the baseline correction.
Radical scavenging activity was expressed as
the inhibition percentage and was calculated:
{(Abs
control
- Abs
sample
)/Abs
control
} x 100
where Abs
control
=absorbance of DPPH radical
in methanol and Abs
sample
=absorbance of the extracts
and pure substances in methanol +DPPH. Scavenging
activities were expressed in g/mL. IC
50
values
(g/mL) was expressed the concentration of sample
necessary to scavenge 50% of DPPH free radicals.
Allelopathic Bioassay
Lactuca sativa (cv Grand Rapids) seeds were
purchased from Isla Pak, RS, Brazil. All undersized
and damaged seeds were discarded. According to
metodology described by Vieira et al. (2005),
germination and growth were conducted in 100 mm
Petri dishes containing 9.0 cm sheet of Whatman no. 1
filter paper as suport. Then, 25 lettuce seeds were
placed per dish with 10 mL of a test (10
-4
, 10
-6
and 10
-8

M) or a control solution (without substance). All
solutions were prepared with deionized water and their
pH values [buffered with 10 mM 2-(N-morpholino)
ethanesulfonic acid, MES] were adjusted to 6.0 - 6.5
with NaOH solution. Concentrations lower than 10
-4

M were obtained by dilution series. All tests were
triplicated. Dishes were covered with Parafilm to
reduce evaporation and incubated in the dark at 25
o
C,
in a controlled-environment growth chamber, for 5
days. After this time, the number of germinated seeds
were counted (a seed was considered to be germinated
when the radicle was at least 0.2 mm long), the
lengths of radicle and shoots were measured (using a
paquimeter). During the measurement process, the
dishes were kept at 4
o
C to avoid subsequent growth.
The osmotic pressure values were measured on a
microsmometer and ranged between 30 and 38
mOsmolar.

26
25
27
O
OH
H
O
7
5
4 3
2
6
Ethyl galate (1) 5-Hydroxymethylfurfural (2) Galic acid (3)
COOCH
2
CH
3
OH
OH
HO
1
2
4
6
7
8 9
COOH
OH
OH
HO
1
2
4
6
7
O CH
2
OH
OH
O H
OH
OH
6
4
3
1
2
O
OH
OH
OH
CH
2
OH
CH
2
OH
2
3
4
5
6
1
Methyl shikimate (4) -D-Frutopyranose (5) -D-Frutofuranose (6)
1
O
9
10
Oleic acid (10) -Sitosterol (11) Stigmasterol (12)
H OH
OH
O
H
HO
HO
H
HO
H
H
OH
O
H
HO
HO
H
HO
H
H
OH
H
HO
3
1
29
30
19
28
17
27
14
26
8
10
25
23 24
20
-D-Glucose (7) -D-Glucose (8) Lupeol (9)

1
3
5 6
18
19
20
21
29
26
27
25
22
23
1
3 5
6
18
19
20
21 22
23
29
H
H H
H
H H
HO
HO
1
2
3
4
5
6
7
8
CO
2
CH
3
HO
HO
HO



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.
Inhibition (%) Extracts
/fractions
(1 g/mL)
(10 g/mL) (100 g/mL)
IC
50
(g/mL)
EE 0.0 63.7 94.4 28.36.6
G-1 5.4 51.3 96.7 9.36.3
G -1E 16.6 95.5 96.9 2,40.8
T1 3.6 16.9 91.7 22.22.5
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 13.02.8 202.8 nt
S. typhymurium 11.01.41 200.0 nt
E. coli 12.02.8 231.4 nt
C. freundi 11.00.0 220.7 nt
B. cereus 11.02.8 241.4 nt
L. monocytogenes 18.02.8 301.4 nt
P. aeruginosa 10.01.4 142.1 nt
C. albicans 0 nt 252.1
nt =not tested;
a
Values 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.
-30
-20
-10
0
10
20
30
40
Radical

L
e
n
g
t
h

(
%

c
o
n
t
r
o
l
)
1.0 mg/ mL
0.2 mg/ mL
0.04 mg/ mL
EE
G-1
EE G-1
Shoot


Figure 3. Effect of ethereal fraction (G1-E), ethereal insoluble
fraction (T1) and G-3, 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 (solution without
substance), zero representing an observed value identical to the
control, a positive value representing stimulation and a negative
value representing inhibition.
-75
-60
-45
-30
-15
0
15
30
1.0 mg/ mL
0.2 mg/ mL
0.04 mg/ mL
0.008 mg/ mL
0.0016 mg/ mL

R
a
d
i
c
a
l
G1-E T1
-30
-15
0
15
30
L
e
n
g
t
h

(
%

c
o
n
t
r
o
l
)

S
h
o
o
t
G1-E T1 G1-E T1 G-3
G1-E T1 G-3

Figure 4. Effect of ethyl galate (1), hydroxymethylfurfural (2)
and galic acid (3), isolated 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 (solution without
substance), zero representing an observed value identical to the
control, a positive value representing stimulation and a negative
value representing inhibition.
-40
-30
-20
-10
0
10
20
10
-3
M
10
-5
M
10
-7
M
L
e
n
g
t
h

(
%

c
o
n
t
r
o
l
)

Shoot
3 2 1
3 2
1
Radical

Data Analysis
The effect on germination and growth are
given as percent differences from control, and consist
of the differences (in cm) between mean values of
seeds with tested compounds and mean values for
control (seeds grown without addition of tested
compounds)/ mean values for control x 100. Thus,
zero represents the control, positive values represent
stimulation of the studied parameter and negative
values represent inhibition. The data were evaluated
by using Students t-test and the differences between
the experiment and control were significant at a value
of P 0.05. The inhibitory and stimulatory activities,
compared to those of the control, are shown in
Figures 2, 3 and 4.
Antimicrobial Bioassay
Samples were tested in duplicate by disc diffusion
method in agar with minor modifications (Lana et al.,
2003). Microorganisms used were Staphylococcus
aureus ATCC 29212, Salmonella typhimurium
ATCC 14028, Escherichia coli ATCC 25922,
Citrobacter freundi ATCC 8090, Bacillus cereus
ATCC 11778, Listeria monocytogenes ATCC 15313,
Pseudomonas aeruginosa ATCC 27853 and Candida
albicans ATCC 18804. For minimum inhibitory
concentration test, carried out in duplicate, samples
were serially diluted starting from concentration of
512 to 5.0 g/ml for each test microorganism. Tubes
were incubated for 18 hours at 35c. The results are
shown in Table 2
A total of twelve compounds were isolated
from the pequi fruit epicarp and external mesocarp
parts. Their structures were determined based on the
analysis of spectroscopic data, especially NMR, and
literature data comparison. The pure compounds were
identified as ethyl gallate (1) (Ceruks et al., 2007), 5-
hydroxymethylfurfural (2) (Kuo et al., 2002), gallic
acid (3) (Souza Filho

et al., 2006) and methyl
shikimate (4) (Liu et al., 2004; Adrio et al., 1997).
Additionally, mixtures containing -D-
fructopyranose (5) and -D-fructofuranose (6)
(Sobolev et al., 2003; Breitmaier and Voelter, 1987);
- and -D-glucose (7 and 8) (Collins and Ferrier,
1995); lupeol (9) (Mahato and Kundun, 1994) and
oleic acid (10) (Oliveira et al., 2006); -sitosterol
(11), stigmasterol (12) (Goulart et al., 1993) and oleic
acid (10) were obtained. The structures of these
isolated compounds were assigned on the basis of
spectroscopic data, including two-dimensional NMR
methods and by comparison of their spectral data
with values described in the literature. Structures of
compounds 1-12 can be found in Figure 1.
Ethyl gallate (1) was isolated on a very high
yield (14% from the crude ethanol extract, EE),
whereas compounds 2, 3 and 4 were isolated on a
very low yield from EE.



Ethyl gallate and gallic acid were previously
isolated from leaves of Caryocar microcarpum
(Kawanishi and Raffaud, 1986). Oleic acid, -
sitosterol and stigmasterol have already been isolated
from C. microcarpum and C. villosum (Kawanishi
and Raffaud, 1986; Marx et al., 1997).
Crude ethanol extract (EE), fractions G-1
obtained from polyamide column, G-1E (fraction
soluble in ethyl ether, obtained from G-1) and T1
(ethyl ether insoluble residue, obtained from G-1)
were evaluated for their antioxidant activities (Burda
and Oleszek, 2001). Table 1 shows the average
values for DPPH radical scavenging activity in each
tested concentration, and IC
50
values. G1-E presented
the higher IC
50
, that can be associated to the presence
of gallic acid and ethyl gallate in higher
concentrations than in EE and T1. At 100 g/mL, all
extracts tested were more active than BHT, the
reference compound.
The obtained results from allelopathic
evaluation, according to methodology described by
Vieira et al. (2005), of for crude ethanol extract,
fractions and compounds 1, 2 and 3 are shown in
Figures 2, 3 and 4. Both EE and G-1 showed
inhibitory activity on radical growth and stimulatory
activity on shoot growth of L. sativa. G-1 presented
biggest inhibitory effect at 1.0 mg/mL, and also
biggest stimulatory effect at 0.2 mg/mL (Figure 2).
Fractions G-1E, T1 e G-3 (Figure 3) were tested at
concentrations below 0.04 mg/mL, aimed to observe
more stimulation on shoot and radical growth
(Macas et al., 2000). However, only G1-E showed a
slight stimulatory effect on radical growth at 1.6 x 10
-
3
mg/mL and the inhibitory effects were bigger than
those presented for EE and G-1, at analogous
concentrations. On shoot, the best growth stimulatory
effect was observed for G-3, at 8.0 x 10
-3
mg/mL.
The effect of pure compounds 1, 2 and 3 on radicle
and shoot growth of L. sativa was mainly inhibitory
(Figure 4). Gallic acid (3) presented the biggest
inhibitory effect on radicle, at 10
-3
M, and also the
biggest stimulatory effect on shoot, at 10
-7
M.
Crude ethanol extract (EB) and fractions G-1
and G-1E were tested for their antimicrobial activity
(Lana et al., 2003), presenting inhibition zones of 7
mm. Only fraction G-1E presented expressive
activity towards the tested microorganisms,
according to Table 2. C. albicans was not affected by
this fraction in the tested concentration. The
minimum inhibitory concentration (MIC) found for
all tested microorganisms, except for S. typhimurium
and C. albicans was 512 g/mL.
CONCLUSIONS
This work pointed out for the possibility to
use the external mesocarp of pequi fruit as a rich
source of ethyl gallate, since this compound was
isolated in an expressive yield (14 % from crude
ethanol extract). The biological potential of this crude
extract and isolated compounds were also noticeable
(high IC
50
in the antioxidant evaluation of extracts,
and both inhibitory and stimulatory effect on radical
and shoot growth of L. sativa, respectively from
gallic acid), since plant material used is a residue
produced in large scale in Brazil.
AKNOWLEDGEMENTS
To CNPq, for J AT e MADB grants. To
FAPEMIG, for financial help.
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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]
Mara de las M. OLIVA
1
, Emilia BELTRAMINO
1
, Nicols GALLUCCI
1
, Carina CASERO
1
, Julio ZYGADLO
2
, Mirta
DEMO
1
1
Universidad Nacional de Ro Cuarto. Dpto de Microb e Inmunol. Ruta 36. Km 601. Rio Cuarto. Crdoba. Argentina.
2
Instituto
Multidisciplinario de Biologa Vegetal (IMBIV). Cat. Qca. Org. UNC. Crdoba. Argentina.
Abstract
Essential oils are known to exert antimicrobial activity. Differences in the chemical composition of theminfluence this activity. This work intends
to study the variability in the chemical composition and the antimicrobial activity of essential oils obtained fromplants of A. triphylla collected fromdifferent
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 fromLa Paz showed the highest citral/limonene relation and the best antimicrobial activity. Yeasts resulted to be the most sensitive microorganisms,
followed by the Grampositive 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 composicin qumica de los
aceites. El objetivo del presente trabajo fue estudiar la variabilidad en la composicin qumica 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 hidrodestilacin y analizados por
GC-MS. Los estudios antimicrobianos se llevaron a cabo por la tcnica de difusin en disco. Los aceites esenciales presentaron componentes mayoritarios
comunes y presentaron diferencias en la cantidad y calidad del resto de los componentes. La mayor relacin citral/limoneno y la mejor actividad
antimicrobiana fue obtenida con el aceite esencial de La Paz. Las levaduras resultaron ser los microorganismos ms sensibles, seguidos por las bacterias Gram
positivo. El anlisis estadstico mostr diferencias significativas en la actividad antimicrobiana de las distintas muestras. Las diferencias en la actividad
biolgica de cada aceite esencial podra 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 Versin Corregida | Accepted in Corrected Version: December 10, 2009.
Publicado en Lnea | Published Online: December 15, 2009.
Declaracin de intereses | Declaration of interests: Authors have no competing interests.
Financiacin | Funding: We are grateful to SECyT of Universidad Nacional de Ro Cuarto for financial support.
This article must be cited as: Mara de las M. Oliva, Emilia Beltramino, Nicols Gallucci, Carina Casero, J ulio Zygadlo, Mirta Demo. 2010. Antimicrobial activity of essential oils
of Aloysia triphylla (L`Her.) Britton fromdifferent regions of Argentina. Bol LatinoamCaribe 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


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


of Argentine and the relationship with the
antimicrobial activity.
Plant material
The plants of Aloysia triphylla were obtained
in March, 2005, from plants growing in farms
(plantations) located in different regions of
Argentina: from Crdoba Province: Ro Primero and
La Paz, from Salta Province: Las Vias, from
Mendoza Province, from San Luis Province and one
sample from Paraguay Republic.
Essential oils obtention
The EOs were obtained from dried vegetable
material, which was hydrodistilled in a Clevenger-
like apparatus. The oil obtained was dried with
anhydrous sodium sulphate and stored in the freeze
until analysis (De Feo et al., 1998).
Gas Chromatography-FID
The EO were analyzed with a Shimadzu GC-
R1A gas chromatograph equipped with a fused silica
column (30 m x 0.25 mm) coated with CBP-1. The
temperature of the column was programmed from
60
0
C to 240
0
C at 4
0
C/min. The injector and detector
temperatures were at 270
0
C. The gas carrier was He,
at a flow rate of 1 ml/min. Peak areas were measured
by electronic integration. The relative amounts of the
individual components are based on the peak areas
obtained, without FID response factor correction.
Programmed temperature retention index of the
compounds were determined relative to n-alkanes.
GC analysis was still performed using a column
Supelcowax-10 with the same conditions as
described above (Zunino et al., 1998).
Gas Chromatography-Mass Spectrometry
GC-MS analyses were performed on a Perkin
Elmer Q-910 using a 30 m x 0.25 mm capillary
column coated with CBP-1. The temperature of the
column and the injector were the same than those
from GC. The carrier gas was He, at a flow rate of
1ml/min. Mass spectra were recorded at 70 eV. The
oil components were identified by comparison of
their retention indices, mass spectra with those of
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
references mass spectra and retention indices of
volatile compounds. GC-MS analysis was still
performed using a column Supelcowax 10 with the
same conditions as describe above (Adams. 1989).
Microorganisms
The activity of the EOs was tested against the
following microorganisms: Gram positive bacteria:
Staphylococcus aureus ATCC 25923, Staphylococcus
epidermidis (milk), Micrococcus luteus ATCC 9341,
Enterococcus faecalis ATCC 29212, Bacillus cereus
(rice). Gram negative bacteria: Escherichia coli
(water), Proteus mirabilis (urine), Klebsiella
pneumoniae (composting of poultry), and
Pseudomonas aeruginosa (water). The yeasts
Candida albicans (mouth), Rhodotorula sp. (cereal)
and Hansenula sp. (cereal) were used in order to
probe antifungal activity.
Antimicrobial assays
Analysis of the antibacterial activity
The antibacterial studies were carried out
according to De Pooter et al., (1995). The paper disc
diffusion method was used to test the antimicrobial
activity. Tubes containing Triptein Soy Broth (TSB)
inoculated with the microorganisms were incubated
during 18 h, at 37 C. From these tubes ten-fold
dilutions were made, until an OD 0.04 (10
6
cfu/ml)
was reached. The antifungal activity was determined
with the same methodology but using that dilution
with an OD 0.4 (10
6
cfu/ml). The inoculumm
(200l) was spread over plates containing Mueller-
Hinton Agar and a paper filter disc (6mm) was
impregnated with 10l of the EO and placed on the
surface of the media. The plates were left 30 minutes
at room temperature to allow the diffusion of the oil
in the agar; then they were incubated at 37C during
24 hours. After this time the inhibition zone around
the disc was measured with a caliper. Discs with
gentamicine (10 g) were used as positive control.
Analysis of the antifungal activity
Antifungal experiments were performed in
the same way as those with bacteria using Sabouraud
Agar (SA) for the plates. Discs with anfotericine B (2
g/ml) were used as positive controls.


Minimum inhibitory concentration assay (MIC)
The minimum inhibitory concentration
(MIC) was performed according to the method
previously described by De Feo et al., (1998). It was
determined by two-fold dilutions of EOs in dimethyl
sulfoxide (DMSO), placing 10 l of each dilution on
a filter paper disc. The EOs concentration range was
from 900 mg/ml to 7.03 mg/ml. The discs were
placed on the surface of a TSA plate, previously
inoculated with 200 l of each inoculumm, and left at
room temperature to allow the diffusion of the oil.
Then they were incubated at 37
o
C during 24 h. After
this time the inhibition zone around the disc was
measured with a caliper. MIC was defined as the
lowest concentration that inhibited visible growth.
The MIC with fungus was determined in the same
way as with bacteria using Sabouraud Agar (SA) in
the plates. The negative control consisted in a paper
disc impregnated with 10 l of DMSO. The positive
control was a disc impregnated with the antibiotic
gentamicine (10 g) for bacteria. For yeasts,
anfotericine B (2 g/ml) was used.
Statistical analysis
All the experiments were performed in
duplicate and statistical analysis of the data were
performed using GraphPad Prism 4.0 program. A
probability value of p<0.05 was considered
statistically significant.
RESULTS
The chemical composition of the EOs from
Aloysia triphylla collected from Rio Primero, La Paz,
Las Vias, Mendoza, San Luis and Paraguay has
been investigated by means of gas chromatographic
techniques. The EOs average yield in all the samples
was 0.4% (w/v) and the components which were
commonly found in all the EOs samples were:
limonene, neral, geranial, spathulenol and
caryophyllene oxide, with intrinsic variations in the
quantity and quality of the resting terpenes in each
sample (Table 1). The samples analyzed showed that
the EOs from Mendoza had the biggest proportion of
neral and geranial, followed by La Paz. Las Vias
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 -
thujone. The EOs from Paraguay had spathulenol and
caryophyllene oxide while San Luis had D
germacrene and biciclogermacrene (Table1).
The relation between major terpenic
components of an EO has been proposed as a
criterion to identify chemotypes (Muoz-Collazos et.
al., 1993). In this work the rate between citral (neral
+geranial) and limonene has been calculated. The
EOs from La Paz showed the highest citral/limonene
relation (16.9) and Las Vias the least relation (0.73).
The rest of the EOs relations were located between
both of them (Table 1).
The antimicrobial activity of the EOs was
assayed against Gram positive bacteria, Gram
negative bacteria and yeasts. The yeasts resulted to be
the most sensitive microorganisms to the effect of the
EOs, followed by the Gram positive bacteria and
lastly the Gram negative ones. It is interesting to note
that the three yeasts were inhibited by all the EOs,
with average inhibition zones diameters of 22 mm.
(Table 2)
The EOs from La Paz showed the best
antimicrobial activity, inhibiting the growth of all
tested microorganisms, except P. aeruginosa. The
inhibition zones obtained with this oil for B. cereus
(38mm), M. luteus (33mm) and C. albicans are
remarkable. Mendoza`s EOs presented good
inhibition activity against microorganisms with
average diameters of 29 mm against B. cereus. The
EOs from Las Vias, Paraguay and San Luis showed
varied antimicrobial activity, with inhibition zones of
20 mm, 21 mm and 15 mm for B. cereus,
respectively. (Table 2)
Previously, it had been reported good
antimicrobial activity for the EOs obtained from Ro
Primero (Demo, et al. 2005). Taking into
consideration these previous studies, La Paz and Rio
Primero, both located in Crdoba Province, showed
the best inhibition spectrum of all the oily samples.
However there were differences in their chemical
composition.
B. cereus, S. aureus and M. luteus were the
most susceptible Gram positive bacteria to the EOs
action. The Gram negative bacteria E. coli and K.
pneumoniae were inhibited by the EOs from La Paz
and Las Vias.



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 Vias Paraguay Mendoza San Luis
thujene 0,8 tr 0,6 tr tr tr
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
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
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 - - -
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 - - - - -
copaene 0,8 0,5 tr 0,3 tr 0,8
bourbonene 1 1 0,6 0,8 tr 0,9
cubebene 0,2 tr 4,2 tr tr tr
cedrene 3,2 0,9 tr 2,8 tr 3,2
(E) caryophyllene 0,4 0,6 tr 0,7 tr 0,4
humulene 0,6 1,1 tr 0,5 tr 0,6
Cisdihydroterpineol - - tr - - -
curcumene (ar) 0,1 tr 3,3 tr tr tr
germacrene D 4,3 2,3 tr 5,3 tr 6,9
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
curcumene 0,1 0,4 0,5 tr tr 0,8
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 Vias Paraguay Mendoza San Luis
P(<0.05)
MO

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 Vias 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
microorganisms, being inhibited by al the EOs. La
Paz EOs showed the biggest inhibition zones, with
diameters of 39 mm against C. albicans and 34 mm
against Rhodotorula sp, while San Luis showed the
smallest one. The others EOs samples showed
different degrees of inhibition activity against the
yeasts.
In order to analize if the differences found in
the antimicrobial activity could be attributed to the
origin and composition of the EOs, the statistical
analysis was performed. This analysis showed
significative differences in the antimicrobial activity
of the EOs from all the places collected. These
variability was present in all the tested
microorganisms (Table 2).
For Gram positive bacteria the best values
were obtained with La Paz EOs with values of 28.1
mg/ml for S. aureus and S. epidermidis and 7 mg/ml
for M. luteus and B. cereus (Table 3). Gram negative
bacteria were inhibited by pure compounds with the
exception of P. mirabilis that presented a CIM of 450
mg/ml with La Paz EOs.
The best MIC values for C. albicans were
obtained with the EOs of Las Vias (MIC: 7 mg/ml)
and the best MIC values for Rhodotorula sp and
Hansenula sp were obtained with La Paz and
Paraguay EOs (MIC: 7 mg/ml) (Table 3).
DISCUSSION
Differences in the content and composition of
the EOs of A. triphylla have been reported
previously. In these reports, the EOs content ranged
between 0.2 and 1% on dry weight (Gil et al., 2007,
Sartoratto, et al. 2004). In this study, the average
yield obtained with the EOs samples (0.4% (w/v))
was included between these values.


There were chemical differences in the
quantity and quality of the EOs obtained from A.
triphylla collected from different places. However,
all the EOs samples shared the terpenic components
limonene, neral, geranial, espathulenol and
cariophyllene oxide, which were described by other
authors as the characteristically constituents of A.
triphylla EOs (Gil et al., 2007; Pascual et al., 2001;
Stashenko y col., 2003). Each EO sample showed
particular features in the rest of the constituents with
variability in the composition of each one. The EOs
content in vegetable species is influenced by genetic
material, culture conditions, environment, season,
extraction methods, crop and post-crop processing
(Gil et al., 2007; Hussain et al., 2008; Tampieri et al.;
2005). The culture conditions, kind of soil and
climate were particular for each one of the samples
collected. Ro Primero and La Paz are located in the
central region of Argentina, Mendoza and San Luis
are located in the central-west of Argentina while Las
Vias and Paraguay are in the north. Previous studies
on A. triphylla have reported that the production and
composition of the EOs vary according to the part of
the plant, the stage of development and the harvesting
locations of the plant (Gil et al., 2007). Other
investigations support the fact that the differences in
the quantity of the terpenes identified in an EO
obtained from a vegetable collected in the same zone
are due to the environment (Karaman, 2006; Merle et
al., 2004). It was found variability in the terpenic
composition of Ocimun basilicum EOs collected in
different seasons, concluding that the growing season
was affecting the chemical content (Hussain et al.,
2008). It is worth to mention that it was analyzed an
EO sample from La Paz collected the following year
(2006) in order to analyze if variations in the
chemical composition related to the harvesting year
happened and it was found the same terpenic
composition as in 2005 with variations in the quantity
of them. (Data not shown)
Citral and limonene were the major
components identified in these EOs. It has been
reported antimicrobial activity of both of them
(Demo y col. 2001, Di Pasqua et al., 2006; Wolken et
al., 2002). The individual activity of citral and
limonene could be suggesting that the relationship
between them could be determining the antimicrobial
activity. This justifies the study of the rate between
them and the possible relation between this value and
the antimicrobial activity of the EOs. In this work the
EO of A. triphylla from La Paz showed the biggest
rate citral/limonene and the best antimicrobial
activity, while Las Vias EO showed the least
terpenic relation and antimicrobial activity (Table 1
and 2). These results are suggesting that higher rates
between both compounds could be determining a
better antimicrobial ability and a broader microbial
spectrum. Some authors suggested that the
compounds present in the greatest proportions are not
necessarily responsible for the greatest share of the
total activity. The data on the activity of the essential
oils, in some cases are not compatible with those of
the pure constituents in higher percentages Thus, the
involvement of the less abundant constituents should
be considered (Cimanga et al., 2002; Tampieri et al.,
2005; Zygadlo and J uliani. 2000).
Mono and sesquiterpenes and the mixture
between them in the oil, could constitute a barrier to
microbial infections (Cowan. 1999; Lambert et al.,
2001; Tan et al., 1999; Vataru Nakamura et al.,
2004). The biotic and abiotic factors (environment,
specie, chemotype) of the places where specie are
collected have influence on the quantity and quality
of the terpenic composition of the EOs.
Consequently, differences in the EOs yielding and in
the biological activity are observed, being active
against bacteria and fungi or only one of them (De
Pooter et al., 1995; Hess et al., 2007; Zygadlo and
J uliani. 2000). A study made with O. basilicum EOs
with regard to seasonal variations, showed changes in
the antimicrobial activity, attributing these variations
to the different chemical composition of the oils.
Some earlier reports showed that the changes in
chemical composition of an EOs directly affected
their biological activities (Hussain et al., 2008). The
differences in the biological activity of each A.
triphylla EOs could be attributed to the quantity and
quality of the terpenic composition and the possible
associations between them. In addition, it could be
deduced that the antimicrobial activity is not only
dependent on the quality and quantity of the EOs but,
on the particular sensibility of each particular strain.
The inhibitory activity against
microorganisms responsible for human and plant
diseases of EOs from A. triphylla has been described
in other investigations (Demo et al., 2005; Pascual et
al., 2001; Sartoratto, et al. 2004). What is more,
Sartoratto, et al. 2004, described MIC values of A.
tryphilla (LHr.) Britton lower than
chloramphenicol, when it was used as the positive
control antibiotic, showing the antimicrobial potential
of this EO. (Sartoratto, et al. 2004) But, a comparison
of the chemical composition and the antimicrobial
activity of the EOs obtained from A. triphylla


collected in different regions, and the relation
between this two variables, have not been previously
reported.
It is difficult to compare results with others
reported in the literature because of the naturally
varying composition of EOs even in the same species
due to the presence of chemotypes, different harvest
times, different extraction methods, etc. Furthermore,
it is important to consider the different
microbiological tests utilized and the different
sensitivities of the strains (Tampieri et al., 2005). The
information described here clearly shows the
influence of the chemical composition of this EO on
the antimicrobial activity. This is also suggesting that
the genotypical composition of this specie should be
taken into consideration, in order to obtain the ideal
terpenic composition with the best antimicrobial
activity.
The production of EOs and their utilization as
potential therapeutic agents and natural food
preservants could be of economical value. However,
further investigations to establish how components
interact to provide the biological activities are needed
(Hussain et al., 2008).
ACKNOWLEDGEMENTS
Mara de las Mercedes Oliva, Mauro Nicols
Gallucci, Carina Caseros and J ulio Alberto Zygadlo
are researchers from CONICET. We are grateful to
SECyT of Universidad Nacional de Ro Cuarto for
financial support. We thanks to Plantadroga S. A.
Laboratories (Paraguay Republic sample) and Ing.
Alvarez Toledo (Salta sample) for the provission of
A. triphylla.
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27-29.


2010 The Authors
Articulo original | Original article



Estudio farmacobotnico de hojas, cortezas y leos 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 CORTADI
1
, Luisina ANDRIOLO
1
, Mara Noel CAMPAGNA
1
, Mara Laura MARTNEZ
1
, Osvaldo Di SAPIO
1
,
Adriana BROUSSALIS
2
, Martha GATTUSO
1
, Susana GATTUSO
1
.

1
Ctedra de Farmacobotnica. Departamento de Ciencias Biolgicas. Facultad de Ciencias Bioqumicas y Farmacuticas, UNR. Suipacha
531. S2002 LRK. Rosario. Repblica Argentina.
2
Ctedra de Farmacognosia. Facultad de Farmacia y Bioqumica, UBA. Junn 956, 2 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 leos 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 leos fromAlvaradoa 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. leo 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) est representada en Argentina, por 6 gneros y 8 especies, de las cuales 7 son nativas y una introducida; algunas
son utilizadas en medicina popular como tnicas, insecticidas y antiparasitarias. Las hojas se cortaron previa inclusin en parafina y se diafanizaron; las
cortezas y leos se cortaron longitudinal y transversalmente y se maceraron. Se colorearon con Safranina-Fast-green y Violeta de Cresyl. Las observaciones
se realizaron con microscopio ptico y microscopio electrnico de barrido. En este trabajo se han estudiado morfoanatmicamente las hojas, cortezas y leos
de Alvaradoa subovata, Picramnia parvifolia, Picramnia sellowii y Castela coccinea a fin de determinar caracteres diagnsticos para garantizar la identidad y
calidad de estos recursos. En hojas: presencia o ausencia de pelos glandulares, tipo de mesfilo, presencia de muclagos; en cortezas: la peridermis, tipos de
radios, cristales, elementos esclerenquimticos; en leos: tamao y disposicin de los poros, radios, y parnquima axial, entre otros. Se completa la
presentacin con fotomicrografas y claves con el objeto de brindar una adecuada diferenciacin entre las entidades.
Palabras Clave: Alvaradoa subovata; Picramnia parvifolia; Picramnia sellowii; Castela coccinea; Simaroubaceae; caracteres morfoanatmicos.

Recibido | Received: Agosto 4, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: Noviembre 30, 2009.
Publicado en Lnea | Published Online Diciembre 15 2009
Financiacin | Funding: ANPCyT, proyecto PICT BID/2007-1494 y SECYT BIO/2008-200.
This article must be cited as: Adriana Cortadi, Luisina Andriolo, Mara Noel Campagna, Mara Laura Martnez, Osvaldo Di Sapio, Adriana Broussalis, Martha Gattuso, Susana
Gattuso. 2010. Estudio farmacobotnico dehojas, cortezas y leos de Simaroubaceae (sensu lato) de Argentina. Parte I. Alvaradoa subovata Cronquist, Picramnia parvifolia Engl.,
Picramnia sellowii Planch. y Castela coccinea Griseb. Bol LatinoamCaribe Plant Med Aromat 9(1):38 55. {EPub 15 December 2009 }.
*Contactos | Contacts: sgattuso@fbioyf.unr.edu.ar
Cortadi et al. Caracteres morfoanatmicos de especies de Simaroubaceae Part I


INTRODUCCIN
La familia Simaroubaceae incluye 30 gneros y
200 especies de climas tropicales y subtropicales.
Los estudios taxonmicos referidos a las
Simaroubaceae de Amrica fueron abordados por
Cronquist (1944), Pirani (1987). Este mismo autor se
ocupo de las especies de Picramnia de Brasil en un
detallado trabajo (Pirani 1990).
En la Argentina las Simaroubaceae (sensu lato)
estn representadas por una especie introducida
Ailanthus altissima (Mill) Swingle y siete especies
nativas, Castela coccinea Griseb, Castela tweedii
Planch., Picrasma crenata. (Vell.) Engl., Simaba
glabra, Alvaradoa subovata Cronquist, Picramnia
parvifolia Engl. y Picramnia sellowii Planch.,
(Xifreda 1999, Xifreda y Seo, 2006).
Existen datos anatmicos de la madera, (Webber,
1936; ODonell, 1937; Heimsch 1942; Metcalfe y
Chalk 1950, 1972), Wheeler et al. (1986, 1989)
trabajaron con maderas duras, incluyendo al gnero
Picramnia confeccionaron una base de datos con
caracteres anatmicos; morfologa del polen
(Erdtman 1986; Moncada y Machado 1987, estudios
fitoqumicos (Stuhlfauth et al. 1985; Da Silva y
Gottlieb 1987; Simao et al. 1991) y de la estructura
del pericarpio (Fernando y Quinn 1992).
Las hojas, cortezas y leos de algunas de estas
especies de Simaroubaceae (sensu lato) son utilizadas
en la medicina popular para el tratamiento de
enfermedades de la piel: Picramnia sellowii
(Balderrama et al. 2001); vermicida: Picramnia
antidesma y Picrasma exelsa (Kelly y Dickinson
1985) y en trastornos del tracto gastrointestinal:
Ailanthus altissima (Martnez Crovetto, 1981;
Grieve, 1996); Castela tweedii y Picrasma crenata
(Martnez Crovetto, 1981, Simao et al. 1991) y
Castela coccinea (UMSA-Fundacin, 2002, Bourdy
et al. 2004). El leo de Alvaradoa amorphoides se
usa como tnico estomacal y la corteza como
antipruriginosa (Martinez, 1969; Toursarkissian,
1980).
Las Simaroubaceae se caracterizan por la
presencia en todos sus representantes de cuasinoides,
principios amargos, derivados de triterpenos
degradados y alcaloides derivados del triptofano, -
carbolines, canthinones, clasificados en los tipos
estructurales C1-C5, (Simao et al 1991). Los
cuasinoides de acuerdo a su estructura qumica se
dividen en 5 grupos C-18, C-19, C-20, C-22 y C-25.
muchos de stos tienen un amplio rango de
actividades biolgicas in vivo y/o in vitro. Los
cuasinoides de C-20 han sido extensamente
estudiados por sus propiedades biolgicas, dado que
se han encontrado algunos compuestos con marcada
actividad antileucmica (Guo et al. 2005); actividad
antitumoral (Cuendet y Pezzuto 2004, Fukamiya et
al. 1992, Guy Balansard y Hajime Ohigashi 2002,
Ogura et al. 1977, Toyota et al. 1990), el modo de
accin de los cuasinoides en sta actividad fue
encarada por Grieco et al. (1997) y Morre et al.
(1998) entre otros; actividad antimalarica, (Bourdy et
al. 2004, Castro et al. 2006, ONeill et al. 1986, 1987,
1988, Ajaiyeoba et al. 1999, Bertani et al. 2007);
actividad antiviral, (Grieco et al. 1997); actividad
antifeedant-insecticida (Lidert et al. 1987); actividad
antiparasitaria-antiprotozoaria (Camacho et al. 2003,
2004, Guy Balansard y Hajime Ohigashi 2002,
Moncayo 2003, Martnez et al. 2009); actividad
herbisttica-herbicida (Grieco et al. 1997, Siwajinda
et al 2001) y actividad citotxica (Anderson et al
1991, Cordell et al. 1993, Tagahara y Kuo-Hsiung
1993).
Actualmente, el anlisis microgrfico de aquellas
especies biolgicamente activas, es requisito
indispensable para las Farmacopeas herbarias de todo
el mundo, las cortezas y maderas de la flora de
Argentina han sido poco estudiadas, en este contexto,
el objetivo del presente trabajo es estudiar caracteres
morfoanatmicos de hojas, cortezas y leos de
Alvaradoa subovata, Picramnia parvifolia,
Picramnia sellowii y Castela coccinea, de modo de
tener no slo una identificacin morfolgica sino
tambin microgrfica de las mismas, permitiendo la
identificacin de cada especie cuando se encuentren
molturadas.
MATERIALES Y MTODOS
Materiales estudiados
Se emplearon materiales fresco y de los herbarios
BAA, MCNS, SF, SI y UNR, los que son citados
conforme a las siglas respectivas (Holmgren et al.,
1990). El material fresco fue coleccionado por los
autores, en las provincias de Misiones, Corrientes,
Chaco, Santiago del Estero, Tucumn, J ujuy, Salta y
Santa Fe. Para los estudios anatmicos fueron
utilizados siete ejemplares de cada especie.
Se utiliz material de herbario y fresco, a este se
lo fij en FAA (alcohol etlico 70, cido actico
glacial, formaldehdo y agua 50:5:30:15) y al
material de herbario se lo hidrat. Para el estudio de


las hojas, las lminas, se cortaron transversalmente en
la parte media de las mismas con micrtomo tipo
Minot, previa inclusin en parafina (Gattuso y
Gattuso 2002). Para el anlisis de las epidermis,
venacin y micrografa cuantitativa, las lminas
foliares se diafanizaron (Strittmatter, 1973) y se
determinaron los siguientes parmetros: ndice de
estomas (Salisbury, 1927), estomas por milmetro
cuadrado (Timmerman, 1927), ndice de empalizada
(Zorning y Weiss, 1925) y pelos simples por
milmetro cuadrado; para todas estas medidas se
trabaj con objetivo de 40x con un ocular de 10x.
Para la descripcin de la arquitectura foliar se utiliz
la terminologa de Hickey (1973) y para los pelos
phof et al. (1962). Las cortezas y leos se cortaron
con xiltomo en forma transversal y longitudinal
(radial y tangencial), previo ablandado con agua
hirviendo adicionada de una gotas de detergente
comercial; y se maceraron aplicando la tcnica de
Boodle (1916) y se midi con ocular micromtrico la
longitud y el dimetro de los elementos vasales como
as tambin longitud y latitud de fibras.
Las coloraciones empleadas fueron Safranina
alcohlica 80, Safranina-Fast-green (Strittmatter,
1979) y Violeta de Cresyl (Strittmatter, 1980). La
distribucin de los cristales de oxalato de calcio fue
analizada utilizando luz polarizada.
Para la descripcin de los elementos del leo se
us IAWA Committee (1989).
Las ilustraciones son originales y fueron
realizadas con microscopio ptico (MO) Nikon
Alphaphot, con tubo de dibujo. Para los esquemas se
sigui la simbologa de Metcalfe y Chalk (1950). Las
fotomicrografas fueron obtenidas con Microscopio
Carl Zeiss Axiolab y equipo fotogrfico MC 80. Los
detalles de las epidermis en superficie, cortezas y
leos fueron observados con microscopio electrnico
de barrido (MEB) Leitz AMR 1000; en el caso de la
lminas foliares las muestras fueron fijadas en
glutaraldehdo al 4 % deshidratadas en alcoholes
ascendentes, se aplic punto crtico y finalmente se
metaliz con oro paladio (OBrien y McCully, 1981).
Las observaciones morfolgicas se efectuaron con
microscopio estereoscpico Nikon SMZ-U
ZOOM1:1 con tubo de dibujo.
Para los caracteres anatmicos cuantitativos se
calcularon las medias aritmticas (x) con su
correspondiente desvo estndar sobre 10 campos.
Se acondicionaron los ejemplares para la
incorporacin a los herbarios UNR y de la Ctedra de
Farmacobotnica de la Facultad de Ciencias
Bioqumicas y Farmacuticas (UNR). Las
preparaciones histolgicas se hallan depositadas en la
histoteca de dicha Ctedra.
RESULTADOS
La identificacin de estas cuatro especies se
realiza por una combinacin de caracteres
morfolgicos y anatmicos de las partes utilizadas,
los que se presentan en cuadros, figuras y lminas.

DESCRIPCIN MACROSCPICA DE LAS
ESPECIES ESTUDIADAS
Alvaradoa subovata Cronquist. Brittonia 5 (2):134.
1944.
Sinnimos: Alvaradoa amorphoides var. puberulenta
Monach. Lilloa 8: 407. 1942.
Nombres vulgares: Pichi-blanco, Sacha ruda o
Chuquisaca.
Uso vernculo: El leo se utiliza como tnico
estomacal, y la corteza como antipurriginosa.
(Toursarkissian, 1980).
Hojas: compuestas, pecioladas, alternas,
imparipinnadas con 16-24 fololos, de 2-4 cm de
long. x 0,4-1 cm lat., oblongos, alternos, borde
entero, pice retuso, base aguda (Fig.1 A, a).
Corteza: color castao grisceo, escasas grietas
longitudinales, numerosas lenticelas. Fractura entera.
Leo: color amarillo plido, poros apenas visibles,
anillos pocos diferenciables, albura blanquecina.

Picramnia parvifolia Engl. Fl. Bras. 12(2): 242, pl.
49. 1874.
Nombres vulgares: No conocidos.
Uso vernculo: No se registra en la bibliografa
consultada ningn uso medicinal para esta especie.
imparipinnadas de 7-15 fololos, de 2-10 cm de long.
x 0,8-2 cm lat., alternos a subopuestos,
membranceos, oblongo-elptico a oblongo
lanceolados, margen poco revoluto, pice
subacuminado o ms raramente obtuso, base aguda y
asimtrica, a veces oval obtusa (Fig.1 B, b).
Corteza: color pardo rojizo superficie uniforme,
escasas lenticelas. Fractura entera.
Leo: color amarillo brillante, poros imperceptibles,
anillos apenas diferenciables, albura y duramen de
aspecto homogneo

Fig.1. A-V: Caracteres morfo-anatmicos foliares diferenciales. A-D: tipos de hojas, observacin con microscopio estereoscpico. A-C:
compuestas pinadas: A, de 16-24 fololos en A. subovata. B, de 7-15 fololos en P. parvifolia. C, de 9-15 fololos en P. sellowii. D, hoja
simple en C. coccinea. E-V: observacin 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 esquizgena en P. parvifolia. N-U, seccin transversal de las
lminas foliares: N-S, mesfilo dorsiventral: N-O, en A. subovata. P-Q, en P. parvifolia. R-S, P. sellowii. T-U, mesfilo cntrico en C.
coccinea. En todos, esquemas y detalle de lo indicado. Caracter anatmico foliar comn: a-c, D: arquitectura foliar camptdroma
broquiddroma, a-c fololos, 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 anatmicos foliares diferenciales. Observacin con MO. A-C, seccin transversal de la lmina foliar; A-B, mesfilo
dorsiventral: A, A. subovata; B, P. parvifolia. C, mesfilo cntrico en C. coccinea. D-G: vista superficial: D-E en P. sellowii: D, pelos
simples y glandulares (flechas), E, pelo glandular y estomas anomocticos. F, C. coccinea, pelos simples (flechas) y estomas. G, A. subovata,
pelos simples y estomas hundidos (flechas). ce, cavidad esquizgena; e, epidermis; ep, epidermis papilosa; et, estomas; h, hipodermis; m,
muclagos.



Fig.3. A-R: Caracteres anatmicos diferenciales de las cortezas. Observacin con MO. A-H: Representacin esquemtica de la corteza y
detalle de la seccin transversal de las clulas del sber: A y E: C. coccinea. B y F: A. subovata. C y G: P. parvifolia. D y H: P. sellowii.
Macerado de corteza: I: Clulas del sber en vista superficial. J: fibras libriformes. K: parnquima axial. L: clulas de radio. M y N: drusas,
cristales polidricos y estiloides de oxalato de calcio. O: braquiesclereidas. P: fibroesclereidas. Q: macroesclereidas. R: macroesclereidas
con inclusin de cristales polidricos. 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: Seccin transversal de los leos .a-d: detalle de los vasos. Observacin 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 anatmicos diferenciales y comunes del leo. Observacin con MEB. A-D: Seccin transversal del leo: A, A.
subovata poros solitarios con distribucin radial y oblicua. B, P. parvifolia poros solitarios. C, P. sellowii poros solitarios y mltiples
radiales. D, C. coccinea poros solitarios, geminados, racemiformes y mltiples radiales. E-F: seccin longitudinal: E, vasos. F, puntuaciones
areoladas alternas. G-I: Cristales de oxalato de calcio en corteza. Observacin con MEB. G, solitarios polidricos. H, drusa. I, estiloides
(flechas).




Picramnia sellowii Planch. London J. Bot. 5: 578.
1846.
Sinnimos:Picramnia sellowii fo. glabrescens
Chodat & Hassl. Bull. Herb. Boissier, sr.2, 3:800.
1903
Picramnia sellowii fo. hirsuta. Chodat & Hassl. Bull.
Herb. Boissier, sr.2, 3:800. 1903
Picramnia sellowii fo. intermedia Chodat y Hassl.
Bull. Herb. Boissier, sr.2, 3:800. 1903
Picramnia sellowii var. latifolia Engl. Fl. Bras.
12(2):232. 1874.
Picramnia sellowii subsp. spruceana (Engl.) Pirani.
Bol. Bot. Univ. Sao Paulo 12: 132. 1990.
Nombres vulgares: Cedrillo, Cedrillo-na y
Tarir
Uso vernculo: Se utiliza como alterante
(Toursarkissian, 1980).
imparipinnadas de 9-15 fololos, de 4-8 cm de long. x
1-3 cm lat., alternos a subopuestos en la misma hoja,
membranceos a cartceos, oval lanceolados, margen
poco revoluto, pice obtuso, base asimtrica obtusa o
raramente aguda (Fig.1 C, c).
Corteza: color gris pardusco; estras longitudinales y
transversales poco profundas que delimitan pequeas
placas. Abundantes lenticelas con importante reborde
y apertura en cruz. Fractura entera.
Leo: color amarillo pardusco, poros no visibles,
anillos apenas perceptibles, albura y duramen no
diferenciables.

Castela coccinea Griseb. Abh. Konigl. Ges. Wiss.
Gottingen 19: 107. 1874.
Sinnimo:Ximenia americana var. purbens Griseb.
Symb. Fl. Argent. 149. 1879.
Nombres vulgares: Espada, Granadillo,
Meloncillo, Mistol del zorro, Mistol del chivo,
Molle Colorado, Sacha meln y Sacha
meloncillo-
Uso vernculo: La corteza, hojas y raz se utilizan en
contra de la disentera, diarreas y fiebres
intermitentes; tambin se reconoce como tnico
gstrico (Xifreda y Seo, 2006)
Hojas: simples, cortamente pecioladas, alternas, de
1,5-3 cm de long. x 0,5-1 cm lat., oblongas
pinnatinervias, de margen liso, pice redondeado,
base cuneada (FIg.1 D, d).
Corteza: color pardo grisceo a pardo amarillento,
muy rugosa, ligeras estras longitudinales y
transversales cortas, lenticelas prominentes y notable
depsito de lquenes. Fractura fibrosa.
Leo: color castao amarillento, brillante, anillos de
crecimiento medianamente visibles, delimitados por
una franja pardusca. Porosidad difusa, con tendencia
a semicircular. Duramen y albura no diferenciables.
CARACTERES ANATMICOS COMUNES
Hojas
1. Lmina en vista superficial
Arquitectura: camptdroma, broquiddroma
(Fig.1 a, b, c, D) En todas las especies hay de 4 a 5
rdenes de venas, las secundarias son pinnadas,
mientras que las de orden superior son reticuladas.
Las venas marginales forman ojales cerrados con
terminaciones vasculares libres. Las areolas son
poligonales dispuestas al azar, coexistiendo
terminaciones vasculares simples y ramificadas y
rectas o curvas. La red vascular es de densidad
intermedia.
Epidermis: las clulas de la epidermis adaxial
(Fig.1 E, G, I, K) son ligeramente ms grandes que
las de la epidermis abaxial (Fig.1 F, H, J , L),
elongadas sobre los nervios. Los estomas estn
confinados a la epidermis abaxial. En ambas
epidermis se observan escasos pelos simples,
unicelulares, de paredes delgadas, que se ubican con
mayor densidad sobre las nervaduras, siendo la
longitud de los mismos diferente para cada especie
(Fig.1 F, H, J, K, L. Fig.2 D, F, G).
2. Lmina en corte transversal
Ambas epidermis son uniestratificadas. La hoja es
hipostomtica (Fig.1 N, P, R, T). En posicin
subepidrrmica la vena media se halla reforzada por
colnquima de tipo laminar del lado adaxial y
abaxial. El nervio medio est constituido por 5 a 7
haces vasculares colaterales abiertos dispuestos en
arco, acompaados por una vaina conspicua de fibras
(Fig.1 N, P, R, T).
Cortezas
Felodermis pluriestratificada.
Leos
De porosidad difusa (Fig.4 A-D), la
disposicin de los poros es variable segn las
especies. Los miembros de vasos son en su mayora
de contorno circular y se observan escasos elpticos,
presentan placa perforada simple y oblicua, con


apndices (Fig.4 a-d), las puntuaciones
intervasculares son areoladas, de disposicin alterna,
con abertura interna elptica e inclusa (Fig.5 E, F).
El parnquima axial se encuentra presente.
Los radios son no estratificados, las clulas
no dejan espacios intercelulares y son de paredes
medianamente engrosadas.
Las fibras son de dos tipo: 1- libriforme,
dispuestas de manera no estratificada, algunas de
ellas son septadas y 2- fibrotraqueidas de paredes
moderadamente engrosadas.
CARACTERES ANATOMICOS DIFERENCIALES
Tabla 1: Caracteres anatmicos diferenciales de las hojas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Alvaradoa subovata Picramnia parvifolia Picramnia sellowii Castela coccinea
Epidermis abaxial
Clulas de paredes
rectas (Fig.1 F. Fig.2
G)
Clulas de paredes ligeramente sinuosas (Fig.1 H, J , L. Fig.2 E, F)
Tipo
Anomocticos (Fig.1
F)
Anomo y paracticos
(Fig.1 H)
Anomocticos (Fig.1
J . Fig.2 E)
Anomocticos (Fig.1
L. Fig.2F) Estomas
Posicin
Hundidos (Fig.2 G) A nivel
V
i
s
t
a

s
u
p
e
r
f
i
c
i
a
l

Pelos glandulares
Ausentes
Pie uniseriado de
longitud variable y
cabeza pluricelular
(Fig.1 M; Fig.2 D, E)
Ausentes
Cutcula en ambas epidermis Delgada y lisa
Gruesa y lisa (Fig.2
C)
Uniestratificada
Epidermis adaxial
Papilosa (Fig.1 O.
Fig.2 A)
Con algunas clulas
septadas;
mucilaginosas (Fig.2
B)
Uniestratificada
Con hipodermis
mucilaginosa (Fig.1
T, U. Fig.2 C)
Dorsiventral (Fig.1 O, U, S)
Cntrica (Fig.1 T.
Fig.2 C)
2-3 hileras de
empalizada (Fig.1 O.
Fig.2 A)
1-2 hileras de empalizada. (Fig.1 Q, S. Fig.2 B)
2-3 hileras de
empalizada (Fig.1 U.
Fig.2 C)
Mesfilo

Parnquima esponjoso
sin muclagos
Parnquima esponjoso
con muclagos (Fig.2
B)
Parnquima esponjoso sin muclagos
Estructuras secretoras internas Ausentes
Cavidad esquizgena
con gomorresinas
(Fig.1 V; Fig.2 B)
Ausentes
Cristales de oxalato de calcio
Drusas y escasos
estiloides
Drusas y abundantes
cristales solitarios
Drusas
Drusas y escasos
cristales solitarios
Haces de nervios menores

La vaina
parenquimtica no
alcanza ambas
epidermis
La vaina
parenquimtica
alcanza ambas
epidermis (Fig.2 B)
La vaina parenquimtica no alcanza ambas
epidermis
S
e
c
c
i
n

t
r
a
n
s
v
e
r
s
a
l

Epidermis abaxial
Uniestratificada
papilosa
Uniestratificada
mucilaginosa
Uniestratificada



Tabla 2: Caracteres anatmicos diferenciales de las cortezas de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Clulas del sber
Dimensiones variables,
paredes con
engrosamiento mediano
y homogneo (Fig.3 F).
Paredes con
y heterogneo, las
basales lignificadas
(Fig.3 G).
Dimensiones variables,
paredes con
y heterogneo, las
basales en forma de U
o lignificadas (Fig.3 H).
Homogneas, no
aplanadas, de paredes
levemente engrosadas
(Fig.3 E).
Parnquima cortical Abundante (Fig3 B).
Escaso (Fig.3 C, D). Ausente (Fig.3 A).
Radios secundarios 1-3-seriados. 1-2-seriados, raro 3.
1-3-seriados con
ensanchamiento distal.
1-5-seriados con
ensanchamiento distal.
Elementos esclerenquimticos
Braquiesclereidas
aisladas en parnquima
cortical; macro y
braquiesclereidas en el
lmite con el floema
(Fig.3 B).
Fibroesclereidas del
sistema axial dispuestas
en forma de estratos
tangenciales
discontinuos (Fig.3 C).
Braquiesclereidas
aisladas en parnquima
cortical.
Macro, braqui y
fibroesclereidas en el
lmite con el floema.
Fibroesclereidas
aisladas en el floema
funcional (Fig.3 D).
Macro, braqui y
fibroesclereidas en el
lmite con el floema.
Fibras libriformes en el
floema funcional (Fig.3
A).
Cristales de oxalato de calcio
Drusas y escasos
polidricos en
parnquima cortical
(Fig.5 G, H). Estiloides
abundantes en el
parnquima axial (Fig.3
B. Fig.5 I).
Polidricos en serie de
parnquima septado
axial (Fig.3 C).
Drusas y polidricos
abundantes en
parnquima cortical y
escasos en el axial.
Polidricos incluidos en
algunas macro y
braquiesclereidas (Fig.3
D).
Drusas y escasos
polidricos en los radios
secundarios (Fig.3 A).
Tabla 3: Caracteres anatmicos diferenciales de los leos de A. subovata, P. parvifolia, P. sellowii y C. coccinea.
Crecimiento en anillos

Poco marcados.

De transicin gradual Marcados
Disposicin de poros
Solitarios y mltiples
con distribucin radial
y oblicua (Fig.4 A;
Fig.5 A).
Solitarios (Fig.4 B,
Fig.5 B).
Solitarios (escasos) y
mltiples radiales de 4-
10 (Fig.4 C Fig.5 C).
Solitarios, geminados,
racemiformes y
mltiples radiales
cortos de 3-5 (Fig.4 D;
Fig.5 D).
Elemento de vaso con apndice
Apndice pronunciado
(Fig.4, a)
Apndice
medianamente
pronunciado (Fig.4, b)
Apndice muy
pronunciado
(Fig.4, c)
Apndice apenas
pronunciado (Fig.4, d)
Parnquima axial
Escaso, difuso,
apotraqueal.
Escaso, metatraqueal,
de paredes engrosadas.
Casi ausente o apenas
metatraqueal.
Abundante paratraqueal
vasicntrico, aliforme o
en bandas diagonales.
Radios
1-3-seriados,
heterocelulares (Fig.4
A).
Uniseriados (Fig. 4 B, C), homocelulares, con
clulas erectas cuadrangulares y 2-3 seriados,
heterocelulares.
1-7 seriados (Fig.4 D),
homocelulares, con
abundantes cristales de
oxalato de calcio.



CARACTERES CUANTITATIVOS
Los datos cuantitativos obtenidos del estudio anatmico 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.

ndice de estomas 9,020,91 12,811,76 11,041,28 13,362,65
Estomas (mm
2
) 4,500,85 9,702,06 8,202,10 13,702,50
ndice de empalizada 6,631,00 7,331,54 4,050,66 11,452,03
Pelos simple (mm
2
) 3,500,85 2,900,88 3,300,67 3,000,82
Longitud pelos simple (m) 153,6026,61 160,0023,45
543,4057,66
a
203,2021,73
137,6020,80
a
90,603,27
Tabla 5: Resultados cuantitativos del estudio de las cortezas de A. subovata, P. parvifolia; P. sellowii y C. coccinea.
Longitud fibras (m)
359,6045,19
a
656,0069,64
338,4050,74
a
720,0061,51-
421,6047,71
a
876,00121,16-
439,3064,29
a
1082,00176,05
Latitud fibras (m) 22,403,37 24,805,90 24,804,54 11,402,32
Tabla 6: Resultados cuantitativos del estudio de los leos de A. subovata; P. parvifolia; P. sellowii y C. coccinea.

Vasos (mm
2
)
26,506,13 11,601,51 89,6010,71 104,2012,26
Dimetro miembros de vasos (m) 64,807,00 76,006,80
32,006,53
a
56,006,53
32,006,53
a
70,414,51
Longitud miembros de vasos (m)
194,4043,42
a
560,00132,09
422,4047,97 506,4054,00 173,9024,55
unicelulares
8,501,43 7,201,14 12,42,80 16,505,23
Altura radios (n
de clulas
pluricelulares
23,503,37 16,001,94 29,306,75 31,905,97
Longitud fibras xilares (m)
350,4043,79
a
627,2061,30
355,2044,01
a
780,8049,77
347,2041,86
a
736,0057,44
340,9049,83
a
864,0061,18
Latitud fibras xilares (m)
26,405,40 24,005,33 24,804,54 11,601,57
DISCUSIN Y CONCLUSIONES
Las especies de la familia Simaroubaceae (sensu
lato) aqu estudiadas, muestran caracteres morfo-
anatmicos de valor diagnstico que permiten su
reconocimiento cuando se encuentran molturadas,
que es la forma en que se utilizan y/o comercializan.
De las especies estudiadas, P. parvifolia no cuenta,
en la bibliografa consultada, con uso vernculo
reconocido; sin embargo por comunicaciones orales
se sabe que se la comercializa indistintamente con las
otras especies analizadas.
En la bibliografa consultada, las obras clsicas
sobre anatoma de los rganos vegetativos de las
Dicotiledneas (Solereder, 1908; Metcalfe y Chalk,
1972), no se hallaron las entidades estudiadas en el
presente trabajo, todas nativas de Argentina, si se
mencionan los gneros, Alvaradoa, Picramnia y
Castela.
El estudio morfoanatmico de las hojas ha
aportado los siguientes caracteres diagnsticos: pelos
glandulares presentes solo en P. sellowii acordando
con Solereder (1908), quien seala que no ocurren


pelos glandulares en todos los miembros de esta
familia. La estructura del mesfilo es en C. coccnea
cntrica y dorsiventral en las tres restantes,
coincidente con lo indicado por Metcalfe y Chalk
(1972) para el gnero Castela; en esta misma especie
se observa una hipodermis mucilaginosa, mientras
que en P. parvifolia los muclagos estn presentes en
las epidermis, carcter no mencionado por los autores
consultados para estos gneros. Con respecto a las
cavidades secretoras, solo se encontraron en los
parnquimas de la lmina de P. parvifolia; Metcalfe y
Chalk (1972) lo mencionan acompaando a los haces
vasculares en numerosos gneros, indicando que son
poco frecuentes en Picramnia y Alvaradoa, por lo
que coincidimos en esta apreciacin con respecto a
Alvaradoa, no as con Picramnia. Los cristales de
oxalato de calcio constituyen un caracteres valioso
para diferenciar a estas cuatro especies, se presentan
como cristales polidricos solitarios y drusas en C.
coccnea y P. parvifolia, como drusas y estiloides en
A. subovata, acordando con lo mencionado por
Solereder (1908) y Metcalfe y Chalk (1972), mientras
que solo se presentan drusas en P. sellowii. El tamao
y cantidad de los cristales vara segn las especies;
as, en A. subovata los estiloides son escasos, en P.
parvifolia son abundantes los cristales polidricos y
en C. coccinea abundan las drusas; estas
observaciones son indicadas tambin por Solereder
(1908) y Metcalfe y Chalk (1972). No es un dato
menor la extensin de la vaina parenquimtica de los
haces vasculares menores las que se presentan solo
en P. parvifolia.
Para cortezas, Solereder (1908) y Metcalfe y
Chalk (1972), slo se circunscriben a la especie
Ailanthus altisssima, en base a las descripciones de
Mller (1908). Di Sapio et al. (1997) analizan
caracteres anatmicos de cortezas y leos de
Ailanthus altisssima, Quassia amara y Castela
tweedii a fin de contribuir al conocimiento y
delimitacin de las citadas especies.
Existe uniformidad respecto del nmero de
peridermis excepto para Castella coccinea. Las
clulas del sber son muy variables en la constitucin
de sus paredes ya que alternan notablemente estratos
celulares con escaso o fuerte depsito 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 seccin transversal de la corteza muestra, en la
zona lmite entre el parnquima cortical y el floema
funcional en A. subovata, P. sellowii y C. coccinea
un anillo discontinuo constituido por tres tipos
celulares: macro, braqui y fibroesclereidas. En P.
sellowii, las macro y braquiesclereidas muestran
incrustaciones de cristales polidricos de oxalato de
calcio.
En la corteza interna de C. coccinea y P. sellowii
se observ un ensanchamiento distal de los radios
secundarios. Roth (1981) en sus estudios acerca de la
estructura anatmica de la corteza de rboles
tropicales, reconoce la disposicin de las fibras como
el criterio diagnstico de mayor valor, coincidiendo
con esta observacin, se ha encontrado que en C.
coccinea, hay abundantes estratos formados por
fibras libriformes, en P. parvifolia 3-5 estratos de
fibroesclereidas y en P. sellowii aislados estratos de
fibroesclereidas.
Los tipos de cristales de oxalato de calcio
coinciden con los mencionados para los caracteres
histofoliares; en cuanto a la distribucin es tpica para
cada especie, as se observan estiloides en el floema
funcional de A. subovata, para las dos especies de
Picramnia los cristales polidricos se ubican en el
floema funcional siendo ms abundantes en P.
parvifolia y en C. coccinea las drusas y los
polidricos, que son escasos, estn confinados a los
radios secundarios.
La caracterizacin de las cortezas encaradas en
este trabajo es la primera contribucin al respecto, ya
que las mismas no han sido descriptas con
anterioridad.
Del anlisis de los resultados obtenidos sobre las
estructuras de los leos de las especies aqu
estudiadas, se puede afirmar que existen escasas
analogas entre ellas, lo que permite lograr una
adecuada diferenciacin de los mismos. Asimismo, y
a pesar de algunas pequeas discrepancias
observadas, sobretodo en los caracteres referidos al
tamao y nmero de los elementos celulares,
originadas por las distintas condiciones climticas en
sus hbitat, coincidimos con las observaciones
realizadas por ODonell (1937), Heimsch (1942) y
Metcalfe y Chalk (1972), relativo a las descripciones
del leo de las especies de los gneros Alvaradoa,
Picramnia y Castela.


Clave para delimitar las especies por los caracteres morfo-anatmicos de la hoja

A- Hojas compuestas imparipinnadas.
B- 16-24 fololos.
C- Mesfilo dorsiventral, 2-3 hileras de clulas en empalizada, ambas epidermis papilosas,
parnquima esponjoso sin muclagos y sin cavidades secretoras, con drusas y estiloides de
oxalato de calcio. A. subovata
BB- 7-15 fololos.
C- Epidermis adx. y parnquima esponjoso con muclagos, cavidades secretoras, drusas y abundantes
cristales polidricos de oxalato de calcio. P. parvifolia
CC- Epidermis adx. y parnquima esponjoso sin muclagos, sin cavidades secretoras y con drusas de
oxalato de calcio. P. sellowii
AA- Hojas simples.
B- Mesfilo cntrico con 2-3 hileras de clulas en empalizada, parnquima esponjoso sin muclagos
y con drusas y escasos cristales solitarios de oxalato de calcio. Hipodermis mucilaginosa.
C. coccinea

Clave para delimitar las especies por los caracteres anatmicos de la corteza

A- Clulas del sber con engrosamiento homogneo.
B- Paredes de las clulas del sber levemente engrosadas, sin parnquima cortical, drusas de oxalato
de calcio en parnquima de radios secundarios. C. coccinea
BB- Paredes de las clulas del sber medianamente engrosadas, con abundante parnquima cortical
y estiloides de oxalato de calcio en el parnquima axial. A. subovata
AA- Clulas del sber con engrosamiento heterogneo.
B- Clulas basales del sber lignificadas, radios secundarios 1-2 seriados (raro 3), cristales
polidricos en parnquima septado axial. P. parvifolia
BB- Clulas basales del sber engrosadas en U con lignina, radios secundarios 1-3 seriados
con ensanchamiento distal, cristales polidricos de oxalato de calcio incluidos en
macro y braquiesclereidas. P. sellowii

Clave para delimitar las especies por los caracteres anatmicos del leo

A- Poros solitarios.
B- Parnquima axial metatraqueal escaso. P. parvifolia
AA- Poros solitarios y mltiples, radiales.
B- Poros geminados y racemiformes. Parnquima axial paratraqueal vasicntrico, aliforme o
en bandas diagonales. C. coccinea
BB- Parnquima axial metatraqueal, escaso.
C- Radios 1-3 seriados heterocelulares. A. subovata
CC-Radios uniseriados homocelulares y 2-3 seriados heterocelulares. P. sellowii



ANEXO 1:
Materiales estudiados

Alvaradoa subovata Cronquist

ARGENTINA. Prov. Jujuy: Dpto. Valle
Grande, R J ordn. 28-XII-1977, Kiesling y Ulibarry
1634 (SI); Dpto. Ledesma, camino de Ro A. Negra a
Abra de las Caas, 21-III-1972, Legname et al 9153y
9155 (MCNS), Ruta provincial n 83 Parque
Nacional Calilegua Km 8-10 margen norte del ro
San Lorenzo 10-12 Km al NW del pueblo Ledesma
700-900 msm, 20-VI-1998, Novara et al 11083
(MCNS); Dpto. Santa Brbara, Abra de los
Morteros. 26-I-1975, Zuloaga y Deginiani 278 (SI).
Prov. Salta: Dpto. Orn, ruta Prov.18 a 3-4 Km. del
Puente Internacional Argentina-Bolivia. 22 43S
6443W, 1-V-2003, Morrone et al 4536 (SI); Dpto.
Metan, Sierras de Metan Proyecto de Prospeccin
Minera Len Finca Cachari 18-20 Km al W de
Lumbreras 1334 msm, 25 12' 57,3" S 65 06' 44,0"
W, 10-IV-2006, Tolaba et al 4095 y 4116 (MCNS);
Dpto. Capital, Ro Vaqueros 5 Km al W del puente
Ruta 9, 1-III-1982; Novara et al 2546 (MCNS); Dpto.
Chicoana, Quebrada de Tilian 1300 msm, 13-II-
1982, Novara et al 2390 (MCNS), 1400 msm, 2-XII-
1983, Varela y Del Castillo 270 (MCNS), 5-XII-
1986, Palaci 837 (A) (MCNS), 4-XII-1986, Del
Castillo y Varela 970 (MCNS), 30-I-1987, Ortin 65
(MCNS); Quebrada de Scoipe Mal paso pasando
Caada de la Gotera, 19-XII-1980, Novara 1442
(MCNS), a 1 Km de Los Laureles 1400 msm, 27-III-
1984, Varela et al 503 (MCNS), pasando Los
Laureles 2-4 Km antes de Chorro Blanco 1600 msm,
15-XII-1989, Novara et al 9199 (MCNS), El
Infienillo pasando El Nogalar antes de arroyo La
Gotera 1600 msm, 16-XII-1995, Novara et al 10762
(MCNS),en la Quebrada Los Sauces al oeste de la
Ruta 33 Km 16 1400 msm, 30-XI-1997, Novara et al
10955 (MCNS), ruta 33 pasando 1-2 Km Los
Laureles antes de Finca Cerro Grande 1300-1400
msm, 28-II-2008, Lazaro et al 13074 (MCNS); Dpto.
La Via, 2-II-1951, Hunziker 1226 (SI). Prov.
Tucumn: Dpto. burruyac, Rio Nio al Alto de
Medina 1500msm. 7-VII-2007, Ponesa s/n (UNR)
.

Picramnia parvifolia Engl.

ARGENTINA. Prov. Misiones: Dpto. San
Pedro, Arroyo San Pedro, 1-XI-1958, Gamerro y
Toursarkissian 69 (SI). Dpto. Ober, Ober, 5-V-
2007, Oakley et al. s/n (UNR). Prov. Corrientes:
Dpto. Santo Tom, Santo Tom, 7-XII-1997, Mlgura
et al. 1589 (SI)

Picramnia sellowii Planch.

ARGENTINA. Prov.Formosa: s/f. J orgensen
3260 (SI). Prov. Misiones: Dpto. Gral. Belgrano,
Bernardo de Irigoyen 2 Km al S de B. de Irigoyen
sobre naciente del ro Pepir Guaz, 2616S
5338W, 15-X-1996, Morrone et al 1426, (SI); Dpto.
San Ignacio, Pen del Teyucar, 2716 S 5535W,
20-IX-2000, Mlgura et al 2157 (SI); Dpto. Posadas,
Posadas, 17-I-1930, Rodrguez 27 (SI). Prov. Chaco:
Dpto. Bermejo, arroyo Zapirn y ruta 11, 23-XI-
2007, Oakley et al. 53 (UNR). Prov. Corrientes:
Dpto. Loreto, Timb Paso, 5-V-45, Huidobor 2179
(SI).

Castela coccinea Griseb.

ARGENTINA. Prov. Jujuy: Dpto. San
Pedro, Cerritos de San Pedro a 700m, -X-1930,
Venturi 19546 (SI). Prov. Salta: Dpto. Oran, Ro
Piedras, 13-XI-1911, Rodriguez 76 (SI). Prov.
Chaco: Dpto. 1 de Mayo, Colonia Bentez, 30-IX-
1971, Martnez et al 9479 (BAA); Dpto. Chacabuco,
Charata, 24-I-2008, Oakley y Festa 73 (UNR); Dpto.
Resistencia, A 5 Km. al S de Resistencia, 24-IX-
1986, Pire 7707 (UNR). Prov. Santiago del Estero:
Dpto. Robles, Ventura, 11-I-2008, Oakley 66 (UNR).
Prov. Santa Fe: Dpto. Gral. Obligado, prximo a
Berna, 17-XII-2004, Pensiero 6943 (SF); Dpto. Vera,
Las Gamas, 12-I-1989, Pire 2641 (UNR); Dpto. 9 de
Julio, al O de Sta. Margarita, 25-IX-1981, Lewis
3255 (UNR); Ruta 35 El Cuadrado, 25-IX-1981, Pire
3262 (UNR); Dpto. San Javier, Ruta 11 arroyo El
Toba, 14-XII-1982, Pire 1177 (UNR).


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2010 The Authors




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-
aplicarsesi seobtieneel permiso del titular delos derechos deautor Nadaen estalicenciamenoscabao restringelos derechos morales del autor.
Composicin qumica y efecto antibacteriano del aceite esencial de
Aloysia triphylla (LHr.) Britton contra patgenos genito-urinarios
[Chemical composition and antibacterial effects of the essential oil of Aloysia triphylla against genito-
urinary pathogens]
Luis B. ROJAS
1
, Judith VELASCO
2
, Tulia DAZ
3
, Ricardo GIL OTAIZA
4
, Juan CARMONA
5
, Alfredo USUBILLAGA
1
.
1
Instituto de Investigaciones,
2
Departamento de Microbiologa y Parasitologa,
3
Departamento de Bioanlisis Clnico,
4
Ctedra de
Farmacognosia,
5
Jardn de Plantas Medicinales Dr. Lus Ruiz Tern, Facultad de Farmacia y Bioanlisis. Universidad de Los Andes,
Mrida - 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 fromurinary 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 hidrodestilacin de las partes areas 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 evaluacin de la
actividad antibacteriana del aceite esencial por el mtodo de difusin en agar con discos contra aislados clnicos de infecciones del tracto urinario y de
vaginosis bacteriana, revel inhibicin 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 g/ml. Este es el primer reporte sobre el efecto antibacteriano de este aceite
esencial contra patgenos genito-urinarios y la baja dosis observada, sugiere que este aceite podra ser usado en preparaciones farmacuticas 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: Cromatografa de Gases acoplada a Espectrometra de Masas ; CG: Cromatografa de Gases; CIM: Concentracin
Inhibitoria Mnima; ITU: Infeccin del Tracto Urinario ; VB: Vaginosis Bacteriana ; DMSO: Dimetilsulfxido ;BLEE: -Lactamasa de espectro
extensor; SV: Secrecin vaginal; URO: Urocultivo

Recibido | Received: September, 30, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: November 16, 2009.
Financiacin | Funding: This work was financed by el Consejo de Desarrollo Cientfico Humanstico y Tecnolgico (CDCHT Universidad de los Andes, Mrida-Venezuela)
This article must be cited as: Luis B. Rojas, J udith Velasco, Tulia Daz, Ricardo Gil Otaiza, J uan Carmona, Alfredo Usubillaga. 2009. Composicin qumica y efecto
antibacteriano del aceite esencial de Aloysia triphylla (LHr.) Britton contra patgenos genito-urinarios. Bol LatinoamCaribe Plant Med Aromat 9(1): 56 - 62. {EPub 15 Dec
2009}.
*Contactos | Contacts: Email: judithvelasco2005@yahoo.es ; Tel: 0058-2742403568; 00582742403410; Fax: 0058-2742403568.
Rojas et al. Composicin qumica del aceite esencial de A. triphylla y efecto contra patgenos genito-urinarios


INTRODUCCIN
La infeccin del tracto urinario (ITU) es la
invasin, colonizacin y multiplicacin de
microorganismos en el aparato urinario (Lizama et al.,
2005, Ochoa et al., 2005). Es la segunda causa de
infeccin ms frecuente en humanos, constituye un
importante problema de salud que afecta a millones de
personas cada ao (Echeverra et al., 2006).
La vaginosis bacteriana (VB) es una infeccin
cervicovaginal, que resulta de alteraciones en la flora
bacteriana aerobia y anaerobia, con disminucin del
nmero de bacilos de Dderlein con aparicin de un
flujo genital, lo cual se traduce en cambios
fisicoqumicos de las secreciones vaginales. Es una de
las dos infecciones genitales ms frecuentes en las
mujeres con vida sexual activa (Hillier & Holmes,
1999, Caballero et al., 2000, Mota et al., 2008).
A pesar de la amplia disponibilidad de
antibiticos para el tratamiento de las ITU y VB, en
ocasiones la sintomatologa no desaparece, por un
fenmeno creciente y que preocupa a la comunidad
mdica denominado resistencia bacteriana. Cada vez
es ms frecuente el aislamiento de bacterias
multirresistentes como las productoras de -Lactamasa
de espectro extenso (BLEE) (Beigi et al., 2004,
Karlowsky et al., 2006, Gobernado et al., 2007,
Colodner et al., 2008, Gagliotti et al., 2008, Guneysel
et al., 2009).
Frente a esta problemtica, se destaca el importante
papel que han desempeado las plantas como fuente
de sustancias con importante actividad
farmacoteraputica. En tal sentido, a la especie
Aloysia triphylla se le ha descrito actividad
antibacteriana y antifngica (Lpez et al., 2004,
Teixeira et al., 2005, 2007, Sartoratto et al., 2004,
Demo et al., 2005, Duarte 2006, Duarte et al., 2007).
Aloysia triphylla (LHr.) Britton (Aloysia
citriodora [Lam.] H.B.K., es originaria de Sudamrica
y pertenece a la familia Verbenaceae. La especie es
ampliamente utilizada por los pobladores de la zona
para resolver diversos problemas de salud. En trabajos
anteriores se reporta la presencia de un flavonoide, la
luteolin-7-diglucuronida y el compuesto fenlico
verbascoside (Carnat et al., 1995; Skaltsa & Shammas,
1988). La composicin del aceite esencial ha sido
estudiada en diversos pases, la mayora presentan
como componentes mayoritarios citral (neral +
geranial) y limoneno (Montes et al., 1973, zek et al.,
1996, Carnat et al., 1999, Sartoratto et al., 2004,
Gomes et al., 2006, Argyropoulou et al., 2007, ; Daz
et al., 2007, Di Leo Lira et al., 2008), un trabajo
realizado en Marruecos reporta 1,8-cineol y citral
(neral +geranial) (Bellakhdar & ldrissi, 1994), otro en
Argentina presenta a la Myrcenona y alfa-tujona
(Zygadlo et al., 1994) y por ltimo un estudio de
Colombia muestra citral (neral + geranial), nerol y
geraniol como componentes ms abundantes
(J aramillo et al., 2003).
Motivado al incremento de patgenos genito-
urinarios multirresistentes, en el presente estudio se
evalu la actividad antibacteriana del aceite esencial
de A. triphylla contra 9 aislados asociados a ITU, 11
aislados asociados a VB y 5 cepas de referencia por el
mtodo de difusin en agar con discos, adems se
describe su composicin qumica.
MATERIALES Y MTODOS
Material vegetal y extraccin
Las partes areas frescas (1 kg) de A. triphylla,
fueron recolectadas en junio de 2007 en la localidad de
Cacute, Parroquia perteneciente al Municipio Rangel
del Estado Mrida (Venezuela), una muestra (voucher
N J uan Carmona 788) se deposit en el Herbario
MERF Lus Ruiz Tern de la Facultad de Farmacia
y Bioanlisis de la Universidad de Los Andes. La
muestra fue sometida a hidrodestilada durante 4 horas,
usando un aparato tipo Clevenger. El aceite esencial
obtenido se sec sobre sulfato de sodio anhidro y se
guard bajo refrigeracin (4C) en la oscuridad.
Cromatografa de gases (CG)
Un cromatgrafo de gases, modelo Autosystem,
marca Perkin Elmer, equipado con un detector de
llama, fue usado para la determinacin de los ndices
de Kovts, mediante et comparacin con n-alcanos
(C
7
-C
22
). Los anlisis fueron llevados a cabo usando
una columna capilar HP-5 (30 m x 0,25 mm id, con
una pelcula de 0,25 m de espesor). La temperatura
del bloque de inyeccin fue de 200 C. La temperatura
del horno del GC fue programada como sigue:
temperatura inicial 60 C, se aplic luego un
incremento de 4 C.min
-1
hasta una temperatura final
de 260 C. Se us helio como gas portador con un flujo
de 1.0 ml/mina volumen constante (Rojas et al., 1999).
Cromatografa de gases acoplada a espectrometra
de masas (CG-EM)
El anlisis se realiz en un cromatgrafo de gases
Hewlett Packard 6890 serie II acoplado a un detector
de masas Hewlett Packard 5973, equipado con un


inyector automtico HP y una columna capilar HP-
5MS de 30 m de largo (0.25 mm id; con una pelcula
de 0,25 m de espesor). Se us una energa de
ionizacin de 70 eV. Se inyect una muestra de 1.0 l
de 2% de solucin de aceite en n-heptano con un
reparto de 100:1. La identificacin de los componentes
de la esencia fue establecida utilizando la base de
datos Wiley (6
a
Ed.) y comparacin de los ndices de
kovts obtenidos con datos publicados en la literatura
(Adams, 1995). La temperatura del inyector y el
programa de temperatura fueron los mismos usados
para la medicin de los ndices de Kovts.
Anlisis Microbiolgico
Cepas bacterianas
Se sometieron al estudio las cepas bacterianas
descritas en la Tabla N 3, que fueron proporcionadas
por el Laboratorio Clnico Lic. Ana Aparicio y por
el Departamento de Microbiologa y Parasitologa,
Facultad de Farmacia y Bioanlisis de la Universidad
de los Andes. Estas cepas fueron aisladas de pacientes
con ITU y VB en Mrida - Venezuela. Adems, se
incluyeron las cepas de referencia que se mencionan
en la Tabla N 2.
Mtodo antimicrobiano
La evaluacin de la actividad antibacteriana se
realiz de acuerdo al mtodo de difusin en agar con
discos descrita por Velasco et al., 2007 y la
concentracin inhibitoria mnima (CIM) solo contra
los microorganismos que mostraron zonas de
inhibicin. La CIM se determin por dilucin del
aceite con dimetilsulfxido (DMSO) con un rango de
10-160 g/ml, definida como la concentracin ms
baja capaz de inhibir el desarrollo bacteriano (CLSI,
2009). Los ensayos se realizaron por duplicado.
RESULTADOS Y DISCUSIN
Caracterizacin Fitoqumica
Por hidrodestilacin de las hojas frescas de la A.
triphylla se obtuvo un aceite esencial de color
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 bsqueda computarizada en la Librera
Wiley y por comparacin de los ndices de Kovts
obtenidos experimentalmente, con los reportados en la
literatura (Adams, 1995).
De los veintids componentes identificados, 2,7 %
resultaron ser compuestos alifticos insaturados (de
menos de 10 tomos de carbono), el 72,4 %
monoterpenos y el 22,3 % sesquiterpenos.
Los componentes mayoritarios identificados
fueron: geranial (27,3 %), neral (22,5 %), geraniol
(6,2%), bicyclogermacrene (5,2 %) y nerol (4,9 %).
Este aceite guarda estrecha relacin con el aceite
esencial de la misma especie estudiada en Colombia
[geranial (38,1 %), neral (19,3 %), geraniol (5,4%),
nerol (4,7 %) y biciclogermacreno (3,4 %)] (J aramillo
et al., 2003) y los componentes geranial y neral forman
parte de los aceites esenciales de A. triphylla
estudiadas en el mbito mundial (Montes et al., 1973;
Carnat et al., 1999; Sartoratto et al., 2004; Gomes et
al., 2006; Argyropoulou et al., 2007; Daz et al., 2007;
Di Leo Lira et al., 2008). Siendo Colombia un pas
vecino al nuestro, se puede pensar que ambas especies
estn relacionadas botnicamente.
Evaluacin de la actividad antibacteriana
El aceite de A. triphylla mostr fuerte actividad
antibacteriana contra las cepas de referencia,
observndose zonas de inhibicin con dimetros entre
7 y 19 mm y valores de CIM entre 10 a 60 g/mL, sin
embargo, no present actividad contra P. aeruginosa
ATCC 27853 (Tabla N 2). Tambin fue activo contra
todos los patgenos genito-urinarios probados, con
rangos de zona de inhibicin de 9 a 30 mm y valores
de CIM de 10 a 50 g/mL (Tabla N 3). En resumen,
la actividad del aceite contra todas las bacterias
ensayadas mostr valores de CIM de 10 a 60 g/mL y
la dosis de 20 g/mL predomin en el 52 % del total
de microorganismos.
La actividad biolgica observada en el presente
estudio coincide con los resultados obtenidos por
Demo et al., 2005 (Argentina), quienes observaron
actividad antibacteriana del aceite esencial de A.
triphylla contra S. aureus, E. faecalis, E. coli,
Klebsiella sp. y Proteus mirabilis y ausencia de
actividad frente aP. aeruginosa. Sin embargo, difiere
de los hallazgos de Sartoratto et al., 2004, en cuanto a
la actividad contra E. coli.


Tabla N1. Componentes voltiles del aceite esencial deAloysia triphylla en columna capilar HP-5

N Compuesto % IKcal
a

1 1 octen3ol 0,9 977
2 6metil5hepten2ona 1,8 985
3 Limoneno 3,0 1030
4 Eucaliptol 1,4 1033
5 cisocimeno 1,5 1049
6 linalool 0,5 1099
7 cis-crisantenol 0,9 1169
8 mentol 1,3 1187
9 -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 -cariofileno 2,1 1427
16 germacreno-D 4,6 1494
17 -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 composicin del aceite esencial fue determinada por comparacin de los espectros de masas de cada componente con la base de datos
Willey e ndice de Kovts calculado (IKcal).

Tabla 2: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra cepas de referencia
Zona de inhibicin (mm)*
Microorganismos Aceite
esencial
Control positivo
SAM VA GM AZT CAZ
CIM
g/ml
Staphylococcus aureus
(ATCC 6538)
18,750,25 40,750,25 20
Enterococcus faecalis
(ATCC 29212)
17,750,25 20,500,50 60
Escherichia coli
(ATCC 25922)
12,500,50 21,000,00 10
Klebsiella pneumoniae
(ATCC 23357)
7,000,00 29,500,50 30
Pseudomonas
aeruginosa (ATCC
27853)
NA 28,000,00 NP
* Zona de inhibicin en mm, discos 6 mm dimetro, media de dos ensayos, SAM: Sulbactam-Ampicilina(10g/10 g), VA:
Vancomicina(30 g), GM: Gentamicina(10 g), AZT: Aztreonam(30g), CAZ: Ceftazidima(30 g), NA: No activo, NP: No
probado. CIM: Concentracin inhibitoria mnima, rango de concentracin 10-160 g/ml.


Tabla 3: Actividad antibacteriana del aceite esencial de Aloysia triphylla contra patgenos genito-urinarios
Aceite esencial
Microorganismos
Zona de inhibicin
(mm)*
CIM
g/ml
Aislados clnicos de ITU:
Escherichia coli BLEE (URO 1) 9,750,25 20
Escherichia coli (URO 5) 11,000,00 20
Escherichia coli (URO 8) 9,500,50 20
Klebsiella ozaenae (URO 2) 9,000,00 30
Klebsiella ozaenae (URO 4) 10,750,25 10
Klebsiella ozaenae BLEE (URO 7) 9,000,00 50
Klebsiella ozaenae multirresistente (URO 11) 8,000,00 20
Enterobacter aerogenes (URO 6) 8,750,25 30
Proteus mirabilis (URO 9) 7,000,00 10
Aislados clnicos de SV:
Escherichia coli (SV 5) 12,750,25 20
Klebsiella ozaenae (SV 1) 9,750,25 20
Staphylococcus aureus (SV 4) 30,000,00 20
Enterococcus faecalis (SV 2) 14.500,50 20
Enterococcus faecalis (SV 3) 23,000,00 20
Enterococcus faecium (SV 7) 21,750,25 20
Enterococcus faecium (SV 9) 25,000,00 20
*Zona de inhibicin en mm, discos 6 mm dimetro, media de dos ensayos, BLEE: -Lactamasa de espectro extenso. ITU: Infecciones del
tracto urinario; SV: Secrecin vaginal. URO: Urocultivo.; CIM: Concentracin inhibitoria mnima, rango de concentracin 10-160 g/ml.



Recientemente, Duarte et al., 2007 en una
investigacin sobre efecto inhibitorio de aceites
esenciales obtenidos de varias plantas medicinales de
Brasil contra cepas de E. coli diarreognicas, sealan
actividad inhibitoria del aceite de A. triphylla contra
las diferentes categoras de esta especie bacteriana
con valores de CIM de 400 1000 g/ml.
De acuerdo a los valores de la CIM obtenidos se
observa que el aceite esencial de A. triphylla de
Mrida Venezuela fue ms activo que el aceite
esencial de esta especie obtenido en Brasil y
Argentina, contra E. faecium, S. aureus y E coli
(Sartoratto et al., 2004, Demo et al., 2005, Duarte
2006, Duarte et al., 2007). Esta diferencia se puede
atribuir a variacin en la composicin qumica del
aceite, la cual est influenciada por las condiciones
del medio ambiente donde se cultiva la planta, por
otra parte, difieren en algunos casos las metodologas
utilizadas para determinar la actividad antibacteriana.
El efecto antimicrobiano del aceite esencial de A.
triphylla observado en este estudio se podra atribuir
a los monoterpenos oxigenados, que son los
compuestos ms abundantes, geranial 27,3 % y neral
22,5 %, y a los cuales de manera individual se les ha
demostrado actividad contra bacterias grampositivas
y gramnegativas (Onawunmi et al., 1984, Kim et al.,
1995).
Otro hallazgo importante es la actividad
observada contra dos aislados productores de BLEE,
Escherichia coli BLEE (URO 1) y Klebsiella
ozaenae BLEE (URO 7), y una cepa de Klebsiella
ozaenae multirresistente (URO 11), con valores de
CIM de 20 g/ml, 50 g/ml y 20 g/ml,
respectivamente.
CONCLUSIN
Con base al efecto inhibitorio contra bacterias
multirresistentes y la baja dosis de CIM mostradas
por el aceite esencial de A. triphylla, se puede
concluir que este aceite es una fuente importante de
sustancias con alta actividad antibacteriana que
podra ser usado en preparaciones farmacuticas para
el tratamiento de infecciones causadas por estos
microorganismos. Por otra parte, este es el primer
reporte de actividad antibacteriana del aceite esencial
de A. triphylla contra patgenos genito-urinarios.
AGRADECIMIENTOS
Los autores agradecen al Consejo de Desarrollo
Cientfico, Humanstico y Tecnolgico (CDCHT
Universidad de los Andes, Mrida, Venezuela,
Programa ADG, Grupo de investigacin:
Bacteriologa Clnica) por el soporte financiero para
el desarrollo de esta investigacin.
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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 antiestafiloccica]
M. Golam KADER
1
, M. Rowshanul HABIB
1
, Farjana NIKKON
1
, Tanzima YEASMIN
1
,

Mohammad A. RASHID
2
, M.
Mukhlesur RAHMAN
3*
, Simon GIBBONS
3

1
Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh;
2
Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh;
3
Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy, University of London, 2939 Brunswick Square, London WC1N 1AX, UK
Abstract
A sesquiterpene, zederone (1), was isolated fromthe crude ethanolic extract of the rhizomes of Zingiber zerumbet (L.) Smith. It is the first time
report of isolation of this compound fromthe 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 minimuminhibitory concentration (MIC) values were found to be in the range
of 64-128 g/ml.

Keywords: Zingiber zerumbet; Zingiberaceae; Zederone; Antibacterial; Staphylococcus aureus

Resumen
Un sesquiterpeno, zederona (1), fue aislado del extracto crudo metanlico de los rizomas de Zingiber zerumbet (L.) Smith. Esta es la primera vez
que se reporta este compuesto en el gnero Zingiber. Su estructura se estableci tras una serie de anlisis espectrales incluyendo NMR de alto campo (1D y
2D) y espectrometra de masa. La actividad antibacteriana de este compuesto se determine frente a varias cepas multi-frmaco resistentes y meticilina-
resistentes Staphylococcus aureus (SA1199B, ATCC25923, XU212, RN4220 and EMRSA15) y las concentraciones inhibidoras mnimas se encontraron en el
rango de 64-128 g/ml.

Palabras Clave: Zingiber zerumbet; Zingiberaceae; Zederona; Actividad antibacteriana; Staphylococcus aureus.

Recibido | Received: December 01, 2009
Aceptado en Versin Corregida | Accepted in Corrected Version: December 15, 2009
Publicado en Lnea | Published Online: December 17, 2009
Financiacin | Funding: none declared
This article must be cited as:. M. GolamKader, 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 LatinoamCaribe 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.
Zingiberaceae) locally known as Bon Ada is a
vigorous ginger with leafy stems growing to about
1.2 m in height that is widely cultivated throughout
the tropics including Southeast Asia, Korea, India
and Bangladesh for its medicinal properties (Kritikar
and Basu, 1984; Fansworth and Bunyapraphatsara,
1992; Saadiah and Halijah, 1995). The most common
use of Z. zerumbet is as a shampoo and conditioner
for the hair (Burkill, 1966; Petard, 1986). Its
rhizomes are used in traditional medicine for the
treatment of inflammation, swelling, loss of appetite,
lumbago, diabetes, chest pain, rheumatic pains,
bronchitis, dyspepsia and sore throat (Burkill, 1966;
Kritikar and Basu, 1984; Farnsworth and
Bunyapraphatsara, 1992). The juice of the boiled
rhizomes has also been used in indigenous medicine
for worm infestation in children (Petard, 1986).
Previous phytochemical investigations on this plant
have revealed the isolation of several sesquiterpenes,
flavonoids and aromatic compounds (Matthes et al.,
1980; Masuda et al., 1991; Dai et al., 1997; J ang et
al, 2004; J ang et al, 2005). The volatile oil of the
rhizomes has been shown to contain zerumbone,
humulene, camprene -caryophyllene and camphene
(Hasnah, 1991; Srivastava et al., 2000; Bhuiyan et al.,
2009). Zerumbone, a predominant sesquiterpene from
this plant, has been studied intensively for its use as
anti-inflammatory, and in chemoprevention and
chemotherapy strategies (Dai et al., 1997; Kitayama
et al., 2001; Murakami et al., 1999; Murakami et al.,
2002; Tanaka et al., 2001). From the pharmacological
point of view, Z. zerumbet has been reported to
inhibit colon and lung carcinogenesis in mice (Kim et
al., 2009) and CXCL12-induced invasion of breast
and pancreatic tumor cells (Sung et al., 2008),
suppresses phorbol ester-induced expression of
multiple scavenger receptor genes in THP-1 human
monocytic cells and inhibits Epstein-Barr Virus
activation (Vimala et al., 1999; Murakami et al.,
1999). As a part of our research focused on bioactive
compounds from indigenous medicinal plants, we
here report the isolation and identification of a
compound (1) from the ethanolic extract of Zingiber
zerumbet (L.) Smith and its ant-staphylococcal
activity against a series of multi-drug resistant
(MDR) and methicillin resistant Staphylococcus
aureus strains: SA1199B, ATCC25923, XU212,
RN4220 and EMRSA15.
General experimental procedures
NMR spectra (both 1D and 2D) were
acquired on a Bruker Avance 500 MHz NMR (500
MHz for
1
H and 125 MHz for
13
C) spectrometer
using the residual solvent peak as internal standard.
The IR spectra were recorded with a Perkin-Elmer
Lambda spectrophotometer and the Mass spectra
were recorded with HRTOF-MS in positive mode.
The melting point were determined using a Digital
Melting point Apparatus (model IA 8103,
Electrothermal Engineering LTD, Southend-on-Sea,
Essex, UK) and are uncorrected. All solvents used in
this study were of analytical grade and purchased
from BDH and Merck.
Plant materials
Fresh rhizomes of Z. zerumbet were collected
from the hilly areas of Chittagong, Bangladesh in
October 2007 and identified by a taxonomist, Dr.
Mohammed Yusuf, BCSIR Laboratory, Chittagong,
Bangladesh where a voucher specimen (No. 1061) of
this collection has been maintained.
Extraction and isolation of compound (1)
The powdered plant material (800 g) of Z.
zerumbet was extracted with ethanol (4 L) in an
aspirator bottle for a week and then filtered and
concentrated by using a rotary evaporator at 45
o
C
under reduced pressure. The crude ethanol extract
(2.0 g) was subjected to column chromatography
over silica gel (Merck) eluting with petroleum ether
and ethyl acetate of increasing polarity and finally
with ethanol which yielded a total of 105 fractions.
Based on TLC analysis, fractions 15-18 were
combined together and then subjected to preparative
TLC using the solvent system petroleum ether and
ethyl acetate in a ratio of 20:1 to yield compound 1
(8.0 mg; white powder; R
f
0.45 in 5% EtOAc in
petroleum ether). The structure of the compound (1)
was confirmed by analysis of its IR,
1
H-NMR,
13
C-
NMR and TOF- Mass spectral data at The School of
Pharmacy, University of London, UK.
Compound 1: White powder; mp. 58-60
o
C;
IR (KBr): 1662, 1521, 1533, 1558, 914, 861 cm
-1
;
1
H-
NMR (500 MHz, CDCl
3
): 5.49 (1H, dd, J =12.0,
4.0 Hz , H-1), 2.25 (1H, m, H-2a), 2.53 (1H, m, H-
2b), 1.29 (1H, m, H-3a), 2.31 (1H, dt, J=13.0, 3.5
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
(3H, s, H-15);
13
C-NMR (125 MHz, CDCl
3
): 131.4
(C-1), 24.9 (C-2), 38.2 (C-3), 64.2 (C-4), 66.8 (C-5),
192.4 (C-6), 122.5 (C-7), 157.3 (C-8), 42.1 (C-9),
131.3 (C-10), 123.5 (C-11), 138.3 (C-12), 16.0 (C-
13), 15.4 (C-14), 10.5 (C-15); HR-TOF-ESIMS
[M+H]
+
m/z 247.0889.
Bacterial strains
The antibacterial assay was performed
against a panel of multi-drug and methicillin-
resistant strains of Staphylococcus aureus. S. aureus
standard strain ATCC 25923 and tetracycline-
resistant strain XU212 which possesses the TetK
tetracycline efflux protein provided by Dr Edet Udo
(Gibbons and Udo, 2000). Strain SA-1199B which
overexpresses the norA gene encoding the NorA
MDR efflux pump was provided by Professor Glenn
Kaatz (Kaatz et al., 1993). Strain RN4220 which
possesses the MsrA macrolide efflux protein was
provided by Dr J on Cove (Ross et al., 1989).
EMRSA-15 (Richardson and Reith, 1993) was the
generous gift of Dr Paul Stapleton.
Minimum inhibitory concentration (MIC) assay.
All five S. aureus strains were cultured on
nutrient agar (Oxoid) and incubated for 24 h at 37C
prior to MIC determination. Norfloxacin was
purchased from the Sigma Chemical Co. Mueller-
Hinton broth (MHB; Oxoid) was adjusted to contain
20 and 10 mg/l of Ca
2+
and Mg
2+
, respectively. An
inoculum density of 5 x 10
5
cfu of each of the test
organisms was prepared in normal saline (9 g/l) by
comparison with a 0.5 MacFarland standard. MHB
(125 l) was dispensed into 10 wells of a 96 well
microtitre plate (Nunc, 0.3 ml volume per well). A
stock solution of norfloxacin was prepared by
dissolving the antibiotic in DMSO (Sigma) and
dilution in MHB to give a final concentration of
0.625%. A DMSO control was included in all assays.
Compounds were serially diluted into each of
the wells followed by the addition of the bacterial
inoculum and the microtitre plate was incubated at
37C for 18 h. The MIC recorded as the lowest
concentration at which no growth was observed. This
was facilitated by the addition of 20 l of a 5 mg/ml
methanolic solution of 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT; Sigma) to
each of the wells and incubation for 20 minutes. A
blue colouration indicated bacterial growth (Shiu and
Gibbons, 2006).
Identification of compound (1)
Compound (1) was isolated as an amorphous
off-white powder from the crude ethanol extract of
the rhizomes of Zingiber zerumbet (L.) Smith. The
high-resolution TOFMS showed the pseudo
molecular ion, [M+H]
+
at m/z 247.0889,
corresponding to the molecular formula as C
15
H
18
O
3.
The
1
H-NMR spectrum of compound 1 showed a
downfield one proton singlet at 7.10, an olefinic
proton signal at 5.49, another methine signal at
3.81, three methyl proton resonances at 1.35,
1.61, 2.12, and methylene proton resonances between
1.29-3.77 integrating for four protons. The
13
C-
NMR spectrum displayed a total of 15 carbons while
the DEPT-135 and HMQC experiments indicated that
9 out of 15 carbons had attached protons. Analysis of
the
13
C and DEPT135 spectra allowed discernment of
the carbon resonances into three methyls (
C
10.5,
15.4, 16.0), three methylenes (
C
24.9, 38.2 and
42.1), three methines (
C
66.8, 131.4, 138.3), and six
quaternary carbons, including a carbonyl group (
C

192.4). The assignment of all carbons and protons
and thereby the structure of the compound was
resolved by 2D experiments, notably COSY, HMQC
and HMBC experiments. In the COSY experiment,
the olefinic proton at 5.49 (H-1;
C
131.4 from
HMQC) showed strong interaction with H
2
-2 protons
at 2.25 and 2.53 along with a weak connectivity
with the methyl singlet at 1.61 (H
3
-15). The
methylene protons (H
2
-2) showed coupling with H
2
-3
protons at 1.29 and 2.31 in the COSY experiment.
The presence of a methine (66.8;
H
3.81 from
HMQC) and a quaternary (64.2) in the
13
C
experiment confirmed the presence of an epoxide in
the molecule. The C-5 methine proton at 3.81
showed HMBC connectivities over two bonds (
2
J) to
a quaternary carbon at 64.2 (C-4) and the carbonyl
group at 192.4 (C-6). The methyl protons at 1.35 (
C

15.4 from HMQC) revealed
3
J connectivities to
38.2 (C-3) and

66.8 (C-5) and thereby confirmed its
linkage at C-4. The methylene protons at 3.70 and
3.77 (H
2
-9,
C
42.1 from HMQC) showed
2
J
correlations over two bonds to quaternary carbons at
131.3 (C-10) and 157.3 (C-8) and a
3
J interactions
to methyl (
C
16.0), olefinic (
C
131.4, C-1) and
quaternary (
C
122.5, C-7) carbons. Furthermore,
3
J
connectivities from the methyl protons at 1.61 to
131.4 (C-1) and 42.1 (C-9) confirmed its placement

at C-10. The remaining methyl resonance at

2.12
(
C
10.5 from HMQC) showed connectivities to
C

122.5 (C-7) and a methine carbon at 138.3 (C-12)
over three bonds. This allowed the placement of this
methyl group at C-11. Accordingly, compound 1 was
identified as zederone. Its NMR data were in
agreement with those reported previously (Hikino et
al., 1971). Although, it is a known natural product
reported before from a number species of the genus
Curcuma including C. zedoaria (Matthes et al.,
1980), C. aromatic (Phan and Phan 2000), C.
comosa (Qu et al., 2009), C. kwangsiensis ( Zhu et
al., 2009), C. ochrorhiza. (Sirat et al. 2009), C.
xanthorrhiza (Sukari et al., 2008), this is the first
isolation from the genus, Zingiber.
The compound was tested for the
antibacterial activity against a panel of five strains of
Staphylococcus aureus: SA1199B, ATCC25923,
XU212, RN4220 and EMRSA15 and showed weak
activity with minimum inhibitory concentration
(MIC) values in the range of 64-128g/ml (Table 2).
Figure 1. Structure of compound 1
O
14
O
O
1
2
3
4
5
6
7
8
9
10
12
11
13
15

Table 1
1
NMR (500 MHz),
13
C NMR (125 MHz) and HMBC data of compound 1 in CDCl
3.

HMBC Position
H

C

2
J
3
J
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
5.49, dd, J=12.0, 4.0 Hz
2.25, br d; 2.53, m
1.29, m; 2.31, dt, J=13.0, 3.5 Hz
-
3.81, s
-
-
-
3.69, d, J=16.0 Hz; 3.77, d, J=16.0 Hz
-
-
7.10, s
1.61, s
1.35, s
2.12, s
131.4
24.9
38.2
64.2
66.8
192.4
122.5
157.3
42.1
131.3
123.5
138.3
16.0
15.4
10.5
C-2, C-10
C-3
C-2, C-4
-
C-4, C-6
-
-
-
C-8, C-10
-
-
C-11
-
C-4
-
C-9, C-15
-
-
-
C-14
-
-
-
C-7, C-15
-
-
C-7, C-8, C-13
C-7, C-12
C-3, C-5
C-1, C-9
Table 2. MICs of 1 and standard antibiotic in g/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
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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 supercrtico de Saw Palmetto]
Mara 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 (hemogramand 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 desarroll una metodologa para examinar parmetros
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 das, para ensayar
parmetros generales homeostticos (hemograma y perfil bioqumico), as como caractersticas morfolgicas de tejidos involucrados en la respuesta a
xenobiticos (hgado, rin, bazo y ganglios linfticos). Ninguno de los parmetros analizados sufri cambios significativos durante el tratamiento, sugiriendo
que HiPower
presenta un buen perfil de seguridad durante el periodo evaluado. Este mtodo 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 Versin Corregida | Accepted in Corrected Version: December 16, 2009.
Financiacin | Funding: This work was supported by Madreselva Produccin y Desarrollo Ltda. (Santiago, Chile).
This article must be cited as: Names. 2009. Title. Bol LatinoamCaribe 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
Compounds occurring in plants have been
used for millennia to treat diseases; this use has set the
basis for the isolation of such compounds for
therapeutic use. Only recently, however, scientific
research on herbal extracts has begun to fully validate
their therapeutic use and safety. Most of this research
is focused on the therapeutic application of compounds
found in herbal extracts. For instance, extracts from
leaves of plants are rich in antioxidant compounds,
such as polyphenols, which can be useful for the
treatment of pathologies associated to oxidative stress
(e.g. neurodegenerative diseases). Nevertheless,
assessing the safety of herbal extracts is a challenging
endeavour, due to their complex nature. In contrast to
purified compounds, herbal extracts display a number
of different molecules with potentially very different
biological targets; interaction of herbal compounds
with their targets may lead to beneficial and/or toxic
consequences. Therefore, it becomes necessary to
develop a methodology that evaluates the safety
profile of herbal extracts containing more than one
active compound.
The present work is aimed to establish a
methodology for assessing the acute toxicity of a
particular herbal extract of Saw Palmetto (Serenoa
repens W. Bartram). Saw Palmetto belongs to the
Arecaceae (Palmae or Palmaceae) family. It is also
known as Serenoa serrulatum Schultes, Serenoa
serrulata (Michaux) Nichols, or Sabal serrulata
(Michaux) Nutall ex Schultes (Wilt et al., 2000)).
The therapeutic benefits of Saw Palmetto
appear to be related to its fruits, rich in natural oils.
Currently, Saw Palmetto extract is widely used for the
treatment of benign prostate hyperplasia (BPH,
prostate enlargement) (Bent et al., 2006; Gerber and
Fitzpatrick, 2004; Hizli and Uygur, 2007; Wilt et al.,
2000; Wilt et al., 1998). The type and relative
abundance of characteristic compounds (phenolic
compounds, phytosterols, flavonoids, polyprenoids,
sugars, fatty acids, etc.) of the oily extract from Saw
Palmetto depends on the extraction procedure.
Currently, the most used procedures to obtain Saw
Palmetto extracts are: 1) n-hexane (100%) extraction
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 CO
2
(Cristoni et al., 1997).
Although there are several reports regarding
the therapeutical applications of Saw Palmetto extracts
(Bent et al., 2006; Carraro et al., 1996; Lowe and
Fagelman, 2004; Ulbricht et al., 2006; Vallancien and
Pariente, 2001; Wilt et al., 2000), there are few
classical toxicological studies in animals. Only a small
amount of studies have shown significant toxic effects
of a particular Saw Palmetto extract (PC-SPES), which
has been removed from the market (de la Taille et al.,
2000; de la Taille et al., 1999; Small et al., 2000;
Sovak et al., 2002). More recent clinical studies in
humans have found no serious adverse effects of Saw
Palmetto extracts (Avins et al., 2008; Boyle et al.,
2004; Ernst, 2002; Hizli and Uygur, 2007; Willetts et
al., 2003). Some hepatotoxic effects have been
associated to a n-hexane-based Saw Palmetto extract
(Hamid et al., 1997); indeed, a classical toxicity study
in rats has been reported, showing an increase in
oxidative stress associated with the intake of 2X and
5X the maximum dose recommended for humans
(480l/day) for this type of preparation (Singh et al.,
2007). Nonetheless, no toxicological studies have been
reported using the supercritical fluid extract of Saw
Palmetto. Therefore, to perform the first acute
toxicological study in rats of a supercritical fluid Saw
Palmetto extract (commercial name HiPower
),
Sprague-Dawley rats were fed with 1X and 10X the
recommended dose for humans of this product (doses
adjusted for rat metabolism) for 10 days. Rat blood
samples obtained at different time intervals were
analyzed through hemogram and clinical chemistry
parameters; biopsies from liver, spleen, thymus and
lymphatic ganglia were also collected and analyzed for
possible morphological changes indicative of tissue
damage. This study showed no statistically significant
changes compared to normal ranges of any of the
parameters analyzed; in addition, no significant
changes were found in the morphological profile of the
studied tissues. This demonstrates the safety of this
particular Saw Palmetto extract at the doses and
periods tested.
The protocol followed for this study evaluates
general homeostasis and the function of liver and
kidney, organs responsible for xenobiotic
biotransformation and excretion, respectively.
Therefore, the type of study presented here may be
adopted as a method for assessing the acute toxicity of
herbal extracts.
Saw Palmetto extract HiPower

HiPower
is a supercritical fluid extract from Saw

Palmetto fruits and was provided by Madreselva


Desarrollo y Produccin Ltda. (Santiago, Chile). This
extract contains: 3.8% palmitic acid, 1.7% stearic acid,
14.8% oleic acid, 44.2% linoleic acid, and 34.3%
linolenic acid.
Animals
Male Sprague-Dawley rats (200-230g) were
maintained with normal pellet diet (Kimber), access to
water ad libitum, in a 12:12 light/dark cycle at 21C.
Animals were maintained in the vivarium of the
School of Chemical and Pharmaceutical Sciences
(Universidad de Chile, Santiago, Chile). All
procedures were performed according to the protocols
approved by the Ethical Committee of the institution
and to the Guide for the Care and Use of Laboratory
Animals (NRC, USA).
Treatment of rats
Dosage of HiPower
was calculated from the 1X

recommended dose for humans (480l/day), and
considering 70kg for normal human weight and that
rats display a 4-fold higher metabolic rate than
humans. Doses were diluted in sunflower oil for oral
delivery. Rats were distributed in 3 experimental
groups of 40 rats each: two groups that were given 28
(HiPower
1X group) and 280l (HiPower
10X
group) extract/Kg/day, in two oral (gavage) doses,
respectively, and a control group that received
sunflower oil alone in equivalent volumes (Sunflower
oil group). Following 2, 4, 6, 8, and 10 days of daily
treatment, 8 rats from each group were anaesthetized
with ether and sacrificed by exsanguination through
cardiac puncture. Blood samples were used for
hemogram and biochemical profile, and biopsies of
liver, thymus, spleen and lymphatic ganglia were
collected for histopathology analysis.
Hemogram study
It was performed by the Central Laboratory of the
Clinical Hospital of the Universidad de Chile
(Santiago, Chile). Parameters analyzed were red blood
cell count (RBC), hematocrit, haemoglobin, mean
corpuscular volume (MCV), mean corpuscular
haemoglobin (MCH), mean corpuscular haemoglobin
concentration (MCHC), and white blood cell,
lymphocyte and platelet counts.
Clinical chemistry study
It was performed by the Central Laboratory of the
Clinical Hospital of the Universidad de Chile
(Santiago, Chile). Parameters analyzed were: calcium,
phosphorus, glucose, blood ureic nitrogen, cholesterol,
total protein, albumin, total bilirubin, acid phosphatase
(AP), lactate dehydrogenase (LDH), and glutamyl
oxaloacetic transaminase (GOT).
Histopathology studies
These studies were performed by the Cyto-
Histopathology Laboratory BiopsCyt (Santiago,
Chile). Histomorphology studies were performed with
haematoxylin-eosin on the biopsies obtained at the
different intervals during treatment.
Statistical Analyses
Data presented in this study correspond to mean
95% confidence intervals (95% CI). Statistical
significances between means of hemogram and
clinical chemistry data were obtained through
ANOVA. Statistical significances between medians of
data and reference values were obtained through
Wilcoxon Signed Rank tests. Reference values for
adult Sprague-Dawley rats were obtained from
observations from the Institute of Public Health of
Chile (Uribe et al., 1995) or according to Lillie et al.,
1996. All statistical analyses were performed using
GraphPad Prism 5.0. Significances were set at 95%
confidence
RESULTS
1. Effect of HiPower
on hemogram
parameters of Sprague-Dawley rats.
Adult male Sprague-Dawley rats were treated
orally for up to 10 days with vehicle (Sunflower oil
group), 1X (HiPower
1X group), or 10X (HiPower

10X group) the recommended dose of HiPower
for
humans, as detailed in Material and Methods.
Following 2, 4, 6, 8, and 10 days of each treatment,
rats were sacrificed and blood samples were collected
for hemogram analysis.
As shown in Figure 1, HiPower
treatment did
not significantly alter the following parameters,
compared to normal ranges: red blood cells count
(RBC) or hematocrit (Figure 1); haemoglobin or mean
corpuscular haemoglobin (MCHC, Figure 2); white
blood cells count (WBC, Figure 3); and platelet count
(Figure 4). We also found complete absence of
bacilliform white cells, basophiles, eosinophiles,
metamyelocytes, or myelocytes (not shown).

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.

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.



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.

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.

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.



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.

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.



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.



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
particular cohorts displayed medians significantly
different from reference ranges in mean corpuscular
volume (MCV), mean corpuscular haemoglobin
(MCH) and the % lymphocytes (stars in Figures 2
and 3). Two-way ANOVA tests, however, showed
that these differences are not a reflection of
significant deviations compared to the vehicle
(p>0.05).
on clinical chemistry
parameters of Sprague-Dawley rats.
In addition to hemogram analysis, blood
samples were also analyzed for several clinical
chemistry parameters. As shown in Figures 5-8,
HiPower
treatment did not change the following

parameters: calcium or phosphorus (Figure 5); blood
levels of albumin or total proteins (Figure 6); blood
glucose, cholesterol, bilirubin, or urea nitrogen
(Figure 7); and the activity of alkaline phosphatase
(AP, Figure 8).
Wilcoxon Signed Rank tests showed that
some cohorts displayed medians significantly higher
than the upper limit of the normal range for aspartate
aminotransferase activity (stars in Figure 8, middle
panel). Two-way ANOVA tests, however, showed
that these differences are not a reflection of
significant deviations compared to the vehicle
(p>0.05). Also, most cohorts showed medians
significantly lower than the lower limit of the normal
range for lactate dehydrogenase (LDH) activity (stars
in Figure 8, bottom panel). Two-way ANOVA
analysis demonstrated that most of these deviations
were not significantly different from the vehicle
cohort, except for the cohorts treated with 1X
HiPower
(8 and 10 days of treatment, p<0.001) and

10X HiPower
(6 and 8 days of treatment, p<0.001).

on morphological
features of rat tissues.
We also obtained biopsies (hepatic, splenic,
thymic, and lymphatic) from each group at different
time intervals of the treatments. These biopsies were
used to perform histopathology analysis of tissue
sections with haematoxylin-eosin, as detailed in
Material and Methods.
All liver biopsies, regardless of the group or
the time of collection, showed a conserved
histological architecture (Figure 9A-C). We observed
classical structures, such as hepatic lobules with a
central vein (Figure 9A and 9B) and portal triads
constituted by blood vessels and bile ducts (Figure
9C). Hepatocytes displayed a conserved structure,
without noticeable degeneration or necrosis. Most
liver biopsies displayed small hematopoietic loci,
with the same occurrence regardless of the cohort
analyzed or the collection time. Liver parenchyma
displayed no mitosis count per mm
2
in most samples,
with a mitosis count of 1-4 per mm
2
in a few
biopsies, regardless of the cohort or collection time.
Spleen biopsies had also normal architecture
of red or white pulp, regardless of the cohort or
treatment interval analyzed (Figure 9D-F). All
samples displayed hematopoietic activity and a
mitotic count in germinative centers of 0-4 per mm
2
,
regardless of the treatment or collection interval
(Figures 9D and 9E). Vehicle cohort displayed
virtually no apoptotic bodies, while 1X HiPower

and 10X HiPower
cohorts showed some apoptotic

bodies in seldom cases (Figure 9F).
Lymphatic ganglia biopsies from all three
cohorts displayed conserved histological structures
(Figure 9G-I). We found lymphocytic elements and
reticulo-histocytic cells in the lymphoid tissue. Most
biopsies, regardless of the cohort or collection time,
displayed secondary follicles with germinative
centers (Figure 9G). With low frequency, some
sinusal edema was found regardless of the cohort or
the collection time. At the level of lymphoid follicles,
some apoptotic activity was evidenced by the
occurrence of apoptotic bodies in the cytoplasm of
reticulo-histocytic cells (Figures 9H and 9I). The
abundance of apoptotic bodies, however, was not
associated to a cohort or collection time. On the other
hand, at the level of germinative center, we observed
a mitotic count of 2-25 per mm
2
. Variability of
mitotic count was unchanged among cohorts or
collection times.
Histological structures of the thymus were
conserved in all biopsies tested (Figure 9J -L),
regardless of the cohort or the collection time, with
normal architectures of cortex and medulla (Figure
9J ). Normal lymphoid and epithelial (Hassall bodies,
Figure 9K) components were also visualized.
Apoptotic scores did not reveal significant
differences between cohorts or collection times
(Figure 9L). Mitotic count in germinative centers was
1-2 per mm
2
, displaying no significant differences
between cohorts or collection times.


DISCUSSION
Our study was aimed to develop a
methodology that allows the evaluation of the safety
profile of herbal extracts, using a supercritical fluid
Saw Palmetto extract (HiPower
) as a start point. To
this end, we evaluated possible changes in classical
hemogram and clinical chemistry parameters (as a
measure of general homeostasis) from Sprague-
Dawley rats fed for 10 days with two different doses
of HiPower
, compared with rats fed with vehicle

(sunflower oil). We also evaluated potential gross
morphological alterations in key organs involved in
xenobiotic metabolism (to address possible toxicity
associated with xenobiotic biotransformation) and
immune system (to address potential toxic
immunological response). With this purpose, blood
samples and tissue biopsies were collected from rats
at different intervals during the 10-day treatment.
Essentially none of the parameters tested
underwent any statistically significant changes
following this treatment, as assessed by Wilcoxon
Signed Rank test. Although these analyses showed
some deviations of medians from the normal ranges,
most of such deviations were not significantly
different from those of the vehicle cohort. This may
be the reflection of parameters with high inter-
individual dispersion. Noteworthy, lactate
dehydrogenase activity values were lower than the
lower limit of normal ranges for this parameter in all
cohorts, especially at the beginning of the treatment.
Although this parameter reached the normal interval
in the case of the vehicle cohort, it remained under
the lower limit for the 1X HiPower
and 10X
HiPower
cohorts. Since lactate dehydrogenase

activity is a classical marker for liver damage, these
data suggest that HiPower
may display
hepatoprotective activity.
In summary, hemogram and clinical
chemistry data show that the treatment of Sprague-
Dawley rats with a supercritical fluid Saw Palmetto
extract led to undetectable alterations of red blood
cells integrity, haematopoiesis, immune or
inflammatory responses, coagulation, metabolism of
glucose, nucleic acids, proteins, fatty acids or
steroids, and function of liver, spleen, kidney or
parathyroid gland. The latter was also corroborated
by the absence of gross morphological changes in
liver, spleen, lymphatic ganglia or thymus. Therefore,
we concluded that this acute treatment did not lead to
any classic toxicological outcomes, even with doses
that differ in one order of magnitude.
It has been shown that some Saw Palmetto
extracts can be toxic in acute toxicological studies
(Hamid et al., 1997; Singh et al., 2007). On the other
hand, several studies have demonstrated the safety of
these extracts in humans (Avins et al., 2008; Boyle et
al., 2004; Ernst, 2002; Hizli and Uygur, 2007;
Willetts et al., 2003). It is very difficult to address the
safety of a Saw Palmetto extract due to the diversity
of extraction methods (Habib and Wyllie, 2004).
Although this extract appears to be more
concentrated than other commercial Saw Palmetto
extracts, we postulated that the absence of organic
solvents in the supercritical fluid extraction of Saw
Palmetto fruits, leading to the preparation of an
extract with a good safety profile. Due to their oily
nature, it is possible that Saw Palmetto extracts used
for the treatment of benign prostate hyperplasia
contain lipoperoxides; these substances must be
controlled due to their potential for promoting lipid
peroxidation in tissues. Noteworthy, the particular
Saw Palmetto extract used in this study did not lead
to an increase in basal lipoperoxidation of rat liver
microsomes (data not shown), a system routinely
used by our lab to test oxidative damage (Letelier et
al., 2007). The absence of pro-oxidant activity
corroborates the good safety profile of this Saw
Palmetto extract. In light of our data, we can
conclude that HiPower
displays a safety profile of at

least one order of magnitude in dosage.
The general strategy used for the present
study indeed allowed the assessment of potential
alterations in general homeostasis and morphological
features of key organs in xenobiotic
biotransformation (liver) and excretion (kidney).
Therefore, we conclude that this type of studies may
be adopted for future evaluations of safety profiles of
herbal extracts.
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Goldberg H, Neuhaus J , Hudes E, Shinohara K, Kane
C. 2008. A detailed safety assessment of a saw
palmetto extract. Complement Ther Med 16: 147-154.
Bent S, Kane C, Shinohara K, Neuhaus J , Hudes ES,
Goldberg H, Avins AL. 2006. Saw palmetto for
benign prostatic hyperplasia. New Eng J Med 354:
557-566.
Blumenthal M, Busse WR, Goldberg AD, Gruenwald J ,
Hall T, Riggins CW, Klein S, Rister RS. 1998. The
complete German Commission E monographs:
Therapeutic guide to herbal medicines. Ed. Lippincott
Williams & Wilkins, Austin, Texas, pp.74, 201, 432.

Boyle P, Robertson C, Lowe F, Roehrborn C. 2004.
Updated meta-analysis of clinical trials of Serenoa
repens extract in the treatment of symptomatic benign
prostatic hyperplasia. BJ U Int 93: 751-756.
Carraro J C, Raynaud J P, Koch G, Chisholm GD, Di
Silverio F, Teillac P, Da Silva FC, Cauquil J , Chopin
DK, Hamdy FC, Hanus M, Hauri D, Kalinteris A,
Marencak J , Perier A, Perrin P. 1996. Comparison of
phytotherapy (Permixon) with finasteride in the
treatment of benign prostate hyperplasia: a
randomized international study of 1,098 patients.
Prostate 29: 231-240.

Cristoni A, Morazzoni P, Bombardelli E. 1997. Chemical
and pharmacological study on hypercritical CO
2

extracts of Serenoa repens fruits. Fitoterapia 68: 355-
358.
de la Taille A, Buttyan R, Hayek O, Bagiella E, Shabsigh
A, Burchardt M, Burchardt T, Chopin DK, Katz AE.
2000. Herbal therapy PC-SPES: in vitro effects and
evaluation of its efficacy in 69 patients with prostate
cancer. J Urol 164: 1229-1234.
de la Taille A, Hayek OR, Buttyan R, Bagiella E,
Burchardt M, Katz AE. 1999. Effects of a
phytotherapeutic agent, PC-SPES, on prostate cancer:
a preliminary investigation on human cell lines and
patients. BJ U Int 84: 845-850.
Derakhshani P, Geerke H, Bhnert KJ , Engelmann U.
1997. Beeinflussung des Internationalen Prostata-
Symptomen-Score unter der Therapie mit
Sgepalmenfrchteextrakt bei tglicher Einmalgabe.
Der Urologe B 37: 384-391.
Ernst E. 2002. The risk-benefit profile of commonly used
herbal therapies: Ginkgo, St. J ohn's Wort, Ginseng,
Echinacea, Saw Palmetto, and Kava. Ann Intern Med
136: 42-53.
Gerber GS, Fitzpatrick J M. 2004. The role of a lipido-
sterolic extract of Serenoa repens in the management
of lower urinary tract symptoms associated with
benign prostatic hyperplasia. BJ U Int 94: 338-344.
Habib FK, Wyllie MG. 2004. Not all brands are created
equal: a comparison of selected components of
different brands of Serenoa repens extract. Prostate
Cancer Prostatic Dis 7: 195-200.
Hamid S, Rojter S, Vierling J . 1997. Protracted cholestatic
hepatitis after the use of prostata. Ann Intern Med
127: 169-170.
Hizli F, Uygur MC. 2007. A prospective study of the
efficacy of Serenoa repens, tamsulosin, and Serenoa
repens plus tamsulosin treatment for patients with
benign prostate hyperplasia. Int Urol Nephrol 39: 879-
886.
Letelier ME, Entrala P, Lpez-Alarcn C, Gonzlez-Lira
V, Molina-Berros A, Corts-Troncoso J , Jara-
Sandoval J , Santander P, Nez-Vergara L. 2007.
Nitroaryl-1,4-dihydropyridines as antioxidants against
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iron/ascorbate, nitrofurantoin and naphthalene.
Toxicol in Vitro 21: 1610-1618.
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values for young normal Sprague-Dawley rats: weight
gain, hematology and clinical chemistry. Hum Exp
Toxicol 15: 612-616.
Lowe FC, Fagelman E. 2004. Permixon: A review. Curr
Prostate Rep 2: 133-136.
Singh YN, Devkota AK, Sneeden DC, Singh KK,
Halaweish F. 2007. Hepatotoxicity potential of saw
palmetto (Serenoa repens) in rats. Phytomedicine 14:
204-208.
Small EJ , Frohlich MW, Bok R, Shinohara K, Grossfeld
G, Rozenblat Z, Kelly WK, Corry M, Reese DM.
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2010 The Authors

Comunicacin | Communication



Volatile Components from the Leaves of Solanum hypomalacophyllum
Bitter.
[Componentes voltiles de las hojas de Solanum hypomalacophyllum Bitter.]
Alida PREZ COLMENARES
1
, Luis B. ROJAS
1*
, Alfredo USUBILLAGA
1

1
Instituto de Investigaciones, Facultad de Farmacia y Bioanlisis. Universidad de Los Andes, Mrida - Venezuela.

Abstract
The essential oil fromthe leaves of Solanum hypomalacophyllum (Solanaceae) collected in May 2007 at Pramo La Culata (Mrida 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 Pramo La Culata (Estado de Mrida,
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 cido hexadecanoico
(5.2 %).
Palabras Clave: Solanum hypomalacophylllum; Solanaceae; aceite esencial; 5-octen-1-ol; GC-MS anlisis.

Recibido | Received: November, 18, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: J anuary, 10, 2010.
Publicado en Lnea | Published Online J anuary 21, 2010.
Financiacin | Funding: This work was supported by: Consejo de Desarrollo Cientfico, Humanstico y Tecnolgico (CDCHTMridaVenezuela, project FA-380-06-03-A),
FONACIT-PCP (46.255), University of Los Andes-Plan II.
This article must be cited as: Prez Colmenares A, Rojas L B., Usubillaga A. 2010. Volatile Components fromthe Leaves of Solanum hypomalacophyllum. Bol LatinoamCaribe
Plant Med Aromat 9(1):80 83. {EPub 21 J an 2010 }.
*Contactos | Contacts: E-mail: rojasl@ula.ve; Tel: 0058-2742403958 ; Fax: 0058-2742403455.
Prez Colmenares et al. Essential Oil of Solanum hypomalacophyllum


INTRODUCTION
The Solanaceae family comprises about 90
genera and 3.000 species which are widely
distributed in the world. They are a rich source of
active secondary metabolites (Coletto da Silva et al.,
2004). Within this family, the genus Solanum is the
largest and most complex with more than 1500
species(Chowdhury et al., 2007) which yield a great
variety of steroidal saponins and glycoalkaloids of
interest from ecological and human health viewpoints
(Roddick et al., 2001).

Numerous species of Solanum
are known to possess a variety of biological activities
including antimycotic (Gonzales et al., 2004; Singh et
al., 2007), antiviral (Arthan et al., 2002),
molluscicidal (Silva et al., 2006), teratogenic (Keeler
et al., 1990), and cytotoxic properties (Nakamura et
al., 1996; Lu et al., 2009). Previous studies reported
the chemical composition of the essential oil of
several species of Solanum, like S. verbascifolium
(Ma et al., 2006), S. lyratum (Xu et al., 2006), S.
aculeastrum (Koduru et al., 2006) and of S.
pseudocapsicum (Aliero et al., 2006, 2007), but no
reports have been published on the composition of
the volatile constituents of S. hypomalacophyllum
Bitter. This species is a small tree native to the
Venezuelan Andes where it grows wild in humid
places at altitudes between 2150 and 3200 meters.
Previous studies reported the isolation of 4-keto
steroidal alkaloids solaphyllidine,
deacetoxysolaphyllidine, desacetylsolaphyllidine,
spirosolaphyllidine and solamaladine from green
berries and leaves (Usubillaga, 1970, 1984;
Usubillaga et al., 1982; Alarcon et al., 2006).
Therefore, in continuation of our study of the
Venezuelan Solanaceae, we report here the first study
on the essential oil of S. hypomalacophyllum in order
to obtain knowledge on its chemical composition.
Plant Material
Leaves of Solanum hypomalacophyllum were
collected in May 2007, in the area of El Pramo de la
Culata, Mrida State, Venezuela at 2800 m above sea
level. A voucher specimen has been deposited at the
MERF Herbarium (Herbarium of the Faculty of
Pharmacy and Bioanalysis in Mrida, Venezuela).
The botanical identification was made by Forest
Engineer J uan Carmona, MERF Herbarium.
Extraction and analysis of the essential oil
Fresh leaves (2000 g) were cut into small
pieces and submitted to hydrodestillation for 3 h
using a Clevenger-type apparatus. 0.1 mL of essential
oil was obtained that corresponds to a yield of
0.005%
v
/
w
.
The composition of the essential oil was
determined by comparison of the mass spectrum of
each component with Wiley GC/MS library data and
also from retention index (RI) data (Morteza et al.,
2004).
Gas chromatography
GC analyses were performed using a Perkin-
Elmer Autosystem gas chromatograph equipped with
a FID detector and data-handling system. A 5%
phenylmethylpolysiloxane fused-silica capillary
column was used (30 m x 0.25 mm i.d., film
thickness 0.25 m; HP-5, Hewlett-Packard, CA,
USA). The oven temperature was programmed from
60 C to 260 C at 4 C/min. The injector and detector
(Flame ionization detector, FID) temperatures were
200C and 280 C, respectively. The carrier gas was
helium at 0.8 mL/min. The sample (1.0 L) was
injected using a split ratio of 10:1. Retention indices
were calculated by comparing the retention times of
the eluting peaks with those of standard C
8
-C
24
n-
alkanes. The percentage composition of the oil was
calculated by the normalization method from the GC
peak areas.
Gas chromatography mass spectrometry
GC-MS analyses were carried out on a Model
5973 Hewlett-Packard GC-MS system fitted with a
HP-5MS fused silica column (30 m x 0.25 mm i.d.,
film thickness 0.25 m, Hewlett-Packard). The oven
temperature program was the same as that used for
the HP-5 column for GC analysis; the transfer line
temperature was programmed from 150C to 280C;
source temperature, 230C; quadrupole temperature,
150C; carrier gas, helium, adjusted to a linear
velocity of 34 cm/s; ionization energy, 70 eV; scan
range, 40:500 amu; 3.9 scans/s. Sample (1.0 L) was
injected using a Hewlett-Packard ALS injector with a
split ratio of 50:1. The identity of the oil components
was established from their GC retention indices, by
comparison of their MS spectra with those of
standard compounds available in the laboratory, and
by a library search (NIST 05 and Wiley)(Adams,
1995).


Hydrodistillation of the leaves yielded a
yellowish liquid oil in 0.005 %
v
/
w
yield. The
chemical composition of the oil was investigated
using both GC and GC-MS techniques. It was
possible to identify 11 components, which represent
92.4% of the total oil. These compounds with their
retention indices (RI) and relative percentage
concentrations are listed in Table 1 according to the
elution order on the HP-5 column by GC and by
comparison of the fragmentation pattern. The oil was
found to contain alcohols (78.2%), fatty acid (5.2%)
and aldehydes (4.8%) as major components. The
dominant compounds were 5-octen-1-ol (39.7 %),
cis-3-hexenol (20.2 %), 1-hexanol (12.0 %) and
hexadecanoic acid (5.2 %).
Table 1. Percentage composition of the essential oil of the leaves
of Solanum hypomalacophyllum.

Peak N Compounds Area (%) RI
1 Trans-3-hexenol 1.4 844
2 Cis-3-hexenol 20.2 850
3 1-Hexanol 12.0 862
4 Cis-4-heptenal 4.8 898
5 1-heptanol 1.6 967
6 5-octen-1-ol 39.7 1071
7 1-nonanol 1.4 1174
8 1-decanol 1.9 1276
9 -ionone 1.8 1497
10 Butylated-hydroxytoluene 2.4 1523
11 Hexadecanoic acid 5.2 1961
Total 92.4
RI: retention index
CONCLUSION
The chemical composition of the essential oil
from leaves the Solanum hypomalacophyllum contain
5-octen-1-ol (39.7 %), cis-3-hexenol (20.2 %), 1-
hexanol (12.0 %) and hexadecanoic acid (5.2 %). as
major components. These compounds are reported
for the first time in this species.
ACKNOWLEDGMENTS
The authors would like to thank the financial
support of Consejo de Desarrollo Cientfico,
Humanstico y Tecnolgico (CDCHTMrida
Venezuela, project FA-380-06-03-A), FONACIT-
PCP (46.255), University of Los Andes-Plan II and
Forest Engineer J uan Carmona (MERF Herbarium,
Facultad de Farmacia y Bioanlisis) for the
identification of the botanical material.
REFERENCES
Adams R. 1995. Identification of Esential Oil Components
by Gas Chromatography/Mass Spectrometry. (USA).
Alarcon L, Velasco J , and Usubillaga A. 2006.
Determinacin de la Actividad Antibacteriana de los
Alcaloides presentes en los Frutos Verdes del
Solanum hypomalocophyllum Bitter. Rev Latinoam
Qum 34: 13-21.
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Chemical Composition of the Esentail Oil from
Solanum pseudocapsicum. J Biol Sci 9: 1175-1177.
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Arthan D, Svasti J , Kittakoop P, Pittayakhachonwut D,
Tanticharoen M, Thebtaranonth Y. 2002. Antiviral
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the Fruits of Solanum torvum. Phytochemistry 59:
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Alvarez, L. 2004. Antimycotic Spirostanol Saponins
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Keeler R, Baker D, Gaffield W. 1990. Spirosolane-
Containing Solanum Species and Induction of
Congenital Craniofacial Malformations. Toxicon 28:
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Koduru S, Aseku, O, Grierson D, Afolayan A. 2006.
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acueslastrum (Solanaceae). J Essent Oil-Bear Plant 9:
65-66.
Lu Y, Luo J , Huang X, Kong L. 2009. Four New Steroidal
Glycosides from Solanum torvum and their Cytotoxic
Activities. Steroids 74: 95-101.
Ma R., Guo S., Zhu H., Zhang T. 2006. Chemical
Constituents of Esential Oil from Leaves of Solanum
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526-529.
Morteza K, Azadbkht M, Goodarzi A. 2004. The Essential
Oils Composition of Phlomis herba-venti L. Leaves
and Flowers of Iranian Origin. Flavour Fragance J 19:
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Nakamura T, Komori A, Lee Y, Hashimoto F, Yahara S,
Nohara T, Ejima A. 1996. Cytotoxic Activities of
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Usubillaga A. 1970. Structure of solaphyllidine, a novel 4-
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Usubillaga A. 1984. Alkaloids from Solanum
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Usubillaga A, Zabel V, Watson W. 1982. The Revised
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pyrrolin-2-yl)-23,24-dinor-5-cholane-4,22-dione. Acta
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Silva T, Camara C, Agra F, de Carvalho M, Frana M,
Brandoline, S, da Silva L, Braz-Filho R. 2006.
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Fitoterapia 77: 449-452.
Xu S, Wang L, Li R, Liu H. 2006. Analysis of Chemical
Constituents of the Volatile Oil from Solanum lyratum
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Activities. Nat Prod Res 21: 585-590.


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is a supercritical fluid extract from Saw

was calculated from the 1X

1X group) and 280l (HiPower

1X group), or 10X (HiPower

10X cohorts), as detailed in Material and

10X cohorts), as detailed in

10X cohorts), as detailed in Material and Methods. Platelets were

10X cohorts), as detailed in Material and

10X cohorts), as detailed in

treatment did not change the following

(8 and 10 days of treatment, p<0.001) and

(6 and 8 days of treatment, p<0.001).

cohorts showed some apoptotic

, compared with rats fed with vehicle

cohorts. Since lactate dehydrogenase

displays a safety profile of at

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